雷公藤多甙对抗Thy1.1抗体肾炎蛋白尿和足细胞裂隙膜相关分子表达的影响

目的 观察雷公藤多甙（multi—glycoside of Tripterygium wilfordii Hook．f．,GTW）对抗Thy1．1抗体肾炎蛋白尿和足细胞裂隙膜相关分子（nephrin、podocin）表达的影响。方法 经尾静脉一次性注射500μg单克隆抗体（monoclonal antibody,mAb）1—22—3建立大鼠抗Thy1．1抗体肾炎模型。14只大鼠随机分为GTW干预组（简称GTW组）和安慰剂干预组（简称造模组）。实验1中,在注射抗体前3天,经灌胃给予GTW[75mg／（kg·d）]或安慰剂,直至造模后第14天,处死大鼠;实验2中,在注射抗体同时,经灌胃给予GTW[100mg／（kg·d）]或安慰剂,直至造模后第7天,处死大鼠。分别观察14天内或7天内模型鼠24h尿蛋白排泄量（urinary protein excretion,Upro）动态变化。在第14天或第7天,处死大鼠后,取出肾脏,分别观察肾小球形态学变化、肾小球内巨噬细胞浸润、肾小球内nephrin、podocin及其核酸表达。结果 实验1中,GTW抑制抗Thy1．1抗体肾炎蛋白尿和系膜损伤,在第14天,对肾小球内nephrin、podocin免疫荧光染色强度无影响;实验2中,GTW改善抗Thy1．1抗体肾炎蛋白尿、系膜损伤、肾小球内巨噬细胞浸润,而且,在第7天,显著增强肾小球内nephrin、podocin及其核酸表达。结论 在抗Thy1．1抗体肾炎中,nephrin、podocin表达的减弱可能会促进系膜损伤和蛋白尿。GTW改善系膜损伤和蛋白尿的作用,很可能是通过增强nephrin、podocin表达来实现的。     

抗Thy1.1抗体诱导的肾炎模型及其在中药研究中的应用

抗Thy1.1单克隆抗体诱导的抗Thy1.1抗体肾炎模型广泛应用于人类系膜增殖性肾炎的研究领域。该模型的病理特征包括补体依赖性系膜溶解、肾小球内炎症细胞浸润、显著的蛋白尿,以及急性或进展性系膜损伤。文章综述了经静脉注射单克隆抗体1-22-3诱导的可逆性抗Thy1.1抗体肾炎模型和不可逆性抗Thy1.1抗体肾炎模型的病理特征；阐述了肾小球内炎症细胞浸润和各种损伤性细胞因子的变化规律；分析了包括系膜细胞增殖和细胞外基质沉积在内的复杂的系膜损伤病变过程。运用该模型,可以说明雷公藤多苷、柴苓汤等中药对系膜损伤和蛋白尿的药理作用,并且,在分子水平阐明这些药物对各种损伤因子的作用机制。     

Low-dose of multi-glycoside of Tripterygium wilfordii Hook. f., a natural regulator of TGF-β1/Smad signaling activity improves adriamycin-induced glomerulosclerosis in vivo

Ethnopharmacological relevance

Transforming growth factor (TGF)-β1/Smad signaling pathway plays a critical role in the prolonged glomerulosclerosis (GS), which is an important determinant during the progression in chronic kidney disease (CKD). For recent 30 years, multi-glycoside of Tripterygium wilfordii Hook. f. (GTW), an extract from Chinese herbal medicine has been proved clinically effective in improving GS in CKD in China. However, therapeutic mechanisms involved in vivo are still unclear. In this study, we aimed to explain the dose-effects and molecular mechanisms of GTW on GS by regulating TGF-β1/Smad signaling activity in adriamycin (ADR)-induced nephropathy (ADRN).

Materials and methods

Rats with ADRN, created by unilateral nephrectomy and twice adriamycin injections (ADR, 4 mg/kg and 2 mg/kg) within 4 weeks, were divided into four groups, the Sham group, the Vehicle group, the low-dose GTW-treated group, and the high-dose GTW-treated group, and that, sacrificed at the end of the 6th week after administration. Proteinuria, blood biochemical parameters, glomerulosclerotic morphological makers, podocyte shape, and nephrin expression were examined, respectively. Protein expressions of key signaling molecules in TGF-β1/Smad pathway, such as TGF-β1, Smad3, phosphorylated-Smad2/3 (p-Smad2/3), and Smad7, were also evaluated individually.

Results

The results indicated that the characterizations of ADRN involved the typical prolonged GS, a small amount of abnormal proteinuria, and the failing renal function; TGF-β1/Smad signaling molecules, especially Smad3, p-Smad2/3, and Smad7 were activated in vivo, accompanied by the exasperation of glomerulosclerotic lesion; GTW at high-dose (100 mg/kg) and low-dose (50 mg/kg) could slightly ameliorate the prolonged GS and nephrin expression, furthermore, the anti-proliferative action of GTW at high-dose was superior to that at low-dose, but caused the significant liver injury; in ADRN model rats, protein expressions of TGF-β1, p-Smad2/3, and Smad7 in the kidneys could be regulated with the treatment of GTW at low-dose.

