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Chronic hepatitis caused by the hepatitis C virus (HCV) is closely linked with the advancement of liver disease. The research hypothesis suggests that the NS5B enzyme (non-structural 5B protein) of HCV plays a pivotal role in facilitating viral replication within host cells. Hence, the objective of the present investigation is to identify the binding interactions between the structurally diverse phytotherapeutics and those of the catalytic residue of the target NS5B polymerase protein. Results of our docking simulations reveal that compounds such as arjunolic acid, sesamin, arjungenin, astragalin, piperic acid, piperidine, piperine, acalyphin, adhatodine, amyrin, anisotine, apigenin, cuminaldehyde, and curcumin exhibit a maximum of three interactions with the catalytic residues (Asp 220, Asp 318, and Asp 319) present on the Hepatitis C virus NS5B polymerase of HCV. Molecular dynamic simulation, particularly focusing on the best binding lead compound, arjunolic acid (-8.78 kcal/mol), was further extensively analyzed using RMSD, RMSF, Rg, and SASA techniques. The results of the MD simulation confirm that the NS5B-arjunolic acid complex becomes increasingly stable from 20 to 100 ns. The orientation of both arjunolic acid and sofosbuvir triphosphate (standard) within the active site was investigated through DCCM, PCA, and FEL analysis, indicating highly stable interactions of the lead arjunolic acid with the catalytic region of the NS5B enzyme. The findings of our current investigation suggest that bioactive therapeutics like arjunolic acid could serve as promising candidates for limiting the NS5B polymerase activity of the hepatitis C virus, offering hope for the future of HCV treatment.

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Triterpenoids (C30-isoprenoids) represent a major group of natural products with various physiological functions in plants. Triterpenoids and their derivatives have medicinal uses owing to diverse bioactivities. Arjuna (Terminalia arjuna) tree bark accumulates highly oxygenated ß-amyrin-derived oleanane triterpenoids (e.g., arjunic acid, arjungenin, and arjunolic acid) with cardioprotective roles. However, biosynthetic routes and enzymes remain poorly understood. We mined the arjuna transcriptome and conducted cytochrome P450 monooxygenase (P450) assays using Saccharomyces cerevisiae and Nicotiana benthamiana to identify six P450s and two P450 reductases for oxidative modifications of oleanane triterpenoids. P450 assays using oleananes revealed a greater substrate promiscuity of C-2α and C-23 hydroxylases/oxidases than C-28 oxidases. CYP716A233 and CYP716A432 catalyzed ß-amyrin/erythrodiol C-28 oxidation to produce oleanolic acid. C-2α hydroxylases (CYP716C88 and CYP716C89) converted oleanolic acid and hederagenin to maslinic acid and arjunolic acid. CYP716C89 also hydroxylated erythrodiol and oleanolic aldehyde. However, CYP714E107a and CYP714E107b catalyzed oleanolic acid/maslinic acid/arjunic acid, C-23 hydroxylation to form hederagenin, arjunolic acid and arjungenin, and hederagenin C-23 oxidation to produce gypsogenic acid, but at a lower rate than oleanolic acid C-23 hydroxylation. Overall, P450 substrate selectivity suggested that C-28 oxidation is the first P450-catalyzed oxidative modification in the arjuna triterpenoid pathway. However, the pathway might branch thereafter through C-2α/C-23 hydroxylation of oleanolic acid. Taken together, these results provided new insights into substrate range of P450s and unraveled biosynthetic routes of triterpenoids in arjuna. Moreover, complete elucidation and reconstruction of arjunolic acid pathway in S. cerevisiae and N. benthamiana suggested the utility of arjuna P450s in heterologous production of cardioprotective compounds.

