New withanolides from leaves along with alkyl glucoside from roots and stem as well as variations in withanolide contents in leaves of accessions of Withania somnifera, Ashwagandha- the Indian ginseng$.

Based on the major components in the leaves, the ashwagandha has been found to exist in several chemotypic forms in India. From the leaves of various accessions of Withania somnifera, which were maintained in our institute, three new steroids namely, 4-acetoxy-20ß-hydroxy-1-oxo-witha-2,5,24-trienolide (7), 24,25-dihydro-14α-hydroxy withanolide D (9), 5α,6ß,17α,27-tetrahydroxy-1-oxo-witha-2,24-dienolide (12) together with thirteen known withanolides were identified by spectroscopic methods. From the roots and stem of one accession and leaves of another, a new alkyl ester glucoside (4) has also been isolated. The new withanolides 7, 9 and 12 have been tentatively named as withanolide 135 A, withanolide 135B and withanolide 108, respectively.

Synergistic effect of (+)-pinitol from Saraca asoca with ß-lactam antibiotics and studies on the in silico possible mechanism.

Saraca asoca bark has been used in the Ayurvedic system of medicine for female urino-genital disorders. We have recently reported the isolation and characterization of several compounds as markers to develop HPLC profiling of its methanol and aqueous methanol extracts. Now, a HPLC-PDA inactive compound, (+)-pinitol has been isolated and characterized from the bark of this medicinally important tree. Pinitol is a well known bioactive compound for a variety of biological activities, including hypoglycemic and anti-inflammatory activities. A process for the isolation of relatively good concentration of (+)-pinitol from S. asoca bark has been developed and its in vitro anti TNF-α and anti-inflammatory activities against carragenan-induced edema confirmed. While conducting experiments on the possible agonistic activity, it was found that (+)-pinitol showed up to eight fold reduction in the doses of ß-lactam antibiotics. The mechanism of its agonistic activity was studied by docking experiments which showed that different conformations of (+)-pinitol and antibiotics were actually in the same binding site with no significant change in the binding energy. These docking simulations, thus predict the possible binding mode of studied compounds and probable reason behind the synergistic effect of (+)-pinitol along with ß-lactam antibiotics.

Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L. (Dunal).

Withanolides are pharmaceutically important C(28)-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of (13)C label from (13)C(1)-D-glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of (13)C abundance at each carbon of withaferin A from (13)C(1)-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific (13)C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids. Key message The study elucidates significant contribution of DOXP pathway to withanolide biosynthesis. A new connotation of biosynthetic load-based role of DOXP/MVA recruitment in isoprenoid biosynthesis has been proposed.

1,4-Dioxane and ergosterol derivatives from Withania somnifera roots.

The chemical investigation on the n-hexane extract of Withania somnifera roots has yielded octacosane, oleic and stearic fatty acids, stigmasterone, stigmasterol, sitostanone, oleanolic acid along with the ergosterol and 1,4-dioxane derivatives as new compounds. The isolation of alkenyl-1,4-dioxane compound is rare, whereas the ergosterol derivative may have biogenetic significance in the lactone formation in the E ring of withanolides. The presence of a 1,4-dioxane derivative in the nonpolar extract of roots assumes importance as this type of compound has not been reported earlier from W. somnifera. The structures of new compounds were elucidated by spectroscopic methods and chemical transformations.

Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade.

