INTRODUCTION:

Commercial production of PCs using grape pips/seeds has been routinely practiced in Europe since the 1980s, (and is now available in the U.S also). Professor Jacques Masquelier, University of Bordeaux, France, has claimed a significant therapeutic effect of the PCs by means of their potent free-radical scavenging ability. A gist of his abstract says that, the PCs can play a major role in the prevention and cure of a wide range of illnesses.^1,2

PCs are an integral part of the human diet, naturally occurring compounds, found in high concentrations in fruits such as apple, pear, and grapes, and in nuts, seeds, flowers, barks. The fundamental structure of PCs is the phenolic flavan-3-ol nucleus (Figure 1). PCs are a class of phenolic compounds that take the form of oligomers or polymers of polyhydroxy flavan-3-ol units, such as the (+)-catechin and (-)-epicatechin.³

Jacques Masquelier has taken patent for the method of extraction of OPCs from Grape seeds in 1970.⁷The seeds of the grape (Vitis vinifera) are particularly rich source of PCs, and the PCs represent the major type of polyphenols in red wine.

These grape seed PCs are mainly dimers, trimers and highly polymerized oligomers of monomeric catechins.^{4, 5}The catechins (referring to both catechins and epicatechins) have the peculiar property of forming polymers with themselves. When the number of connected catechins is 10 or less they are called oligomers and thus the term used is "oligomeric proanthocyanidins." OPCs When the number of connected catechins is more than 10 the term condensed tannins is generally used. The OPCs is some of the most abundant polyphenolic substances in the plant kingdom. OPCs in nutritional supplements were generally extracted from grape seeds or pine bark. Today, dietary intake of OPCs varies from tens to hundreds of mg/day, depending on geographical and seasonal dietary differences.⁶Due to potent antioxidant activity, OPCs have been the subject of recent research, demonstrating anticarcinogenic, anti-inflammatory, antimicrobial, and vasodilatory properties, making them a potentially valuable therapeutic tool for the treatment of a variety of conditions.

1. Structures of PCs:

PCs are Oligomers and Polymers composed of elementary flavan-3-ol units. The structure variability of PCs depends upon the nature (the stereochemistry at the chiral centers and the hydroxylation pattern) of the flavan-3-ol extension and end units, the location and stereochemistry of the interflavan linkage (IFL) between the monomeric units and the degree of polymerization (DP) ⁷. Additionally, derivatizations such as O-Methylation, O-Acylation, C- and O- Glycosylation are involved in the rearrangement products of PCs ⁸.

As the building blocks of PCs, the flavan-3-ol units have the typical C6-C3-C6 Flavonoid skeletons. The heterocyclic benzopyran ring is referred to as the C ring, the fused aromatic ring as the A ring, and the phenyl constituent as the B ring ⁹(Figure 1). They differ structurally according to the nature of the stereochemistry of the asymmetric carbons on the C rings and the number of hydroxyl groups on the B rings. 2, 3-trans-(+)-Catechin and 2, 3-cis-(-)-epicatechin are stated to be the most usual monomeric units in PCs, which have the opposite stereochemistry of the chiral C3 carbon on the C rings (Figure 2, a and b). As observed in (+)-catechin (2R, 3S) and (-)-epicatechin (2R, 3R), the C2 configuration is almost always R,

Figure 1: Basic Structure and Numbering System of Flavonoids

Figure 1 : Structure of the 2R – Type Flavan – 3 –ols. 1 and 2.

a) 2R, 3S- Flavan - 3 - ol b) 2R, 3R- Flavan - 3 - ol

(+) – Afzelechin R_1, R₂ = H (-) – Epiafzelechin R_1, R₂ = H

(+) - Catechin R₁=OH, R₂ = H (-) - Epicatechin R₁=OH, R₂ = H

(+) - Gallocatechin R₁, R₂ = OH (-) - Epigallocatechin R₁, R₂ = OH

Subgroups of PCs:^{8, 10, 11}.

