David Wing-Shing Cheung

The anti-hypertensive effect of Danshen (Salvia miltiorrhiza) and Gegen (Pueraria lobata) formula in rats and its underlying mechanisms of vasorelaxation

ETHNOPHARMACOLOGICAL RELEVANCE Radix Salviae miltiorrhizae (Danshen) and Radix Puerariae lobatae (Gegen) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating cardiovascular disease. AIMS OF THE STUDY In the present study, an aqueous extract comprising Danshen and Gegen in the ratio of 7:3 (DG) was investigated for its anti-hypertension in vivo and vasodilative activities ex vivo. MATERIALS AND METHODS The anti-hypertensive effect of DG extract was investigated in spontaneously hypertensive rat (SHR) by measuring systolic blood pressure (SBP). Oral administration of DG extract was started at age of 6 weeks and 14 weeks for the preventive and therapeutic studies, respectively. Blood pressure was measured by tail-cuff method biweekly for 12 weeks. The ex vivo vasodilative activities of DG extract, its dependency on endothelium and the involvement of nitric oxide, prostacyclin and potassium channels were investigated using isolated rat aorta ring in organ bath. RESULTS For in vivo study, systolic blood pressure was significantly reduced in DG extract-treated groups (90.2 and 300 mg/kg) as compared with the SHR control in both preventive and therapeutic studies. However, DG extract was unable to suppress or delay the onset of hypertension in the preventive study. For ex vivo study, the results showed that DG extract induced a concentration-dependent relaxation in aorta and persisted response was observed with the removal of endothelium. Besides, pretreatment with a non-selective potassium channel inhibitor tetraethylammonium (TEA) also significantly inhibited DG extract-induced vasodilation. Further investigations on specific potassium channel blockers revealed that ATP-sensitive potassium (K(ATP)) channel inhibitor glibenclamide, inward rectifier potassium (Kir) inhibitor barium chloride and voltage-dependent potassium (K(v)) channel inhibitor 4-aminopyridine, but not BK(Ca) channel inhibitor iberiotoxin, exerted significant inhibition on DG extract-induced vasodilation. CONCLUSIONS The results of in vivo SHR animal model suggested that DG aqueous extract possessed blood pressure lowering effect on both pre- and post-hypertensive rats, which could be explained by its endothelium-independent vasodilation via the opening of K(ATP), Kir and K(v) channels.

The roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen) inhibit atherogenic events: a study of the combination effects of the 2-herb formula.

ETHNOPHARMACOLOGICAL RELEVANCE The roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen) are principle herbs of Chinese herbal formulae which have long been used to treat cardiovascular diseases. AIM OF STUDY The present study validated the anti-atherogenic effects of three extracts, Danshen alone (DE), Gegen alone (GE) as well as DGE and interpreted their combination effects statistically. MATERIALS AND METHODS The anti-atherogenic effects of the three extracts were studied in three assays with regards to inflammation, foam cell formation and vascular smooth muscle cell (vSMC) proliferation using lipopolysaccharide (LPS)-induced nitric oxide production model, macrophage foam cell formation model and platelet-derived growth factor (PDGF)-induced vSMC proliferation model, respectively. The combination effects of DGE were statistically analyzed using combination index (CI) and fixed-ratio experimental design. RESULTS The anti-atherogenic effects of the three extracts including anti-inflammation, anti-foam cell formation and anti-vSMC proliferation were demonstrated in this study. Their combination effects in anti-inflammation, anti-foam cell formation and anti-vSMC proliferation were found to be synergistic, additive and antagonistic, respectively. CONCLUSIONS This study provided scientific support for the combination use of DGE on atherosclerosis and presented one of the first applications of statistical interpretations of the combination effects of the 2-herb formula.

A herbal formula containing roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen) inhibits inflammatory mediators in LPS-stimulated RAW 264.7 macrophages through inhibition of nuclear factor κB (NFκB) pathway.

