Evidence of the Regulatory Effect of Ginkgo biloba Extract on Skin Blood Flow and Study of its Effects on Urinary Metabolites in Healthy humans

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Subjects
• Study design and procedure
• Study substances
• Blood flow measurements
• Metabolic fingerprinting
• Statistical methods
• Results
• Discussion
• Acknowledgements
• References

Abstract

Ginkgo biloba extract has been advocated for the improvement of blood circulation in circulatory disorders. This study investigated the effect of the Gingko biloba extract EGb 761 on skin blood flow in healthy volunteers and accompanying changes in urinary metabolites. Twenty-seven healthy middle-aged subjects participated in a randomized, double-blind, placebo-controlled, crossover study. Subjects received 240 mg/d EGb 761 or placebo for periods of 3 weeks. Skin blood flow was measured on the forefoot using laser Doppler flowmetry and changes in urinary metabolites were identified by a combination of nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis (MVDA). These measurements were performed on 24-h urine samples collected at the end of the intervention periods. Following EGb 761 treatment, overall mean skin blood flow was significantly reduced as compared with placebo. Remarkably, the change of skin blood flow after EGb 761 intervention was proportionally related to blood flow after placebo treatment: subjects showed either an increased, decreased or unaltered skin blood flow. NMR/MDVA analyses showed that urinary metabolic patterns differed depending on the change in baseline blood flow after treatment with EGb 761. The present findings substantiate that EGb 761 has a multi-directional modulating action on blood flow in healthy subjects and support findings of a vasoregulatory role of this extract. Moreover, the results indicate that metabolic fingerprinting provides a powerful means to identify biochemical markers that are associated with functional changes.

Abbreviations

MDVA:multivariate data analysis

N-PLS:partial least squares

NMR:nuclear magnetic resonance

pu:perfusion units

Introduction

Extracts from the green leaves of the Ginkgo biloba tree have been applied as a traditional Chinese medicine for several thousands of years and have been claimed to exert beneficial effects on cerebral insufficiency (mainly Alzheimer’s disease), circulatory diseases, cardiovascular function, hearing and vision problems [1], [2]. Nowadays, standardized extracts of the leaves are widely sold in Europe and the United States and the number of scientific studies on G. biloba is rapidly increasing.

Beneficial effects of G. biloba on blood circulation have mainly been reported in clinical populations. Several studies demonstrated improvement in pain-free walking in subjects with intermittent claudication after treatment with G. biloba [3]. Most clinical trials used dosages of 120 - 240 mg/d. In subjects with hemorrheological disorders, acute beneficial effects on blood flow were shown after intravenous injection as well as after oral ingestion of 113 - 120 mg G. biloba extracts [4]. Besides effects on circulation, also decreased erythrocyte rigidity and aggregation, decreased platelet aggregation, and a decrease in fibrinogen were reported in these studies [5]. Moreover, in vitro studies demonstrated that G. biloba not only enhances blood flow, but that it also acts as a vasomodulator [1], [6].

As the extract is easily available, people without circulatory disorders may also get interested in G. biloba’s claimed health benefits. In a recent study in young healthy subjects, an increase in forearm blood flow was observed after 3 weeks of active treatment with 7.2 mg terpenoids and 28.8 mg flavonoid glycosides [7]. No further thorough studies on the effects of G. biloba in healthy subjects have been published.

Metabolic fingerprinting, which is a method utilizing nuclear magnetic resonance spectroscopy (NMR) and multivariate data analysis (MVDA), was applied to investigate biochemical variation in relation to changes in skin blood flow after EGb 761 treatment as expressed in altered urine composition. NMR is a very suitable technique to analyse biological fluids because it provides both quantitative as well as qualitative metabolic information [8]. Nevertheless, due to the complexity of the mixture of metabolites contained in these fluids, MVDA is needed to find significant similarities and differences in metabolic profiles. It visualizes the correlation between variables (e. g., thousands of signals in NMR spectra) in relation to a target variable such as health status or treatment. The contemporary technique of metabolic fingerprinting is a very sensitive measurement tool for diagnosing and monitoring effects of nutrient or drug interventions [8], [9] and its holistic approach harmonizes with the study on a complex herbal product.

