Modulation of TCA cycle enzymes and electron transport chain systems in experimental lung cancer

The modulatory effect of Withania somnifera along with paclitaxel on tricarboxylic acid (TCA) cycle key enzymes and electron transport chain complexes were investigated against lung cancer induced by benzo(a)pyrene in Swiss albino mice. Decreased activities of TCA cycle key enzymes such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) in lung cancer bearing animals were observed. Upon W. somnifera along with paclitaxel administration the above biochemical changes were inclined towards normal control animal values. Activities of mitochondrial enzymes and electron transport complexes were analyzed in the experimental groups to determine the efficiency of energy production. This study further confirms the chemotherapeutic effect of W. somnifera along with paclitaxel which is found to be more effective in the treatment of lung cancer. Thus these results are consistent with our hypothesis that the combination chemotherapy of W. somnifera along with paclitaxel as a promising chemotherapeutic agent.     

Suppressive effect of Withania somnifera root powder on experimental gouty arthritis: An in vivo and in vitro study

The effect of Withania somnifera L. Dunal root powder on paw volume and serum lysosomal enzyme activities was investigated in monosodium urate crystal-induced rats. The levels of beta-glucuronidase and lactate dehydrogenase were also measured in monosodium urate crystal incubated polymorphonuclear leucocytes (PMNL). A significant increase in the level of paw volume and serum lysosomal enzymes was observed in monosodium urate crystal-induced rats. The increased beta-glucuronidase and lactate dehydrogenase level were observed in untreated monosodium urate crystal incubated polymorphonuclear leucocytes. On treatment with the W. somnifera root powder (500/1000 mg/kg body weight), the above changes were reverted back to near normal levels. W. somnifera also showed potent analgesic and antipyretic effect with the absence of gastric damage at different dose levels in experimental rats. For comparison purpose, non-steroidal anti-inflammatory drug (NSAID) indomethacin was used as a standard. These results provide evidence for the suppressive effect of W. somnifera root powder by retarding amplification and propagation of the inflammatory response without causing any gastric damage.     

Molecular characterization of India Ginseng Withania somnifera (L) using ISSR markers

Molecular Biology Reports - Ashwagandha (Withania somnifera (L.) Dunal), popularly known as Indian ginseng or winter cherry is a multipurpose plant of immense therapeutic value in the ayurvedic and...     

Phytochemical and genetic analysis in selected chemotypes of Withania somnifera

The main active components and genetic profile of 15 selected accessions of Withania somnifera Dunal. were analysed. Ethanolic extract of the dried roots/leaves of the plant was concentrated under pressure at 50+/-5 degrees C and was analysed for main compounds (withanolides and withaferin A) by HPLC. All the main components were found to be present in accessions (AGB 002, AGB 009, RSS 009, RSS 033). Correlation of these main components with their genetic factors, was undertaken using AFLP (amplified fragment length polymorphism) markers. Among 64 primers 7 yielded optimum polymorphism. A total of 913 polymorphic peaks were generated using these primers. Jaccard's similarity coefficient indicated that accessions having almost the same active compounds clustered together. The present study demonstrates that AFLP can be successfully used to resolve the correlation of AFLP data with the presence of secondary metabolites.     

Antibacterial efficacy of Withania somnifera (ashwagandha) an indigenous medicinal plant against experimental murine salmonellosis

In the present study, we evaluated the antibacterial activity of ashwagandha [Withania somnifera L. Dunal (Solanaceae; root and leaves)], an Indian traditional medicinal plant against pathogenic bacteria. Both aqueous as well as alcoholic extracts of the plant (root as well as leaves) were found to possess strong antibacterial activity against a range of bacteria, as revealed by in vitro Agar Well Diffusion Method. The methanolic extract was further subfractionated using various solvents and the butanolic sub-fraction was found to possess maximum inhibitory activity against a spectrum of bacteria including Salmonella typhimurium. Moreover, in contrast to the synthetic antibiotic (viz. chloramphenicol), these extracts did not induce lysis on incubation with human erythrocytes, advocating their safety to the living cells. Finally, the antibacterial efficacy of the extracts isolated from plant (both root and leaves) was determined against experimental salmonellosis in Balb/C mice. Oral administration of the aqueous extracts successfully obliterated salmonella infection in Balb/C mice as revealed by increased survival rate as well as less bacterial load in various vital organs of the treated animals.     