ETHNOPHARMACOLOGICAL RELEVANCE The herbal formula DG, containing roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen), has long history in treating cardiovascular diseases. It has been shown to be able to reduce intima-media thickening in coronary patients in our previous clinical study. Since intima-media thickening is the hallmark of atherosclerotic disease, the etiology of which is inflammation of the arterial wall, the mechanism underlying the effect of DG may be related to its anti-inflammatory activities. AIM OF STUDY The present study aims to determine the anti-inflammatory activity of DG and elucidate its underlying mechanisms with regards to its molecular basis of action. MATERIALS AND METHOD The anti-inflammatory effect of DG was studied by using lipopolysaccharide (LPS)-stimulated activation of nuclear factor κB (NFκB) pathway and subsequent production of inflammatory mediators, including nitric oxide (NO), prostaglandin E(2) (PGE(2)), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and macrophage chemotactic protein-1 (MCP-1), in mouse RAW 264.7 macrophages. RESULTS The present study demonstrated that DG could suppress the production of NO and PGE(2) through the inhibition of iNOS and COX-2 genes. DG could also inhibit the production of IL-1β, IL-6 and MCP-1, but not TNF-α, through the inhibition of respective mRNA expressions. Further investigations showed the inhibitory effect of DG on activation of IKKα/β and degradation of IκBα, thus preventing nuclear translocation of NFκB. All these results suggested the inhibitory effects of DG on the production of inflammatory mediators through the inhibition of the NFκB pathway. CONCLUSIONS The inhibitory effects of DG on the production of inflammatory mediators by LPS-stimulated RAW 264.7 macrophages, are accomplished by inhibiting the nuclear translocation of NFκB through inactivating IKKα/β and preventing degradation of IκBα.

Effect of Dietary Cocoa Tea (Camellia ptilophylla) Supplementation on High-Fat Diet-Induced Obesity, Hepatic Steatosis, and Hyperlipidemia in Mice

Recent studies suggested that green tea has the potential to protect against diet-induced obesity. The presence of caffeine within green tea has caused limitations. Cocoa tea (Camellia ptilophylla) is a naturally decaffeinated tea plant. To determine whether cocoa tea supplementation results in an improvement in high-fat diet-induced obesity, hyperlipidemia and hepatic steatosis, and whether such effects would be comparable to those of green tea extract, we studied six groups (n = 10) of C57BL/6 mice that were fed with (1) normal chow (N); (2) high-fat diet (21% butterfat + 0.15% cholesterol, wt/wt) (HF); (3) a high-fat diet supplemented with 2% green tea extract (HFLG); (4) a high-fat diet supplemented with 4% green tea extract (HFHG); (5) a high-fat diet supplemented with 2% cocoa tea extract (HFLC); and (6) a high-fat diet supplemented with 4% cocoa tea extract (HFHC). From the results, 2% and 4% dietary cocoa tea supplementation caused a dose-dependent decrease in (a) body weight, (b) fat pad mass, (c) liver weight, (d) total liver lipid, (e) liver triglyceride and cholesterol, and (f) plasma lipids (triglyceride and cholesterol). These data indicate that dietary cocoa tea, being naturally decaffeinated, has a beneficial effect on high-fat diet-induced obesity, hepatomegaly, hepatic steatosis, and elevated plasma lipid levels in mice, which are comparable to green tea. The present findings have provided the proof of concept that dietary cocoa tea might be of therapeutic value and could therefore provide a safer and cost effective option for patients with diet-induced metabolic syndrome.

