Innate endophytic fungus,Aspergillus terreus as biotic elicitor of withanolide A in root cell suspension cultures of Withania somnifera

Molecular Biology Reports - In the present study, root cell suspension cultures of W. somnifera were elicited with mycelial extract (1% w/v) and culture filtrate (5% v/v) of their native endophytic...     

Effect of Withania somnifera on glycosaminoglycan synthesis in carrageenin-induced air pouch granuloma

The effect of W. somnifera on glycosaminoglycan synthesis in the granulation tissue of carrageenin-induced air pouch granuloma was studied. W. somnifera was shown to exert significant inhibitory effect on incorporation of 35S into the granulation tissue. The uncoupling effect on oxidative phosphorylation (ADP/O ratio reduction) was also observed in the mitochondria of granulation tissue. Further, Mg2+ dependent ATPase activity was found to be influenced by W. somnifera. W. somnifera also reduced the succinate dehydrogenase enzyme activity in the mitochondria of granulation tissue.     

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.     

Purification and characterization of a novel glucosyltransferase specific to 27beta-hydroxy steroidal lactones from Withania somnifera and its role in stress responses

Sterol glycosyltransferases catalyze the synthesis of diverse glycosterols in plants. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a novel 27beta-hydroxy glucosyltransferase was purified to near homogeneity from cytosolic fraction of W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme showed activity with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols/withanolides with beta-OH group at C-17, C-21 and C-27 positions. It transferred glucose to the alkanol at C-25 position of the lactone ring, provided an alpha-OH was present at C-17 in the sterol skeleton. A comparable enzyme has not been reported earlier from plants. The enzyme is distinct from the previously purified W. somnifera 3beta-hydroxy specific sterol glucosyltransferase and does not glucosylate the sterols at C-3 position; though it also follows an ordered sequential bisubstrate reaction mechanism, in which UDP-glucose and sterol are the first and second binding substrates. The enzyme activity with withanolides suggests its role in secondary metabolism in W. somnifera. Results on peptide mass fingerprinting showed its resemblance with glycuronosyltransferase like protein. The enzyme activity in the leaves of W. somnifera was enhanced following the application of salicylic acid. In contrast, it decreased rapidly on exposure of the plants to heat shock, suggesting functional role of the enzyme in biotic and abiotic stresses.     

黄花蒿培养细胞中青蒿素合成代谢的体外调节

黄花蒿培养细胞通过两步培养积累青蒿素．第１步在含有０．２～０．４ｍｇ／Ｌ６－苄基氨基嘌呤（６－ＢＡ）和３～４ｍｇ／Ｌ吲哚乙酸（ＩＡＡ）的Ｎ６培养基中进行细胞的增殖培养,第２步将培养好的细胞转入含０．２～０．４ｍｇ／Ｌ６－ＢＡ和０．２～０．４ｍｇ／ＬＩＡＡ的改良Ｎ６培养基中进行青蒿素的合成．青蒿素的合成量为１９０μｇ／ｇ干细胞左右．当在第２步培养中加入青蒿素合成前体青蒿酸,青蒿素合成量比仅靠激素诱导提高了３倍多．青蒿素的合成途径是植物固醇合成途径的分支途径,当在青蒿素合成过程即第２步培养中加入固醇生物合成抑制剂双氯苯咪唑和氯化氯胆碱处理,可使代谢向合成青蒿素的方向移动,青蒿素合成量明显提高．经２００ｍｇ／Ｌ氯化氯胆碱处理２ｄ,黄花蒿细胞合成青蒿素量为３７２μｇ／ｇ干细胞;经２０ｍｇ／Ｌ双氯苯咪唑处理４ｄ,黄花蒿细胞合成青蒿素量为１５４０μｇ／ｇ干细胞,比靠激素诱导提高了８倍多,与诱导脱分化细胞的黄花蒿叶中所含的青蒿素（３０００μｇ／ｇ干细胞）处于同一个数量级．以上结果表明：在通过植物激素调节可以合成青蒿素的黄花蒿培养细胞中,缺乏青蒿素合成前体是青蒿素合成量低的重要原因．因此,在青蒿素合成的过程中通过体外调节,     

Enhancement of antitumor effect of paclitaxel in combination with immunomodulatory Withania somnifera on benzo(a)pyrene induced experimental lung cancer

The current experimental work deals with the immunomodulatory studies on the extract of Withania somnifera (L.) Dunal root powder against benzo(a)pyrene induced lung cancer in male Swiss albino mice. In our previous study, we reported the antioxidant and anticarcinogenic effect of W. somnifera (L.) Dunal along with paclitaxel. Immune dysfunction has been found to be associated with cancer and chemotherapy. Benzo(a)pyrene induced cancer animals were treated with 400mg/kg bodyweight of W. somnifera (L.) Dunal extract for 30 days significantly alters the levels of immunocompetent cells, immune complexes and immunoglobulins. Based on the data, the carcinogen as well as the paclitaxel affects the immune system, the toxic side effects on the immune system is more reversible and more controllable by W. somnifera (L.) Dunal. These results concluded the immunomodulatory activity of W. somnifera (L.) Dunal extract, which is a known immunomodulator in indigenous medicine.     

