叶下珠高效液相色谱指纹图谱研究

目的:建立叶下珠药材的高效液相色谱指纹图谱.方法:采用反相高效液相色谱法,以柯里拉京为内参比峰.色谱条件为Kromasil C18柱(4.6 mm × 250 mm,5μm),流动相乙腈-体积分数为0.1％的磷酸水,梯度洗脱,流速1.0 mL·min-1,柱温30℃,检测波长280 nm.采用SPSS 16.0软件进行系统聚类分析;采用国家药典委员会颁布的中药色谱指纹图谱相似度评价系统(2009版)进行相似度评价.结果:在色谱指纹图谱中,确立了21个共有峰,指认了其中6个色谱峰,系统聚类分析将14批叶下珠药材分为两类,建立了叶下珠药材的共有模式,在14批叶下珠药材中有12批样品的指纹图谱相似度在0.90以上.结论:该方法简便、准确、重复性好,为评价叶下珠药材的质量提供了依据.     

纤维素酶辅助提取叶下珠黄酮的工艺优选

目的:优选纤维素酶辅助提取叶下珠黄酮提取工艺条件,为叶下珠的高效利用提供依据。方法:采用单因素试验研究料液比、提取温度、提取时间和酶浓度对叶下珠黄酮提取得率的影响,通过正交试验优选纤维素酶辅助提取叶下珠黄酮的工艺条件。结果:纤维素酶辅助提取叶下珠黄酮的最佳工艺条件为酶质量浓度4.25 g.L-1,提取温度40℃,料液比1∶30,提取时间1.5 h。在此条件下,黄酮的提取得率为1.51%。结论:采用纤维素酶辅助提取叶下珠黄酮,提取效果较好,该方法具有较好的应用前景。     

内部沸腾法提取叶下珠没食子酸

目的:优选内部沸腾法提取叶下珠没食子酸工艺条件.方法:用少量乙醇溶液为解吸剂润湿叶下珠粉末,使没食子酸充分解吸,加一定量热提取剂,使渗透到叶下珠组织内部的乙醇沸腾,强化提取过程.选取乙醇体积分数、解吸时间、乙醇用量、提取时间和提取温度等5个因素,4水平进行正交试验,采用高效液相色谱法测定没食子酸含量.结果:内部沸腾法提取叶下珠没食子酸的最佳工艺条件为1.6倍量60％乙醇解吸30 min,80℃提取15 min.结论:在最佳提取工艺条件下,叶下珠没食子酸提取得率可达0.970％,该法有较好的应用前景.     

余甘子对肿瘤细胞抑制作用及免疫调节的研究

目的 :运用中药血清药理学的研究方法,研究余甘子对S180荷瘤小鼠肿瘤存活率、主要免疫器官及红细胞免疫调节能力的影响。 方法 :余甘子采用鲜果榨汁经冷冻干燥保存。建立小鼠肿瘤模型,模型小鼠随机分组,小鼠末次给药后摘眼球取血,分离红细胞、淋巴细胞,进行肿瘤红细胞淋巴细胞混合花环实验;观察不同剂量给药组对小鼠肿瘤抑制率及脾、胸腺指数的影响。 结果 :与模型对照组比较,余甘子大剂量组红细胞促淋巴细胞黏附肿瘤细胞能力及抑制肿瘤存活率具极显著差异性;与阳性对照组比较,各剂量组对免疫器官的影响均具显著差异性。 结论 :余甘子在抑制肿瘤生长的同时,对免疫器官有极好的保护作用。     

In vitro and in vivo anti-hepatitis B virus activities of the lignan niranthin isolated from Phyllanthus niruri L.

Ethnopharmacological relevance

Niranthin is a lignan isolated from Phyllanthus niruri L. This plant has long been used in folk medicine for liver protection and antihepatitis B in many Asian countries. This study was designed to evaluate the anti-hepatitis B virus activity of niranthin using HepG2.2.15 cells and duck hepatitis B virus (DHBV) infected ducks as in vitro and in vivo models.

Materials and methods

Niranthin was isolated from Phyllanthus niruri L. (Euphorbiaceae) by extraction and chromatographic procedures and the anti-hepatitis B virus activity was evaluated both in vitro and in vivo. The human HBV-transfected liver cell line HepG2.2.15 was used in vitro assay. And the in vivo anti-hepatitis B virus activity was evaluated on the expression of HBV replication, HBsAg, HBeAg, ALT and AST on day 0, 7, 14, 17 after niranthin was dosed intragastricly (i.g.) once a day for 14 days at the dosages of 25, 50 and 100 mg/kg/day in the duck hepatitis B virus (DHBV) infected ducks.