The hepatoprotective effect of the combination use of Fructus Schisandrae with statin--A preclinical evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE Fructus Schisandrae is traditionally used as a liver-toning Chinese herb. Recent studies suggested Fructus Schisandrae could prevent high-fat diet-induced hepatic steatosis as well as improving anti-oxidative status within the liver, which is a proposed mechanism against statin-induced liver toxicity. AIM The aim of the present study was to determine if the combination use of Atorvastatin (AS) and Fructus Schisandrae aqueous extract (FSE) could (a) exert potent therapeutic effects not only on high-fat diet-induced hyperlipidemia, but also on hepatomegaly (enlarge of liver size) and hepatic steatosis (fatty liver); and (b) reduce side effects caused by intake of statin alone including increased incidence of elevated liver enzymes and liver toxicity in Sprague Dawley rats. MATERIALS AND METHODS We studied 5 groups of Sprague Dawley rats that were given the following treatment for 8 weeks: (i) Normal-chow diet; (ii) High-fat diet (contains 21% fat and 0.15% cholesterol); (iii) High-fat diet (contains 21% fat and 0.15% cholesterol)+0.3% Atorvastatin; (iv) High-fat diet (contains 21% fat and 0.15% cholesterol)+0.45% FSE; (v) High-fat diet (contains 21% fat and 0.15% cholesterol)+0.3% Atorvastatin+0.45% FSE. After 8 weeks of treatment, body weight, adipose tissue and liver mass were measured, and liver and plasma lipid levels were determined to evaluate to effect of FSE with or without AS treatment on diet-induced obesity, hyperlipidemia and hepatic steatosis. Liver enzyme activities, anti-oxidative status and membrane permeability transition were also assessed to determine if FSE could reduce the side effects induced by AS. RESULTS From the results, FSE treatment alone resulted in significant inhibitory effect on diet-induced increase in: (a) body weight; (b) fat pad mass (epididymal, perirenal and inguinal fat); (c) liver weight; (d) total liver lipid; (e) liver triglyceride and cholesterol levels; and (f) plasma lipid levels, suggesting FSE has a potential preventive beneficial effect on weight control and lipid metabolism in Sprague Dawley rats with diet-induced obesity. However, FSE supplementation exerted no further beneficial effect on diet-induced metabolic syndrome when it is combined with AS treatment, compared with rats given AS-treatment alone. At the dose of 0.45%, dietary FSE supplementation resulted in: (a) reduced liver enzymes (ALT and AST) levels; (b) reduced macrophage infiltration (CD68); (c) improved liver glutathione levels (anti-oxidative status); (d) reduced liver reactive oxidative species; (e) a trend to reduce calcium-induced membrane permeability transition within the liver. Most importantly, these improvements induced by FSE treatment were not only observed in the livers of rats given high-fat-diet, but also in high-fat-fed rats with atorvastatin-induced hepatotoxicity. CONCLUSIONS Taken together, these data suggested FSE has a potential beneficial effect on weight control and lipid metabolism in Sprague Dawley rats with diet-induced obesity, and the combination use of FSE with AS could significantly prevent liver toxicity and anti-oxidative status induced by AS alone.

Mechanisms of the dilator action of the Erigerontis Herba on rat aorta

ETHNOPHARMACOLOGICAL RELEVANCE Erigerontis Herba is widely used as a traditional Chinese medicine and is commonly used for neuroprotection and vascular protection. AIM OF STUDY In this study, the vasodilator effects of Erigerontis Herba (DZXX) were investigated using rat isolated aorta rings. MATERIAL AND METHOD The involvement of endothelium in the vasorelaxation was studied by comparing response of endothelium-intact and endothelium-denuded aorta rings which precontracted with U46619. The involvement of K(+) channels was studied by pretreatment of the aorta rings with various K(+) channel inhibitors. The involvement of Ca(2+) channel was studied by incubating aorta rings with Ca(2+)-free solution, primed with U46619 prior to elicit contraction by addition of Ca(2+) solution. RESULTS DZXX (0.2-2mg/ml) induced a concentration-dependent relaxation on U44619-precontracted aorta rings with EC50 of 0.354±0.036mg/ml. Removal of endothelium or pretreatment with a BKCa inhibitor iberiotoxin, KIR inhibitor barium chloride or Kv inhibitor 4-aminopyridine produced no effect on the DZXX-induced vasorelaxation. However, pretreatment with a KATP inhibitor glibenclamide or a non-selective K(+) channel inhibitor tetraethylammonium produced significant inhibition on the DZXX-induced vasorelaxation by 29.9% and 21.3%, respectively. Pretreatment with DZXX (0.4, 1.2 and 2mg/ml) produced a concentration-dependent inhibition on Ca(2+)-induced vasoconstriction. CONCLUSIONS These results suggest that the vasodilator effect of DZXX was endothelium-independent, mediated by decreasing the influx of Ca(2+) by calcium channel inhibition and increasing the influx of K(+) by opening of a KATP channel.

