A Validated and Densitometric HPTLC Method for the Quantification of Withaferin-A and Withanolide-A in Different Plant Parts of Two Morphotypes of Withania somnifera

A simple, sensitive and accurate high performance thin layer chromatographic (HPTLC) method has been developed for the estimation of withaferin-A and withanolide-A in different plant parts such as, leaf, root, stem and fruit of two morphotypes of Withania somnifera. HPTLC of W. somnifera methanolic extract was performed on Si 60 F₂₅₄ (20 cm × 20 cm) plates with toluene:ethyl acetate:formic acid (5:5:1), as mobile phase. Quantitative evaluation of the plate was performed in the absorption-reflection mode at 530 nm. The method was validated for precision, repeatability, and accuracy. The average recovery of withaferin-A and withanolide-A in two levels were 96.0 and 96.7%, showing the excellent reproducibility of the method. The calibration curves were linear for both in the range of 200–3,200 ng. The technique has been applied, for the first time, for the estimation of withaferin-A and withanolide-A in different parts of the two morphotypes of Withania somnifera. The method is simple, precise, specific, sensitive and accurate and can be used for routine analysis as well as for quality control of raw materials and herbal formulations.     

Assessment of Chemopreventive Potential of the Plant Extracts against Liver Cancer Using HepG2 Cell Line

The edible parts of the plants Camellia sinensis, Vitis vinifera and Withania somnifera were extensively used in ancient practices such as Ayurveda, owing to their potent biomedical significance. They are very rich in secondary metabolites such as polyphenols, which are very good antioxidants and exhibit anti-carcinogenic properties. This study aims to evaluate the anti-cancerous properties of these plant crude extracts on human liver cancer HepG2 cells. The leaves of Camellia sinensis, Withania somnifera and the seeds of Vitis vinifera were collected and methanolic extracts were prepared. Then, these extracts were subjected to DPPH, α- amylase assays to determine the antioxidant properties. A MTT assay was performed to investigate the viability of the extracts of HepG2 cells, and the mode of cell death was detected by Ao/EtBr staining and flow cytometry with PI Annexin- V FITC dual staining. Then, the protein expression of BAX and BCl2 was studied using fluorescent dye to determine the regulation of the BAX and BCl2 genes. We observed that all the three extracts showed the presence of bioactive compounds such as polyphenols or phytochemicals. The W. somnifera bioactive compounds were found to have the highest anti-proliferative activity on human liver cancer cells.     

Correlation between heavy metal contents and antioxidant activities in medicinal plants grown in copper mining areas

Three commonly used medicinal plants, e.g., Adhatoda vasica, Cassia fistula, and Withania somnifera grown in two contrasting environmental conditions, namely from copper mining site and from control site corresponding to soil not contaminated with Cu, to understand correlations between high Cu bioaccumulation in medicinal plants on their antioxidant activities. Concentrations of some essential metals, e.g., Cr, Mn, Fe, Cu, Zn, and Se in the leaves of these plants were measured by instrumental neutron activation analysis. The Cu levels in the samples from mining site were in the range of 32.6 to 57.2?mg/kg, which were 5?C7 folds higher than the control samples, while Cr levels were about 2-folds higher in the mining site. Speciation studies of Cr revealed negligible content of toxic hexavalent Cr. Antioxidant assay of these plants from both the sampling sites, measured as total phenolic content, total flavonoid content, 2,2??-diphenyl-1-picrylhydrazyl, free radical scavenging ability, and chelating ability with ferrous ions exhibited maximum activity for A. vasica, while that of W. somnifera was minimum. However, the variations in the antioxidant activities for each medicinal plant species from mining site and control site did not reveal significant differences.     

