M. Gabriel Paulraj

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.

Immunomodulatory potential of Enicostema axillare (Lam.) A. Raynal, a traditional medicinal plant

ETHNOPHARMACOLOGICAL RELEVANCE Enicostema axillare (Lam.) A. Raynal., (Gentianaceae) has been used in traditional Indian system of medicine as depurative and for the treatment of psoriasis, intermittent fever and cancer. Ethnobotanical survey conducted in Theni District indicated a high consensus for this plant as blood purifier and to treat dermatopathy and venereal infections. The present study evaluated the immunomodulatory activity of the methanol extract of Enicostema axillare. MATERIALS AND METHODS In vivo immunomodulatory activity of Enicostema axillare methanol extract (100 and 200mg/kg b.w) was evaluated by assessing its effect on the total and differential leukocyte count, organ weight, hemaggultinating antibody titer, plaque forming cells, quantitative hemolysis of SRBC and delayed type hypersensitivity. Sheep red blood cells (SRBC, 5×10(8)cells/0.1mL) were used to immunize the animals. In vitro immunomodulatory potential of the extract was studied using peritoneal macrophages by evaluating its effects on NBT reduction, NO production and cytokine release. RESULTS The animals treated with Enicostema axillare methanol extract showed a significant (P≤0.05) increase in weight of the thymus and spleen. The total leukocyte and lymphocyte count was increased significantly (P<0.005) by the treatment. There was no significant alteration in neutrophil count. A dose dependent increase in antibody titer value was observed. A decreased response to DTH reaction induced by SRBC was recorded. A potential phagocytic response was seen on treatment with the extracts at 10 and 25μg/mL. The extract inhibited the release of pro-inflammatory cytokines and production of NO significantly in a dose dependent manner. CONCLUSION These findings suggested that the methanol extract of Enicostema axillare acted on both humoral and cell mediated immune functions and decreased the release of pro-inflammatory cytokines in the peritoneal macrophages.

Documentation and quantitative analysis of local ethnozoological knowledge among traditional healers of Theni district, Tamil Nadu, India.

ETHNOPHARMACOLOGICAL RELEVANCE This study investigated the use of animals among the traditional healers in Theni district of Tamil Nadu, India. The data regarding the medicinal animals/animal products were documented and their usages were analyzed quantitatively. MATERIALS AND METHODS Based on free list interviews with the traditional healers, we documented the medicinal usage of animals/animal products and calculated the indices such as informant consensus factor (Fic) to determine the consensus over the species for an illness category, as well as the Index Agreement on Remedies (IAR) to determine the extent of potential utilization of each species. RESULTS In this study, 69 medicinal animals/animal products were documented with the help of standardized questionnaires among the local healers. The results were tabulated and Fic value for each illness category was calculated. Three illness categories viz., jaundice (milk of Capra aegagrus hircus), orthopedics (egg white and meat of Gallus gallus domesticus) and pediatrics (milk of Equus africanus asinus) had got high Fic values. Fifteen illness categories had moderate Fic values. Highly cited animals in these illness categories were: Rusa unicolor (antiemetic), Reticulitermes spp. (diabetes), flesh of Varanus benghalensis (oral ailments), milk (eye ailments, fever) and urine (antidote) of Homo sepians, meat of Trachypithecus johnii (respiratory ailments), various parts of C. aegagrus hircus (blood ailments, coolants, diarrhea, pulmonary and urinary ailments), flesh of Chamaeleon zeyalnica (neural ailments), meat of Passer domesticus (aphrodisiac), curd and dung of Bos primigenius taurus (dermatological ailments), meat of G. domesticus (musculo-skeletal disorders, analgesic), meat of Lissemys punctata (hemorrhoids), and Pherthima posthuma (psychological ailments). Six illness categories had low Fic values. CONCLUSION This study indicated that the animals are still being used by the local healers of Theni district, to treat various illnesses. Cross-disciplinary approaches to explore the full potential of animal-derived medicines will help to improve the health of local people.

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.

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

Abstract 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.

