Ginkgo biloba extract improves coronary blood flow in healthy elderly adults: role of endothelium-dependent vasodilation.

Introduction

Ginkgo biloba extract (GBE) has a broad spectrum of pharmacological activities, which allows it to be in adequacy to the numerous potential therapeutic applications, including conditions associated with cognitive impairment, depression problem, liver and kidney injury, cerebral vascular insufficiency, myocardial ischemia and peripheral arterial occlusive disease (Dubey et al., 2004; Zhang et al., 2004; Zhu et al., 2004; Deng et al., 2006; Welt et al., 2007; Naidu et al., 2000). The results of recent studies support new indications for GBE in the treatment of ageing damages (Dong et al., 2004). Advancing age decreases the capacity of vascular endothelium to generate bioactive NO, even in healthy subjects, which contributes to the development of endothelial dysfunction and abnormal regulation of coronary blood flow (Matz and Andriantsitohaina, 2003; Ishida et al., 2003). However, little is yet known about the pharmacological actions of GBE on endothelial dysfunction and coronary blood flow in healthy elderly adults. Some studies showed that GBE caused vasodilation through the mechanism involving endothelium derived nitric oxide (NO) (Chen et al., 1997; Delaflotte et al., 1984; Kubota et al., 2006a, 2006b), and increased coronary flow in isolated guinea pig and rat hearts (Liebgott et al., 2000; Chatterjee and Gabard, 1982). We hypothesized that GBE might increase coronary flow by a way of improving endothelial function in humans with ageing. To test this assumption, the present study was designed to test the effects of GBE on distal left anterior descending coronary artery (LAD) blood flow and endothelium-dependent brachial artery flow-mediated dilation (FMD) in healthy elderly adults, and evaluate whether a link may exist between the changes in coronary artery flow and endothelial function.

Materials and methods

Drug

The GBE injectable solution (Ginaton) used in this study was manufactured by Chi Sheng Chemical Corp. Ltd. (Hsinchu, Taiwan, China). The Ginaton injectable solution is an aqueous solution which contains 17.5 mg GBE for injection (extraction agent acetone) per 5 ml ampoule. The extract is standardized to 24% ginkgo flavone glycosides (corresp. to 4.2mg ginkgo flavone glycosides per 5ml ampoule) and less than 1 ppm ginkgolic acids. The concentration of terpene lactones (ginkgolides and bilobalide) in the solution is about 2.3% less than that in EGb 761. EGb 761, a Ginkgo extract of Schwabe Comp. (Germany) is standardized on 22-27% flavonoid glycosides, 5-7% terpene lactones (2.8-3.4% ginkgolides A, B and C, and 2.6-3.2% bilobalide) and less than 5 ppm ginkgolic acids. Thus the composition of Ginaton injectable solution is with some quantitative deviations similar to that of EGb 761.

Subjects

The study subjects had angiographically normal coronary arteries, and those had a chronic health condition, regular smokers, renal, hepatic and thyroid diseases, hypertension, blood clotting disorders, peripheral vascular disease, drug and alcohol abuse problems were eliminated from the study. The investigation was carried out in compliance with institutional guidelines and complies with the principles outlined in the 1964 Declaration of Helsinki, and was approved by the Ethics Committee of Hebei Medical University. Written informed consent was obtained from all subjects before enrollment into the study.

Study design

The study protocol was undertaken with a double-blinded, randomized, and placebo-controlled design. Ginaton injectable solution was dissolved in 250ml physiologic saline solution. Thereafter, subjects received either GBE (0.7 mg/min) or placebo (physiologic saline solution) for 120min. LAD blood flow and brachial artery FMD were measured using Doppler echocardiography (Sequoia 512, Acuson, California, USA) before drug administration and during the last 20min of the infusion period. To avoid possible errors caused by variable arterial compliance, all measurements were made by a single ultrasonographer blinded to treatment assignment. Average parameters were calculated from 3 consecutive cardiac cycles.

