BannerBio Nutraceuticals Inc.

GRAPE SEED EXTRACT
Scavenge Free Radicals and Protect Cardiovascularity

1. Background
　Proanthocyanidins, also known as condensed tannins, are a class of biologically active flavonoids that has been called "super antioxidants". Typically concentrated in the bark of trees and in the outer shells of seeds, proanthocyanidins serve to protect plants against oxidative elements such as oxygen and sunshine. Grape seeds are a particularly rich source of proanthocyanidins.
　　Grape seed extract, concentrated from the seeds of grape (Vitis vinifera L.), has a high content of compounds known as oligomeric proanthocyanidins (OPCs), which are made up of proanthocyanidin monomers. In grape seed extract there are basically two proanthocyanidin monomers called catechin and epicatechin. Each of them binds at either the alpha or beta position on their molecular structures to form oligomers or polymers. In addition, catechin and epicatechin can also form numerous esters from gallic acid as well as various glycosides and peptides.
　　A growing body of scientific evidence suggests that grape seed extract possesses a broad spectrum of biological, pharmacological and chemo-protective properties against free radicals and oxidative stress. Grape seed extract, best known for its anti-oxidant, has been shown to have significantly greater antioxidant activity than vitamins C, E or beta-carotene. 1

Figure 1：OPC free radical scavenging capacity is greater than either vitamin E or vitamin because it has more free radical quenching stations to neutralize free radicals.

　　It has also been reported that grape seed extract enhances cardiovascular health,2 inhibits growth and formation of certain types of cancer,3 improves visual performance in humans,4 reduces symptoms of premenstrual syndrome, 5reduces symptoms in gastric ulcers,6 and protects cells against drug, chemical and environmental pollutants toxicity.7

2. Functional components of Grape Seed Extract
　　Grape seed extract is made up of one kind of polyphenol compounds including gallic acid, catechin, epicatechin, catechin-3-gallate, epicatechin-3-gallate and proanthocyanidins, The majority of which are proanthocyanidins, which account for the most dominant among the effective components.
　　Proanthocyanidins are polyphenol compounds condensed through the link models of C4→C8 or C4→C6 bond by monomers of (+)-catechin, (-)-epicatechin, (-)-epicatechin-3-gallate etc.. According to the difference of polymerization degree, proanthocyanidins are also sorted into oligomeric proathocyanidin (OPC) and polymeric proathocyanidin (PPC). Commonly, Proanthocyanidins with 2-7 polymerization degree was named OPC, bioactivity and water-solubility of which are the best.

Figure 2：the chemical structures of main functional components in Grape Seed Extract

3. Function of Grape Seed Extract
3.1BannerBio Grape Seed Extract increases the life span of Drosophila melanogaster
　　According to the free radical theory of aging, free radicals are a primary cause of many degenerative diseases and accelerated aging. Consequently, antioxidant and/or free radicals scavengers may retard the aging process. Dr Winter has discovered that regular consumption of ginkgo may extend both the average and the maximum life span in rats.8 Likewise, it has been reported recently that long-term supplementation with melatonin as antioxidant has a life-extending effect on Drosophila melanogaster.9 It would be interesting to repeat such study using grape seed extract since it is best known as a powerful antioxidant to scavenge free radicals.
　　As shown in Figure 4, BannerBio Grape Seed Extract, when added daily to the nutrition medium at appropriate concentrations [0.03%, 0.08% in the diet (w/w), Group I and II], significantly increased the life span of Drosophila melanogaster (Oregon wild strain) regardless of sex.10 In the case of the concentration of 0.08% (Group II), the percentage increase in the BannerBio ?Grape Seed Extract fed female flies was 20% in median life span, 12% in maximum life span and 12% in average life span relative to the controls. Similar results were also observed in the experiments of the 200 male flies .

Figure 4: Effects of BannerBio ?Grape Seed Extract on the Life Span of Drosophila melanogaster

4.2 BannerBio Grape Seed Extract decreases MDA and increases SOD activity in rats
　　In order to further investigate antioxidant effects of BannerBio Grape Seed Extract in vivo, a study was carried out in Sprague Dawley rats (body weight 530*50 g). After BannerBio ?Grape Seed Extract was administered intragastrically to the rats at different concentrations (8.3, 33.3 and 50.0 mg/kg body weight, respectively) for 30 consecutive days, the lipid peroxidation (measured as malondialdehyde, MDA) and the activity of the antioxidant enzyme superoxide dismutase (SOD) were evaluated. In comparison with the controls, a significant decrease in serum MDA (Group II and III, Figure 5a) and a significant increase in serum SOD activity were observed in administered rats (Group I and II, Figure 5b).10 This effect was not accompanied by changes in body weight.