Results

In the human HBV-transfected liver cell line HepG2.2.15, the secretion of HBsAg and HBeAg were significantly decreased after treatment with niranthin for 144 h, with IC₅₀ values for HBsAg of 15.6 µM, IC₅₀ values for HBeAg of 25.1 µM. In DHBV-infected ducklings, niranthin significantly reduced the serum DHBV DNA, HBsAg, HBeAg, ALT and AST. Furthermore, analysis of the liver pathological changes confirmed the hepatoprotective effect of niranthin.

Conclusion

The experimental data demonstrated that niranthin exhibits anti-hepatitis B virus activity both in vitro and in vivo.     

Effect of Rhaphidophora korthalsii methanol extract on human peripheral blood mononuclear cell (PBMC) proliferation and cytolytic activity toward HepG2

The study of bioactivity of natural product is one of the major researches for drug discovery. The aim of this finding was to study the proliferation effect of Rhaphidophora korthalsii methanol extract on human PBMC and subsequently the cytotoxic effect of activated PBMC toward HepG2 human hepatocellular carcinoma. In this present study, MTT assay, cell cycle study and Annexin 5 binding assay were used to study the immunomodulatory and cytotoxic effects. In vitro cytotoxic screening of Rhaphidophora korthalsii methanol extract showed that the extract was non-toxic against hepatocellular carcinoma (HepG2). In contrast, the extract was able to stimulate the proliferation of human PBMC at 48 h and 72 h in MTT assay and cell cycle progress study. The application of immunomodulator in tumor research was studied by using MTT microcytotoxicity assay and flow cytometric Annexin V. Results indicated that pre-treated PBMC with Rhaphidophora korthalsii methanol extract induced the highest cytotoxicity (44.87 ± 6.06% for MTT microcytotoxicity assay and 51.51 ± 3.85% for Annexin V) toward HepG2. This finding demonstrates that Rhaphidophora korthalsii methanol extract are potent to stimulate the cytotoxic effect of immune cells toward HepG2.     

Antioxidant and hepatoprotective activity of Fagonia schweinfurthii (Hadidi) Hadidi extract in carbon tetrachloride induced hepatotoxicity in HepG2 cell line and rats

Ethnopharmacological relevance

The whole plant of Fagonia schweinfurthii (Hadidi) Hadidi (Family: Zygophyllaceae) is used in variety of diseases including hepatic ailments in deserts and dry areas of India.

Aim of the study

To evaluate antioxidant and hepatoprotective activity of ethanolic extract from Fagonia schweinfurthii (Hadidi) Hadidi (FSEE) in carbon tetrachloride (CCl₄) induced hepatotoxicity in HepG2 cell line and rats.

Materials and methods

In vitro antioxidant activity was determined by DPPH, ABTS radicals and hydrogen peroxide methods. In vitro cytotoxicity and hepatoprotective potential of FSEE were evaluated using HepG2 cells. Based on the cytotoxicity assay, FSEE (50, 100, 200 µg/ml) was assessed for hepatoprotective potential against CCl₄ induced toxicity in HepG2 cell line by monitoring cell viability, aspartate aminotransferase (AST), alanine aminotransaminase (ALT), lactate dehydrogenase (LDH) leakage, lipid peroxidation (LPO) and glutathione level (GSH). Further, in vivo hepatoprotective activity of FSEE was evaluated against CCl₄ induced hepatotoxicity in male Wistar albino rats. Rats were pre-treated with FSEE (200 mg, 400 mg kg⁻¹ day⁻¹ p.o.) for 7 days followed by a single dose of CCl₄ (1.0 ml/kg, i.p.) on 8th day. Silymarin was used as positive control. After 24 h of CCl₄ administration, various biochemical parameters like aspartate aminotransferase (AST), alanine aminotransaminase (ALT), alkaline phosphatase (ALP), total bilirubin (TB) and total protein (TP) levels were estimated in serum. The antioxidant parameters like superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione (GSH) content and malondialdehyde (MDA) level in the liver homogenate were determined. Histopathological changes in the liver of different groups were also studied.