Assessment of LAD blood flow

Coronary artery flow was assessed with transthoracic Doppler echocardiography (TTDE) using a 6-MHz transducer before and after trial therapy in the same manner as described previously (Daimon et al., 2005). Briefly, the transducer was placed at the fourth or fifth intercostal space between the cardiac apex and the parasternal area, and the anterior interventricular groove was visualized in the short-axis view. Then, the transducer was rotated to visualize the long axis of the groove, and color flow mapping was applied to visualize the distal LAD blood flow. At last, flow velocity was measured by pulsed TTDE, placing a 2.5-mm wide sample volume on the visualized flow signals. Transducer position and direction were adjusted to make the Doppler beam as parallel as possible to the LAD blood flow, and angle correction was performed for each Doppler measurement. Pulsed Doppler parameters of LAD blood flow, including the maximal diastolic peak velocity (MDPV), maximal systolic peak velocity (MSPV), and diastolic time velocity integral (DTVI), were measured using the analyzer incorporated in the ultrasound system.

Measurement of endothelium-dependent brachial artery FMD

Endothelial function in the form of endothelium-dependent brachial artery FMD was measured in the subject's right arm with an 8-MHz transducer (Celermajer et al., 1994). Brachial artery diameter was measured at baseline and 60s after the release of forearm blood pressure cuff which was inflated for 4 min at the pressure of 40 mmHg greater than systolic blood pressure. The diameter change caused by endothelium-dependent FMD was expressed as percentage change relative to that at the initial scan at rest.

Statistical analyses

All the data are presented as mean [+ or -] SD for continuous variables and frequency (percentage) for categorical variables. Differences of clinical characteristics, brachial artery vasodilator responses and LAD blood flow were evaluated and analyzed between the 2 study groups (GBE and control) at baseline by independent Student's t tests for continuous variables and by [chi square] tests for categorical variables. Differences between the groups over time were calculated by 2-way repeated-measures analysis of variance with group as the nested variable. Pearson's correlations were calculated for estimating the linear relations between continuous variables. p<0.05 was considered statistically significant.

Results

The clinical characteristics of the 2 study groups are listed in Table 1. Sixty subjects were randomly assigned to GBE group (n = 30) or control group (n = 30). There were no significant differences in age, gender, body mass index, heart rate, and blood pressure between the 2 groups on baseline. In addition, there were no complications caused by GBE therapy, which was well tolerated by all enrolled subjects, and no subject withdrew after study initiation.

As shown in Table 2, LAD blood flow velocity parameters were similar between the 2 study groups before treatment. After the treatment, MDPV, MSPV. and DTVI of LAD blood flow were significantly increased in the GBE group compared with the placebo group (19.16 [+ or -] 13.91% vs. 0.30 [+ or -] 2.55%, 17.76 [+ or -] 14.56% vs. 0.53 [+ or -] 2.32%, and 21.73 [+ or -] 16.13% vs. 0.81 [+ or -] 2.33%, MDPV, MSPV, and DTVI improvement from baseline, respectively, p < 0.01). Multivariate linear regression for estimating MDPV, MSPV, and DTVI improvement involved MDPV, MSPV, and DTVI at baseline, age, gender, heart rate, systolic blood pressure, diastolic blood pressure, brachial artery FMD, as well as the treatment group. GBE treatment was a significant predictor for MDPV, MSPV, and DTVI improvement and increased the percentages of the explained variance in 69.2, 64.4% and 67.8%, respectively.

The total study population had brachial artery FMD of 7.14 [+ or -] 2.45%. As shown in Table 2, there were no significant differences in baseline brachial artery FMD between the 2 groups. GBE treatment resulted in a significant improvement in brachial artery FMD (7.21 [+ or -] 2.52% vs. 11.28 [+ or -] 3.95%, p < 0.01). However, the finding was not evident in the control group (7.07 [+ or -] 2.43% vs. 7.14 [+ or -] 2.43%; p > 0.05). GBE increased brachial artery FMD by 56.03% (p < 0.01). At the end of the trial, FMD was significantly greater in the GBE-treated group compared with the control group (11.28 [+ or -] 3.95%% vs. 7.14 [+ or -] 2.43%, respectively, p < 0.01). Multivariate linear regression for estimating increase in brachial artery FMD included brachial artery FMD at baseline, age, gender, heart rate, systolic blood pressure, diastolic blood pressure, as well as the treatment group. GBE treatment was a significant predictor of increased brachial artery FMD and increased the percentages of the explained variance in 80.9%. Responses of increasing in brachial artery FMD were correlated with the percentage change in MDPV, MSPV, and DTVI of LAD blood flow following treatment with GBE (r = 0.538, 0.366, or 0.573, respectively, p < 0.01, p < 0.05, and p < 0.01; Figs. 1 A, B and C).