Figure 5: Effects of BannerBio ?Grape Seed Extract on MDA and Activity of SOD in Rat Sera

4.3 BannerBio Grape Seed Extract is effective in the treatment of human chloasma

　　Thirty female volunteers with chloasma (aged 30-52) were given an oral supplementation of 4 capsules each containing 50 mg BannerBio ?Grape Seed Extract in two doses per day. After one month of consecutive treatment, objective skin color evaluation was performed instrumentally. As shown in Figure 6, a significant improvement in the de-pigmentation and decrease in the chloasma
area were observed after the treatment.11 None of the volunteers reported any side effects from the treatment .

Figure6: Effects of BannerBio ?Grape Seed Extract Treatment on Human Chloasma

4.4 BannerBio Grape Seed Extract improves learning and memory in NIH mice

　　An improvement in learning and memory deficiencies associated with aging was reported in animals treated with pine bark extract.12 Similar experiments were repeated with BannerBio ?Grape Seed Extract in order to examine the efficacy of BannerBio ?Grape Seed Extract on learning and memory in rodent animals. NIH mice (body weight 30-35 g) were studied by step-down passive avoidance test, Y-maze and Morris water maze paradigms.13
　　In the step-down passive avoidance task, mice were randomly divided into 5 groups (n = 10). Separate groups were administrated orally with saline, piracetam and BannerBio ?Grape Seed Extract at different doses for 7 days, ending 1 hr before training. 24 hr after training the step-down latency (SDL) and step-down errors (SDE) were measured. It was found that the BannerBio ?Grape Seed Extract administration, like the piracetam administration (300 mg/kg.bw), significantly prolonged the SDL and decreased the SDE when compared with the saline control (Figure 7).13

Figure 7: Effects of BannerBio ?Grape Seed Extract on Step-Down Performance of Mice
　　In the Y-maze performance task, mice were randomly divided into 5 groups (n = 10). Separate groups were administrated orally with saline, piracetam and BannerBio ?Grape Seed Extract at different doses for 10 days, ending 1 hr before training. On day 10 each mouse was given 10 training trials. One day later, shortterm memory was tested by recording the errors made by the mice in a shock-motivated visual discrimination Y-maze. As shown in Table 1, the BannerBio ?Grape Seed Extract and piracetam treated mice made significantly few errors than the saline treated mice.13

Table 1: Effects of BannerBio ?Grape Seed Extract on Learning and Memory in Mice (Y-maze Test)

　　Effects of BannerBio ?Grape Seed Extract administration on spatial acquisition and memory were investigated in the Morris water-maze. NIH mice were administrated orally with BannerBio Grape Seed Extract at different doses or saline or piracetam for 19 consecutive days, ending 1 hr before training. On day 14 the mice were placed in the water to swim freely for 2 min to familiarize the environment. From day 15 to day 19 the mice were given four training trials per day and the learning/acquisition ability was measured by the amount of time to locate the submerged platform (SPL). On day 19 a spatial-probe trial was performed with the platform removed and the mice placed randomly in the water. CPT (crossing platform times) was measured by counting the number of times the mice crossed the quadrant containing the platform within 2 minutes. The CPP (crossing platform percentage) was calculated as the ratio of CPT to the total times the mice crossed all quadrants. 10 days later (day 29), the same experiments were repeated with and without the platform.
　　When compared to saline treated mice, mice treated with BannerBio Grape Seed Extract and piracetam showed a significant decline in the SPL on day 29 (Table 2).13 Similarly, significant improvements in memory retrieval at the spatial probe trials were also noted in mice treated with BannerBio ?Grape Seed Extract and piracetam, as evidenced by the significant increases of CPT
and CPP (Figure 8).13

Table 2: Effects of BannerBio ?Grape Seed Extract on the Water Maze Performance (SPL in seconds)

Figure 8: Effects of BannerBio ?Grape Seed Extract on the Water Maze Performance

4. Stability of Grape Seed Extract

　　No loss of potency was found after 3 months of storage at the temperature of 37~40 oC and relative humility of 75%,10 indicating BannerBio ?Grape Seed Extract is extremely stable on the shelf. The product is also very soluble in water (insoluble substances < 1% when 1 g/100ml).