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ETHNOPHARMACOLOGICAL RELEVANCE: Neuroinflammation is involved in the pathogenesis of depression disorder by activating microglia cells, increasing proinflammatory cytokines, effecting serotonin synthesis and metabolism, and neuronal apoptosis and neurogenesis. Arjunolic acid (ARG) is a triterpenoid derived from the fruits of Akebia trifoliata for treating psychiatric disorders in TCM clinic, which exhibits anti-inflammatory and neuroprotective effects. However, its anti-depressive effect and underlying mechanism are unknown. AIM OF THE STUDY: The aim of this study is to explore the effect of arjunolic acid on depression and its possible mechanisms. METHODS: Intraperitoneal injection of LPS in mice and LPS stimulated-BV2 microglia were utilized to set up in vivo and in vitro models. Behavioral tests, H&E staining and ELISA were employed to evaluate the effect of arjunolic acid on depression. RT-qPCR, immunofluorescence, molecular docking and Western blot were performed to elucidate the molecular mechanisms. RESULTS: Arjunolic acid dramatically ameliorated depressive behavior in LPS-induced mice. The levels of BDNF and 5-HT in the hippocampus of the mice were increased, while the number of iNOS + IBA1+ cells in the brain were decreased and Arg1+IBA1+ positive cells were increased after arjunolic acid treatment. In addition, arjunolic acid promoted the polarization of BV2 microglia from M1 to M2 type. Notably, drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA) and molecular docking technologies identified SIRT1 as the target of arjunolic acid. Moreover, after SIRT1 inhibition by using EX-527, the effects of arjunolic acid on ameliorating LPS-induced depressive behavior in mice and promoting M2 Microglia polarization were blocked. In addition, arjunolic acid activated AMPK and decreased Notch1 expression, however, inhibition of AMPK, the effect of arjunolic acid on the downregulation of Notch1 expression were weaken. CONCLUSIONS: This study elucidates that arjunolic acid suppressed neuroinflammation through modulating the SIRT1/AMPK/Notch1 signaling pathway. Our study demonstrates that arjunolic acid might serve as a potiential anti-depressant.

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OBJECTIVE: The impact of the anti-depressant therapy on gonadal function has been recognized and discussed over the years. However, data to supplement our understanding of the impact of arjunolic acid (AA) therapies in protecting against FXT-induced gonadal dysfunction is lacking clear scientific evidence. Hence, this study aimed to investigate the possible effect of AA on fluoxetine-induced altered testicular function in rats. METHODS: After 14 days acclimatization, Thirty-six (36) adult male rats were randomly divided into 6 groups (n=6). Rats in groups 1 received normal saline (10mL/kg); groups 2 & 3 were given AA (1.0mg/kg body weight) and AA (2.0mg/kg body weight), respectively; whereas, rats in group 4 were given FXT (10mg/kg/p.o/day), and groups 5 & 6 were given a combination of FXT (10mg/kg) + AA (1.0mg/kg body weight); and FXT (10mg/kg) + AA (2.0mg/kg body weight), respectively. RESULTS: The results shows that FXT significantly altered testicular steroidogenic enzymes (3ß-HSD and 17ß-HSD) and proton pump ATPase (Na+/K+ ATPase, Ca2+ ATPase and H+ ATPase) activities, as well as testicular architecture when compared with controls. More so, FXT caused oxido-inflammation and apoptosis, as evidence by increases in MDA, MPO, TNF-α, IL-1ß, Caspase 3 and p53. However, AA at a different dose significantly ameliorated the destructive impacts of FXT on steroidogenic enzymes, proton pump ATPase as well as increased Bcl-2, SOD, CAT, GSH and improved testicular architecture in rats. CONCLUSIONS: AA reverses fluoxetine-induced alterations in testicular steroidogenic enzymes and membrane-bound ionic pump through suppression of oxido-inflammatory stress and apoptosis.