Conclusion

This study farther demonstrated that the low-dose of GTW, as a natural regulator in vivo, could effectively and safely ameliorate the prolonged GS in FSGS model, via the potential molecular mechanisms involving the reduction of ECM components and the suppression of TGF-β1 over-expression, as well as the bidirectional regulation of TGF-β1/Smad signaling activity.     

Qi-Dan Fang ameliorates adriamycin-induced nephrotic syndrome rat model by enhancing renal function and inhibiting podocyte injury

Ethnopharmacological relevance

Nephrotic syndrome (NS) is a clinical syndrome with a variety of causes, mainly characterized by heavy proteinuria. Podocyte injury plays a key role in proteinuria, one of the principal means for the control of NS is to prevent podocyte injury. Qi-Dan Fang consists of two of the most extensively applied herbal remedies among Traditional Chinese Medicine (TCM) (Radix Astragali Mongolici and Radix Salviae Miltiorrhizae, with a weight ratio of 5:1) which are specifically used for the treatment of various kidney diseases. In previous studies, we found that Qi-Dan Fang provides improvement to patients with adriamycin-induced nephrotic syndrome by alleviating proteinuria and serum lipid. The aim of this study is to study the efficiency of Qi-Dan Fang on NS model rat with renal dysfunction and podocyte injury, something which has not been carried out yet.

Materials and methods

The rats were divided into Normal, Model, Jin Gui Shen Qi Pill (4.12 g/kg), Qi-Dan Fang (3.09, 6.17 and 12.34 g/kg/d) groups, they were each given a single tail intravenous injection of Adriamycin (6.0 mg/kg) except for the Normal group and were orally administered dosages of Qi-Dian Fang and Jin Gui Shen Qi pills once daily for 7 weeks. Following the treatment, the content of cystation C (CysC), blood urea nitrogen (BUN), serum creatinine (Scr) were measured with an autobiochemical analyser. The pathomorphological changes to the glomeruli, the mRNA expressions of nephrin, podocin, CD2AP genes and p53, bax, bcl-2 proteins expressions were also carried out to probe the effects of Qi-Dan Fang.

Results

(1) Qi-Dan Fang treatment raised the level of CysC in blood serum while lowering the content of BUN and Scr in the adriamycin-induced nephrotic syndrome rat model; (2) Long-term administration of Qi-Dan Fang was able to ameliorate pathomorphological change of glomeruli and repair the organization structure of Glomerulus; (3) Qi-Dan Fang could increase the mRNA expression of nephrin, podocin and CD2AP genes, down-regulate the expression of p53, bax proteins, while increased bcl-2 protein to protect the podocyte and restore Glomerular selective filtration function.

Conclusions

Results of our present studies reveal that Qi-Dan Fang is able to enhance renal function, inhibit podocyte injury to provide improvements to the Adriamycin-induced nephrotic syndrome.     

Zhen-wu-tang, a blended traditional Chinese herbal medicine, ameliorates proteinuria and renal damage of streptozotocin-induced diabetic nephropathy in rats

Ethnopharmacological relevance

Zhen-wu-tang (ZWT) is a blended traditional Chinese medicine specifically used for various kidney diseases.

Aim of the study

The present study is to evaluate the effects of ZWT on diabetic nephropathy and investigate the potential anti-diabetic mechanisms.

Materials and methods

Streptozotocin (STZ)-induced diabetic rats were orally administrated ZWT (80,320 mg/kg b.w.) once a day, for a period of 12 weeks. Body weight, urinary volume, urine protein and blood biochemical parameters were measured every 3 weeks. At the end of the observation period, kidneys were isolated for histology, angiotensin II content determination, real time PCR and Western blot analyses.

Results

ZWT (320 mg/kg b.w.) prevented body weight loss, reduced polyurea, urinary protein excretion, serum creatinine and blood urea nitrogen, although it did not alter the hyperglycemia. It ameliorated glomerular hypertrophy and expansion of the mesangial area, swell and effacement of foot process. It also suppressed the increased expression of angiotensin II, nephrin and podocin.

Conclusions

Data from this study suggest that ZWT possess a protective effect on renal damage of STZ-induced diabetic nephropathy in rats, by suppressing hyperactivity of renal renin-angiotensin system that turns in to modulate renal nephrin and podocin expressions, thereby protecting podocyte from injury.     

Comparison of toxic reaction of Tripterygium wilfordii multiglycoside in normal and adjuvant arthritic rats

Aim of the study

Tripterygium wilfordii multiglycoside (GTW), an authorized Chinese patent drug, is used for treatment of rheumatoid arthritis and other immune disease. This study was to determine whether GTW induced different toxic reactions in adjuvant arthritis rats (AA rats) compared to those in normal rats.

