Golden berry leaf extract containing withanolides suppresses TNF-α and IL-17 induced IL-6 expression in HeLa Cells.

Inflammation, characterized by the overexpression of IL-6 in various tissues, has been reported as a symptom of coronavirus disease 2019. In this study, we established an experimental system for overexpression of IL-6 in HeLa cells stimulated by TNF-α and IL-17, along with identification of anti-inflammatory materials and components from local agricultural, forestry, and fishery resources. We constructed a library of extracts from natural sources, of which 111 samples were evaluated for their anti-inflammatory activities. The MeOH extract of Golden Berry (Physalis peruviana L) leaf was found to exhibit strong anti-inflammatory properties (IC50 = 4.97 µg/mL). Preparative chromatography identified two active constituents, 4ß-hydroxywithanolide E (4ß-HWE) (IC50 = 183 nM) and withanolide E (WE) (IC50 = 65.1 nM). Withanolides are known anti-inflammatory ingredients of Withania somnifera, an Ayurvedic herbal medicine. P. peruviana leaves containing 4ß-HWE and WE should be considered as useful natural resources for anti-inflammatory products.

High performance thin layer chromatography based chemo profiling of Ashvagandharishta and its antidepressant activity.

Withania somnifera (L.) has long been used as a traditional rasayana herb against a variety of human ailments. This research presents a high performance thin layer chromatography based chemo profiling of Ashvagandharishta and its antidepressant activity. The in-house formulation was made using a fermentation process according to the Indian Pharmacopoeia. Physiochemical standardization of the formulation was performed using different quality control parameters such as total ash, acid insoluble ash, alcohol soluble extract value and water soluble extract value. A column chromatography and high performance thin layer chromatography method was used to isolate and estimation of withanolide-A, withaferin-A & ß-sitosterol from the root of W. somnifera. In addition. The antidepressant effect of different formulations were carried out by force swimming test in albino mice. The thiobarbituric acid reactive substances (TBARS) and Glutathione (GSH) assay was used to find out the oxidative stress. W. somnifera root has been standardized macroscopically, microscopically, physico-chemically according to the guidelines of the Ayurvedic Pharmacopoeia. The qualitative and quantitative analysis was performed using high performance thin layer chromatography and it was performed on each formulation and found the content of withanolide-A and -sitosterol in the in-house formulation is higher while withaferin-A is rather contained in the decoction. The antidepressant effect showed that the immobility time was lowest in the case of the standard formulation followed by in house formulation, while the increase in glutathione and the reduction in thiobarbituric acid reactive substances levels revealed the antioxidant nature of the formulation. In conclusion, based on the above results, we can conclude that Ashvagandharishta could be a breakthrough for the treatment of depression in the future.

Traditional Horticulture Practices Increase the Production of Selected Withanolides in Withania Somnifera (L.) Dunal-A RP-UFLC Analysis.

The study evaluates the effect of two traditional horticulture treatments mentioned in Vrikshayurveda, a text from ancient India on the science of plant life, namely Kunapa jala (KJ) and Pancha gavya (PG) on the production of Withaferin A (WFA), withanolide A (WIA) and Withanolide B (WIB) in Withania somnifera (L) Dunal. Leaves and roots of W. somnifera were collected from different treated groups viz. control, KJ, PG, farmyard manure (FYM) and inorganic fertilizer (NPK). Reverse phase ultra-flow liquid chromatography (RP-UFLC) method was developed, validated for simultaneous detection of WFA, WIA and WIB. Statistical analysis of data was performed by ANOVA and tested for significance by the Dunnett multiple comparison test and data were expressed as mean ± standard deviation (SD). Results revealed, leaves possessed highest WFA content and roots possessed highest content of WIA and WIB. PG treated leaves were observed highest WFA (18.29 mg/g) and roots were observed highest WIA (19.63 mg/g) and WIB (1.36 mg/g). Conclusively, RP-UFLC method for simultaneous detection of withanolides has been developed and validated to evaluate the effect of traditional horticulture treatments. It is concluded that the enhanced production of withanolides can be achieved by the application of PG when compared to NPK application.

