Isodon eriocalyx and its bioactive component Eriocalyxin B enhance cytotoxic and apoptotic effects of gemcitabine in pancreatic cancer.

BACKGROUND: Pancreatic cancer, associated with poor prognosis and low survival rate, has been the fourth leading cause of cancer-related death in the US. Although gemcitabine (Gem) is the first-line chemotherapeutic drug in the management of pancreatic cancer, the median survival extension is only 1.5 months, indicating unsatisfactory clinical results. Therefore, exploring agents that can enhance the anti-cancer activity of Gem would be an attractive strategy. PURPOSE: Our previous studies have demonstrated that eriocalyxin b (EriB), an ent­kaurane diterpenoid isolated from Isodon eriocalyx (Dunn.) Hara, possesses anti-pancreatic cancer effects, thus acting as a potential therapeutic agent. In this study, we further investigated whether EriB or the ethanol extract of I. eriocalyx (Isodon) could potentiate the cytotoxic activity of Gem in human pancreatic adenocarcinoma cells. In addition, the mechanism associated with their effects was also studied. METHODS: The anti-proliferation effect was assessed by MTT assay and Ki-67 immunostaining. The combination effect (addition, synergism and antagonism) of various agents was calculated by the Calcusyn software (Biosoft), utilizing the T.C. Chou Method. Apoptosis was detected using Annexin V and PI double staining followed by quantitative flow cytometry. Protein expression regulated by various treatments was analyzed by western blotting. RESULTS: The combination index revealed that Gem and EriB (or Isodon extract) had synergistic anti-proliferative effect. Both cellular apoptotic and anti-proliferative effects of Gem were significantly increased after combination with EriB (or Isodon extract). The underlying mechanisms involved in the combination effects were elucidated, which include: (1) increased activation of the caspase cascade; (2) reduction of PDK1 and AKT phosphorylation; (3) induction of JNK phosphorylation by Isodon and Gem combination. CONCLUSION: Gem and EriB (or Isodon extract) taken together in combination regulated PDK1/AKT1/caspase and JNK signaling and promoted apoptosis synergistically, which may contribute to the much increased anti-proliferative activity compared to either agent alone.

Eriocalyxin B-induced apoptosis in pancreatic adenocarcinoma cells through thiol-containing antioxidant systems and downstream signalling pathways.

Thiol-containing antioxidant systems play an important role in regulating cellular redox homeostasis. Several anti-cancer agents act by targeting these systems by inducing the production of reactive oxygen species (ROS). Our earlier studies have shown that Eriocalyxin B (EriB), a diterpenoid isolated from Isodon eriocalyx, possesses anti-pancreatic tumour activities in vitro and in vivo. The present study further demonstrated that only thiol-containing antioxidants, N-acetylcysteine (NAC) or dithiothreitol (DTT), inhibited EriB-induced cytotoxicity and apoptosis. EriB suppressed the glutathione and thioredoxin antioxidant systems, thus increasing the intracellular ROS levels and regulating the MAPK, NFκB pathways. Treatment with EriB depleted the intracellular thiol-containing proteins in CAPAN-2 cells. In vivo studies also showed that EriB treatment (2.5 mg/kg) reduced the pancreatic tumour weights significantly in nude mice with increased superoxide levels. Taken together, our results shed important new light on the molecular mechanisms of EriB acting as an apoptogenic agent and its therapeutic potential for pancreatic cancer.

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.

Mechanisms of the cerebral vasodilator actions of isoflavonoids of Gegen on rat isolated basilar artery.

