Anti-inflammatory

  1. Protects DNA from harmful molecular scavengers that disrupt and damage the DNA.
  2. Dilates blood vessels to increase blood flow and allows more oxygen to reach the outer extremities of the body.
  3. Reduces inflammation produced from stress. Stress causes hyperactive adrenal glands in the brain and balanced glands are essential for optimal brain function.
  4. Detoxifies airborne pollutants.
  5. Provides an ample supply of healthy fats supplied from vegetable extracts within the super formula. Healthy fats reduce unwanted cravings and leave you feeling more satisfied.
  6. Increased energy.
  7. Balances of gut flora and important enzymes used to break down food, resulting in better digestion
  8. Provides an excellent source of fiber and minerals
  9. Increases brain function

Broccoli Extract

Broccoli Sulforaphane

Combat Depression and Anxiety. Inflammation has been recognized as one of the causes of depression. By reducing inflammation, sulforaphane can help combat depression.

Repeated SFN administration reverses depression– and anxiety-like behaviors in chronically stressed mice, likely by inhibiting the hypothalamic-pituitary-adrenal (HPA) axis and inflammatory responses to stress.

In another study, it was shown that Nrf2 deficiency in mice results in depressive-like behavior, while the induction of Nrf2 by sulforaphane form broccoli has antidepressant-like effects.

The endogenous protein, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), is a member of the TNF superfamily and has been shown to induce apoptosis in cancer cells but not normal cells; for this reason, TRAIL has become a recognized target for cancer therapy (Ashkenazi et al., 2008). TRAIL binds to TRAIL-R1 or TRAIL-R2, two death domain-containing receptors, also called DR4 and DR5, to trigger apoptosis. Unfortunately, a considerable range of cancer cells, especially some highly malignant tumors, are resistant to induction of apoptosis by TRAIL. Resistance to TRAIL can occur at different points in the signaling pathways of TRAIL-induced apoptosis, including mutations in the death receptors, defects in the molecules involved in formation of the death-inducing signaling complex (DISC), dysregulation of DISC activation by TRAIL receptor antagonists or by over-expression of cellular FLICE-like inhibitory protein (cFLIP) (Merino et al., 2007). Therefore, developing strategies to overcome such resistance are important for the successful use of TRAIL for cancer therapy.

A large number of studies have revealed that combination of TRAIL with chemotherapy or radiotherapy significantly enhances cytotoxicity to tumors or reverses the resistance to monotherapy (Merino et al., 2007). However, the use of conventional chemotherapeutic drugs or radiotherapy in association with TRAIL could be limited due to their severe toxic side effects and their potential to induce cell death processes in both malignant and non-malignant cells (Meurette et al., 2006). In recent years, efforts have been focused on the development of biologically based strategies to enhance the anti-tumor activity of TRAIL without the toxic side effects of chemotherapy or radiotherapy (Ashkenazi et al., 2008). Among these, the flavonoids and glucosinolates, which are ‘natural antioxidants’ known to scavenge free radicals, have emerged as promising compounds to overcome resistance of cancer cells to TRAIL, while having little or no effect on normal cells (Ishibashi and Ohtsuki, 2008). Resveratrol which is found in numerous plant species, including mulberries, peanuts and grapes can also enhance TRAIL-induced

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apoptosis in prostate, colon and melanoma cancer cells (Ishibashi and Ohtsuki, 2008). Resveratrolinduced sensitization to TRAIL was shown to occur through multiple mechanisms including cFLIP downregulation in melanoma cells (Ivanov et al., 2008) or redistribution of agonistic TRAIL receptors within lipid rafts in colon carcinoma cells (Delmas et al., 2004). Other antioxidants found to enhance the apoptotic properties of TRAIL include Broccoli sulphoraphane, indole-3-carbinol, apigenin and quercetin (Ishibashi and Ohtsuki, 2008).

Induction of procaspase-3, procaspase-8 and procaspase-9 cleavage followed by Bid truncation and cytochrome c release from the mitochondria is probably the most frequent mechanism by which these antioxidants are found to enhance TRAIL-induced apoptosis. Strikingly, in a large number of reports, TRAIL-induced sensitization by flavonoids or sulphoraphane was also associated with production of reactive oxygen species (ROS). Likewise, sulphoraphane (from broccoli) was found to enhance TRAILinduced apoptosis in a prostate cancer cell line (PC-3) through the generation of intracellular ROS, leading to collapse of mitochondrial membrane potential, activation of caspase-3 and caspase-9, and upregulation of DR4 and DR5 (Ishibashi and Ohtsuki, 2008). Another study showed that resveratrol in combination with TRAIL resulted in generation of ROS, translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins into the cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. More recently, ROSinduced up-regulation of DR5 was also reported to account for TRAIL-induced sensitization by baicalein (Taniguchi et al., 2008). The common theme from these studies, with a range of antioxidants in combination with TRAIL, is that generation of ROS seems to trigger signal transduction culminating in cell cycle arrest and/or apoptosis.

Sulforaphane from broccoli

helps the body detoxify airborne pollutants, pesticides, and heavy metals, by activating detoxification systems, mainly the Phase II enzymes.

In a farming community exposed to airborne pollutants, with a high risk of hepatocellular carcinoma, broccoli sprouts enhanced the detoxication of airborne pollutants and reduced the risks of cancer.

Sulforaphane

induces Phase II enzymes in the upper airway of human subjects (R). Phase II enzymes have important protective effects against diesel exhaust particles (DEP), ozone, and tobacco smoke.

Sulforaphane

reduces the pro-inflammatory and pro-allergic effects typically caused by exposure to diesel exhaust particle (R).

Sulforaphane

protects human white blood cells (lymphocytes) from pesticide-induced DNA damage.

Aflatoxin binds DNA and causes liver cancer. rats.

Sulforaphane

reduces the binding of aflatoxin to DNA in

Sulforaphane

inhibits the mutagenicity caused by heterocyclic amines (cooked food mutagens).

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Cadmium reduces testosterone, sperm motility, sperm count, and increases sperm deformity in mice. SFN improved sperm quality, testosterone, and antioxidant levels.

Olive Hydroxytyrosol Extract

Hydroxytyrosol

is the main antioxidant of virgin olive oil and, therefore, a potent free radical scavenger. As free radicals are the precursors of many health problems, numerous scientific studies have shown that the consumption of olive polyphenols, and especially Hydroxytyrosol (HTY), promotes positive health effects.

To understand the scope of Hydroxytyrosol and why we use it in the Pink formulation, first we have to explain what antioxidants and free radicals are.

Free radicals are very reactive chemical species, and harmful to the body, when they are in a situation of imbalance, that is, when their number increases what our body can eliminate with its endogenous antioxidants.

An excess of oxidants against antioxidants causes the appearance of oxidative stress and cellular damage, a situation that is aggravated under certain circumstances such as intense physical exercise, stress, pollution, smoking, alcoholism, poor nutrition and exposure to solar radiation. Many of the most common human diseases are the result of the action of free radicals against cells, proteins, etc.

Antioxidants, in turn, are compounds that inhibit or retard the oxidation of certain molecules, preventing the initiation or propagation of oxidative reactions in chain caused by free radicals. As consequence, the contribution of antioxidants to the body is fundamental to protect cells against oxidative stress.

Polyphenols, source of antioxidants:

Experimental evidence from human and animal studies (both in vivo and in vitro) shows that Hydroxytyrosol and other phenolic compounds of olive oil have antioxidant biological activities.

Hydroxytyrosol, is a highly bioavailable phytochemical with a high antioxidant capacity, responsible for most of the benefits of olive polyphenols in health.

According to plenty of scientific studies around the world, the main properties attributed are:

Prevention of cardiovascular diseases (CVD). The cardioprotective capacity of Hydroxytyrosol is endorsed by the EFSA with a health claim for its inhibitory activity on the oxidation of LDL lipoproteins, which leads to diseases such as atherosclerosis. HT also inhibits platelet aggregation that occurs in thrombosis and protects from cell death induced by reactive oxygen intermediates (as it happens in situations of ischemia).

Anti-inflammatory. HTY has been shown to inhibit the production of pro-inflammatory compounds caused by human leukocytes such as leukotrienes.

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Anti-aging. The antioxidant capacity of Hydroxytyrosol is superior to other antioxidants such as vitamin C and E. It plays an important role in delaying cellular aging processes as blocking the appearance of external signs such as wrinkles, sagging and spots. And due to the fact that HTY contains lipophilic and hydrophilic properties, Hydroxytyrosol turns to be a molecule very suitable for cosmetic use. Antimicrobial. Because it has potent antimicrobial properties against different types of bacteria, viruses and fungi.

Anticancer. For its chemo preventive properties (try to reduce the risk, delay the cancer or prevent it from returning). This is because HT can inhibit the initiation and promotion phases of carcinogenesis. This prevents DNA damage induced by different genotoxic species and induces cell death in different tumor lines, such as breast and colon cancer.

Protection against UV radiation. Hydroxytyrosol is able to prevent damage on melanoma cells when humans are exposed to harmful UV radiation. This is because HT reduces, significantly, the breakdown of DNA strands caused by UVB radiation. The reason is the capacity of this molecule to inhibit the proliferation of reactive species caused by incident radiation.

Prevention of neurodegenerative diseases. Several studies suggest that reactive oxygen species (ROS) are involved in the etiology of many degenerative diseases such as cardiovascular, diabetes, cancer or Alzheimer’s. In this case, Hydroxytyrosol exerts an antioxidant protection against the oxidative damage induced by several types of reactive species of oxygen and nitrogen in the nervous tissue.

Protection against ARMD (age-related macular degeneration). This is a disease related to age and other harmful effects such as smoking, which causes blindness in people over 55 years. Hydroxytyrosol properties such as antioxidant, anti-inflammatory and protection of the pigment epithelium, minimize the appearance and development of this disease.

Citrulline Extract

L-citrulline

is an amino acid normally made by the body. The body converts L-citrulline to L-arginine, another type of amino acid. L-arginine improves blood flow. It does so by creating nitric oxide (NO), a gas that helps dilate blood vessels. This amino is used in our Pink formulation because it has many health benefits, including increased strength and stamina, improvement in erectile dysfunction, and improved heart health.

Benefits of L-Citrulline

1) L-Citrulline Decreases Fatigue and Muscle Soreness

2) L-Citrulline May Increase Exercise Capacity

3) L-Citrulline Reduces Erectile Dysfunction

4) L-Citrulline Increases Nitric Oxide

5) L-Citrulline May Increase Blood Flow

6) L-Citrulline Decreases Blood Pressure

7) L-Citrulline Improves Heart Function

8) L-Citrulline May Increase Growth Hormone

9) L-Citrulline May Boost Immunity

10) L-Citrulline May Help Treat Sickle Cell Anemia

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11) L-Citrulline Helps Treat Urea Cycle Disorders

Below I have shortened the NO Sycle form the long version.

Short version L-citrulline is one of the three amino acids in the urea cycle (the other two being L-arginine and Lornithine). The urea cycle converts a toxic compound called ammonia into urea, which is then eliminated from the body through urine. Therefore, I like to use L-citrulline in our Pink formula rather than beet root extracts to increase Nitric Oxide.

After ingestion, L-citrulline is converted to L-arginine by the kidneys and is released into the bloodstream by the kidneys so that it is available to the whole body.

L-arginine, a precursor to nitric oxide. Nitric oxide synthase produces nitric oxide from L-arginine. Nitric oxide plays a crucial role in the immune system and heart and blood vessel health. Many of the benefits of L-citrulline are due to L-arginine and nitric oxide.

L-Citrulline

is also formed as a by-product in the production of nitric oxide from arginine. It can be recycled back to arginine in what is known as the citrulline-NO cycle.

Long version for science geeks.

L-citrulline is converted to L-arginine by argininosuccinate synthase. L-arginine is in turn responsible for citrulline’s therapeutic effects. Many of L-arginine’s activities, including its possible anti-atherogenic actions, may be accounted for by its role as the precursor to nitric oxide or NO. NO is produced by all tissues of the body and plays very important roles in the cardiovascular system, immune system and nervous system. NO is formed from L-arginine via the enzyme nitric oxide synthase or synthetase (NOS), and the effects of NO are mainly mediated by 3′,5′ -cyclic guanylate or cyclic GMP. NO activates the enzyme guanylate cyclase, which catalyzes the synthesis of cyclic GMP from guanosine triphosphate or GTP. Cyclic GMP is converted to guanylic acid via the enzyme cyclic GMP phosphodiesterase.

NOS is a heme-containing enzyme with some sequences similar to cytochrome P-450 reductase. Several isoforms of NOS exist, two of which are constitutive and one of which is inducible by immunological stimuli. The constitutive NOS found in the vascular endothelium is designated eNOS and that present in the brain, spinal cord and peripheral nervous system is designated nNOS. The form of NOS induced by immunological or inflammatory stimuli is known as iNOS. iNOS may be expressed constitutively in select tissues such as lung epithelium.

All the nitric oxide synthases use NADPH (reduced nicotinamide adenine dinucleotide phosphate) and oxygen (O2) as cosubstrates, as well as the cofactors FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), tetrahydrobiopterin and heme. Interestingly, ascorbic acid appears to enhance NOS activity by increasing intracellular tetrahydrobiopterin. eNOS and nNOS synthesize NO in response to an increased concentration of calcium ions or in some cases in response to calcium-independent stimuli, such as shear stress. In vitro studies of NOS indicate that the Km of the enzyme for L-arginine is in the micromolar range. The concentration of L-arginine in endothelial cells, as well as in other cells, and in plasma is in the millimolar range. What this means is that, under physiological conditions, NOS is saturated with its L-arginine substrate. In other words, L-arginine would not be expected to be ratelimiting for the enzyme, and it would not appear that supraphysiological levels of L-arginine which could

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occur with oral supplementation of the amino acid would make any difference with regard to NO production. The reaction would appear to have reached its maximum level. However, in vivo studies have demonstrated that, under certain conditions, e.g. hypercholesterolemia, L-arginine could enhance endothelial-dependent vasodilation and NO production.

A study of 18 men with fatigue found that man reported a reduction in fatigue after phosphate, which is a form of energy reserves in the muscles.

L-citrulline

reduced

exercise.

L-Citrulline

fatigue and increased ATP levels. Every also increased the production of creatine

Another study (DB-RCT) of 41 men showed that citrulline malate reduced fatigue. Those receiving citrulline were able to do more reps per set of bench presses and experienced less soreness after

exercise.

A study (DB-RCT) on 22 men found that compared to placebo.

Mulberry Extract

L-citrulline

increased cycling speed and reduced muscle fatigue

Mulberry (Morus alba) is a small tree native to northern China. During ancient times its primary uses were to feed silkworms and treat various illnesses.

Morus alba contains many unique, active compounds and flavonoids that give it its broad range of benefits. Mulberry can have its leaves and roots prepared in a tea and its berries are eaten whole or made into wine.

Constituents of Morus Alba Morus alba contains many natural polyphenols and flavonoids, which have antioxidants, antiinflammatory, anti-cancer, antibacterial, and anti-aging properties.

Compared to the stems and fruits, the leaves contain more phenolic compounds and flavonoids.

Some of the compounds in

Morus alba

include:

Rutin Apigenin Luteolin Morin Isoquercitrin Anthocyanins Resveratrol (and its derivatives, like oxyresveratrol) Caffeic acid Gallic acid Umbelliferone Chlorogenic acid Kuwanon C and G Additionally, Morus alba contains many constituents that are unique to the plant:

Moracin has anti-inflammatory, antioxidant, and anticancer properties

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Albanol A

is toxic to cancer cells and can induce cell death (apoptosis) Albosteroid has antioxidant and anti-ulcer properties

Health Benefits of Morus Alba

1) Morus Alba Is Anti-Inflammatory

2) Morus Alba May Boost the Immune System

3) Morus Alba Improves Heart Health

4) Morus Alba Helps Treats Diabetes

5) Morus Alba May Lower Cancer Risk

6) Morus Alba May Reduce Fatigue

7) Morus Alba Is Antimicrobial

8) Morus Alba May Improve Memory and Learning

9) Morus Alba May Help with Anxiety

10) Morus Alba Might Help Treat Depression

11) Morus Alba Might Improve the Stress Response

12) Morus Alba Might Help Prevent Obesity

Ellagic Acid Extract

An antioxidant, which means that it targets disease-causing free radicals throughout the body. In certain clinical studies, ellagic acid has even been shown to slow the growth of tumors.

Benefits of

Ellagic

Acid

Ellagic

is purported to have anti-cancer properties. It’s been shown to cause cell death in cancer cells in laboratory settings, although it has limited cancer-fighting evidence in all human trials performed thus far.

acid

Supporters of

ellagic

acid

believe the compound comes with the following benefits:

Reduces the Effect of Estrogen in Promoting the Growth Of Breast Cancer Cells In Tissue Cultures Helps the Liver Break Down and Remove Cancer-causing Substances (Carcinogens) From the Blood Reduces the Risk of Heart Disease, Birth Defects, And Liver Problems Encourages Wounds to Heal More Quickly

Artichoke Extract

Artichoke

come from the globe artichoke (Cynara Scolymus) or the cardoon (Cynara Cardunculus) plants. It has been historically used to treat stomach problems, gout, and diabetes.

extracts

Artichoke

has a high mineral content. It also contains vitamin C, fiber, inulin, and polyphenols. Soil, climate, source, and part of the plant all affect its mineral content. The edible parts, or the artichoke heart, contain more nutrients than the leaves. Polyphenolic compounds with medicinal effects are mainly found in the leaves and are responsible for the active principles of the plant.

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Artichoke

contains luteolin, caffeoylquinic acid, chlorogenic acid, apigenin, sterols, and inulin. It also contains potassium, magnesium, calcium, sodium, zinc, copper, and manganese.

extract

Artichoke

are rich in caffeoylquinic acid and luteolin. The edible parts (artichoke also rich in caffeoylquinic acids and various luteolin derivatives.

Leaf

extracts

heart) are

The main polyphenols in luteolin 7-O-glucoside.

artichoke

extract

are chlorogenic acid, cynarin, luteolin 7-O-rutinoside, and

Luteolin is an antioxidant, which helps prevent inflammation and cancer.

Caffeoylquinic acid is another antioxidant. It helps form bonds with toxic compounds, which helps lower toxicity. Cynarin is another antioxidant compound found in artichoke. It stimulates bile production, which helps the gut digest fats and absorbs vitamins from food. Inulin is a sweet-tasting indigestible starchy substance found in artichoke. It is a prebiotic, which mean it can increase the number of beneficial microorganisms in the intestines. Cynarin, a natural compound found in artichoke, stimulates bile production. This helps accelerate gut movement and also support fat digestion and vitamin absorption. Artichoke extract also helps with digestion by stopping muscle spasms in the gut. Artichoke aids digestive health and can help patients suffering from indigestion, bloating, nausea, and heartburn (collectively called dyspepsia) and irritable bowel syndrome.

In a study, researchers treated 247 patients with functional dyspepsia with either globe artichoke leaf extract or placebo (DB-RCT). The artichoke group improved dyspepsia symptoms compared to placebo.

The globe artichoke extract was especially effective in easing fullness and reducing flatulence. However, the artichoke extract was barely more effective than the placebo in reducing the pain and nausea caused by indigestion. Irritable Bowel Syndrome (IBS) is a gastrointestinal disorder that affects 22% of the population. Some symptoms include abdominal pain, diarrhea, flatulence, and constipation (R).

Many factors contribute to IBS: abnormal gut movement, imbalanced intestinal microbes, genetic factors, inflammation, and immune disorders, among others. Artichoke has both preventive and curative effects on IBS. It balances the intestinal microbiota and also stops muscle spasms.

Artichoke

Balances the Gut Flora Inulin, a starchy substance found in artichoke, balances the intestinal microbiota. In a controlled, double-blind crossover study, daily consumption of inulin derived from globe artichoke extract increased beneficial microorganisms in the gut of healthy adult volunteers.

Most of the inulin’s effects are caused by bacterial metabolites. The metabolites use the inulin for energy. The metabolites, such as SCFA, then acidifies the colonic environment. This is beneficial for healthy bacteria such as bifidobacteria and lactobacilli. The acidic environment also stops the growth of potentially harmful species.

Artichoke

extract

globe

artichokes,

artichoke

extract

also supports healthy gut movement. Dichloromethane and ethyl acetate extracts of along with cynaropicrin, stop muscle spasms in the gut. In guinea pig intestines, the constituents inhibited spasmic gut contractions. This reduces abdominal pain and

diarrhea.

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In a study of IBS patients who took globe artichoke leaf extract, the patients had a significant reduction in IBS symptoms. 96% of the patients rated the extract as better than or equivalent to previous IBS therapies, such as antidiarrheals, laxatives, and anti-depressants.

In another study, 208 patients with both IBS and dyspepsia were administered globe artichoke leaf extract capsules. After two months of treatment, IBS symptoms decreased significantly. The patients also reported less constipation and diarrhea.

Blueberry Anthocyanin Extract

Blueberries

are a small, blue-purple fruit that belong to the genus vaccinium, which also includes cranberries and bilberries. Blueberries are a popular food and frequently supplemented. The antioxidant and anthocyanin content of blueberries makes them particularly effective at reducing cognitive decline, supporting cardiovascular health, protecting the liver, and reducing liver fat buildup.

Blueberries

also have a potential nootropic effect. They have been found to improve cognition in people undergoing cognitive decline, but there is also some rodent evidence that suggests blueberries can improve cognition in healthy young people as well. They also have a role to play in promoting the growth of nervous tissue and reducing neurological inflammation.

Anthocyanins

enhance heart health, according to a 2010 report published in Nutrition Reviews. The report’s authors note that anthocyanins appear to improve cholesterol levels and blood sugar metabolism, as well as fight oxidative stress (a process known to play a role in heart disease).

Dietary intake of anthocyanins also helps prevent high blood pressure (a major risk factor for heart disease), according to a 2011 study from the American Journal of Clinical Nutrition.

Preliminary research suggests that anthocyanins may protect against obesity. In a 2008 study from the Journal of Agricultural and Food Chemistry, scientists found that mice fed an anthocyanin-enriched high-fat diet for eight weeks gained less weight than mice fed a high-fat diet without anthocyanins.

Anthocyanins

may aid in the prevention of breast cancer, according to a laboratory study published in Phytotherapy Research in 2010. In test-tube experiments, scientists showed that anthocyanins extracted from blueberries helped inhibit the growth of breast cancer cells.

Health Benefits

1) Blueberries Can Help Improve Brain Function

2) Blueberries Can Decrease the Risk for Heart Disease

3) Blueberries Can Potentially Lower Blood Pressure

4) Blueberries Can Help Manage Diabetes

5) Blueberries Can Help Maintain Healthy Bones

6) Blueberries May Prevent Different Forms of Cancer

7) Blueberries Can Potentially Increase Lifespan

8) Topical Blueberry Extract Can Reduce Wrinkles

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9) Blueberries Can Contribute to Weight Loss

10) Blueberry Husks with Probiotics Help Alleviate Inflammatory Bowel Disease

11) Blueberries Can Potentially Prevent Urinary Tract Infections

12) Blueberries Can Help Detoxify Heavy Metals

Anthocyanins

have multiple beneficial and protective effects on the body, being one of the most important for the prevention of oxidation or damage to biological structures like fats, proteins, and nucleic acids. These effects are attributed to their chemical structure.

