Hepatoprotective effect of bisbenzylisoquinoline alkaloid tiliamosine from Tiliacora racemosa in high-fat diet/diethylnitrosamine-induced non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is one of the aggressive forms of non-alcoholic fatty liver disease (NAFLD) and is a potential risk factor of HCC. This study reports the curative effect of tiliamosine on NASH. Tiliamosine was isolated from Tiliacora racemosa Colebr. (Menispermaceae) and its structure was confirmed by studying the physical and spectroscopic data. The effects of tiliamsoine on lipid accumulation and lipotoxicity were evaluated using palmitate-oleate induced steatosis in HepG2 cells. The in vivo efficacy of tiliamosine was evaluated using HFD fed, DEN induced non-alcoholic steatohepatitis Wistar rats. In HepG2 cells, tiliamosine did not affect the cell viability up to 100 µM concentration and showed GI25 value of 264.28 µM. The treatment with tiliamsoine significantly lowered the ORO concentration by 44.17% and triglyceride accumulation by 69.32% at 50 µM concentration (P < 0.005). It also reduced the leakage of LDH and transaminases in PO-BSA induced HepG2 cells. The treatment with tiliamsoine significantly decreased the plasma levels of transaminases, phosphatase and LDH (P < 0.05) in HFD-DEN induced steatohepatitis. The histology and the immunohistochemistry of the hepatic sections were in accordance with the biochemical findings. Preliminary molecular analysis indicated that the hepatic FXR expression was upregulated and TNFα expression was downregulated by the treatment with tiliamsoine. This study provided preliminary evidence on the use of tiliamosine for the treatment of NASH.

Hepatoprotective effect of lawsone on rifampicin-isoniazid induced hepatotoxicity in in vitro and in vivo models.

The Drug-induced liver injury is one of the common unfavourable impacts, which seriously affects any drug therapy. This study documented the hepatoprotective efficacy of lawsone, the major bioactive naphthoquinone present in Lawsonia inermis L. (Lythraceae) using in vitro and in vivo models. Lawsone was isolated from the leaves of L. inermis and its structure was confirmed using spectroscopic data. In-vitro antioxidant effect of lawsone was evaluated using ABTS assay. Hepatoprotective effect of lawsone was determined with RIF-INH treated HepG2 cells and Wistar rats. Administration of RIF-INH reduced the viability of the HepG2 cells and the treatment with lawsone significantly restored the viability of the cells even at lower concentration (7.5 µM). The other parameters such as the leakage of transaminases and MDA levels were also significantly reduced by the treatment with lawsone. Oral administration of lawsone to the animals did not show any toxicity up to 2 g/kg b.w. concentration. Treatment with lawsone to the RIF-INH administered animals significantly lowered the serum transaminases levels. The ratio of albumin to globulin was improved and the level of bilirubin was lowered. This study indicated the hepatoprotective effect of lawsone; detailed investigations will give deeper understanding of the application of lawsone for hepatoprotection.

Quantitative ethnomedicinal survey of medicinal plants given for cardiometabolic diseases by the non-institutionally trained siddha practitioners of Tiruvallur district, Tamil Nadu, India.

ETHNOBOTANICAL RELEVANCE: The burden of cardiometabolic diseases such as dyslipidemia, hyperglycemia, hypertension, visceral obesity and atherosclerotic cardiovascular diseases and the use of traditional medicine for the management of such diseases are high in India; hence there is a need to document and analyze such therapies. AIM OF THE STUDY: This study documented and analyzed the medicinal plants prescribed for cardiometabolic diseases by the non-institutionally trained siddha practitioners of Tiruvallur district of Tamil Nadu, India. METHODOLOGY: The field survey was conducted between December 2014 to November 2015. Successive free listing assisted with field-walks was used to interview the informants. After assessing the sampling sufficiency using rarefaction curve analysis, indices such as Informant Consensus Factor (Fic) and Index of Agreement on Remedies (IAR) were calculated for the data. The indicators of informant's medicinal plant knowledge such as Shannon's index, equitability index, etc., were regressed with the demographic profile of the informants. RESULTS: For this study 70 non-institutionally trained Siddha medical practitioners were approached; the data from 36 practitioners who were treating cardiometabolic diseases were documented. This study recorded the use of 188 species which were used to prepare 368 formulations to treat illnesses categorized under cardiometabolic diseases. In this, 53.04% claims were singletons. Regression analysis showed that single species dominance was reduced and the diversity of medicinal plants was increased with the increase in the age and experience. Increase in the years of formal education increased the equitability in the uses. The plants such as Nelumbo nucifera Gaertn. (cardiovascular diseases), Allium sativum L. (dyslipidemia), Cuminum cyminum L. (hypertension), Macrotyloma uniflorum Verdc. (obesity) and Azadirachta indica A. Juss. (type 2 diabetes) were the highly cited medicinal plants. CONCLUSION: This survey has identified the plants most commonly used by Siddha practitioners of Tiruvallur district, Tamil Nadu, India for cardiometabolic diseases. The prevalence of chronic, non-communicable metabolic illnesses such as type 2 diabetes, hypertension and obesity are increasing worldwide due to the rapid changes in the lifestyle. These ailments require a life-long care and in such instances, people tend to use complementary therapies in most cases, alongside with conventional therapies. In view of the high use of traditional therapies for treating cardiometabolic illnesses, this study supports the need for more research to evaluate the potential benefits of the treatments and to identify any safety concern.