ETHNOPHARMACOLOGICAL RELEVANCE: Gegen (root of Pueraria lobata) is used in traditional Chinese medicine for treatment of cardiovascular diseases. In this study, the relaxant actions of three of its isoflavonoids; puerarin, daidzein, and daidzin, were investigated on rat-isolated cerebral basilar artery. MATERIALS AND METHODS: Rat basilar artery rings were precontracted with 100 nM U46619. Involvement of endothelium-dependent mechanisms was investigated by mechanical removal of the endothelium and inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX) enzymes. Adenylyl cyclase- and guanylyl cyclase-dependent pathways were investigated using their respective inhibitors 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536) and 1H-[1,2,4]oxadiazolo [4,3-[alpha]]-quinoxalin-1-one (ODQ). K(+) channels were investigated by pretreatment of the artery rings with various K(+) channel inhibitors, and Ca(2+) channels were investigated in artery rings incubated with Ca(2+)-free buffer and primed with 100 nM U46619 for 5 min prior to adding CaCl(2) to elicit contraction. RESULTS: Puerarin, daidzein, and daidzin produced concentration-dependent relaxation of the artery rings with concentration that produced 50% inhibition (IC(50)) of 304 ± 49 µM, 20 ± 7 µM, and 140 ± 21 µM, respectively. Removal of the endothelium produced no change on their vasorelaxant responses except the maximum response (I(max)) to puerarin was inhibited by 28%. The NOS inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 100 µM) also produced 45% inhibition on the puerarin-induced vasorelaxant response, but not the COX inhibitor flurbiprofen (10 µM). SQ22536 (100 µM) and ODQ (100µM) did not affect the vasodilator responses to puerarin, daidzein and daidzin, but glibenclamide (1µM), tetraethylammonium (TEA, 100mM) or a combination of K(+) channel inhibitors (100nM iberiotoxin+1mM 4-aminopyridine+100 µM barium chloride+1 µM glibenclamide+100mM TEA) reduced their I(max). The contractile response to CaCl(2) was attenuated by 61% and 34% in the presence of daidzein and daidzin, respectively, whereas, puerarin did not significantly affect the contraction. CONCLUSIONS: The vasorelaxant action of daidzein and daidzin involved opening of K(+) channels and inhibition of Ca(2+) influx in the vascular smooth muscle cells. There is no evidence supporting involvement of endothelium-derived relaxing factors (EDRFs) in their actions. In contrast, puerarin produced vasodilatation via an endothelium-dependent mechanism involving nitric oxide production and an endothelium-independent pathway mediated by the opening of K(+) channels. The cerebral vasodilator activities of all these three isoflavonoids may be beneficial to patients with obstructive cerebrovascular diseases.

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.

Mechanisms of the relaxant effect of a danshen and gegen formulation on rat isolated cerebral basilar artery.

AIM OF THE STUDY: Danshen (root of Salvia miltiorrhiza) and gegen (root of Pueraria lobata) are two herbs used in traditional Chinese medicine, most commonly for their putative cardioprotective and anti-atherosclerotic effects. In this study, the actions of a danshen and gegen formulation (DG; ratio 7:3) were investigated on rat-isolated cerebral basilar artery. MATERIALS AND METHODS: Rat basilar artery rings were precontracted with 100 nM U46619. Involvement of endothelium-dependent mechanisms was investigated by mechanical removal of the endothelium; K(+) channels were investigated by pretreatment of the artery rings with various K(+) channel inhibitors, and Ca(2+) channels were investigated in artery rings incubated with Ca(2+)-free buffer and primed with 100 nM U46619 for 5 min prior to adding CaCl(2) to elicit contraction. RESULTS: DG produced concentration-dependent relaxation of the artery rings with an IC(50) of 895±121 µg/ml. Mechanical removal of the endothelium or pretreatment with the BK(Ca) channel inhibitor iberiotoxin (100 nM), the K(V) channel inhibitor 4-aminopyridine (1 mM), or the K(IR) channel inhibitor barium chloride (100 µM), all had no effect on the DG-induced response (P>0.05 for all). However, pretreatment with the K(ATP) channel inhibitor glibenclamide (1 µM), the non-selective K(+) channel inhibitor tetraethylammonium (TEA, 100 mM), or a combination of all the K(+) channel inhibitors (iberiotoxin+4-aminopyrindine+barium chloride+glibenclamide+TEA) produced significant inhibition on the DG-induced response (P<0.01 for all); its maximum vasorelaxant effect (Imax) was reduced by 37, 24, and 30%, respectively. Preincubation of the artery rings with DG for 10 min produced concentration-dependent (1, 3 and 7 mg/ml) and total inhibition on the CaCl(2)-induced vasoconstriction. CONCLUSIONS: These findings suggest the vasorelaxant effect of DG on rat basilar artery is independent of endothelium-derived mediators, whereas, inhibition of Ca(2+) influx in the vascular smooth muscle cells is important, and a minor component is mediated by the opening of K(ATP) channels. DG could be a useful cerebroprotective agent in some patients with occlusive cerebrovascular disease.