Anthocyanins

produce a wide range of colors in plant tissues. Anthocyanins neutralize the potentially harmful effects of free radicals in the body. Anthocyanins can be directly incorporated into (endothelial)

cells, protecting them from oxidative damage.

Although anthocyanins can directly inhibit oxidative stress, anthocyanins can also detoxify heavy metals (chelation) and exert their effects through direct protein binding. Lab tests have demonstrated that anthocyanins stimulate detoxifying enzymes and antioxidation in cultured cells.

Anthocyanins

in blueberries have been associated with memory and thinking improvement as well as the mitigation of neurodegeneration. Anthocyanins can cross the blood-brain barrier and travel to specific areas of the brain (hippocampus) that are associated with learning and memory. They inhibit several enzymes in the brain (JNKs, ASK1, and p38 pathways), which decreases inflammation and promotes neuronal health. Anthocyanins enhance brain function by stimulating the growth and development of neurons (through changes in synaptic plasticity and neurogenesis).

Wheatgrass Extract

Wheatgrass

extract is derived from freshly cut, young, vital wheatgrass sprouts. Using a proprietary process that enables almost 100% retention of bioactive results in a stable extract that lasts for many years. The process takes about three months to complete under strictly controlled conditions. As a result, wheatgrass extract contains higher antioxidant levels than fresh wheatgrass juice.

Wheatgrass extract

is a slightly brown, clear liquid, unlike green fresh wheatgrass juice. It is brown because there is no chlorophyll remaining in the extract. (Instead of “chlorophyll”, think “Grass Juice Factor.”) Wheatgrass extract does not have the odor and taste of fresh wheatgrass but still retain full biological activity in it. Many people find it difficult to tolerate the odor and taste of fresh wheatgrass juice. Wheatgrass extract is well received by all ages including young children. Wheatgrass extract is shelf-stable for at least two years at room temperature. Fresh wheatgrass juice only lasts a few days if refrigerated.

In 2006 a group of Indian scientists (Kulkarni et. al.) performed an important study prompted by the knowledge that the search is on for plant products with high antioxidant activities Antioxidant levels (phenolic and flavonoid compounds) of water-based (i.e. wheatgrass juice) and alcohol-based extractions of wheatgrass were measured from plants grown under different growing conditions over periods from 6 to 15 days. These conditions included tap water, tap water with nutrients, soil and tap water and soil with nutrients.

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Wheatgrass extract

contains higher antioxidant levels than fresh juice As you know, antioxidants are a much-discussed topic.

Basically, as oxygen is used up by the body’s normal metabolic processes, potentially damaging chemicals called ‘free radicals’ are produced. Environmental chemicals can also increase free radical levels. In turn, the free radicals attack other cells in the body, oxidizing them and causing damage. This oxidizing process is somewhat similar to when the cut surface of an apple turns brown when exposed to the air.

Pineapple Bromelain Extract

Bromelain, found in pineapple extracts, is an anti-inflammatory enzyme. It helps reduce pain, has anticancer activity, and improves digestive health. In addition, it even helps with weight loss. The composition of bromelain depends on the method of purification and the source of the pineapple

extract.

Fruit

Stem

bromelain

bromelain

is prepared using cooled pineapple juice, which goes through ultra-filtration.

is made when

pineapple stems

are centrifuged, filtered, lyophilized, and freeze-dried.

Bromelain

is a mixture of different thiol endopeptidases, which are enzymes that break down proteins. It also contains other enzymes such as phosphatase, glucosidase, peroxidase, cellulase, escharase, and different protease inhibitors. Protease inhibitors stop the protease enzymes from breaking down proteins. Bromelain’s anti-inflammatory action and inhibition of platelet aggregation are likely due to its protease (protein-digesting) activity. However, it inhibits tumor growth and help heal burns by other means than digesting proteins.

Commercially available bromelain is evaluated according to its proteolytic activity and other effects on health, like its anti-inflammatory effects and tumor inhibition.

Health Benefits of Bromelain

1) Bromelain Helps with Weight Loss

2) Bromelain Helps the Immune System

3) Bromelain Reduces Inflammation

4) Bromelain Helps Stop Cancer Growth

5) Bromelain Helps Treat Arthritis

6) Bromelain Treats Allergy Symptoms

7) Bromelain Reduces Pain and Swelling

8) Bromelain Improves Intestinal Health

9) Bromelain Protects the Heart

10) Bromelain Helps Heal Skin

11) Bromelain Potentially Reduces the Risk of Alzheimer’s Disease

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Bromelain

helps with weight loss due to its effects on fat (adipose) tissue. In rat cell cultures, stem bromelain administration inhibited the formation (differentiation) of fat cells. It does this by increasing genes (C/EBPα and PPARγ) that are needed for fat cell formation. Also, it blocked Akt/mTOR signaling (transmission) and increased TNF-α levels in mature fat cells. This caused fat cells to self-destruct. TNF-α induces the breakdown of fats (lipolysis). All of these factors combined together help prevent and address obesity. Bromelain supports healthy immune responses. Bromelain activates these inflammatory proteins IL-1β, IL-6, IFN-γ in the presence of cellular stress, but reduce the levels of these proteins when there is too much inflammation.

In mice cell cultures, bromelain both inhibited and enhanced T cell responses. Under normal conditions, bromelain enhanced the T cell response to help the immune system stay healthy. At the same time, it inhibited IL-2 production in mice. These conflicting effects are due to the different kinds of proteases in bromelain. On the other hand, when T cells are inappropriately activated during autoimmune or infectious diseases, bromelain inhibits T cell activation. This helps fight against immune diseases.

Bromelain

decreases the majority of pro-inflammatory mediators and is a powerful anti-inflammatory agent. Cyclooxygenase-2 (COX-2) is a major contributor to inflammation. It helps with the synthesis of prostaglandin E2 (PGE-2), which is a pro-inflammatory fat (lipid). PGE-2 also suppresses the immune system and promotes tumor progression. Bromelain reduces COX-2 and PGE-2 levels in mouse and human cell cultures. When inflammation causes the overproduction of proinflammatory cytokines, bromelain reduces IL-1β, IL-6 and TNF-α secretion. For example, bromelain reduces IFN-γ and TNF-α production in inflammatory bowel disease (IBD).

Bromelain

also lowers the production of TGF-β, another major contributor of inflammation.

In mouse cell cultures, the proteases in bromelain inhibited ERK-2 transmission. This inhibition blocks cytokine production and helps prevent inflammation.

Acai Berry Extract

There are many healthy berries you can add to your diet. The acai berry is one of the healthiest berries you will ever find. Acai is an indigenous berry found in the rain forests of the Amazon. This fruit has been heralded for centuries as a healing, immune-stimulating, energy-boosting fruit. Research reveals this antioxidant-rich berry may help suppress and repair oxidative damage. [1] Let’s take a look at twelve health benefits of acai berries.

Acai

promotes Heart Health. Acai Berries like red wine, research shows that acai berries are extremely high in anthocyanins, a type of antioxidant that supports balanced cholesterol levels. They are also rich in plant sterols that provide cardio-protective benefits, including supporting circulation, improving overall blood composition, and relaxing the blood vessels.

Berries

Acai

resists Harmful Organisms. Research has shown that consuming harmful organisms. However, I recommend oregano oil for this task.

Berries

acai

extract

may help fight

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Acai

aid in weight loss. Known by nutritionists as a superfood, acai may help us not only lose weight but maintain a healthy weight. One interesting study found that pulp from the acai berry had the ability to reduce fat deposits in study participants.

Berries

Acai

promotes Skin Health. Acai oil is a great natural alternative to chemical based skin care products. Currently, many beauty products contain acai oil because of the oil’s high antioxidant content. When eaten, the berries provide nutrition that can give your skin a healthy glow. In fact, Brazilians have been eating acai berries for centuries to promote skin health.

Berries

Acai

can help digestion. Acai may also aid in keeping the digestive system clean and functional. In the human body, the berries have powerful detoxification capacities and are a source of dietary fiber. Of course, there are many other high fiber foods that can do the same thing, including other types of berries.

Berries

Acai

reduces irritation. Acai berries contain properties that may prevent the irritation in the lungs typically associated with respiratory distress and swelling.

Berries

Acai

improve cellular health. On a general level, the anthocyanins found in acai play a role in the body’s cellular protection system, helping to keep cells strong against the invasion of free radicals.

Berries

One study found that polyphenolic compounds extracted from acai reduced the proliferation of malfunctioning cells by up to 86%. It is thought that acai berry contains phytochemicals that can disrupt cell mutation at a molecular level, killing the affected cells before they multiply. Acai berries are not a cure for any disease but, hopefully, more research will yield more positive discoveries.

Extremely high in many forms of phytochemicals, acai berries may slow or reverse aging processes as they relate to oxidative damage. In fact, the berries are one of the best sources of antioxidants; one berry has ten times as many antioxidants as grapes and twice as many as blueberries.

Acai

boost energy. Due to its overall health benefits, taking acai extract can lead to an increased overall level of energy and stamina, and may aid to combat fatigue and exhaustion. Whenever you need a boost, simply eat a handful of berries and you will be ready to go in no time!

Berries

Preliminary research studies show that

acai

may improve cognition and promote healthy brain aging.

Vitamin B12 Cyanocobalamin

Vitamin B12 is part of the Vitamin B complex. It is considered to be a pain killing vitamin. It helps DNA production, cardiovascular support, and energy metabolism.

Research

Broccoli Extract

Behav Brain Res. 2016 Mar 15;301:55-62. doi: 10.1016/j.bbr.2015.12.030. Epub 2015 Dec 22.

Broccli Sulforaphane produces antidepressant- and anxiolytic-like effects in adult mice.

Wu S1, Gao Q2, Zhao P3, Gao Y4, Xi Y5, Wang X5, Liang Y5, Shi H6, Ma Y7.

Increasing evidence suggests that depression is accompanied by dysregulation of neuroimmune system. Sulforaphane (SFN) is a natural compound with antioxidative, anti-inflammatory and neuroprotective activities. The present study aims to investigate the effects of SFN on depressive- and anxiety-like behaviors as well as potential neuroimmune mechanisms in mice. Repeated SFN administration (10mg/kg, i.p.) significantly decreased the immobility time in the forced swimming test (FST), tail suspension test (TST), and latency time to feeding in the novelty suppressed feeding test (NSF), and increased the time in the central zone in the open field test (OPT). Using the chronic mild stress (CMS) paradigm, we confirmed that repeated SFN (10mg/kg, i.p.) administration significantly increased sucrose preference in the sucrose preference test (SPT), and immobility time in the FST and TST of mice subjected to CMS. Also, SFN treatment significantly reversed anxiety-like behaviors (assessed by the OPT and NSF) of chronically stressed mice. Finally, ELISA analysis showed that SFN administration blocked the increase in the serum levels of corticosterone (CORT), adrenocorticotropic hormone (ACTH), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in chronically stressed mice. In summary, these findings demonstrated that SFN has antidepressant- and anxiolytic-like activities in stressed mice model of depression, which likely occurs by inhibiting the hypothalamic-pituitary-adrenal (HPA) axis and inflammatory response to stress. These data support further exploration for developing SFN as a novel agent to treat depression and anxiety disorders.

J Nutr Biochem. 2017 Jan;39:134-144. doi: 10.1016/j.jnutbio.2016.10.004. Epub 2016 Oct 11.

Prophylactic effects of sulforaphane on depression-like behavior and dendritic changes in mice after inflammation.

Zhang JC1, Yao W1, Dong C1, Yang C1, Ren Q1, Ma M1, Han M1, Wu J1, Ushida Y2, Suganuma H2, Hashimoto K3.

Author information Abstract Inflammation plays a role in the pathophysiology of depression. Sulforaphane (SFN) from broccoli, an isothiocyanate compound derived from broccoli, is a potent activator of the NF-E2-related factor-2 (Nrf2), which plays a role in inflammation. In this study, we examined whether the prevention effects of SFN in lipopolysaccharide (LPS) induced depression-like behavior in mice. Pretreatment with SFN significantly blocked an increase in the serum tumor necrosis factor-α (TNF-α) level and an increase in microglial activation of brain regions after a single administration of LPS (0.5 mg/kg). Furthermore, SFN significantly potentiated increased serum levels of IL-10 after LPS administration. In the tail-suspension test and forced swimming test, SFN significantly attenuated an increase of the immobility time after LPS administration. In addition, SFN significantly recovered to control levels for LPS-induced alterations in the proteins such as brain-derived neurotrophic factor, postsynaptic density protein 95 and AMPA receptor 1 (GluA1) and dendritic spine density in the brain regions. Finally, dietary intake of 0.1% glucoraphanin (a glucosinolate precursor of SFN) food during the juvenile and adolescence could prevent the onset of LPS-induced depression-like behaviors and dendritic spine changes in the brain regions at adulthood. In conclusion, these findings suggest that dietary intake of SFN-rich broccoli sprout has prophylactic effects on inflammation-related depressive symptoms. Therefore, supplementation of SFN-rich broccoli sprout could be prophylactic vegetable to prevent or minimize the relapse by inflammation in the remission state of depressed patients.

Psychoneuroendocrinology. 2013 Oct;38(10):2010-22. doi: 10.1016/j.psyneuen.2013.03.020. Epub 2013 Apr 23.

Nrf2 participates in depressive disorders through an anti-inflammatory mechanism.

Martín-de-Saavedra MD1, Budni J, Cunha MP, Gómez-Rangel V, Lorrio S, Del Barrio L, Lastres-Becker I, Parada E, Tordera RM, Rodrigues AL, Cuadrado A, López MG.

Author information Abstract A causative relationship between inflammation and depression is gradually gaining consistency. Because Nrf2 participates in inflammation, we hypothesized that Nrf2 could play a role in depressive disorders. In this study, we have observed that Nrf2 deletion in mice results in: (i) a depressive-like behavior evaluated as an increase in the immobility time in the tail-suspension test and by a decrease in the grooming time in the splash test, (ii) reduced levels of dopamine and serotonin and increased levels of glutamate in the prefrontal cortex, (iii) altered levels of proteins associated to depression such as VEGF and synaptophysin and (iv) microgliosis. Furthermore, treatment of Nrf2 knockout mice with the antiinflammatory drug rofecoxib reversed their depressive-like behavior, while induction of Nrf2 by sulforaphane from broccolli, in an inflammatory model of depression elicited by LPS, afforded antidepressant-like effects. In conclusion, our results indicate that chronic inflammation due to a deletion of Nrf2 can lead to a depressive-like phenotype while induction of Nrf2 could become a new and interesting target to develop novel antidepressive drugs.

Mol Nutr Food Res. 2017 Feb;61(2). doi: 10.1002/mnfr.201600194. Epub 2016 Nov 30.

Sulforaphane epigenetically enhances neuronal BDNF expression and TrkB signaling pathways.

Kim J1, Lee S1, Choi BR2, Yang H1, Hwang Y1, Park JH3,4, LaFerla FM5, Han JS2, Lee KW1,4,6,7, Kim J6,8. Author information Bionomic Solutions

Abstract SCOPE:

Brain-derived neurotrophic factor (BDNF) is a neurotrophin that supports the survival of existing neurons and encourages the growth and differentiation of new neurons and synapses. We investigated the effect of sulforaphane from broccoli, a hydrolysis product of glucoraphanin present in Brassica vegetables, on neuronal BDNF expression and its synaptic signaling pathways.

METHODS AND RESULTS:

Mouse primary cortical neurons and a triple-transgenic mouse model of Alzheimer’s disease (3 × Tg-AD) were used to study the effect of sulforaphane. Sulforaphane enhanced neuronal BDNF expression and increased levels of neuronal and synaptic molecules such as MAP2, synaptophysin, and PSD-95 in primary cortical neurons and 3 × Tg-AD mice. Sulforaphane elevated levels of synaptic TrkB signaling pathway components, including CREB, CaMKII, ERK, and Akt in both primary cortical neurons and 3 × Tg-AD mice. Sulforaphane increased global acetylation of histone 3 (H3) and H4, inhibited HDAC activity, and decreased the level of HDAC2 in primary cortical neurons. Chromatin immunoprecipitation analysis revealed that sulforaphane increased acetylated H3 and H4 at BDNF promoters, suggesting that sulforaphane regulates BDNF expression via HDAC inhibition.

CONCLUSION:

These findings suggest that sulforaphane has the potential to prevent neuronal disorders such as Alzheimer’s disease by epigenetically enhancing neuronal BDNF expression and its TrkB signaling pathways.

Vascul Pharmacol. 2016 Oct;85:29-38. doi: 10.1016/j.vph.2016.07.004. Epub 2016 Jul 8.

Sulforaphane induces neurovascular protection against a systemic inflammatory challenge via both Nrf2-dependent and independent pathways. Holloway PM1, Gillespie S2, Becker F3, Vital SA1, Nguyen V1, Alexander JS1, Evans PC4, Gavins FNE5.

Author information Abstract Sepsis is often characterized by an acute brain inflammation and dysfunction, which is associated with increased morbidity and mortality worldwide. Preventing cerebral leukocyte recruitment may provide the key to halt progression of systemic inflammation to the brain. Here we investigated the influence of the anti-inflammatory and anti-oxidant compound, sulforaphane (SFN) on lipopolysaccharide (LPS)-induced cellular interactions in the brain. The inflammatory response elicited by LPS was blunted by SFN administration (5 and 50mg/kg i.p.) 24h prior to LPS treatment in WT animals, as visualized and quantified using intravital microscopy. This protective effect of SFN was lost in Nrf2-KO mice at the lower dose tested, however 50mg/kg SFN revealed a partial effect, suggesting SFN works in part independently of Nrf2 activity. In vitro, SFN reduced neutrophil recruitment to human brain endothelial cells via a down regulation of E-selectin and vascular cell adhesion molecule 1 (VCAM-1). Our data confirm a fundamental dose-dependent role of SFN in limiting cerebral inflammation. Furthermore, our data demonstrate that not only is Nrf2 in part essential in mediating these neuroprotective effects, but they occur via down-regulation of E-selectin and VCAM-1. In conclusion, SFN may provide a useful therapeutic drug to reduce cerebral inflammation in sepsis..

Published online 2012 Apr 27. doi: 10.1371/journal.pone.0036216 PMCID: PMC3338688 Modulation of Experimental Herpes Encephalitis-Associated Neurotoxicity through Sulforaphane Treatment Scott J. Schachtele, Shuxian Hu, and James R. Lokensgard* Maria Gasset, Editor Abstract Reactive oxygen species (ROS) produced by brain-infiltrating macrophages and neutrophils, as well as resident microglia, are pivotal to pathogen clearance during viral brain infection. However, unchecked free radical generation is also responsible for damage to and cytotoxicity of critical host tissue bystander to primary infection. These unwanted effects of excessive ROS are combated by local cellular production of antioxidant enzymes, including heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (Gpx1). In this study, we showed that experimental murine herpes encephalitis triggered robust ROS production, as well as an opposing upregulation of the antioxidants HO-1 and Gpx1. This antioxidant response was insufficient to prevent tissue damage, neurotoxicity, and mortality associated with viral brain infection. Previous studies corroborate our data supporting astrocytes as the major antioxidant producer in brain cell cultures exposed to HSV-1 stimulated microglia. We hypothesized that stimulating opposing antioxidative responses in astrocytes, as well as neurons, would mitigate the effects of ROS-mediated neurotoxicity both in vitro and during viral brain infection in vivo. Here, we demonstrate that the addition of sulforaphane, a potent stimulator of antioxidant responses, enhanced HO-1 and Gpx1 expression in astrocytes through the activation of nuclear factor-E2related factor 2 (Nrf2). Additionally, sulforaphane treatment was found to be effective in reducing neurotoxicity associated with HSV-stimulated microglial ROS production. Finally, intraperitoneal injections of sulforaphane into mice during active HSV infection reduced neuroinflammation via a decrease in brain-infiltrating leukocytes, macrophage- and neutrophil-produced ROS, and MHCII-positive, activated microglia. These data support a key role for astrocyte-produced antioxidants in modulating oxidative stress and neuronal damage in response to viral infection. Bionomic Solutions

Int Immunopharmacol. 2017 Apr;45:74-78. doi: 10.1016/j.intimp.2017.01.034. Epub 2017 Feb 10.

Sulforaphane improves outcomes and slows cerebral ischemic/reperfusion injury via inhibition of NLRP3 inflammasome activation in rats.

Yu C1, He Q1, Zheng J2, Li LY1, Hou YH1, Song FZ3.

Abstract Ischemia/reperfusion (I/R) injury has been correlated with systemic inflammatory response. In addition, NLRP3 has been suggested as a cause in many inflammatory processes. Sulforaphane (SFN) is a naturally occurring isothiocyanate found in cruciferous vegetables, such as broccoli and cabbage. While recent studies have demonstrated that Sulforaphane has protective effects against cerebral ischemia/reperfusion injury, little is known about how those protective effects work. In this study, we focus our investigation on the role and process of Sulforaphane in the inhibition of NLRP3 inflammasome activation, as well as its effect on brain ischemia/reperfusion injury. Adult male Sprague-Dawley rats were injected with Sulforaphane (5 or 10mg/kg) intraperitoneally at the beginning of reperfusion, after a 60min period of occlusion. A neurological score and infarct volume were assessed at 24h after the administration of Sulforaphane. Myeloperoxidase (MPO) activity was measured at 24h to assess neutrophil infiltration in brain tissue. ELISA, RT-PCR and Western blot analyses were used to measure any inflammatory reaction. Sulforaphane treatment significantly reduced infarct volume and improved neurological scores when compared to a vehicle-treated group. Neutrophil infiltration was significantly higher in the vehicle-treated group than in the Sulforaphane treatment group. Sulforaphane treatment inhibits NLRP3 inflammasome activation and the downregulation of cleaved caspase-1, while reducing IL-1β and IL-18 expression. The inhibition of inflammatory response with Sulforaphane treatment improves outcomes after focal cerebral ischemia. This neuroprotective effect is likely exerted by Sulforaphane inhibited NLRP3 inflammasome activation caused by the downregulation of NLRP3, the induction of cleaved caspase-1, and thus the reduction of IL-1β and IL-18.

J Neuroinflammation. 2016; 13: 41.

Published online 2016 Feb 16. doi: 10.1186/s12974-016-0505-y PMCID: PMC4754839 Hyperammonemia induces glial activation, neuroinflammation and alters neurotransmitter receptors in hippocampus, impairing spatial learning: reversal by sulforaphane Vicente Hernández-Rabaza, Andrea Cabrera-Pastor, Lucas Taoro-González, Michele Malaguarnera, Ana Agustí, Marta Llansola, and Vicente Felipocor responding author Abstract Background Patients with liver cirrhosis and minimal hepatic encephalopathy (MHE) show mild cognitive impairment and spatial learning dysfunction. Hyperammonemia acts synergistically with inflammation to induce cognitive impairment in MHE. Hyperammonemia-induced neuroinflammation in hippocampus could contribute to spatial learning impairment in MHE. Two main aims of this work were: (1) to assess whether chronic hyperammonemia increases inflammatory factors in the hippocampus and if this is associated with microglia and/or astrocytes activation and (2) to assess whether hyperammonemia-induced neuroinflammation in the hippocampus is associated with altered membrane expression of glutamate and GABA receptors and spatial learning impairment. There are no specific treatments for cognitive alterations in patients with MHE. A third aim was to assess whether treatment with sulforaphane enhances endogenous the anti-inflammatory system, reduces neuroinflammation in the hippocampus of hyperammonemic rats, and restores spatial learning and if normalization of receptor membrane expression is associated with learning improvement.