Results

The FSEE possessed strong antioxidant activity in vitro. The results indicated that CCl₄ treatment caused a significant decrease in cell viability. The CCl₄-induced changes in the HepG2 cells were significantly ameliorated by treatment of the FSEE. FSEE significantly prevented CCl₄ induced elevation of AST, ALT, ALP, TB, and CCl₄ induced decrease in total protein in rats. FSEE treated rat liver anti-oxidant parameters (SOD, CAT, MDA and GSH,) were significantly antagonized for the pro-oxidant effect of CCl₄. Histopathological studies also supported the protective effect of FSEE.

Conclusion

The results of this study revealed that FSEE has significant hepatoprotective activity. This effect may be due to the ability of the extract to inhibit lipid peroxidation and increase in the anti-oxidant enzymatic activity.     

Antidiabetic and antioxidant effect of various fractions of Phyllanthus simplex in alloxan diabetic rats

Aim of the study

To evaluate the antidiabetic and antioxidant effects of various fractions of Phyllanthus simplex on alloxan induced diabetes in rats.

Materials and methods

Hypoglycemic effect of Phyllanthus simplex fractions was evaluated in normal and diabetic rats. Diabetes was induced by intraperitoneal injection of alloxan monohydrate (120 mg/kg). Normal and diabetic rats were divided into different groups (six rats each group) and orally administered with petroleum ether (P.E.) (200 and 400 mg/kg), ethyl acetate (EtOAc) (100 and 200 mg/kg), methanol (125 and 250 mg/kg), water fraction (150 and 300 mg/kg) and glibenclamide (10 mg/kg) for 21 days. Blood samples were collected from overnight fasted normal rats on day 21, from overnight fasted diabetic rats at 7, 14 and 21 days of treatment and analyzed for blood glucose level. On day 22 blood samples were collected from diabetic rats to estimate biochemical parameters, rats were sacrificed by single stunning and tissues were excised to measure their antioxidant and glycogen status.

Results

In the normoglycemic rats, MeOH (125 and 250 mg/kg) and aqueous fractions (150 and 300 mg/kg) showed a significant (P < 0.05) hypoglycemic effect on day 21. In diabetic control rats, MeOH (125 and 250 mg/kg) and aqueous fractions (150 and 300 mg/kg) showed significant antihyperglycemic effect (P < 0.001). The active fractions (MeOH and aqueous) of Phyllanthus simplex also increased the body weight of diabetic rats significantly compared to the control group. The active fractions were able to normalize the marked alterations in antioxidant enzymes and antioxidant parameters levels in liver and kidney. Treatment with the active fractions also normalized the diabetic induced hyperlipidemia and liver glycogen.

Conclusions

These results demonstrate the antidiabetic and antioxidant potential of fractions of Phyllanthus simplex and suggests that the plant may have therapeutic value in diabetes and related complications.     

Six weeks oral gavage of a Phyllanthus acidus leaf water extract decreased visceral fat,the serum lipid profile and liver lipid accumulation in middle-aged male rats

Ethnopharmacological relevance

Advancing age is associated with an increased accumulation of visceral fat and liver lipid which is then responsible for an age-related risk for cardiovascular disease. Looking after ourselves well with suitable micronutrients could prevent disease or prolong our healthy cardiovascular functions. In Thai traditional medicine, leaves of Phyllanthus acidus (PA) have been used for many purposes including as an antihypertensive agent and to provide relief from a headache caused by hypertension. We aimed to investigate the effects of a chronic oral administration of PA extracts to middle-aged (12–14 months) rats on their body weight, food intake, body fats, liver and kidney functions, fasting blood glucose and lipid profiles, liver lipid accumulation and on blood pressure.

Materials and methods

Three different kinds of PA extracts were used: (1) a PA water extract, (2) a heated PA water extract, and (3) an n-butanol fraction of the PA water extract, prepared from fresh leaves of Phyllanthus acidus. The rats were orally gavaged with the three PA extracts at 1.0 g/kg body weight or, as a control, with distilled water once a day for 6 weeks. Fasting blood sugar, lipid profile and ALP, SGOT, SGPT, BUN and creatinine levels were measured by enzymatic methods. Liver lipid accumulation was measured using oil red O staining on fresh thin cryostat liver tissue sections. The animal basal blood pressure and heart rate were measured in anesthetized rats via a common carotid artery using a polygraph.