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BACKGROUND AND AIMS: Crohn's disease (CD) is characterized by an overabundance of epithelial cell death and an imbalance in microflora, both of which contribute to the dysfunction of the intestinal barrier. Arjunolic acid (AA) has anti-apoptotic effects and regulates microbiota efficacy. The objective of this study was to assess the impact of the treatment on colitis resembling Crohn's disease, along with exploring the potential underlying mechanism. METHODS: CD animal models were created using Il-10-/- mice, and the impact of AA on colitis in mice was evaluated through disease activity index, weight fluctuations, pathological examination, and assessment of intestinal barrier function. To clarify the direct role of AA on intestinal epithelial cell apoptosis, organoids were induced by LPS, and TUNEL staining was performed. To investigate the potential mechanisms of AA in protecting the intestinal barrier, various methods including bioinformatics analysis and FMT experiments were employed. RESULTS: The treatment for AA enhanced the condition of colitis and the function of the intestinal barrier in Il-10-/- mice. This was demonstrated by the amelioration of weight loss, reduction in tissue inflammation score, and improvement in intestinal permeability. Moreover, AA suppressed the apoptosis of intestinal epithelial cells in Il-10-/- mice and LPS-induced colon organoids, while also reducing the levels of Bax and C-caspase-3. In terms of mechanism, AA suppressed the activation of TLR4 signaling in Il-10-/- mice and colon organoids induced by LPS. In addition, AA increased the abundance of short-chain fatty acid-producing bacteria in the stool of Il-10-/- mice, and transplantation of feces from AA-treated mice improved CD-like colitis. CONCLUSIONS: The results of our study demonstrate that AA has a protective effect on the intestinal barrier in Crohn's disease-like colitis by preventing apoptosis. Additionally, this groundbreaking study reveals the capacity of AA to hinder TLR4 signaling and alter the makeup of the intestinal microbiome. The findings present fresh possibilities for treating individuals diagnosed with Crohn's disease. AA offers a hopeful novel strategy for managing Crohn's disease by obstructing crucial pathways implicated in intestinal inflammation and enhancing the gut microbiota.

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Rhoicissus tridentata is one of the most frequently used plants in preparing Isihlambezo, a herbal drink consumed by many South African women to induce labour and tone the uterus in pregnancy. This study aimed to identify the uteroactive compounds in this plant. Chromatographic purification of the methanol and water extracts from the roots yielded eight compounds, i.e. morin 3-O-α-L-rhamnopyranoside, trans-resveratrol 3-O-ß-glucopyranoside, a mixture of asiatic and arjunolic acids, quercetin 3-O-rhamnopyranoside, catechin, ß-sitosterol, and linoleic acid. All compounds were evaluated for their uterotonic effects using uterine smooth muscle isolated from stilboestrol-primed Sprague-Dawley rats. The mixture of asiatic and arjunolic acids showed the highest activity with EC50 of 0.02129 µg/mL for amplitude. These results validate the use of R. tridentata in ethnomedicine to facilitate labour in childbirth. Morin 3-O-α-L-rhamnopyranoside and trans-resveratrol 3-O-ß-glucopyranoside caused a relaxation of the uterine muscle, which suggests that some compounds in R. tridentata possess opposing activities.

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BACKGROUND: Arjunolic acid (AA) is a potent phytochemical with multiple therapeutics effects. In this study, AA is evaluated on type 2 diabetic (T2DM) rats to understand the mechanism of ß-cell linkage with Toll-like receptor 4 (TLR-4) and canonical Wnt signaling. However, its role in modulating TLR-4 and canonical Wnt/ß-catenin crosstalk on insulin signaling remains unclear during T2DM. Aim The current study is aimed to examine the potential role of AA on insulin signaling and TLR-4-Wnt crosstalk in the pancreas of type 2 diabetic rats. METHOD: Multiple methods were used to determine molecular cognizance of AA in T2DM rats, when treated with different dosage levels. Histopathological and histomorphometry analysis was conducted using masson trichrome and H&E stains. While, protein and mRNA expressions of TLR-4/Wnt and insulin signaling were assessed using automated Western blotting (jess), immunohistochemistry, and RT-PCR. RESULTS: Histopathological findings revealed that AA had reversed back the T2DM-induced apoptosis and necrosis caused to rats pancreas. Molecular findings exhibited prominent effects of AA in downregulating the elevated level of TLR-4, MyD88, NF-κB, p-JNK, and Wnt/ß-catenin by blocking TLR-4/MyD88 and canonical Wnt signaling in diabetic pancreas, while IRS-1, PI3K, and pAkt were all upregulated by altering the NF-κB and ß-catenin crosstalk during T2DM. CONCLUSION: Overall results, indicate that AA has potential to develop as an effective therapeutic in the treatment of T2DM associated meta-inflammation. However, future preclinical research at multiple dose level in a long-term chronic T2DM disease model is warranted to understand its clinical relevance in cardiometabolic disease.