Neurosci Lett. 2009 Aug 28; 460(2): 103–107.

Published online 2009 Apr 15. doi: 10.1016/j.neulet.2009.04.028 PMCID: PMC2700200 NIHMSID: NIHMS110906 SULFORAPHANE IMPROVES COGNITIVE FUNCTION ADMINISTERED FOLLOWING TRAUMATIC BRAIN INJURY Pramod K. Dash,* Jing Zhao, Sara A. Orsi, Min Zhang, and Anthony N. Moore Author information ► Copyright and License information ► The publisher’s final edited version of this article is available at Neurosci Lett Abstract Recent studies have shown that sulforaphane, a naturally occurring compound that is found in cruciferous vegetables, offers cellular protection in several models of brain injury. When administered following traumatic brain injury (TBI), sulforaphane has been demonstrated to attenuate blood-brain barrier permeability and reduce cerebral edema. These beneficial effects of sulforaphane have been shown to involve induction of a group of cytoprotective, Nrf2-driven genes, whose protein products include free radical scavenging and detoxifying enzymes. However, the influence of sulforaphane on post-injury cognitive deficits has not been examined. In this study, we examined if sulforaphane, when administered following cortical impact injury, can improve the performance of rats tested in hippocampal- and prefrontal cortex-dependent tasks. Our results indicate that sulforaphane treatment improves performance in the Morris water maze task (as indicated by decreased latencies during learning and platform localization during a probe trial) and reduces working memory dysfunction (tested using the delayed match-to-place task). These behavioral improvements were only observed when the treatment was initiated 1 hr, but not 6 hr, post-injury. These studies support the use of sulforaphane in the treatment of TBI and extend the previously observed protective effects to include enhanced cognition.

Neurobiol Aging. 2016 Feb;38:1-10. doi: 10.1016/j.neurobiolaging.2015.10.016. Epub 2015 Oct 23.

Sulforaphane exerts its anti-inflammatory effect against amyloid-β peptide via STAT-1 dephosphorylation and activation of Nrf2/HO-1 cascade in human THP-1 macrophages. Bionomic Solutions

An YW1, Jhang KA1, Woo SY1, Kang JL2, Chong YH3.

Abstract Alzheimer’s disease (AD) is the most common neurodegenerative disorder worldwide, accounting for most cases of dementia in elderly individuals, and effective therapies are still lacking. This study was designed to investigate the anti-inflammatory properties of sulforaphane against Aβ1-42 monomers in human THP-1 microglia-like cells. The results showed that sulforaphane preferentially inhibited cathepsin B- and caspase-1-dependent NLRP3 inflammasome activation induced by mostly Aβ1-42 monomers, an effect that potently reduced excessive secretion of the proinflammatory cytokine interleukin-1β (IL-1β). Subsequent mechanistic studies revealed that sulforaphane mitigated the activation of signal transducer and activator of transcription-1 induced by Aβ1-42 monomers. Sulforaphane also increased nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, which was followed by upregulation of heme-oxygenase 1 (HO-1). The anti-inflammatory effect of sulforaphane on Aβ1-42-induced IL-1β production was diminished by small interfering RNA-mediated knockdown of Nrf2 or HO-1. Moreover, sulforaphane significantly attenuated the levels of microRNA-146a, which is selectively upregulated in the temporal cortex and hippocampus of AD brains. The aforementioned effects of sulforaphane were replicated by the tyrosine kinase inhibitor, herbimycin A, and Nrf2 activator. These results indicate that signal transducer and activator of transcription-1 dephosphorylation, HO-1 and its upstream effector, Nrf2, play a pivotal role in triggering an anti-inflammatory signaling cascade of sulforaphane that results in decreases of IL-1β release and microRNA-146a production in Aβ1-42-stimulated human microglia-like cells. These findings suggest that the phytochemical sulforaphane has a potential application in AD therapeutics.

Am J Alzheimers Dis Other Demen. 2015 Mar;30(2):183-91. doi: 10.1177/1533317514542645. Epub 2014 Jul 13.

Sulforaphane ameliorates neurobehavioral deficits and protects the brain from amyloid β deposits and peroxidation in mice with Alzheimer-like

lesions.

Zhang R1, Miao QW1, Zhu CX1, Zhao Y1, Liu L1, Yang J1, An L2.

Abstract Alzheimer’s disease (AD) is a common neurodegenerative disease in the elderly individuals and its effective therapies are still unavailable. This study was designed to investigate the neuroprotection of sulforaphane (SFN) in AD-lesion mice induced by combined administration of dgalactose and aluminium. Results showed that SFN ameliorated spatial cognitive impairment and locomotor activity decrease in Morris water maze and open field test, respectively. And attenuated numbers of amyloid β (Aβ) plaques in both hippocampus and cerebral cortex of AD-lesion mice were detected by immunohistochemistry. According to spectrophotometry and quantitative reverse-transcriptase polymerase chain reaction results, a significant increase in carbonyl group level and obvious decreases in both activity and messenger RNA expression of glutathione peroxidase were found in brain of AD-lesion mice compared with control, but not in SFN-treated AD-lesion mice. In conclusion, SFN ameliorates neurobehavioral deficits and protects the brain from Aβ deposits and peroxidation in mice with Alzheimer-like lesions, suggesting SFN is likely a potential phytochemical to be used in AD therapeutics.

Amyloid. 2013 Mar;20(1):7-12. doi: 10.3109/13506129.2012.751367. Epub 2012 Dec 19.

Amelioration of Alzheimer’s disease by neuroprotective effect of sulforaphane in animal model.

Kim HV1, Kim HY, Ehrlich HY, Choi SY, Kim DJ, Kim Y.

Abstract Pathophysiological evidences of AD have indicated that aggregation of Aβ is one of the principal causes of neuronal dysfunction, largely by way of inducing oxidative stresses such as free radical formation. We hypothesized that the known antioxidative attribute of SFN could be harnessed in Alzheimer’s treatment. SFN is an indirect, potent antioxidant derived from broccoli that has previously been found to stimulate the Nrf2-ARE pathway and facilitate several other cytoprotective mechanisms. In this study, administration of SFN ameliorated cognitive function of Aβinduced AD acute mouse models in Y-maze and passive avoidance behavior tests. Interestingly, we found that the therapeutic effect of SFN did not involve inhibition of Aβ aggregation. While the exact mechanism of interaction of SFN in AD has not yet been ascertained, our results suggest that SFN can aid in cognitive impairment and may protect the brain from amyloidogenic damages.

J Neurochem. Author manuscript; available in PMC 2015 May 1.

Published in final edited form as:

J Neurochem. 2014 May; 129(3): 539–547.

Published online 2014 Jan 18. doi: 10.1111/jnc.12647 PMCID: PMC3997618 NIHMSID: NIHMS553897 Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential therapeutic reagent for Huntington’s disease Yanying Liu, Casey L. Hettinger, Dong Zhang, Khosrow Rezvani, Xuejun Wang, and Hongmin Wang Abstract The ubiquitin-proteasome system (UPS) is impaired in Huntington’s disease, a devastating neurodegenerative disorder. Sulforaphane, a naturally-occurring compound, has been shown to stimulate UPS activity in cell cultures. To test whether sulforaphane enhances UPS function in vivo, we treated UPS function reporter mice ubiquitously expressing the green fluorescence protein (GFP) fused to a constitutive degradation signal that promotes its rapid degradation in the conditions of a healthy UPS. The modified GFP is termed GFPu. We found that both GFPu and ubiquitinated protein levels were significantly reduced and the three peptidase activities of the proteasome were increased in the brain and peripheral tissues of the mice. Interestingly, sulforaphane treatment also enhanced autophagy activity in the brain and the liver. To further examine whether SFN promotes mutant huntingtin (mHtt) degradation, we treated HD cells with sulforaphane and found that sulforaphane not only enhanced mHtt degradation but also reduced mHtt cytotoxicity. Sulforaphane-mediated mHtt degradation was mainly through the UPS pathway since the presence of a proteasome inhibitor abolished this effect. Taken together, these data indicate that sulforaphane activates Bionomic Solutions

protein degradation machineries in both the brain and peripheral tissues and may be a therapeutic reagent for HD and other intractable disorders.

J Neurochem. 2015 Dec;135(5):932-42. doi: 10.1111/jnc.13361. Epub 2015 Oct 8.

Sulforaphane is anticonvulsant and improves mitochondrial function.

Carrasco-Pozo C1,2, Tan KN1, Borges K1.

Abstract The nuclear factor erythroid 2-related factor 2 pathway (Nrf2) has been previously identified to protect the brain against various impacts. Here, we investigated the effect of the Nrf2 activator sulforaphane in various seizure models and hippocampal mitochondrial bioenergetics. We found that daily injections of sulforaphane for 5 days elevated the seizure thresholds to 6 Hz stimulation and fluorothyl-, but not pentylenetetrazoleinduced tonic seizures and protected mice against pilocarpine-induced status epilepticus (SE). Also, sulforaphane increased the antioxidant defences within hippocampal formations and blood plasma. In addition, sulforaphane treatment reduced the extent of hippocampal lipid peroxidation 24 h post-SE and protected hippocampal mitochondria against SE-induced reduction in state 2 and uncoupler-stimulated state 3 respiration. SE-mediated partial loss of rotenone-sensitive and complex II-driven respiration was reduced, consistent with the enhanced activities of complexes I and II in sulforaphane-treated SE mice. In mitochondria isolated from both no SE and SE mice, sulforaphane increased state 3 respiration and respiration linked to ATP synthesis, which may contribute to its anticonvulsant and antioxidant effects by providing more ATP for cellular vital and protective functions. However, sulforaphane did not prevent SE-induced hippocampal cell death. In conclusion, sulforaphane and/or Nrf2 activation are viable anticonvulsant strategies, which are antioxidant and enhance mitochondrial function, especially the ability to produce ATP. Sulforaphane was anticonvulsant in two acute mouse models of epilepsy and protected mice against pilocarpine-induced status epilepticus (SE). We also found antioxidant effects of sulforaphane in mouse plasma and hippocampal formations, exhibited by increased catalase and superoxide dismutase (SOD) activity, as well as increased abilities of hippocampal mitochondria to produce ATP. These effects likely underlie sulforaphane’s anticonvulsant mechanisms of action.

Clin Immunol. Author manuscript; available in PMC 2010 Mar 1.

Published in final edited form as:

Clin Immunol. 2009 Mar; 130(3): 244–251.

Published online 2008 Nov 22. doi: 10.1016/j.clim.2008.10.007 PMCID: PMC2668525 NIHMSID: NIHMS96740 Oral Sulforaphane increases Phase II antioxidant enzymes in the human upper airway Marc A. Riedl, M.D., M.S.,1 Andrew Saxon, M.D.,1 and David Diaz-Sanchez, Ph.D2 Author information ► Copyright and License information ► The publisher’s final edited version of this article is available at Clin Immunol Background Cellular oxidative stress is an important factor in asthma and is thought to be the principle mechanism by which oxidant pollutants such as ozone and particulates mediate their pro-inflammatory effects. Endogenous Phase II enzymes abrogate oxidative stress through the scavenging of reactive oxygen species and metabolism of reactive chemicals.

Inhal Toxicol. 2007;19 Suppl 1:177-82.

Antioxidant enzyme induction: a new protective approach against the adverse effects of diesel exhaust particles.

Wan J1, Diaz-Sanchez D.

Abstract Exposure to airborne particulate pollutants such as diesel exhaust particles (DEPs) has been associated with allergic respiratory disorders, including asthma and allergic rhinitis. In this communication, we review recent advances in the mechanism by which DEPs elicit their harmful effects and the protective role of antioxidants. Reactive oxidative species (ROS) are believed to play a key role in cellular damage after exposure to DEPs. Numerous reports demonstrate that both proinflammatory and anti-inflammatory products are induced by DEPs via the activation of transcription factors. DEPs trigger multiple signaling pathways, which lead to DNA damage and cell apoptosis, inflammatory response, and antioxidant defense. Recent studies both in vitro and in mice show that antioxidants could alleviate the allergic inflammatory effects of DEPs. Human in vivo models suggest that the important phase II enzymes GSTM1 and GSTP1 modify the adjuvant effect of diesel exhaust particles on allergic inflammation. We have shown that the induction of phase II enzymes by the chemical sulforaphane can block DEP-induced enhanced immunoglobulin (Ig) E production in B cells and DEP-induced proinflammatory cytokine production in epithelial cells. These findings suggest that overexpression of antioxidant enzymes could constitute a powerful potential chemopreventive approach against adverse effects induced by oxidant pollutants such as DEPs.

J Environ Sci Health B. 2009 Sep;44(7):657-62. doi: 10.1080/03601230903163624.

Evaluation of protective effects of sulforaphane on DNA damage caused by exposure to low levels of pesticide mixture using comet assay. Topè AM1, Rogers PF. Bionomic Solutions

Abstract The objective of the study was to evaluate the potential risk of DNA damage due to exposure to a mixture of the most widely used pesticides, namely endosulfan, chlorpyriphos and thiram at an environmentally relevant concentration (5 microM each) and the DNA protective capacity of sulforaphane (SFN) (10-30 microg/mL). DNA damage in human lymphocytes was ascertained with Single Cell Gel Electrophoresis (SCGE), also called Comet Assay. For positive control, H(2)O(2) at 100 mM was used. The pesticide mixture produced DNA damage at the concentration used in the lymphocytes. SFN was able to offer a statistically significant (P < 0.01), concentration-dependant protection to DNA damage between 1020 microg/mL in both the pre-incubation and co-incubation strategies. The results indicate that exposure to low levels of these pesticide mixtures can induce DNA damage, and the presence of SFN in diet may reduce the incidence of genetic damage, especially in farm workers. However, it is not clear whether SFN is involved in quenching of the free radicals generated by the pesticide mixture or it is involved in DNA repair mechanism.

Toxicol Sci. 2011 May; 121(1): 57–62.

Published online 2011 Jan 28. doi: 10.1093/toxsci/kfr026 PMCID: PMC3080186 Sulforaphane-Mediated Reduction of Aflatoxin B1-N7-Guanine in Rat Liver DNA: Impacts of Strain and Sex Jeannette L. A. Fiala,*†,1 Patricia A. Egner,‡,1 Nirachara Wiriyachan,§¶,1 Mathuros Ruchirawat,§ Kevin H. Kensler,‡ Gerald N. Wogan,*† John D. Groopman,‡ Robert G. Croy,*† and John M. Essigmann*†,2 Abstract Aflatoxin B1 (AFB1) is a DNA-binding toxin that contributes to the burden of liver cancer in tropical areas. AFB1-DNA adducts are powerful biomarkers that discern individual and population risk from exposure to this carcinogen. The discovery of concordance between the metabolic pathways of the male Fischer rat and humans allowed data from rats to guide the development of chemoprevention strategies employed in clinical trials in high-risk regions. In this study, the variables of strain and sex are studied in the rat model, as a step toward understanding how ethnic differences and sex influence DNA adduct formation and the induction of enzymes by chemoprotective agents. Sulforaphane (SF), which induces phase II enzymes including glutathione S-transferases (GSTs), was evaluated for its ability to induce GST activity and reduce the AFB1DNA adducts in livers of both sexes of two rat strains that differ in susceptibility to AFB1 hepatocarcinogenesis. A dose-dependent relationship was found for SF for both induction of GST and reduction in of AFB1-N7-guanine in both Fischer (sensitive to AFB1) and Sprague-Dawley rats (relatively resistant). Sprague-Dawley rats exhibited the greatest increase in GST levels and the largest reduction in AFB1-N7-guanine in liver DNA. Males and females of each strain were also compared to determine if the ability of SF to induce GST and reduce AFB1-N7-guanine correlated with gender differences in sensitivity to AFB1 carcinogenesis. No gender-specific responses to SF were observed. These results support the view that SF induction of liver GST activity may play a role in its chemoprotective activity.

Indian J Exp Biol. 2003 Mar;41(3):216-9.

Inhibition of mutagenicity of food-derived heterocyclic amines by sulforaphane–a constituent of broccoli.

Shishu1, Kaur IP.

Abstract Sulforaphane, a constituent of broccoli was investigated for its antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines). These include imidazoazaarenes such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); pyridoindole derivatives such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2); and, dipyridoimidazole derivative such as 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-1). Tests were carried out by Ames Salmonella/reversion assay using Salmonella typhimurium TA98 (frame shift mutation sensitive) and TA100 (base pair mutation sensitive) bacterial strains in the presence of Aroclor 1254-induced rat liver S9. Results of these in vitro antimutagenicity studies strongly suggest that sulforaphane is a potent inhibitor of the mutagenicity induced by imidazoazaarenes such as IQ, MeIQ and MeIQx (approximately 60% inhibition) and moderately active against pyridoindole derivatives such as Trp-P-1 and Trp-P-2 (32-48% inhibition), but ineffective against dipyridoimidazole derivative (Glu-P-1) in TA 100.

Int J Mol Sci. 2016 Oct; 17(10): 1703.

Published online 2016 Oct 11. doi: 10.3390/ijms17101703 PMCID: PMC5085735 Sulforaphane Prevents Testicular Damage in Kunming Mice Exposed to Cadmium via Activation of Nrf2/ARE Signaling Pathways Shu-Hua Yang,1,† Miao Long,1,† Li-Hui Yu,1 Lin Li,1 Peng Li,1 Yi Zhang,1 Yang Guo,1 Feng Gao,1 Ming-Da Liu,2,* and Jian-Bin He1,* Athanasios Salifoglou, Academic Editor Abstract Sulforaphane (SFN) is a natural and highly effective antioxidant. Studies suggest that SFN protects cells and tissues against cadmium (Cd) toxicity. This study investigated the protective effect of SFN against oxidative damage in the testes of Kunming mice exposed to cadmium, and explored the possible molecular mechanisms involved. Cadmium greatly reduced the serum testosterone levels in mice, reduced sperm motility, total sperm count, and increased the sperm deformity rate. Cadmium also reduces superoxide dismutase (T-SOD) and glutathione (GSH) levels and increases malondialdehyde (MDA) concentrations. SFN intervention improved sperm quality, serum testosterone, and antioxidant levels. Both mRNA and protein expression of mouse testicular nuclear factor-erythroid 2-related factor 2 (Nrf2) was reduced in cadmium-treated group. Furthermore, the downstream genes of Nrf2, glutathione peroxidase (GSH-Px), γ-glutamyl cysteine synthetase (γ-GCS), heme oxygenase-1 (HO- Bionomic Solutions

1), and NAD(P)H:quinone oxidoreductase-1 (NQO1) were also decreased in cadmium-treated group. SFN intervention increases the expression of these genes. Sulforaphane prevents cadmium-induced testicular damage, probably via activation of Nrf2/ARE signaling.

PLoS One. 2015; 10(9): e0138771.

Published online 2015 Sep 24. doi: 10.1371/journal.pone.0138771 PMCID: PMC4581733 Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity Wei Wang,1 Yan He,2 Guodong Yu,1 Baolong Li,3 Darren W. Sexton,1 Thomas Wileman,1 Alexandra A. Roberts,4 Chris J. Hamilton,4 Ruoxi Liu,5 Yimin Chao,5 Yujuan Shan,6,* and Yongping Bao1,* Ilya Ulasov, Editor Abstract The potential cytotoxicity of cadmium selenide (CdSe) quantum dots (QDs) presents a barrier to their use in biomedical imaging or as diagnostic and therapeutic agents. Sulforaphane (SFN) is a chemoprotective compound derived from cruciferous vegetables which can up-regulate antioxidant enzymes and induce apoptosis and autophagy. This study reports the effects of SFN on CdSe QD-induced cytotoxicity in immortalised human hepatocytes and in the livers of mice. CdSe QDs induced dose-dependent cell death in hepatocytes with an IC50 = 20.4 μM. Pre-treatment with SFN (5 μM) increased cell viability in response to CdSe QDs (20 μM) from 49.5 to 89.3%. SFN induced a pro-oxidant effect characterized by depletion of intracellular reduced glutathione during short term exposure (3–6 h), followed by up-regulation of antioxidant enzymes and glutathione levels at 24 h. SFN also caused Nrf2 translocation into the nucleus, up-regulation of antioxidant enzymes and autophagy. siRNA knockdown of Nrf2 suggests that the Nrf2 pathway plays a role in the protection against CdSe QD-induced cell death. Wortmannin inhibition of SFN-induced autophagy significantly suppressed the protective effect of SFN on CdSe QD-induced cell death. Moreover, the role of autophagy in SFN protection against CdSe QD-induced cell death was confirmed using mouse embryonic fibroblasts lacking ATG5. CdSe QDs caused significant liver damage in mice, and this was decreased by SFN treatment. In conclusion, SFN attenuated the cytotoxicity of CdSe QDs in both human hepatocytes and in the mouse liver, and this protection was associated with the induction of Nrf2 pathway and autophagy.

Toxicol Appl Pharmacol. Author manuscript; available in PMC 2013 Jul 28.

Published in final edited form as:

Toxicol Appl Pharmacol. 2012 Dec 15; 265(3): 292–299.

Published online 2012 Sep 6. doi: 10.1016/j.taap.2012.08.028 PMCID: PMC3725323 NIHMSID: NIHMS483535 Sulforaphane prevents pulmonary damage in response to inhaled arsenic by activating the Nrf2-defense response Yi Zheng,a,b Shasha Tao,b Fangru Lian,c Binh T. Chau,d Jie Chen,a Guifan Sun,a Deyu Fang,e R. Clark Lantz,d,f and Donna D. Zhangb,f,* Exposure to arsenic is associated with an increased risk of lung disease. Novel strategies are needed to reduce the adverse health effects associated with arsenic exposure in the lung. Nrf2, a transcription factor that mediates an adaptive cellular defense response, is effective in detoxifying environmental insults and prevents a broad spectrum of diseases induced by environmental exposure to harmful substances. In this report, we tested whether Nrf2 activation protects mice from arsenic-induced toxicity. We used an in vivo arsenic inhalation model that is highly relevant to low environmental human exposure to arsenic-containing dusts. Two-week exposure to arsenic-containing dust resulted in pathological alterations, oxidative DNA damage, and mild apoptotic cell death in the lung; all of which were blocked by sulforaphane (SF) in an Nrf2-dependent manner. Mechanistically, SF-mediated activation of Nrf2 alleviated inflammatory responses by modulating cytokine production. This study provides strong evidence that dietary intervention targeting Nrf2 activation is a feasible approach to reduce adverse health effects associated with arsenic exposure.