A standardized root extract of Withania somnifera and its major constituent withanolide-A elicit humoral and cell-mediated immune responses by up regulation of Th1-dominant polarization in BALB/c mice

The effects of graded doses of a chemically standardized aqueous alcoholic (1:1) root extract (AGB) of Withania somnifera on the immune system of SRBC immunized BALB/c mice were investigated. Mice were administrated AGB orally for 15 days. AGB stimulated cell mediated immunity, IgM and IgG titers reaching peak value with 30 mg/kg b.wt. Flow cytometric analysis of lymphocyte surface markers of T cells (CD3(+), CD4(+) and CD8(+)) and B cells (CD19(+)) indicated prominent enhancement in proliferation and differentiation of lymphocytes. The extract selectively, induced type 1 immunity because it guided enhanced expression of T helper cells (Th)1 cytokines interferon (IFN)-gamma and interleukin (IL)-2 while Th2 cytokine IL-4 observed a moderate decline. Confirmation of Th1 polarization was obtained from augmented levels of IgG2a over IgG1 in the blood sera of AGB treated groups. Withanolide-A, a major constituent of AGB appeared responsible for Th1 skewing effect of the extract as it significantly increased the levels of Th1 cytokines, decreased moderately IL-4 and significantly restored the selective dexamethasone inhibition of Th1 cytokines in mouse splenocytes cultures in vitro. In addition, AGB also strongly activated macrophage functions ex vivo and in vitro indicated by enhanced secretion of nitrite, IL-12 and TNF-alpha. In contrast IL-10 remained unchanged again suggesting that AGB critically influenced Th1 profile of the cytokines. The studies suggested that AGB supports predominantly Th1 immunity with increase in macrophage functions. The standardized root extract of no toxicological consequences might therefore, find useful applications against the intracellular pathogens and in the management of immune suppressed diseases.     

Stabilization of Membrane Bound Enzyme Profiles and Lipid Peroxidation by Withania Somnifera Along with Paclitaxel on Benzo(a)pyrene Induced Experimental Lung Cancer

The present study was aimed to evaluate the therapeutic effects of Withania somnifera along with paclitaxel on lung tumor induced by benzo(a)pyrene in male Swiss albino mice. The levels of ATPase enzymes and lipid peroxidation were evaluated in lung cancer bearing mice, in erythrocyte membrane and tissues. The extent of peroxidation was estimated by measuring the thiobarbituric acid-reactive substances. Simultaneously the activities of different ATPases (Na⁺/K⁺-ATPases, Mg²⁺-ATPases and Ca²⁺-ATPases) were determined. The alterations of these enzyme activities in membrane and tissues were indicative of the tumor formation caused by benzo(a)pyrene (50 mg/kg body weight, orally) in cancer bearing animals. The activities of these enzymes were reversed to near normal control values in animals treated with Withania somnifera (400 mg/kg b.wt, orally) along with paclitaxel (33 mg/kg b.wt, i.p). Treatment with Withania somnifera along with paclitaxel altered these damage mediated through free radicals, and the treatment displays the protective role of these drugs by inhibiting free radical mediated cellular damages. Over, based on the data providing a correlation Withania somnifera along with paclitaxel provide stabilization of membrane bound enzyme profiles and decreased lipid peroxidation against benzo(a)pyrene induced lung cancer in mice.     

Immunomodulatory effect of Withania somnifera supplementation diet in the giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila

The effect of Withania somnifera extract supplementation diets on innate immune response in giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila was investigated. The bacterial clearance efficiency significantly increased in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet against pathogen from weeks 1-4 as compared to the control. The innate immune parameters such as, phenoloxidase activity, superoxide anion level, superoxide dismutase activity, nitrate, and nitrite concentrations were significantly enhanced in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet from weeks 1-4 against pathogen. The total hemocyte counts (THC) significantly increased in prawn fed with 0.1% and 1.0% doses diet from weeks 1-4 against pathogen as compared to the control. These results strongly suggested that administration of W. somnifera through supplementation diet positively enhances the innate immune system and enhanced survival rate in M. rosenbergii against A. hydrophila infection.     