Withaferin A (WA) is present abundantly in Withania somnifera, a well-known Indian medicinal plant. Here we demonstrate how WA exhibits a strong growth-inhibitory effect on several human leukemic cell lines and on primary cells from patients with lymphoblastic and myeloid leukemia in a dose-dependent manner, showing no toxicity on normal human lymphocytes and primitive hematopoietic progenitor cells. WA-mediated decrease in cell viability was observed through apoptosis as demonstrated by externalization of phosphatidylserine, a time-dependent increase in Bax/Bcl-2 ratio; loss of mitochondrial transmembrane potential, cytochrome c release, caspases 9 and 3 activation; and accumulation of cells in sub-G0 region based on DNA fragmentation. A search for the downstream pathway further reveals that WA-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2 and HSP27 in leukemic cells. The RNA interference of p38MAPK protected these cells from WA-induced apoptosis. The RNAi knockdown of p38MAPK inhibited active phosphorylation of p38MAPK, Bax expression, activation of caspase 3 and increase in Annexin V positivity. Altogether, these findings suggest that p38MAPK in leukemic cells promotes WA-induced apoptosis. WA caused increased levels of Bax in response to MAPK signaling, which resulted in the initiation of mitochondrial death cascade, and therefore it holds promise as a new, alternative, inexpensive chemotherapeutic agent for the treatment of patients with leukemia of both lymphoid and myeloid origin.

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.

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 (14)C from [2-(14)C]-acetate and [U-(14)C]-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.

Selective reactivity of 2-mercaptoethanol with 5beta,6beta-epoxide in steroids from Withania somnifera.

2-Mercaptoethanol reacts selectively with the 5beta,6beta-epoxy steroids isolated from Withania somnifera substituting the epoxide by a six-membered oxyethylene-2'-thio ring whereas it failed to show such reactivity on 6alpha,7alpha-epoxy withasteroids. The structure of the product has been elucidated by spectroscopic methods, especially applying extensive 2D NMR methods. The anticancer activity of withaferin A was lost in the reaction product indicating that its activity is also linked to the free 5beta,6beta-epoxide functional group.

Analysis of withanolides in root and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection.

A reversed-phase HPLC method for the simultaneous analysis of nine structurally similar withanolides, namely, 27-hydroxy withanone, 17-hydroxy withaferin A, 17-hydroxy-27-deoxy withaferin A, withaferin A, withanolide D, 27-hydroxy withanolide B, withanolide A, withanone and 27-deoxywithaferin A, has been developed using a linear binary gradient solvent system comprising methanol and water containing 0.1% acetic acid. Both photodiode array and evaporative light scattering detection were used to profile the extract compositions and to quantify the withanolides therein. Homogeneity and purity of each peak was ascertained by comparative evaluation of the on-line UV spectra of the eluted compounds with those of the reference compounds. The method has been validated with respect to various parameters of performance quality including computation regression analysis based on calibration curves, peak resolution factor, asymmetry factor, tailing factor, RSD (%) of retention time and peak area response, limit of quantivation, limit of detection, precision and recovery. The developed method has been applied to the analysis of leaf and root tissues of Withania somnifera for withanolide content.

Extraction of bioactive principles from Mucuna pruriens seeds.

Mucuna pruriens (L.) DC. Syn. M. prurita Hook. (Papilionaceae) is used in male impotency, as aphrodisiac, in sexual debility, and as nervine tonic. It also possesses anti-parkinson property, possibly due to the presence of L-DOPA. In the present study, attempts were made to develop the suitable method(s) for extraction of L-DOPA/other active components from the seeds using different solvents. The various extracts were also screened for their neuroprotective and antioxidant activities. In addition, TLC and HPLC fingerprinting of the extracts for amino acid components were also developed for preliminary and sophisticated analysis. The L-DOPA could be obtained in good yield on extraction with EtOH-H2O mixture (1:1) using ascorbic acid as protector. Interestingly, n-propanol extract, which contained negligible amount of L-DOPA, had shown significant neuroprotective activity, suggesting that some components, other than L-DOPA, might also be responsible for anti-Parkinson property of seeds. The extract (MW-0100) containing mainly amino acids and water-ethanol extract (1:1) (MWEL-1299) showed promising antioxidant activity (EC50 = 2.5 microg) against DPPH radicals. MWEL-1299 also exhibited encouraging results against 1-methyl-4-phenylpyridinium ion (MPP+) toxicity. The TLC fingerprinting may be used to authenticate the plant material in herbal industry.