Olive Hydroxytyrosol Extract

Deiana M, Incani A, Rosa A, Atzeri A, Loru D, Cabboi B, Paola Melis M, Lucas R, Morales JC, Assunta Dessì M. Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H(2)O(2) induced oxidative damage. Chem Biol Interact. 2011 Sep 30; 193(3):232-9

Bullon P, Quiles JL, Morillo JM, Rubini C, Goteri G, Granados-Principal S, Battino M, Ramirez-Tortosa M. Gingival vascular damage in atherosclerotic rabbits: hydroxytyrosol and squalene benefits. Food Chem Toxicol. 2009 Sep.; 47(9):2327-31.

Carluccio, M. A., Siculella, L., Ancora, M. A., Massaro, M.et al., Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals. Arterioscler Thromb.Vasc. Biol. 2003, 23, 622–629

Castañer O, Covas MI, Khymenets O, Nyyssonen K, Konstantinidou V, Zunft HF, de la Torre R, Muñoz-Aguayo D, Vila J, Fitó M. Protection of LDL from oxidation by olive oil polyphenols is associated with a downregulation of CD40-ligand expression and its downstream products in vivo in humans. American Journal of Clin Nutr. 2012 May; 95(5):1238-44.

Hedya Jemai†, Abdelfattah El Feki‡ and Sami Sayadi*† † Laboratoire des Bioprocédés, Pôle d’Excellence Régionale AUF, (PER-LBP) Centre de Biotechnologie de Sfax, B.P. 1177, 3018 Sfax, Tunisia Bionomic Solutions

‡ Laboratoire d’Ecophysiologie animale, Faculté des Sciences de Sfax, B.P. 802, 3018 Sfax, Tunisia J. Agric. Food Chem., 2009, 57 (19), pp 8798–8804 DOI: 10.1021/jf901280r Publication Date (Web): September 2, 2009 Copyright © 2009 American Chemical Society *Corresponding author. Tel/Fax: 216 74 874 452. E-mail: sami.sayadi@cbs.rnrt.tn.

Cite this:J. Agric. Food Chem. 57, 19, 8798-8804 Abstract This study was designed to test the antidiabetic and antioxidative activities of olive leaf oleuropein and hydroxytyrosol. Diabetes in Wistar rats was induced by intraperitoneal injections of alloxan. The serum glucose and cholesterol, hepatic glycogen, the thiobarbituric acid-reactive substances (TBARS), and the components of hepatic and serum antioxidant system were examined. Diabetic rats showed hyperglycemia, hypercholesterolemia, increased lipid peroxidation, and depletion in the antioxidant enzymes activities. The administration, for 4 weeks, of oleuropein and hydroxytyrosol rich extracts, leading to 8 and 16 mg/kg body weight of each compound, significantly decreased the serum glucose and cholesterols levels and restored the antioxidant perturbations. These results suggested that the antidiabetic effect of oleuropein and hydroxytyrosol might be due to their antioxidant activities restraining the oxidative stress which is widely associated with diabetes pathologies and complications.

Elisa Tripoli et al. The phenolic compounds of 2005;18:98-112.

olive oil: structure, biological activity and beneficial effects on human health. Nutr Res Rev

Li J, Yang G, Wang S, Jiang L, Liu X, Geng C, Zhong L, Chen M. The protective effects of damage in HepG2 cells. Toxicol Mech Methods. 2012.

hydroxytyrosol

against ortho-phenylphenol-induced DNA

Felipe Alconchel-Gago1, Abel Santamaría, Isaac Túnez. Antioxidant effect of oleic acid and Huntington’s disease. Med. 2014; 99: (792): 60-64

hydroxytyrosol

in an experimental model similar to

Giuseppe Bisignano, Antonio Tomaino, Rossella Lo Cascio, Giuseppe Crisafi, Nicola Uccella, Antonella Saija First published: August 1999 DOI: 10.1211/0022357991773258 Department Farmaco-Biologico, University of Messina, Contrada Annunziata, 98168 Messina, Italy.

Abstract Secoiridoides (oleuropein and derivatives), one of the major classes of polyphenol contained in olives and olive oil, have recently been shown to inhibit or delay the rate of growth of a range of bacteria and microfungi but there are no data in the literature concerning the possible employment of these secoiridoides as antimicrobial agents against pathogenic bacteria in man.

In this study five ATCC standard bacterial strains (Haemophilus influenzae ATCC 9006, Moraxella catarrhalis ATCC 8176, Salmonella typhi ATCC 6539, Vibrio parahaemolyticus ATCC 17802 and Staphylococcus aureus ATCC 25923) and 44 fresh clinical isolates (Haemophilus influenzae, eight strains, Moraxella catarrhalis, six strains, Salmonella species, 15 strains, Vibrio cholerae, one strain, Vibrio alginolyticus, two strains, Vibrio parahaemolyticus, one strain, Staphylococcus aureus, five penicillin-susceptible strains and six penicillin-resistant strains), causal agents of intestinal or respiratory tract infections in man, were tested for in-vitro susceptibility to two olive (Olea europaea) secoiridoides, oleuropein (the bitter principle of olives) and hydroxytyrosol (derived from oleuropein by enzymatic hydrolysis and responsible for the high stability of olive oil). The minimum inhibitory concentrations (MICs) calculated in our study are evidence of the broad antimicrobial activity of hydroxytyrosol against these bacterial strains (MIC values between 0.24 and 7.85 μg mL−1 for ATCC strains and between 0.97 and 31.25 μg mL−1 for clinically isolated strains). Furthermore, oleuropein also inhibited (although to a much lesser extent) the growth of several bacterial strains (MIC values between 62.5 and 500 μg mL−1 for ATCC strains and between 31.25 and 250 μg mL−1 for clinical isolates); oleuropein was ineffective against Haemophilus influenzae and Moraxella catarrhalis.

These data indicate that in addition to the potential employment of its active principles as food additives or in integrated pest-management programs, Olea europaea can be considered a potential source of promising antimicrobial agents for treatment of intestinal or respiratory tract infections in man.

Friedman M, Rasooly R, Do PM, Henika PR. The Olive Compound 4-Hydroxytyrosol Inactivates Staphylococcus aureus Bacteria and Staphylococcal Enterotoxin A (SEA). J Food Sci. 2011 Oct; 76(8):M558-63.

Maria C. et al. Hydroxytyrosol, a phenolic compound from virgin olive oil, prevents macrophage activation. Naunyn-Schmied Arch Pharmacol.

2005;371:457-465.

Effect of Hydroxytyrosol Found in Extra Virgin Olive Oil on Oxidative DNA Damage and on Low-Density Lipoprotein Oxidation Okezie I. Aruoma*†‡, Monica Deiana‡§, Andrew Jenner‡, Barry Halliwell‡‖, Harparkash Kaur‡⊥, Sebastiano Banni§, Francesco P. Corongiu§, M. Assunta Dessí§, and Robert Aeschbach# Bionomic Solutions

Department of Clinical Analysis, Toxicology and Bromatology, Faculty of Pharmaceutical Sciences, University of São Paulo-Ribeirão Preto, Avenida do Cafè s/n-Monte Alegre, CEP 14040-903, Ribeirão Preto-São Paulo, Brazil; Pharmacology Group, University of London King’s College, Manresa Road, London SW3 6LX, United Kingdom; Dipartimento di Biologia Sperimentale, Sezione Patologia Sperimentale, Universitá degli Studi di Cagliari, Cittadella Universitaria, Km 4.500 SS 554, 09040 Monserrato, Cagliari, Italy; Department of Biochemistry, National University of Singapore, Kent Ridge Crescent, Singapore 119260; Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex HA1 3UJ, United Kingdom; and Nestlé Research Centre, Vers-Chez-les Blanc, CH-1000 Lausanne 26, Switzerland J. Agric. Food Chem., 1998, 46 (12), pp 5181–5187 DOI: 10.1021/jf980649b Publication Date (Web): November 21, 1998 Copyright © 1998 American Chemical Society Abstract Hydroxytyrosol found in extra virgin olive oil strongly inhibited low-density lipoprotein oxidation stimulated by 2,2‘-azobis(2-amidinopropane) hydrochloride (AAPH), suggesting the ability to scavenge the AAPH-derived peroxyl radicals. Hydroxytyrosol inhibited iron-dependent phospholipid liposome peroxidation at low concentrations (IC50 = 50 ± 1.3 μM). In similar experiments, the calculated, IC50 values for other antioxidants compared are 1.5 ± 0.05 μM (carnosol), 2.25 ± 0.08 μM (carnosic acid), 65 ± 2.6 μM (Trolox C), and 250 ± 10 μM (vitamin E). Hydroxytyrosol and ascorbate reduced copper(II) ions to their copper(I) prooxidant form, but this was not reflected by their abilities to induce oxidative DNA damage in the complex copper−phenanthroline. Only high, nonphysiological, millimolar concentrations of pure hydroxytyrosol weakly stimulated copper-dependent chemical modification to DNA bases. The prooxidant (redox actions on metal ions) concentrations in vitro may never be achieved in vivo (following consumption of extra virgin olive oil). Thus, hydroxytyrosol may represent a useful diet-derived antioxidant depending on its bioavailability.

Maiuri MC, De Stefano D, Di Meglio P, Irace C, Savarese M, Sacchi R, Cinelli MP, Carnuccio R. Hydroxytyrosol, a phenolic compound from virgin olive oil, prevents macrophage activation. Naunyn Schmiedebergs Arch Pharmacol. 2005 Jun; 371(6):457-65

Oxidative DNA Damage Is Prevented by Extracts of Olive Oil, Hydroxytyrosol, and Other Olive Phenolic Compounds in Human Blood Mononuclear Cells and HL60 Cells Roberto Fabiani Patrizia Rosignoli Angelo De Bartolomeo Raffaela Fuccelli Maurizio Servili Gian Francesco Montedoro Guido Morozzi The Journal of Nutrition, Volume 138, Issue 8, 1 August 2008, Pages 1411–1416, https://doi.org/10.1093/jn/138.8.1411 Published: 01 August 2008 Article history Abstract Our aim in this study was to provide further support to the hypothesis that phenolic compounds may play an important role in the anticarcinogenic properties of olive oil. We measured the effect of olive oil phenols on hydrogen peroxide (H2O2)-induced DNA damage in human peripheral blood mononuclear cells (PBMC) and promyelocytic leukemia cells (HL60) using single-cell gel electrophoresis (comet assay). Hydroxytyrosol [3,4-dyhydroxyphenyl-ethanol (3,4-DHPEA)] and a complex mixture of phenols extracted from both virgin olive oil (OO-PE) and olive mill wastewater (WW-PE) reduced the DNA damage at concentrations as low as 1 μmol/L when coincubated in the medium with H2O2 (40 μmol/L). At 10 μmol/L 3,4-DHPEA, the protection was 93% in HL60 and 89% in PBMC. A similar protective activity was also shown by the dialdehydic form of elenoic acid linked to hydroxytyrosol (3,4-DHPEA-EDA) on both kinds of cells. Other purified compounds such as isomer of oleuropein aglycon (3,4-DHPEA-EA), oleuropein, tyrosol, [p-hydroxyphenyl-ethanol (p-HPEA)] the dialdehydic form of elenoic acid linked to tyrosol, caffeic acid, and verbascoside also protected the cells against H2O2-induced DNA damage although with a lower efficacy (range of protection, 25–75%). On the other hand, when tested in a model system in which the oxidative stress was induced by phorbole 12-myristate 13acetate-activated monocytes, p-HPEA was more effective than 3,4-DHPEA in preventing the oxidative DNA damage. Overall, these results suggest that OO-PE and WW-PE may efficiently prevent the initiation step of carcinogenesis in vivo, because the concentrations effective against the oxidative DNA damage could be easily reached with normal intake of olive oil.

Raederstorff, D., Antioxidant activity of olive polyphenols in humans: a review. Int. J. Vitam Nutr. Res. 2009, 79, 152– 165.

Zhongo Liu, et al. Hydroxytyrosol protects retinal pigment epithelial cells from acrolein-induced oxidative stress and mitochondrial dysfunction. J. Neurochem. 2007;103:2690-2700.

Xiaomei Zhang et al. Hydroxytyrosol inhibits pro-inflammatory cytokines, iNOS, and COX-2 expression in human monocytic cells. NaunynSchmied Arch Pharmacol. 2009;379:581-586.

Human absorption and metabolism of oleuropein and hydroxytyrosol ingested as olive (Olea europaea L.) leaf extract Authors Martin de Bock, Eric B. Thorstensen, José G. B. Derraik, Harold V. Henderson, Paul L. Hofman, Wayne S. Cutfield First published: 14 June 2013Full publication history DOI: 10.1002/mnfr.201200795 View/save citation Cited by (CrossRef): 58 articles Check for updates Citation tools Abstract Phenolic compounds derived from the olive plant (Olea europaea L.), particularly hydroxytyrosol and oleuropein, have many beneficial effects in vitro. Olive leaves are the richest source of olive phenolic compounds, and olive leaf extract (OLE) is now a popular nutraceutical taken either as Bionomic Solutions

liquid or capsules. To quantify the bioavailability and metabolism of oleuropein and hydroxytyrosol when taken as OLE, nine volunteers (five males) aged 42.8 ± 7.4 years were randomized to receive either capsulated or liquid OLE as a single lower (51.1 mg oleuropein, 9.7 mg hydroxytyrosol) or higher (76.6 mg oleuropein, 14.5 mg hydroxytyrosol) dose, and then the opposite strength (but same formulation) a week later. Plasma and urine samples were collected at fixed intervals for 24 h post-ingestion. Phenolic content was analyzed by LC-ESI-MS/MS. Conjugated metabolites of hydroxytyrosol were the primary metabolites recovered in plasma and urine after OLE ingestion. Peak oleuropein concentrations in plasma were greater following ingestion of liquid than capsule preparations (0.47 versus 2.74 ng/mL; p = 0.004), but no such effect was observed for peak concentrations of conjugated (sulfated and glucuronidated) hydroxytyrosol (p = 0.94). However, the latter peak was reached earlier with liquid preparation (93 versus 64 min; p = 0.031). There was a gender effect on the bioavailability of phenolic compounds, with males displaying greater plasma area under the curve for conjugated hydroxytyrosol (11 600 versus 2550 ng/mL; p = 0.048). All conjugated hydroxytyrosol metabolites were recovered in the urine within 8 h. There was wide inter-individual variation. OLE effectively delivers oleuropein and hydroxytrosol metabolites to plasma in humans.

Citrulline Extract

Br J Sports Med. 2002 Aug;36(4):282-9.

Citrulline/malate promotes aerobic energy production in human exercising muscle.

Bendahan D1, Mattei JP, Ghattas B, Confort-Gouny S, Le Guern ME, Cozzone PJ.

Abstract BACKGROUND:

Previous studies have shown an antiasthenic effect of citrulline/malate (CM) but the mechanism of action at the muscular level remains unknown.

OBJECTIVE:

To investigate the effects of CM supplementation on muscle energetics.

METHODS:

Eighteen men complaining of fatigue but with no documented disease were included in the study. A rest-exercise (finger flexions)-recovery protocol was performed twice before (D-7 and D0), three times during (D3, D8, D15), and once after (D22) 15 days of oral supplementation with 6 g/day CM. Metabolism of the flexor digitorum superficialis was analysed by (31)P magnetic resonance spectroscopy at 4.7 T.

RESULTS:

Metabolic variables measured twice before CM ingestion showed no differences, indicating good reproducibility of measurements and no learning effect from repeating the exercise protocol. CM ingestion resulted in a significant reduction in the sensation of fatigue, a 34% increase in the rate of oxidative ATP production during exercise, and a 20% increase in the rate of phosphocreatine recovery after exercise, indicating a larger contribution of oxidative ATP synthesis to energy production. Considering subjects individually and variables characterising aerobic function, extrema were measured after either eight or 15 days of treatment, indicating chronological heterogeneity of treatment induced changes. One-way analysis of variance confirmed improved aerobic function, which may be the result of an enhanced malate supply activating ATP production from the tricarboxylic acid cycle through anaplerotic reactions.

J Strength Cond Res. 2010 May;24(5):1215-22. doi: 10.1519/JSC.0b013e3181cb28e0.

Citrulline malate enhances athletic anaerobic performance and relieves muscle soreness.

Pérez-Guisado J1, Jakeman PM.

Abstract The purpose of the present study was to determine the effects of a single dose of citrulline malate (CM) on the performance of flat barbell bench presses as an anaerobic exercise and in terms of decreasing muscle soreness after exercise. Forty-one men performed 2 consecutive pectoral training session protocols (16 sets). The study was performed as a randomized, double-blind, 2-period crossover design. Eight grams of CM was used in 1 of the 2 training sessions, and a placebo was used in the other. The subjects’ resistance was tested using the repetitions to fatigue test, at 80% of their predetermined 1 repetition maximum (RM), in the 8 sets of flat barbell bench presses during the pectoral training session (S1-4 and S1′-4′). The p-value was 0.05. The number of repetitions showed a significant increase from placebo treatment to CM treatment from the third set evaluated (p <0.0001). This increase was positively correlated with the number of sets, achieving 52.92% more repetitions and the 100% of response in the last set (S4′). A significant decrease of 40% in muscle soreness at 24 hours and 48 hours after the pectoral training session and a higher percentage response than 90% was achieved with CM supplementation. The only side effect reported was a feeling of stomach discomfort in 14.63% of the subjects. We conclude that the use of CM might be useful to increase athletic performance in high-intensity anaerobic exercises with short rest times and to relieve postexercise muscle soreness. Thus, athletes undergoing intensive preparation involving a high level of training or in competitive events might profit from CM.

PMID: 20386132 DOI: 10.1519/JSC.0b013e3181cb28e0

J Int Soc Sports Nutr. 2016 Feb 19;13:6. doi: 10.1186/s12970-016-0117-z. eCollection 2016.

Oral L-citrulline supplementation enhances cycling time trial performance in healthy trained men: Double-blind randomized placebo-controlled 2-way crossover study.

Suzuki T1, Morita M1, Kobayashi Y2, Kamimura A1. Bionomic Solutions

Abstract BACKGROUND:

Many human studies report that nitric oxide (NO) improves sport performance. This is because NO is a potential modulator of blood flow, muscle energy metabolism, and mitochondrial respiration during exercise. L-Citrulline is an amino acid present in the body and is a potent endogenous precursor of L-arginine, which is a substrate for NO synthase. Here, we investigated the effect of oral L-citrulline supplementation on cycling time trial performance in humans.

METHODS:

A double-blind randomized placebo-controlled 2-way crossover study was employed. Twenty-two trained males consumed 2.4 g/day of Lcitrulline or placebo orally for 7 days. On Day 8 they took 2.4 g of L-citrulline or placebo 1 h before a 4-km cycling time trial. Time taken to complete the 4 km cycle, along with power output/VO2 ratio (PO/VO2), plasma nitrite and nitrate (NOx) and amino acid levels, and visual analog scale (VAS) scores, was evaluated.

RESULTS:

L-Citrulline supplementation significantly increased plasma L-arginine levels and reduced completion time by 1.5 % (p < 0.05) compared with placebo. Moreover, L-citrulline significantly improved subjective feelings of muscle fatigue and concentration immediately after exercise.

. Biochem Biophys Res Commun. 2000 Sep 7;275(3):715-9.

Regulation of nitric oxide production by arginine metabolic enzymes.

Mori M1, Gotoh T.

Abstract Nitric oxide (NO) is synthesized from arginine by NO synthase (NOS), and the availability of arginine is one of the rate-limiting factors in cellular NO production. Citrulline, which is formed as a by-product of the NOS reaction, can be recycled to arginine by successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), forming the citrulline-NO cycle. AS and sometimes AL have been shown to be coinduced with inducible NOS (iNOS) in various cell types including activated macrophages, vascular smooth muscle cells, glial cells, neuronal PC12 cells, and pancreatic beta-cells. Cationic amino acid transporter (CAT)-2 is induced in activated macrophages but not in PC12 cells. On the other hand, arginase can downregulate NO production by decreasing intracellular arginine concentrations. iNOS and arginase activities are regulated reciprocally in macrophages by cytokines, and this may guarantee the efficient production of NO. In contrast, iNOS and arginase isoforms (type I and II) are coinduced in lipopolysaccharide (LPS)-activated macrophages. These results indicate that NO production is modulated by the uptake, recycling, and degradation of arginine.

Bovine bronchial epithelial cells metabolize L-arginine to L-citrulline: Possible role of nitric oxide synthase Author panelR.A.RobbinsF.G.HamelA.A.FloreaniG.L.GossmanK.J.NelsonS.BelenkyI.Rubinstein https://doi.org/10.1016/0024-3205(93)90232-RGet rights and content Abstract The conversion of L-arginine to L-citrulline is catalyzed by nitric oxide synthase (NOS), and results in the release of nitric oxide (NO). We hypothesized that bronchial epithelial cells metabolize L-arginine to L-citrulline. We found that cell lysates obtained from unstimulated, cultured bovine bronchial epithelial cells (BBECs) converted L-[3H]arginine to L-[3H]citrulline. This conversion was attenuated by three competitive NOS inhibitors and modulated by lipopolysaccharide and cigarette smoke extract (p<0.01, all comparisons). These data demonstrate that BBECs metabolize L-arginine to L-citrulline and implicate a role for the L-arginine:NOS biosynthetic pathway in modulating airway responses.

Isolation and determination of arginine and citrulline Author links open overlay panelEvelyn L.Oginsky https://doi.org/10.1016/S0076-6879(57)03434-5 Determination of arginine can be achieved by colorimetric and microbiological method. In colorimetric method arginine in the sample is decomposed by arginase to ornithine and urea, and the latter is assayed colorimetrically. In microbiological arginine is assayed by measurement of the growth response of bacteria for which the amino acid is an essential nutrient. Arginine can be prepared from protein hydrolyzates, or from enzymatic synthesis, and isolated as the flavianate, which is then hydrolyzed to the hydrochloride. The procedure described in this chapter is for the large-scale isolation of L-arginine from gelatin, which involves isolation of the flavianate, conversion of flavianate to hydrochloride, and recrystallization of arginine hydrochloride. The melting point of L-arginine hydrochloride isolated by this procedure was reported as 222 °. The determination of citrulline is based on the formation of a colored reaction product with diacetyl monoxime in acid solution. The L-isomer of citrulline can be formed from L-arginine by arginine desimidase, and can be isolated via the copper salt.

Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts Authors Rudolf Tischner, Mary Galli, Yair M. Heimer, Sarah Bielefeld, Mamoru Okamoto, Alyson Mack, Nigel M. Crawford First published: 25 July 2007Full publication history DOI: 10.1111/j.1742-4658.2007.05950.x Cited by (CrossRef): 21 articles Check for updates Citation tools N. M. Crawford, Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA Bionomic Solutions

Abstract There are many reports of an arginine-dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified. To measure nitric oxide synthase activity, many studies have relied on a citrulline-based assay that measures the formation of l-citrulline from l-arginine using ion exchange chromatography. In this article, we report that when such assays are used with protein extracts from Arabidopsis, an arginine-dependent activity was observed, but it produced a product other than citrulline. TLC analysis identified the product as argininosuccinate. The reaction was stimulated by fumarate (> 500 µm), implicating the urea cycle enzyme argininosuccinate lyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argininosuccinate. These results indicate that caution is needed when using standard citrulline-based assays to measure nitric oxide synthase activity in plant extracts, and highlight the importance of verifying the identity of the product as citrulline.