The intra-observer variability in measurements of MDPV, MSPV, and DTVI of LAD blood flow in our laboratory was 4.1%, 2.3%, and 3.2%, respectively. Meanwhile, the intraobserver variability in measurements of brachial artery diameter was 2.8%.

[FIGURE 1 OMITTED]

Discussion

Coronary endothelial function can be measured via intra-arterial infusion of pharmacologic substances that enhance the release of NO. The disadvantage of these methods is their invasive nature, which generally makes them unsuitable for serial studies of progression or reversibility. Recently, numerous clinical studies have demonstrated that the brachial artery FMD is thought to correlate modestly with the endothelium function of coronary arteries (Teragawa et al., 2005), which can be measured with high-resolution ultrasound. In addition, advancements in TTDE have enabled direct visualization and evaluation of coronary artery flow velocity, especially in the distal LAD (Daimon et al., 2005). The two methods can be performed non-invasively and repeatedly; thus, we used them in the present study to investigate the effect of GBE on coronary flow and endothelial function in healthy elderly adults.

This study demonstrated for the first time that GBE effectively improves endothelium vasomotor function in brachial artery and LAD blood flow in healthy elderly adults, which was agreed with previous reports. For example, Chatterjee and Gabard (1982) have demonstrated that GBE (5 to 37mg/ml) increased coronary flow of isolated guinea pig heart in a dose-dependent manner. Investigations showed that in vitro perfusion with GBE significantly recovered coronary flow in rat heart (Liebgott et al., 2000). Meanwhile, recent studies showed that GBE could induce endothelium-dependent relaxation. Nishida and Satoh (2003) demonstrated that GBE had the concentration-dependent vasorelaxant effect on rat aortic ring strips. Similarly, Delaflotte et al. (1984) showed the endothelium-dependent vasorelaxation effects of GBE on isolated rabbit aorta. Furthermore, it was demonstrated that GBE restore impaired endothelial dependent vasodilation induced by acetylcholine in spontaneously hypertensive rats (Kubota et al., 2006b). Chen et al. (1998) confirmed that the vasorelaxation effects of GBE on rabbit aortic rings and porcine basilar arteries were mediated via the NO pathway. Thus, these findings suggested that GBE may participate in the beneficial effects on endothelium-dependent relaxation and coronary blood flow with ageing.

Studies have shown that GBE produced relaxation in vascular strips in an endothelium-dependent manner. Meanwhile, Krieglstein et al. (1986) found the mechanism of GBE that increased cerebral artery blood flow was due to the release of endothelial-derived NO. Moreover, Zhou et al. (2006) investigated that NO-dependent coronary vasorelaxation would be involved with the actions of GBE. Because endothelium is an active participant in the regulation of coronary flow, one might speculate that the GBE-improved age-related endothelial dysfunction may alter the physiological regulation of coronary blood flow. Our results showed that GBE resulted in significant improvement in brachial artery endothelial function in healthy elderly adults, and the improvement might correlate to the increases of LAD blood flow in MDPV, MSPV, and DTVI. This phenomenon supported the hypothesis and suggested that at least part of the positive effect of GBE on coronary flow was mediated by the improvement of endothelial function.