Results

Results showed that after 6 weeks of treatment using gavaged heated PA extract and PA n-butanol extract there were no changes in any of the parameters studied. However, the initial PA water extract caused a slight decrease in the animal body weight with no change in food intake. No changes were observed in the liver and kidney functions (serum ALP, SGOT, SGPT, BUN and creatinine did not change), nor did the fasting blood sugar or triglyceride levels differ significantly. Serum cholesterol, HDL and LDL levels, as well as visceral and subcutaneous adipose tissue and liver lipid accumulation were significantly decreased compared to that of the control group. There were no differences found in the basal systolic and diastolic blood pressure and the basal heart rate between the PA water extract treatment and the control group.

Conclusions

These results indicated that the PA water extract had an effect on lipid metabolisms that resulted in a decrease of the serum lipid profile, visceral and subcutaneous fat, as well as on liver lipid accumulation in middle-aged rats. The active component that is responsible for these effects is likely to be a water soluble substance(s) and is heat labile. As a consequence of these beneficial effects of the PA water extract, it would be a good choice for further development for use as a nutraceutical or health product to prevent and/or to slow down the development of obesity and/or cardiovascular disease.     

Anti-hyperalgesic activity of corilagin,a tannin isolated from Phyllanthus niruri L. (Euphorbiaceae)

Ethnopharmacological relevance

Corilagin (β-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-d-glucose) is a tannin isolated from Phyllanthus niruri (Euphorbiaceae). This plant is well known for their therapeutic purposes to treat several diseases associated with dolorous process and are used in several ethno-medicines in tropical and subtropical countries.

Aim of the study

This study was designed to evaluate the anti-hyperalgesic activity of corilagin using chemically and thermally based nociception models in mice.

Materials and methods

Corilagin was isolated from Phyllanthus niruri (Euphorbiaceae) by extraction and chromatographic procedures and the anti-hyperalgesic activity was evaluated by using writhing, formalin, capsaicin, glutamate and hot plate tests in mice.

Results

Corilagin presented activity in acetic acid model with the ID₅₀ calculated value of 6.46 (3.09–13.51) being about 20.6 fold more potent than acetylsalicylic acid. It also exhibited activity against the first phase of formalin test with ID₅₀ value of 18.38 (15.15–22.59) μmol/kg. In the capsaicin and glutamate models, corilagin demonstrated significant activity at the 3 mg/kg.

Conclusion

The experimental data demonstrated that corilagin exhibits anti-hyperalgesic activity that may be due to interaction with the glutamatergic system.     

Apoptotic activity of ethanol extract from Styrax Japonica Siebold et al Zuccarini in HepG2 cells

Ethnopharmacological relevance

Styrax japonica Siebold et al Zuccarini (SJSZ) has been used to heal inflammation and bronchitis as an herbal plant in Korea.

Aims of the study

The purpose of the present study is to determine whether the ethanol (EtOH) extract of SJSZ induces the programmed cell death (apoptosis) in human hepatoma HepG2 cells.

Materials and methods

It was evaluated cytotoxicity using MTT assay, amount of intracellular reactive oxygen species (iROS) and Ca²⁺ using fluorescence. Activities of apoptotic relevant factors [Bid, cytochrome c, caspase-9, -3, and poly-(ADP-ribose) polymerase (PARP)] were measured by Western blot.

Results

The results in this study indicated that ethanol extract of SJSZ (75 μg/ml) stimulates to increase amount of iROS, Ca²⁺, and the apoptotic relevant factors [Bid, cytochrome c, caspase-9, -3, and poly-(ADP-ribose) polymerase (PARP) in the HepG2 cells.

Conclusion

The results in this study indicated that ethanol extract of SJSZ (75 μg/ml) induces programmed cell death (apoptosis) in the HepG2 cells. Therefore, we speculate that ethanol extract of SJSZ could be used for healing of hepatocarcinoma as one of chemotherapeutic agents.     

Comparative in vitro bioactivities of tea extracts from six species of Ardisia and their effect on growth inhibition of HepG2 cells

Aim of the study

Ardisia species, notably A. compressa, are used in some regions of the world as food or in traditional medicine for prevention and treatment of certain health conditions including liver disease. We investigated the chemical composition and relative anticancer potential of six Ardisia species [A. japonica (AJ), A. escallonioides (AES), A. mamillata (AM), A. compressa (AC), A. crenata (ACR), and A. elliptica (AE)].