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A series of new phenyl acetylene and isoxazole analogues of arjunolic acid were designed, synthesized and evaluated (3-8) for their tyrosinase and alpha glucosidase inhibitory potential. All the tested analogues exhibited stronger inhibitory activity than the standard drug or parent compound. Of these, compound (7) displayed the most potent tyrosinase inhibitory action with IC50 (14.3 ± 7.6) of about three folds more than the standard drug, kojic acid (41.5 ± 1.0). Further, compound (8) (14.5 ± 0.15) possessed the potent alpha glucosidase inhibitory action with IC50 value comparable to that of standard, acarbose (10.4 ± 0.06). Henceforth, compounds (7) and (8) are promising candidates for further studies.

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Lipopolysaccharide (LPS) is a glycolipid component of the cell wall of Gram-negative bacteria, which induces multiple organ dysfunctions, eventually leading to septic shock and death. Arjunolic acid (AA) has been shown to have therapeutic benefits against various organ pathophysiologies, although its role in sepsis remains unclear. Here, we evaluated the effects of AA on LPS-induced free radical production and cardiotoxicity. Male albino mice were allocated to four groups: normal, 1.5 µg/30 g b.w. of LPS (LPS), 20 mg/kg b.w. AA with LPS (AA+LPS) and 20 mg/kg b.w. of AA (AA). Subsequently, blood and heart samples were harvested for biochemical and histopathological examinations. Pretreatment with AA attenuated LPS-induced increased serum levels of cardiac troponin I, lactate dehydrogenase and creatine kinase. In the meantime, AA pretreatment before LPS resulted in a significant increase in endogenous antioxidants (superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione) and a significant decrease in the level of lipid peroxidation product (malondialdehyde) in the heart as compared to the LPS group, while cardiac cytochrome c activity were significantly increased. In addition, in the AA-pretreated mice, C-reactive protein and proinflammatory cytokines (interlukin-1 and tumor necrosis factor-alpha) were significantly reduced, and anti-inflammatory cytokines (interleukin-4 and -10) were significantly increased in cardiac tissues as compared to the LPS-treated animals. Furthermore, prior administration of AA to LPS exposed mice led to a significant a significant decrease in heart caspase-3, -8, and -9 as compared to the LPS group. Interestingly, AA was also able to improve LPS-induced histopathological changes in the cardiomyocytes. In conclusion, these in vivo findings indicate that AA may be a promising cardioprotective agent against LPS-stimulated cardiotoxicity, at least in part, through upregulation of cardiac antioxidants, reduction of lipid peroxidation, and inhibition of inflammation and cardiac cell death.

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ETHNOPHARMACOLOGICAL RELEVANCE: Cyclocarya paliurus (CP) is a traditional Chinese herb and possesses a variety of biological activities including anti-hyperglycemia, anti-hyperlipidemia, antioxidant and anti-inflammation. Arjunolic acid (AA) is an abundant and bioactive ingredient in CP that shows significant protection against many metabolic diseases such as diabetic complication. Diabetic retinopathy (DR) is a serious complication of diabetes and may lead to vision loss. However, the protective effects and underlying mechanisms of AA against DR is not still understood. AIM OF THE STUDY: We aimed to investigate whether AA activates AMPK/mTOR/HO-1 regulated autophagy pathway to alleviate DR. MATERIALS AND METHODS: In the study, the STZ-induced diabetic model of rats was established, and AA with 10 and 30 mg/kg dosages was given orally for ten weeks to investigate their effect on retinal injury of DR. H2O2-induced ARPE-19 cells were applied to evaluate anti-apoptosis and anti-oxidant effect of AA. RESULTS: The results revealed that AA could prevent STZ-induced weight loss and increase the retinal thickness and nuclei counts. The level of HO-1 protein was upregulated both in vivo and in vitro. In addition, AA prevented retinal damage and cell apoptosis through the AMPK-mTOR-regulated autophagy pathway. Furthermore, anti-apoptosis capacity, as well as the expression of HO-1 and LC3 protein, were effectively locked by AMPK inhibitor dorsomorphin dihydrochloride (compound C). CONCLUSIONS: This finding implies that AA may be a promising candidate drug by protecting retinal cells from STZ-induced oxidative stress and inflammation through the AMPK/mTOR/HO-1 regulated autophagy pathway.

