Phytochemical profiling of polyphenols and thyroid stimulatory activity of Ficus religiosa leaf extract in 6-propyl-thiouracil-induced hypothyroid rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant, Ficus religiosa (L.) from the family Moraceae, has been extensively used in Ayurveda and Unani. Traditionally this plant is known for the treatment of constipation, liver diseases and neurological disorders that are related to hypothyroidism. AIM OF THE STUDY: This study was primarily designed to evaluate the effect of Ficus religiosa leaf (FL) extract in ameliorating hypothyroidism in rats and to identify the major bioactive compounds in the test extract that might be responsible for the thyroid-altering activity. In addition, the probable mechanism underlying the thyroid regulation of the main FL constituents were analyzed by molecular docking. MATERIALS AND METHODS: Adult female Wistar rats were used. LC-ESI-MS/MS was performed to identify the compounds present in the extract. HPLC analysis of FL extract was also performed. A pilot study was made using 3 doses of FL extract. Out of 50, 100, and 200 mg/kg, 100 mg/kg appeared to be the most effective one as it could increase thyroid hormones and decreased TSH levels. In the final experiment, propyl-thiouracil (PTU)-induced hypothyroid rats were orally treated with FL extract (100 mg/kg) or L-thyroxine (100 µg/kg, i.p.) daily for 28 consecutive days. On 29th day, all rats were sacrificed and the serum levels of triiodothyronine (T3), thyroxine (T4), thyrotropin (TSH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and hepatic 5' deiodinase-1(5'D1) were estimated by ELISA. Liver marker enzymes (alanine aminotransferase, ALT and aspartate aminotransferase, AST); total cholesterol (TC) and triglycerides (TG); hepatic lipid peroxidation (LPO) and the activities of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH) content were estimated in liver tissues. RESULTS: LC-MS-MS analyses of the leaf extract identified 11 compounds including the three major compounds, betulinic acid (BA), chlorogenic acid (CGA), and quinic acid (QA). While the PTU treatment decreased the levels of thyroid hormones and 5'D1 activity, it increased the TSH, ALT, AST, TNF-α, IL-6, TC, and TG levels. Furthermore, hepatic LPO significantly increased with a decrease in reduced GSH, SOD, CAT, and GPx. However, FL treatment in PTU-induced animals nearly reversed these adverse effects and improved liver function by decreasing ALT, AST, hepatic LPO and increasing the levels of antioxidants. FL not only improved the liver histology, but also suppressed the inflammatory cytokines, TNF-α and IL-6 in PTU-induced animals. A molecular docking study towards the understanding of the thyroid stimulatory mechanism of action revealed that BA, CGA, and QA might have augmented thyroid hormones by interacting with the thyroid hormone receptor (TRß1) and TSH receptor (TSHR). CONCLUSION: For the first time, we report the pro-thyroidal potential of Ficus religiosa leaf extract. We postulate that its main bioactive compounds, BA, CGA, and QA involved in this action may serve as novel thyroid agonists in ameliorating hypothyroidism.

Antitumor Activity of Choerospondias axillaris Fruit Extract by Regulating the Expression of SNCAIP and SNCA on MDA-MB-231 Cells.

OBJECTIVE: Cancer is a huge problem of disease globally. Today, the percentage of people die from cancer is more than a combination of various diseases. In females, most common types of malignancies that occur are breast and cervical. The present focus has been shifted on medicinal plants as a form of therapy and there is a constant need to identify new therapeutic agents. Choerospondias axillaris (C. axillaris), an underutilized fruit, has been used in the remedy of various diseases. In the present communication, we evaluated the molecular mechanism of C. axillaris methanol extract in regulating cell death in human breast cancer cells (MDA-MB-231). METHODS: Methanol extract of C. axillaris was prepared and compounds were screened by Gas chromatography-mass spectrometry. The effect of fruit extract was determined on MDA-MB-231 cells by MTT ((3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay and to analyse the molecular mechanism of human breast cancer cells after treating with fruit extract, protein profiling study was performed by two-dimensional gel electrophoresis. RESULTS: A total 9 differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS/MS) analysis. Among 9 identified proteins, synphilin-1 protein was found to be significantly downregulated, validated by western blot and RT-qPCR analysis. Possible interacting partners of synphilin-1 (SNCAIP) were analyzed for their possible role in cancer by the in-silico method. CONCLUSION: Our data implicate that the presence of bioactive compound(s) in C. axillaris fruits might play an important role in inhibiting the proliferation of breast carcinoma cells and Synphilin-1 protein may play a role of apoptotic function.