Withaferin A—A Promising Phytochemical Compound with Multiple Results in Dermatological Diseases

Withaferin A (WFA) was identified as the most active phytocompound of the plant Withania somnifera (WS) and as having multiple therapeutic/ameliorating properties (anticancer, antiangiogenic, anti-invasive, anti-inflammatory, proapoptotic, etc.) in case of various diseases. In drug chemistry, WFA in silico approaches have identified favorite biological targets, stimulating and accelerating research to evaluate its pharmacological activity—numerous anticancer effects manifested in various organs (breast, pancreas, skin, colon, etc.), antivirals, anti-infective, etc., which are not yet sufficiently explored. This paper is a synthesis of the most relevant specialized papers in the field that are focused on the use of WFA in dermatological diseases, describing its mechanism of action while providing, at the same time, details about the results of its testing in in vitro/in vivo studies.     

Effect of Environmental Pollution on Heavy Metals Content of Withania Somnifera

Heavy metals were investigated in the medicinal plant Withania somnifera as well as of the soil it was grown in using atomic absorption spectrophotometer. The plant samples were collected from three different locations of N.W.F.P, Pakistan. The plant parts including roots, stem, leaves and fruits were found to have the quantity of heavy metals corresponding to their content in the soil. The purpose of the study is to create awareness among people about the proper use and collection of medicinal plants containing high levels of heavy metals and their adverse health affects.     

Withanolide production by fungal endophyte isolated from Withania somnifera

Withanolides, the secondary metabolite from Withania species is used in Ayurvedic medicine and now proved to have potential use in treating cardiovascular, Alzheimer’s disease, etc. Its production in plants varies between genotypes with very low yield. For improved industrial commercialisation, there is a need to increase its production. Endophytic fungi are symbiotically associated with plants and can synthesise the same bioactive compounds and natural products as their host plant. There are no reports available on withanolide-producing (endophytic) fungi. The present study identified an endophytic fungus (Taleromyces pinophilus) from leaves of Withania somnifera which produces withanolides in the medium. The structure of withanolide was confirmed by ¹H NMR, LC-MS analyses and quantified by HPLC analysis. The fungus produces high amount of withanolide when compared to leaf and root of W. somnifera. The fungus can be exploited to produce the withanolide to meet its demand.     

HPTLC analysis of Withaferine A from an herbal extract and polyherbal formulations

Withania somnifera has been used in Ayurvedic medicine for treatment of depression and inflammation, and as an aphrodisiac. It contains many phytochemicals such as Withaferine A, withanine, anahygrine, tropine, and withanolides. Of these, withaferine A is considered to be the most active compound. Withaferine A was estimated in herbal extract and polyherbal formulations by high performance thin layer chromatography (HPTLC). As there is no official HPTLC protocol for quantitation of the above phytochemicals, an attempt was made to quantify withaferine A in herbal extract and polyherbal formulations produced from Withania somnifera. Precoated silica gel G (aluminium backed) plates were used as stationary phase and toluene:ethyl acetate: formic acid (50 : 15 : 5) was used as mobile phase. Detection and quantification were performed by densitometry at λ 213 nm. The linear range was 1 μg to 3 μg. This HPTLC method was found to be reproducible, accurate, and precise.     

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.     

Anticancer Properties of Highly Purified l-Asparaginase from Withania somnifera L. against Acute Lymphoblastic Leukemia

Withania somnifera L. has been traditionally used as a sedative and hypnotic. The present study was carried out for the purification, characterization, and in vitro cytotoxicity of l-asparaginase from W. somnifera L. l-Asparaginase was purified from the fruits of W. somnifera L. up to 95% through chromatography. The purified l-asparaginase was characterized by size exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and 2D PAGE. The antitumor and growth inhibition effect of the l-asparaginase was assessed using [3-(4, 5-dimethyl-thiazol-2yl)-2, 5-diphenyl-tetrazolium bromide] (MTT) colorimetric dye reduction method. The purified enzyme is a homodimer, with a molecular mass of 72 ± 0.5 kDa, and the pI value of the enzyme was around 5.1. This is the first report of the plant containing l-asparaginase with antitumor activity. Data obtained from the MTT assay showed a LD₅₀ value of 1.45 ± 0.05 IU/ml. W. somnifera L. proved to be an effective and a novel source of l-asparaginase. Furthermore, it shows a lot of similarity with bacterial l-asparaginases EC-2.     