Withanolides from the genus Physalis: a review on their phytochemical and pharmacological aspects.

OBJECTIVES: Withanolides are a group of modified C28 ergostane-type steroids with a C-22, C-26 δ-lactone side chain or a C-23, C-26 γ-lactone side chain. They enjoy a limited distribution in the plant kingdom and predominantly occur in several genera of Solanaceae. Of which, the genus Physalis is an important resource for this type of natural molecules. The present review aims to comprehensively illustrate the structural characteristics and classification of withanolides, and particularly focus on the progression on phytochemical and pharmacological aspects of withanolides from Physalis ranging from January 2015 to June 2019. KEY FINDINGS: Approximately 351 natural withanolides with novel and unique structures have so far been identified from genus Physalis, mainly isolated from the species of P. angulata and P. peruviana. Withanolides demonstrated diverse biological activity, such as anticancer, anti-inflammatory, antimicrobial, immunoregulatory, trypanocidal and leishmanicidal activity. Their observed pharmacological functions supported the uses of Physalis species in traditional or folk medicines. SUMMARY: Due to their unique structure skeleton and potent bioactivities, withanolides are regarded to be promising drug candidates, particularly for developing anticancer and anti-inflammatory agents. Further investigations for discovering novel withanolides of genus Physalis, exploiting their pharmacological values and evaluating their potency as therapeutic agents are significant work.

Development and Validation of a Novel, Rapid Gradient HPLC Method for Simultaneous Estimation of Bioactive Marker Compounds in a Mixture of Convolvulus pluricaulis, Withania somnifera and Bacopa monnieri Extracts.

The use of herbs as medicine is an ancient form of healthcare known to mankind. Standardization of herbal medicines is however a challenging task and is the major bottleneck in their acceptance as the primary therapeutic option. The aim of this study was to develop and validate a simple, rapid HPLC method for standardizing the mixture of extracts of three Medhya Rasayanas (neurotonic), Convolvulus pluricaulis, Withania somnifera and Bacopa monnieri. Simultaneous estimation of the respective bioactive markers of these plants viz., scopoletin, withaferin A, bacoside A 3, bacopaside II, jujubogenin and bacosaponin C has been reported for the first time. The method was developed using Waters Hybrid X-Bridge shield with BEH technology 2.5 µm, 4.6 × 75 mm column and validated according to ICH guidelines. The 20 minutes run time makes the method eco-friendly. The method was linear over a range of 12.5-400 ng/10 µL for scopoletin and 62.5-2,000 ng/10 µL for withaferin A, bacoside A 3, bacopaside II, jujubogenin and bacosaponin C with detection limits of 8.0, 48.3, 30.4, 40.7, 15.6 and 18.9 ng/10 µL and quantification limits of 24.5, 146.5, 92.2, 123.4, 47.4 and 57.4 ng/10 µL, respectively. The correlation coefficient for each analyte was >0.999. The intra-day and inter-day precision was <2%. These results confirmed the precision, accuracy and robustness of the proposed method.

Liquid chromatography-tandem mass spectrometry to assess the pharmacokinetics and tissue distribution of withaferin A in rats.

Withaferin A (WA) is a bioactive ingredient in the medicinal Indian herb Withania somnifera (WS). In this study, we developed a rapid and accurate Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) method to determine the concentration of WA in rat plasma and tissue following intravenous (i.v., 4.5 mg/kg) and oral (i.g, 0.5, 1.5 and 4.5 mg/kg) administration. WA was isolated on a Hypurity C18 (50 × 4.6 mm, 5 µm) column by isocratic elution at a flow rate of 0.5 mL/min using acetonitrile and water as the mobile phase (35:65, v/v). The retention time was 4 min. Ethyl acetate containing 5% ascorbic acid was used as the extraction solvent through simple liquid-liquid extraction (LLE). Withanolide A (WLD) was used as the internal standard (IS). Quantification was performed through multiple reaction monitoring (MRM) modes of m/z 471.1 → 281 for WA and m/z 488.1 → 263 for IS in the positive-ion mode. This revealed no significant effects of the WA concentration or administration route on the T1/2. The distribution of WA in the various tissues was in the order: stomach > heart > lung > kidney > small intestinal > spleen > following i.g administration (4.5 mg/kg). These data provide valuable insight into the clinical parameters of WA.