Materials and methods

Antioxidant capacity, DNA human topoisomerase II catalytic inhibition, and cytotoxicity on human liver cancer cells (HepG2) were determined in vitro in tea extracts of the 6 Ardisia species evaluated. Selected pure phenolic compounds present in Ardisia species were also evaluated.

Results

AC showed the highest topoisomerase II catalytic inhibition (IC₅₀ = 12 μg/ml) and cytotoxicity (IC₅₀ = 117 μg/ml) against HepG2 cells, followed by ACR and AJ. Total polyphenols ranged from 21 to 72 mg equivalents of gallic acid (GA)/g solid extract (SE). LC–MS analysis revealed the presence of GA, quercetin derivatives, ardisenone, ardisiaquinone, ardisianone, bergenin, norbergenin, and embelin. However, neither total polyphenol concentration nor antioxidant capacity correlated with anticancer capacity. Significant HepG2 cytotoxicity was also achieved by bergenin (IC₅₀ = 18 μM) and embelin (IC₅₀ = 120 μM). AC, bergenin, embelin, and quercetin showed a tendency to accumulate cells in the G1 phase and reduced G2/M leading to apoptosis.

Conclusions

Although the mechanism is not entirely clear, AC, ACR, and AJ are the Ardisia species with the greatest anticancer potential against liver cancer cells in vitro and deserve further investigation.     

Antiproliferative and apoptosis inducing activity of Markhamia tomentosa leaf extract on HeLa cells

Ethnopharmacological relevance

Markhamia tomentosa (Benth) K. Schum ex. Engl. (Bignoniaceae), a tree widely dispersed in West Tropical Africa, is used traditionally to treat various diseases as it possesses antimicrobial, antioxidant, analgesic, anticancer and anti-inflammatory activities.

Materials and methods

This study evaluates the cytotoxic effect and underlying mechanisms of the ethanolic extract of Markhamia tomentosa on HeLa and MCF-7 cancer cell lines and non-cancerous Vero cell line. Brine shrimp lethality test was used for preliminary screening. Cytotoxicity was determined using the MTT assay and IC₅₀ was calculated. Effect of Markhamia tomentosa on the cell cycle was monitored by flow cytometry and the apoptosis-induction capability confirmed by exposure of phosphatidylserine to the outer leaflet of the plasma membrane. Loss of mitochondrial membrane potential was analysed by flow cytometry using JC-1.

Results

Markhamia tomentosa was toxic to brine shrimps with LD₅₀ of 31.62 µg/ml. Cell viability and growth of HeLa cells was inhibited by the extract with an IC₅₀ of 189.1±1.76 µg/ml at 24 h post treatment. However, no cytotoxic effect was observed in MCF-7 and Vero cell lines. The extract induced cell cycle arrest in HeLa cells in the G₀/G₁ phase resulting in cell death after 24 h exposure. Induction of apoptosis in HeLa cells was substantiated by Annexin V-FITC/PI double staining showing phosphatidylserine translocation and depolarisation of the mitochondrial membrane potential by flow cytometry of JC-1 stained cells.

Conclusion

The ethanolic extract of Markhamia tomentosa induces G₀/G₁ in HeLa cells followed by induction of the intrinsic pathway of apoptosis.     

地黄寡糖改善HepG2细胞胰岛素抵抗的分子机制研究

目的研究地黄寡糖改善HepG2细胞胰岛素抵抗的分子机制。方法运用RT-PCR技术,检测地黄寡糖对胰岛素抵抗HepG2细胞内过氧化物酶体增殖物激活受体α(PPAR-α)、胰岛素受体(IR)、葡萄糖转运蛋白4(GLUT4)和2(GLUT2)基因表达的影响。结果地黄寡糖能够促进胰岛素抵抗HepG2细胞中PPAR-α、IR、GLUT4 mRNA的表达;能够明显降低GLUT2基因mRNA的表达。结论地黄寡糖对HepG2胰岛素抵抗具有明显的改善作用,其机制可能与激活PPAR-α、调节HepG2内IR及GLUT2 mRNA的表达有关。     

