Synthesis and in-vitro anti-HIV-1 evaluation of novel pyrazolo[4,3-c]pyridin-4-one derivatives.

In our continuing efforts to find novel anti-HIV compounds, we have synthesized sixteen novel pyrazolo[4,3-c]pyridin-4-one derivatives. All the synthesized compounds were screened for anti-HIV activity against HIV-1VB59 (R5, subtype C). Compounds 12a-12c and 12e were also tested against HIV-1UG070 (X4, subtype D) in TZM-bl cell line. Compound 12c was found to be the most active against HIV-1VB59 and HIV-1UG070 with IC50 value 3.67 µM and 2.79 µM, and therapeutic indices 185 and 243, respectively. The lead compound 12c inhibited the HIV-192/BR/018 (R5, subtype B) and drug resistant isolates, NIH-119 (X4/R5, subtype B) and NARI-DR (R5, subtype C) effectively. The activity of the lead compound was further confirmed by PBMC assays. The molecular docking data showed that the most active compound 12c binds in the non-nucleoside binding pocket of HIV-1 reverse transcriptase, which was confirmed by the ToA assay. Thus the study indicated that 12c may be considered as a NNRTI and further explored as a lead for anti-HIV drug development.

Synthesis of C-2 and C-3 substituted quinolines and their evaluation as anti-HIV-1 agents.

A plenty of natural products and synthetic derivatives containing quinoline moiety have been reported to possess various pharmacological activities. Quinolines such as 2-styrylquinolines and 8-hydroxyquinolines are extensively studied for their anti-HIV-1 activity and found to act mainly through HIV-1 integrase enzyme inhibition. In continuation of our efforts to search for newer anti-HIV-1 molecules, thirty-one quinoline derivatives with different linkers to ancillary phenyl ring were synthesized and evaluated for in vitro anti-HIV-1 activity using TZM-bl assays. Compound 31 showed higher activity in TZM-bl cell line against HIV-1VB59 and HIV-1UG070 cell associated virus (IC50 3.35 ±â€¯0.87 and 2.57 ±â€¯0.71 µM) as compared to other derivatives. Compound 31 was further tested against cell free virus HIV-1VB59 and HIV-1UG070 (IC50 1.27 ±â€¯0.31 and 2.88 ±â€¯1.79 µM, TI 42.20 and 18.61, respectively). This lead molecule also showed good activity in viral entry inhibition assay and cell fusion assay defining its mode of action. The activity of compound 31 was confirmed by testing against HIV-1VB51 in activated peripheral blood mononuclear cells (PBMCs). Binding interactions of 31 were compared with known entry inhibitors.

Finger millet arabinoxylan protects mice from high-fat diet induced lipid derangements, inflammation, endotoxemia and gut bacterial dysbiosis.

Arabinoxylan (AX), a non-starch polysaccharide extracted from cereals such as wheat, rice and millets, is known to impart various health promoting effects. Our earlier study suggested that finger millet (FM) could ameliorate high fat diet (HFD)-induced metabolic derangements. The present study is aimed to evaluate the effect of FM-AX supplementation, a key bioactive from finger millet, on HFD-induced metabolic and gut bacterial derangements. Male Swiss albino mice were fed with normal chow diet (NPD) or HFD (60%kcal from fat) for 10 weeks. FM-AX was orally supplemented at doses of 0.5 and 1.0g/kg bodyweight on every alternate day for 10 weeks. Glucose tolerance, serum hormones, hepatic lipid accumulation and inflammation, white adipose tissue marker gene expression, adipocyte size and inflammation; metagenomic alterations in cecal bacteria; cecal short chain fatty acids and colonic tight junction gene expressions were studied. FM-AX supplementation prevented HFD-induced weight gain, alerted glucose tolerance and serum lipid profile, hepatic lipid accumulation and inflammation. Hepatic and white adipose tissue gene expressions were beneficially modulated. Further, AX supplementation prevented metagenomic alterations in cecum; improved ileal and colonic health and overall prevented metabolic endotoxemia. Present work suggests that AX from finger millet can be developed as a nutraceutical for the management of HFD- induced obesity.

Sameerpannag Ras Mixture (SRM) improved neurobehavioral deficits following acute ischemic stroke by attenuating neuroinflammatory response.