5. Nutrition analysis

6. Safety of Grape Seed Extract
　　Recently a series of safety studies were performed to investigate the safety of grape seed extract. The LD50 (median lethal dose) was found to be greater than 4 g/kg in the male and female rats combined in the oral acute toxicity study, demonstrating a lack of toxicity and supporting its use in various foods.14 Likewise, BannerBio Grape Seed Extract was examined for its acute oral toxicity using NIH mice (body weight 18-22 g). The LD50 of BannerBio ?Grape Seed Extract was determined to be 5.625 g/kg body weight (Bliss' method) when fed once via by oral gavage to fasted male and female mice.13 Similarly, mutagenic studies of BannerBio Grape Seed Extract were carried out using NIH mice (doses at 1.25~5 g/mg body weight) and Salmonella typhimurium (0.5~5000 ?g/plate) and negative results were obtained in the Ames test, mouse micronucleus test and mouse sperm abnormality test.10 At the same time a sub-chronic oral toxicity study was carried out in the 80 Sprague Dawley rats (body weight 85~100g). It was found that intragastrical administration of BannerBio ?Grape Seed Extract as a dietary admixture at levels of 0.5, 1.0 and 2.0 g/kg.bw to the rats for 30 days did not induced any noticeable signs of toxicity.15

7. Daily dosage of Grape Seed Extract
Recommend daily dose of Grape Seed Extract: 200mg-400mg。

8. Packaging
Interior packaging：dual-layer polyethylene plastic bag
Exterior packaging：cardboard box
25kg/drum，fine powder。

9. Storing Method
Store in a cool, dry place and keep away from direct strong light.。

References
1. Bagchi D et al., Oxygen free radical scavenging abilities of vitamins C and a grape seed proanthocyanidin extract in vitro, Res Commun Mol Pathol Pharmacol 1997, 95(2): 179-89.

2. Yamakoshi J et al., Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits, Atherosclerosis 1999, 142: 139-49.

3. Joshi SS et al., The cytotoxic effects of a novel grape seed proanthocyanidin extract on cultured human cancer cells. Proc Am Assoc Cancer Res 1998, 39: 227.

4. Corbe C et al., Light vision and chorioretinal circulation; Study of the effect of procyanidolic oligomers. J Fr Ophthalmol 1998, 11: 453-60.

5. Amsellem M et al., Endotelon in the treatment of venolymphatic problems in premenstrual syndrome. Multicentered study on 165 patients. Tempo Medical 1987, 282.

6. Saito M et al., Aiti-ulcer activity of grape seed extract and procyanidins. J Agri Food Chem 1998, 46: 1460-4.

7. Bagchi D et al., Protection against drug- and chemical-induced multi-organ toxicity by a novel IH636 grape seed proanthocyanidin extract. Drugs Exp Clin Res 2001, 27(1): 3-15.

8. Winter JC, The effects of an extract of ginkgo biloba on cognitive behavior and longevity in the rat. Physiol Behav 1998, 63: 425-33.

9. Bonilla E et al., Extension of life span and stress resistance of Drosophila melanogaster by long-term supplementation with melatonin, Exp Gerontol 2002, 37(5): 629-38.

10. Data on company file. The study was performed independently in the Guangdong Provincial Center for Disease Control and Prevention, Guangzhou China. The experiment results have been accepted by Ministry of Public Health of China as documentation required for health food application.

11. Data on company file. The human open clinical trial was carried out in Xiyuan Hospital of China Academy of Traditional Chinese Medicine, Beijing China. The data have been accepted by Ministry of Public Health of China as documentation required for health food application.

12. Liu FJ et al., Pycnogenol enhances immune and hemopoietic function in senescence-accelerated mice, Cell Mol Life Sci 1998, 54: 1168-72.

13. Tan Y et al., Improving Effect on Learning and Memory and Acute Oral Toxicity of Grape Seed Extract, submitted for publication 2002.

14. Yamakoshi J et al., Safety evaluation of proanthocyanidinrich extract from grape seeds, Food and Chemical Toxicology 2002, 40: 599-607.

15. Data on company file. The study was performed independently in the Sun Yat-sen University of Medical Sciences, Guangzhou China. The experiment results have been accepted by Ministry of Public Health of China as documentation required for health food application.

Product Standard
BANNERBIO Grape Seed Extract- Proanthocyanidins

　　This product is extracted with aqueous and water from Grape seed, the uncontaminated seed of Vitis vinifera L. from Europe. It includes more than 95% Proanthocyanidins.

Appearance	Brown Red Powder
Odor	Light Aromatic
Taste	Astringent
Proanthocyanidins	≥ 95% (Bate-Smith)
Mesh Size	80 mesh
Loss on Drying	≤ 5%
Ash	≤1%
Heavy Metals	≤ 10 ppm
Arsenic	≤ 2 ppm
Total Plate Count	< 1000 cfu/g
Fungi	< 100 cfu/g
Escherichia Coli	Negative
Salmonella	Negative