Anti-inflammatory flavanol glycosides from Saraca asoca bark.

Saraca asoca (Roxb.) de Wilde, a common tree of India, is popularly used in the Ayurvedic and modern herbal systems of medicine for genito-urinary problems of women. Considering the reported antimicrobial or anti-inflammatory effect of S. asoca bark against such infections, we studied the anti-inflammatory activity-guided isolation of active compounds from methanol extract. The methanol extract of bark has yielded 10 compounds out of which 3'-deoxyepicatechin-3-O-ß-D-glucopyranoside (6) and 3'-deoxycatechin-3-O-α-L-rhamnopyranoside (8) have been found to be in vitro and in vivo active. 3',5-Dimethoxy epicatechin (3), 3'-deoxyepicatechin-3-O-ß-D-glucopyranoside (6), 3'-deoxycatechin-3-O-α-L-rhamnopyranoside (8) and epigallocatechin (9) are being reported for the first time from S. asoca.

Unusually sulfated and oxygenated steroids from Withania somnifera.

Four (1, 8-10) and six known (2-7) withanolides were isolated from the leaves of Withania somnifera. Among the new compounds, 10 possessed the rare 3-O-sulfate group with the saturation in A ring and 9 contained unusual 1,4-dien-3-one group. Compound 8 did not have usual 2,3 unsaturation in A ring while 1 had the rare C-16 double bond. The structures of all the compounds were elucidated by spectroscopic methods and chemical transformation.

Alkaloidal constituents of Mucuna pruriens seeds.

The seeds of Mucuna pruriens (L.) DC. after chemical analysis afforded four tetrahydroisoquinoline alkaloids which have been isolated for the first time from M. pruriens. Out of them, two are new whose structures have been elucidated by spectroscopic methods.

Antibacterial activity of essential oils of edible spices, Ocimum canum and Xylopia aethiopica.

UNLABELLED: The essential oils of 2 Cameroonian spices, namely, Xylopia aethiopica and Ocimum canum, were chemically investigated and screened for their antibacterial activity. The essential oils were analyzed by means of GC, GC/MS, and NMR. X. aethiopica oil contained myrtenol (12%), a monoterpenoid in highest concentration. The essential oil of O. canum belonged to the known linalool (44%) rich chemotype. The results of the antibacterial screening against the food spoiling bacteria revealed a significant and broad spectrum of activity for these essential oils. The present material of X. aethiopica, which is having myrtenol in relatively higher concentration, has shown moderate antibacterial activity. The bioassay-guided fractionation of Ocimum canum oil through flash chromatography showed that minor compounds, namely, α-terpineol, chavicol, chavibetol, and trans-p-mentha-2,8-dien-ol, significantly contributed for the overall activity observed. Hence, these results evidenced the possible potential of the essential oil of O. canum as a suitable antibacterial for controlling food-borne pathogens whereas the X. aethiopica oil has moderate possibility. PRACTICAL APPLICATION: There is a strong global demand for the microbe-free, safe, and healthy foods. In this study, we showed that the essential oil of O. canum (wild basil) can be used as antibacterial for food items. Also, we showed that a value addition in the antibacterial potential of O. canum oil can be done by processing the essential oil through flash chromatographic separations.

Epoxide group relationship with cytotoxicity in withanolide derivatives from Withania somnifera.

Withania somnifera is one of the highly reputed medicinal plants of India. Its steroidal constituents exist in the form of two major substitution patterns, viz. withaferin A (1) and withanone (5). Withaferin A with oxidation at carbons 4, 5, and 6 is considered as an active type, especially as anticancer, whereas the withanones with oxidation at carbons 5, 6, and 7 rarely show any activity. We prepared a series of derivatives with modifications at carbons 5, 6, and 7 in ring B of these withanolides to study the role of the epoxide group towards the cytotoxic property of these bioactive steroids. We have converted withanolides into the respective thiiranes, amino alcohols and alcohols by selective reactions at the epoxide ring and were evaluated for in vitro anticancer activity against four cancer cell lines to study the structure activity relationships. The transformations of the epoxide group in withanolides of the withaferin A type showed moderate reduction in their cytotoxicity whereas the almost inactive withanones have shown some improvements in their alcohol derivatives.