The aims of our study were to evaluate the effects of G. biloba extract EGb 761 on skin blood flow in healthy, middle-aged volunteers, and to study its effects on urinary metabolites.

Materials and Methods

Subjects

Twenty-seven Caucasian non-smoking male (n = 10) and female (n = 17) subjects aged 55 - 74 years entered the study after giving their written informed consent. They were recruited from the pool of volunteers of TNO Nutrition and Food Research (Zeist, The Netherlands) and through advertisements in the local newspapers. All volunteers underwent a screening procedure that included a health and lifestyle questionnaire, physical examination and a routine blood clinical chemistry profile. Subjects had no metabolic or endocrine disease and no history of medical or surgical events that may have affected the results, including cardiovascular disease, skin diseases or hypertension. Subjects who were on anticoagulant therapy and/or chronically used vasoactive agents were excluded. Use of dietary supplements was not allowed. The study was approved by the TNO Medical Ethics Committee and complied with Good Clinical Practice guidelines.

Study design and procedure

The study was a randomized, placebo-controlled, double-blind study using a crossover approach with two treatments and two study periods. Subjects were stratified by age and gender. The order of supplementation with EGb 761 or placebo was counterbalanced across subjects: one-half started with a 3-week period of EGb 761 intake followed by a 3-week period of placebo intake, whereas the other half of the subjects received the treatments in the reverse order. Both treatment periods were separated by a 2-week wash-out period. After each treatment period, subjects went through a test day. On the evening before the test days and on the test days no alcohol was allowed. Coffee was not allowed on the test days. After arrival, subjects acclimatized for 15 minutes at ca. 24 °C and a relative humidity of ca. 50 % in a climate-controlled room. The subjects were seated comfortably with both legs at a right angle to the upper part of the body. Conversation and movement were avoided. In the morning of both test days, subjects started urine collection during 24 h.

Study substances

The test substance was Ginkgo biloba extract EGb 761 standardized to 24 % flavonoid glycosides and 6 % terpenoids (Tavonin®, Dr. Willmar Schwabe BV, Alkmaar, The Netherlands). The remainder comprised various commonly used fillers, such as microcrystalline cellulose. Microcrystalline cellulose was used as a placebo (Avicel®, Fagron, The Netherlands). Tablets of 40 mg Tavonin® were powdered and packed into capsules by a pharmacist. EGb 761 and placebo were supplied in similar capsules to meet the double-blindness of the study. Total consumption of EGb 761 was 240 mg per day, which was divided over 3 capsules each containing 80 mg. Capsules had to be ingested with breakfast, lunch and dinner, respectively. The contents of the capsules, including flavonoids and terpenoids were determined with high performance liquid chromatography (HPLC) and diode array detection, and gas chromatography/mass spectrometry (GC/MS), respectively. Analyses revealed 7.0 % terpenoids (mean 5.6 mg per 80 mg) and 25.6 % flavonoid glycosides (mean 20.5 mg per 80 mg). Terpenoids consisted of 2.3 % bilobalide, 1.9 % ginkgolide A, 0.6 % ginkgolide B, 0.7 % ginkgolide J and 1.5 % ginkgolide C. Flavonoid glycosides consisted of 13.0 % quercetin glycosides, 9.7 % kaempferol glycosides and 2.9 % isorhamnetin glycosides (data not shown).

Compliance was checked by counting the number of returned capsules and the reported deviations from intake of capsules registered by the subjects in a diary. After both treatment periods, safety was evaluated by the number and nature of adverse events as well as by hematology and blood clinical chemistry profile.

Blood flow measurements

Skin blood flow was assessed on the forefoot using a laser Doppler flowmeter with a 780 nm laser (PeriFlux System 5000, Perimed AB, Stockholm, Sweden). A standard probe was used with a fiber separation of 250 μm. The technique has been described in detail elsewhere [10]. Calibration of the instrument was done according to the instructions of the manufacturer. The optic cable was fixed relative to the foot as well as the probe that was fixed with a double adhesive ring approximately 2 cm proximal to the basis of the second and third toe. After registration of baseline blood flow for a minimum of 2 min, the arterial perfusion of the forefoot was occluded for 3 min by inflating a blood pressure cuff to 220 mm Hg that was placed just below the knee. Some researchers refer to this value as the biological zero. Thereafter, cuff pressure was quickly reduced to zero and the blood flow was recorded for the following 3 min. Data from the laser Doppler instrument were recorded using the software package Perisoft for Windows (Perimed AB, Stockholm, Sweden). Analysis of the recorded curves was performed off-line.