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.     

In vitro withanolide production by Withania somnifera L. cultures

In vitro multiple shoots, root, callus and cell suspension cultures of Withania somnifera exhibited the potentiality to produce pharmacologically active withanolides. Multiple shoots cultures exhibited an increase in withanolide A accumulation compared to shoots of the mother plant. In vitro generated root cultures as well as callus and suspension cultures also produced withanolides albeit at lower levels.     

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.     

Genetic variation and relationship among and within Withania species as revealed by AFLP markers.

Withania somnifera is an important medicinal plant, and its anticancerous properties have been attributed to various classes of withanolide compounds. The objective of the present study was to investigate the inter- and intraspecific genetic variation present in 35 individuals of W. somnifera and 5 individuals of W. coagulans using AFLP (amplified fragment length polymorphism) marker technique. The information about genetic variation determined from AFLP data for 40 individuals was employed to estimate similarity matrix value based on Jaccard's coefficient. The similarity values were further used to construct a phenetic dendrogram revealing the genetic relationships. The dendrogram generated by UPGMA (unweighted pair group method of arithmetic averages) distinguished W. somnifera from W. coagulans and formed two major clusters. These two main clusters shared a similarity coefficient of 0.3, correlating with the high level of polymorphism detected. The dendrogram further separated W. somnifera into three subclasses corresponding to Kashmiri and Nagori groups and an intermediate type. The AFLP profile of Kashmiri individuals was distinct from that of the Nagori group of plants. The intermediate genotype was distinct as it shared bands with both the Kashmiri and Nagori individuals, even though it was identified as a Kashmiri morphotype. Furthermore, the intermediate type shared a similarity coefficient of 0.8 with the Kashmiri individuals. The present work revealed low levels of variation within a population though high levels of polymorphism were detected between Nagori and Kashmiri populations. The ability of AFLP markers for efficient and rapid detection of genetic variations at the species as well as intraspecific level qualifies it as an efficient tool for estimating genetic similarity in plant species and effective management of genetic resources.     

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.     

Abscisic acid (ABA) treatment increases artemisinin content in <Emphasis Type="Italic">Artemisia annua</Emphasis> by enhancing the expression of genes in artemisinin biosynthetic pathway

Artemisinin, a sesquiterpene lactone endoperoxide derived from Artemisia annua L., is the most effective antimalarial drug. In an effort to increase the artemisinin production, abscisic acid (ABA) with different concentrations (1, 10 and 100 μM) was tested by treating A. annua plants. As a result, the artemisinin content in ABA-treated plants was significantly increased. Especially, artemisinin content in plants treated by 10 μM ABA was 65% higher than that in the control plants, up to an average of 1.84% dry weight. Gene expression analysis showed that in both the ABA-treated plants and cell suspension cultures, HMGR, FPS, CYP71AV1 and CPR, the important genes in the artemisinin biosynthetic pathway, were significantly induced. While only a slight increase of ADS expression was observed in ABA-treated plants, no expression of ADS was detected in cell suspension cultures. This study suggests that there is probably a crosstalk between the ABA signaling pathway and artemisinin biosynthetic pathway and that CYP71AV1, which was induced most significantly, may play a key regulatory role in the artemisinin biosynthetic pathway.     

Hypoglycemic, diuretic and hypocholesterolemic effect of winter cherry (Withania somnifera, Dunal) root

Hypoglycemic, diuretic and hypocholesterolemic effects of roots of W. somnifera (ashvagandha) were assessed on human subjects. Six mild NIDDM subjects and six mild hypercholesterolemic subjects were treated with the powder of roots of W. somnifera for 30 days. Suitable parameters were studied in the blood and urine samples of the subjects along with dietary pattern before and at the end of treatment period. Decrease in blood glucose was comparable to that of an oral hypoglycemic drug. Significant increase in urine sodium, urine volume, significant decrease in serum cholesterol, triglycerides, LDL (low density lipoproteins) and VLDL (very low density lipoproteins) cholesterol were observed indicating that root of W. somnifera is a potential source of hypoglycemic, diuretic and hypocholesterolemic agents. Clinical observations revealed no adverse effects.     