Cerebral vasodilator properties of Danshen and Gegen: A study of their combined efficacy and mechanisms of actions

Danshen and Gegen are two commonly used Chinese herbal medicines for treatment of cardiovascular diseases. The aim of the present study was to elucidate the combination effects of these two herbs on cerebral vascular tone and their underlying mechanisms of actions. Basilar artery rings were obtained from rats and precontracted with U46619. Cumulative administrations of aqueous extracts of Danshen, Gegen, or the two herbs combined (DG; ratio 7:3) produced concentration-dependent relaxation of the artery rings. Statistical analysis on these findings produced a combination index (CI) of 1.041 at ED50, which indicates the two herbs produced additive vasodilator effects when used as a combined decoction. Removal of the endothelium had no effect on the vasodilator properties of Danshen, Gegen, and DG. However, their maximum effects (Imax) were significantly blunted by a KATP channel inhibitor glibenclamide, a non-selective K(+) channel inhibitor tetraethylammonium (TEA), and by a combination of K(+) channel inhibitors (glibenclamide+TEA+iberiotoxin+4-aminopyridine+barium chloride). In addition, Danshen, Gegen, and DG produced augmentation of KATP currents and inhibited Ca(2+) influx in vascular smooth muscle cells isolated from rat basilar arteries. Furthermore, these agents inhibited CaCl2-induced contraction in the artery rings. In conclusion, the present study showed that Danshen and Gegen produced additive vasodilator effects on rat cerebral basilar arteries. These effects were independent of endothelium-derived relaxant factors (EDRF), but required the opening of KATP channels and inhibition of Ca(2+) influx in the vascular smooth muscle cells. It is suspected that the cerebral vasodilator effects of Danshen and Gegen produced either on their own or in combination, can help patients with obstructive cerebrovascular diseases.

The beneficial potential of polyphenol-enriched fraction from Erigerontis Herba on metabolic syndrome

ETHNOPHARMACOLOGICAL RELEVANCE Erigerontis Herba is a Chinese herb that is traditionally used to treat cardiovascular disease. Recent literatures suggested that it could exert beneficial effects on various cardiovascular metabolic risk factors including hypertension and hyperlipidemia in order to exert its cardio-protective effects. AIM Erigerontis Herba contains a variety of flavonoids and polyphenols that are bioactive. The aim of the present study was to investigate the cardio-protective effects of the total polyphenols of Erigerontis Herba (EHP), particularly on the metabolic parameters which could contribute to metabolic syndrome including obesity, hepatic steatosis, hyperlipidemia and hypertension. MATERIALS AND METHODS C57Bl/6 metabolic syndrome mice model was used to determine the effects of EHP on metabolic syndrome. High-fat diet-induced metabolic syndrome in C57Bl/6 mice is an animal model which mimics human metabolic syndrome. The model is achieved by high-fat diet feeding to C57Bl/6 mice for 8 weeks. In our study, the mice were divided into 3 groups and fed for 8 weeks with: 1) normal chow (N); 2) high-fat diet (HF); or 3) high-fat diet supplemented with 2% EHP (HF+EHP). Various parameters such as body weight, adipose tissue weight and liver weight were measured. Liver and plasma lipid levels were also determined. In addition, the effect of EHP on vasodilation in Sprague Dawley rats was also determined using ex vivo aortic ring model. RESULTS Various types of adipose tissues weights were significantly lowered in HF+EHP vs HF mice. Hepatic lipid levels were also significantly decreased by EHP vs HF. For plasma lipid (including TC and TG), EHP exerted no significant effects on plasma lipid levels. To understand the mechanisms as to how EHP regulated lipid metabolism via liver, various hepatic gene expressions were also measured using real-time PCR. The results showed that EHP regulated the expressions of Cyp7α1, CD36 and PPAR-γ. EHP showed significant vasodilative effects in both intact aortas and endothelium-removed aortas. Further mechanistic studies indicated that EHP dilated aorta endothelium-dependently through nitric oxide synthase (NOS) pathway, and endothelium-independently through BKca, Kv and Kir channels. In addition to the vasodilative effects, EHP could also inhibit aorta contraction through Ca(2+) channel. CONCLUSIONS EHP exerted promising effects on diet-induced obesity and hepatic steatosis in C57Bl/6 mice model. It also exerted significant vasodilative effect ex vivo, suggesting the potential of EHP to be developed as a dietary supplement for metabolic syndrome.