Mulberry Extract

Chin J Nat Med. 2016 Jan;14(1):17-30. doi: 10.3724/SP.J.1009.2016.00017.

Phytochemistry, pharmacology, and clinical trials of Morus alba.

Chan EW1, Lye PY2, Wong SK3.

Abstract The present review is aimed at providing a comprehensive summary on the botany, utility, phytochemistry, pharmacology, and clinical trials of Morus alba (mulberry or sang shu). The mulberry foliage has remained the primary food for silkworms for centuries. Its leaves have also been used as animal feed for livestock and its fruits have been made into a variety of food products. With flavonoids as major constituents, mulberry leaves possess various biological activities, including antioxidant, antimicrobial, skin-whitening, cytotoxic, anti-diabetic, glucosidase inhibition, anti-hyperlipidemic, anti-atherosclerotic, anti-obesity, cardioprotective, and cognitive enhancement activities. Rich in anthocyanins and alkaloids, mulberry fruits have pharmacological properties, such as antioxidant, anti-diabetic, anti-atherosclerotic, anti-obesity, and hepatoprotective activities. The root bark of mulberry, containing flavonoids, alkaloids and stilbenoids, has antimicrobial, cytotoxic, antiinflammatory, and anti-hyperlipidemic properties. Other pharmacological properties of M. alba include anti-platelet, anxiolytic, anti-asthmatic, anthelmintic, antidepressant, cardioprotective, and immunomodulatory activities. Clinical trials on the efficiency of M. alba extracts in reducing blood glucose and cholesterol levels and enhancing cognitive ability have been conducted. The phytochemistry and pharmacology of the different parts of the mulberry tree confer its traditional and current uses as fodder, food, cosmetics, and medicine. Overall, M. alba is a multifunctional plant with promising medicinal properties.

Am J Chin Med. 2012;40(1):163-75.

Mulberry leaf extract stimulates glucose uptake and GLUT4 translocation in rat adipocytes.

Naowaboot J1, Pannangpetch P, Kukongviriyapan V, Prawan A, Kukongviriyapan U, Itharat A.

Abstract Mulberry (Morus alba L.) leaf tea is promoted for its health benefits and the control of diabetes in Asian nations. The blood glucose lowering activity of mulberry leaf extract (MA) has been proven; however, the molecular basis underlying this effect remains unclear. The aim of the present work is to elucidate its mechanism of the antihyperglycemic action, by examining the effect of MA on glucose uptake and the translocation of glucose transporter 4 protein (GLUT4) to the plasma membrane of adipocytes isolated from diabetic rats. The incubation of adipocytes with 5-45 μg/ml MA resulted in 31-54% increase of glucose uptake in a dose-dependent manner. This glucose uptake enhancing effect was inhibited by the phosphoinositol 3-kinase (PI3-K) inhibitor, wortmannin (100 nM). The GLUT4 protein on the plasma membrane fraction of adipocytes was markedly increased after treatment with 15 μg/ml MA extract. Interestingly, gallic acid, one of the phenolic compounds found in MA extract, increased glucose uptake and enhanced the translocation of GLUT4 at concentrations comparable to the amount of gallic acid in the effective concentration ranges of MA. Thus, it is likely that gallic acid contributes, at least in part, to its antihyperglycemic activity. The present results suggest that the antihyperglycemic action of MA is mediated by increasing glucose uptake via the activation of PI3-K signaling pathway and translocation of GLUT4 to the plasma membrane. These findings are the first molecular evidence supporting the mulberry tea as herbal medicine for diabetic patients.

PMID: 22298456 DOI: 10.1142/S0192415X12500139

J Sci Food Agric. 2010 Nov;90(14):2386-92. doi: 10.1002/jsfa.4096.

Effect of flavonol glycoside in mulberry (Morus alba L.) leaf on glucose metabolism and oxidative stress in liver in diet-induced obese mice. Katsube T1, Yamasaki M, Shiwaku K, Ishijima T, Matsumoto I, Abe K, Yamasaki Y.

Abstract BACKGROUND:

Mulberry therapies on type 2 diabetic patients or streptozotocin-induced diabetic rats have been reported to improve fasting blood glucose levels. We investigated the effects of dietary consumption of mulberry-leaf powder and purified quercetin 3-(6-malonylglucoside), the quantitatively major flavonol glycoside in mulberry leaves, on glucose and lipid metabolism in high-fat diet-induced obese mice. Male C57BL/6J mice aged 8 weeks were assigned to three groups (control, mulberry leaf powder (MLP), and quercetin 3-(6-malonylglucoside) (Q3MG)) and treated with their respective diets for 8 weeks.

RESULTS:

We found that dietary supplementation of 10 g MLP kg(-1) or 1 g Q3MG kg(-1) in high-fat diet effectively suppressed blood glucose levels. We also noted increased expression of glycolysis-related genes and suppression of thiobarbituric acid reactive substances concentrations in the liver of Q3MG group compared to control mice.

CONCLUSION: Bionomic Solutions

Dietary consumption of Q3MG, the quantitatively major flavonol glycoside in mulberry leaves, improved hyperglycemia in obese mice and reduced oxidative stress in the liver.

2010 Society of Chemical Industry PMID: 20648552 DOI: 10.1002/jsfa.4096

Int J Biol Sci. 2011; 7(6): 715–728.

Published online 2011 Jun 4.

PMCID: PMC3119844 Protective Effects of Morus alba Leaves Extract on Ocular Functions of Pups from Diabetic and Hypercholesterolemic Mother Rats H.I.H. El-Sayyad,1,M.A. El-Sherbiny,2 M.A. Sobh,3 A.M. Abou-El-Naga,1 M.A.N. Ibrahim,1 and S.A. Mousa4 Abstract Phytotherapy is frequently considered to be less toxic and free from side effects than synthetic drugs. Hence, the present study was designed to investigate the protective use of crude water extract of Morus alba leaves on ocular functions including cataractogenesis, biochemical diabetic and hypercholesterolemic markers, retinal neurotransmitters and retinopathy of rat pups maternally subjected to either diabetes and/or hypercholesterolemia. Application of crude water extract of Morus alba resulted in amelioration of the alterations of maternal serum glucose, LDL, HDL, total cholesterol and creatine phosphokinase activity as well as retinal neurotransmitters including acetylcholine (ACE), adrenaline (AD), nor-adrenaline (NAD), serotonin (5-HT), histamine (HS), dopamine (DA) and gamma amino butyric acid (GABA). The retina of pups of either diabetic and/or hypercholesterolemia mothers exhibited massive alterations of retinal neurotransmitters. The alterations of retinal neurotransmitters were correlated with the observed pathological alterations of retinal pigmented epithelium, photoreceptor inner segment and ganglion cells and increased incidence of DNA fragmentation and apoptosis cell death. However, protection with Morus alba extract led to amelioration of the pathological alterations of retinal neurons and estimated neurotransmitters. Furthermore, a striking incidence of cataract was detected in pups of either diabetic and/or hypercholesterolemic mothers. Highest cataractogenesis was observed in pups of combined treated groups. Our data indicate that experimental maternal diabetes alone or in combination with hypercholesterolemia led to alteration in the ocular structures of their pups, with an increasing incidence of cataract and retinopathy, and the effects of the extract might be attributed to the hypoglycaemic, antihypercholesterolemic and anti-oxidative potential of flavonoids, the major components of the plant extract.

Mulberry

Fruit

Extract

Protects against Memory Impairment and Hippocampal Damage in Animal Model of Vascular Dementia.

Kaewkaen P1, Tong-Un T, Wattanathorn J, Muchimapura S, Kaewrueng W, Wongcharoenwanakit S.

Abstract Nowadays, the preventive strategy of vascular dementia, one of the challenge problems of elderly, has received attention due to the limitation of therapeutic efficacy. In this study, we aimed to determine the protective effect and possible mechanism of action of mulberry fruit extract on memory impairment and brain damage in animal model of vascular dementia. Male Wistar rats, weighing 300-350 g, were orally given mulberry extract at doses of 2, 10 and 50 mg/kg at a period of 7 days before and 21 days after the occlusion of right middle cerebral artery (Rt.MCAO). It was found that rats subjected to mulberry fruits plus Rt.MCAO showed the enhanced memory, the increased densities of neuron, cholinergic neuron, Bcl-2-immunopositive neuron together with the decreased oxidative stress in hippocampus. Taken all data together, the cognitive enhancing effect of mulberry fruit extract observed in this study might be partly associated with the increased cholinergic function and its neuroprotective effect in turn occurs partly via the decreased oxidative stress and apoptosis. Therefore, mulberry fruit is the potential natural cognitive enhancer and neuroprotectant. However, further researches are essential to elucidate the possible active ingredient.

Regulation of obesity and lipid disorders by herbal extracts from Morus alba, Melissa officinalis, and Artemisia capillaris in high-fat diet-induced obese mice.

Lee J1, Chae K, Ha J, Park BY, Lee HS, Jeong S, Kim MY, Yoon M.

Erratum in J Ethnopharmacol. 2008 Mar 28;116(3):576.

Abstract Melissa officinalis L. (Labiatae), Morus alba L. (Moraceae), and Artemisia capillaris Thunb. (Compositae) are suggested to be involved in the regulation of hyperlipidemia. We hypothesized that Ob-X, a mixture of three herbs, Morus alba, Melissa officinalis and Artemisia capillaris [corrected] improves lipid metabolism, body weight gain and adiposity and that peroxisome proliferator-activated receptor alpha (PPARalpha) is associated with these events. Mice fed a high-fat diet for 12 weeks exhibited increases in body weight gain and adipose tissue mass compared with mice fed a low fat diet. However, feeding a high-fat diet supplemented with Ob-X significantly reduced these effects. Ob-X treatment also decreased the circulating levels of triglycerides and total cholesterol, and inhibited hepatic lipid accumulation. Ob-X supplementation was found to increase the hepatic mRNA levels of PPARalpha target enzymes responsible for fatty acid beta-oxidation. Moreover, Ob-X elevated the endogenous expression of a luciferase reporter gene containing three copies of a PPAR response element (PPRE) in NMu2Li liver cells. These data demonstrate that Ob-X regulates body weight gain, adipose tissue mass, and lipid metabolism in part through changes in the expression of hepatic PPARalpha target genes.

PMID: 18023310 DOI: 10.1016/j.jep.2007.09.029

Genetics of Insulin Resistance and the Metabolic Syndrome.

Brown AE1, Walker M2.

Abstract Insulin resistance and the metabolic syndrome are complex metabolic traits and key risk factors for the development of cardiovascular disease. They result from the interplay of environmental and genetic factors but the full extent of the genetic background to these conditions remains incomplete. Large-scale genome-wide association studies have helped advance the identification of common genetic variation associated with insulin resistance and the metabolic syndrome, and more recently, exome sequencing has allowed the identification of rare variants associated Bionomic Solutions

with the pathogenesis of these conditions. Many variants associated with insulin resistance are directly involved in glucose metabolism; however, functional studies are required to assess the contribution of other variants to the development of insulin resistance. Many genetic variants involved in the pathogenesis of the metabolic syndrome are associated with lipid metabolism.

PMID: 27312935 PMCID: PMC4911377 DOI: 10.1007/s11886-016-0755-4

Prev Nutr Food Sci. 2013 Dec; 18(4): 256–262.

doi: 10.3746/pnf.2013.18.4.256 PMCID: PMC3925215 Analysis of Functional Constituents in Mulberry (Morus alba L.) Twigs by Different Cultivars, Producing Areas, and Heat Processings Sang Won Choi,1 Yeon Jeong Jang,1 Yu Jin Lee,1 Hyun Hee Leem,2 and Eun Ok Kim3 Abstract Four functional constituents, oxyresveratrol 3ʹ-O-β-D-glucoside (ORTG), oxyresveratrol (ORT), t-resveratrol (RT), and moracin (MC) were isolated from the ethanolic extract of mulberry (Morus alba L.) twigs by a series of isolation procedures, including solvent fractionation, and silica-gel, ODS-A, and Sephadex LH-20 column chromatographies. Their chemical structures were identified by NMR and FABMS spectral analysis. Quantitative changes of four phytochemicals in mulberry twigs were determined by HPLC according to cultivar, producing area, and heat processing. ORTG was a major abundant compound in the mulberry twigs, and its levels ranged from 23.7 to 105.5 mg% in six different mulberry cultivars. Three other compounds were present in trace amounts (<1 mg/100 g) or were not detected. Among mulberry cultivars examined, “Yongcheon” showed the highest level of ORTG, whereas “Somok” had the least ORTG content. Levels of four phytochemicals in the mulberry twigs harvested in early September were higher than those harvested in early July. Levels of ORTG and ORT in the “Cheongil” mulberry twigs produced in the Uljin area were higher than those produced in other areas. Generally, levels of ORTG and ORT in mulberry twigs decreased with heat processing, such as steaming, and microwaving except roasting, whereas those of RT and MC did not considerably vary according to heat processing. These results suggest that the roasted mulberry twigs may be useful as potential sources of functional ingredients and foods.

Chin J Nat Med. 2016 Jan;14(1):17-30. doi: 10.3724/SP.J.1009.2016.00017.

Phytochemistry, pharmacology, and clinical trials of Morus alba.

Chan EW1, Lye PY2, Wong SK3.

Abstract The present review is aimed at providing a comprehensive summary on the botany, utility, phytochemistry, pharmacology, and clinical trials of Morus alba (mulberry or sang shu). The mulberry foliage has remained the primary food for silkworms for centuries. Its leaves have also been used as animal feed for livestock and its fruits have been made into a variety of food products. With flavonoids as major constituents, mulberry leaves possess various biological activities, including antioxidant, antimicrobial, skin-whitening, cytotoxic, anti-diabetic, glucosidase inhibition, anti-hyperlipidemic, anti-atherosclerotic, anti-obesity, cardioprotective, and cognitive enhancement activities. Rich in anthocyanins and alkaloids, mulberry fruits have pharmacological properties, such as antioxidant, anti-diabetic, anti-atherosclerotic, anti-obesity, and hepatoprotective activities. The root bark of mulberry, containing flavonoids, alkaloids and stilbenoids, has antimicrobial, skin-whitening, cytotoxic, anti-inflammatory, and anti-hyperlipidemic properties. Other pharmacological properties of M. alba include anti-platelet, anxiolytic, anti-asthmatic, anthelmintic, antidepressant, cardioprotective, and immunomodulatory activities. Clinical trials on the efficiency of M. alba extracts in reducing blood glucose and cholesterol levels and enhancing cognitive ability have been conducted. The phytochemistry and pharmacology of the different parts of the mulberry tree confer its traditional and current uses as fodder, food, cosmetics, and medicine. Overall, M. alba is a multi-functional plant with promising medicinal properties.

PMID: 26850343 DOI: 10.3724/SP.J.1009.2016.00017

Ellagic Acid Extract

Exp Dermatol. 2010 Aug;19(8):e182-90. doi: 10.1111/j.1600-0625.2009.01044.x.

Dietary compound ellagic acid alleviates skin wrinkle and inflammation induced by UV-B irradiation.

Bae JY1, Choi JS, Kang SW, Lee YJ, Park J, Kang YH.

Abstract Ellagic acid, a polyphenol compound present in berries and pomegranate, has received attention as an agent that may have potential bioactivities preventing chronic diseases. This study examined photoprotective effects of ellagic acid on collagen breakdown and inflammatory responses in UV (ultraviolet)-B irradiated human skin cells and hairless mice. Ellagic acid attenuated the UV-B-induced toxicity of HaCaT keratinocytes and human dermal fibroblasts. Non-toxic ellagic acid markedly prevented collagen degradation by blocking matrix metalloproteinase production in UV-B-exposed fibroblasts. Anti-wrinkle activity of ellagic acid was further investigated in hairless mice exposed to UV-B, in which it attenuated UV-B-triggered skin wrinkle formation and epidermal thickening. Topical application of 10 micromol/l ellagic acid diminished production of pro-inflammatory cytokines IL-1beta and IL-6, and blocked infiltration of inflammatory macrophages in the integuments of SKH-1 hairless mice exposed to UV-B for 8 weeks. In addition, this compound mitigated inflammatory intracellular cell adhesion molecule-1 expression in UV-B-irradiated keratinocytes and photoaged mouse epidermis. These results demonstrate that ellagic acid prevented collagen destruction and inflammatory responses caused by UV-B. Therefore, dietary and pharmacological interventions with berries rich in ellagic acid may be promising treatment strategies interrupting skin wrinkle and inflammation associated with chronic UV exposure leading to photoageing. Bionomic Solutions

Ellagic acid

in pomegranate suppresses resistin secretion by a novel regulatory mechanism involving the degradation of intracellular resistin protein in adipocytes Author links open overlay panelYasukoMakino-WakagiYukihiroYoshimuraYukiUzawaNobuhiroZaimaTatsuyaMoriyamaYukioKawamura https://doi.org/10.1016/j.bbrc.2011.12.067 Abstract Resistin, an adipocytokine, is considered the link between obesity and type 2 diabetes. Pomegranate is a rich source of compounds used to treat metabolic diseases including type 2 diabetes. In this study, we found that consumption of pomegranate fruit extract (PFE) predominantly reduced the serum resistin levels in ovariectomized mice, an animal model with elevated resistin levels in serum and upregulated resistin mRNA expression in white adipose tissue. Moreover, the PFE significantly reduced the secretion and intracellular protein levels of resistin in differentiated murine 3T3-L1 adipocytes, but it did not alter resistin mRNA expression. When de novo protein synthesis was inhibited by the protein synthesis inhibitor cycloheximide, the intracellular resistin protein levels were drastically reduced by the PFE, suggesting that the PFE promoted the degradation of resistin at the protein level. We also found that ellagic acid (EA), a main component of pomegranate, had the same effects on the secretion and intracellular protein level of resistin. These results suggest that EA in pomegranate suppresses resistin secretion by a novel mechanism involving the degradation of intracellular resistin protein in adipocytes.

Highlights Pomegranate fruit extract (PFE) suppressed the elevated serum resistin in mice.

PFE reduced resistin secretion without affecting the mRNA expression in 3T3-L1 cells. It was suggested that intracellular resistin protein was degraded by PFE treatment. Ellagic acid was identified as an active component of PFE.

Our study provides a novel mechanism for suppressing resistin secretion.

Adv Nutr. 2016 Sep; 7(5): 961–972.

Published online 2016 Sep 7. doi: 10.3945/an.116.012575 PMCID: PMC5015040 Improvements in Metabolic Health with Consumption of Ellagic Acid and Subsequent Conversion into Urolithins: Evidence and Mechanisms1,2 Inhae Kang,3 Teresa Buckner,3 Neil F Shay,4 Liwei Gu,5 and Soonkyu Chung3 Abstract Ellagic acid (EA) is a naturally occurring polyphenol found in some fruits and nuts, including berries, pomegranates, grapes, and walnuts. EA has been investigated extensively because of its antiproliferative action in some cancers, along with its anti-inflammatory effects. A growing body of evidence suggests that the intake of EA is effective in attenuating obesity and ameliorating obesity-mediated metabolic complications, such as insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, and atherosclerosis. In this review, we summarize how intake of EA regulates lipid metabolism in vitro and in vivo and delineate the potential mechanisms of action of EA on obesity-mediated metabolic complications. We also discuss EA as an epigenetic effector, as well as a modulator of the gut microbiome, suggesting that EA may exert a broader spectrum of health benefits than has been demonstrated to date. Therefore, this review aims to suggest the potential metabolic benefits of consumption of EA-containing fruits and nuts against obesity-associated health conditions.

Introduction Obesity and its associated metabolic complications, such as type 2 diabetes, have reached epidemic proportions in the United States (1). Studies show an inverse relation between an increased intake of fruits and vegetables and the incidence of obesity (2, 3), emphasizing the significance of fruit and vegetable intake in the pathogenesis of obesity. The metabolic benefit of fruit and vegetable intake likely is dependent on the synergistic effects of numerous dietary bioactive components, including fiber, vitamins, minerals, and polyphenolic compounds. Dietary polyphenols have received attention for their ability to modulate lipid and glucose metabolism (4, 5). In addition to the health benefits from flavonoids, it is also notable that several nonflavonoid polyphenolic compounds are considered to be important to metabolic health, including resveratrol, unique to grapes and red wine (6); curcumin, a strong antioxidant from turmeric (7); and now perhaps ellagic acid (EA; 2,3,7,8tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione)6, found in berries.