Studies on oral bioavailability and first-pass metabolism of withaferin A in rats using LC-MS/MS and Q-TRAP.

Withaferin A (WA) is one of the major bioactive steroidal lactones with extensive pharmacological activities present in the plant Withania somnifera. The absolute oral bioavailability of WA remains unknown and human-related in vitro data are not available. Therefore, in the present study, the absolute oral bioavailability of WA in male rats and the in vitro screening of absorption factors by Q-trap and LC-MS/MS analysis were conducted to explore possible clinical properties of WA. The developed and validated analytical methods were successfully applied to the pharmacokinetic studies and in vitro measurement of WA. The oral bioavailability was determined to be 32.4 ± 4.8% based on intravenous (5 mg/kg) and oral (10 mg/kg) administrations of WA in male rats. The in vitro results showed that WA could be easily transported across Caco-2 cells and WA did not show as a substrate for P-glycoprotein. Moreover, the stability of WA was similar between male rat and human in simulated gastric fluid (stable), in intestinal microflora solution (slow decrease) and in liver microsomes (rapid depletion, with a half-life of 5.6 min). As such, the first-pass metabolism of WA was further verified by rat intestine-liver in situ perfusion, revealing that WA rapidly decreased and 27.1% remained within 1 h, while the content of three major metabolites (M1, M4, M5) identified by Q-trap increased. This perfusion result is consistent with the oral bioavailability results in vivo. The first-pass metabolism of WA might be the main barrier in achieving good oral bioavailability in male rats and it is predicted to be similar in humans. This study may hold clinical significance.

Isolation, characterization and DFT studies of epoxy ring containing new withanolides from Withania coagulans Dunal.

A new withanolide named as withacoagulin J (1) along with a known withanolide H (2) from Withania coagulans Dunal are reported in this paper. The isolated compounds were elucidated by using 1D-NMR (1H NMR, 13C NMR) and 2D-NMR including homonuclear (COSY, NOESY) and heteronuclear (HSQC, HMBC); along with Mass spectrometry, UV-Visible and IR spectroscopic techniques. The molecular formula based on Fast-Atom Bombardment Mass Spectrometry [FAB-MS (M + 1)] for 1 and 2 were deduced as C28H37O5 and C28H39O6 with m/z values 453.2624 and 471.6041, respectively. The quantum mechanical studies of both compounds are based on DFT calculations. The DFT studies show band gaps of 4.86 and 4.83 eV for 1 and 2, respectively. The band gaps of 1 and 2 reflect high stability and resistivity towards oxidation-reduction reactions. The energies of HOMO and LUMO for compound 1 are -6.11 and -1.25 eV and for compound 2: -6.47 and -1.64 eV respectively. Theoretical and experimental FTIR data closely match for both the compounds which support the high accuracy of the computational protocol selection. Other parameters such as bond lengths, bond angles and dihedral angles for both compounds are also studied.

Cytotoxic Withanolides from the Roots of Indian Ginseng ( Withania somnifera).

Withania somnifera, commonly known as "Indian ginseng" or "ashwagandha", is popular as a functional food because of its diverse purported therapeutic efficacies including invigorating, improvement of cognitive ability, and stress release activities. Chemical investigation of the MeOH extract of W. somnifera roots combined with LC/MS-based analysis resulted in the identification of six new withanolides, withasilolides A-F (1-6), as well as seven known compounds (7-13). The structures of the new compounds were established by application of spectroscopic methods, including 1D and 2D NMR, HRMS, and ECD measurements. The cytotoxicity of the isolated compounds was evaluated against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15). Compounds 1, 2, 4, 6, and withanone (11) each showed cytotoxicity for one or more of the four cancer cell lines used.