Materials and methods

To prepare arthritic rat model, male Sprague-Dawley (SD) rats were immunized by injecting complete Freund's Adjuvant into right hind footpad. And then, GTW was given to rats intragastrically at dosage of 7 or 105 mg kg⁻¹ day⁻¹ from day 15 to day 28 after immunization. Routine clinical parameters and histopathologic changes of liver, kidney and testis were examined. Metabolic profiling in serum of groups was analyzed by LC-MS. A principal component analysis (PCA) and partial-least-squares discriminate analysis (PLS-DA) were carried out combined with mass spectrometry (MS) data set. All the quantitative data were performed by two-way ANOVA analysis following Student's t-test.

Results and conclusions

Treatment with GTW at both doses could diminish the right and left hind paws swelling. There was slight lipoid degeneration in hepatic tissue of normal rats treated by high dose of GTW, but there were not distinctly pathological changes in hepatic tissue of AA rats treated by GTW. Compared normal rats administered with GTW, no statistically significant difference in the serum alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN) levels were observed. However, the serum aspartate aminotransferase (AST) level was significant decreased in AA rats under exposure GTW compared with normal rats in the same conditions (p < 0.05), which indicated that GTW could offer a different liver toxic reaction in normal and AA rats. The metabolic analysis showed that a clear separation of PCA and PLS-DA score spot in normal rats, but not separation was seen in AA rats perturbed with low dosage GTW. The result indicated low dosage GTW might arouse a general toxic in normal rats but not in AA rats. The biomarker analysis showed that the level of lysophosphatidylcholines (LPCs) was down-regulated, but the level of ursodeoxycholic acid (UDCA) and chenodexycholic acid (CDCA) was up-regulated in AA rats compared with normal rats under exposure GTW. According to pathway analysis of metabolic markers, we conceived that LPC, UDCA and CDCA were the critical intermediates of choline and fatty acid metabolism. And the lipid metabolism was a correlative outcome of GTW induced toxicity in the liver in physiological condition animals. Taken together, GTW could induce different toxic reactions between normal and AA rats, and the lipid metabolism might be part of the mechanism for the hepatic lipidosis or the other liver injury.     

嘌呤霉素肾病模型肾组织微小RNA的差异性表达及雷至胶囊的干预作用

目的:观察嘌呤霉素氨基核苷(puromycin aminonucleoside,PAN)肾病模型鼠肾组织微小RNA(microRNAs,miR-NAs)差异性表达的特征及其与足细胞裂隙膜(slid diaphragm,SD)关键结构分子nephrin,podocin和骨架蛋白synaptopodin表达的关系;阐明雷至胶囊(Leizhi capsule,LZC)在体内通过调控模型鼠肾组织miRNAs差异性表达而改善足细胞nephrin,podocin,synaptopodin表达,减少蛋白尿的机制.方法:将50只雄性Wistar大鼠随机分为空白组(A)、模型组(B)、雷至胶囊组(C,5 mL·kg-1·d-1)、雷公藤多苷组(D,10mL·kg-1 ·d-1)和缬沙坦(E,7.5 mL· kg-1·d-1)组.除A组外,其余各组大鼠一次性经颈静脉注射PAN(100 mg·kg-1)而建立PAN肾病模型.造模后第2天灌胃给药,A,B组大鼠用生理盐水干预,共10 d.造模前及造模后第3,9天称量各组大鼠体重,并检测24h尿蛋白排泄量(urinary protein ration,Upro).造模后第11天,处死全部大鼠,采集血液和肾组织,观察血清生化指标血清白蛋白(albumin,Alb),血清肌酐(serum creatinine,Scr),血清尿素氮(blood urea nitrogen,BUN),肾小球超微结构(足细胞足突形态)以及肾组织dicer酶,nephrin,podocin,synaptopodin表达;同时,借助生物芯片(biochip)技术,分析肾皮质miRNAs差异性表达的特征,并且,借助荧光实时定量聚合酶链反应(Real-time PCR)验证mo-miR-23a,rno-miR-300-3p,rno-miR-24,rno-miR-30c等的差异性表达量.结果:在PAN诱导下,模型鼠出现蛋白尿、肾功能减退、低白蛋白血症、足细胞足突融合;在模型鼠肾组织中,dicer酶影响足细胞nephrin,podocin,synaptopodin表达;上调rno-miR-23a,mo-miR-300-3p表达,下调mo-miR-24,rno-miR-30c表达;差异性表达的miRNAs包括rno-miR-24,rno-miR-30c,rno-miR-23a,rno-miR-300-3p等.雷至胶囊能改善PAN肾病模型鼠的一般情况、蛋白尿、血清BUN、足细胞足突融合;还能减少模型鼠肾组织dicer酶表达,增加足细胞nephrin,podocin和synaptopodin表达;减弱在模型鼠肾组织中上调的rno-miR-23a,rno-miR-300-3p,增强在模型鼠肾组织中下调的rno-miR-24,rno-miR-30c等.结论:PAN在体内通过dicer酶/miRNAs差异性表达途径而影响模型鼠肾组织miRNAs表达,干预足细胞nephrin,podocin,synaptopodin表达,破坏足细胞结构和功能,产生蛋白尿;雷至胶囊可以减少PAN肾病模型鼠蛋白尿,其机制可能是干预dicer酶/miRNAs差异性表达途径,调控足细胞nephrin,podocin,synaptopodin表达,改善足细胞的结构与功能.     