Pharmacological investigation on the wound healing effects of Radix Rehmanniae in an animal model of diabetic foot ulcer.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Rehmanniae (RR) has a very long history of usage in traditional Chinese medicine and is usually one of the principal herb found in many herbal formulae used in diabetic foot ulcer. AIM OF THE STUDY: RR aqueous extract was investigated for its wound healing effects in a diabetic foot ulcer rat model and its detailed mechanism of actions. MATERIALS AND METHODS: A previously established diabetic foot ulcer rat model was used to assess the effect of RR extract on wound area reduction, tissue regeneration and angiogenesis. Carrageenan-induced inflammation rat model was used for inflammation study; and diabetic control was evaluated using a neonatal streptozotocin-induced diabetic rat model. RESULTS: In the RR treated group, a trend of reduction of the wound area was observed from days 8 to 18 and a significant difference (as compared with control group) was found on day 8. The ulcer healing effect of RR extract was further supported by better developed scars and epithelialization as well as good formation of capillaries with enhanced VEGF expression. Carrageenan-induced inflammation was also significantly alleviated with RR extract. CONCLUSIONS: Our results demonstrated for the first time that Radix Rehmanniae was effective in promoting diabetic foot ulcer healing in rats through the processes of tissue regeneration, angiogenesis and inflammation control, but not glycemia control. The present study provided scientific basis to support the traditional use of Radix Rehmanniae in diabetic foot ulcer.

Radix Astragali and Radix Rehmanniae, the principal components of two antidiabetic foot ulcer herbal formulae, elicit viability-promoting effects on primary fibroblasts cultured from diabetic foot ulcer tissues.

Over 194 million people suffer from diabetes worldwide. The improper control of diabetes may result in diabetic foot ulcer or even amputation. Herbal medicine provides a means for treating diabetic foot ulcers for a large population in developing countries. The wound healing-enhancing activities of the principal herbs, Radix Astragali (RA) and Radix Rehmanniae (RR) in two clinically efficacious Chinese herbal formulae were studied in primary fibroblasts from diabetic foot ulcer patients. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that RA and RR significantly enhanced the viability of fibroblasts isolated from foot ulcers of diabetic patients, even from those with no response to insulin treatment. The results in this study indicate that fibroblast viability enhancement effects of RA and RR likely underlie the healing effects of F1 and F2 in diabetic foot ulcers.

In vitro antidiabetic activities of five medicinal herbs used in Chinese medicinal formulae.

Fructus Corni, Fructus Schisandrae Chinensis, Poria, Rhizoma Alismatis and Rhizoma Dioscoreae are commonly used in traditional Chinese medicine for diabetes treatment. They are also the component herbs of an antidiabetic foot ulcer formula with demonstrated clinical efficacy. Although some of these herbal extracts were previously shown to possess in vivo antidiabetic effects (i.e. lowering blood glucose levels), the underlying mechanisms remain elusive. The objective of this study is to investigate the possible antidiabetic mechanisms of these individual herbs, using a systematic study platform which includes four in vitro tissue models: glucose absorption into intestinal brush border membrane vesicles (BBMV), gluconeogenesis by rat hepatoma cell line H4IIE, glucose uptake by human skin fibroblasts cell line Hs68 and mouse adipocytes 3T3-L1. All tested herbs showed significant in vitro antidiabetic effects in at least two models. Fructus Schisandrae Chinensis, Poria, Rhizoma Alismatis and Rhizoma Dioscoreae showed significant inhibitory effects in the BBMV glucose uptake assay. All tested herbs showed significant stimulatory effects to the glucose uptake of Hs68 and 3T3-L1 cells, except Poria and Rhizoma Dioscoreae which were not effective to Hs68 and 3T3-L1 respectively. However, none of the tested herbs inhibited hepatic gluconeogenesis. In conclusion, the five herbs exhibited distinct antidiabetic mechanisms in vitro and hence our investigations provided scientific evidence to support the traditional usage of these herbs for diabetic treatment in medicinal formulae.