Fungal endophytes attune withanolide biosynthesis in Withania somnifera, prime to enhanced withanolide A content in leaves and roots.

Endophytes have been reported from all plant species from different parts of tissue including root, stem and leaves. Here we report, three fungal endophytes, Aspergillus terreus strain 2aWF (2aWF), Penicillium oxalicum strain 5aWF (5aWF), and Sarocladium kiliense strain 10aWF (10aWF) from Withania somnifera, which could enhance withanolides content in leaf and root. Upon treatment with the above endophytes to 4 weeks old plants in field conditions, W. somnifera elicited withanolide A content (97 to 100%) in leaves without considerable changes in withaferin A content. Furthermore, withanolide A content in roots of 5aWF and 10aWF endophyte treated W. somnifera plants increased up to 52% and 65% respectively. Incidentally, expression profile of withanolide and sterol biosynthetic pathway genes HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1 and CYP710A1 were significantly upregulated in 2aWF, 5aWF and 10aWF fungal endophyte treated plants. Besides, modulation of withanolide biosynthetic pathway genes, fungal endophytes also induce a host resistant related gene, NPR1 resulting in 2, 4 and 16 fold expression levels in 2aWF, 10aWF and 5aWF endophyte treatments respectively, compared to control plants. Overall, our results illustrate that application of native-fungal endophytes 2aWF (96.60%), 5aWF (95%) and 10aWF (147%) enhances plant biomass in addition to withanolide content.

Quantification of withaferin-A and withanolide-A in diploid (n = 12) and tetraploid cytotypes (n = 24) of "Rassbhary", Physalis angulata L.

During the present study an analytical method based on reverse-phase high performance liquid chromatography photodiode array detection method was developed for simultaneous determination of withaferin-A and withanolide-A in plant parts of two cytotypes (diploid n = 12 & tetraploid n = 24) of Physalis angulata. All the plant parts were extracted in different solvent solutions i.e., acidic [HCl] methanol (i.e., methanol containing 0.3% of HCl), methanol, n-hexane, chloroform. Both the compounds were comparatively analysed. The results revealed that tetraploid cytotype (n = 24) showed the higher composition of both the reference compounds. The method is simple, rapid and provides better resolution can be easily applied to the quantitative analyses of withanolides in plant matrices.

A multi-analytical platform based on pressurized-liquid extraction, in vitro assays and liquid chromatography/gas chromatography coupled to high resolution mass spectrometry for food by-products valorisation. Part 1: Withanolides-rich fractions from goldenberry (Physalis peruviana L.) calyces obtained after extraction optimization as case study.

In this work, a multi-analytical platform that allows obtaining and characterizing high-added value compounds from natural sources is presented, with a huge potential in traditional medicine, natural products characterization, functional foods, etc. Namely, the proposed multi-analytical platform is based on the combination of pressurized liquid extraction (PLE), liquid chromatography (LC) and gas chromatography quadrupole time-of-flight mass spectrometry GC-q-TOF-MS(/MS), in vitro assays and modelling tools for guiding extraction optimization. As case study, goldenberry or cape gooseberry fruit (Physalys peruviana L.) was selected. In particular, the potential of P. peruviana calyces, an important by-product of goldenberry processing, as promising source of bioactive compounds was evaluated. Selection of the most suitable solvent for PLE was based on the Hansen solubility parameters (HSP) approach using 4ß-hydroxywithanolide E (4ßHWE) and withanolide E (WE) as target compounds due to their bioactive potential. A surface response methodology was further applied for the optimization of the PLE parameters: temperature (50, 100 and 150 °C) and solvent composition (% EtOH in the mixture EtOH/EtOAc). The effects of the independent variables on extraction yield, withanolides content (4ßHWE and WE), total phenolic content (TPC), total flavonoids content (TFC) and antioxidant activity (EC50 and TEAC) were evaluated in order to obtain withanolide-rich extracts from P. peruviana calyces. The extract obtained under optimal conditions (at 125 °C and 75% EtOH v/v) exhibited satisfactory extraction yield (14.7%) and moderate antioxidant activity (with an EC50 value of 77.18 µg mL-1 and 1.08 mM trolox g-1), with 4ßHWE and WE concentrations of 8.8 and 2.3 mg g-1, respectively. LC-q-TOF-MS/MS analysis of the extract allowed the quantitation of 4ßHWE and WE and the tentative identification of several other withanolides structures. The obtained results demonstrate the great potential of this multi-analytical approach for developing valorisation strategies of food by-products under sustainable conditions, to obtain bioactive-enriched extracts with potential medicinal or health-promoting properties.