雷公藤多甙对实验性系膜增殖性肾炎蛋白尿及系膜损伤的影响

目的 观察雷公藤多甙（multi-glycoside of tripterygium wilfordii Hook．f．,GTW）在体内对实验性系膜增殖性肾炎蛋白尿及系膜损伤的影响。方法 运用单克隆抗体（monoclonal antibody,mAb）1-22-3建立大鼠可逆性抗Thy1．1抗体肾炎（简称“抗Thy1．1肾炎”）模型,以GTW干预,并设立对照组。比较24h尿蛋白排泄量（urinary protein excretion,Upro）、肾功能（serum creatinine,SCr;blood urea nitrogen,BUN）、血浆总蛋白（total plasma protein,TP）以及肾小球形态学变化,并检测肾组织中增殖因子（platelet-derived growth factor-BB,PDGF-BB;transforming growth factor-β,TGF-β）mRNA表达水平。结果 GTW抑制抗Thy1-1肾炎模型Upro和系膜损伤;抗Thy1．1肾炎模型肾组织中增殖因子（PDGF-BB、TGF-β）mRNA表达水平与正常组比较,分别为其2．84倍与1．64倍,GTW使PDGF-BB mRNA过高表达水平下调33．1％,与对照组比较,差异有显著性（P〈0．05）。结论 GTW可减少系膜增殖性肾炎模型蛋白尿,抑制系膜细胞增殖和细胞外基质沉积;这些作用可能与下调肾组织增殖因子（PDGF-BB）mRNA的表达有关。     

Optimized project of traditional Chinese medicine in treating chronic kidney disease stage 3: a multicenter double-blinded randomized controlled trial

Ethnopharmacological relevance

Stage 3 is the key phase of chronic kidney disease. Traditional Chinese medicine (TCM) has been used for the treatment of chronic kidney disease. But a large sample trial is desirable.

Materials and methods

A total of 578 Chinese patients with primary glomerulonephritis in CKD stage 3 were randomly assigned to three groups: patients received TCM (TCM group), benazepril (Ben group), TCM combined with benazepril (TCM + Ben group). Patients were followed up for 24 weeks. The primary endpoint was the time to the composite of 50% increased of serum creatinine, end stage renal disease or death.

Results

eGFR in the TCM and the TCM + Ben group were improved (week 24 vs. baseline, P < 0.05) while eGFR in the Ben group was decreased (week 24 vs. baseline, P > 0.05). 24 h urinary protein excretion (UP) and urinary albumin/creatinine (UAlb/Cr) were decreased in the TCM + Ben (week 24 vs. baseline, P < 0.05) and the Ben group (week 24 vs. baseline, P > 0.05). UP and UAlb/Cr were increased in the TCM group to week 12, then were stable (week 24 vs. baseline, P < 0.05). The hemoglobin in the TCM group was also improved (week 24 vs. baseline, P < 0.05). The accumulative survival rate in the TCM + Ben group was higher than that in the TCM group and the Ben group (P = 0.044). Side effects in the TCM group were the lowest in these groups (P < 0.05). The patients with dry cough in the TCM + Ben group and the Ben group were increased as compared with the TCM group (P < 0.05). Hyperkalemia happened less frequently in the TCM group as compared with the other two groups (P = 0.052).

Conclusions

For the patients with CKD stage 3, TCM can improve eGFR and hemoglobin with lower side effects. Benazepril significantly decreased the proteinuria. Chinese medicine integrated with benazepril can ameliorate renal function and decrease proteinuria synergistically.     

MACC5对大鼠抗Thy1.1系膜增生性肾炎防治作用的研究

目的 :观察中草药有效成分 MACC5对 SD大鼠抗 Thy1.1系膜增生性肾炎实验模型的防治作用 ,探讨其作用机理。方法 :用 SD大鼠胸腺细胞免疫新西兰白兔 ,制备兔抗大鼠胸腺细胞免疫血清 ( ATS) ,再将 ATS经尾静脉注射给SD大鼠 ,诱导其发生系膜增生性肾炎 ( Ms PGN) ,然后腹腔注射 MACC5 ,观察其尿量、尿蛋白定量改变 ,并用 H E染色法检测系膜增殖程度 ,用免疫组化法 ( LSAB)检测其肾小球内细胞增殖核抗原 ( PCNA)的表达。结果 :MACC5两试验组尿量( 2 4小时 )均多于对照组 ( P<0 .0 1) ;MACC5 3.0 m L试验组尿蛋白定量少于对照组 ( P<0 .0 1) ;MACC5两试验组肾小球细胞数均少于对照组 ( P<0 .0 1) ;MACC5两试验组 PCNA的表达均少于对照组 ( P<0 .0 1)。结论 :MACC5对大鼠 Ms PGN实验模型具有利尿、降低尿蛋白和促进肾小球系膜损伤修复的作用。     