Metabolic fingerprinting

Prior to NMR spectroscopic analysis, 1 mL urine samples were lyophilized and reconstructed in 1 mL of sodium phosphate buffer (pH 6.0, made up with D₂O) containing 1 mM sodium trimethylsilyl-[2,2,3,3-² H ₄]-1-propionate (TMSP) as internal standard. NMR spectra were recorded in triplicate in a fully automated manner on a Varian UNITY 400 MHz spectrometer using a proton NMR set-up operating at a temperature of 293 K. The spectra were processed using the standard Varian software. Subsequently, the NMR data reduction file was imported into Winlin (V1.10, TNO, The Netherlands). Minor variations from comparable signals in different NMR spectra were adjusted and lines were fitted without loss of resolution. To correct for urinary dilution, the data were auto-scaled so that small and large peaks contribute similarly to the final study result. Where needed endogenous and exogenous metabolites of EGb 761 were eliminated from the NMR spectra leading to more universal EGb 761 related changes. The resulting data set was used to perform MVDA.

Statistical methods

Data on blood flow were log-transformed because they were not normally distributed. Individual peak blood flow response was calculated by subtracting the blood flow during occlusion. Mean baseline blood flow, and mean blood flow during and after occlusion, following placebo and EGb 761 treatment were compared by ANOVA using the SAS statistical software package (V8.1; SAS Institute Inc., North Carolina, USA). All statistics were evaluated at a significance level of 5 % (two-sided) and data are reported as means ± SD.

The NMR data were autoscaled in Matlab (Version 6.5; The MathWorks Inc., Natick, MA, USA), after which a supervised MVDA technique was applied (partial least squares; N-PLS) to visualize metabolic differences in the NMR spectra. N-PLS is an explorative MVDA technique [9] in which the goal is to describe the response variables (NMR data) by means of information about the input variables (treatment: placebo or EGb 761; effect on blood flow: decrease, no effect or increase). The resulting latent variables were quantified for each of the urinary NMR spectra and the first latent variable (LV1) was plotted versus the second latent variable (LV2) to visualize clustering. A factor spectrum, or metabolic fingerprint, was then used to correlate the position of clusters in the score plot to the original NMR signals in the spectra.

Results

Twenty-seven volunteers completed the study. Mean age was 61 ± 4 years ranging from 55 to 74 years, and mean body mass index was 25.8 ± 2.6 kg/m² ranging from 22.3 to 31.0 kg/m². During the first treatment period, six men and eight women ingested placebo, whereas four men and nine women ingested EGb 761. In the second treatment period, treatments were reversed. Overall compliance to consume three capsules containing test substances daily was 98 %. No adverse events were reported that were possibly related to treatment with EGb 761. Safety parameters were considered normal.

Skin blood flow values did not correlate with the age of the subject, but blood flow was significantly different after EGb 761 treatment compared with placebo. As is shown in Table [1], mean baseline blood flow was significantly reduced from 10.4 ± 6.7 perfusion units (pu) to 7.1 ± 4.4 pu (p < 0.01) after treatment with EGb 761 compared with placebo. In addition, peak blood flow after occlusion was significantly lower after treatment with EGb 761 (20.9 ± 16.8 pu) compared with placebo (29.1 ± 18.9 pu) (p < 0.01). No difference was found in blood flow during arrest by the inflated cuff. After treatment with EGb 761, 17 subjects showed a decrease of baseline blood flow, three subjects showed no change, and seven subjects showed a slight increase. Subjects with the highest baseline blood flow (as measured after treatment with placebo) demonstrated the highest decrease in blood flow after treatment with EGb 761 (correlation coefficient -0.81) (Fig. [1]). A similar pattern was also seen for peak values after occlusion (correlation coefficient -0.74) (data not shown).