Generally, the term PCs refers to the release of anthocyanidins from extension positions after being boiled with strong mineral acid. Correspondingly, procyanidins designate oligomers and polymers with 3’, 4’-dihydroxyl pattern ((+)-catechin and/or (-)-epicatechin units) extension units, while propelargonidins or prodelphinidins designate oligomers and polymers with extension units of 4’-hydroxyl pattern ((+)-afzelechin and/or (-)-epiafzelechin units) or 3’, 4’, 5’- trihydroxyl pattern ((+)-gallocatechin and/or (-)-epigallocatechin), respectively. Therefore, PCs can thus be classified, according to the differences in hydroxylation patterns, into several subgroups:

1. Propelargonidins (3,4’,5,7-hydroxyl).

2. Procyanidins (3,3’,4’,5,7-hydroxyl).

3. Prodelphinidins(3,3’,4’,5,5’,7-hydroxyl).

4. Proguibourtinidins (3,4’,7-hydroxyl).

5. Profisetinidins (3,3’,4’,7-hydroxyl).

6. Prorobinetinidins (3,3’,4’,5’,7-hydroxyl).

7. Proteracacidins (4’,7,8-hydroxyl).

8. Promelacacidins(3’,4’,7,8-hydroxyl).

9. Proapigeninidins (4’,5,7-hydroxyl) and

10. Proluteolinidins (3’,4’,5,7-hydroxyl).

PCs are linked between the C4 position of the upper unit and the C8 or C6 position of the lower unit, and the type of IFL can be either α or β type. Generally, the IFL between C4 and C8 position are stereochemically predominant in procyanidins and prodelphinidins, as the C4→C8 IFL and the C4→C6 IFL are usually present in a ratio of 3:1⁸, but in 5-deoxy PCs, the C4→C6 IFL is predominant ^12,13. Oligomeric and polymeric PCs which are composed of flavan-3-ol units linked mainly through C4→C8 and/or C4→C6 IFL are categorized as B-type PCs (Figure 3). Among the dimers, procyanidins B1, B2, B3 and B4 are linked by the C4→C8 IFL and are the most frequently occurring in plants, whereas procyanidins B5, B6, B7 and B8 are linked by the C4→C6 IFL and are also widespread ¹⁴. On the other hand, the flavan-3-ol units can also be doubly linked by an additional ether bond between C2 position of the upper unit and the oxygen at C7 or C5 position of the lower unit. The oligomers or polymers which contain both C2β→O→7 ether-type IFL and C4→C8 or C4→C6 IFL, as well as those which contain both C2β→O→5 ether-type IFL and C4→C6 IFL are categorized as A-type PCs (Figure 4),which are found in various plant origin, such as Grapes, Peanut, and Cranberry, etc.¹⁵. The different structures of the B-type procyanidins (B₁-B₈, and C₁) and A-type procyanidins (A₁-A₂) are shown in Figure 3 and 4.

The degree of polymerization (DP) is another variable factor for the structure complexity of PCs. The mean degree of polymerization (mDP) is used to evaluate the Molecular Weight of the PCs. The (mDP) of PCs in grape seeds is from 2.3 to 16.7. ^16,17

The presence or absence of modifications of the monomeric flavan-3-ol units further enhances the structure complexity of PCs in Grape seed. Various methyl, acyl or glycosyl substituents of the monomeric units of PCs all occur in natural products (Grape seed) ¹⁸. The 3-hydroxyl group of the flavan-3-ol units of B-type PCs in Grape seeds is usually esterified with gallic acid ⁷. A recent report shows evidence for the existence of galloylated A-type procyanidins and also (-)-epicatechin gallate in Grape seeds ^17,19. Further more, gallic acid substituted (+)-catechin and (+)-gallocatechin also exist in Grape.^20,21

2. Biochemistry:

PCs were high-molecular weight oligomers or polymers of a basic flavan-3-ol unit, with an average degree of polymerization. PCs mixtures from grapes were a combination of dimers, trimers, tetramers, oligomers, and polymers. The reducing capacity of OPCs was thought to be proportional to weight concentration rather than the degree of polymerization.^22,
23 The polymeric nature of PCs was unique among polyphenols; they complex and precipitate proteins and inhibit enzymes involved in vascular tissue degradation. The ability of OPCs to complex proteins was referred to as astringency and is responsible for the "puckery" sensation when tea or red wine comes in contact with saliva and buccal tissue.²⁴