Drier Climatic Conditions Increase Withanolide Content of Withania coagulans Enhancing Its Inhibitory Potential Against Human Prostate Cancer Cells.

Prostate cancer is one of the major causes of cancer-related deaths in men and there is a growing interest in identifying natural compounds for its management. We analyzed bioactive withanolides in Withania coagulans from 11 different sites in Pakistan and evaluated the antiprostate cancer activities of leaf extracts from two sites with the greatest amounts. Total withanolide concentration differed by ~ 17-fold between sites, ranging from 1.01 ± 0.01 mg/g dry weight (mean ± SE) at Jand to 16.83 ± 0.02 mg/g at Mohmand Agency. Different tissues varied in their total withanolide content with roots having the least (0.42 ± 0.07 mg/g dry weight) and leaves the most (2.45 ± 0.45 mg/g). We found strong inverse correlations between site annual precipitation versus withanolide amounts in fruits (r = - 0.84, P = 0.001), leaves (r = - 0.88, P < 0.001), roots (r = - 0.91, P < 0.001), and total (r = - 0.89, P < 0.001), but not stems (r = - 0.20, P = 0.556). Extracts made from Mianwali and Mohmand Agency leaves possessed high anticancer activity in terms of increased induction of apoptosis and decreased cell viability, cell proliferation, invasion, and migration of different prostate cancer cell lines. These results are useful for the selection of withanolide-rich germplasm with potent anticancer properties.

Determination of permeability, plasma protein binding, blood partitioning, pharmacokinetics and tissue distribution of Withanolide A in rats: A neuroprotective steroidal lactone.

Preclinical Research & Development Withanolide A (WA), a steroidal lactone is a major bioactive constituent of Withania somnifera (L.) with remarkable neuropharmacological activity. In this study, we investigated the permeability, plasma protein binding (PPB), blood partitioning, intravenous (i.v.), and oral pharmacokinetics as well as i.v. tissue distribution (TD) of pure WA in a rat model. The PPB, RBCs partitioning, and permeability of WA were determined by Ultra Performance Liquid Chromatography (UPLC) method. However, the pharmacokinetics and TD of WA were evaluated by validated and sensitive liquid chromatography coupled mass spectrometry (LC-ESI-MS/MS) method. The PPB and permeability of WA were determined by equilibrium dialysis and parallel artificial membrane permeability assay method, respectively. The results demonstrated that WA has high PPB and passive permeability. Furthermore, WA was found to have fast equilibration between RBCs and plasma. Following i.v. (2 mg/kg) and per-oral (25 mg/kg) administration of WA, the max concentration (Cmax ) in plasma was found as 85.53 ± 6.54 and 48.04 ±5.78 ng/mL, respectively. The TD study results indicated that WA has a rapid and wide TD. The maximum concentration in various tissues was found in following order: Clung > Cliver > Ckidney ≈ Cspleen > Cheart > Cbrain . The preclinical in vitro, as well as pharmacokinetics and TD results, are anticipated to support the future preclinical and clinical application of WA.