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BACKGROUND: Type 2 diabetes mellitus (T2DM) is a worldwide health issue primarily due to failure of pancreatic ß-cells to release sufficient insulin. PURPOSE: The present work aimed to assess the antidiabetic potential of arjunolic acid (AA) isolated from Terminalia arjuna in type 2 diabetic rats. STUDY DESIGN: After extraction, isolation and purification, AA was orally administered to type 2 diabetic Sprague Dawley rats to investigate antidiabetic effect of AA. METHOD: T2DM was induced via single intraperitoneal injection of streptozotocin-nicotinamide (STZ-NIC) in adult male rats. After 10 days, fasting and random blood glucose (FBG and RBG), body weight (BW), food and water intake, serum C-peptide, insulin and glycated hemoglobin (HbA1c) was measured to confirm T2DM development. Dose dependent effects of orally administered AA (25 and 50 mg/kg/day) for 4 weeks was investigated by measuring BW variation, fasting and postprandial hyperglycemia, oral glucose tolerance test (OGTT), and levels of serum HbA1c, serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), serum and pancreatic C-peptide, insulin, growth differentiation factor 15 (GDF-15), serum and pancreatic inflammatory cytokines. RESULTS: The oral administration of AA in preclinical model of T2DM significantly normalized FBG and RBG, restored BW, controlled polyphagia, polydipsia and glucose tolerance. In addition, AA notably reduced serum HbA1c, TC, TG, LDL with non-significant increase in HDL. On the other hand, significant increase in serum and pancreatic C-peptide and insulin was observed with AA treatment, while serum and pancreatic GDF-15 were non-significantly altered in AA treated diabetic rats. Moreover, AA showed dose dependent reduction in serum and pancreatic proinflammatory cytokines including TNF-α, IL-1ß and IL-6. CONCLUSION: For the first time our findings highlighted AA as a potential candidate in type 2 diabetic conditions.

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Minimizing the adverse effects of chemotherapeutic agents remains a therapeutic challenge. Cisplatin (CP) induces hepatotoxicity through activation of oxidative stress, inflammation, and apoptosis cascades which is considered a significant drawback. Thus, this study aimed to highlight the possible hepatoprotective role of arjunolic acid (Arj) in a rat model of CP-induced hepatotoxicity. Four groups of rats were included; the normal control group, Arj control group, CP group which was injected with 7.5 mg/kg CP intraperitoneally to induce hepatotoxicity, and the treated group (Arj + CP), which was orally administered 20 mg/kg Arj for 10 days with a CP hepatotoxic dose on day 5. Blood and liver tissues were assembled for analysis at the end of the study. Pretreatment with Arj exhibited a marked improvement in liver function as well as histopathology when compared with the CP group. Moreover, Arj suppressed the oxidative stress in hepatic tissue by significantly decreasing malondialdehyde and nitric oxide contents along with markedly elevating the levels of superoxide dismutase, catalase, and reduced glutathione when compared with CP injected rats. Attenuation of hepatic inflammation and apoptosis was also reported with Arj treatment through the marked reduction in the proinflammatory cytokine tumor necrosis factor α level as well as the apoptotic marker caspase-3 protein expression in comparison to the CP group. This study explored for the first time the Arj hepatoprotective effect against CP-induced hepatotoxicity through its antioxidant, anti-inflammatory, and antiapoptotic activities.