ETHNOPHARMACOLOGICAL RELEVANCE: Cerebral ischemic stroke is one of the leading causes of death and long-term disability worldwide. Unfortunately, due to the failure of most of drugs in clinical trials recently, attentions have moved towards the traditional system of medicines including Ayurveda. In Ayurveda, Sameerpannag Ras (SR) is a mineral and metallic origin based formulation which has been used for the treatment of arthritis and chronic systematic inflammatory disorder. The current study was designed to investigate the neuroprotective effects of Sameerpannag Ras Mixture (SRM), on the neurobehavioral dysfunction and associated neuroinflammation, induced by transient Internal Carotid Artery Occlusion (ICAO) in mice model. MATERIALS AND METHODS: In the present study, mice were treated with Sameerpannag Ras Mixture (SRM) at the dose of 40mg/kg body weight by oral gavages for 3 and 7days respectively, twice a day, after the induction of ICAO for 90min followed by reperfusion. The efficacy of SRM was examined by scoring neurological behavioral deficit using the standard neurological deficit score (NDS), grip strength and rotarod performance tests at different time intervals of post-ICAO. RESULTS: Post-ischemic treatment with Sameerpannag Ras Mixture (SRM) at 40mg/kg significantly reduced Neurological Deficit Score and improved the motor coordination at different time intervals post-ICAO. The analysis of RT-qPCR data showed that transient cerebral ischemia could induce the inflammatory response genes in the affected striatal region of ICAO group, as compared to sham group, on day3 and day7 post-ICAO. Interestingly, SRM treatment showed marked improvement in the ischemia-induced neurobehavioral deficits by attenuating ischemia-induced neuroinflammatory response at both gene and protein level. CONCLUSION: The present study suggests that Sameerpannag Ras Mixture (SRM) treatment ameliorates behavioral outcomes after mild ischemia through the suppression of a number of inflammatory response genes involved in neuronal damage. This is the first report of the molecular mechanism underlying the significant neuroprotective action of the Ayurvedic drug, Sameerpannag Ras Mixture (SRM), using a mild stroke in mice model.

Screening of six Ayurvedic medicinal plants for anti-obesity potential: An investigation on bioactive constituents from Oroxylum indicum (L.) Kurz bark.

ETHNOPHARMACOLOGICAL RELEVANCE: As an effort to identify newer anti-obesity lead(s) we have selected 13 plant materials from the six plant species which have been reported in Indian Ayurvedic medicine as remedy against complications affecting glucose and lipid homeostasis. AIM OF THE STUDY: In vitro screening of six Indian Ayurvedic medicinal plants on anti-adipogenic and pancreatic lipase (PL) inhibition potential followed by bioactivity guided isolation from most active plant material. MATERIALS AND METHODS: In vitro anti-adipogenic assay using 3T3-L1 preadipocytes and pancreatic lipase (PL) inhibition assay were performed for hexanes, dichloromethane, ethyl acetate and methanolic extracts of all the plant materials. Bioactivity guided isolation approach was used to identify active constituent for anti-adipogenesis and PL inhibition assay. Inhibition of lipid accumulation and adipogenic transcription factor was measured by oil Red 'O' staining and quantitative real-time PCR method respectively. RESULTS: Ethyl acetate extract of Oroxylum indicum bark was found to be most active in screening of anti-adipogenesis (59.12±1.66% lipid accumulation as compared to control at 50µg/mL dose) and PL inhibition (89.12±6.87% PL inhibition at 250µg/mL dose) assays. Further, three bioactive flavonoids were isolated and identified as oroxylin A, chrysin and baicalein from O. indicum bark. Oroxylin A, chrysin, and baicalein were inhibited lipid accumulation in 3T3-L1 preadipocytes (75.00±5.76%, 70.21±4.23% and 77.21±5.49% lipid accumulation respectively in comparison to control at 50µM dose) and PL enzyme (69.86±2.96%, 52.08±2.14% and 45.06±2.42% PL inhibition respectively at 250µg/mL dose). In addition, oroxylin A and chrysin also inhibited PPARγ and C/EBPα, major adipogenic transcription factors, in 3T3L-1 preadipocytes during adipogenesis process at 50µM dose. CONCLUSION: The present study augurs the anti-obesity potential of well practiced Ayurvedic herb O. indicum and its flavonoids.

Effect of mahanimbine, an alkaloid from curry leaves, on high-fat diet-induced adiposity, insulin resistance, and inflammatory alterations.