We and others have demonstrated that EA is a promising dietary nonflavonoid polyphenolic compound that could modulate the development of obesity and the associated metabolic consequences. EA occurs naturally in some fruits, such as berries (strawberries and red and black raspberries), tree nuts (walnuts, pistachios, cashews, acorns, and pecans), pomegranates, and muscadine grapes (8–12). In particular, berries provide EA in the form of ellagitannins, which constitute ∼60% (red raspberry) to 80% (cloudberry) of the total phenolic compounds present (13). There is emerging evidence that EA may decrease symptoms of chronic metabolic diseases, including dyslipidemia, insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease. Despite the growing amount of information on EA, a definitive mechanism of action has not been established. This may be attributed to the complexity of EA metabolism, which is governed by various factors, including 1) the dietary source of EA and the EA-to-ellagitannin ratio, 2) the existence of other phytochemicals in a specific food, 3) the absorption rate, 4) interactions with gut microbes, and 5) the health status of the host. Therefore, an integrative view that considers all of the above is required for a complete assessment of a potential beneficial role of EA-containing foods on obesity-mediated chronic diseases. The objective of this review is to consolidate recent advances in EA research related to obesity and its metabolic consequences, especially from the most recently published reports. In this review, we first address EA absorption in the digestive system, summarizing how EA enters circulation, then separately delineate the metabolic effects of EA in vitro and in vivo. For in vitro studies, we included studies that examined the effects of EA on lipid metabolism in adipocytes or hepatocytes, but excluded studies in breast- or colon-cancer cells. For in vivo studies, we included studies that used obesity-prone rodent models with dietary supplementation with pure EA or EA-enriched polyphenolic extracts. To be included in this review, cited reports indicated that EA or ellagitannins were the primary polyphenolic component, or demonstrated that EA or ellagitannins were the most responsible polyphenol for target outcomes. Bionomic Solutions

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García-Villalba R, Beltran D, Espin JC, Selma MV, Tomas-Barberan FA. Time course production of urolithins from ellagic acid by human gut microbiota. J Agric Food Chem 2013;61:8797–806. [PubMed] González-Barrio R, Edwards CA, Crozier A. Colonic catabolism of ellagitannins, ellagic acid, and raspberry anthocyanins: in vivo and in vitro studies. Drug Metab Dispos 2011;39:1680–8. [PubMed] Espín JC, González-Barrio R, Cerdá B, López-Bote C, Rey AI, Tomás-Barberán FA. Iberian pig as a model to clarify obscure points in the bioavailability and metabolism of ellagitannins in humans. J Agric Food Chem 2007;55:10476–85. [PubMed] Cerdá B, Espín JC, Parra S, Martínez P, Tomás-Barberán FA. The potent in vitro antioxidant ellagitannins from pomegranate juice are metabolised into bioavailable but poor antioxidant hydroxy–6H–dibenzopyran–6– one derivatives by the colonic microflora of healthy humans. Eur J Nutr 2004;43:205–20. 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Kang I, Kim Y, Tomas-Barberan FA, Espin JC, Chung S, Urolithin A. C and D, but not iso-Urolithin A and Urolithin B, attenuate triglyceride accumulation in human cultures of adipocytes and hepatocytes. Mol Nutr Food Res 2016;60:1129–38. [PubMed] Lei F, Zhang XN, Wang W, Xing DM, Xie WD, Su H, Du LJ. Evidence of anti-obesity effects of the pomegranate leaf extract in high-fat diet induced obese mice. Int J Obes (Lond) 2007;31:1023–9. [PubMed] Monika P, Geetha A. The modulating effect of Persea americana fruit extract on the level of expression of fatty acid synthase complex, lipoprotein lipase, fibroblast growth factor-21 and leptin–a biochemical study in rats subjected to experimental hyperlipidemia and obesity. Phytomedicine 2015;22:939–45. [PubMed] Ratnam DV, Chandraiah G, Meena AK, Ramarao P, Kumar MN. The co-encapsulated antioxidant nanoparticles of ellagic acid and coenzyme Q10 ameliorates hyperlipidemia in high fat diet fed rats. J Nanosci Nanotechnol 2009;9:6741–6. [PubMed] Cao K, Xu J, Pu W, Dong Z, Sun L, Zang W, Gao F, Zhang Y, Feng Z, Liu J. Punicalagin, an active component in pomegranate, ameliorates cardiac mitochondrial impairment in obese rats via AMPK activation. Sci Rep 2015;5:14014. [PMC free article] [PubMed] Kang I, Espin JC, Carr T, Tomas-Barberan FA, Chung S. Raspberry seed flour attenuates high sucrose diet-mediated hepatic stress and adipose tissue inflammation. J Nutr Biochem 2016;32:64–72. [PubMed] Yoshimura Y, Nishii S, Zaima N, Moriyama T, Kawamura Y. Ellagic acid improves hepatic steatosis and serum lipid composition through reduction of serum resistin levels and transcriptional activation of hepatic PPARa in obese, diabetic KK-A(y) mice. Biochem Biophys Res Commun 2013;434:486–91. [PubMed] Panchal SK, Ward L, Brown L. Ellagic acid attenuates high-carbohydrate, high-fat diet-induced metabolic syndrome in rats. Eur J Nutr 2013;52:559–68. [PubMed] Kannan MM, Quine SD. Ellagic acid inhibits cardiac arrhythmias, hypertrophy and hyperlipidaemia during myocardial infarction in rats. Metabolism 2013;62:52–61. [PubMed] Ahad A, Ganai AA, Mujeeb M, Siddiqui WA. Ellagic acid, an NF-kappaB inhibitor, ameliorates renal function in experimental diabetic nephropathy. Chem Biol Interact 2014;219:64–75. [PubMed] Rani UP, Kesavan R, Ganugula R, Avaneesh T, Kumar UP, Reddy GB, Dixit M. Ellagic acid inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and prevents atheroma formation in streptozotocin-induced diabetic rats. J Nutr Biochem 2013;24:1830–9. [PubMed] Neyrinck AM, Van Hee VF, Bindels LB, De BF, Cani PD, Delzenne NM. Polyphenol-rich extract of pomegranate peel alleviates tissue inflammation and hypercholesterolaemia in high-fat diet-induced obese mice: potential implication of the gut microbiota. Br J Nutr 2013;109:802–9. [PubMed] Song Y, Park HJ, Kang SN, Jang SH, Lee SJ, Ko YG, Kim GS, Cho JH. Blueberry peel extracts inhibit adipogenesis in 3T3–L1 cells and reduce high-fat diet-induced obesity. PLoS One 2013;8:e69925. [PMC free article] [PubMed] Noh J, Kim Y, Gang G, Yang K, Lee H, Oh W, Song K, Lee C. Chestnut (Castanea crenata) inner shell extract inhibits development of hepatic steatosis in C57BL/6 mice fed a high-fat diet. Food Chem 2012;121:437–42.

Wang J, Rong X, Um IS, Yamahara J, Li Y. 55-week treatment of mice with the unani and ayurvedic medicine pomegranate flower ameliorates ageing-associated insulin resistance and skin abnormalities. Evid Based Complement Alternat Med 2012;2012:350125. [PMC free article] [PubMed] USDA Food and Drug Administration, Center for Drug Evaluation and Research [Internet]. Guidance for industry on estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers [updated 2005 July 22] [cited 2016 May 1]. Available from: http://www.fda.gov/downloads/Drugs/…/Guidances/UCM078932.pdf. Bionomic Solutions

Tasaki M, Umemura T, Maeda M, Ishii Y, Okamura T, Inoue T, Kuroiwa Y, Hirose M, Nishikawa A. Safety assessment of ellagic acid, a food additive, in a subchronic toxicity study using F344 rats. Food Chem Toxicol 2008;46:1119–24. [PubMed] Ziccardi P, Nappo F, Giugliano G, Esposito K, Marfella R, Cioffi M, D’Andrea F, Molinari AM, Giugliano D. Reduction of inflammatory cytokine concentrations and improvement of endothelial functions in obese women after weight loss over one year. Circulation 2002;105:804–9. [PubMed] Frühbeck G, Gomez-Ambrosi J, Muruzabal FJ, Burrell MA. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation. Am J Physiol Endocrinol Metab 2001;280:E827–47. [PubMed] Roberts CK, Sindhu KK. Oxidative stress and metabolic syndrome. Life Sci 2009;84:705–12. [PubMed] Winand J, Schneider YJ. The anti-inflammatory effect of a pomegranate husk extract on inflamed adipocytes and macrophages cultivated independently, but not on the inflammatory vicious cycle between adipocytes and macrophages. Food Funct 2014;5:310–8. [PubMed] Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA. The hormone resistin links obesity to diabetes. Nature 2001;409:307–12. [PubMed] Yu YM, Chang WC, Wu CH, Chiang SY. Reduction of oxidative stress and apoptosis in hyperlipidemic rabbits by ellagic acid. J Nutr Biochem 2005;16:675–81. [PubMed] Ding Y, Zhang B, Zhou K, Chen M, Wang M, Jia Y, Song Y, Li Y, Wen A. Dietary ellagic acid improves oxidant-induced endothelial dysfunction and atherosclerosis: role of Nrf2 activation. Int J Cardiol 2014;175:508–14. [PubMed] Chen B, Tuuli MG, Longtine MS, Shin JS, Lawrence R, Inder T, Michael Nelson D. Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts. Am J Physiol Endocrinol Metab 2012;302:E1142–52. [PMC free article] [PubMed] Chao CY, Mong MC, Chan KC, Yin MC. Anti-glycative and anti-inflammatory effects of caffeic acid and ellagic acid in kidney of diabetic mice. Mol Nutr Food Res 2010;54:388–95. [PubMed] Wu Q, Suzuki M. Parental obesity and overweight affect the body-fat accumulation in the offspring: the possible effect of a high-fat diet through epigenetic inheritance. Obes Rev 2006;7:201–8. [PubMed] Boqué N, de la Iglesia R, de la Garza AL, Milagro FI, Olivares M, Banuelos O, Soria AC, Rodriguez-Sanchez S, Martinez JA, Campion J. Prevention of diet-induced obesity by apple polyphenols in Wistar rats through regulation of adipocyte gene expression and DNA methylation patterns. Mol Nutr Food Res 2013;57:1473–8. [PubMed] Kiss AK, Granica S, Stolarczyk M, Melzig MF. Epigenetic modulation of mechanisms involved in inflammation: influence of selected polyphenolic substances on histone acetylation state. Food Chem 2012;131:1015–20.

Yun JM, Jialal I, Devaraj S. Epigenetic regulation of high glucose-induced proinflammatory cytokine production in monocytes by curcumin. J Nutr Biochem 2011;22:450–8. [PMC free article] [PubMed] Dolinoy DC, Weidman JR, Waterland RA, Jirtle RL. Maternal genistein alters coat color and protects Avy mouse offspring from obesity by modifying the fetal epigenome. Environ Health Perspect 2006;114:567–72. [PMC free article] [PubMed] Howard TD, Ho SM, Zhang L, Chen J, Cui W, Slager R, Gray S, Hawkins GA, Medvedovic M, Wagner JD. Epigenetic changes with dietary soy in cynomolgus monkeys. PLoS One 2011;6:e26791. [PMC free article] [PubMed] Bäckhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, Semenkovich CF, Gordon JI. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 2004;101:15718–23. [PMC free article] [PubMed] Bäckhed F, Manchester JK, Semenkovich CF, Gordon JI. Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. Proc Natl Acad Sci USA 2007;104:979–84. [PMC free article] [PubMed] Anhê FF, Roy D, Pilon G, Dudonne S, Matamoros S, Varin TV, Garofalo C, Moine Q, Desjardins Y, Levy E, et al. A polyphenol-rich cranberry extract protects from diet-induced obesity, insulin resistance and intestinal inflammation in association with increased Akkermansia spp. population in the gut microbiota of mice. Gut 2015;64:872–83. [PubMed] Li Z, Henning SM, Lee RP, Lu QY, Summanen PH, Thames G, Corbett K, Downes J, Tseng CH, Finegold SM, et al. Pomegranate extract induces ellagitannin metabolite formation and changes stool microbiota in healthy volunteers. Food Funct 2015;6:2487–95. [PubMed] Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature 2006;444:1022–3. [PubMed] Bionomic Solutions

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Artichoke Extract

Br J Nutr. 2010 Oct;104(7):1007-17. doi: 10.1017/S0007114510001571. Epub 2010 Jul 1.

A double-blind, placebo-controlled, cross-over study to establish the bifidogenic effect of a very-long-chain inulin extracted from globe artichoke (Cynara scolymus) in healthy human subjects.

Costabile A1, Kolida S, Klinder A, Gietl E, Bäuerlein M, Frohberg C, Landschütze V, Gibson GR.

Abstract There is growing interest in the use of inulins as substrates for the selective growth of beneficial gut bacteria such as bifidobacteria and lactobacilli because recent studies have established that their prebiotic effect is linked to several health benefits. In the present study, the impact of a very-long-chain inulin (VLCI), derived from globe artichoke (Cynara scolymus), on the human intestinal microbiota compared with maltodextrin was determined. A double-blind, cross-over study was carried out in thirty-two healthy adults who were randomised into two groups and consumed 10 g/d of either VLCI or maltodextrin, for two 3-week study periods, separated by a 3-week washout period. Numbers of faecal bifidobacteria and lactobacilli were significantly higher upon VLCI ingestion compared with the placebo. Additionally, levels of Atopobium group significantly increased, while Bacteroides-Prevotella numbers were significantly reduced. No significant changes in faecal SCFA concentrations were observed. There were no adverse gastrointestinal symptoms apart from a significant increase in mild and moderate bloating upon VLCI ingestion. These observations were also confirmed by in vitro gas production measurements. In conclusion, daily consumption of VLCI extracted from globe artichoke exerted a pronounced prebiotic effect on the human faecal microbiota composition and was well tolerated by all volunteers.

PMID: 20591206 DOI: 10.1017/S0007114510001571

Plant Foods Hum Nutr. 2015 Dec;70(4):441-53. doi: 10.1007/s11130-015-0503-8.

Pharmacological Studies of Artichoke Leaf Extract and Their Health Benefits.

Ben Salem M1, Affes H2,3, Ksouda K1, Dhouibi R1, Sahnoun Z1, Hammami S1, Zeghal KM1.

Abstract Artichoke (Cynara scolymus) leaf extract was one of the few herbal remedies which the clinical and experimental trials have complemented each other. Both experimental and clinical effects have been verified through extensive biomedical herbal remedy research. Specifically, antioxidant, choleretic, hepatoprotective, bile-enhancing and lipid-lowering effects have been demonstrated, which corresponded with its historical use. Ongoing research seems to indicate that artichoke indeed have medicinal qualities. Most significant appears to be its beneficial effect on the liver. In animal studies, liquid extracts of the roots and leaves of artichoke have demonstrated an ability to protect the liver, with possibly even to help liver cells regenerate. Although research is not yet conclusive, scientists were optimistic that its long-standing use in humans for digestive and bowel problems was indeed justified. It may also play a role in lowering cholesterol and thus help to prevent heart disease. Boiled wild artichoke reduced postprandial glycemic and insulinemic responses in normal subjects but has no effect on metabolic syndrome patients. This article intended to review the wide ranging pharmacological effects of artichoke leaf extract.

PMID: 26310198 DOI: 10.1007/s11130-015-0503-8

Pharm Biol. 2013 Sep;51(9):1104-9. doi: 10.3109/13880209.2013.777931. Epub 2013 Jun 7.

Protective effect of artichoke (Cynara scolymus) leaf extract against lead toxicity in rat.

Heidarian E1, Rafieian-Kopaei M.

Abstract Artichoke, Cynara scolymus L. (Asteraceae), has many natural antioxidants and multiple pharmacological actions. Recent studies have shown that it has antitoxic activity.

OBJECTIVE:

Lead (Pb) is a dangerous environmental toxicant that induces a broad range of dysfunctions in human. This study evaluated the protective effect of the hydroethanolic extract of artichoke against altered biochemical parameters in rats fed with lead-containing diet.

RESULTS:

Serum lead, triglyceride (TG), VLDL, ALT, AST, ALP and MDA levels decreased significantly (p < 0.05) in the artichoke-treated group (35.85, 38.26, 38.38, 21.90, 12.81, 26.86 and 46.91%, respectively) compared to lead-intoxicated rats without treatment. No significant change was observed in serum lead, ALP and ALT between artichoke and vitamin C-treated groups (p > 0.05). Furthermore, the liver histopathology in rats treated with artichoke showed a mild degree of lymphocyte infiltration that was relatively comparable to the control and vitamin C-treated groups. Bionomic Solutions

DISCUSSION AND CONCLUSION:

These results clearly show that the artichoke extract in lead-poisoned rats has suitable chelating properties for the reduction of blood lead levels. PMID: 23745593 DOI: 10.3109/13880209.2013.777931

J Food Sci. 2012 Feb;77(2):C244-52. doi: 10.1111/j.1750-3841.2011.02531.x. Epub 2012 Jan 17.

Polyphenol compounds in artichoke plant tissues and varieties.

Negro D1, Montesano V, Grieco S, Crupi P, Sarli G, De Lisi A, Sonnante G.

Abstract Polyphenol compounds, particularly caffeoylquinic acids and flavonoids, were measured in different tissues and developmental stages of 6 artichoke varietal types diffused in the Mediterranean region. Flower heads were subdivided into external, intermediate, internal bracts, and receptacle, while leaves were collected at the vegetative and productive stages. The main polyphenols detected were chlorogenic acid, cynarin, luteolin 7-O-rutinoside, and luteolin 7-O-glucoside. “Violet de Provence” artichoke proved to retain the highest content of total phenols. Single polyphenols accumulated preferentially in specific parts of capitula. In leaves, most polyphenols were detected in the productive stage of the plant. Altogether, results provide useful indications for the promotion of artichoke as nutraceutical food and for the extraction of healthpromoting substances in particular tissues/stages of the artichoke plant.

PRACTICAL APPLICATION:

We describe the content of phenolic compounds in various artichoke tissues, developmental stages, and varieties. Results indicate that artichoke leaves represent an important source of these health-promoting compounds, also useful for phytopharmaceutical applications. A wider utilization of specific artichoke types should be strongly encouraged not only as a food for the fresh market, as they are now used, but also for the pharmaceutical industry, since the content of phenolic compounds is abundant both in the heads and in the leaves.

PMID: 22251096 DOI: 10.1111/j.1750-3841.2011.02531.x

Food Chem. 2013 Dec 1;141(3):2269-77. doi: 10.1016/j.foodchem.2013.04.066. Epub 2013 May 2.

Extensive characterisation of bioactive phenolic constituents from globe artichoke (Cynara scolymus L.) by HPLC-DAD-ESI-QTOF-MS. Abu-Reidah IM1, Arráez-Román D, Segura-Carretero A, Fernández-Gutiérrez A.

Abstract The aim of this work was to characterise the phenolic compounds in artichoke (hearts) by using HPLC coupled to DAD-ESI-QTOF-MS, which proved useful in characterising 61 phenolic and other polar compounds. Notably, of the 61 compounds characterised, 34 new phenolic compounds with their isomers have been tentatively characterised in artichoke for the first time, namely: 3 hydroxybenzoic acids, 17 hydroxycinnamic acids, 4 lignans, 7 flavones, 2 flavonols, and 1 phenol derivative. Moreover, a total of 28 isomers of previously described phenolics have also been detected. The data compiled from the qualitative polyphenol characterisation indicate that the artichoke extract analysed (Blanca de Tudela variety) could be regarded as a bioactive functional food and as a promising source of antioxidant phenolic compounds.

PMID: 23870957 DOI: 10.1016/j.foodchem.2013.04.066

Efficacy of artichoke leaf extract in the treatment of patients with functional dyspepsia: a six-week placebo-controlled, double-blind, multicentre trial Authors

G. Holtmann,

B. Adam,

S. Haag,

W. Collet,

E. Grünewald,

T. Windeck First published: 20 November 2003Full publication history DOI: 10.1046/j.1365-2036.2003.01767.x Cited by (CrossRef): 64 articles Check for updates Citation tools Dr G. Holtmann, University of Essen, Department of Internal Medicine, Division of Gastroenterology and Hepatology, Hufelandstrasse 55, 45122 Essen, Germany.

Summary Background : This study aimed to assess the efficacy of artichoke leaf extract (ALE) in the treatment of patients with functional dyspepsia (FD). Methods : In a double-blind, randomized controlled trial (RCT), 247 patients with functional dyspepsia were recruited and treated with either a commercial ALE preparation (2 × 320 mg plant extract t.d.s.) or a placebo. The primary efficacy variable was the sum score of the patient’s weekly rating of the overall change in dyspeptic symptoms (four-point scale). Secondary variables were the scores of each dyspeptic symptom and the quality of life (QOL) as assessed by the Nepean Dyspepsia Index (NDI).

Results : Two hundred and forty-seven patients were enrolled, and data from 244 patients (129 active treatment, 115 placebo) were suitable for inclusion in the statistical analysis (intention-to-treat). The overall symptom improvement over the 6 weeks of treatment was significantly greater with ALE than with the placebo (8.3 ± 4.6, vs. 6.7 ± 4.8, P < 0.01). Similarly, patients treated with ALE showed significantly greater improvement in the global quality-of-life scores (NDI) compared with the placebo-treated patients (− 41.1 ± 47.6 vs. − 24.8 ± 35.6, P < 0.01). Conclusion : The ALE preparation tested was significantly better than the placebo in alleviating symptoms and improving the disease-specific quality of life in patients with functional dyspepsia.

Phytother Res. 2001 Feb;15(1):58-61.

Artichoke leaf extract reduces symptoms of irritable bowel syndrome in a post-marketing surveillance study. Walker AF1, Middleton RW, Petrowicz O. Bionomic Solutions

Abstract Irritable bowel syndrome (IBS) is a problem reported to affect 22% of the general population. It is characterized by abdominal pain and altered bowel habit but has so far defied elucidation of its pathogenesis and proved difficult to treat. There is a growing body of evidence which indicates therapeutic properties for artichoke leaf extract (ALE). Dyspepsia is the condition for which the herb is specifically indicated, but the symptom overlap between dyspeptic syndrome and IBS has given rise to the notion that ALE may have potential for treating IBS as well. A subgroup of patients with IBS symptoms was therefore identified from a sample of individuals with dyspeptic syndrome who were being monitored in a post-marketing surveillance study of ALE for 6 weeks. Analysis of the data from the IBS sub-group revealed significant reductions in the severity of symptoms and favourable evaluations of overall effectiveness by both physicians and patients. Furthermore, 96% of patients rated ALE as better than or at least equal to previous therapies administered for their symptoms, and the tolerability of ALE was very good. These results provide support for the notion that ALE has potential value in relieving IBS symptoms and suggest that a controlled trial is justified. PMID: 11180525

J Agric Food Chem. 2004 Dec 1;52(24):7272-8.

Phenolic compounds from the leaf extract of artichoke (Cynara scolymus L.) and their antimicrobial activities.

Zhu X1, Zhang H, Lo R.

Abstract A preliminary antimicrobial disk assay of chloroform, ethyl acetate, and n-butanol extracts of artichoke (Cynara scolymus L.) leaf extracts showed that the n-butanol fraction exhibited the most significant antimicrobial activities against seven bacteria species, four yeasts, and four molds. Eight phenolic compounds were isolated from the n-butanol soluble fraction of artichoke leaf extracts. On the basis of high-performance liquid chromatography/electrospray ionization mass spectrometry, tandem mass spectrometry, and nuclear magnetic resonance techniques, the structures of the isolated compounds were determined as the four caffeoylquinic acid derivatives, chlorogenic acid (1), cynarin (2), 3,5-di-Ocaffeoylquinic acid (3), and 4,5-di-O-caffeoylquinic acid (4), and the four flavonoids, luteolin-7-rutinoside (5), cynaroside (6), apigenin-7rutinoside (7), and apigenin-7-O-beta-D-glucopyranoside (8), respectively. The isolated compounds were examined for their antimicrobial activities on the above microorganisms, indicating that all eight phenolic compounds showed activity against most of the tested organisms. Among them, chlorogenic acid, cynarin, luteolin-7-rutinoside, and cynaroside exhibited a relatively higher activity than other compounds; in addition, they were more effective against fungi than bacteria. The minimum inhibitory concentrations of these compounds were between 50 and 200 microg/mL.

PMID: 15563206 DOI: 10.1021/jf0490192

Blueberry Anthocyanin Extract

Ghosh D, Konishi T. “

Anthocyanins and anthocyanin-rich extracts: role in diabetes and eye function.” Asia Pac J Clin Nutr. 2007;16(2):200-8.

Mazza GJ. “

Anthocyanins

and heart health.” Ann Ist Super Sanita. 2007;43(4):369-74.

Nutr Neurosci. 2005 Apr;8(2):111-20.

Anthocyanins in aged blueberry-fed rats are found centrally and may enhance memory.

Andres-Lacueva C1, Shukitt-Hale B, Galli RL, Jauregui O, Lamuela-Raventos RM, Joseph JA.

Abstract Research has shown that fruits and vegetables containing high levels of polyphenolics (flavonoids) display high total antioxidant activity. Our laboratory found that various fruit and vegetable extracts, particularly blueberry (BB), were effective in reversing age-related deficits in neuronal signaling and behavioral parameters following 8 weeks of feeding, possibly due to their polyphenolic content. However, it was unclear if these phytonutrients were able to directly access the brain from dietary BB supplementation (BBS). The present study examined whether different classes of polyphenols could be found in brain areas associated with cognitive performance following BBS. Thus, 19 month old F344 rats were fed a control or 2% BB diet for 8-10 weeks and tested in the Morris water maze (MWM), a measure of spatial learning and memory. LC-MS analyses of anthocyanins in the diet and subsequently in different brain regions of BBS and control rats were carried out. Several anthocyanins (cyanidin-3-O-beta-galactoside, cyanidin-3-O-beta-glucoside, cyanidin-3-O-beta-arabinose, malvidin-3-O-beta-galactoside, malvidin-3-O-betaglucoside, malvidin-3-O-beta-arabinose, peonidin-3-O-beta-arabinose and delphinidin-3-O-beta-galactoside) were found in the cerebellum, cortex, hippocampus or striatum of the BBS rats, but not the controls. These findings are the first to suggest that polyphenolic compounds are able to cross the blood brain barrier and localize in various brain regions important for learning and memory. Correlational analyses revealed a relationship between MWM performance in BBS rats and the total number of anthocyanin compounds found in the cortex. These findings suggest that these compounds may deliver their antioxidant and signaling modifying capabilities centrally.

