Abhishek Niranjan

DEVELOPMENT AND OPTIMIZATION OF HPLC-PDA-MS-MS METHOD FOR SIMULTANEOUS QUANTIFICATION OF THREE CLASSES OF FLAVONOIDS IN LEGUME SEEDS, VEGETABLES, FRUITS, AND MEDICINAL PLANTS

Certain plants of the daily human diet are an important source of beneficially healthy flavonoids, possessing significant pharmacological activities against various diseases. Flavonoids display an array of chemical diversity based on relative positioning of benzopyrano moiety and aromatic ring. Due to structural diversity of these molecules, simultaneous quantification of these molecules in plant materials becomes difficult. In the present study, a rapid, simple, and sensitive, High Performance Liquid Chromatography-Photo Diode Array Mass Spectrometry (HPLC-PDA-MS-MS) method was developed for the simultaneous determination of three different classes of flavonoids,namely, isoflavonoids, flavanones, and flavonols in legume seeds, vegetables, fruits, and medicinal plants. The method was evaluated for validation parameters, such as linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), specificity, selectivity, and compound stability. The developed method was applied on extracts of different plant samples for quantification of the flavonoids daidzein, genistein, naringenin, quercetin, kaempferol, and rutin. Method developed in this study can be used in the quality control and standardization of plant extracts as well as herbal drugs and formulations. Supplemental materials are available for this article. Go to the publishers online edition of the Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.

Selenite modulates the level of phenolics and nutrient element to alleviate the toxicity of arsenite in rice (Oryza sativa L.).

Arsenic (As) contamination of paddy rice is a serious threat all over the world particularly in South East Asia. Selenium (Se) plays important role in protection of plants against various abiotic stresses including heavy metals. Moreover, arsenite (AsIII) and selenite (SeIV) can be biologically antagonistic due to similar electronic configuration and sharing the common transporter for their uptake in plant. In the present study, the response of oxidative stress, phenolic compounds and nutrient elements was analyzed to investigate Se mediated As tolerance in rice seedlings during AsIII and SeIV exposure in hydroponics. Selenite (25µM) significantly decreased As accumulation in plant than As (25µM) alone treated plants. Level of oxidative stress related parameters viz., reactive oxygen species (ROS), lipid peroxidation, electrical conductivity, nitric oxide and pro-oxidant enzyme (NADPH oxidase), were in the order of As>As+Se>control>Se. Selenium ameliorated As phytotoxicity by increased level of phenolic compounds particularly gallic acid, protocatechuic acid, ferulic acid and rutin and thiol metabolism related enzymes viz., serine acetyl transferase (SAT) and cysteine synthase (CS). Selenium supplementation enhanced the uptake of nutrient elements viz., Fe, Mn, Co, Cu, Zn, Mo, and improved plant growth. The results concluded that Se addition in As contaminated environment might be an important strategy to reduce As uptake and associated phytotoxicity in rice plant by modulation of phenolic compounds and increased uptake of nutrient elements.

SIMULTANEOUS SEPARATION AND QUANTIFICATION OF TARGETED GROUP OF COMPOUNDS IN PSORALEA CORYLIFOLIA L. USING HPLC-PDA-MS-MS

A rapid, sensitive, and simple high performance liquid chromatography photo diode array mass spectrometry (HPLC-PDA-MS-MS) method was developed for the simultaneous determination of nine compounds, namely, psoralen (furocoumarin), daidzein, genistein (isoflavonoids), daidzin and genestin (isoflavone glycosides), biochanin A (methoxylated isoflavone), and quercetin, kaempferol, and rutin (flavonols). Chromatographic separation of these nine molecules was performed on RP C18 column within 20 min. Elution was accomplished by the application of trifluoroacetic acid (0.05%) in water and methanol in gradient system with flow rate of 1.0 mL/min. PDA and mass spectrometry was employed for qualitative and quantitative analysis. The method is economical in terms of the time expended and the amount of solvent used for each analysis. The calibration curves were linear and ranged between 0.994 to 0.999; limit of detection (LOD) and quantification (LOQ) ranged between 1.02 to 1.18 and 2.23 to 3.34 and recovery ranged between 91.76% to 97.41%. The validated method was successively used to separate and quantitative measurement of these compounds in plant parts of Psoralea corylifolia. The developed method may be used in the quality control and standardization of plant extracts as well as herbal drugs and formulations. Supplemental materials are available for this article. Go to the publishers online edition of Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.