An in vivo investigation on the wound-healing effect of two medicinal herbs using an animal model with foot ulcer.

BACKGROUND/AIM: Over 190 million people in the world suffer from diabetes mellitus. Diabetics are 25 times more likely to have a leg amputated because of unhealing foot ulcers. Herbal medicine has been used in China to salvage the ulcerated limb. With the aim to study the efficacy of two commonly used herbs for ulcer healing, namely Radix Astragali and Radix Rehmanniae, a good animal model needs to be developed for a proper in vivo investigation. METHODS: Firstly, a diabetic animal model was established by streptozotocin injection. Then standard wounds were created on the feet of the diabetic rats. Digital photographs were taken and analyzed by a novel image analysis software. RESULTS: The average ulcer area in the Radix Rehmanniae treatment group was 11.45 mm2, which was significantly smaller than the 15.12 mm2 in the water treatment group (p = 0.04). Radix Astragali, on the other hand, was found to have no significant effect on ulcer shrinkage. CONCLUSION: Further investigation is needed for the identification of the active principles of Radix Rehmanniae.

Investigation of the effects of Chinese medicine on fibroblast viability: implications in wound healing.

Diabetes mellitus has been a clinical problem for hundreds of years. Over 194 million people suffer from this disease worldwide. Improper control of diabetes may result in diabetic foot ulcer or even amputation. Granulation formation is an important issue essential for ulcer healing. The CRL-7522 fibroblast cell line and primary fibroblasts from a diabetic foot ulcer patient were used to model the wound healing enhancing activities of two clinically efficacious Chinese herbal formulae, Formula 1 (F1) and Formula 2 (F2) and their component herbs. Results showed that the two formulae and four of their component herbs, Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis and Rhizoma Atractylodis Macrocephalae significantly enhanced CRL-7522 cell viability. However, these component herbs showed compromised effects on the viability of primary fibroblasts cultured from the ulcerous tissue of a diabetic patient. Interestingly, F1 and F2 enhanced the viability of primary cultured fibroblasts from the diabetic patient even in the face of insulin resistance. These results further support the previously reported clinical efficacies of the two formulae on healing diabetic foot ulcers.

Pharmacological investigations of the anti-diabetic effect of Cortex Moutan and its active component paeonol.

Cortex Moutan (CM, root bark of Paeonia suffruticosa Andr.) is one of the common herbs found in anti-diabetic traditional Chinese medicine formulae. To study the potential anti-diabetic mechanisms of CM, four in vitro models (intestinal brush border membrane vesicles (BBMV), rat hepatoma cell line H4IIE, human skin fibroblasts cell line Hs68 and mouse adipocytes 3T3-L1) were used. CM showed significant in vitro anti-diabetic effects by inhibiting glucose uptake of BBMV and enhancing glucose uptake into Hs68 and 3T3-L1 cells. Using bioassay-guided fractionation, paeonol was confirmed to be one of the active constituents for inhibiting BBMV glucose uptake. With neonatal-streptozotocin diabetic rats, paeonol (200 and 400mg/kgbody wt.) was found to improve oral glucose tolerance in vivo. To the best of our knowledge, this is the first report on the anti-diabetic effect of paeonol.

Influence of an anti-diabetic foot ulcer formula and its component herbs on tissue and systemic glucose homeostasis.