Hypoglycemic effect of bitter melon compared with metformin in newly diagnosed type 2 diabetes patients

Ethnopharmacological relevance

Bitter melon (Momordica charantia L.) has been widely used as an traditional medicine treatment for diabetic patients in Asia. In vitro and animal studies suggested its hypoglycemic activity, but limited human studies are available to support its use.

Aim of study

This study was conducted to assess the efficacy and safety of three doses of bitter melon compared with metformin.

Materials and methods

This is a 4-week, multicenter, randomized, double-blind, active-control trial. Patients were randomized into 4 groups to receive bitter melon 500 mg/day, 1000 mg/day, and 2000 mg/day or metformin 1000 mg/day. All patients were followed for 4 weeks.

Results

There was a significant decline in fructosamine at week 4 of the metformin group (−16.8; 95% CI, −31.2, −2.4 μmol/L) and the bitter melon 2000 mg/day group (−10.2; 95% CI, −19.1, −1.3 μmol/L). Bitter melon 500 and 1000 mg/day did not significantly decrease fructosamine levels (−3.5; 95% CI −11.7, 4.6 and −10.3; 95% CI −22.7, 2.2 μmol/L, respectively).

Conclusions

Bitter melon had a modest hypoglycemic effect and significantly reduced fructosamine levels from baseline among patients with type 2 diabetes who received 2000 mg/day. However, the hypoglycemic effect of bitter melon was less than metformin 1000 mg/day.     

Treatment of high fat diet induced type 2 diabetes in C57BL/6J mice by two medicinal plants used in traditional treatment of diabetes in the east of Algeria

Aim of the study

Hydro-alcoholic extracts of Centaurium erythraea Rafn (CE), Gentianaceae and Artemisia herba-alba Asso (AHA), Asteraceae, medicinal plants used in traditional treatment of diabetes in north-eastern Algeria, were tested in established type 2 diabetes induced with a standardized high fat diet (HFD) in mice.

Materials and methods

After confirmation of diabetes (17th week), plant extracts were administered orally by gavage at a dose of 2 g/kg daily for 18 weeks to male C57BL/6J mice fed HFD. Animals were weighed, food intake and plasma glucose measured weekly, insulin and lipid profile at study end.

Results

At 35 weeks, groups treated with AHA or CE vs. HFD control had a significant reduction in mean (±SD) fasting blood glucose concentrations (143.8 ± 23.9 and 139.5 ± 14.2 vs. 229.0 ± 20.8 mg/dL, p < 0.05, respectively), triglyceride (18.9 ± 11.1 and 16.0 ± 6.5 vs. 62.8 ± 18.3 mg/dL, p < 0.05), total cholesterol (1.2 ± 0.1 and 1.2 ± 0.3 vs. 1.8 ± 1.1 g/L, p < 0.05) and serum insulin concentrations (1.7 ± 0.7 and 0.9 ± 0.7 vs. 3.3 ± 14.3 ng/mL, p < 0.05). Plant extracts also markedly reduced insulin resistance as compared to HFD controls (AHA: 15.6 ± 9.1, CE: 9.0 ± 7.7 vs. HFD control 38.5 ± 30.3, p < 0.05). The plant extracts decreased calorie intake and had little effect on body weight or HDL-cholesterol.

Conclusion

AHA has already been shown to have a antihyperglycaemic and antihyperlipidemic effect but this is the first demonstration of an effect of AHA and CE on established HFD-induced diabetes.     

Extracts of Arisaema rhizomatum C.E.C. Fischer attenuate inflammatory response on collagen-induced arthritis in BALB/c mice

Aim of the study

Arisaema rhizomatum C.E.C. Fischer (ARCF), called as “Xuelijian”, a local herb just growing in China, has been used as a traditional ethnic Chinese medicine for long because of its remarkable activity to alleviate pain and inflammation for patients suffering from rheumatism among the people with weak side-effect. However, rare study on the anti-arthritic activity of ARCF has been reported in vivo. The aim of this study is to investigate the protective effect of the herb on collagen-induced arthritis in mice and explore the potential immunological mechanisms.