RNAi-mediated gene silencing of WsSGTL1 in W.somnifera affects growth and glycosylation pattern

Sterol glycosyltransferases (SGTs) belong to family 1 of glycosyltransferases (GTs) and are enzymes responsible for synthesis of sterol–glucosides (SGs) in many organisms. WsSGTL1 is a SGT of Withania somnifera that has been found associated with plasma membranes. However its biological function in W.somnifera is largely unknown. In the present study, we have demonstrated through RNAi silencing of WsSGTL1 gene that it performs glycosylation of withanolides and sterols resulting in glycowithanolides and glycosylated sterols respectively, and affects the growth and development of transgenic W.somnifera. For this, RNAi construct (pFGC1008-WsSGTL1) was made and genetic transformation was done by Agrobacterium tumefaciens. HPLC analysis depicts the reduction of withanoside V (the glycowithanolide of W.somnifera) and a large increase of withanolides (majorly withaferin A) content. Also, a significant decrease in level of glycosylated sterols has been observed. Hence, the obtained data provides an insight into the biological function of WsSGTL1 gene in W.somnifera.

High-performance thin-layer chromatographic analysis for the simultaneous quantification of four phenolic compounds in green, red, and black fruits of Trapa natans var. bispinosa Roxb. (singhara)

The fruits of Trapa natans var. bispinosa Roxb. (singhara) of nutraceutical importance are commercially consumed in food commodities. It is nutritious while having several biological activities. In the present study, a simple, rapid, cost-effective, and sensitive highperformance thin-layer chromatography (HPTLC) method was developed for the simultaneous determination of four phenolic compounds viz. gallic (phenolic acid), caffeic acid (hydroxycinnamic acid), quercetin, and kaempferol (flavonols) in seeds and pericarp of green-, red-, and black-colored singhara fruits. The method is economical in terms of the time taken and the amount of solvent used for analysis. Simultaneous separation and quantification of compounds were achieved on HPTLC precoated silica gel 60 F254 aluminum plates using mobile phase of toluene-ethyl acetate-formic acid (13:11:2). Densitometric determination was carried out at λmax 282 nm. The calibration curves were linear ranging between 0.996 and 0.999; the limits of detection and quantification ranged between 86.8–135.0 ng µL−1 and 263.2–409.2 ng µL−1 and recovery ranged between 96.4% and 98.5%. The validated method was successively used to analyze the above compounds in fruit parts of singhara. The amount of phenolic compounds ranged from 0.04% kaempferol (green pericarp) to 2.11% gallic acid (red pericarp). The developed method may be used in the quality control and standardization of plant extracts as well as herbal drugs and formulations having polyphenols. As this study reveals the presence of specific phenolic compounds in green, red, and black pericarp of singhara, the agrowaste shall be a good source for isolation of the above compounds for industrial use.

Development and Validation of HPTLC Densitometric Method for Identification and Quantification of Geraniol in Palmarosa Oil

India is the main producer of palmarosa oil obtained from rosha grass (Cymbopogan martini var. motia) of family Graminae. The essential oil obtained from the grass is rich in geraniol content. The oil is commercially obtained by hydro-steam distillation of rosha grass. Oil of palmarosa and its separated fraction geraniol are widely used in perfumery industry. The palmarosa oil is valued due to geraniol and largely used as base for fine perfumery. In this article, a simple, rapid, cost-effective and accurate method using high-performance thin-layer chromatographic (HPTLC) method has been developed for separation, identification and quantification of geraniol in palmarosa oil. Separation and quantification of palmarosa are achieved by HPTLC using mobile phase of toluene-ethylacetate (92.5:7.5) followed by separation on precoated silica gel 60 F254 aluminium plates and densitometric determination carried out after derivatization with vanillin-sulfuric acid reagent at λmax, 400 nm, in absorption-reflectance mode. The calibration curves were linear in the range of (1–7 µg) and all the distinguished bands were observed at 400 nm. The method was also evaluated for different validation parameters such as linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), specificity, selectivity and sample stability. The amount of geraniol varied from 78.3% to 78.9% by HPTLC and 78.2% to 78.7% by gas chromatographyflame-ionization detector (GC-FID).