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Apo lipoprotein-E (APOE) encoded by APOE gene, is a plasma glycoprotein of 34.15 kDa and has a significant genetic association in coronary artery disease (CAD) progression. The silent epidemic of different cardiovascular diseases including CAD challenges novel therapeutic alternatives to prevent to treat chronic conditions of the disease and its associated complications. It is believed that natural phyto compounds and extracts have been a potential source of treating health conditions and have been practiced since several decades. The aim of the study is to identify phyto compounds having significant cardio protective activity targeting APOE4. Since protein-ligand interactions play a leading role in structure-based drug design, with the help of molecular docking, we selected 20 phyto chemicals present in different plants and investigated their binding affinity against targeted APOE isoforms. Among all selected phytoc ompounds, arjunolic acid, from Terminalia arjuna plant was found as promising candidate for developing therapeutic against APOE4 activated CAD. Findings from the present work could be further studied for clinical evaluations on human to adopt strategies and reduce the prevalence and mortality. Arjunolic acid derivatives can be used as a source of new medication or development of novel compounds in the treatment of CAD.

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ETHNOPHARMACOLOGICAL RELEVANCE: Plumeria rubra L. (Apocynaceae) is a deciduous, commonly ornamental, tropical plant grown in home premises, parks, gardens, graveyards, because of its beautiful and attractive flowers of various colours and size. The different parts of the plant are used traditionally to treat various diseases and conditions like leprosy, inflammation, diabetic mellitus, ulcers, wounds, itching, acne, toothache, earache, tongue cleaning, pain, asthma, constipation and antifertility. AIM OF THE REVIEW: The main aim of this review is to provide an overview and critically analyze the reported ethnomedical uses, phytochemistry, pharmacological activities and toxicological studies of P. rubra and to identify the remaining gaps and thus supply a basis for further investigations. The review also focuses towards drawing attention of people and researchers about the wide spread pharmaceutical properties of the plant for its better utilization in the coming future. MATERIAL AND METHODS: All the relevant data and information on P. rubra was gathered using various databases such as PubMed, Springer, Taylor and Francis imprints, NCBI (National Center for Biotechnology Information), Science direct, Google scholar, Chemspider, SciFinder, research and review articles from peer-reviewed journals and unpublished data such as Phd thesis, etc. Some other 'grey literature' sources such as webpages, ethnobotanical books, chapters, wikipedia were also studied. RESULTS: More than 110 chemical constituents have been isolated from P. rubra including iridoids, terpenoids, flavonoids and flavonoid glycosides, alkaloids, glycosides, fatty acid esters, carbohydrates, animo acids, lignan, coumarin, volatile oils, etc. The important chemical constituents responsible for pharmacological activities of the plant are fulvoplumierin, plumieride, rubrinol, lupeol, oleanolic acid, stigmasterol, taraxasteryl acetate, plumieride-p-E-coumarate, rubranonoside, rubrajalellol, plumericin, isoplumericin, etc. The plant possess a wide range of pharmacological activities present namely antibacterial, antiviral, anti-inflammatory, antipyretic, antidiabetic, hepatoprotective, anticancer, anthelmintic, antifertility and many other activities. CONCLUSION: P. rubra is a valuable medicinal source and further study in this topic can validate the traditional and ethnobotanical use of the plant. However, many aspects of the plant have not been studied yet. The pharmacological activity of active chemical constituent isolated from the plant is proven only for a couple of activities hence, lack of bio-guided isolation strategies is observed. Further studies on bioavailability, pharmacokinetics, mechanism of action and structural activity relationship studies of isolated pure compounds will contribute more in understanding their pharmacological effects. Higher doses of plant extracts are administered to experimental animals, therefore their toxicity and side effects in humans are needed to be thoroughly studied, although no side effect or toxicity is seen or observed in experimental animals. Studies are also essential to investigate the long term in vivo toxicity and clinical efficacy of the plant.

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Arjunolic acid (AA) a plant derived pentacyclic triterpenoid which showed effective anticancer activity against MCF-7 and HeLa cells as well as no significant toxic effect was observed against normal lymphocytes. In the current study the self assemble property of arjunolic acid gives an extra emphasis on anticancer activity which was proved by several fluorescence studies like ROS generation, EtBr/AO and DAPI staining. At a selected dose of 50µg/ml AA disrupt the redox balance inside the cancer cells by producing reactive oxygen species. The apoptotic event was mediated by two key regulator proteins TNF-α and NF-κß which was proved here. The increment of the pro-inflammatory cytokines indicates the ROS mediated pathway of cancer cell apoptosis.