Delivery of chitosan/dsRNA nanoparticles for silencing of wing development vestigial (vg) gene in Aedes aegypti mosquitoes.

RNA interference (RNAi) has been used as a gene silencing strategy by the introduction of long double stranded RNA (dsRNA) for the control of pest insects. The aim of the present study was to examine whether the expression of vg gene which is responsible for wing development, can be repressed by chitosan/dsRNA based nanoparticles in Aedes aegypti. The vestigial gene (vg) was amplified from adult mosquito and cloned in pLitmus28i vector. Genetically engineered recombinant plasmid was transformed into RNase III deficient strain for synthesis of bacterially expressed dsRNA. Nanoparticles were prepared via electrostatic interaction between cationic polymer chitosan and anionic nucleic acids (dsRNA). The formation of chitosan/dsRNAnanoparticles and their size were confirmed by Atomic force microscopy (AFM). Chitosan/dsRNA mediated knockdown of Enhanced Green Fluorescence Protein (EGFP) was demonstrated in Sf21 cells. Further, we tested whether such an approach could be used to target vg gene in Ae. aegypti. The results showed that chitosan/dsRNA caused significant mortality, delayed growth development and caused adult wing-malformation. A qRT-PCR analysis confirmed that the chitosan/dsRNA mediated transcriptional level was downregulated. Our findings suggest that vg gene intervention strategies through RNAi can emerge as viable option for pest control.

Swertiamarin attenuates inflammation mediators via modulating NF-κB/I κB and JAK2/STAT3 transcription factors in adjuvant induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease that leads to pannus formation followed by severe joint destruction, characterized by synovial hyperplasia, inflammation and angiogenesis. Swertiamarin is a secoiridoid glycoside that is used as an anti-inflammatory compound, mainly found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in Indian system of traditional medicine. In the present study, the effect of swertiamarin was evlauated in experimental adjuvant arthritis animal model by the estimation of biochemical (paw thickness, lysosomal enzymes, and urinary degradative products) parameters, proinflammatory cytokines and enzymes along with histopathological and radiographic observations. The proteins of phosphorylated NF-κB/IκB and JAK2/STAT3 transcription factors were also quantified from experimental animals as well as LPS induced RAW 264.7 macrophage cells. In in silico analysis, swertiamarin was docked with proinflammatory enzymes to confirm its potential. The administration of swertiamarin (2, 5, 10mg/kg bw) significantly (P⩽0.05) inhibited the levels of paw thickness, lysosomal enzymes and increased the body weight of experimental animals in a dose dependent manner. In molecular analysis, the treatment decreased the release of proinflammatory cytokines (IL1, TNF, IL-6) and proangiogenic enzymes (MMPs, iNOS, PGE2, PPARγ and COX-2); and also significantly (P⩽0.05) increased the levels of antiinflammatory proteins (IL-10, IL-4) when compared to the disease groups. The swertiamarin treatment significantly (P⩽0.05) inhibited the release of NF-κB p65, p-IκBα, p-JAK2 and p-STAT3 signaling proteins levels on both experimental animals and LPS induced cells. Histopathological and radiological analysis evidenced the curative effect of swertiamarin on bone destruction. The docking studies of swertiamarin on proinflammatory enzymes supported the results from the in vivo experiments. Thus the swertiamarin inhibited the development of arthritis by modulating NF-κB/IκB and JAK2/STAT3 signaling. These findings suggested that swertiamarin acted as an anti-rheumatic agent.

Swertiamarin ameliorates inflammation and osteoclastogenesis intermediates in IL-1ß induced rat fibroblast-like synoviocytes.