Advancing age decreases the capacity of vascular endothelium to generate bioactive NO, a function of endothelial nitric oxide synthase-derived NO production and NO inactivation by reactive oxygen species, even in healthy subjects, which contributes to the development of endothelial dysfunction. Multiple clinical and experimental studies have shown that endothelium-dependent vasodilation progressively declines in the coronary and brachial arteries of angiographically normal individuals with ageing, and in different elderly animal species (Egashira et al., 1993; Hinschen et al., 2001; Matz et al., 2000). Considering increased endothelial superoxide production is inherent to the ageing process (Taddei et al., 2001; Csiszar et al., 2002), it is suggested that oxidative damage plays a key role in development of endothelial dysfunction in healthy elderly adults. Ginkgo biloba has been used therapeutically in traditional Chinese medicine for about 5000 years. The most important effect of GBE is scavenging of oxygen free radicals. Several studies have shown the potential benefits of GBE on the vascular endothelium through its protective roles against free radical injury. Pretreatment with EGb 761 has been shown to reduce free radical induced oxidative damage in patients undergoing coronary bypass surgery (Pietri et al., 1997). Additionally, Zhou et al. (2006) proved that ginkgolide A, a major constituent of Ginkgo biloba, effectively prevents homocysteine-induced endothelial dysfunction via reverse free radical production in porcine coronary arteries. Therefore, these studies support our results and provide a mechanism that GBE may improve endothelium-dependent relaxation in healthy elderly adults through its antioxidant effects. Meanwhile, these studies imply an important future therapeutic strategy of using antioxidant-based pharmacotherapy to counteract the detrimental effects of ageing.

Conclusion

We conclude that treatment with GBE increases LAD blood flow in MDPV. MSPV, DTVI and improves brachial artery FMD in healthy elderly adults. Meanwhile, the increased response in MDPV, MSPV, DTVI might relate to the improved endothelium-dependent vasodilatory capacity. The results suggest that GBE treatment in healthy elderly adults had beneficial effect on coronary flow, at least partially through endothelium-dependent vasodilation. This study implies an important future therapeutic strategy of using GBE to counteract the detrimental effects of ageing.

Acknowledgment

This study was supported by the Scientific and Technological Project of Hebei province, PR China, No. 99276129D.

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Yuzhou Wu (a,*), Shuqin Li (b,*), Wei Cui (a), Xiuguang Zu (a), Jun Du (a), Fengfei Wang (a)

(a) Department of Cardiology, Second Hospital of Hebei Medical University, Shijiazhuang 050000, PR China

(b) Department of Pathophysiology, Hebei Medical University, Shijiazhuang, PR China

*Corresponding authors. Tel.: [phone].

E-mail addresses: [e-mail]@126.com (Y. Wu).

Table 1. Baseline characteristics of study patients

                               Control             GBE
                               group               group
Variables                      (n = 30)            (n = 30)

Age, years                      56 [+ or -] 8       54 [+ or -] 10
Male                            15                  16
Body mass index, kg/[m.sup.2]   21.6 [+ or -] 2.2   22.2 [+ or -] 2.8
Heart rate, beats/min           72 [+ or -] 11      71 [+ or -] 14
Systolic blood pressure, mmHg  118.5 [+ or -] 9.1  119.6 [+ or -] 9.2
Diastolic blood pressure,       75.2 [+ or -] 8.3   76.4 [+ or -] 8.7
  mmHg

Data are given as mean [+ or -] SD.

Table 2. Effects of GBE on LAD blood flow velocity and brachial artery
FMD in healthy elderly adults

                          Saline
            Baseline             Post-medication

MDPV, cm/s  30.87 [+ or -] 9.65  30.98 [+ or -] 9.74
MSPV, cm/s  16.07 [+ or -] 5.63  16.16 [+ or -] 5.66
DTVI, cm/s  21.07 [+ or -] 6.00  21.21 [+ or -] 5.90
FMD, %       7.07 [+ or -] 2.43   7.14 [+ or -] 2.43

                         GBE
            Baseline              Post-medication

MDPV, cm/s  30.20 [+ or -] 10.44  36.03 [+ or -] 13.05* (#)
MSPV, cm/s  15.77 [+ or -] 5.87   18.26 [+ or -] 6.11* (#)
DTVI, cm/s  20.57 [+ or -] 6.40   24.98 [+ or -] 8.03* (#)
FMD, %       7.21 [+ or -] 2.52   11.28 [+ or -] 3.95* (#)

Values are means [+ or -] SD. *Significant difference between GBE and
saline (p < 0.01), (#) significant difference from baseline (p < 0.01).
MDPV = maximal diastolic peak velocity; MSPV = maximal systolic peak
velocity; DTVI = diastolic time velocity integral; FMD = flow-mediated
dilation.