Spices and condiments, small but an integral part of the daily diet, are known to affect physiological functions. This study evaluated the effects of mahanimbine, a major carbazole alkaloid from Murraya koenigii (curry leaves), against progression of high-fat diet (HFD)-induced metabolic complications in mice (male and female). Mahanimbine at 2 mg/kg (HFD + LD) and 4 mg/kg (HFD + HD) of body weight was administered daily along with HFD feeding for 12 weeks. At the end of the study, male HFD + LD and HFD + HD groups showed 51.70 ± 3.59% and 47.37 ± 3.73% weight gain, respectively, as compared with 71.02 ± 6.04% in HFD fed mice whereas female HFD + LD and HFD + HD groups showed 24.31 ± 1.68% and 25.10 ± 2.61% weight gain as compared with HFD group with 36.69 ± 3.60% of weight gain. Mahanimbine prevented HFD-induced hyperlipidemia and fat accumulation in adipose tissue and liver along with the restricted progression of systemic inflammation and oxidative stress. Moreover, mahanimbine treatment improved glucose clearance and upregulated the expression of insulin responsive genes in liver and adipose tissue. Male and female mice showed different traits in development of HFD-induced metabolic disturbances; however, mahanimbine treatment exerted similar effects in both the sexes. In addition, mahanimbine lowered the absorption of dietary fat resulting in dietary fat excretion. In conclusion, daily consumption of mahanimbine and thereby curry leaves may alleviate development of HFD-induced metabolic alterations. © 2016 BioFactors, 43(2):220-231, 2017.

Oleanolic acid analogs as NO, TNF-α and IL-1ß inhibitors: synthesis, biological evaluation and docking studies.

A series of oleanolic acid analogs, characterized by structural modifications at position C-3 and C-28 of oleanane skeleton were synthesized and assessed for antiinflammatory potential towards lipopolysaccharide (LPS) induced nitric oxide (NO) production in macrophages. Results revealed that all the synthesized analogs of oleanolic acid inhibit NO production with an IC50 of 2.66-41.7 µM as compared to the specific nitric oxide synthase (NOS) inhibitor, L-NAME (IC50=69.21 and 73.18 µM on RAW 264.7 and J774A.1 cells, respectively) without affecting the cell viability when tested at their half maximal concentration. The most potent NO inhibitors (2, 8, 9 and 10) at a concentration of 20 µg/mL also demonstrated mild inhibition (27.9-51.9%) of LPS-induced tumor necrosis factor alpha (TNF-α) and weak inhibition (11.1-37.5%) towards interleukin 1-beta (IL-1ß) production in both the cells. The present study paves a direction that analogs of oleanolic acid can be employed as a lead in the development of potent NO inhibitors. Molecular docking studies also showed that 10 (with top Goldscore docking pose 19.05) showed similar interaction as that of co-crystallized inhibitor and, thereby, helps to design the potent inhibitors of TNF-α.

Synthesis of new heterocyclic lupeol derivatives as nitric oxide and pro-inflammatory cytokine inhibitors.

A series of heterocyclic derivatives including indoles, pyrazines along with oximes and esters were synthesized from lupeol and evaluated for anti-inflammatory activity through inhibition of lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 and J774A.1 cells. All the synthesized molecules of lupeol were found to be more active in inhibiting NO production with an IC50 of 18.4-48.7 µM in both the cell lines when compared to the specific nitric oxide synthase (NOS) inhibitor, L-NAME (IC50=69.21 and 73.18 µM on RAW 264.7 and J774A.1 cells, respectively). The halogen substitution at phenyl ring of indole moiety leads to potent inhibition of NO production with half maximal concentration ranging from 18.4 to 41.7 µM. Furthermore, alkyl (11, 12) and p-bromo/iodo (15, 16) substituted compounds at a concentration of 20 µg/mL exhibited mild inhibition (29-42%) of LPS-induced tumor necrosis factor alpha (TNF-α) and weak inhibition (10-22%) towards interleukin 1-beta (IL-1ß) production in both the cell lines. All the derivatives were found to be non-cytotoxic when tested at their IC50 (µM). These findings suggest that the derivatives of lupeol could be a lead to potent inhibitors of NO.

Antioxidant activity of a new phenolic glycoside from Lagenaria siceraria Stand. fruits.

The antioxidant properties of different extracts of Lagenaria siceraria (bottle gourd) fruit were evaluated. In the process, a new phenolic glycoside (E)-4-hydroxymethyl-phenyl-6-O-caffeoyl-ß-d-glucopyranoside (1) was isolated and identified together with 1-(2-hydroxy-4-hydroxymethyl)-phenyl-6-O-caffeoyl-ß-d-gluco-pyranoside (2), protocatechuic acid (3), gallic acid (4), caffeic acid (5) and 3,4-dimethoxy cinnamic acid (6). Their structures were elucidated by extensive NMR experiments including (1)H-(1)H (COSY) and (1)H-(13)C (HMQC and HMBC) spectroscopy and chemical evidences. The antioxidant potential of the compound 1 and 2 was tested in different in vitro assay systems such as free radical scavenging assay, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, superoxide scavenging activity, reducing power assay and linoleic acid peroxidation assay.