Materials and methods

CIA was induced in male BALB/c mice by been subcutaneously injected type II bovine collagen (CII) for twice. The combined MeOH extract (ME) of ARCF rhizome was successively partitioned into four fractions with petroleum ether (PE), ethyl acetate (EE), n-butyl alcohol (n-BE) and water (WE). After the second collagen immunization, mice were administered orally with different doses of ME, EE and n-BE (ME 130, 261, 522 mg kg⁻¹; EE 10.2, 20.4, 40.8 mg kg⁻¹; n-BE 52, 104, 208 mg kg⁻¹) every other day for 3 weeks. The progression of edema of paws and knee joints was inspected by using a vernier calliper every 3 days from the 10th day after the first injection to the end of the experiment. The spleen index was measured and the knee joint destruction was observed by pathological sections. Levels of inflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-33 (IL-33 or IL-1F11) and rheumatoid factor (RF) in serum were measured by ELISA.

Results

Administration of ME, EE and n-BE significantly suppressed paws and joints swelling and reduced the spleen indexes. Pathological examination demonstrated that ARCF effectively protected anklebone and cartilage from being eroded versus vehicle-treated mice. Moreover, the serum levels of inflammatory cytokines TNF-α, IL-1β, IL-6, IL-33 and RF were markedly lowered in ARCF treated groups compared with the control group (p < 0.05).

Conclusion

Our studies demonstrate that administration of ARCF is obviously suppressed the progression of CIA. The anti-arthritic effectiveness of ARCF will make the herb a strong candidate for further clinical trials on RA patients.     

Cytochrome P450 inhibitory potential of Triphala--a Rasayana from Ayurveda

Ethnopharmacological relevance

‘Triphala’ is one of the age-old, most commonly used polyherbal preparation from Ayurveda as Rasayana drug.

Aim of the study

This study was aimed at evaluating the effect of ‘Triphala’ on drug modulating enzymes to assess its safety through its potential to interact with co-administered drugs.

Materials and methods

The cytochrome P450 inhibitory effect of ‘triphala’ formulation was investigated on rat liver microsomes using CYP450-CO complex assay and on individual isoform such as CYP3A4 and 2D6 using fluorescence screening. RP-HPLC method was developed to standardize ‘triphala’ and its individual components using gallic acid as analytical marker compound.

Results

RP-HPLC analysis demonstrated the presence of gallic acid (4.30 ± 2.09 mg/g) in the formulation. The formulation showed 23% inhibition of the rat liver microsomes through CYP450-CO complex assay which is comparatively less when compared with the individual components. Further, the effect of standardized formulation dissolved in ethanol showed CYP3A4 and CYP2D6 inhibitory activity at the IC₅₀ values of 119.65 ± 1.91 μg/ml and 105.03 ± 0.98 μg/ml respectively. Gallic acid was also found to inhibit both the isoforms at the IC₅₀ values of 87.24 ± 1.11 μg/ml and 92.03 ± 0.38 μg/ml respectively.

Conclusions

Various concentrations of the formulation and its individual components showed significantly less inhibitory activity (p < 0.001) on individual isoforms when compared with the positive control. Assessment on the in vitro effect of ‘triphala’ on drug modulating enzymes has important implications for predicting the likelihood of herb-drug interactions if these are administered concomitantly.     

Prevention of the progression of adjuvant induced arthritis by oral supplementation of Indian fresh water mussel (Lamellidens marginalis) aqueous extract in experimental rats

Aim of this study

Mussel is well accepted as food all over India. Beside for its nutritive value, people residing in Kosi river basin, Bihar, India, consume a preparation of soup, made from the footpad of molluscan species, with the belief that it gives relief from signs and symptoms of joint pain and related problems. This study was designed to explore the preventive activity of Indian fresh water mussel (Lamellidens marginalis) aqueous extract oral supplementation in experimental arthritis model.

Materials and methods

Arthritis was induced in male albino rats by intradermal injection of Freund's complete adjuvant in right hind footpad. Lamellidens marginalis extract (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) peroral supplementation started from the 1st day after adjuvant injection and was continued for the subsequent 13 days. Severity of arthritis was evaluated from paw diameter, ankle diameter, paw weight, urinary hydroxyproline, glucosamine level, serum interleukin-1β, IL6, IL10, CINC1, TNFα level, lysosomal enzyme levels and from histopathological assessment.

Results

Lamellidens marginalis extract supplementation significantly (p < 0.05) decreased paw diameter, ankle diameter, and paw weight in treated groups (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) as compared with arthritic group. Urinary hydroxyproline, glucosamine level, serum IL1β, IL6, CINC1, TNFα, IL10 and lysosomal enzyme levels were restored significantly (p < 0.05) in treated groups (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) as compared to arthritic group. Synovial membrane damage and neutrophil infiltration in histopathological examination was restored significantly by LME supplementation as compared to arthritic group.

Conclusions

Thus, it might be concluded that experimental animals supplemented with Lamellidens marginalis extract were protected against the severity of disease progression in adjuvant induced arthritis.     