PMID: 16053243 DOI: 10.1080/10284150500078117

Basu A, Rhone M, Lyons TJ. “Berries: emerging impact on cardiovascular health.” Nutr Rev. 2010 Mar;68(3):168-77.

Cassidy A, O’Reilly ÉJ, Kay C, Sampson L, Franz M, Forman JP, Curhan G, Rimm EB. “Habitual intake of flavonoid subclasses and incident hypertension in adults.” Am J Clin Nutr. 2011 Feb;93(2):338-47. Bionomic Solutions

J Agric Food Chem. Author manuscript; available in PMC 2011 Apr 14.

J Agric Food Chem. 2010 Apr 14; 58(7): 3996–4000.

doi: 10.1021/jf9029332 PMCID: PMC2850944 NIHMSID: NIHMS168597 Blueberry Supplementation Improves Memory in Older Adults ROBERT KRIKORIAN,*† MARCELLE D SHIDLER,† TIFFANY A NASH,†# WILHELMINA KALT,‡ MELINDA R VINQVIST-TYMCHUK,‡ BARBARA SHUKITTHALE,§ and JAMES A JOSEPH§ The publisher’s final edited version of this article is available at J Agric Food Chem Abstract The prevalence of dementia is increasing with expansion of the older adult population. In the absence of effective therapy, preventive approaches are essential to address this public health problem. Blueberries contain polyphenolic compounds, most prominently anthocyanins, which have antioxidant and anti-inflammatory effects. In addition, anthocyanins have been associated with increased neuronal signaling in brain centers mediating memory function as well as improved glucose disposal, benefits that would be expected to mitigate neurodegeneration. We investigated the effects of daily consumption of wild blueberry juice in a sample of nine older adults with early memory changes. At 12 weeks, we observed improved paired associate learning (p = 0.009) and word list recall (p = 0.04). In addition, there were trends suggesting reduced depressive symptoms (p = 0.08) and lower glucose levels (p = 0.10). We also compared the memory performances of the blueberry subjects with a demographically-matched sample who consumed a berry placebo beverage in a companion trial of identical design and observed comparable results for paired associate learning. The findings of this preliminary study suggest that moderate-term blueberry supplementation can confer neurocognitive benefit and establish a basis for more comprehensive human trials to study preventive potential and neuronal mechanisms.

Prior RL, Wu X, Gu L, Hager TJ, Hager A, Howard LR. “Whole berries versus berry anthocyanins: interactions with dietary fat levels in the C57BL/6J mouse model of obesity.” J Agric Food Chem. 2008 Feb 13;56(3):647-53.

Faria A, Pestana D, Teixeira D, de Freitas V, Mateus N, Calhau C. “Blueberry anthocyanins breast cancer cell lines.” Phytother Res. 2010 Dec;24(12):1862-9. doi: 10.1002/ptr.3213.

Ann Neurol. Author manuscript; available in PMC 2013 Jul 1.

Ann Neurol. 2012 Jul; 72(1): 135–143.

and pyruvic acid adducts: anticancer properties in

Published online 2012 Apr 26. doi: 10.1002/ana.23594 PMCID: PMC3582325 NIHMSID: NIHMS366552 Dietary intake of berries and flavonoids in relation to cognitive decline Elizabeth E. Devore, ScD,1 Jae Hee Kang, ScD,1 Monique M.B. Breteler, MD, PhD,2 and Francine Grodstein, ScD1 Objective Berries are high in flavonoids, especially anthocyanidins, and improve cognition in experimental studies. We prospectively evaluated whether greater long-term intakes of berries and flavonoids are associated with slower rates of cognitive decline in older women.

Greater intakes of blueberries and strawberries were associated with slower rates of cognitive decline (e.g., for a global score averaging all six cognitive tests, for blueberries: p-trend=0.014 and mean difference=0.04 [95% CI=0.01, 0.07] comparing extreme categories of intake; for strawberries: p-trend= 0.022 and mean difference=0.03 [95% CI=0.00, 0.06] comparing extreme categories of intake), after adjusting for multiple potential confounders. These effect estimates were equivalent to those we find for approximately 1.5 to 2.5 years of age in our cohort, indicating that berry intake appears to delay cognitive aging by up to 2.5 years. Additionally, in further supporting evidence, greater intakes of anthocyanidins and total flavonoids were associated with slower rates of cognitive decline (p-trends= 0.015 and 0.053, respectively, for the global score).

Interpretation Higher intake of flavonoids, particularly from berries, appears to reduce rates of cognitive decline in older adults.

Genes Nutr. 2007 Dec; 2(3): 257–273.

Published online 2007 Oct 16. doi: 10.1007/s12263-007-0056-z PMCID: PMC2474943 The interactions of flavonoids within neuronal signalling pathways Abstract Emerging evidence suggests that dietary phytochemicals, in particular flavonoids, may exert beneficial effects in the central nervous system by protecting neurons against stress-induced injury, by suppressing neuroinflammation and by promoting neurocognitive performance, through changes in synaptic plasticity. It is likely that flavonoids exert such effects in neurons, through selective actions on different components within a number of protein kinase and lipid kinase signalling cascades, such as phosphatidylinositol-3 kinase (PI3K)/Akt, protein kinase C and mitogenactivated protein kinase. This review details the potential inhibitory or stimulatory actions of flavonoids within these pathways and describes how such interactions are likely to affect cellular function through changes in the activation state of target molecules and/or by modulating gene expression. Although, precise sites of action are presently unknown, their abilities to: (1) bind to ATP binding sites on enzymes and receptors; (2) modulate the activity of kinases directly; (3) affect the function of important phosphatases; (4) preserve neuronal Ca2+ homeostasis; and (5) modulate signalling cascades lying downstream of kinases, are explored. Future research directions are outlined in relation to their precise site(s) of action within the signalling pathways and the sequence of events that allow them to regulate neuronal function in the central nervous system. Bionomic Solutions

Flavonoids

display potent antioxidant capacity in vitro [138–140]. However, during absorption flavonoids are extensively metabolised resulting in a significant alteration in their redox potentials. It has become clear that the bioactive forms of flavonoids in vivo are not those forms found in plants. For example, the majority of flavonoid glycosides, and in some instances the aglycones, present in plant-derived foods, are extensively conjugated and metabolised during absorption (reviewed in [47, 51, 162, 163, 180]). In particular, there is much evidence for the extensive phase I de-glycosylation and phase II metabolism of the resulting aglycones to glucuronides, sulphates and O-methylated forms during transfer across the small intestine [157, 162] and then again in the liver. Further transformation has been reported in the colon where the enzymes of the gut microflora degrade flavonoids to simple phenolic acids [146]. In addition, flavonoids may undergo at least three types of intracellular metabolism: (1) Oxidative metabolism, (2) P450-related metabolism and (3) conjugation with thiols, particularly GSH [158, 160]. Circulating metabolites of flavonoids, such as glucuronides, sulphates and conjugated O-methylated forms, or intracellular metabolites like flavonoid-GSH adducts, have greatly reduced antioxidant potential [160]. Indeed, studies have indicated that although such conjugates and metabolites may participate directly in plasma antioxidant reactions and in scavenging reactive oxygen and nitrogen species in the circulation, their effectiveness is reduced relative to their parent aglycones [41, 122, 152, 170, 189].

In order to understand whether flavonoids and their metabolic derivatives are capable of direct neuroprotective effects, it is crucial to ascertain whether they are able to access the central nervous system. In order for flavonoids to enter into the brain, they must first cross the blood brain barrier (BBB). The function of the BBB includes protection of the brain from xenobiotics and the general maintenance of the brain’s microenvironment [2, 193]. Studies by Youdim et al. indicated that certain flavonoids were able to penetrate the BBB in relevant in vitro and in situ models [193, 195, 196]. In these studies, the flavanones, hesperetin, naringenin and their relevant in vivo metabolites, as well as some dietary anthocyanins, cyanidin-3-rutinoside and pelargonidin-3-glucoside, were able to traverse the BBB. Furthermore, it was demonstrated that the uptake of the relatively lipophilic flavanones, naringenin and hesperetin, was much greater than for other flavonoids, such as epicatechin, epicatechin metabolites, anthocyanins and their glucuronidated metabolites, which are more polar in nature. This study suggested that the potential for flavonoid penetration is dependent on compound lipophilicity [193]. Accordingly, it is plausible that the uptake of the less polar methylated metabolites, such as the methylated epicatechin metabolites (formed in the small intestine and liver), may be greater than the parent aglycone. For the same reason, the more polar flavonoid glucuronidated metabolites, which seem to have low BBB permeability values

[193], may not be able to access the brain. However, evidence exists to suggest that certain drug glucuronides may cross the BBB [1] and exert pharmacological effects [94, 164], suggesting that there may be a specific uptake mechanism for glucuronides in vivo. It has also been suggested that apart from the components’ lipophilicity, the ability of flavonoids to enter the brain may be influenced by their interactions with specific efflux transporters expressed in the BBB. One such transporter is P-glycoprotein [195], which plays an important role in drug absorption and brain uptake [107]. In the study conducted by Youdim et al. [195], the differences between naringenin and quercetin flux into the brain in situ was attributed not only to their differences in lipophilicity, but also to their ability to act as efflux transporter substrates.

There is also evidence from animal feeding studies to suggest that flavonoids may access the brain. The tea flavanol EGCG has been reported to access the brain after oral administration to mice [166]. Furthermore, oral ingestion of pure epicatechin resulted in the detection of epicatechin glucuronide and 3ʹ-O-methyl-epicatechin glucuronide in rat brain tissue [3]. Anthocyanidins have also been detected in the brain after oral administration [53, 168], with several anthocyanidins being identified in different regions of rat brain after the animals were fed with blueberry

[12]. Such flavonoid localisation has been correlated with increased cognitive performance, suggesting a central neuroprotective role of these components.

It is clear that the concentrations of flavonoids and their metabolite forms accumulated in vivo, for example in the plasma or in organs such as the brain [3] are lower (high nM, low μM) than those recorded for small molecule antioxidant nutrients such as ascorbic acid and α-tocopherol

[67]. In addition, these in vivo forms will mainly be metabolites possessing lower antioxidant potential relevant to parent compounds. Therefore, the beneficial effects of flavonoid metabolites in vivo are unlikely to result by their ability to out-compete antioxidants such as ascorbate, which are present at higher concentrations (high μM to mM). For example, flavonoids have been shown to protect neurons against oxidative stress more effectively than ascorbate even when the latter was used at tenfold higher concentrations [150], which supports a non-antioxidant mechanism of action. If such an antioxidant mechanism is unlikely in vivo, there must be other potential routes by which they exert beneficial effects at the cellular and tissue level. Over the last 5–10 years evidence has accumulated to suggest that the cellular effects of flavonoids may be mediated by their interactions with specific proteins central to intracellular signalling cascades [148]. In the next section we highlight the potential for these polyphenols and their metabolites to interact at various points within the mitogen activated protein kinase (MAP kinase) signalling pathway and the phosphoinositide 3-kinase (PI3 kinase/Akt) signalling cascade.

Flavonoid interactions with neuronal signalling cascades Flavonoids have been shown to exert modulatory effects in neurons through selective actions at different components of a number of protein kinase and lipid kinase signalling cascades, such as the PI3 kinase (PI3K)/Akt, tyrosine kinase, protein kinase C (PKC) and mitogen-activated protein kinase (MAP kinase) signalling pathways [7, 61, 93, 116, 149, 159, 178] (Fig. 2). Inhibitory or stimulatory actions at these pathways are likely to profoundly affect cellular function by altering the phosphorylation state of target molecules and/or by modulating gene expression. Although selective inhibitory actions at these kinase cascades may be beneficial in cancer, proliferative diseases, inflammation and neurodegeneration they could be detrimental during development particularly in the immature nervous system when protein and lipid kinase signalling regulates survival, synaptogenesis and neurite outgrowth. In the mature brain, post-mitotic neurones utilise MAP kinase and PI3K cascades in the regulation of key functions such as synaptic plasticity and memory formation [106, 167] (Fig. 2), t

Neurobiol Aging. 2008 Nov;29(11):1680-9. Epub 2007 May 23.

A blueberry-enriched diet provides cellular protection against oxidative stress and reduces a kainate-induced learning impairment in rats. Duffy KB1, Spangler EL, Devan BD, Guo Z, Bowker JL, Janas AM, Hagepanos A, Minor RK, DeCabo R, Mouton PR, Shukitt-Hale B, Joseph JA, Ingram DK.

Abstract Young male Fischer-344 rats were fed a diet containing 2% blueberry (BB) extract or control diet for at least 8 weeks and then received bilateral hippocampal injections of kainic acid (KA 200 ng/0.5 microl) or phosphate buffered saline (PBS). One week later rats were trained in one-way active footshock avoidance in a straight runway followed the next day by training in a footshock motivated 14-unit T-maze with documented sensitivity to hippocampal glutamatergic manipulations. Based on analyses of several performance variables, KA-treated rats exhibited clearly Bionomic Solutions

impaired learning performance; however, the BB diet significantly reduced this impairment. Supporting the behavioral findings, stereological assessment of CA1 pyramidal neurons documented greater neuronal loss in KA-treated controls compared to KA-treated rats on the BB diet. In an in vitro experiment, FaO cells grown in medium supplemented with serum from BB-fed rats had enhanced viability after exposure to hydrogen peroxide. These findings suggest that BB supplementation may protect against neurodegeneration and cognitive impairment mediated by excitotoxicity and oxidative stress.

PMID: 17524525 DOI: 10.1016/j.neurobiolaging.2007.04.002

Wheatgrass Extract

Antiproliferative, apoptotic and antioxidant activities of wheatgrass (Triticum aestivum L.) extract on CML (K562) cell line Authors: OYA SENA AYDOS, ASLIHAN AVCI, TÜLİN ÖZKAN, AYNUR KARADAĞ, EBRU GÜRLEYİK, BUKET ALTINOK, ASUMAN SUNGUROĞLU Abstract: Plant-based diet supplements help the prevention and therapy of several kinds of cancer because they contain micronutrients, a class of substances that have been shown to exhibit chemopreventive and chemotherapeutic activities. In the present study the effects and oxidant/antioxidant status of aqueous and ethanol extracts of wheatgrass were tested in human chronic myeloid leukemia CML (K562) cell line. Materials and methods: K562 cell lines were treated with 10% (w/v) concentration of aqueous and ethanol wheatgrass extracts. Cytotoxicity and apoptosis were determined morphologically and by MTT and DNA laddering. MDA level and CAT, SOD, and ADA activities of the cell lines were measured. Results: Both preparations inhibited the growth of leukemia cells in a time-dependent manner. The most apoptotic and antiproliferative effect was seen in the cell line treated with aqueous extract at 48 h (P < 0.001). Increases in MDA level and CAT and SOD activities were observed. Conclusion: Wheatgrass extract has an antioxidant activity, inhibits proliferation of leukemia cells, and induces apoptosis; thus, this finding may represent a novel therapeutic approach for the treatment of CML.

Meyer KA, Kushi LH, Jacobs DR Jr, Slavin J, Sellers TA, Folsom AR. Carbohydrates, dietary fiber, and incident type 2 diabetesin older women. Am J Clin Nutr 2000; 71: 921-30.

Nicodemus KK, Jacobs DR Jr, Folsom AR. Whole and refined grain intake and risk of incident postmenopausal breast cancer (United States). Cancer Causes Control 2001; 12: 917-25

Kulkarni SD, Tilak JC, Acharya R, Rajurkar NS, Devasagayam TPA, Reddy AVR. Evaluation of the antioxidant activity of wheatgrass (Triticum aestivum L.) as a function of growth under different conditions. Phytother Res 2006; 20: 218-27. Hänninen O, Rauma AL, Kaartinen K, Nenonen M. Vegan dietin physiological health promotion. Acta Physiol Hung 1999; 86: 171-80.

Lai CN, Dabney BJ, Shaw CR. Inhibition of in vitro metabolic activation of carcinogens by

wheat

sprout

extracts. NutrCancer 1978; 1: 27-30.

Lai CN. Chlorophyll: the active factor in wheat sprout extracts inhibiting the metabolic activation of carcinogens in vitro.Nutr Cancer 1979; 1: 1921.

Peryt B, Szymczyk T, Lesca P. Mechanism of antimutagenicity of

wheat

sprout

extracts. Mutat Res 1992; 269: 201-15.

Falcioni G, Fedeli D, Tiano L, Calzuola I, Mancinelli L, Marsili V, Gianfranceschi G. Antioxidant activity of Inhibition of DNA oxidative damage. J Food Sci 2002; 67: 2918-22.

wheat

sprouts

extracts

in vitro:

Calzuola I, Marsili V, Gianfranceschi GL. Synthesis of antioxidants in

wheat

sprouts. J. Agric Food Chem 2004; 52:5201-6.

Blokhina O, Virolainen E, Fagerstedt K. Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann Bot 2003;91: 179-94.

Dahle LK, Hill EG, Hollman RT. Th e thiobarbituric acid reaction and the autoxidations of polyunsaturated fatty acid methyl esters. Arch Biochem Biophys 1962; 98: 253-61.

Wheat Grass Juice in the Treatment of Active Distal Ulcerative Colitis: A Randomized Double-blind Placebo-controlled Trial E. Ben-Arye, E. Goldin, D. Wengrower, A. Stamper, R. Kohn & E. Berry Pages 444-449 | Published online: 08 Jul 2009 Abstract Background: The use of wheat grass ( Triticum aestivum ) juice for treatment of various gastrointestinal and other conditions had been suggested by its proponents for more than 30 years, but was never clinically assessed in a controlled trial. A preliminary unpublished pilot study suggested efficacy of wheat grass juice in the treatment of ulcerative colitis (UC). Methods: A randomized, double-blind, placebocontrolled study. One gastroenterology unit in a tertiary hospital and three study coordinating centers in three major cities in Israel. Twenty-three patients diagnosed clinically and sigmoidoscopically with active distal UC were randomly allocated to receive either 100 cc of wheat grass juice, or a matching placebo, daily for 1 month. Efficacy of treatment was assessed by a 4-fold disease activity index that included rectal bleeding and number of bowel movements as determined from patient diary records, a sigmoidoscopic evaluation, and global assessment by a physician. Results. Twenty-one patients completed the study, and full information was available on 19 of them. Treatment with wheat grass juice was associated with significant reductions in the overall disease activity index ( P = 0.031) and in the severity of rectal bleeding ( P = 0.025). No Bionomic Solutions

serious side effects were found. Fresh extract of wheat grass demonstrated a prominent tracing in cyclic voltammetry methodology, presumably corresponding to four groups of compounds that exhibit anti-oxidative properties. Conclusion. Wheat grass juice appeared effective and safe as a single or adjuvant treatment of active distal UC.

Aebi H. Catalase: methods of enzymatic analysis. New York: Academic Press; 1974. 15. Durak İ, Canbolat O, Kaçmaz M, Özgen G, Öztürk HS. Antioxidant interference in superoxide dismutase activity methods using superoxide radical as substrate. Clin Chem Lab Med 1998; 36: 407-8.

Di Bacco A, Keeshan K, McKenna SL, Cotter TG. Molecular abnormalities in chronic myeloid leukemia: deregulation of cell growth and apoptosis. Oncologist 2000; 5: 405-15.

Reed JC. Mechanism of apoptosis avoidance in cancer. Curr Opin Oncol 1999; 11: 68-75.

Senderowicz AM. Targeting cell cycle and apoptosis for the treatment of human malignancies. Curr Opin Cell Biol 2004;16: 670-8.

Han DH, Lee MJ, Kim JH. Inhibition of cancer cell proliferation and suppression of TNF-induced activation of NFkappaB by edible berry juice. Anticancer Res 2006; 26: 3601-6.

Surh YJ, Hurh YJ, Kang JY, Lee E, Kong G, Lee SJ. Resveratrol,an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Cancer Lett 1999; 140:1-10.

Boivin D, Blanchette M, Barrette S, Moghrabi A, Béliveau R. Inhibition of cancer cell proliferation and suppression of TNF-induced activation of NFkappaB by edible berry juice. Anticancer Res 2007; 27: 937-48.

Karadag A, Ozkan T, Altinok B, Aydos S, Sunguroglu A. Antiproliferative and apoptotic effects of leukemia (CML) cell line. Planta Med 2007; 73: 991-3.

Pineapple Bromelain Extract

wheatgrass

extracts

on chronic myeloid

Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey L. Mynott, Andrew Ladhams, Pierre Scarmato and Christian R. Engwerda J Immunol September 1, 1999, 163 (5) 2568-2575; Abstract Recently, it has emerged that extracellular proteases have specific regulatory roles in modulating immune responses. Proteases may act as signaling molecules to activate the Raf-1/extracellular regulated kinase (ERK)-2 pathway to participate in mitogenesis, apoptosis, and cytokine production. Most reports on the role of protease-mediated cell signaling, however, focus on their stimulatory effects. In this study, we show for the first time that extracellular proteases may also block signal transduction. We show that bromelain, a mixture of cysteine proteases from pineapple stems, blocks activation of ERK-2 in Th0 cells stimulated via the TCR with anti-CD3ε mAb or stimulated with combined PMA and calcium ionophore. The inhibitory activity of bromelain was dependent on its proteolytic activity, as ERK-2 inhibition was abrogated by E-64, a selective cysteine protease inhibitor. However, inhibitory effects were not caused by nonspecific proteolysis, as the protease trypsin had no effect on ERK activation. Bromelain also inhibited PMA-induced IL-2, IFN-γ, and IL-4 mRNA accumulation, but had no effect on TCR-induced cytokine mRNA production. This data suggests a critical requirement for ERK-2 in PMA-induced cytokine production, but not TCR-induced cytokine production. Bromelain did not act on ERK-2 directly, as it also inhibited p21ras activation, an effector molecule upstream from ERK-2 in the Raf1/MEK/ERK-2 kinase signaling cascade. The results indicate that bromelain is a novel inhibitor of T cell signal transduction and suggests a novel role for extracellular proteases as inhibitors of intracellular signal transduction pathways.