Inhibition of thermal induced protein denaturation of extract/fractions of Withania somnifera and isolated withanolides

This study describes the in vitro inhibition of protein denaturation of extract/fractions of Withania somnifera and isolated withanolides including 20β hydroxy-1-oxo(22R)-witha-2,5,24 trienolide (1), (20R,22R-14α,20α)-dihydroxy-1-oxowitha-2,5,16,24 tetraenolide (2). The results showed that the extract/fractions of the plant evoked profound inhibitory effect on thermal-induced protein denaturation. The chloroform fraction caused the most dominant attenuation of 68% at 500 μg/mL. The bioactivity-guided isolation from chloroform fraction led to the isolation of compounds 1 and 2 that showed profound protein inhibition with 78.05% and 80.43% effect at 500 μg/mL and thus strongly complimented the activity of extract/fractions. In conclusion, extract/fractions of W. somnifera possessed strong inhibition of protein denaturation that can be attributed to these isolated withanolides.     

Probing of Metabolites in Finely Powdered Plant Material by Direct Laser Desorption Ionization Mass Spectrometry

Natural products continue to serve as an important source of novel drugs since the beginning of human history. High-throughput techniques, such as MALDI-MS, can be techniques of choice for the rapid screening of natural products in plant materials. We present here a fast and reproducible matrix-free approach for the direct detection of UV active metabolites in plant materials without any prior sample preparation. The plant material is mechanically ground to a fine powder and then sieved through different mesh sizes. The collected plant material is dispersed using 1 μL solvent on a target plate is directly exposed to Nd:YAG 335 nm laser. The strategy was optimized for the analysis of plant metabolites after study of the different factors affecting the reproducibility and effectiveness of the analysis, including particle sizes effects, types of solvents used to disperse the sample, and the part of the plant analyzed. Moreover, several plant species, known for different classes of metabolites, were screened to establish the generality of the approach. The developed approach was validated by the characterization of withaferin A and nicotine in the leaves of Withania somnifera and Nicotiana tabacum, respectively, through comparison of its MS/MS data with the standard compound. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques were used for the tissue imaging purposes. This approach can be used to directly probe small molecules in plant materials as well as in herbal and pharmaceutical formulations for fingerprinting development.      

Functional Characterization of a Flavonoid Glycosyltransferase Gene from Withania somnifera (Ashwagandha)

Glycosylation of flavonoids is mediated by family 1 uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs). Until date, there are few reports on functionally characterized flavonoid glycosyltransferases from Withania somnifera. In this study, we cloned the glycosyltransferase gene from W. somnifera (UGT73A16) showing 85–92 % homology with UGTs from other plants. UGT73A16 was expressed as a His₆-tagged fusion protein in Escherichia coli. Several compounds, including flavonoids, were screened as potential substrates for UGT73A16. HPLC analysis and hypsochromic shift indicated that UGT73A16 transfers a glucose molecule to several different flavonoids. Based on kinetic parameters, UGT73A16 shows more catalytic efficiency towards naringenin. Here, we explored UGT73A16 of W. somnifera as whole cell catalyst in E. coli. We used flavonoids (genistein, apigenin, kaempferol, naringenin, biochanin A, and daidzein) as substrates for this study. More than 95 % of the glucoside products were released into the medium, facilitating their isolation. Glycosylation of substrates occurred on the 7- and 3-hydroxyl group of the aglycone. UGT73A16 also displayed regiospecific glucosyl transfer activity towards 3-hydroxy flavone compound, which is the backbone of all flavonols and also for a chemically synthesized compound, not found naturally. The present study generates essential knowledge and molecular as well as biochemical tools that allow the verification of UGT73A16 in glycosylation.     