Antidiabetic activity of alcoholic stem extract of Nervilia plicata in streptozotocin-nicotinamide induced type 2 diabetic rats

Ethnopharmacological relevance

Nervilia plicata (Orchidaceae) has long been used in the antidiabetic medicinal preparations of traditional healers of Wayanad (Kerala), but recuperative potential of the plant was remained undefined. We demonstrated the regenerative potential of the plant extract on kidney affected by type 2 diabetes besides lowering blood glucose.

Aim of the study

The aim of the current study was to investigate the recuperative and regenerative potential of alcoholic stem extract of Nervilia plicata on streptozotocin-nicotinamide induced type 2 diabetic models.

Materials and methods

Non insulin dependent diabetes mellitus (NIDDM) was induced in overnight fasted rats by intramuscular injection (IMI) of 60 mg/kg STZ and 120 mg/kg of nicotinamide after 5 min interval. Blood glucose was assessed by a glucometer, serum urea and creatinine levels were determined by diacetylmonooxime method and Jaffe reaction respectively. Kidney sections were taken and stained with Masson's tri-dye and Periodic Acid Schiff (PAS) and examined for structural changes. Also lipid peroxidation product (LPP) levels were determined as thio barbituric acid reactive substance levels (TBARS) method.

Results

On administration of 5 mg/kg of plant extract, blood glucose levels of the NIDDM rats showed 62.00 and 76.29% decrease in the blood glucose levels on day 0 and day 30 respectively. Damages caused to the kidney tissue were negligible or not seen. Serum urea and creatinine levels showed 61.49 and 70.96% decrease on day 30. LPP levels of kidney and pancreas showed 70.58 and 77.41% decrease respectively.

Conclusion

These results demonstrate significant antidiabetic and regenerative potential of the Nervilia plicata, justifying the use of plant in the indigenous system of medicine. Isolation and characterisation of the compound(s) playing pivotal role in the cure would open new vistas in the therapy of type 2 diabetes.     

Toxicogenomic analysis of the gene expression changes in rat liver after a 28-day oral Tripterygium wilfordii multiglycoside exposure

Ethnopharmacological relevance

Tripterygium wilfordii multiglycoside (GTW), which is an extract derived from Tripterygium wilfordii Hook.f., has been used for the treatment of rheumatoid arthritis and other immune diseases in China. However, its potential hepatotoxicity has not been completely investigated.

The aim of the study

The aim of the study was to determine the hepatotoxicity of GTW in Wistar rats and to investigate the underlying cellular mechanism further by microarray analysis.

Materials and methods

Doses of GTW at 60, 100 and 120 mg/kg/day were administered by oral gavage for subchronic toxicity in Wistar rats. Changes in the hepatic gene expression were identified with oligonucleotide microarrays at the 100-mg/kg/day dose level to study the hepatotoxic mechanism of GTW.

Results and conclusions

A number of changes in the body weight and food consumption, absolute and relative liver weight, biochemical analysis and histopathology were observed after the subacute exposure to GTW, and a dose-dependent hepatotoxicity was observed. A total of 1312 genes were found to be significantly altered (2-fold, P < 0.05), including 582 up-regulated genes and 730 down-regulated genes. According to our biological pathway analysis, the GTW resulted in aberrant gene expression in metabolic pathways and the peroxisome proliferator-activated receptor (PPAR) signaling pathway and cellular stress. Real-time PCR analyses of several genes verified these results. Consequently, our gene expression microarray study will be useful for future GTW hepatotoxicity studies.     

Inhibitory effect of hydro-methanolic extract of seed of Holarrhena antidysenterica on alpha-glucosidase activity and postprandial blood glucose level in normoglycemic rat

Ethnopharmacological relevance

The present experiment was conducted to search out the effect of hydro-methanolic extract of seed of Holarrhena antidysenterica on intestinal α-glucosidase activity in dose dependent manner and on the management of postprandial hyperglycemia in starch loaded rats.

Materials and methods

Activity of intestinal α-glucosidase was measured by in vitro method. Fasting blood glucose level was determined by single touch glucometer. Total phenol and flavonoids of seed extract of Holarrhena antidysenterica were estimated using gallic acid and quercetin standard curves, respectively.

Results

The degree of elevation in blood glucose level after starch administration was significantly (p < 0.05) less by the extract in respect to the control. The said extract also inhibited α-glucosidase activity having an IC₅₀ of 0.52 mg/ml. Phytochemical study revealed that the extract is rich in phenolic compounds (60.23 mg of gallic acid equivalent/g of extract) and flavonoids (360.23 mg of quercetin equivalent/g of the extract).

Conclusion

The extract exerts its antihyperglycemic effect by retarding the carbohydrate absorption from intestine through the inhibition in α-glucosidase activity and therefore resists postprandial hyperglycemia.     

Cytotoxic, apoptotic and anti-α-glucosidase activities of 3,4-di-O-caffeoyl quinic acid, an antioxidant isolated from the polyphenolic-rich extract of Elephantopus mollis Kunth

Ethnopharmacological relevance

The decoction of the whole plant of Elephantopus mollis Kunth. is traditionally consumed to treat various free radical-mediated diseases including cancer and diabetes.