Explorative N-PLS pointed out that a visible separation between placebo versus EGb 761 treated subjects could be made based on ratios of the concentrations of metabolites which were characteristic for each group (Fig. [2] A). Subjects treated with EGb 761 are positioned in the two right quadrants, whereas subjects treated with placebo are positioned in the two left quadrants. The NMR signals of urinary metabolites responsible for the difference between the groups can be visualized in a metabolic fingerprint (Fig. [2] B). Metabolites that were more abundant in urine of subjects treated with placebo are presented in the negative direction whereas metabolites that were overall more abundant in the EGb 761 treated subjects are presented in the positive direction. Metabolites that could be assigned to the most prominent signals using the in-house database with NMR spectra were amongst others glutamine/glutamate (δ = 2.25 - 2.45 in positive direction), tryptophan (δ = 7.21 - 7.40 in positive direction), glutathione (δ = 2.47 - 2.67 in negative direction), sugars and amino acids (δ = 3.38 - 4.60 in negative direction), and nicotinate, nicotinamides (δ = 7.97 - 9.12 in negative direction).

It appeared that urinary metabolite patterns are associated with different blood flow responses irrespective of the treatment. A metabolic fingerprint with NMR signals was derived that represents the differences between the subjects showing a decrease in blood flow versus subjects showing an increase or no effect (Fig. [3]). Signals in the positive direction indicate metabolites that were more abundant in subjects showing a decrease in blood flow upon treatment.

Metabolites that were more abundant in urine of subjects showing an increase in blood flow are presented as signals in the negative direction. Metabolites that could be assigned to the signals using the in-house database with NMR spectra were amongst others arginine, proline, lysine, ornithine (δ = 1.38 - 2.83 in positive direction), sugars and amino acids (δ = 3.38 - 4.60 in negative direction), phenylalanine, tyrosine, cinnamic acid (δ = 6.48 - 7.48 in positive direction), nicotinate, nicotinamides (δ = 7.97 - 9.12 in positive direction).

Discussion

The purpose of this study was to investigate the effects of EGb 761 on skin blood flow in healthy middle-aged subjects and to evaluate whether these effects are accompanied by metabolic changes in urine. Daily intake of 240 mg EGb 761 for 3 weeks resulted in a mean decrease of skin blood flow. A clear correlation was found between baseline blood flow after placebo and the difference in baseline blood flow after EGb 761 versus placebo. Seventeen subjects with the highest resting blood flow values demonstrated a decrease in flow after treatment with EGb 761; in three subjects with an average blood flow, no change was observed after EGb 761; the seven subjects with the lowest resting blood flow values demonstrated a slight increase after treatment with EGb 761. These data suggest that EGb 761 exerts dilatory or constrictive effects on blood vessels probably according to the physiological/pathological condition. Such modulating actions have been shown in vitro [1], [11], but in humans this is much more difficult to demonstrate. To our knowledge, this is the first study providing evidence of this regulatory effect in vivo.

In addition, metabolic fingerprinting pointed out that urinary metabolites differed depending on the direction of EGb 761-induced changes from baseline blood flow, rather than being related to the intervention. Obviously, EGb 761 exerted differential effects between subjects, implying that G. biloba may be efficient or not efficient according to the condition of the subject. Our results suggest different biological profiles between (groups of) individuals that may be related to the individual metabolism. This is an important and intriguing finding.

NMR shifts for a number of endogenous metabolites could be indicated in both the up- and down-fields in the NMR spectra. Clear differences were observed between the signals of metabolites that were affected due to change in blood flow after intervention. In the literature, various mechanisms for the vasodilatory actions of G. biloba have been proposed. G. biloba can influence vascular tone by the sympathetic nervous system directly, by acting on neuromediator release, and indirectly, by inhibiting catecholamine degradation by catechol O-methyltransferase [12] and monoamine oxidase [13]. Catecholamines such as norepinephrine, epinephrine and dopamine are derived from the amino acid tyrosine, which is in turn the product of phenylalanine. Catecholamines can stimulate vasoconstriction in the (sub)cutaneous vascular beds by alpha-adrenergic mechanisms [14]. It may be hypothesized that the higher amounts of phenylalanine and tyrosine that were observed in subjects showing a decrease in blood flow after EGb 761 treatment, are (partly) involved in this effect. In addition, also a number of other amino acids were more abundantly present in these subjects compared to subjects showing a decrease in blood flow. These changes in the levels of various amino acids may be involved in the broad spectrum of pharmacological activities of G. biloba, but no specific role can be attributed to these compounds with regard to blood flow regulation. Only arginine may be interesting because it is the precursor of nitric oxide (NO) which participates in the regulation of resting vascular tone [15]. In this view, it has been demonstrated that G. biloba can induce NO production as well as scavenge the free radical form of NO, illustrating its multidirectional regulatory action [16]. With respect to the NMR spectra comparing urinary metabolites after EGb 761 and placebo treatment, also other compounds were more abundant after supplementation with EGb 761, such as glutamate that is a common excitatory neurotransmitter in the central nervous system. Previous findings that stress-elevated alterations in brain neurotransmitters can be normalized upon administration of G. biloba suggest a possible mechanism to account for Ginkgo’s neuroprotective mechanism [17].