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BACKGROUND: Arjunolic acid (AA) is a potent phytochemical with wider pharmacological activities. Despite potential medicinal properties on various in vitro and in vivo studies, there is still a dearth of scientific data related to its safety profile and toxicological parameters. The current study aimed to investigate acute toxicity of AA in normal female Sprague Dawley rats. METHODS: In this study, AA was administered orally at an individual dose of 300 and 2000 mg/kg body weight to group 1 and 2 respectively, while group 3 served as normal control. All the animals were observed for 2 weeks to determine any behavioral and physical changes. On day 15, blood was collected for hematological and biochemical investigation, later animals from all the three groups were euthanized to harvest and store essential organs for histopathological analysis. Four different staining techniques; hematoxylin and eosin, Masson trichrome, Periodic acid Schiff and Oil O Red were used to investigate any alterations in different tissues through microscopical observation. RESULTS: The results of the study showed no morbidity and mortality at two different dosage of AA treatment. Daily food & water intake, body weight, relative organ weight, hematological and biochemical parameters were detected to be normal with no severe alteration seen through microscopical investigation in the structure of harvested tissues. Our findings support the safety profile of AA, which was well tolerated at higher dose. Thus, an in-detail study on the subacute disease model is warranted.

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Continuation of prolonged treatment against arsenicosis with conventional chelating therapy is a global challenge. The present study was intended to evaluate the defensive effect of arjunolic acid against arsenic-induced oxidative stress and female reproductive dysfunction. Wistar strain adult female rats were given sodium arsenite (10 mg/kg body weight) in combination with arjunolic acid (10 mg/kg body weight) orally for two estrous cycles. Electrozymographic analysis explored that arjunolic acid co-treatment counteracted As3+-induced ROS production in uterine tissue by stimulating the activities of endogenous enzymatic antioxidants. Arjunolic acid was able to enhance the protection against mutagenic uterine DNA breakage, necrosis, and ovarian-uterine tissue damages in arsenicated rats by improving the ovarian steroidogenesis. The mechanisms might be coupled with the augmentation of antioxidant defense system, partly through the elimination of arsenic with the involvement of S-adenosyl methionine pool where circulating levels of vitamin B12, folic acid, and homocysteine play critical roles as evidenced from our present investigation.

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BACKGROUND: Mutations play a major role in the pathogenesis and development of several chronic degenerative diseases including cancer. It follows, therefore that antimutagenic compound may inhibit the pathological process resulting from exposure to mutagens. Investigation of the antimutagenic potential of traditional medicinal plants and compounds isolated from plant extracts provides one of the tools that can be used to identify compounds with potential cancer chemopreventive properties. The aim of this study was to isolate and characterise the compounds responsible for the antimutagenic activity of Combretum microphyllum. METHODS: The methanol leaf extract of C. microphyllum was evaluated for antimutagenicity in the Ames/microsome assay using Salmonella typhimurium TA98. TA100 and TA102. Solvent-solvent fractionation was used to partition the extracts and by using bioassay-guided fractionation, three compounds were isolated. The antimutagenic activity of the three compounds were determined in the Ames test using Salmonella typhimurium TA98, TA100 and TA102. The antioxidant activity of the three compounds were determined by the quantitative 2,2-diphenyl-1-picrylhydrazyl (DPPH)-free radical scavenging method. The cytotoxicity was determined in the MTT assay using human hepatocytes. RESULTS: A bioassay-guided fractionation of the crude extracts for antimutagenic activity led to the isolation of three compounds; n-tetracosanol, eicosanoic acid and arjunolic acid. Arjunolic acid was the most active in all three tested strains with a antimutagenicity of 42 ± 9.6%, 36 ± 1.5% and 44 ± 0.18% in S. typhimurium TA98, TA100 and TA102 respectively at the highest concentration (500 µg/ml) tested, followed by eicosanoic acid and n-tetracosanol. The antioxidant activity of the compounds were determined using the quantitative 2,2 diphenyl-1-picryhydrazyl (DPPH)-free radical scavenging method. Only arjunolic acid had pronounced antioxidant activity (measured as DPPH-free scavenging activity) with an EC50 value of 0.51 µg/ml. The cytotoxicity of the isolated compounds were determined in the MTT assay using human hepatocytes. The compounds had low cytotoxicity at the highest concentration tested with LC50 values >200 µg/ml for n-tetracosanol and eicosanoic acid and 106.39 µg/ml for arjunolic acid. CONCLUSIONS: Based on findings from this study, compounds in leaf extracts of C. microphyllum protected against 4-NQO and MMC induced mutations as evident in the Ames test. The antimutagenic activity of arjunolic acid may, at least in part, be attributed to its antioxidant activity resulting in the detoxification of reactive oxygen species produced during mutagenesis.