天王补心丸中生地黄、玄参、天冬和麦冬水提液对大鼠肝CYP450酶含量及其亚型CYP3A, CYP2E1和CYP1A2活性的影响

目的:研究天王补心丸中滋阴类药味生地黄、玄参、天冬和麦冬水提物对大鼠肝肝药酶(CYP450)含量及对亚型CYP3A,CYP2E1,CYP1A2活性的影响,探讨CYP450在天王补心丸代谢中的作用.方法:大鼠灌胃给予生地黄、玄参、天冬和麦冬水提物7d后取肝脏称重并制备肝微粒体,紫外分光光度法测定肝微粒体细胞色素b5(Cytb5),P450的含量及CYP3A的活性,高效液相法测定CYP2E1和CYP1A2的活性.结果:与空白对照组比较,各给药组大鼠肝指数无显著变化;生地黄组CYP450含量极显著减少(P＜0.01),CYP3A活性极显著增加(P＜0.01),CYP1A2活性显著增加(P＜0.05);玄参组CYP450含量减少(P＜0.05),CYP3A和CYP1A2活性均极显著增加(P＜0.01);天冬组Cytb5含量增加(P＜0.05),CYP2E1活性增加(P＜0.05),CYP1A2活性极显著增加(P＜0.01);麦冬组cytb5,CYP450含量无统计学差异,CYP3A活性增加(P＜0.05),CYP2E1活性增加(P＜0.05),CYP1A2活性极显著增加(P＜0.01).结论:天王补心丸中生地黄、玄参降低大鼠肝脏CYP450酶含量并均诱导CYP3A和CYP1A2活性,天冬诱导CYP2E1和CYP1A2活性,麦冬诱导CYP3A,CYP2E1和CYP1A2活性.由于抑制CYP450活性,减少对其他药代谢,滋阴药组在天王补心丸全方配伍中可增强其他各功能组比如补血养血、宁心安神类药味的作用,为探讨天王补心丸的配伍机制提供了理论基础.     

Mechanisms of antihyperuricemic effect of Phyllanthus niruri and its lignan constituents

Ethnopharmacological relevance

Phyllanthus niruri Linn. (Euphorbiaceae) is used as folk medicine in South America to treat excess uric acid. Our initial study showed that the methanol extract of Phyllanthus niruri and its lignans were able to reverse the plasma uric acid of hyperuricemic animals.

Aim of the study

The study was undertaken to investigate the mechanisms of antihyperuricemic effect of Phyllanthus niruri and its lignan constituents.

Material and methods

The mechanisms were investigated using xanthine oxidase assay and uricosuric studies in potassium oxonate- and uric acid-induced hyperuricemic rats.

Results

Phyllanthus niruri methanol extract exhibited in vitro xanthine oxidase inhibition with an IC₅₀ of 39.39 μg/mL and a moderate in vivo xanthine oxidase inhibitory activity. However, the lignans display poor xanthine oxidase inhibition in vitro and a relatively weak in vivo inhibitory activity at 10 mg/kg. On the other hand, intraperitoneal treatment with Phyllanthus niruri methanol extract showed 1.69 folds increase in urinary uric acid excretion when compared to the hyperuricemic control animals. Likewise, the lignans, phyllanthin, hypophyllanthin and phyltetralin exhibited up to 2.51 and 11.0 folds higher in urinary uric acid excretion and clearance, respectively. The co-administration of pyrazinamide with phyllanthin exhibited a significant suppression of phyllanthin's uricosuric activity resembling that of pyrazinamide with benzbromarone.

Conclusions

The present study showed that the antihyperuricemic effect of Phyllanthus niruri methanol extract may be mainly due to its uricosuric action and partly through xanthine oxidase inhibition, whereas the antihyperuricemic effect of the lignans was attributed to their uricosuric action.     

Anti-tumor activity and relative mechanism of ethanolic extract of Marsdenia tenacissima (Asclepiadaceae) against human hematologic neoplasm in vitro and in vivo

Ethnopharmacological relevance

Marsdenia tenacissima, a traditional Chinese medicinal herb, endemic to Yunnan Province is widely used to treat cough, asthma, expectorant, esophageal cancer, gastric cancer, lung cancer, and hepatocellular carcinoma. The aim of this study was to evaluate in vitro and in vivo anti-hematologic neoplasm potential of the ethanolic extract of this herb (crude ethanolic extract of Marsdenia tenacissima, CME) and by using different assays to elucidate its possible mechanism of action.