Anti-inflammatory activity of nicotine isolated from Brassica oleracea in rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease, associated with chronic inflammation of synoviocytes. Tumor necrosis factor α (TNF-α) plays a crucial role in the pathogenesis of RA through pro-inflammatory cytokines. Nicotine, an alkaloid used as herbal medicine, often worked as an anti-inflammatory agent. In the present study, we tried to uncover the anti-inflammatory impact of nicotine against RA. MATERIALS AND METHODS: Nicotine was isolated from Brassica oleracea, purified by high profile/phase liquid chromatography (HPLC). In-silico docking was carried out using bioinformatics tools SwissADME (absorption, distribution, metabolism and excretion), PASS, and Drug-induced Gene Expression Profile (DIGEP)-Pred to determine drug likeliness of nicotine. The in-vitro study was performed in TNFα-induced SW982 synoviocytes by qPCR. mRNA expression of pro-inflammatory cytokines (TNF, IL6, IL1ß) and proteins (TRAF2, P50, P65) were analyzed followed by validation of P65 (RELA), pP65, IkBα by Western blot analysis. RESULTS: Nicotine compound was extracted from Brassica oleracea and purified by HPLC method (Rt values at 2.67 min). The physicochemical, pharmacokinetic properties and drug-likeliness of nicotine were studied by in-silico analysis. In-vitro studies revealed that nicotine lowers the expression of inflammatory cytokines (TNF, IL6, IL1ß) and proteins (TNF receptor-associated factor 2 (TRAF2), P50, P65) at 1 µg/ml in TNFα-induced SW982 cells. CONCLUSION: Nicotine from natural sources (Brassica oleracea) has been found to be an effective anti- inflammatory compound at a low dosage; thus, identifying the role of nicotine present in the natural sources as a therapeutic option for RA, may be recommended as remedial drug instead of synthetic drug.

Exogenous miRNA: A Perspective Role as Therapeutic in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease that causes joint deformation. Till now several studies has been carried out promising its cure, but curing has not yet achieved to the satisfactory levels. Herbal approach to treat disease by a cross-kingdom mechanism via exogenous miRNA is an emerging trend to target associated genes with RA pathogenesis as a therapeutic potential. The concept of acquired/exogenous miRNA into pathophysiological prospect provides an opportunity to explore inter-species kingdom like regulation of plant miRNAs on human health. The change in gene expression was attributed by a short 22-24 nucleotide long sequence that binds to its complementary region to suppress/silence the gene expression. This makes exogenous miRNA a novel approach for targeted therapy to treat complex chronic inflammatory diseases. Here, aim of the review was to address significance of plant derived miRNA based targeted therapy to regulate inflammation in RA.

A mechanistic insight of phytoestrogens used for Rheumatoid arthritis: An evidence-based review.