Protective effect of Withania somnifera (Solanaceae) on collagen glycation and cross-linking

Modification of collagen such as non-enzymatic glycation and cross-linking plays an important role in diabetic complications and age-related diseases. We evaluate the effect of Withania somnifera on glucose-mediated collagen glycation and cross-linking in vitro. Extent of glycation, viscosity, collagen-linked fluorescence and pepsin solubility were assessed in different experimental procedures to investigate the effect of W. somnifera. Tail tendons obtained from rats (Rattus norvegicus) weighing 250-275 g were incubated with 50 mM glucose and 100 mg of metformin or Withania root powder or ethanolic extract of Withania under physiological conditions of temperature and pH for 30 days. Formation of advanced glycation end products (AGE) was measured by fluorescent method whereas the cross-linking of collagen was assessed by pepsin digestion and viscosity measurements. Tendon collagen incubated with glucose showed an increase in glycation, AGE and cross-linking of collagen. The collagen incubated with W. somnifera and metformin ameliorates these modifications. The ethanolic extract of Withania showed more prominent effect than Withania root powder. The activity of ethanolic extract of Withania is comparable to metformin, a known antiglycating agent. In conclusion, Withania could have therapeutic role in the prevention of glycation induced pathogenesis in diabetes mellitus and aging.     

On the action and mechanism of withaferin-A from Withania somnifera, a novel and potent melanin dispersing agent in frog melanophores

The present work was carried out to determine the effects of lyophilized root extracts of Withania somnifera along with pure withaferin-A, on the isolated skin melanophores of frog, Rana tigerina which are disguised type of smooth muscle cells and offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The lyophilized extract of W. somnifera and its active ingredient withaferin-A induced powerful dose-dependent physiologically significant melanin dispersal effects in the isolated skin melanophores of R. tigerina, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of W. somnifera and its active ingredient withaferin-A got highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of W. somnifera and withaferin-A is mediated by cholino-muscarinic like receptors having similar properties.     

Indian medicinal plants as antiradicals and DNA cleavage protectors

Celastrus paniculatus L. (Celastraceae) (CP), Picrorhiza kurroa L. (Scrophulariaceae) (PK) and Withania somnifera L. (Solanaceae) (WS) are Indian medicinal plants having a remarkable reputation, as a factor of health care, among the indigenous medical practitioners. The plants exhibit varying degrees of therapeutic value some of which useful in the treatment of cognitive dysfunction, epilepsy, insomnia, rheumatism, gout, dyspepsia. In this work, we have investigated the free radical scavenging capacity of methanolic extracts from CP, PK, WS and the effect on DNA cleavage induced by H2O2 UV-photholysis. In addition, we investigated whether these plant extracts are capable of reducing the hydrogen peroxide-induced cytotoxicity and DNA damage in human non-immortalized fibroblasts. These extracts showed a dose-dependent free radical scavenging capacity and a protective effect on DNA cleavage; methanolic extracts from PK was more active than extracts from CP and WS. These results were confirmed by a significant protective effect on H2O2-induced cytoxicity and DNA damage in human non-immortalized fibroblasts. These antioxidant effects of active principle of CP, PK and WS may explain, at least in part, the reported anti-stress, immunomodulatory, cognition-facilitating, anti-inflammatory and antiaging effects produced by them in experimental animal and in clinical situations and may justify the further investigation of their other beneficial biological properties.     

Withanolide A is inherently de novo biosynthesized in roots of the medicinal plant Ashwagandha (Withania somnifera)

Ashwagandha ( Withania somnifera Dunal., Solanaceae) is one of the most reputed medicinal plants of Ayurveda, the traditional medical system. Several of its traditionally proclaimed medicinal properties have been corroborated by recent molecular pharmacological investigations and have been shown to be associated with its specific secondary metabolites known as withanolides, the novel group of ergostane skeletal phytosteroids named after the plant. Withanolides are structurally distinct from tropane/nortropane alkaloids (usually found in Solanaceae plants) and are produced only by a few genera within Solanaceae. W. somnifera contains many structurally diverse withanolides in its leaves as well as roots. To date, there has been little biosynthetic or metabolism-related research on withanolides. It is thought that withanolides are synthesized in leaves and transported to roots like the tropane alkaloids, a group of bioactive secondary metabolites in Solanaceae members known to be synthesized in roots and transported to leaves for storage. To examine this, we have studied incorporation of ¹⁴C from [2-¹⁴C]-acetate and [U-¹⁴C]-glucose into withanolide A in the in vitro cultured normal roots as well as native/orphan roots of W. somnifera . Analysis of products by thin layer chromatography revealed that these primary metabolites were incorporated into withanolide A, demonstrating that root-contained withanolide A is de novo synthesized within roots from primary isoprenogenic precursors. Therefore, withanolides are synthesized in different parts of the plant (through operation of the complete metabolic pathway) rather than imported.     