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Cardiac hypertrophy and associated heart fibrosis remain a major cause of death worldwide. Phytochemicals have gained attention as alternative therapeutics for managing cardiovascular diseases. These include the extract from the plant Terminalia arjuna, which is a popular cardioprotectant and may prevent or slow progression of pathological hypertrophy to heart failure. Here, we investigated the mode of action of a principal bioactive T. arjuna compound, arjunolic acid (AA), in ameliorating hemodynamic load-induced cardiac fibrosis and identified its intracellular target. Our data revealed that AA significantly represses collagen expression and improves cardiac function during hypertrophy. We found that AA binds to and stabilizes the ligand-binding domain of peroxisome proliferator-activated receptor α (PPARα) and increases its expression during cardiac hypertrophy. PPARα knockdown during AA treatment in hypertrophy samples, including angiotensin II-treated adult cardiac fibroblasts and renal artery-ligated rat heart, suggests that AA-driven cardioprotection primarily arises from PPARα agonism. Moreover, AA-induced PPARα up-regulation leads to repression of TGF-ß signaling, specifically by inhibiting TGF-ß-activated kinase1 (TAK1) phosphorylation. We observed that PPARα directly interacts with TAK1, predominantly via PPARα N-terminal transactivation domain (AF-1) thereby masking the TAK1 kinase domain. The AA-induced PPARα-bound TAK1 level thereby shows inverse correlation with the phosphorylation level of TAK1 and subsequent reduction in p38 MAPK and NF-κBp65 activation, ultimately culminating in amelioration of excess collagen synthesis in cardiac hypertrophy. In conclusion, our findings unravel the mechanism of AA action in regressing hypertrophy-associated cardiac fibrosis by assigning a role of AA as a PPARα agonist that inactivates non-canonical TGF-ß signaling.

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The optimization and microwave assisted extraction of stem bark of Terminalia arjuna, quantitative estimation of the marker compounds arjunic acid and arjunolic acid using HPTLC and the evaluation of free radical scavenging activity has been performed in this study. The central composite design was used for optimization and the values of parameters for optimized batch of microwave assisted extraction were 1000W (Power), 3 minutes (Time) and 1/120 (Solid/solvent ratio). The solvent system to carry out the HPTLC was toluene: acetic acid: ethyl acetate (5: 5: 0.5) and quantitative estimation was done using standard equations obtained from the marker compounds. The in-vitro free radical scavenging activity was performed spectrophotometrically using ascorbic acid as standard. The value of estimated percentage yield of arjunic acid and arjunolic acid was 1.42% and 1.52% which upon experimentation was obtained as 1.38% and 1.51% respectively. The DPPH assay of the different batches of microwave assisted extraction and marker compounds taken suggested that the marker compounds arjunic acid and the arjunolic acid were responsible for the free radical scavenging activity as the batch having the maximum percentage yield of the marker compounds showed best free radical scavenging effect as compared to standard ascorbic acid. The IC₅₀ value of the optimized batch was found to be 24.72 while that of the standard ascorbic acid was 29.83. Hence, the yield of arjunic acid and arjunolic acid has direct correlation with the free radical scavenging activity of stem bark extract of Terminalia arjuna and have potential to serve as active lead compounds for free radical scavenging activity.

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