Assessment of the potential therapeutic benefits offered by naturally occurring phytoestrogens necessitate inspection of their potency and sites of action in impeding the chronic, systemic, autoimmune, joint destructing disorder Rheumatoid arthritis (RA). Possessing structural and functional similarity with human estrogen, phytoestrogen promisingly replaces the use of hormone therapy in eradicating RA symptoms with their anti-inflammatory, anti-oxidative, anti-proliferative, anti-angiogenesis, immunomodulatory, joint protection properties abolishing the harmful side effects of synthetic drugs. Scientific evidences revealed that use of phytoestrogens from different chemical categories including flavonoids, alkaloids, stilbenoids derived from different plant species manifest beneficial effects on RA through various cellular mechanisms including suppression of pro-inflammatory cytokines in particular tumor necrosis factor (TNF-α), interleukin(IL-6) and nuclear factor kappa B (NF-κB) and destructive metalloproteinases, inhibition of oxidative stress, suppressing inflammatory signalling pathways, attenuating osteoclastogenesis ameliorating cartilage degradation and bone erosion. This review summarizes the evidences of different phytoestrogen treatment and their pharmacological mechanisms in both in vitro and in vivo studies along with discussing clinical evaluations in RA patients showing phytoestrogen as a promising agent for RA therapy. Further investigations and more clinical trials are mandatory to clarify the utility of these plant derived compounds in RA prevention and in managing oestrogen deficient diseases in patients.

Evaluation of Anti-inflammatory Effects of Choerospondias axillaris Fruit's Methanolic Extract in Synoviocytes and CIA Rat Model.

BACKGROUND: Rheumatoid Arthritis (RA) is an autoimmune, systemic disease mainly affecting joints. Presently, there is no specific treatment/ drug available for curing RA except few supportive medicines. Therefore, the focus has been shifted to medicinal plants for the treatment of such diseases. Choerospondias axillaris commonly known as Lupsi/Lapsi and has been reported to have several properties for the treatment of various diseases. OBJECTIVE: The present study has been conducted to explore the anti-inflammatory effects of Choerospondias axillaris fruit extract on Synoviocytes (FLS) and Collagen-Induced Arthritis (CIA) rat model. METHODS: Methanolic extract of the Choerospondias axillaris fruit was used for determining phytochemical, antioxidant and anti-inflammatory properties. Antioxidant activity of Choerospondias axillaris fruit was determined by free radicals scavenging assays and bioactive compounds were identified via LC-MS/MS analysis. Anti-inflammatory effect was investigated in RA and Osteo Arthritis (OA) primary cells and also in Collagen Induced Arthritis (CIA) rat models. Further, the medicinal properties of anti-inflammatory bioactive compounds were supported by docking studies. RESULTS: In-vitro and in-vivo studies showed significant decrease in the levels of inflammatory cytokines. Docking analysis revealed that quercetin inhibits TNF-α having -9.1 kcal/mol binding energy and 10.13 µM inhibitory constant. Quercetin also inhibits IL-6 having -6.6 kcal/mol binding energy and 21.9 µM inhibitory constant. CONCLUSION: Observed results suggest that the underutilized fruit Choerospondias axillaris can be used to reduce the inflammation of inflammatory diseases like RA.

Comparative Molecular Characterization to Reveal Surface Behaviour of Non-proteolytic Bromelain Mutants.

BACKGROUND: Rheumatoid Arthritis (RA) is a chronic autoimmune disease that results in the systemic inflammation principally affecting the capsule covering the articulating ends of the synovial joints. Pharmacological treatment involving analgesics and anti-inflammatory drugs including steroids suppresses the symptoms and has no effect on disease progression. However, disease modifying anti rheumatic drugs (DMARD) used for the treatment were still analysed for their long-term effects. METHODS: Bromelain has been widely used as phytotherapeutic drug owing to its anti-inflammatory, analgesic, anti-tumor and fibrinolytic properties. Bromelain refers to the combination of thiol proteases available in the extract of Ananas comosus. The fibrinolytic property confers to the reduced pannus development and hence the prevention of disease progression. RESULTS: It had been inferred that the observed clinical significance may not be solely accounted for its proteolytic property but also may be due to its hormone-like behaviour (i.e.) non-canonical interactions to initiate the signal transduction pathway. The hormone-like behaviour has been studied in cell models, suggesting that bromelain acts at system level. In the present study, molecular behaviour of the wild-type and mutant proteins has been studied by simulating the predicted structure in an aqueous system. The comparative study of the mutants revealed that the mutant with both C26A and H158F mutations has the similar surface properties compared to the other mutants and can be used in studying the non-enzymatic interactions. CONCLUSION: Thus, this study may prove to be a tool in experimental studies to understand the hormone-like behaviour and in the construction of oral immunogenic synthetic peptides for treating inflamed conditions.