Proteases contribute to effective immune responses, although their role is commonly thought of as degradative, such as in the destruction of cell targets by lymphocytes and NK cells (1), or the cleavage of cellular substrates during apoptosis (2). Recently, however, it has become apparent that proteases also convey hormone-like signals and stimulate intracellular signal transduction via specific cell surface receptors (3). Thrombin, for example, a protease involved in the blood coagulation cascade, is mitogenic in many cells and activates the Raf-1/extracellular regulated kinase-2 (ERK-2)2 pathway leading to DNA synthesis, cell growth, proliferation, and differentiation (4). Thrombin activation is mediated via proteinase-activated receptor (PAR)-1, a member of the family of PARs (4). The proteases, plasmin, granzyme A, and cathepsin G also activate PAR-1 and stimulate signal transduction (5, 6, 7). Bromelain is a mixture of cysteine proteases obtained from pineapple stems (Ananus comosus) (8) that has varied stimulatory effects on leukocyte populations in vitro. It has been shown to increase CD2-mediated T cell activation, enhance Ag-independent binding to monocytes (9), and increase IFN-γ-dependent TNF-α, IL-1β, and IL-6 production in PBMC (10). Bromelain-treated RBC are also used in murine studies of the role of CD5+ B cells in autoimmunity (11). Bromelain’s effects have previously been attributed to its degradative action at cell surfaces, whereby it either removes surface molecules or, alternatively, reveals ones that already exist on cell membranes, thereby altering receptor-ligand interactions (9, 12, 13). Recently, however, we showed that bromelain also exerted effects independent to that mediated by the removal of cell surface molecules (14). We showed that bromelain inhibited the action of cyclic nucleotide agonists and calcium agonists that directly stimulate intracellular signaling pathways and bypass cell surface receptors in their action (14). Therefore, to further investigate the possible hormone-like effects of bromelain on intracellular signaling, we studied its effects on TCR/CD3 signaling and IL-2 production. Bionomic Solutions

Biomed Rep. 2016 Sep; 5(3): 283–288.

Published online 2016 Jul 18. doi: 10.3892/br.2016.720 PMCID: PMC4998156 Potential role of bromelain in clinical and therapeutic applications Vidhya Rathnavelu,1 Noorjahan Banu Alitheen,2 Subramaniam Sohila,3 Samikannu Kanagesan,4 and Rajendran Ramesh5 Abstract Pineapple has been used as part of traditional folk medicine since ancient times and it continues to be present in various herbal preparations. Bromelain is a complex mixture of protease extracted from the fruit or stem of the pineapple plant. Although the complete molecular mechanism of action of bromelain has not been completely identified, bromelain gained universal acceptability as a phytotherapeutic agent due to its history of safe use and lack of side effects. Bromelain is widely administered for its well-recognized properties, such as its antiinflammatory, antithrombotic and fibrinolytic affects, anticancer activity and immunomodulatory effects, in addition to being a wound healing and circulatory improvement agent. The current review describes the promising clinical applications and therapeutic properties of bromelain.

PLoS One. 2012; 7(1): e30831.

Published online 2012 Jan 24. doi: 10.1371/journal.pone.0030831 PMCID: PMC3265525 Inhibition of Adipogenesis and Induction of Apoptosis and Lipolysis by Stem Bromelain in 3T3-L1 Adipocytes Sandeep Dave, Naval Jit Kaur, Ravikanth Nanduri, H. Kitdorlang Dkhar, Ashwani Kumar, and Pawan Gupta* Abstract The phytotherapeutic protein stem bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBPα and PPARγ independent of C/EBPβ gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2), fatty acid synthase (FAS), lipoprotein lipase (LPL), CD36, and acetyl-CoA carboxylase (ACC) were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBM’s ability to repress PPARγ expression seems to stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt–TSC2–mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα upregulation compromised cell viability of mature adipocytes (via apoptosis) and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B), and GTP binding protein Giα1, as well as sustained expression of hormone sensitive lipase (HSL). These data indicate that SBM, together with all-trans retinoic-acid (atRA), may be a potent modulator of obesity by repressing the PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes.

Cell Immunol. 2001 May 25;210(1):66-75.

Bromelain modulates T cell and B cell immune responses in vitro and in vivo.

Engwerda CR1, Andrew D, Ladhams A, Mynott TL.

Abstract The ability to modulate immune responses is a major aim of many vaccine and immunotherapeutic development programs. Bromelain, a mixture of cysteine proteases, modulates immunological responses and has been proposed to be of clinical use. However, the identity of the immune cells affected by bromelain and the specific cellular functions that are altered remain poorly understood. To address these shortcomings in our knowledge, we have used both in vitro and in vivo immunological assays to study the effects of bromelain. We found that bromelain enhanced T cell receptor (TCR) and anti-CD28-mediated T cell proliferation in splenocyte cultures by increasing the costimulatory activity of accessory cell populations. However, despite increased T cell proliferation, bromelain concomitantly decreased IL-2 production in splenocyte cultures. Additionally, bromelain did not affect TCR and CD28-induced proliferation of highly purified CD4+ T cells but did inhibit IL-2 production by these cells. In vivo, bromelain enhanced T-cell-dependent, Ag-specific, B cell antibody responses. Again, bromelain induced a concomitant decrease in splenic IL-2 mRNA accumulation in immunized mice. Together, these data show that bromelain can simultaneously enhance and inhibit T cell responses in vitro and in vivo via a stimulatory action on accessory cells and a direct inhibitory action on T cells. This work provides important insights into the immunomodulatory activity of bromelain and has important implications for the use of exogenous cysteine proteases as vaccine adjuvants or immunomodulatory agents.

PMID: 11485354 DOI: 10.1006/cimm.2001.1807

Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey L. Mynott, Andrew Ladhams, Pierre Scarmato and Christian R. Engwerda J Immunol September 1, 1999, 163 (5) 2568-2575; Abstract Recently, it has emerged that extracellular proteases have specific regulatory roles in modulating immune responses. Proteases may act as signaling molecules to activate the Raf-1/extracellular regulated kinase (ERK)-2 pathway to participate in mitogenesis, apoptosis, and cytokine production. Most reports on the role of protease-mediated cell signaling, however, focus on their stimulatory effects. In this study, we show for the first time that extracellular proteases may also block signal transduction. We show that bromelain, a mixture of cysteine proteases from pineapple stems, blocks activation of ERK-2 in Th0 cells stimulated via the TCR with anti-CD3ε mAb, or stimulated with combined PMA and calcium ionophore. The inhibitory activity of bromelain was dependent on its proteolytic activity, as ERK-2 inhibition was abrogated by E-64, a selective cysteine protease inhibitor. However, inhibitory effects were not caused by nonspecific proteolysis, as the protease trypsin had no effect on ERK activation. Bromelain also inhibited PMA-induced IL-2, IFN-γ, and IL-4 mRNA accumulation, but had no effect on TCR-induced cytokine mRNA production. This data suggests a critical requirement for ERK-2 in PMA-induced cytokine production, but not TCR-induced cytokine production. Bromelain did not act on ERK-2 directly, as it also inhibited p21ras activation, an effector molecule upstream from ERK-2 in the Raf1/MEK/ERK-2 kinase signaling cascade. The results indicate that bromelain is a novel inhibitor of T cell signal transduction and suggests a novel role for extracellular proteases as inhibitors of intracellular signal transduction pathways. Bionomic Solutions

Clin Rheumatol. 2004 Oct;23(5):410-5. Epub 2004 Jul 24.

Oral enzyme combination versus diclofenac in the treatment of osteoarthritis of the knee–a double-blind prospective randomized study.

Akhtar NM1, Naseer R, Farooqi AZ, Aziz W, Nazir M.

Abstract The aim of this study was to compare the efficacy and safety of an oral enzyme-rutosid combination (ERC) containing rutosid and the enzymes bromelain and trypsin, with that of diclofenac in patients with osteoarthritis (OA) of the knee. A total of 103 patients presenting with painful episodes of OA of the knee were treated for 6 weeks in two study centers in a randomized, double-blind, parallel group trial. Altogether, 52 patients were treated in the ERC group and 51 patients were treated in the diclofenac group. Primary efficacy criteria were Lequesne’s Algofunctional Index (LFI) and a ‘complaint index’, including pain at rest, pain on motion and restricted function. The efficacy criteria were analyzed by applying the Wilcoxon-Mann-Whitney test that provides the Mann-Whitney estimator (MW) as a measure of relevance. Noninferiority was considered to be proven if the lower bound of the 97.5% one-sided confidence interval (CI-LB) was higher than MW = 0.36 (benchmark of not yet relevant inferiority). Both treatments resulted in clear improvements. Within the 6-week observation period, the mean value of the LFI decreased from 13.0 to 9.4 in the ERC group and from 12.5 to 9.4 in the diclofenac group. Non-inferiority of ERC was demonstrated by both primary criteria, LFI (MW = 0.5305; CI-LB = 0.4171) and complaint index (MW = 0.5434; CI-LB = 0.4296). Considerable improvements were also seen in secondary efficacy criteria, with a slight tendency towards superiority of ERC. The global judgment of efficacy by physician resulted in at least good ratings for 51.4% of the ERC patients, and for 37.2% of the diclofenac patients. In the majority of patient’s tolerability was judged in both drug groups as very good or good. The current study indicates that ERC can be considered as an effective and safe alternative to NSAIDs such as diclofenac in the treatment of painful episodes of OA of the knee. Placebo-controlled studies are now needed to confirm these results.

PMID: 15278753 DOI: 10.1007/s10067-004-0902-y

Am J Physiol Heart Circ Physiol. Author manuscript; available in PMC 2008 Nov 10.

Published in final edited form as:

Am J Physiol Heart Circ Physiol. 2008 Mar; 294(3): H1365–H1370.

Published online 2008 Jan 11. doi: 10.1152/ajpheart.01005.2007.

PMCID: PMC2581828 NIHMSID: NIHMS72341 Bromelain induces cardioprotection against ischemia-reperfusion injury through Akt/FOXO pathway in rat myocardium Bela Juhasz,1,6 Mahesh Thirunavukkarasu,1 Rima Pant,1 Lijun Zhan,1 Suresh Varma Penumathsa,1 Eric R. Secor, Jr.,2 Sapna Srivastava,1 Utpal Raychaudhuri,3 Venugopal P. Menon,4 Hajime Otani,5 Roger S. Thrall,2 and Nilanjana Maulik1 The publisher’s final edited version of this article is available free at Am J Physiol Heart Circ Physiol Abstract Bromelain (Br), a proteolytic enzyme extracted from the stem of the pineapple, is known to possess anti-inflammatory activity and has been shown to reduce blood viscosity, prevent the aggregation of blood platelets, and improve ischemia-reperfusion (I/R) injury in a skeletal muscle model. We investigated the capacity of Br to limit myocardial injury in a global I/R model. Adult male Sprague-Dawley rats were divided into two groups: control (PBS) and Br at 10 mg/kg in PBS administered via intraperitoneal injection (twice/day) for 15 consecutive days. On day 16, the hearts were excised and subjected to 30 min of global ischemia followed by 2 h of reperfusion. Br treatment showed higher left ventricular functional recovery throughout reperfusion compared with the controls [maximum rate of rise in intraventricular pressure (dP/dtmax), 2,225 vs. 1,578 mmHg/s at 2 h reperfusion]. Aortic flow was also found to be increased in Br treatment when compared with that in untreated rats (11 vs. 1 ml). Furthermore, Br treatment reduced both the infarct size (34% vs. 43%) and the degree of apoptosis (28% vs. 37%) compared with the control animals. Western blot analysis showed an increased phosphorylation of both Akt and FOXO3A in the treatment group compared with the control. These results demonstrated for the first time that Br triggers an Akt-dependent survival pathway in the heart, revealing a novel mechanism of cardioprotective action and a potential therapeutic target against I/R injury.

Exp Neurol. 2001 Feb;167(2):385-92.

Alpha 2-macroglobulin-mediated degradation of amyloid beta 1–42: a mechanism to enhance amyloid beta catabolism.

Lauer D1, Reichenbach A, Birkenmeier G.

Institute of Biochemistry, University of Leipzig, Liebigstrasse 16, 04103 Leipzig, Germany.

Abstract Peptides derived from proteolytic degradation of the amyloid precursor protein, e.g., amyloid beta (A beta), are considered to be central to the pathology of Alzheimer’s disease (AD). Soluble A beta is present in measurable concentrations in cerebrospinal fluid and blood. There are indications that soluble A beta present in circulation can cross the blood-brain barrier via transcytosis mediated by brain capillary endothelial cells. It implies that A beta originating from circulation may contribute to vascular and parenchymal A beta deposition in AD. Enhancing of A beta catabolism mediated by proteolytic degradation or receptor-mediated endocytosis could be a key mechanism to maintain low concentrations of soluble A beta. To launch A beta clearance we have exploited the A beta-degrading activity of diverse alpha 2-macroglobulin (alpha 2-M)-proteinase complexes. Complexes with trypsin, alpha-chymotrypsin, and bromelain strongly degrade (125)I-A beta 1–42 whereas complexes with endogenous proteinases, e.g., plasmin and prostate-specific antigen, were not effective. A beta degradation by the complexes was not inhibited by alpha 1-antichymotrypsin and soybean trypsin inhibitor which normally would inactivate the free serine proteinases. A prerequisite for A beta degradation is its binding to specific binding sites in alpha 2-M that may direct A beta to the active site of the caged proteinase. Ex vivo, enhanced degradation of (125)I-A beta 1–42 in blood could be achieved upon oral administration of high doses of proteinases to volunteers. These results suggest that up-regulation of A beta catabolism could probably reduce the risk of developing AD by preventing A beta accumulation in brain and vasculature.

PMID: 11161627 DOI: 10.1006/exnr.2000.7569 Bionomic Solutions

Int J Pharm. 2005 Jan 6;288(1):141-50. Epub 2004 Nov 19.

Improved paracellular uptake by the combination of different types of permeation enhancers.

Guggi D1, Bernkop-Schnürch A.

Abstract This study had the purpose to improve the paracellular uptake of drugs by combining the thiomer/reduced glutathione (GSH) permeationenhancing system with a proteolytic enzyme. Due to the covalent binding of 2-iminothiolane to chitosan the thiomer chitosan-TBA (chitosan-4thiobutylamidine) was obtained. Permeation studies were performed with freshly excised intestinal mucosa of guinea pigs mounted in Ussingtype chambers using on the one hand the low-molecular size marker flurescein (Na-Flu) and on the other hand the high-molecular size marker FITC-dextran. Apparent permeability coefficient (P(app)) as well as enhancement ratios (=P(app) permeation-enhancing system/P(app) control) were calculated. Trypsin, papain and bromelain displayed a permeation-enhancing effect for Na-Flu on the small intestinal mucosa. Enhancement ratios of 1.84, 1.63 and 1.78 were identified for 2% trypsin, 0.5% papain and 2% bromelain solutions, respectively. However, only bromelain could guarantee a significant permeation enhancement of FITC-dextran with a P(app) of 4.45+/-0.44 x 10(-6) cm/s representing an enhancement ratio of 1.57. A similar enhancement of FITC-dextran permeation was reached by the use of the chitosan-TBA (0.5%)/GSH (5%) system. Moreover, an additive permeation-enhancing effect of the chitosan-TBA/GSH system in combination with bromelain (2%) was observed, leading to a maximum P(app) of 5.91+/-0.51 x 10(-6) cm/s, which corresponds to an enhancement ratio of 2.1. According to these results, the combination of the thiomer/GSH system with bromelain might represent a new promising strategy in order to raise the in vivo efficacy of noninvasive administered hydrophilic macromolecular drugs.

PMID: 15607266 DOI: 10.1016/j.ijpharm.2004.09.023

Acai Berry Extract

Chin, Young-Won, et al. “Lignans and Other Constituents of the Fruits of Euterpe Oleracea (Açai) with Antioxidant and Cytoprotective Activities.” Journal of Agricultural and Food Chemistry 56.17 (2008): 7759–7764. Web.

Rosso, Veridiana Vera de, et al. “Determination of Anthocyanins from Acerola (DC.) and Açai Composition and Analysis 21.4 (2008): 291–299. Web. 3 Mar. 2017.

Oxid Med Cell Longev. 2016;2016:8940850. Epub 2016 Oct 3.

Neuroprotective Effects of Açaí (Euterpe oleracea Mart.) against Rotenone In Vitro Exposure.

( Mart.) by HPLC–PDA–MS/MS.” Journal of Food

Machado AK1, Andreazza AC2, da Silva TM3, Boligon AA4, do Nascimento V5, Scola G2, Duong A6, Cadoná FC7, Ribeiro EE5, da Cruz IB8. Abstract Neuropsychiatric diseases, such as bipolar disorder (BD) and schizophrenia (SCZ), have a very complex pathophysiology. Several current studies describe an association between psychiatric illness and mitochondrial dysfunction and consequent cellular modifications, including lipid, protein, and DNA damage, caused by cellular oxidative stress. Euterpe oleracea (açaí) is a powerful antioxidant fruit. Açaí is an Amazonian palm fruit primarily found in the lowlands of the Amazonian rainforest, particularly in the floodplains of the Amazon River. Given this proposed association, this study analyzed the potential in vitro neuropharmacological effect of Euterpe oleracea (açaí) extract in the modulation of mitochondrial function and oxidative metabolism. SH-SY5Y cells were treated with rotenone to induce mitochondrial complex I dysfunction and before and after we exposed the cells to açaí extract at 5 μg/mL. Treated and untreated cells were then analyzed by spectrophotometric, fluorescent, immunological, and molecular assays. The results showed that açaí extract can potentially increase protein amount and enzyme activity of mitochondrial complex I, mainly through NDUFS7 and NDUFS8 overexpression. Açaí extract was also able to decrease cell reactive oxygen species levels and lipid peroxidation. We thus suggest açaí as a potential candidate for drug development and a possible alternative BD therapy. PMID: 27781077 PMCID: PMC5066013 DOI: 10.1155/2016/8940850

Souza, Melina Oliveira de, et al. “The Hypocholesterolemic Activity of Açaí (Mart.) Is Mediated by the Enhanced Expression of the ATP-Binding Cassette, Subfamily G Transporters 5 and 8 and Low-Density Lipoprotein Receptor Genes in the Rat.” Nutrition Research 32.12 (2012): 976–984. Web. 3 Mar. 2017.

Feio, Claudine A, et al. “Euterpe Oleracea (Açai) Modifies Sterol Metabolism and Attenuates Experimentally-Induced Atherosclerosis.” Journal of Atherosclerosis and Thrombosis 19.3 (2012): n.pag. Web. 3 Mar. 2017.

J Pharm Biomed Anal. 2017 Sep 5;143:204-213. doi: 10.1016/j.jpba.2017.05.045. Epub 2017 Jun 3.

A UHPLC method for the rapid separation and quantification of anthocyanins in acai berry and dry blueberry extracts.

Fibigr J1, Šatínský D2, Solich P1.

Abstract The presented work describes the development and validation of a rapid UHPLC-UV method using a core-shell particle column with a pentafluorophenyl stationary phase for the separation and quantitative analysis of the six anthocyanins in acai berry and dry blueberry extracts. The anthocyanins (cyanidin-3-glucoside, cyanidin-3-rutenoside, delphinidin-3-galactoside, delphinidin-3-glucoside, delphinidin-3-rutenoside, and peonidin-3-glucoside) were separated and analyzed in 5min. The chromatographic separation was performed on a Kinetex PFP (150×2.1mm) core-shell column with a particle size of 1.7μm at a temperature of 50°C. Acetonitrile was used as mobile phase B and 5% formic acid, filtrated through a 0.22μm filter, as mobile phase A. They were delivered at a flow rate of 0.55mLmin-1 according to the elution gradient program. The detection wavelength was set at 520nm. A solid-liquid extraction with a solution of methanol and a 5% water solution of formic acid (25+75v/v) using an ultrasonic bath was chosen for the preparation of the available commercial samples of food supplements with a content of acai berry extract and blueberry extract. Under optimal chromatographic conditions, the method was validated. Recoveries for all analyzed anthocyanins Bionomic Solutions

were 97.8-102.6% and the relative standard deviation ranged from 0.4% to 3.0% for within-day and from 0.6% to 3.1% for between-day repeatability. The limits of detection were in the range of 0.11-0.14μgmL-1.

PMID: 28605682 DOI: 10.1016/j.jpba.2017.05.045 Rocha, A. P. M., et al. “Endothelium-Dependent Vasodilator Effect of Mart. (Açaí) Extracts in Mesenteric Vascular Bed of the Rat.” Vascular Pharmacology 46.2 (2007): 97–104. Web. 3 Mar. 2017.

Heinrich, Michael, Tasleem Dhanji, and Ivan Casselman. “Açai ( Mart.)—A Phytochemical and Pharmacological Assessment of the Species’ Health Claims.” Phytochemistry Letters 4.1 (2011): 10–21. Web. 3 Mar. 2017.

Sousa Pereira, Izabelle de, et al. “The Consumption of Acai Pulp Changes the Concentrations of Plasminogen Activator Inhibitor-1 and Epidermal Growth Factor (EGF) in Apparently Healthy Women.” Nutricion hospitalaria (2015): n.pag. Web. 3 Mar. 2017.

Edna, dos Santos R., et al. “Preparation and Characterization of the Nutritive Value of Flour Made from Acai (Euterpe oleracea, Mart.) Seeds.” The Natural Products Journal. Bentham Science Publishers, 1 Sept. 2014. Web. 3 Mar. 2017.

Moura, Roberto Soares de, et al. “Effects of Euterpe Oleracea Mart. (AÇAÍ) Extract in Acute Lung Inflammation Induced by Cigarette Smoke in the Mouse.” Phytomedicine 19.s 3–4 (2012): 262–269. Web. 3 Mar. 2017.

Schauss, Alexander G., et al. “Antioxidant Capacity and Other Bioactivities of the Freeze-Dried Amazonian Palm Berry, Euterpe Oleraceae Mart. (Acai).” Journal of Agricultural and Food Chemistry 54.22 (2006): 8604–8610. Web.

“Açai (Euterpe oleracea).” Texas A&M University. 12 Jan. 2006. Web. 3 Mar. 2017.

Laslo, Mara, et al. “A Botanical Containing Freeze Dried Açai Pulp Promotes Healthy Aging and Reduces Oxidative Damage in Sod1 Knockdown Flies.” AGE 35.4 (2012): 1117–1132. Web.

Carvalho-Peixoto, Jacqueline, et al. “Consumption of Açai ( Euterpe Oleracea Mart.) Functional Beverage Reduces Muscle Stress and Improves Effort Tolerance in Elite Athletes: A Randomized Controlled Intervention Study.” Applied Physiology, Nutrition, and Metabolism 40.7 (2015): 725733. Web.

Alqurashi, Randah M, et al. “Consumption of a Flavonoid-Rich Açai Meal Is Associated with Acute Improvements in Vascular Function and a Reduction in Total Oxidative Status in Healthy Overweight Men1,2 .” The American Journal of Clinical Nutrition 104.5 (2016): 1227–1235. Web. 3 Mar. 2017.

Poulose, Shibu M., and Barbara Shukitt-Hale. “Functional Role of Walnuts and Açaí Fruits on Brain Health.” Tropical and Subtropical Fruits: Flavors, Color, and Health Benefits. Boston, MA: American Chemical Society (ACS), Jan. 2013. 171–187. Web.

Carey, Amanda N., et al. “Dietary Supplementation with the Polyphenol-Rich Açaí Pulps (Euterpe Oleracea Mart.) Improves Cognition in Aged Rats and Attenuates Inflammatory Signaling in BV-2 Microglial Cells.” Nutritional Neuroscience (2015): 1–8. Web.

Vitamin B12 Cyanocobalamin

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