Gene Expression Profiling on the Molecular Action of Danshen-Gegen Formula in a Randomized Placebo-Controlled Trial of Postmenopausal Women with Hypercholesterolemia

The Danshen-Gegen formula (DG) is a traditional Chinese herbal formula which has long been used to treat cardiovascular disease. DG was found to be a cardiovascular tonic in our recent research. However, a comprehensive investigation of the molecular mechanism of DG in cardiovascular disease has not been performed. The aim of this study was to clarify the transcriptional profiling of genes modulated by DG on postmenopausal women by using DNAmicroarray technology. We obtained 29 whole blood samples both from DG-treated and placebo-treated subjects. Blood lipid profile and intima-media thickness (IMT) were measured. Affymetrix GeneChip was used to identify differentially expressed genes (DEGs), followed by validation by the real-time PCR method. The results showed that DG-treated group has a significant improvement in IMT and lipid profile as compared to placebo-treated group. For the genomic study, the DG-treated group has a higher number of DEGs identified as compared to the placebo-treated group. Two important biological processes of “regulation of systemic arterial blood pressure by hormone” and “regulation of smooth muscle proliferation” have been identified by GePS in the DG-treated group. No significant biological process and cellular components were identified in the placebo-treated group. This genomic study on the molecular action of DG in postmenopausal women gathered sufficient molecular targets and pathways to reveal that DG could improve neointima thickening and hypertension.

Evaluating Efficacy and Safety of Combination Medication of Atorvastatin and a Herbal Formula Containing Salvia miltiorrhiza and Pueraria lobata on Hyperlipidemia

Despite being a potent hypolipidemic drug, atorvastatin (AS) possesses certain adverse effects. Using AS and an herbal formula (Danshen and Gegen, DG) in combination may achieve potentiated hypolipidemic effects and also reduce its adverse effects. Hence, this study aimed to investigate the efficacy and safety of an AS and DG combination on high‐fat diet‐induced hyperlipidemia. Treatment outcomes were assessed by measuring parameters including body weight, adipose tissue, liver, total cholesterol, triglyceride, and low‐density and high‐density lipoprotein cholesterol. Measurements of adverse effects were achieved by determining aspartate aminotransferase (AST), alanine transaminase (ALT), and creatine kinase (CK). Danshen and Gegen, as well as AS alone, reduced body weight, adipose tissue, liver weight, liver fat vacuoles, total liver lipids, total cholesterol, triglyceride, low‐density lipoprotein cholesterol, and high‐density lipoprotein cholesterol in high‐fat diet‐fed mice but increased AST, ALT, and CK. A combination of AS and DG was able to enhance reduced effects on the aforementioned parameters in relation to hyperlipidemia over AS or DG alone. It also reduced the elevation of AST, ALT, and CK induced than by AS or DG alone. Results demonstrated that an AS and DG combination resulted in stronger hypolipidemic effects than with AS or DG alone. Additionally, DG might attenuate adverse effects of AS on the liver and skeletal muscle. Copyright