Extraction Optimization for Phenolic- and Withanolide-Rich Fractions from Withania somnifera Roots: Identification and Quantification of Withaferin A, 12-Deoxywithastromonolide, and Withanolide A in Plant Materials and Marketed Formulations Using a Reversed-Phase HPLC-Photodiode Array Detection Method.

Background: Both the roots and leaves of Withania somnifera are products of commerce. They contain active compounds of therapeutic value and mostly different withanolides. Several pharmacological activities of W. somnifera have links to one or more withanolides. The presence of phenolic compounds in extracts could play a vital role in the reduction of blood glucose levels in diabetic subjects. Objective: The present study was carried out for the selection of a solvent to prepare extracts rich in phenolics, withaferin A (WA), 12-deoxywithastromonolide (12WD), and withanolide A (WDA). A simple, rapid HPLC method was also developed for the identification and quantification of WA, 12WD, and WDA. Methods: The extraction efficiency of aqueous alcoholic solvents including hexane, chloroform, ethyl acetate, and methanol were compared for three selected withanolides and total phenolic content. The contents of WA, 12WD, and WDA and total phenolics were determined in the extracts. The quality of nine formulations containing W. sominfera were also compared in terms of the content of WA, 12WD, and WDA and total phenolics. Results: The maximum extract yield and the total withanolide and phenolic content were obtained from aqueous alcoholic compositions at 50:50 (v/v), 70:30 (v/v), and 100:0 (v/v), respectively. In the case of organic solvents, chloroform and ethyl acetate yielded the highest concentrations of phenolics and three withanolides, respectively. The total phenolic content in formulations was in the range of 1.84-3.13%, and total withanolide content showed wide variability. Conclusions: The outcome of the present investigation could be utilized for the selection of extraction solvents to prepare W. somnifera-enriched extracts and their quality monitoring by using the developed and validated HPLC-Photodiode array detection method. Highlights: A process for preparation of phenolics and withanolides (withaferin A, 12-deoxywithastramonolide and withanolide A) enriched extracts of Withania somnifera. Simple and rapid HPLC method was also developed and validated as per the ICH guidelines for identification and quantification of three major withanolides. The developed HPLC method was applied to analyze the quality of extracts and marketed herbal products (mono, as well as poly constituents). Optimized extraction process could be utilized for upscaling process development in preparation of enriched extracts from Withania somnifera, crop improvement, bio-prospection studies and quality control.

Untargeted metabolomics reveals specific withanolides and fatty acyl glycoside as tentative metabolites to differentiate organic and conventional Physalis peruviana fruits.

The agronomic production systems may affect the levels of food metabolites. Metabolomics approaches have been applied as useful tool for the characterization of fruit metabolome. In this study, metabolomics techniques were used to assess the differences in phytochemical composition between goldenberry samples produced by organic and conventional systems. To verify that the organic samples were free of pesticides, individual pesticides were analyzed. Principal component analysis showed a clear separation of goldenberry samples from two different farming systems. Via targeted metabolomics assays, whereby carotenoids and ascorbic acid were analyzed, not statistical differences between both crops were found. Conversely, untargeted metabolomics allowed us to identify two withanolides and one fatty acyl glycoside as tentative metabolites to differentiate goldenberry fruits, recording organic fruits higher amounts of these compounds than conventional samples. Hence, untargeted metabolomics technology could be suitable to research differences on phytochemicals under different agricultural management practices and to authenticate organic products.

Validated thin-layer chromatographic method for the identification and monitoring of the effect of the extraction method on the yield and phytochemical constituents of Egyptian Withania somnifera leaves.

A sensitive, reliable, simple and rapid thin-layer chromatographic method has been developed for routine analysis of withanolide S content for the purpose of quality control assessment of chemotype III of Withania somnifera. The new method was used first to compare the accumulation of withanolide S in different parts of the plant, which was found to be the highest in the leaves extract (0.21% w/w). Second, to investigate different extraction parameters that improve the extraction efficiency of withanolides from the leaves using conventional and ultrasound-assisted extraction methods. The extraction efficiency was expressed via total withanolide content and withanolide S content.