Complications of diabetes impose major public health burdens worldwide. The positive effect of a Radix Astragali-based herbal preparation on healing diabetic foot ulcers in patients has been reported. Formula 1 is also referred as the 'Herbal drink to strengthen muscle and control swelling'. This formula contains six Chinese medical herbs, including Radix Astragali, Radix Rehmanniae, Rhizoma Smilacis Chinensis, Rhizoma Atractylodis Macrocephalae, Radix Polygoni Multiflori Preparata, and Radix Stephania Tetrandrae. Three of these herbs (Radix Astragali, Radix Rehmanniae, Rhizoma Atractylodis Macrocephalae) are commonly used in different anti-diabetic formulae of Chinese medicine. The objective of the current study is to use an interdisciplinary approach to test the hypothesis that Formula 1 and its components influence tissue and systemic glucose homeostasis. In vitro and in vivo models have been established including: (1) glucose absorption into intestinal brush border membrane vesicles (BBMV); (2) gluconeogenesis by H4IIE hepatoma cells; (3) glucose uptake by 3T3-L1 adipocytes and Hs68 skin fibroblasts; (4) normalization of glycaemic control in a diabetic rat model. The results of in vitro studies indicated that all herbal extracts can modify cellular glucose homeostasis. Since Formula 1 and Rhizoma Smilacis Chinensis extracts demonstrated potent effects on modifying glucose homeostasis in multiple tissues in vitro, they were further studied for their anti-diabetic activities in vivo using a streptozotocin (STZ)-induced diabetic rat model. The results showed that Formula 1 and Rhizoma Smilacis Chinensis extracts did not significantly improve oral glucose tolerance or basal glycaemia in diabetic rats. In conclusion, the anti-diabetic foot ulcer Formula 1 contains ingredients active in modifying tissue glucose homeostasis in vitro but these biological activities could not be associated with improved glycaemic control of diabetes in vivo.

Immunomodulatory effects of a traditional Chinese medicine with potential antiviral activity: a self-control study.

Traditional Chinese medicine (TCM) has been used for prevention and treatment of severe acute respiratory syndrome (SARS) in Hong Kong during the outbreak in spring 2003. We investigated the immunomodulating effects of an innovative TCM regimen derived from two herbal formulas (Sang Ju Yin and Yu Ping Feng San) for treating febrile diseases. Thirty-seven healthy volunteers were given the oral TCM regimen daily for 14 days. Peripheral venous blood samples were taken on days 0, 15 and 29 for hematology, biochemistry and immunology tests, including the measurement of blood lymphocyte subsets and plasma T-helper lymphocyte types 1 and 2 cytokines and receptor. After 3 months, 23 of the volunteers participated in a control study without TCM treatment for the same time course of blood tests. Two volunteers withdrew on day 2, due to headache and dizziness. All others remained well without any side effects. No participants showed significant changes in their blood test results, except that the T-lymphocyte CD4/CD8 ratio increased significantly from 1.31 +/- 0.50 (mean +/- SD) on day 0 to 1.41 +/- 0.63 on day 15 (p < 0.02), and reduced to 1.32 +/- 0.47 on day 29 (p < 0.05). In the control study, there were no changes in the CD4/CD8 ratio. The transient increase in CD4/CD8 ratio was likely due to the TCM intake. We postulate that the administration of the innovative TCM may have beneficial immunomodulatory effects for preventing viral infections including SARS.

Cytotoxic activities of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma cells by induction of apoptosis.

Coriolus versicolor (CV), also known as Yunzhi, is one of the commonly used Chinese medicinal herbs. Although recent studies have demonstrated its antitumour activities on cancer cells in vitro and in vivo, the exact mechanism is not fully elucidated. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized aqueous ethanol extract prepared from Coriolus versicolor on a B-cell lymphoma (Raji) and two human promyelocytic leukemia (HL-60, NB-4) cell lines using a MTT cytotoxicity assay, and to test whether the mechanism involves induction of apoptosis. Cell death ELISA was employed to quantify the nucleosome production resulting from nuclear DNA fragmentation during apoptosis. The present results demonstrated that CV extract at 50 to 800 microg/ml dose-dependently suppressed the proliferation of Raji, NB-4, and HL-60 cells by more than 90% (p < 0.01), with ascending order of IC50 values: HL-60 (147.3 +/- 15.2 microg/ml), Raji (253.8 +/- 60.7 microg/ml) and NB-4 (269.3 +/- 12.4 microg/ml). The extract however did not exert any significant cytotoxic effect on normal liver cell line WRL (IC50 > 800 microg/ml) when compared with a chemotherapeutic anticancer drug, mitomycin C (MMC), confirming the tumour-selective cytotoxicity. Nucleosome productions in HL-60, NB-4 and Raji cells were significantly increased by 3.6-, 3.6- and 5.6-fold respectively upon the treatment of CV extract, while no significant nucleosome production was detected in extract-treated WRL cells. The CV extract was found to selectively and dose-dependently inhibit the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.