Inhibition of aflatoxin B production of Aspergillus flavus, isolated from soybean seeds by certain natural plant products

AIMS: The inhibitory effect of cowdung fumes, Captan, leaf powder of Withania somnifera, Hyptis suaveolens, Eucalyptus citriodora, peel powder of Citrus sinensis, Citrus medica and Punica granatum, neem cake and pongamia cake and spore suspension of Trichoderma harzianum and Aspergillus niger on aflatoxin B(1) production by toxigenic strain of Aspergillus flavus isolated from soybean seeds was investigated. METHODS AND RESULTS: Soybean seed was treated with different natural products and fungicide captan and was inoculated with toxigenic strain of A. flavus and incubated for different periods. The results showed that all the treatments were effective in controlling aflatoxin B(1) production. Captan, neem cake, spore suspension of T. harzianum, A. niger and combination of both reduced the level of aflatoxin B(1) to a great extent. Leaf powder of W. somnifera, H. suaveolens, peel powder of C. sinensis, C. medica and pongamia cake also controlled the aflatoxin B(1) production. CONCLUSIONS: All the natural product treatments applied were significantly effective in inhibiting aflatoxin B(1) production on soybean seeds by A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: These natural plant products may successfully replace chemical fungicides and provide an alternative method to protect soybean and other agricultural commodities from aflatoxin B(1) production by A. flavus.     

Purification and physico-kinetic characterization of 3beta-hydroxy specific sterol glucosyltransferase from Withania somnifera (L) and its stress response

Sterol glycosyltransferases catalyze the synthesis of diverse glycosteroids in plants, leading to a change in their participation in cellular metabolism. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a cytosolic sterol glucosyltransferase was purified 3406 fold to near homogeneity from W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme was active with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols and phytosterols with 3beta-OH group. It showed a low level of activity with flavonoids and isoflavonoids. The enzyme gave maximum K(cat)/K(m) value (0.957) for 24-methylenecholesterol that resembles aglycone structure of pharmacologically important sitoindosides VII and VIII from W. somnifera. The enzyme follows ordered sequential bisubstrate mechanism of reaction, in which UDP-glucose and sterol are the first and second binding substrates. This is the first detailed kinetic study on purified plant cytosolic sterol glucosyltransferases. Results on peptide mass fingerprinting and substrate specificity suggested that the enzyme belongs to the family of secondary metabolite glucosylating glucosyltransferases. The enzyme activity exhibited a rapid in vivo response to high temperature and salicylic acid treatment of plants, suggesting its physiological role in abiotic and biotic stress.     

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.     

Synthesis of artemisinin dimers using the Ugi reaction and their in vitro efficacy on breast cancer cells

The Ugi four-component reaction was used to prepare a series of artemisinin monomers and dimers. We found that the endoperoxide group in artemisinin remains intact during the reaction. The new artemisinin dimers showed potent anti-cancer activity against two human breast cancer cell lines, MDA-MB-231 and BT-474. One of the Ugi artemisinin dimers showed an IC₅₀ value of 12 nM when tested on BT474 cells, more than 600 times more potent than artesunate. Furthermore, the same Ugi artemisinin dimer showed a low toxicity when tested on MCF10A, a nontumorigenic cell line, resulting in a selectivity index of more than 8000.     

Stress induced neuron degeneration and protective effects of Semecarpus anacardium Linn. and Withania somnifera Dunn. in hippocampus of albino rats: an ultrastructural study

Effects of herbal formulations were studied on hippocampal neuron cell bodies. Study was carried out in adult Swiss albino rats. Experimental rats (E) were divided into three groups. Group E1 rats were given immobilization stress for 14 hr/day for 30 days. Rats in E2 and E3 group were given daily single dose (40 mg/kg/body wt.) of alcoholic extract of S. anacardium and W. somnifera. After 1 hr giving the plant extract, the rats were subjected to stress. Treatment continued for 14 hr for 30 days. Control rats were kept in complete nonstress condition. Ultrastructural characteristics of neuron cell bodies in hippocampal sublayer (CA1-CA4 and Dg) was studied in rats of E1, E2 and E3 groups and compared with control. Results of the present study demonstrated, that both CA2 and Dg, 85% of neuron cell bodies exhibited degenerating characteristics, (which includes karyorrhexis, membrane blebbing, chromatin condensation, chromatin fragmentation and intracellular spacing). Interestingly, after the treatment with S. ancardium cells demonstrating degenerating characteristics was significantly reduced (80%) as compared to treatment with W. somnifera. Study suggests that probably both the herbal drugs have cytoprotective properties.