Anti-HIV activity of Indian medicinal plants.

Acquired immunodeficiency syndrome patients face great socio-economic difficulties in obtaining treatment. There is an urgent need for new, safe, and cheap anti-HIV agents. Traditional medicinal plants are a valuable source of novel anti-HIV agents and may offer alternatives to expensive medicines in future. Various medicinal plants or plant-derived natural products have shown strong anti-HIV activity and are under various stages of clinical development in different parts of the world. The present study was directed towards assessment of anti-HIV activity of various extracts prepared from Indian medicinal plants. The plants were chosen on the basis of similarity of chemical constituents with reported anti-HIV compounds or on the basis of their traditional usage as immunomodulators. Different extracts were prepared by Soxhlet extraction and liquid-liquid partitioning. Ninety-two extracts were prepared from 23 plants. Anti-HIV activity was measured in a human CD4+ T-cell line, CEM-GFP cells infected with HIV-1NL4.3. Nine extracts of 8 different plants significantly reduced viral production in CEM-GFP cells infected with HIV-1NL4.3. Aegle marmelos, Argemone mexicana, Asparagus racemosus, Coleus forskohlii, and Rubia cordifolia demonstrated promising anti-HIV potential and were investigated for their active principles.

Synthesis of 9-substituted derivatives of berberine as anti-HIV agents.

Naturally occurring protoberberine alkaloids, berberine and berberrubine along with 9-substituted derivatives of berberine were assessed for the anti-human immunodeficiency virus (HIV) activity. Berberine was found to be the most active compound with an EC(50) of 0.13 µM against HIV-1 NL4.3 virus in CEM-GFP cell lines. Berberrubine and two other compounds were found to be less active than berberine, at the same time they were less toxic than berberine. Enzyme based assay suggested that the anti-HIV activity of berberine and its analogs might be due to RTase inhibitory activity and some additional mechanisms.

Design and synthesis of caffeoyl-anilides as portmanteau inhibitors of HIV-1 integrase and CCR5.

Designing multi-functional ligands is a recent strategy by which multiple targets can be inhibited by a single entity. A series of caffeoyl-anilide compounds based on structures of various integrase and CCR-5 inhibitors have been designed and synthesized as anti-HIV agents in the present study. Most of the compounds exhibited potent anti-HIV activity at micromolar concentration in CEM-GFP CD4+ T cells infected with HIV-1NL4.3 virus. Compound 14 showed a lower EC(50) and better TI as compared to AZT. Mechanism based studies suggest that these compounds inhibit either one or in some cases, both the targets. The experimental data and the docking results showed that these compounds are potential inhibitors for both HIV-1 IN and CCR5.

Antiobesity and lipid lowering effects of Murraya koenigii (L.) Spreng leaves extracts and mahanimbine on high fat diet induced obese rats.

The dichloromethane (MKD) and ethyl acetate (MKE) extracts of Murraya koenigii leaves significantly reduced the body weight gain, plasma total cholesterol (TC) and triglyceride (TG) levels significantly when given orally at a dose of 300 mg/kg/day to the high fat diet (HFD) induced obese rats for 2 weeks. The observed antiobesity and antihyperlipidemic activities of these extract are correlated with the carbazole alkaloids present in them. Mahanimbine (1) when given orally (30 mg/kg/day) also significantly lowered the body weight gain as well as plasma TC and TG levels. These findings demonstrate the excellent pharmacological potential of mahanimbine to prevent obesity.

Effects of Chlorophytum arundinaceum, Asparagus adscendens and Asparagus racemosus on pro-inflammatory cytokine and corticosterone levels produced by stress.

Chlorophytum arundinaceum, Asparagus adscendens and Asparagus racemosus are used in the Indian traditional system of medicine for improving the general state of health and for stress-related immune disorders. The effects of the methanol and aqueous extracts of the tuberous roots of these plants were examined in an experimental mouse model of stress, induced by swimming. The extracts were shown to exert an inhibitory effect on pro-inflammatory cytokines, namely interleukin 1ß and tumour necrosis factor α, and on the production of nitric oxide in mouse macrophage cells RAW 264.7 stimulated by lipopolysaccharide in vitro. Similar inhibition was also observed in the production of interleukin 2 in EL 4 lymphoma cells stimulated by concanavalin A. Corticosterone levels in serum and adrenal glands were measured. The findings suggest that these plants may be beneficial in the management of stress and inflammatory conditions.