5,6-de-epoxy-5-en-7-one-17-hydroxy withaferin A, a new cytotoxic steroid from Withania somnifera L. Dunal leaves.

From the leaves of Withania somnifera L. Dunal, a new withasteroid named as 5,6-de-epoxy-5-en-7-one-17-hydroxy withaferin A (6) was isolated along with several known compounds, namely 16ß-acetoxy-6α,7α-epoxy-5α-hydroxy-1-oxowitha-2,17(20),24-trienolide (1), withanone (2), 16-en-27-deoxy withaferin A (3), 27-deoxy withaferin A (4), withaferin A (5), withanolide D (7) and 27-hydroxy withanone (8). Its structure was determined using spectroscopic methods, namely IR, (1)H NMR, (13)C NMR, COSY, HMBC and HRMS. Among the known compounds, 16ß-acetoxy-6α,7α-epoxy-5α-hydroxy-1-oxowitha-2,17(20),24-trienolide (1) was previously reported from the roots of W. somnifera. Now, it has been isolated from the leaves, as well. The cytotoxic activity of the new steroid was carried out using the MTT assay against a panel of cancer cell lines, namely MCF-7 (breast), WRL-68 (liver), PC-3 (prostate) and CACO-2 (colon). The results showed that the new compound possesses strong cytotoxic activity against liver and breast cancer with an IC50 of 1.0 µg/mL and a moderate activity against colon (IC50 3.4 µg/mL) and prostate (IC50 7.4 µg/mL) cancer cells.

ANN-QSAR model for virtual screening of androstenedione C-skeleton containing phytomolecules and analogues for cytotoxic activity against human breast cancer cell line MCF-7.

The present study deals with the development of an artificial neural network based quantitative structure activity relationship (QSAR) model for virtual screening of active compounds which contain androstenedione carbonskeleton or their similar skeleton at the core. An empirical data modeling (with fitted data mapping) has been performed on the basis of bioassay record for human breast cancer cell line MCF7. The whole experimental data set was considered as test set. Standard feed-forward back-propagation neural network technique was applied to build the model. Leave-One- Out (LOO) cross-validation was performed to evaluate the performance of the model. The mapped model became the basis for selection best mapped compounds followed by development of Pharmacophore specific secondary QSAR model. In the present study, two best mapped molecules '4beta-hydroxy Withanolide-E' and '7, 8-Dehydrocalotropin' were used for development of the secondary QSAR model. These secondary-QSAR models were resulted with R2 LOOCV value 0.9845 and 0.9666 respectively. Docking studies, in silico phamacokinetic and toxicity analysis was also done for selected compounds. The screened compounds CID_73621, CID_16757497, CID_301751, CID_390666 and CID_46830222 were found with promising binding affinity value with aromatase with reference to the co-crystallized control compound androstenedione. Due to excellent extent of variance coverage in ANN based QSAR map model, it can be used as a robust non-linear QSAR model for androstenedione carbon-skeleton containing molecules and the protocol can be used to derive secondary QSAR models for other compounds set.

Tropine forming tropinone reductase gene from Withania somnifera (Ashwagandha): biochemical characteristics of the recombinant enzyme and novel physiological overtones of tissue-wide gene expression patterns.

Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ~60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[(14)C]-sucrose to orphan shoot (twigs) and [(14)C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones.

Aromatase and glycosyl transferase inhibiting acridone alkaloids from fruits of Cameroonian Zanthoxylum species.