Structural bioinformatics-based identification of putative plant based lead compounds for Alzheimer Disease Therapy

Plant based lead compounds have been historically incredible as a source of therapeutic agents for various complex disorders including Alzheimer’s disease (AD). AD is one of the leading neurodegenerative disorder in which the underlying risk factors remain largely unclear and presently, there is no disease modifying treatment available. Despite its potential, to date only few compounds have entered for clinical trials. Herein, we described the identification of plant based lead compounds for treatment of AD through an integrative approach of pharmacokinetics and structure bioinformatics approach. In particular we performed screening of lead compounds from 3 traditional medicinal plants namely Withania somnifera, Bacopa monnieri and Morus alba, which are known to have potential for treatment of neurodegenerative disease. We retrieved a total of 210 plant based compounds of which 21 compounds were screened based on their pharmacokinetic properties. Further, Docking study against 7 known AD associated targets were carried out to identify the binding sites and direct interacting residues. In addition we investigate the stable and reliable binding mechanism of top such plant compounds against 3 targets through molecular docking followed by Molecular Dynamic(MD) simulation. The results obtained in the study revealed that 3 drug compounds namely Morusin (MRSN), Withanone (WTHN) and 27-Hydroxywithanolide B (HWTHN) were identified as putative lead compounds against mono amine oxidase (MAOB), Beta-secretase 1(BACE1) and phosphodiesterase 4D.     

Constituents of Withania somnifera Dun—XIII : The withanolides of chemotype III

Nine new steroidal lactones of the withanolide series (withanolides E-M) have been isolated from the leaves of Withania somnifera Dun (Solanaceae) growing in the southern coastal plane of Israel. This population of W. somnifera constitutes a new chemotype and is designated as chemotype III. The following structures have been assigned to seven of the above compounds:Withanolide G, 20α-hydroxy-1-oxo-20R,22R-witha-2,5,8(14),24-tetraenolide (1); withanolide H, 20α,27-dihydroxy-1-oxo-20R,22R-witha-2,5,8(14),24-tetraenolide (2); withanolide I, 20α-hydroxy-1-oxo-20R,22R-witha-3,5,8(14),24-tetraenolide (3); withanolide J, 17α,20α-dihydroxy-1-oxo-20S,22R-witha-2,5,8(14),24-tetraenolide (4); withanolide K, 17α,20α-dihydroxy-1-oxo-20S,22R-witha-3,5,8(14),24-tetraenolide (5); withanolide L, 17α,20α-dihydroxy-1-oxo-20S,22R-witha-2,5,14,24-tetraenolide (6); withanolide M, 17α,20α-dihydroxy-1-oxo-14α,15α-epoxy-20S,22R-witha-2,5,24-trienolide (7).     

Determination of mycotoxins,alkaloids, phytochemicals,antioxidants and cytotoxicity in Asiatic ginseng (Ashwagandha,Dong quai, <Emphasis Type="Italic">Panax ginseng</Emphasis>)

Mycotoxins and selected hazardous alkaloids in the medicinal plants (Panax ginseng, Angelica sinensis, and Withania somnifera) and dietary supplements were determined. Purine alkaloids were found in majority of samples; however, isoquinoline alkaloids were less abundant than indole. The predominant alkaloids appear to be caffeine (purine group), harman (indole group) and berberine (isoquinoline). Examined medicinal plants and dietary supplements were contaminated by mycotoxins (especially ochratoxin A 1.72–5.83 µg kg^?1), and many species of mold (e.g. Cladosporium, Eurotium, Aspergillus, Rhizopus, Penicillium). MTT cytotoxicity tests revealed that plant and supplements extracts exhibited medium or high cytotoxicity (only Dong quai—low). Moreover, antioxidant activity, total phenolics content and selected phytochemicals were analyzed by spectrophotometric and chromatographic methods. Quercetin and rutin were predominant flavonols (1.94-9.51 and 2.20–7.28 mg 100 g^?1, respectively). Analysis of phenolic acids revealed—gallic acid, as the most abundant, except Panax ginseng, where ferulic acid was prevailing. The results were analyzed by chemometric methods (cluster analysis, ANOVA).     