OBJECTIVE AND DESIGN: Rheumatoid arthritis is a chronic inflammatory and autoimmune disease that leads to aggressive joint cartilage and bone destruction. Swertiamarin is a secoiridoid glycoside found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in the Indian system of traditional medicine. In the present study, the potential of swertiamarin was evaluated in IL-1ß induced fibroblast-like synoviocytes (FLS). METHODS: The FLS were isolated from Freund's Complete Adjuvant induced arthritic (AA) rats and cultured with IL-1ß. The normal FLS and AA-FLS were cultured and used for subsequent experiment in fibroblastic morphology form. The efficacy of swertiamarin (10-50 µg/ml) was evaluated on mRNA and protein expression levels of inflammatory and osteoclastogenesis mediators. The efficacy was also evaluated on p38 MAPKα levels with time course studies (2, 4, 6, 8 and 12 h). RESULTS: IL-1ß induced cell proliferation (149.46 ± 13.73 %) and NO production (162.03 ± 11.03%) in AA-FLS; treatment with swertiamarin controlled proliferation (82.77 ± 4.22%) and NO production (82.06 ± 3.91% at 50 µg/ml) in a dose-dependent manner. It also significantly (P < 0.05) modulated the expression of apoptotic marker (caspase 3), proinflammation mediators (TNFα, IL-6, PGE2, COX-2, iNOS, MMPs) and osteoclastogenic mediator (RANKL) at both the mRNA and protein levels. Treatment with swertiamarin inhibited the levels of p38 MAPKα in a dose-dependent manner and also significantly (P < 0.05) attenuated the release of the same in time dependent mode. CONCLUSION: These findings suggest that treatment with swertiamarin attenuated IL-1ß induced FLS, and it revealed anti-inflammatory potential by attenuating aggressive FLS.

Protective effect of Enicostema axillare on mutagenicity of Salmonella typhimurium by increasing free radical scavenging abilities.

CONTEXT: Enicostema axillare A. Raynal (Gentianaceae) has been used in the traditional Indian system of medicine as a depurative and for the treatment of skin diseases, tumors, intermittent fever, and helminthiasis. OBJECTIVE: E. axillare was investigated for antimutagenic and antioxidant effects. MATERIALS AND METHODS: The antioxidant and antimutagenic activities of E. axillare fractions were determined by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging assay and Ames test using Salmonella typhimurium tester strains TA98 and TA100 against direct-acting mutagens, such as sodium azide (NaN3), 4-nitro-O-phenylene diamine (NPD), and mutagen needing activation, such as 2-aminofluorene (2AF). Toxicity study of these fractions was also performed. RESULTS AND DISCUSSION: The ethyl acetate fraction showed maximum antimutagenic effect by 88.25 and 84.46% (preincubation) and 85.13 and 84.47% (coincubation) of inhibition against NaN3 and NPD-induced mutagenicity, respectively. Inhibition of S9-dependent mutagens such as 2AF was higher than direct-acting mutagens by the ethyl acetate fraction of E. axillare. It showed 90.25 and 92.00% of inhibition in the preincubation and coincubation experiments. The ethyl acetate fraction showed higher total antioxidant capacity (24.79 ± 0.29 µg) and low IC50 value for DPPH radical scavenging assay (192.27 ± 3.67 µg). The overall effect of E. axillare fractions was found to be in the order: ethyl acetate > methanol > hexane in these assays. In subacute toxicity study, with oral administration of these fractions, no marked biochemical and histopathologic changes were observed. CONCLUSION: The significant antimutagenic and antioxidant activities of E. axillare might provide a scientific validation for the traditional use of this plant.

Indirubin potentiates ciprofloxacin activity in the NorA efflux pump of Staphylococcus aureus.

Staphylococcus aureus is a common multidrug-resistant organism in hospital-acquired infections, and the NorA efflux pump mechanism facilitates resistance to quinone compounds. In India, Wrightia tinctoria R. Br. leaves have traditionally been used to treat skin diseases and have been explored for antibacterial and efflux pump inhibition (EPI) compounds. In this study, indirubin isolated from the chloroform extract of W. tinctoria R. Br. leaves was tested for its antibacterial activity against clinically important Gram-positive and Gram-negative bacteria and the minimum inhibitory concentration (MIC) was determined using broth microdilution. The EPI properties of indirubin were investigated using Staphylococcus aureus SA1199B, and its synergistic effects were tested with ciprofloxacin. Indirubin showed antibacterial activity against both the type strain and drug-resistant S. aureus; the MIC was determined to be 12.5 mg/l for S. aureus and 25 mg/l for Staphylococcus epidermidis. However, it synergistically (fractional inhibitory concentration index = 0.45) potentiated the activity of ciprofloxacin, probably by inhibiting the NorA efflux pump. Indirubin exhibited EPI activity nearly comparable to that of reserpine by 4-fold reduction in ciprofloxacin MIC. Our results suggest that the natural compound indirubin could be used in future therapeutic applications as a potential EPI.