Chemical changes during fermentation of Abhayarishta and its standardization by HPLC-DAD.

Abhayarishta is an Ayurvedic formulation prepared traditionally by the fermentation of the decoction of Terminalia chebula (pericarp), Vitis vinifera (fruits), Embelia ribes (fruits) and Madhuca indica (flowers). In the present communication, chemical changes occurring during fermentation in Abhayarishta have been studied for the purpose of its standardization. An HPLC-DAD method for quantitative estimation of selected marker constituents in the formulation has been developed and validated. A comparison of decoction and final processed formulation revealed that major polyphenolics (chebulagic and chebulinic acid) of T. chebula were hydrolyzed to their respective monomers and, consequently, there was an increase in the amount of chebulic acid, gallic acid, ellagic acid and ethyl gallate after fermentation. 5-Hydroxymethyl furfural (5-HMF) was also found in the formulation. Thus, emphasis is laid upon consideration of processing methods of formulation which has been lacking in the standardization of most of Ayurvedic formulations.

RP-HPLC analysis of Jirakadyarishta and chemical changes during fermentation.

Jirakadyarishta, an Ayurvedic formulation prepared by the fermentation of a decoction of Cuminum cyminum (seeds) is traditionally used for intestinal disorders. RP-HPLC analysis of the decoction and the final processed formulation revealed that apigenin-7-O-[galacturonide (1 --> 4)-O-glucoside] and luteolin-4'-O-glucoside-7-O-galacturonide) were the two major constituents of the decoction of C. cyminum. Selective hydrolysis of 7-O-glucosides of luteolin and apigenin during fermentation resulted in an increase in the amount of luteolin and apigenin. The 4'-O-glucoside-7-O-galacturonide of luteolin and galacturonide derivative of apigenin were not hydrolyzed during fermentation. Monomeric phenolics, together with 5-hydroxymethyl furfural (5-HMF), were also introduced into the formulation through the jaggery and other plant materials during fermentation. This communication highlights the importance of the ancient processing methods used in Ayurveda.

Anti-HIV diterpenes from Coleus forskohlii.

Various extracts of the aerial parts of Coleus forskohlii (Labiatae) were prepared and evaluated at their non cytotoxic concentration against HIV-1 NL4-3. Chloroform, ethyl acetate and n-butanol extracts showed 45.6, 66.5 and 37.7% inhibition of HIV, respectively in CEM-GFP cells infected with HIV-1(NL4-3) at 5 microg/mL. Four diterpenes, 1-deoxyforskolin, 1,9-dideoxyforskolin, forskolin and isoforskolin were isolated from the chloroform extract and tested against the virus. Six semi-synthetic derivatives of forskolin have been prepared to study SAR. 1-Deoxyforskolin and forskolin were found to be active against HIV(NL4-3). This is first report of anti HIV activity of this plant and its isolated constituents.

Pancreatic lipase inhibitory alkaloids of Murraya koenigii leaves.

In the continuing search for newer pancreatic lipase inhibitors from plants, a total of 63 extracts from 21 different plants were screened to study their pancreatic lipase (PL) inhibitory activity in vitro. All three extracts (DCM, EtOAc and MeOH) of Murraya koenigii (L.) Spreng leaves (Rutaceae) exhibited antilipase activity greater than 80%. Further, bioactivity guided fractionation of the EtOAc extract led to the isolation of four alkaloids, namely mahanimbin, koenimbin, koenigicine and clausazoline-K, with IC50 values of 17.9 microM, 168.6 microM, 428.6 microM and <500 microM, respectively. This study reports for the first time the PL inhibitory potential of carbazole alkaloids from plants.

Effect of Symplocos racemosa Roxb. on gonadotropin release in immature female rats and ovarian histology.

In the present study we are reporting in vivo effect of aqueous extracts of Symplocos racemosa Roxb. (Fam. Symplocaceae) on serum FSH and LH levels in immature female Sprague-Dawley rats under basal conditions. Symplocos racemosa is used in Indian System of Medicine (ISM) for various female disorders. Aqueous extract on oral administration significantly stimulated serum FSH level (P < 0.016) along with the rise in serum LH level (P < 0.001). Moreover, histopathological studies revealed enhanced folliculogenesis, presence of mature follicles and detached oocytes, which are result of increased FSH and LH levels. Further, an increase in the ovary weight of treated animals was found due to observed FSH surge. These results are in concordance with the traditional use of the drug for female disorders.