Aim of the study

This study was initiated to determine whether the most effective antioxidant compound isolated from the whole plant of Elephantopus mollis can also contribute to its claimed traditional values as anticancer and antidiabetes agents.

Materials and methods

An active antiradical phenolic compound (3,4-di-O-caffeoyl quinic acid) was isolated from the methanol extract (with the highest in polyphenolic content) and their antioxidant activities were compared using four different assays, that are DPPH, FRAP, metal chelating, and β-carotene bleaching tests. The compound was also evaluated for its cytotoxic activity, apoptotic induction and anti-glucosidase efficacies using methylene blue, DeadEnd™ assay and α-glucosidase assays, respectively.

Results

The compound acted as a greater primary antioxidant than its methanol extract, by having higher ferric reducing activity (EC₅₀ 2.18 ± 0.05 μg/ml), β-carotene bleaching activity (EC₅₀ 23.85 ± 0.65 μg/ml) and DPPH scavenging activity (EC₅₀ 68.91 ± 5.44 μg/ml), whereas the methanol extract exhibited higher secondary antioxidant activity as a metal chelator with lower EC₅₀ value (49.39 ± 3.68 μg/ml) than the compound. Cytotoxicity screening of this compound exhibited a remarkable dose-dependent inhibitory effect on NCI-H23 (human lung adenocarcinoma) cell lines (EC₅₀ 3.26 ± 0.35 μg/ml) and was found to be apoptotic in nature based on a clear indication of DNA fragmentation. This compound also displayed a concentration-dependent α-glucosidase inhibition with EC₅₀ 241.80 ± 14.29 μg/ml.

Conclusions

The findings indicate the major role of 3,4-di-O-caffeoyl quinic acid to antioxidant capacities of Elephantopus mollis extracts. The compound also exerted apoptosis-mediated cytotoxicity and α-glucosidase inhibitory effects and is thus a promising non toxic agent in treating cancer and type 2 diabetes mellitus.     

Potential of a Khaya ivorensis -Alstonia boonei extract combination as antimalarial prophylactic remedy

Ethnopharmacological relevance

The decoction of the combined stem barks of Khaya ivorensis A. Chev. (Meliaceae) and Alstonia boonei De Wild (Apocynaceae) has a history of use in traditional medicine of central Cameroon for malaria treatment but also for the prevention of the disease.

Aim of the study

The purpose of this investigation was to determine the antiplasmodial activity of Khaya ivorensis (K) and Alstonia boonei (A) preparations in the murine malaria model Plasmodium berghei/Anopheles stephensi, to estimate their prophylactic potential and to assess acute and sub-acute toxicity of the formulations prepared according to the traditional recipes.

Materials and methods

Aqueous extracts from the stem-bark of the two plants were prepared and tested separately and in combination. BALB/c mice were treated for 9 days and challenged on day 3 by exposure to mosquitoes infected with Plasmodium berghei. Treatment doses ranged between 200 and 400 mg/kg/day, corresponding approximately to the dosage applied by traditional healers to cure malaria patients or prevent the disease. Parasitemia reduction in treated animals was calculated from Giemsa smear counts, of two replicate experiments. To estimate acute toxicity in terms of median lethal dose (LD50), geometrically increasing doses were administered to mice. Sub-acute toxicity of the herbal combination (KA) was investigated by administering the same doses as in the antiplasmodial activity test for a period of 14 days, followed by 14 days of recovery observation. Locomotor activity (Open Field Test), body weight, liver and kidney morphology were monitored.

Results

The combination KA was found to exhibit antiplasmodial activity in the murine malaria model. In mice treated with the combination remedy at a dosage of 200 mg/kg/day, parasitemia values of 6.2% ± 1.7 and 6.5% ± 0.8 were recorded, compared to 10.8% ± 1.3 and 12.0% ± 4.0 in controls (p < 0.01). Doubling the dosage of the extracts did not significantly increase parasite suppression. When extracts of K and A were administered separately at a dosage of 400 mg/kg, a reduction in parasitemia was still obtained, but it did not reach statistical significance. Toxicity studies yielded comforting results: the LD50 was estimated to be greater than 2779.5 mg/kg. Moreover, mice exposed to the fourteen-day repeated-dose toxicity test (sub-acute toxicity test) did not display weight loss, liver or kidney morphological modifications, significant alterations in locomotor activity or any other sign of illness.

Conclusion

The antiplasmodial activity and the wide dose interval between the therapeutic dosage and the toxic dosage exhibited by the KA herbal combination in the murine malaria model argue in favor of its use as an antimalarial prophylactic remedy. It remains to be demonstrated by human clinical trials whether the combination remedy, when taken by inhabitants during malaria transmission season, can reduce parasite density and lead to a reduction of malaria episodes in the community.