Withanolides from Withania somnifera roots

Two new and seven known withanolides along with beta-sitosterol, stigmasterol, beta-sitosterol glucoside, stigmasterol glucoside, alpha+beta glucose were isolated from the roots of Withania somnifera. Among the known compounds, Viscosa lactone B, stigmasterol, stigmasterol glucoside and alpha+beta glucose are being reported from the roots of W. somnifera for the first time. One of the new compounds contained the rare 16beta-acetoxy-17(20)-ene the other contained unusual 6alpha-hydroxy-5,7alpha-epoxy functional groups in the withasteroid skeleton. The structures were elucidated by spectroscopic methods and chemical transformations.     

NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa

Dextrans are the main exopolysaccharides produced by Leuconostoc species. Other dextran-producing lactic acid bacteria include Streptococci, Lactobacilli, and Weissella species. Commercial production and structural analysis has focused mainly on dextrans from Leuconostoc species, particularly on Leuconostoc mesenteroides strains. In this study, we used NMR spectroscopy techniques to analyze the structures of dextrans produced by Leuconostoc citreum E497 and Weissella confusa E392. The dextrans were compared to that of L. mesenteroides B512F produced under the same conditions. Generally, W. confusa E392 showed better growth and produced more EPS than did L. citreum E497 and L. mesenteroides B512F. Both L. citreum E497 and W. confusa E392 produced a class 1 dextran. Dextran from L. citreum E497 contained about 11% alpha-(1-->2) and about 3.5% alpha-(1-->3)-linked branches whereas dextran from W. confusa E392 was linear with only a few (2.7%) alpha-(1-->3)-linked branches. Dextran from W. confusa E392 was found to be more linear than that of L. mesenteroides B512F, which, according to the present study, contained about 4.1% alpha-(1-->3)-linked branches. Functionality, whether physiological or technological, depends on the structure of the polysaccharide. Dextran from L. citreum E497 may be useful as a source of prebiotic gluco-oligosaccharides with alpha-(1-->2)-linked branches, whereas W. confusa E392 could be a suitable alternative to widely used L. mesenteroides B512F in the production of linear dextran.     

Chlorinated and diepoxy withanolides from Withania somnifera and their cytotoxic effects against human lung cancer cell line

Phytochemical studies on the aerial parts of Withania somnifera L. Dunal. (Solanaceae) led to the isolation of a chlorinated steroidal lactone (27-acetoxy-4β,6α-dihydroxy-5β-chloro-1-oxowitha-2,24-dienolide), a diepoxy withanolide (5β,6β,14α,15α-diepoxy-4β,27-dihydroxy-1-oxowitha-2,24-dienolide), and withaferin A. Their structures were elucidated by using spectroscopic techniques. All three compounds exhibited a growth inhibition and cytotoxic activity against human lung cancer cell line (NCI-H460), with withaferin A being the most potent (GI(50)=0.18 μg/mL and LC(50)=0.45 μg/mL) among three compounds tested.     

Protective effect of Withania somnifera Dunal on the behavioral and biochemical alterations in sleep-disturbed mice (Grid over water suspended method)

Sleep disruption involves extensive changes in physiological function, including EEG, motor, metabolic, autonomic processes physiological homeostasis and psychological balance that are necessary for physical health. Benzodiazepines are the most widely used drugs for the sleep related problems in spite of their limitations and side effects. Objective of the study was to investigate the protective effect of W. somnifera on the behavioral and biochemical alterations in sleep disturbed mice. Pretreatment with W. somnifera root extract (100. 200 mg/kg) and diazepam (0.5 mg/kg) significantly protected reduction in body weight, improved the reduced locomotor activity and anxiety levels in animals. Biochemical studies also revealed that W. somnifera (100 and 200 mg/kg) and diazepam (0.5 mg/kg) pretreatment for five days decreased significantly lipid peroxidation, nitrites levels and improved catalase, and reduced glutathione levels. Co-administration of W. somnifera (100 mg/kg) with diazepam (0.5 mg/kg) improved significantly all the biochemical parameters as compared to their effect per se. Preliminary results suggest that Withania root extract can be used in the management sleep loss and associated oxidative stress.     

