High ORAC
GRAPE SEED EXTRACT

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1. Background
　　Proanthocyanidins, also known as condensed tannins, are a class of biologically active flavonoids that have 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 particularly rich sources of proanthocyanidins.
　　Grape seed extract (GSE), 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, accounting for the most dominant effect of GSE.
　　A growing body of scientific evidence suggests that GSE possesses a broad spectrum of biological, pharmacological and chemo-protective properties against free radicals and oxidative stress. GSE, best known for its anti-oxidant contents, has been shown to have significantly greater antioxidant activity than vitamin C, E or beta-carotene.1

Figure 1：OPC free radical scavenging capacity is greater than either vitamin E or vitamin C because it has more free radical quenching stations to neutralize free radicals.
　　Also, it has been reported that GSE enhances cardiovascular health, inhibits development and progression of certain types of cancer, improves visual performance in humans, reduces symptoms of premenstrual syndrome, reduces symptoms in gastric ulcers, and protects cells from toxicity effects of drug, chemical and environmental pollutants toxicity.2,3,4,5,6,7
　　As one of the earliest natural extract producers in China, BannerBio began to produce GSE in 1999. At that time, the flagship product of the company is 95% proanthocyanidins specification. In 2004, BannerBio developed its second-generation grape seed extract which contains 90% polyphenols and is highly water soluble. In 2006, Bannerbio successfully launched the new generation of grape seed extract, High ORAC GSE product, which has the highest ORAC value among all grape seed products on the market.

2. Brief introduction of ORAC

2.1 Free radicals and antioxidants
　　Free radicals are reactive species (oxidants). Excess production or malfunctioning of the radical scavenging mechanisms (oxidative stress) in the physiological system can lead to severe harmful effects such as damage of critical biomolecules. This is where antioxidants take their dominant roles. They serve to scavenge free radicals and render them harmless. Reducing exposure to free radical initiators and increasing dietary antioxidant intake are advisable strategies for maintaining optimal Redox status.
　　Dr. Ronald Prior, an early collaborator of the Brunswick Laboratories of the USDA demonstrated the measurement of oxygen radical absorption capacity of different foods and natural substances. This assay is called Oxygen Radical Absorbance Capacity (ORAC) assay. This is also one of the most sensitive and reliable methods for measuring antioxidant capacity.

2.2 Concept of ORAC assay
　　ORAC assay has been regarded as the golden standard assessment of antioxidant capacity, particularly for natural products such as botanical ingredients and consumer food products. Some of the world's largest nutrition companies use ORAC to promote the antioxidant benefits of their products.
　　According to the chemical reactions involved, major antioxidant capacity assays can be roughly divided into two categories: (1) hydrogen atom transfer (HAT) reaction based assays and (2) single electron transfer (ET) reaction based assays. ET-based assays measure an antioxidant's reducing capacity, and the HAT-based assays measure the hydrogen atom donating capacity. The hydrogen atom transfer is a key step in the radical chain reaction. Therefore, the HAT-based assays are more relevant to the radical chain-breaking antioxidant capacity. Among all the HAT-based assays, ORAC adopted an AUC (Area under Curve) technique to quantify antioxidant capacity. The advantage of the AUC approach is that it applies equally well for both antioxidants that exhibit distinct lag phases and those samples that have no lag phases. This approach unifies the lag time method and initial rate method, and it is particularly useful for food samples, which often contain multiple ingredients and have complex reaction kinetics.
　　Therefore, the ORAC assay has been broadly applied in academics and the food and supplement industry as the method of choice to quantify antioxidant capacity. In fact, an antioxidant database has been generated applying the ORAC assay in combination with the total phenols assay.

2.3 Principle and characters of ORAC assay
　　The ORAC assay depends on the free radical damage to a fluorescent probe through the change in its fluorescence intensity. The change of fluorescence intensity is an index of the degree of free radical damage. In the presence of antioxidant, the inhibition of free radical damage by an antioxidant, which is reflected in the protection against the change of probe fluorescence in the ORAC assay, is a measure of its antioxidant capacity against the free radical (Figure 2).
　　The uniqueness of the ORAC assay is that the reaction is driven to completion and the quantitation is achieved using "area under the curve" (AUC) (Figure 3). In particular, the AUC technique allows ORAC to combine both inhibition time and inhibition percentage of the free radical damage by the antioxidant into a single quantity (Figure 4) .

Figure 2: Principle of the ORAC assay	Figure 3: Antioxidant activity of tested sample expressed as the net area under the curve (AUC)
Figure 4: Calculation of ORAC values. AUC=0.5+F1/F0+F2/F0+F3/F0+.......+Fn/F0; Relative ORAC value = [(AUCSample-AUCBlank) / (AUCstandard-AUCBlank) x (molarity of standard/molarity of sample) Note: The basic knowledge about ORAC and the figures listed here are cited from http://www.brunswicklabs.com/

3. Characteristics of BannerBio High ORAC GSE
3.1 High ORAC value
　　ORAC value of BannerBio high ORAC GSE product is 18782 μmol TE/g. We obtained the ORAC values for our GSE samples from authoritative Brunswick Laboratory as supported by the following scanned certificate.