Antifeedant activity and toxicity of two alkaloids from Adhatoda vasica Nees leaves against diamondback moth Plutella xylostella (Linn.) (Lepidoptera: Plutellidae) larvae

Two natural alkaloids viz., Vasicine acetate and 2-Acetyl benzylamine, isolated from Adhatoda vasica leaves, showed antifeedant, larvicidal and moult inhibiting properties against diamondback moth Plutella xylostella in laboratory experiments. Maximum antifeedant activity of 98.5% was recorded at 1000 ppm concentration of Vasicine acetate treatment, whereas as 2-Acetyl benzyl amine recorded only 71.4% antifeedant activity at 1000 ppm concentration. Azadirachtin treatment presented 82% antifeedant activity at the highest concentration (1000 ppm). Both the active compounds of A. vasica showed lethal toxicity on larvae and pupae. The highest larvicidal and pupicidal activities were recorded in 2-Acetyl benzylamine treatment at 125 ppm concentration. The two A. vasica compounds also affected the normal growth and development and moulting process of P. xylostella. Final moulting of larvae into pupae was disrupted by the treatments, which resulted in larval–pupal intermediates and abnormal pupae. Treatments also produced small-size pupae and malformed adults with poorly developed wings.

Ameliorative effect of Drynaria quercifolia (L.) J. Sm., an ethnomedicinal plant, in arthritic animals

Drynaria quercifolia (L.) J. Sm., is an ethnomedicinal plant used widely in Tamil Nadu to treat arthritis. The present study was aimed to evaluate the traditional claim of D. quercifolia rhizome water extract in adjuvant induced arthritic animals. Anti-arthritic effect was studied by assessing the levels of lysosomal enzymes, protein bound carbohydrates, urinary degradative collagen and serum cytokines on control and adjuvant induced arthritis. The paw swelling and body weight were also analyzed. The levels of ROS and lysosomal enzymes in neutrophils of control and adjuvant induced animals were also estimated. D. quercifolia rhizome water extract at doses of 100 and 200mg/kg reduced the paw thickness and elevated the mean body weight of arthritic rats. The treatment with extract showed a significant reduction in the levels of plasma and liver lysosomal enzymes as well as protein bound carbohydrates and urinary degradative collagen levels. The treatment reduced the levels of ROS and lysosomal enzymes in neutrophils significantly. The significant reduction in the levels of serum pro-inflammatory cytokines (TNF-α and IL-1β) and the increment in the levels of anti-inflammatory cytokine (IL-10) were also observed by the treatment. The present study supports the traditional claim of using D. quercifolia to treat rheumatism.

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 informants medicinal plant knowledge such as Shannons 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.

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.

Swertiamarin, a natural steroid, prevent bone erosion by modulating RANKL/RANK/OPG signaling

Abstract Bone erosion is a central feature of rheumatoid arthritis (RA) that is characterized by the infiltration of the synovial lining by osteoclasts and lymphocytes. In the present study, swertiamarin a major secoiridoid glycoside was evaluated for anti‐osteoclastogenic property to prevent bone erosion in Freunds complete adjuvant (FCA) induced in‐vivo model, in‐vitro osteoblast and osteoclasts as well as in co‐culture system and in‐silico molecular docking analysis. The swertiamarin treatment decreased the expression of TRAP, RANKL, and RANK levels and increased the levels of OPG levels significantly in both in vitro and in vivo models. In in vitro, the compound treatment significantly increased the cell proliferation and ALP levels in osteoblast cells; the high proliferation (153.8600 ± 5.23%) and ALP release (165.6033 ± 4.13%) were observed at 50 &mgr;g/ml concentration of swertiamarin treatment. At the same time the treatment decreased the TRAP positive cells in osteoclast cells; the high reductions of TRAP positive cells (39.32 ± 3.19%) were observed at 50 &mgr;g/ml of swertiamarin treatment. The treatment modulated the levels of pro‐inflammatory cytokines, MMPs and NF‐&kgr;B levels in osteoblast and osteoclast co‐culture system. In in silico analysis swertiamarin had affinity towards the proteins RANK, RANKL and OPG residues with low binding energy −4.5, −3.92 and −5.77 kcal/mol respectively. Thus, the results of this study revealed the anti‐osteoclastogenic activity of swertiamarin on the prevention of bone destruction. HighlightsSwertiamarin was confirmed as anti‐osteoclastogenic effect by in‐silico analysis on bone mediated proteins.The swertiamarin reciprocally regulate the expression of TRAP, RANKL, RANK levels and OPG levels.The swertiamarin modulated the levels of pro‐inflammatory cytokines, MMPs and NF‐&kgr;B levels.Swertiamarin treatment revealed the prevention of bone destruction in animal models.