Materials and methods

The cytotoxicity of CME on tumor cells and peripheral blood mononuclear cells (PBMCs) was evaluated using MTT and apoptosis assays. We also tested the effect of CME on colony formation inhibition and cell cycle distribution of tumor cells. The protein expressions of Cyclin D1, Bax, Bcl-2, caspase-3 and caspase-9 were detected through Western blotting. In vivo anti-tumor effect was evaluated by measuring tumor volume changes, measuring tumor weight, evaluation of tumor microvessel density (MVD) and TUNEL staining by using immunohistochemistry staining in tumor models of nude mice.

Results

Marsdenia tenacissima ethanolic extract exhibited effects of proliferation inhibition and induction of apoptosis on human hematologic neoplasm tumor cells in vitro, as well as hematologic neoplasm growth in vivo.

Conclusion

This study clearly indicated that the ethanolic extract of Marsdenia tenacissima displayed strong anti-tumor effects against hematologic neoplasm cells and could induce tumor cells apoptosis in vitro and in vivo, and also had a significant anti-angiogenic effect in vivo against tumor cell apoptosis. Its multi-mechanism of action might be associated with the cell cycle (G0/G1) arrest, induction of apoptosis through up-regulation protein expressions of Bax, caspase-9 and caspase-3 genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes, a decrease in tumor microvessel density and an increase of TUNEL-positive cells in vivo. These findings provided the molecular theoretical basis of clinical application.     

藏药余甘子药材及其鞣质部位的指纹图谱评价研究

建立不同产地藏药余甘子药材及其鞣质部位HPLC指纹图谱。采用Diamonsil C₁₈（4.6 mm×250 mm,5 μm）色谱柱,甲醇-0.2%冰醋酸水溶液为流动相梯度洗脱,流速1 mL·min^-1,柱温30 ℃,检测波长260 nm。提取13个色谱峰为药材指纹图谱共有峰,11个色谱峰为鞣质部位指纹图谱共有峰,采用对照品指认了5个主要色谱峰,运用相似度评价软件对所收集的8个不同产地藏药余甘子药材及其鞣质部位进行系统比较与归类,8个余甘子药材相似度在0.763~0.993,鞣质部位相似度在0.903~0.991,通过聚类分析、主成分分析将不同产地的余甘子药材和鞣质部位分别聚为3类。该法重复性好,简便可靠,为不同产地藏药余甘子药材及其鞣质部位质量控制和评价提供依据。     

垂盆草醇提物抑制HepG2细胞STAT-3信号通路诱导细胞凋亡的研究

目的:研究垂盆草醇提物对STAT-3信号通路的影响,及其诱导细胞凋亡的分子机制。方法:MTT法检测垂盆草醇提物对HepG2细胞增殖的影响;流式细胞术分析结合FITC-Annexin V/PI双标记检测对肝癌细胞凋亡的影响,免疫印迹试验检测细胞凋亡Caspase-3,Caspase-9,PARP,P-STAT-3(Tyr705),STAT-3,Bcl-2,Mcl-1相关蛋白表达水平。结果:垂盆草醇提物可明显抑制HepG2细胞的增殖,诱导HepG2细胞凋亡,且抑制作用呈剂量依赖效应。垂盆草醇提物作用后,抑制STAT-3信号转导通路,下调Mcl-1和Bcl-2的表达,引起凋亡相关蛋白Caspase-3,Caspase-9的降解/活化及PARP的降解,并呈剂量依赖效应。结论:垂盆草醇提物通过抑制STAT-3信号转导通路和Mcl-1和Bcl-2的表达,进而抑制HepG2细胞的增殖,诱导其凋亡。     

叶下珠指纹图谱及模式识别研究

目的建立叶下珠HPLC指纹图谱评价体系。方法采用Diamonsil C18(250 mm×4.6 mm,5μm)色谱柱,乙腈-0.2%冰醋酸水溶液为流动相梯度洗脱,检测波长270 nm,体积流量1.0 m L/min。测定9个不同产地叶下珠指纹图谱,采用中药色谱指纹图谱相似度评价系统(2004A版)计算相似度,并用对照品和液质联用技术指认色谱峰。结果建立了叶下珠指纹图谱共有模式,标定了11个共有峰,指认了其中8个主要色谱峰,用聚类分析和主成分分析对指纹图谱进行模式识别。结论将指纹图谱和模式识别结合起来对叶下珠进行质量控制是一种有效的评价方法,为叶下珠的全面质量控制提供了基础。