3.2 High OPC content
　　The OPC content in BannerBio high ORAC GSE product is very high, more than double in quantity compared to common GSE products currently available on the market. The high quality of BannerBio high ORAC GSE product is of guaranteed quality. The chromatograms below show the profiles of BannerBio high ORAC GSE product and a European product (notable for its best quality in Europe), respectively. From the chromatograms,it is evidential that they have comparable profiles (Figure5).

Figure 5: HPLC chromatogram of BannerBio high ORAC GSE product (A) and European GSE product (B)

3.3 High water solubility
　　BannerBio high ORAC GSE has high degree of water solubility, up to 8%, namely 8g samples completely dissolved in 100ml water to give a clear solution. In contrast, most GSE products on the market only reach solubility of 1-2% as illustrated in the following pictures.

From right to left, 1st = control (distilled water), 2nd = high ORAC GSE sample, 3rd and 4th = common GSE samples. All samples were prepared form the same amounts of GSE.

From left to right, 1st = control (distilled water), 2nd = high ORAC GSE sample, 3rd and 4th = common GSE samples. Preparation of the GSE samples was based on the criterion of same ORAC value. More than double the amounts of extract used in the high ORAC GSE sample were required for the latter two samples.

 

4. Stability of BannerBio high ORAC GSE
　　BannerBio carried out experiment on the stability of BannerBio high ORAC GSE over long period of storage under room conditions (25oC, 60% humidity). Several parameters including ORAC value, monomer, OPC and water contents were used as quality indices. No significant changes in these parameters were detected, indicating that BannerBio high ORAC GSE is extremely stable on shelf.

5. Nutrition analysis

 

6. Safety of BannerBio high ORAC GSE
　　A recent study determined the LD50 (oral administration) of proanthocyanidin-rich GSE to be greater than 4 g/kg in male and female rats, indicating that GSE is safe for consumption, e.g. as a food ingredient.8 Likewise, BannerBio high ORAC GSE was examined for its acute oral toxicity using NIH mice (body weight 18-22g). The LD50 of BannerBio high ORAC GSE was determined to be 5.625 g/kg body weight (Bliss' method) when fed once via by oral gavage to fast male and female mice. In addition, mutagenicity test was also conducted for BannerBio high ORAC GSE using NIH mice (1.25~5 g/mg body weight) and Salmonella typhimurium (Ames test) (0.5~5000 g/plate). Negative results were obtained in the Ames test, mouse micronucleus test and mouse sperm abnormality test. At the same time, a sub-chronic oral toxicity study was carried out in 80 Sprague Dawley rats (body weight 85~100g). It was found that intragastrical administration of BannerBio high ORAC GSE as a dietary admixture at levels of 0.5, 1.0 and 2.0 g/kg bw for 30 days did not induce any noticeable signs of toxicity in the rats.

7. Applications of high ORAC GSE
　　ORAC value Enhancement of high ORAC GSE, one important breakthrough, has been producing profound influences in the functions and applications of the Natural Plant Extract. It's certain that coming into being of high ORAC GSE would extend and magnify its functional intensity and fields to the most degree. For instance, the good water solubility and light color guarantee it can be applied into much more high-end product such as high-rank beverages, food, cosmetics, medical products, etc.

8. Daily dosage of BannerBio high ORAC GSE
　　The daily recommended dosage of common GSE products is about 200-400mg. Recommended daily dosage of BannerBio high ORAC GSE is 100-200mg.

9. Packaging
Interior packaging：Dual-layer polyethylene plastic bag
Exterior packaging：Cardboard box
25 kg/drum，fine powder

10. Storage
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. Yamakoshi J, et al., Safety evaluation of proanthocyanidinrich extract from grape seeds, Food Chem Toxicol 2002, 40: 599-607.

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Product Standard
BANNERBIO High ORAC GSE

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　　This product is extracted with ethanol and water from Grape seed, the uncontaminated seed of Vitis vinifera L. from Europe with Traits and virtues of highly solubilization, high Polyphenol content and high ORAC value, namely, completely dissolve in water at the concentration of 4%, less than 2% water-insoluble substances in the extract at 1% concentration, more than 90% Polyphenol and more than 18000 μmol TE/g of ORAC value.

Appearance	Light Red Powder
Odor	Light Aromatic
Taste	Light Astringent
Polyphenol	≥90% (Folin-C, GAE)
ORAC(Oxygen Radical Absorbance Capacity)	≥18000 μmol TE/g
Solubility in water, 4%	100% Solubility
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

　　BannerBio Nutraceuticals Inc., striving for the research, development and production of the new functional active ingredients for general health & life quality

* From product planning to OEM - for any help, suggestion, additional information or assistance or any chance for development and cooperation please contact us:

BannerBio Nutraceuticals Inc.
1/F 25th Building No.5 Kezhi West Road, Hi-Tech Industrial Park,
Nan shan District, Shenzhen 518057, China
Tel：0086-755-26504699, 26504868, 26504869
Fax：0086-755-26504870
Http: //www.bannerbio.com
Code：518057
Market Service: sales@bannerbio.com
Customer Service: tech@bannerbio.com

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Established Date: January 30, 2007