Effect of Coriolus versicolor supplemented diet on innate immune response and disease resistance in kelp grouper Epinephelus bruneus against Listonella anguillarum

The effect of Coriolus versicolor extract supplemented diets on innate immune response and disease resistance in kelp grouper, Epinephelus bruneus against Listonella anguillarum, is reported. Kelp grouper were divided into four groups of 25 each and fed with C. versicolor enriched diets at 0% (control), 0.01%, 0.1%, and 1.0% level. After 30 days of feeding, all fish were injected interaperitoneally (i.p.) with 50 μl of L. anguillarum (4.7 × 10⁷ CFU) to investigate the immune parameters at weeks 1, 2, and 4. The reactive oxygen species and reactive nitrogen species production were significantly enhanced in fish fed with 0.1% and 1.0% supplementation diets from weeks 1-4 when compared to the non enriched diet fed and infected control. The phagocytic activity significantly increased with 0.1% and 1.0% diets on weeks 2 and 4. The leucocyte myeloperoxidase content, lysozyme activity, and total protein level significantly increased when fed with 0.1% and 1.0% supplementation diets from weeks 1-4. The cumulative mortality was 35% and 45% in 1.0% and 0.1% enriched diet fed groups whereas it was 55% and 80% in 0.01% and 0% groups respectively. The present results suggest that diets enriched with C. versicolor at 0.1% or 1.0% level positively enhance the innate immune system and affords protection from L. anguillarum.     

Protective effects of solvent fractions of Mentha spicata (L.) leaves evaluated on 4-nitroquinoline-1-oxide induced chromosome damage and apoptosis in mouse bone marrow cells

Spearmint leaves (Mentha spicata L.) contain high levels of antioxidants that are known to protect against both exogenous and endogenous DNA damage. In this study, the protective effects of the hexane fraction (HF), chloroform fraction (CF) and ethyl acetate fraction (EAF) in an ethanol extract from M. spicata were evaluated against 4-nitroquinoline-1-oxide (4-NQO) induced chromosome damage and apoptosis in bone marrow cells of Swiss albino mice. Two (EAF; 80 and 160 mg/ kg body weight - bw) or three (HF and CF; 80, 160 and 320 mg/ kg bw) doses of solvent fractions or vehicle control (25% DMSO in water) were administered orally for five consecutive days. Upon the sixth day, 4-NQO was injected intraperitoneally. The animals were killed the following day. Other control groups were comprised of animals treated with either the vehicle control or the various doses of solvent fractions, but with no 4-NQO treatment. 4-NQO induced micro-nucleated polychromatic erythrocytes (MnPCEs) in all the test groups. However, pre-treatment of animals with the solvent fractions significantly reduced the 4-NQO-induced MnPCEs as well as the percentage of apoptotic cells. The reduction of both MnPCE and apoptosis was more evident following the pre-treatment of animals with 160 mg/kg bw EAF.     

Trichinella spiralis: modulation of experimental autoimmune encephalomyelitis in DA rats

Helminth infection has a potent systemic immunomodulatory effect on the host immune response, which also affects the development of autoimmune diseases. We investigated the dose-dependent influence of Trichinella spiralis infection on experimental autoimmune encephalomyelitis (EAE). Our model of concomitant T. spiralis infection and EAE demonstrates that established infection of Dark Agouti (DA) rats with the parasite causes amelioration of the clinical course of induced EAE in a dose-dependent way. Infection with T. spiralis L1 stage muscle larvae (TSL1) reduced the severity of the autoimmune disease as judged by lower maximal clinical score, cumulative index, duration of illness and degree of mononuclear cell infiltration in T. spiralis infected animals compared to control, EAE-induced group. This study provides a valuable model of worm infection to investigate helminth-induced regulatory mechanisms for optimal benefit to the host.