Figure 3:- Structure of B-Type Procyanidine

PROCYANIDINE B₁ PROCYANIDINE B₂

PROCYANIDINE B₃ PROCYANIDINE B₄

PROCYANIDINE B₅ PROCYANIDINE B₆

PROCYANIDINE B₇PROCYANIDINE B₈

PROCYANIDINE C₂

3. Bioavailability:

One of the key issues relating to the use of any nutritional supplement or medication was the ability of the substance to get into the body and be utilized at the cellular level. In order to demonstrate the bioavailability of the OPCs by Isotopic labeling technique.²⁵ The results of this study showed that the PCOs were rapidly absorbed into virtually all of the tissue of mammals (including the brain), and that there was a concentration in tissues high in glycosaminoglycans, namely connective tissues in the skin and organ systems, basement membranes of blood vessels, and cartilage.

4. Pharmacokinetics:

Human studies of polyphenol absorption were limited and results have varied depending on the structure and solubility of the phenolic compound. Available research has demonstrated the acidic environment of the human stomach does not readily degrade PCs; therefore, absorption rates in the upper GIT were not high. It appears, however, that even the low amounts observed in urine after an oral dose (usually <25% of original dose) are enough to significantly increase plasma/ serum antioxidant capacity.^26-28 OPCs reaching the colon undergo extensive degradation by the colonic flora. The metabolites and biological properties of this process have not yet been explored, but it has been suggested they may also have direct antioxidant and protective effects on colonic tissue.^{23, 29}

5. Biological Properties:

a. Free Radical Scavenging and Antioxidant activity.OPCs are primarily known for their Free Radical (FR) Scavenging and Antioxidant activity. The FR Scavenging abilities of PCs have been well documented and command the most attention.^30-33 In vivo studies have shown grape seed PCs extract is a better FR Scavenger and inhibitor of oxidative tissue damage than vitamin C, vitamin E succinate, vitamin C and vitamin E succinate combined, and beta carotene.³² Moreover, in vitro experimental results have demonstrated PCs have specificity for the hydroxyl radical, ^30,31 in addition to having the ability to non-competitively inhibit the activity of xanthine oxidase, a major generator of free radicals,³⁰elastase, collagenase, hyaluronidase, and beta-glucuronidase.³⁴

b. Anti-inflammatory: This effects may be due to inhibition of peroxide generation by macrophages.^35,36 In addition, animal studies demonstrate OPCs from grape seed significantly inhibit formation of proinflammatory cytokines, interleukin 1-beta, and tumor necrosis factor-alpha.³⁷

c. Antimutagenic/Anticarcinogenic: OPCs possess natural antimutagenic properties when exposed to certain strains of bacteria.³⁸

d. Antimicrobial Effects: Flavonoids and associated polyphenols, particularly OPCs, elicit an inhibitory effect on human immunodeficiency virus (HIV). A possible mechanism may be inhibition of gene expression regulating virus binding to cell receptors on normal lymphocytes, thus preventing infection.³⁹

e. Vascular Conditions: The use of grape seed OPCs to treat various vascular disorders, including varicose veins, venous insufficiency, capillary fragility, and retinopathies. Several clinical trials have confirmed the beneficial effects of OPC use in treating vascular disorders.^40-43

f. Skin Conditions, skin cancer: Grape seed PCs decreased tumor numbers and reduced the malignancy of papillomas.⁴⁴ ultraviolet damage to skin, dietary PCs may protect against carcinogenesis and provide supplementation for sunscreen protection.⁴⁵

g. Cardiovascular Disease: OPCs in red wine offer protection by reducing LDL oxidation, inhibiting cyclooxygenase and lipoxygenase in platelets and macrophages, and decreasing thrombotic events,⁴⁶ reduces the incidence of coronary heart disease,^{47, 48}increase plasma high-density lipoprotein (HDL) cholesterol and plasma apolipoprotein A-I concentrations in humans.⁴⁹OPCs in decreased lipid peroxidation, increased plasma antioxidant levels, and improved resistance of LDL to oxidation. ⁵⁰