BACKGROUND: Zanthoxylum zanthoxyloides and Z. leprieurii fruits are commonly used in traditional system of medicine for diarrhea, pain, wound healing, etc. in Cameroon, Africa. Z. leprieurii fruits have been chemically studied for its bioactive compounds whereas the investigation on Z. zanthoxyloides fruits is lacking. RESULTS: After a detailed chemical analysis of the fruits of Z. leprieurii and Z. zanthoxyloides, a series of new acridone alkaloids, namely, 3-hydroxy-1,5,6-trimethoxy-9-acridone (1), 1,6-dihydroxy-3-methoxy-9-acridone (2), 3,4,5,7-tetrahydroxy-1-methoxy-10-methyl-9-acridone (3), 4-methoxyzanthacridone (8), 4-hydroxyzanthacridone (9), 4-hydroxyzanthacridone oxide (2,4') (10) have been isolated. The known acridones which have been characterized are, helebelicine A (4), 1-hydroxy-3-methoxy-10-methyl-9-acridone (5), 1,3-dihydroxy-4-methoxy-10-methyl-9-acridone (6) and tegerrardin A (7). The in vitro antibacterial and cytotoxic screening of these acridones reveal that compound 3 has a moderate antibacterial activity (MIC 125 µg/mL) against Micrococcus luteus and Pseudomonas aeruginosa while compound 1 shows a moderate cytotoxic effect (IC50 of 86 µM) against WRL-68 (liver cancer cell line). Furthermore, the molecular modeling of these acridones predicted the structural basis for their mode of action and binding affinity for aromatase, quinone reductase and WAAG, a glycosyltransferase involved in bacterial lipopolysaccharide synthesis. Computational approaches, quantitative SAR and modeling studies predicted that acridones 1, 2, 3, 4, 9 and 10 were the inhibitors of glycosyltransferase while 1, 8, 9 and 10, the inhibitors of aromatase. CONCLUSIONS: A total of 10 acridones have been isolated out of which 6 are new (1, 2, 3, 8, 9 and 10). Alkaloids 8, 9 and 10, having novel tetracyclic acridone structure with new carbon skeleton, have now been named as zanthacridone. The quantitative SAR and molecular modeling studies suggested that the compounds 1, 9 and 10 are inhibitors of both aromatase and glycosyltransferase.

Withanolide A biogeneration in in vitro shoot cultures of ashwagandha (Withania somnifera DUNAL), a main medicinal plant in Ayurveda.

Multiple shoot cultures of two experimental lines of Withania somnifera plants (RS-Selection-1 and RS-Selection-2) were established using nodal segments as explants. The hormonal combinations of benzyl adenine and kinetin not only influenced their morphogenetic response but also differentially modulated the level of biogeneration of withanolide A in the in vitro shoots of the two lines. Interestingly, withanolide-A, that was hardly detectable in the aerial parts of field-grown Withania somnifera (explant source), accumulated considerably in the in vitro shoot cultures of the plant. The productivity of withanolide A in the cultures varied considerably (ca. 10-fold, 0.014 to 0.14 mg per gram fresh weight) with the change in the hormone composition of the culture media as well as genotype used as source of the explant. The shoot culture of RS-Selection-1 raised at 1.00 ppm of BAP and 0.50 ppm of kinetin displayed the highest concentration of withanolide A in the green shoots of 0.238 g per 100 g dry weight tissue. This was a more analytical concentration keeping in view the isolation yields so far reported from the dried roots of the field-grown plant (ca. 0.015 g per 100 g dry weight), even if isolation losses are considered during purification. The enhanced de novo biogenesis of withanolide A in shoot cultures was corroborated with radiolabel incorporation studies using [2-(14)C] acetate as a precursor. Production of withaferin A was also found in the in vitro shoot cultures. As this compound is a predominant withanolide of native shoots as well and has been already reported to be accumulated in in vitro shoot cultures, its biogeneration observed in these shoot cultures is not discussed in detail.