Genome-wide identification and functional annotation of miRNAs in anti-inflammatory plant and their cross-kingdom regulation in Homo sapiens.

MicroRNAs (miRNAs) are newly discovered non-coding small (~17-24 nucleotide) RNAs that regulate gene expression of its target mRNA at the post-transcriptional levels. In this study, total 12,593 ESTs of Curcuma longa were taken from database of expressed sequence tags (dbEST) and clustered into 2821 contigs using EGassembler web server. Precursor miRNAs (pre-miRNAs) were predicted from these contigs that folded into stem-loop structure using MFold server. Thirty-four mature C. longa miRNAs (clo-miRNAs) were identified from pre-miRNAs having targets involved in various important functions of plant such as self-defence, growth and development, alkaloid metabolic pathway and ethylene signalling process. Sequence analysis of identified clo-miRNAs indicated that 56% miRNAs belong to ORF and 44% belong to non-ORF region. clo-mir-5 and clo-mir-6 were established as the conserved miRNAs, whereas clo-mir-20 was predicted to be the most stable miRNA. Phylogenetic analysis carried out by molecular evolutionary genetics analysis (MEGA) software indicated close evolutionary relationship of clo-mir-5075 with osa-MIR5075. Further, identified clo-miRNAs were checked for their cross-kingdom regulatory potential. clo-mir-14 was found to regulate various gene transcripts in humans that has been further investigated for its biostability in foetal bovine serum (FBS). The results indicated higher degree of stability of clo-mir-14 (48 h) in FBS. Thus, contribution of this miRNA to the cellular immune response during the inflamed condition of rheumatoid arthritis and adequate stability may make it a good choice for the therapeutic agent in near future.

Trigonelline isolated from fenugreek seed protects against isoproterenol-induced myocardial injury through down-regulation of Hsp27 and αB-crystallin.

OBJECTIVE: Protective effects of trigonelline (TRG) isolated from fenugreek seed were evaluated in isoproterenol (ISO)-induced myocardial dysfunctions in adult rats and a proteomic approach was applied to understand its mechanism of action. METHODS: In a preliminary experiment, effects of TRG at 20, 40, and 80 mg/kg for 20 d were studied in ISO-induced (100 mg/kg) adult rats. As 40 mg/kg was found the most effective concentration, in the final experiment, effects of 40 mg/kg of the test drug were investigated using different indices including cardiac marker enzymes, lipid peroxidation, antioxidants, cardiac histology, and electrocardiogram. Proteomic analyses were also done in cardiac myocytes. RESULTS: ISO administration increased serum levels of cardiac markers (creatine kinase-MB, glutamate pyruvate transaminase, and lactate dehydrogenase) and exhibited a positive reaction in the TROP-T test. It also increased the cardiac lipid peroxidation and decreased the cellular antioxidants. Proteomic data revealed nine protein spots, seven were down-regulated and two up-regulated. The overexpressions of two small stress proteins, heat shock protein (Hsp)27 and αB crystallin were confirmed by Western blot analysis. All these alterations were restored to nearly normal values in 40 mg/kg of TRG-pretreated animals, suggesting its cardioprotective effects, which were further confirmed by histologic examinations and triphenyl tetrazolium chloride staining assay. CONCLUSION: For the first time, our study revealed the down-regulation of Hsp27 and αB-crystallin and (CaMKII delta) isoform by TRG. As the test compound prevented the ISO-induced myocardial injury, its therapeutic use may further be explored.