RP-HPLC Separation of Isomeric Withanolides: Method Development, Validation and Application to In situ Rat Permeability Determination.

Withanolides are the group of active chemical constituents of Withania somnifera (L.) Dunal. Withaferin A, withanolide A and withanone presents three of the biologically most active constituents of this herb. These steroidal lactones are isomers of each other and thus, pose significant difficulty in their separation. In present study, a simple, specific and reliable RP-HPLC method has been developed and validated for their separation and simultaneous quantification. Separation was carried out on Lichrocart Purospher STAR RP-18e column (250 × 4.5 mm, 5 µm) using mobile phase, methanol and 0.01 M ammonium acetate buffer (pH 5) in the ratio 60:40, v/v. The calibration curves were linear (r2 > 0.99) for all the three compounds across concentration range of 1.56-50 µg/mL. The lower limit of quantification for all the analytes was 1.56 µg/mL. The intra-day and inter-day accuracy was between 88.65% and 110.66% and coefficient of variation was between 0.55 and 10.12. The analytes were stable under different storage conditions. The developed method was successfully applied to analyze the samples for simultaneous determination of permeability of the three withanolides in rats using in situ single-pass intestinal perfusion model. Withanolide A and withanone were found to be high permeability compounds while withaferin A could not be detected.

Comparative study of withanolide production and the related transcriptional responses of biosynthetic genes in fungi elicited cell suspension culture of Withania somnifera in shake flask and bioreactor.

Ashwagandha (Withania somnifera) is one of the most reputed medicinal plants in the traditional medicinal system. In this study, cell suspension culture of W. somnifera was elicited with cell homogenates of fungi (A. alternata, F. solani, V. dahliae and P. indica) in shake flask and the major withanolides like withanolide A, withaferin A and withanone were analysed. Simultaneously expression levels of key pathway genes from withanolides biosynthetic pathways were also checked via quantitative PCR in shake flask as well as in bioreactor. The results show that highest gene expression of 10.8, 5.8, 4.9, and 3.3 folds were observed with HMGR among all the expressed genes in cell suspension cultures with cell homogenates of 3% P. indica, 5% V. dahliae, 3% A. alternata and 3% F. solani, respectively, in comparison to the control in shake flask. Optimized concentration of cell homogenate of P. indica (3% v/v) was added to the growing culture in 5.0-l bioreactor under optimized up-scaling conditions and harvested after 22 days. The genes of MVA, MEP and withanolides biosynthetic pathways like HMGR, SS, SE, CAS, FPPS, DXR and DXS were up-regulated by 12.5, 4.9, 2.18, 4.65, 2.34, 1.89 and 1.4 folds, respectively in bioreactor. The enhancement of biomass (1.13 fold) and withanolides [withanolide A (1.7), withaferin A (1.5), and withanone (1.5) folds] in bioreactor in comparison to shake flask was also found to be in line with the up-regulation of genes of withanolide biosynthetic pathways.

Metabolic profiles of physalin A in rats using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Physalin A, one of the major active components isolated from the calyces of Physalis alkekengi var. franchetii is considered to be a promising natural product due to its anti-inflammatory and excellent antitumor activities. Until now, only one paper is available from our group concerning identification of two sulfonate metabolites from rat feces after physalin A treatment. All the other researches related to physalin A were focused on its extraction, separation and biological activities. In this research, a rapid and reliable ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS) method was developed and employed for the comprehensive study of the metabolism of physalin A in vivo for the first time. A total of 24 proposed metabolites were identified in plasma, bile, urine and feces of rats after oral administration of physalin A. The results indicated that sulfonation, reduction and hydroxylation were the major metabolic pathways of physalin A in vivo. Furthermore, this research provides scientific and reliable support for full understanding of the metabolism of physalin A and the results could help to elucidate the safety and efficacy of physalin A, as well as other physalins.