Simultaneous quantification of six phenolic compounds in various parts of Moringa oleifera Lam. using high-performance thin-layer chromatography

Fresh pods of Moringa oleifera with nutraceutical importance are widely consumed in food commodities as vegetables. It is nutritious and it also has several biological activities. In the present study, a simple, rapid, cost-efective, and sensitive high-performance thin-layer chromatography (HPTLC) method was applied for the simultaneous determination of six phenolic compounds, viz., gallic (phenolic acid), p-coumaric, cafeic acid (hydroxycinnamic acid), chlorogenic acid (cinnamic acid derivative), quercetin and kaempferol (flavonols) in flowers, pods, leaves, twigs, and seeds of M. oleifera. Simultaneous separation and quantification of compounds were achieved on HPTLC pre-coated silica gel 60 F254 aluminum plates using the mobile phase toluene—ethyl acetate—formic acid (14:10:1). Densitometric determination was carried out at λmax 282 nm. The calibration curves were linear, ranged between 0.984 and 0.998; the limit of detection and quantification ranged between 110.8 ng mL-1 and 142.3 ng mL-1, and 301.6 ng µL-1 and 410.8 ng µL-1; and recovery ranged between 96.2% and 97.9%. The validated method was successively used to analyze the above compounds in the plant parts of M. oleifera. The amount of the total phenolic content and specifc phenolic compounds ranged from 4.86 mg g-1 (gallic acid equivalent [GAE]) to 14.79 mg g-1 (GAE) and 0.007% quercetin (fower and fower with pods) to 0.099% gallic acid (pods of 15 days). This study reveals that the presence of specific phenolic compounds in M. oleifera shall be a good source for the isolation of the above-mentioned compounds for industrial use.

In-vitro propagation and identification of phenol compounds of potential medicinal value in the moss Oxystegus stenophyllus (Mitt.) Gangulee