h. Retinopathies/Ophthalmologic Conditions: PCs improved visual acuity, contrast sensitivity in patients with ocular stress due to video display unit use, and visual performance after glare exposure.^{51, 52} In the case of retinopathy OPCs significantly improved vascular lesions, microaneurisms, and exudates associated with diabetic retinopathy.⁵³

i. Cancer: OPCs may up-regulate certain apoptosispromoter genes and down-regulate apoptosis-inhibitor genes in cancerous cells.⁵⁴

j. HIV Infection: Grape seed PCs elicit an inhibitory effect on HIV infection in vitro. Recent research indicates chemokine receptors 3 and 5 expression on Th-2 lymphocytes is a prerequisite for HIV infection of the central nervous system.⁵⁵ OPC extract may prevent binding of the HIV virus to cell receptor sites on normal white blood cells, thereby preventing infection.³⁹

k. Wound Healing: The induction of vascular endothelial growth factor (VEGF) is a crucial step in the re-epithelialization phase of skin repair.^56,
57Extracts of grape seed PCs appear to stimulate the expression of VEGF in cultured keratinocytes, making OPCs a potential therapeutic tool in dermal wound healing.^{58, 59}

l. Protection from Drug Toxicity: Animal studies indicate OPC administration is beneficial in preventing hepatic and renal toxicity in instances of acetaminophen and other drug poisonings. This protection may be attributable to detoxification of cytotoxic free radicals or facilitation of DNA repair.^{60, 61} A study in rats with experimentally-induced myoglobinuric acute renal failure showed OPC administration to be of benefit in reducing blood urea nitrogen and serum creatinine; histological improvement was also noted.⁶²

m. Systemic Lupus Erythematosus: In a pilot study of patients with systemic lupus erythematosus (SLE), showed reductions in spontaneous lymphocyte apoptosis, T-lymphocyte activation, reduced generation of reactive oxygen species, lower erythrocyte sedimentation rates, and a decrease in the SLE disease activity index compared to the placebo group. The mechanism may be attributable to OPCs' antioxidant effect, resulting in reduced inflammatory activity.⁶³

n. Asthma: A significant reduction in serum leukotriene levels was observed in patients receiving OPCs and a significant improvements in asthma symptom scores.⁶⁴

Figure 4:- Structure of A-Type Procyanidine

PROCYANIDINE A₃ PROCYANIDINE A₂

6. Drug Interactions:

There are no known interactions between OPC extracts and other medications; however, data from in vitro and human studies indicate OPCs have an inhibitory effect on platelet aggregation similar to aspirin.^65,66 Therefore, caution is suggested in patients taking anticoagulant medication.

7. Side Effects and Toxicity:

OPCs have an excellent safety profile, with no known side effects, toxicity, or drug interactions. Rat studies have demonstrated OPCs to be nonmutagenic and nontoxic at high levels. The no-observed-adverse-effect level (NOAEL) of a chronic toxicity study of grape seed extract in rats was 1400-1500 mg/kg body wt/day,⁶⁷ which translates to 93-100 g grape seed extract daily for an average 150-pound adult. Human safety and toxicity studies for OPCs are limited, but no side effects are reported in the literature.

8. Dosage, Precautions and Interactions:

Suggested dosages for OPCs generally range from 50-150 mg daily, although in some studies dosages of 300 mg daily were used. A dosage of 1 mg/kg/body wt has also been suggested. In a12-month study, the safety of dietary intake of OPCs in a dose of 100mg/kg/day was demonstrated in rodents.⁶⁸ The US National Center for Complementary and Alternative medicine (NCCAM) reports that oral administration of OPCs was well tolerated in people over 8 weeks of a clinical trial.⁶⁹In one completed clinical trial, OPCs did not alleviate the hardening of breast tissue in female patients undergoing radiation therapy to treat breast cancer.⁷⁰

ACKNOWLEDGEMENTS:

The Authors are grateful to the Chairman, Prof. T.V.Narayana, and the Principal. Prof. R.Ramesh of Dr.H.L.T.College of Pharmacy. Bangalore (Rural), for providing all necessary facilities for this work and constant encouragement.

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Received on 02.05.2009 Modified on 05.07.2009

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 628-633

Oligomeric Proanthocyanidines: Grape Seed Extract

ACKNOWLEDGEMENTS: