THE JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINE Volume 9, Number 5, 2003, pp. 625–629 © Mary Ann Liebert, Inc.
ORIGINAL PAPERS
Comparison of Antioxidant Activity in Commercial Ginkgo biloba Preparations DAVID MANTLE, Ph.D., F.R.S.C., F.R.C.Path.,1 RICHARD M. WILKINS, Ph.D., M.S., M.I. (Biol.),2 and MUHAMED ASIM GOK, M.B., B.S., F.R.C.S.(I)3 ABSTRACT Aim: To compare the relative levels of antioxidant activity in vitro in Ginkgo biloba samples (in tablet or capsule form) from different commercial suppliers, to determine whether some brands may be more efficacious in their potential to increase endogenous antioxidant activity, and thereby counter oxidative stress related disorders. Design: Antioxidant activity of the above sample extracts was determined in vitro against the ABTS?1 (2-29-amino-bis-3-ethylbenzthiazoline-6-sulfonic acid) radical, relative to Trolox (watersoluble vitamin E analogue) antioxidant standards, using an established assay procedure. Outcome measures: The relative antioxidant activity of G. biloba sample extracts was expressed in terms of millimoles per liter of Trolox equivalent (TE) for the initial extract, mmol TE per whole tablet, nmol TE per mg tablet, and nmol TE per mg ginkgo content. Results: Data (as mean 6 standard deviation (SD) from 4 separate estimations) obtained in this study showed a considerable variation (approximately 50-fold) in the level of antioxidant activity in preparations from different suppliers, particularly when compared on an equivalent (i.e., nmol TE/mg ginkgo) basis. Of the 18 products investigated, the highest level of antioxidant activity (whether expressed as mmol TE per whole tablet or nmol TE/mg ginkgo) was obtained for Pharma Nord Bio-Biloba (Pharma Nord, Morpeth, UK) tablets (p , 0.05, Dunnett’s statistical test). Conclusions: Some of the apparent variation in antioxidant activity of the various products investigated can be accounted for in terms of the physical nature of the G. biloba (i.e., dried leaf powder or standardized concentrated extract) used in tablet formulation. However, even when comparing products based on concentrated extract, the data demonstrated that there is still a considerable variation in antioxidant activity. Presumably this may result from differences in the manufacturing process between suppliers, which in turn may limit the efficacy of these preparations in the prevention or treatment of disease. INTRODUCTION
F
ree radicals are highly reactive transient chemical species formed in all cells as unwanted byproducts of normal aerobic metabo-
lism. Cells are protected from free-radical–induced damage by a variety of endogenous or dietary radical scavenging proteins, enzymes, and chemical compounds. Cellular damage arising from an imbalance between free-radi-
1 Department of Agricultural and Environmental Science and 2 School of Biology, University of Newcastle-uponTyne, Newcastle-upon-Tyne, UK. 3 Department of Surgery, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, UK.
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cal–generating and –scavenging systems (oxidative stress) has been implicated in the pathogenesis of a wide range of human disorders (Halliwell and Gutteridge, 1990). In this regard, the importance of the link between disease and diet has been recognized; epidemiologic studies have demonstrated that individuals who regularly consume fruit and vegetables gain protection against a number of disorders, particularly cardiac disease, cancer, and stroke (Block et al., 1992; Frei, 1995). These advantages are thought to result from the dietary content of plant-derived antioxidant compounds, including vitamins, flavonoids, and carotenoids (Eastwood, 1999; Pietta, 2000; Rice-Evans, 2001). This has led to the marketing of plant extracts in tablet form as a dietary supplement, to boost endogenous antioxidant levels and prevent disease. Particular interest has focused on Ginkgo biloba, which has a long history of use in Traditional Chinese Medicine, for example to counteract the effects of aging on brain function in the elderly. The efficacy of G. biloba extract EGB 761 (a mixture containing 24% flavone glucosides and 6% terpenoids) in the stabilization or improvement of symptoms in patients with mild/moderate Alzheimer’s dementia or vascular dementia has been reported in several clinical studies (Mantle et al., 2000a). The role of free radicals in the pathogenesis of Alzheimer’s dementia (Tabet et al., 2000), and evidence of antioxidant activity of G. biloba extract in protecting brain proteins against oxidative damage (Siddique et al., 2000) have been described previously. The objective of the present investigation was therefore to compare the relative levels of antioxidant activity in vitro in commercially available G. biloba samples (in tablet or capsule form) from various manufacturers, in order to determine whether some brands may be more efficacious in their potential to increase endogenous antioxidant activity and prevent disease. MATERIALS AND METHODS G. biloba samples in commercially available tablet/capsule form were solubilized via homogenization in extraction buffer (1:10 w/v in 50 mM phosphate-buffered saline, pH 7.4), using an Ultra-Turrax homogenizer (IKA Works,
Wilmington, DE) for 2 3 5 seconds, at 10,000 revolutions per minute (rpm). The homogenates were centrifuged (3000g, 10 minutes) to remove any insoluble residue, and the supernatants retained for antioxidant analysis. Antioxidant levels were determined in vitro as described previously (Rice-Evans & Miller, 1994). Briefly, chemical reaction between metmyoglobin (2.5 mM), ABTS (2-29-amino-bis3-ethylbenzthiazoline-6-sulfonic acid, 150 mM) and hydrogen peroxide (75 mM) in phosphatebuffer (50 mmol, pH 7.4) produces a relatively long-lived, colored free radical species (ABTS?1), the rate of formation of which can be quantified spectrophotometrically (734 nm at 30°C, 1 cm path length cell). G. biloba extracts are added (10–30 mL) to the reaction medium above (total volume 1 mL), and the extent to which formation of the ABTS?1 radical is suppressed (via competitive inhibition) is a measure of the antioxidant capacity of a given sample. A quantitative relation exists between the absorbance at 734 nm after 6 minutes and the antioxidant status of the test sample, determined relative to Trolox (a water-soluble vitamin E analogue) antioxidant standards (0–2.5 mmol), and expressed in terms of mM Trolox equivalent (TE).
RESULTS The comparative levels of antioxidant activity in the various G. biloba samples are shown in Table 1, expressed in terms of mM TE for the initial extract, mmol TE per whole tablet, nmol TE per mg tablet, and nmol TE per mg G. biloba (data shown as mean 6 standard deviation (SD) from four separate experiments). It is apparent from Table 1 that there is a considerable variation in the level of antioxidant activity in preparations from different manufacturers, particularly when compared on an equivalent (i.e., nanomoles of TE/mg of G. biloba) basis. The highest level of antioxidant activity, whether expressed in terms of micromoles of TE per whole tablet or nmol of TE per mg G. biloba, was obtained with Pharma Nord BioBiloba tablets (Pharma Nord, Morpeth, UK; p , 0.05, Dunnett’s statistical test). It is of note that there was relatively little difference in effective
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GINKGO BILOBA ANTIOXIDANT ACTIVITY TABLE 1. COMPARISON
OF
A NTIOXIDANT A CTIVITY IN VITRO
Manufacturer (G. biloba content/tablet) Pharma Norda Bio-Biloba (100 mg) Viridian b G. biloba (60 mg) Natures Aidc G. biloba concentrate (60 mg) Bootsd One-a-day (120 mg) Lichtwer Pharmae Ginkyo (50 mg) Ginkyo High Strength (120 mg) Holland & Barrettf G. biloba (30 mg) G. biloba (60 mg) Healthspang Ginkgo concentrated (60 mg) Ginkgo one-a-day (25 mg) Questh Ginkgo (150 mg) Ferrosani Ginkgo High Strength (120 mg) Ginkgo Idoloba (40 mg) Seredrin j Ginkgo High Strength (120 mg) Sagak Ginkgo (120 mg) Health Aidl Ginkgo vital (500 mg) Gerrard Housem Ginkgo leaf (150 mg) Good ‘n’ Naturaln G. biloba (250 mg) FSCo G. biloba (500 mg) Tru Health G. biloba (400 mg) Zip vitp G. biloba (400 mg) Solgarq G. biloba (520 mg) a Morpeth,
COMMERCIAL G INKGO
BILOBA
PREPARATIONS
Antioxidant activity (mean 6 SD, n 5 4) Tablet Initial (1; 10 w/v) extract (mmol TE) (mmol TE/tablet) (nmol TE/mg tablet) (nmol TE/mg G. biloba) 30.9 6 1.6
132 6 6.8
309 6 16.1
1326 6 68.7
19.5 6 1.1
68.3 6 3.8
195 6 11.2
1138 6 64.2
12.8 6 05
64.0 6 25.0
128 6 5.0
1067 6 41.7
30.4 6 1.8
120 6 7.1
304 6 18.0
1006 6 59.6
15.8 6 0.9 14.3 6 0.6
42.7 6 2.3 92.9 6 3.8
158 6 9.3 143 6 5.9
853 6 48.6 774 6 32.5
9.95 6 0.4 13.1 6 1.0
25.9 6 1.04 45.6 6 3.5
99.5 6 4.0 131 6 10
852 6 34.5 764 6 58.3
21.0 6 1.4 3.01 6 0.2
50.4 6 3.4 12.6 6 1.0
210 6 14.0 30.1 6 2.3
840 6 56.0 504 6 38.6
25.8 6 2.0
123 6 9.5
258 6 20.0
826 6 64.0
17.5 6 1.5 15.0 6 0.7
75.3 6 6.5 64.6 6 3.0
175 6 15.2 150 6 7.1
627 6 53.7 537 6 25.1
12.8 6 0.5
53.8 6 2.1
128 6 5.1
448 6 17.5
10.5 6 0.3
47.3 6 1.35
105 6 3.0
394 6 11.3
14.1 6 0.8
94.5 6 5.4
141 6 8.3
189 6 10.7
3.10 6 0.4
16.7 6 2.2
31.0 6 4.0
111 6 14.4
4.61 6 0.3
25.6 6 1.6
46.1 6 2.9
103 6 6.7
3.03 6 0.3
17.1 6 1.6
30.3 6 3.2
34.2 6 3.3
3.81 6 0.3
13.7 6 1.1
38.1 6 3.0
34.2 6 2.7
3.33 6 0.2
13.2 6 0.8
33.3 6 2.1
33.3 6 2.1
3.31 6 0.2
13.9 6 0.8
33.1 6 2.0
26.7 6 1.6
UK. UK. c Preston, UK d Nottingham, UK e Slough, UK. f Nuneaton, UK. g Guernsey, UK. h Birmingham, UK. i London, UK j Bracknell, UK k Folkstone, UK. l Harrow, UK m Brighton, UK n Dublin, OH o Great Yarmouth, UK p Rugely, UK q Tring, UK SD, standard deviation; TE, Trolox equivalent. b Daventry,
IN
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antioxidant activity between standard and high-strength variations of products from individual manufacturers (i.e. Healthspan, Guernsey, UK; Lichtwer Pharma, Bucks, UK; Ferrosan, Soborg, Denmark; Holland & Barrett, Warwickshire, UK), when expressed as nanomoles of TE per milligram of G. biloba.
DISCUSSION Free radicals are reactive, short-lived chemical species characterized by the presence of unpaired electrons (conventionally denoted by a point suffix). In relatively few instances, free radicals may perform beneficial functions, such as in the destruction of pathogens during phagocytic activation, or in the regulation of vascular tone. In general, however, free radicals are unwanted byproducts of normal aerobic cellular metabolism, with the potential to damage the various intracellular organelles or components (nucleic acids, lipids, proteins) on which normal cell function depends. In biologic systems, the hydroxyl (OH?) and superoxide (O2? 2 ) radicals are considered to be the most physiologically important primary-tissue–damaging free radicals (Cheeseman and Slater, 1993). The principal source of free radical generation results from leakage of electrons from the mitochondrial respiratory chain (i.e., from intermediate electron carriers onto molecular oxygen) during oxidative metabolism to generate adenosine triphosphate (ATP). It has been estimated that up to 5% of total electron flux results in the formation of free radicals, which in a typical human corresponds to the production of approximately 2 kg of O2? 2 per annum (Chance et al., 1979). Other sources of free radicals include the generation of O2? 2 via the action of oxidase enzymes during the metabolism of purines (xanthine oxidase), catecholamines (monoamine oxidase), prostanoids (lipoxygenase) and xenobiotics (cytochrome P450), as well as via the reaction of metal ions (Fe21, Ca21) with hydrogen peroxide to generate OH? radicals. Cells are protected from free-radical–induced damage by a variety of endogenous radical scavenging antioxidant proteins, enzymes, and chemical (water or lipid soluble) compounds. These include metal ion sequestering
proteins such as transferrin or caeruloplasmin, enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, biothiols such as glutathione, and vitamins C and E. Cellular damage arising from an imbalance between these free radical generating and scavenging systems (oxidative stress) has been implicated in the pathogenesis of a wide range of disorders, including neurodegenerative disorders, cardiovascular disease, cancer, and aging (Halliwell, 1996). This is based on evidence from diseased tissues for increased levels of free radicals (measured by electron spin resonance spectroscopy), increased levels of free-radical–induced oxidative damage products of DNA, lipids or proteins (measured by highperformance liquid chromatography [HPLC]), or decreased levels of antioxidants (Therond et al., 2000). As noted in the Introduction, the link between disease and diet, particularly the antioxidant content of the latter, has been recognized, leading to the marketing of plant extracts in tablet form as dietary supplements. In this regard, G. biloba is currently the best-selling phytomedicine on the European market. G. biloba needs to be taken in a concentrated form to be effective (typical recommended daily dose, 120–240 mg standard concentrated extract), because tea made from the leaves, for example, does not contain enough active ingredients. G. biloba has been used in Traditional Chinese Medicine to counter the effects of aging on brain function, and has been applied in Western medicine for the treatment of a range of disorders, particularly those associated with impaired blood flow (McKenna et al., 2001). G. biloba extracts are reported to have various pharmacologic actions, including platelet-activating factor antagonist and neurotransmitter agonist activities (Yoshikawa et al., 1999). However, it is as an antioxidant that recent research on G. biloba has focused. Some of the variation in antioxidant activity of the various preparations shown in Table 1 can be accounted for in terms of the physical nature of the G. biloba used in tablet or capsule formation (as stated by the manufacturer’s product label). Most manufacturers use a standardized G. biloba extract (typically 50–120 mg per tablet/capsule) equivalent to a 50-fold concentration of dried leaf
GINKGO BILOBA ANTIOXIDANT ACTIVITY
powder, containing 24% flavone glycosides and 6% terpene lactones, the major antioxidant components and less than 5 parts per million (ppm) of the allergen ginkgolic acid. However, some suppliers use only unconcentrated dried leaf powder (typically 400–500 mg per tablet/capsule), and it is these preparations that show the lower levels of antioxidant activity/mg of G. biloba and high amounts of ginkgolic acid (Kressman et al., 2002). Even when comparing levels of antioxidant activity for tablets/capsules based on the concentrated extract above, there is still considerable variation between preparations. Presumably such variations must result from differences in the manufacturing process between suppliers, which in turn may limit the relative efficacy of these preparations when used to prevent or treat disease. In this regard, fresh G. biloba leaf extract (Mantle et al., 2000b) has an antioxidant activity of 150 nmol of TE/mg dry weight (equivalent to 7500 nmol of TE/mg for a 50-fold concentrated extract), so that even the most active G. biloba preparation listed in Table 1 would appear to have lost a substantial proportion of initial antioxidant activity during the manufacturing process. Direct correlation of in vitro antioxidant activity with detailed active compound quantification, determined via HPLC analysis (as opposed to manufacturer’s stated label active compound content) would be of value in further elucidating this matter. However, such HPLC analytical data, where determined by manufacturers as part of the quality control method, are not generally available because of commercial confidentiality considerations, and would need to be independantly establihed as part of a subsequent investigation. We would emphasize that the present investigation relates only to the relative antioxidant activity (determined against ABTS?1 in vitro) of commercially available G. biloba preparations, and comparative aspects of activities against other radical species in vitro/in vivo, or other biologic activities (anticoagulant, anti-inflammatory, etc.) have yet to be determined. REFERENCES Block G, Patterson B, Subar A. Fruit, vegetables and cancer prevention: A review of the epidemiological evidence. Nutr Cancer 1992;18:1–29.
629 Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979;59:527–605. Cheeseman KH, Slater TF. An introduction to free radical biochemistry. Br Med Bull 1993;49:481–493. Eastwood MA. Interaction of dietary antioxidants in vivo: How fruit and vegetables prevent disease. Q J Med 1999;92:527–530. Frei B. Cardiovascular disease and nutritional antioxidants: Role of low-density lipoprotein oxidation. Crit Rev Food Sci Nutr 1995;35:83–98. Halliwell B. Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional intake in humans. Free Radic Res 1996;25:57–74. Halliwell B, Gutteridge JMC. Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol 1990;186:1–85. Kressmann S, Muller WE, Blume HH. Pharmaceutical quality of different Ginkgo biloba brands. J Pharm Pharmacol 2002;54:661–669. Mantle D, Pickering AT, Perry EK. Medicinal plant extracts for the treatment of dementia. CNS Drugs 2000a;13:201–213. Mantle D, Eddeb F, Pickering AT. Comparison of relative antioxidant activities of British medicinal plant species in vitro. J Ethnopharmacol 2000b;72:47–51. McKenna DJ, Jones K, Hughes K. Efficacy, safety and use of Ginkgo biloba in clinical and preclinical applications. Altern Ther Health Med 2001;7:70–86. Pietta PG. Flavonoids as antioxidants. J Nat Prod 2000;63:1035–1042. Rice-Evans C. Flavonoid antioxidants. Curr Med Chem 2001;8:797–807. Rice-Evans C, Miller NJ. Total antioxidant status in plasma and body fluids. Methods Enzymol 1994;234:279–293. Siddique MS, Eddeb F, Mantle D, Mendelow AD. Extracts of Ginkgo biloba and Panax ginseng protect brain proteins from free radical induced oxidative damage in vitro. Acta Neurochir 2000;76:87–90. Tabet N, Mantle D, Orrell M. Free radicals as mediators of toxicity in Alzheimer’s disease: A review and hypothesis. Adv Drug React Toxicol Rev 2000;19: 127–152. Therond P, Rousselot D, Spraul A, Conti M, Legrand A. Biomarkers of oxidative stress: An analytical approach. Curr Opin Clin Nutr Metab Care 2000;3:373–384. Yoshikawa T, Naito Y, Kondo M. Ginkgo biloba leaf extract: Review of biological actions and clinical applications. Antioxid Redox Signal 1999;1:469–4.
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