Further mechanisms for the vasodilatory actions of G. biloba appear to involve the inhibition of Ca²⁺ influx through Ca²⁺ channels and the activation of NO release, and might be in part due to the inhibitions of Ca²⁺ activated K⁺ current and prostacyclin release [18], [19]. Quercetin has been shown to be one of the principal ingredients of G. biloba for inducing relaxation of precontracted isolated blood vessels [20].

G. biloba extract can both counteract the phenomena resulting from vascular contraction and restore circulation in areas subject to vasomotor insufficiency, but whether these regulating actions also play a role in normal physiological situations is not clear. Nevertheless, changes in skin blood flow after EGb 761 treatment may result from the combined activities of several adaptive mechanisms. The dual effect is in line with the bi-directional modulator theory of herbal medicine [21].

In conclusion, the results of our study give evidence of the regulatory effects of G. biloba extract EGb 761 on skin blood flow. Our findings fit in with other studies that reported therapeutic effects, selective effects or effects that counter predicted outcomes [2], [21]. Our data cannot be explained completely, but some interesting findings have been raised: effects of G. biloba on skin blood flow in healthy humans may be either inhibitory or enhancing which may be related to individual metabolism. Healthy subjects under normal conditions likely are functioning close to optimum conditions and are probably less or not influenced by improvement than in case of an impaired (baseline) condition. Moreover, the integration of metabolic data provides a holistic approach to study biochemical effects of complex products (nutraceuticals, herbs) on function of the intact system and, more specifically, enables the characterization of key metabolic effects during changes in skin blood flow.

Acknowledgements

The authors gratefully thank Wouter H.J. Vaes for analysis of terpenoids and flavonoid glycosides in study substances, and Elly J. Spies-Faber for carrying out NMR measurements.

References

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  PubMedSearch in Google Scholar