Bryophytes frequently grow in mixed populations and are strongly attached to the substrate by rhizoids. This, together with their small size and low biomass, often makes purification for biochemical analysis difficult, time consuming and, in the commercial context, hardly worthwhile since it is generally assumed that they do not contain compounds of practical value. However, there is now growing awareness that they are a reservoir of new, natural products or secondary compounds, many of which have interesting biological properties. These properties can be classified as: antifungal, antimicrobial, cytotoxic, antitumor, cardiotonic, molluscicidal, piscicidal, superoxide anion radical release inhibition and 5-lipoxygenase, calmodulin, hyaluronidase, and cyclooxygenase inhibition (Sabovljevic et al., 2009; Singh et al., 2011). Riccardin from Riccardia multifida (L.) Gray, and Marchantin A from Marchantia paleacea Bertol., M. polymorpha L., and M. tosana Steph., all show cytotoxicity against the KB cells (Asakawa et al., 1983a,b). Spjut et al. (1986) tested 184 species of mosses and 23 species of liverworts for antitumor activity and found that extracts of 43 species were active, while those of 75 species were toxic to mice. Bryophytes have been used as herbal medicines in China, India, and among Native Americans since ancient times. They are extremely rich in oligosaccharides, polysaccharides, sugar alcohols, amino acids, terpenoids, phenols, glycosides, and fatty acids (Toyota, 1994; Asakawa, 2007). Polyphenols are a major group of plant secondary metabolites with antioxidant properties and activity against various types of stress, including allergies, ulcers, tumours, platelet aggregation, and cardiovascular disease; they also reduce the risk of cancer (Block, 1992; Hertog et al., 1993; Niranjan and Tewari, 2008; Niranjan et al., 2011). These plant secondary metabolites are usually found in plants used as nutritional supplements for health promotion and disease prevention. An important protective effect of these compounds is reduction of oxidative damage, mediated by lipid peroxidation, which in living systems is strongly associated with induction of mutagenesis, carcinogenesis, ageing, & atherosclerosis (Escarpa & Gonzalez, 2000; Revilla & Ryan, 2000; Tsao & Yang, 2003). Thus bryophytes could, in theory, be used for medicinal purposes, providing that sufficient quantities of the desired species are available. In vitro propagation of bryophytes is probably the most appropriate route to large scale biomass production. A large number of secondary compounds have already been isolated from bryophytes grown in vitro (Wilschke et al., 1989; Morais & Becker, 1990; Nagagawara et al., 1992; Ono et al., 1992, Adam & Becker, 1994; Becker, 1994; Kunz et al., 1994). In this account, we compare the phenolic substances present in Oxystegus stenophyllus (Mitt.) Gangulee, a species not known to have been screened previously, grown in vitro and in vivo in pots. Oxystegus stenophyllus was collected from Kilbury (Nainital), western Himalaya, India, 8 April 2011, (alt ca 6863 ft), Leg. A. K. Asthana and Vinay Sahu. Voucher specimens were deposited in the Bryophyte Herbarium, National Botanical Research Institute, Lucknow (LWG — 252386). The moss was grown axenically from spores sown on Hoagland no.2 basal salt medium. In vitro cultured plants with welldeveloped gametophores were successfully transferred to soil pots in the moss house at the CSIR-National Botanical Research Institute, Lucknow where they produced dense colonies within a few weeks. Secondary metabolite production of the plants grown both in vivo and in vitro was analysed by HPLC analysis using the filtrate from 200 mg of air-dried plant material soaked in 80% methanol overnight. Separation followed by qualitative and quantitative analysis of polyphenols was performed using HPLCUV (Shimadzu LC-10A, Japan) equipped with dual

Biochemical composition of Betula utilis D. Don bark, collected from high altitudes of Indian Himalayas

Betula utilis (family Betulaceae) is an important medicinal plant that grows in high altitudes of Himalayan region of India. The present study is aimed to determine the biochemical composition in the bark of Betula utilis using HPLC, HPTLC and GCMS methods. The optimization of solvent system for maximum extraction yield has been carried out. Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and Antioxidant Activity (AA) were estimated in various solvent extracts. Maximum extractive yield (14.56%), TPC (14.47 mg/g), TFC (20.10 mg/g) in 75% ethanol and scavenging activity (92.89%) were found in 50% ethanol. Pentacyclic triterpenoids (betulin, tupeol, oleanolic acid, beta-sitosterol) were characterized and quantified using HPLC, HPTLC and GCMS found close within limits. Betulin was found maximum in ethanol.

Extraction of polyphenols from Trewia nudiflora L. and its antioxidant activity

Total phenoliccontent(TPC), specific polyphenols (Gallic, ellagic and protocatechuic acid),antioxidant activity (AOA), free radical scavenging activity (FRSA), lipid peroxidation (LPO), inhibition of nitroblue tetrazolium chloride (NBT) reduction caused by superoxide anions and reducing power (RP) through various extraction optimization parameters using solvents of different polarity, time, temperature and pH variation were evaluated in fruits, leaves, twigs and bark of Trewia nudiflora. The extraction parameters exhibited a broad range of TPC inplant parts varying from 7.9–668.6mg/g gallic acid equivalent(GAE), AOA 14.6–82.9% and RP 0.47–7.2ASE/ml. FRSA, measured as IC50 invarious antioxidant assays ranged from 0.014–4.1mg/ml. The extracts of all plant parts showed significant protective effect against Fentons reaction on supercoiled plasmid DNA pUC 18 assayed by agarose gel electrophoresis. Fruits were found to possess the highest TPC, AOA and FRSA in all the tested models, with reduction in the activity, in order of fruits > leaves > twigs > bark.