Optimization of Elicitation Conditions with Methyl Jasmonate and Salicylic Acid to Improve the Productivity of Withanolides in the Adventitious Root Culture of Withania somnifera (L.) Dunal

Adventitious root cultures derived from leaf derived callus of Withania somnifera (L.) Dunal were treated with methyl jasmonate and salicylic acid independently. Biomass accumulation, culture age, elicitation period, and culture duration were optimized for higher withanolides production in the two best-responding varieties collected from Kolli hills (Eastern Ghats) and Cumbum (Western Ghats) of Tamil Nadu, India. Between the two elicitors, salicylic acid (SA) improved the production of major withanolides (withanolide A, withanolide B, withaferin A, and withanone) as well as minor constituents (12-deoxy withastramonolide, withanoside V, and withanoside IV) in the Kolli hills variety. Treatment of root biomass (11.70?g FW) on 30-day-old adventitious root cultures with 150???M SA for 4?h elicitor exposure period resulted in the production of 64.65?mg?g^?l dry weight (DW) withanolide A (48-fold), 33.74?mg?g^?l DW withanolide B (29-fold), 17.47?mg?g^?l DW withaferin A (20-fold), 42.88?mg?g^?l DW withanone (37-fold), 5.34?mg?g^?l DW 12-deoxy withastramonolide (nine fold), 7.23?mg?g^?l DW withanoside V (seven fold), and 9.45?mg?g^?l DW withanoside IV (nine fold) after 10?days of elicitation (40th day of culture) when compared to untreated cultures. This is the first report on the use of elicitation strategy on the significant improvement in withanolides production in the adventitious root cultures of W. somnifera.     

In Vitro Alpha-Amylase and Alpha-Glucosidase Inhibitory Activity and In Vivo Antidiabetic Activity of Withania frutescens L. Foliar Extract

Withania frutescens L. is a wild perennial woody plant used by the local population for diverse therapeutic purposes. This work aims to study for the first time the potential inhibitory effect of this plant hydroethanolic extract on α-amylase and α-glucosidase activities using in vitro methods and its antidiabetic and antihyperglycemic activities using alloxan-induced diabetic mice as a model for experimental diabetes. Two doses were selected for the in vivo study (200 and 400 mg/kg) and glibenclamide, a well-known antidiabetic drug (positive control) in a subacute study (28 days) where the antihyperglycemic activity was also assessed over a period of 12 h on diabetic mice. The continuous treatment of diabetic mice with the extract of Withania frutescens for 4 weeks succeeded to slowly manage their high fasting blood glucose levels (after two weeks), while the antihyperglycemic test result revealed that the extract of this plant did not control hyperglycemia in the short term. No toxicity signs or death were noted for the groups treated with the plant extract, and it shows a protective effect on the liver and kidney. The in vitro assays demonstrated that the inhibition of alpha-amylase and alpha-glucosidase might be one of the mechanisms of action exhibited by the extract of this plant to control and prevent postprandial hyperglycemia. This work indicates that W. frutescens have an important long term antidiabetic effect that can be well established to treat diabetes.     

Proton induced X-ray emission (PIXE) technique for determining multi-element composition of transformed hairy root cultures of Boerhaavia diffusa L.: an important medicinal herb

Therapeutically important inorganic elements in Agrobacterium rhizogenes-mediated genetically transformed hairy root cultures (HRCs) of a pharmaceutically significant herb Boerhaavia diffusa were quantified using proton induced X-ray emission technique. This was compared with that of roots from the naturally grown donor plant. Two macro-elements (Ca & K) and eight different trace elements namely V, Cr, Mn, Fe, Co, Cu Zn, and Ni were detected and their content was determined. In HRCs of a transformed rhizoclone, calcium and potassium had values which were significantly higher than that of in vivo roots. The concentrations of several trace elements, which are known to have a positive implication in human healthcare, were found to be either comparable (Fe) to that in the natural root samples or higher (Mn, Zn, Cr, Cu, Co) in the transformed rhizoclone. The genetically transformed HRCs can thus serve as a fast-proliferating renewable resource of medicinally useful minerals targeting specific diseases.