Dr. L. Roza

Physiological Sciences Department

TNO Nutrition and Food Research

P.O. Box 360

3700 AJ Zeist

The Netherlands

Phone: +31-30-694-4966

Fax: +31-30-694-4928

Email: roza@voeding.tno.nl

References

• 1 Diamond B J, Shiflett S C, Feiwel N, Matheis R J, Noskin O, Richards J A. et al . Ginkgo biloba extract: mechanisms and clinical indications.  Arch Phys Med Rehabil. 2000;  81 668-77
  PubMedSearch in Google Scholar
• 2 Christen Y. From clinical observations to molecular biology: Ginkgo biloba extract EGB 761, a success for reverse pharmacology.  Curr Top Nutraceutical Res. 2003;  1 59-72
  PubMedSearch in Google Scholar
• 3 Pittler M H, Ernst E. Ginkgo biloba extract for the treatment of intermittent claudication: a meta-analysis of randomized trials.  Am J Med. 2000;  108 276-81
  CrossrefPubMedSearch in Google Scholar
• 4 Jung F, Mrowietz C, Kiesewetter H, Wenzel E. Effect of Ginkgo biloba on fluidity of blood and peripheral microcirculation in volunteers.  Arzneimittelforsch. 1990;  40 589-93
  PubMedSearch in Google Scholar
• 5 Witte S, Anadere I, Walitza E. Verbesserung der Hämorheologie durch Ginkgo-biloba-Extrakt. Verminderung eines kardiovaskularen Risikofaktors.  Fortschr Med. 1992;  13 247-50
  PubMedSearch in Google Scholar
• 6 McKenna D J, Jones K, Hughes K. Efficacy, safety, and use of Ginkgo biloba in clinical and preclinical applications.  Altern Ther Health Med. 2001;  7 70-90
  PubMedSearch in Google Scholar
• 7 Mehlsen J, Drabaek H, Wiinberg N, Winther K. Effects of a Ginkgo biloba extract on forearm haemodynamics in healthy volunteers.  Clin Physiol Funct Imagin. 2002;  22 375-8
  PubMedSearch in Google Scholar
• 8 Lindon J C, Nicholson J K, Holmes E, Everett J R. Metabonomics: metabolic processes studied by NMR spectroscopy of biofluids.  Concepts Magn Reso. 2002;  12 289-320
  PubMedSearch in Google Scholar
• 9 Lamers R -J, DeGroot J, Spies-Faber E J, Jellema R H, Kraus V B, Verzijl N. et al . Identification of disease- and nutrient-related metabolic fingerprints in osteoarthritic guinea pigs.  J Nutr. 2003;  133 1776-80
  PubMedSearch in Google Scholar
• 10 Nilsson G E, Tenland T, Oberg P A. Evaluation of a laser Doppler flowmeter for measurement of tissue blood flow.  IEEE Trans Biomed Eng. 1980;  27 597-604
  PubMedSearch in Google Scholar
• 11 Auguet M, Delaflotte S, Hellegouarch A, Clostre F. Pharmacological bases of the vascular impact of Ginkgo biloba extract.  Presse Med. 1986;  15 1524-8
  PubMedSearch in Google Scholar
• 12 Auguet M, DeFeudis F V, Clostre F, Deghenghi R. Effects of an extract of Ginkgo biloba on rabbit isolated aorta.  Gen Pharmacol. 1982;  13 225-30
  PubMedSearch in Google Scholar
• 13 Logani S, Chen M C, Tran T, Le T, Raffa R B. Actions of Ginkgo biloba related to potential utility for the treatment of conditions involving cerebral hypoxia.  Life Sci. 2000;  67 389-96
  PubMedSearch in Google Scholar
• 14 Damas J, Garbacki N, Liegeois J F, Juchmes J. Control of cutaneous blood vessels.  Rev Med Liege. 2001;  56 846-9
  PubMedSearch in Google Scholar
• 15 Wu G, Morris S M. Arginine metabolsim: nitric oxide and beyond.  Biochem J. 1998;  336 1-17
  CrossrefPubMedSearch in Google Scholar
• 16 Pietri S, Maurelli E, Drieu K, Culcasi M. Cardioprotective and anti-oxidant effects of the terpenoid constituents of Ginkgo biloba extract (EGb 761).  J Mol Cell Cardiol. 1997;  29 733-42
  CrossrefPubMedSearch in Google Scholar
• 17 Johns L, Sinclair A J, Davies J A. Effects of bilobalide on hypoxia/hypoglycemia-stimulated glutamate efflux from rat cortical brain slices.  Neurochem Res. 2002;  27 369-71
  CrossrefPubMedSearch in Google Scholar
• 18 Kleijnen J, Knipschild P. Ginkgo biloba.  Lancet. 1992;  340 1136-9
  CrossrefPubMedSearch in Google Scholar
• 19 Nishida S, Satoh H. Mechanisms for the vasodilations induced by Ginkgo biloba extract and its main constituent, bilobalide, in rat aorta.  Life Sci. 2003;  72 2659-67
  CrossrefPubMedSearch in Google Scholar
• 20 Kubota Y, Tanaka N, Umegaki K, Takenaka H, Mizuno H, Nakamura K. et al . Ginkgo biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level.  Life Sci. 2001;  69 2327-36
  CrossrefPubMedSearch in Google Scholar
• 21 Christen Y, Maixent J M. What is Ginkgo biloba extract EGb 761? An overview - from molecular biology to clinical medicine.  Cell Mol Biol. 2002;  48 601-11
  PubMedSearch in Google Scholar

Dr. L. Roza

Physiological Sciences Department

TNO Nutrition and Food Research

P.O. Box 360

3700 AJ Zeist

The Netherlands

Phone: +31-30-694-4966

Fax: +31-30-694-4928

Email: roza@voeding.tno.nl