Satyanshu Kumar

Novel chemical constituents with anti-inflammatory activity from the leaves of Sesbania aculeata

From the hexane and ethyl acetate extracts of the leaves of Sesbania aculeata, three novel chemical compounds were isolated and fully characterized as compound 1, (ceramide type); compound 2, (cerebroside type) and compound 3 as a triterpene acid 3-O-α-L-rhamnopyranoside along with nine known compounds (Tricontanol, Lauric acid, Palmitic acid, Heptadecanoyl-1-tridecanoic acid, β-sitosterol, stigmasterol, poriferasterol glucoside, ononitol and pinitol). The anti-inflammatory potential of all three compounds were evaluated using in vitro target based anti-inflammatory activity in LPS-stimulated macrophages. TNF-α is one of the mediators of various chronic inflammatory disorders and treatment of hexane leaf extract (HL), Ethyl acetate leaf extract (EAL) and compounds 1, 2 and 3 at a dose of 10 μg/mL showed significant (P<0.001) inhibition of TNF-α, a pro-inflammatory cytokine. IL-6 was significantly (P<0.05) inhibited by compound 1 and HL at a dose of 10 μg/mL as compared with vehicle treatment. In-vitro cell cytotoxicity study using MTT assay revealed that these compounds were non toxic to the normal cells.

Extraction optimization of gallic acid, (+)-catechin, procyanidin-B2, (–)-epicatechin, (–)-epigallocatechin gallate, and (–)-epicatechin gallate: their simultaneous identification and quantification in Saraca asoca☆

The objective of the present investigation was to optimize extraction conditions for maximum recovery of bioactive phenolics from different parts of Saraca asoca. Extraction recovery was optimized using a mixture of methanol and water in different proportions. For identification and quantification of six analytes, a rapid reversed phase ultra-performance liquid chromatography (UPLC) photo diode array detection method was developed. UPLC separation was achieved in a gradient elution mode on a C18 column with acetonitrile and aqueous phosphoric acid (0.1%, pH = 2.5). Extraction solvent for maximum recovery of analytes varied depending on the nature of matrices. The developed UPLC method was validated in accordance with International Council for Harmonisation (ICH) guidelines. Wide linearity range, sensitivity, accuracy, short retention time, and simple mobile phase composition implied that the method could be suitable for routine analysis of all six analytes with high precision and accuracy. The uniqueness of this study is the determination of the distribution of these compounds in the various parts of S. asoca.

Validated HPLC method for identification and quantification of p-hydroxy benzoic acid and agnuside in Vitex negundo and Vitex trifolia

A high performance liquid chromatography coupled with photodiode array detection method was developed for the identification and quantification of p-hydroxy benzoic acid and agnuside in the extracts of Vitex negundo and Vitex trifolia. The separation was achieved using acetonitrile and O-phosphoric acid–water (0.5%, v/v) as the mobile phase in an isocratic elution mode. Mean retention times of standard p-hydroxy benzoic acid and agnuside were 6.14 and 11.90 min respectively. The developed method was validated as per the ICH guidelines for limit of detection, limit of quantification, linearity, accuracy and precision. Good linearity (r2≥0.999) was observed for both the compounds in wide concentration range. Relative standard deviation values for intra-day and inter-day precision studies were less than 2%. The analytical recoveries of p-hydroxy benzoic acid and agnuside by the developed HPLC method were 93.07% and 106.11% respectively. Two compounds were identified and quantified in leaves and bar extracts of V. negundo and V. trifolia using the developed HPLC method.

A validated HPLC-PDA method for identification and quantification of two bioactive alkaloids, ephedrine and cryptolepine, in different Sida species

A simple, rapid, accurate and reproducible reverse-phase HPLC method has been developed for the identification and quantification of two alkaloids ephedrine and cryptolepine in different extracts of Sida species using photodiode array detection. Baseline separation of the two alkaloids was achieved on a Waters RP-18 X-terra column (250 × 4.6 mm, 5 µm) using a solvent system consisting of a mixture of water containing 0.1% Trifluoroacetic acid (TFA) and acetonitrile in a gradient elution mode with detection at 210 and 280 nm for ephedrine and cryptolepine, respectively. The calibration curves were linear in a concentration range of 10-250 µg/mL for both the alkaloids with correlation coefficient values >0.99. The limits of detection and quantification for ephedrine and cryptolepine were 5 and 10 µg/mL and 2.5 and 5 µg/mL, respectively. Relative standard deviation values for intra-day and inter-day precision were 1.22 and 1.04% for ephedrine and 1.71 and 2.06% for cryptolepine, respectively. Analytical recovery ranged from 92.46 to 103.95%. The developed HPLC method was applied to identify and quantify ephedrine and cryptolepine in different extracts of Sida species.

A validated high-performance liquid chromatography method for determination of tannin-related marker constituents gallic acid, corilagin, chebulagic acid, ellagic acid and chebulinic Acid in four Terminalia species from India.

A validated rapid HPLC-PDA method was developed for identification and quantification of five tannin-related constituents gallic acid (GA), corilagin (CL), chebulagic acid (CB), ellagic acid (EA) and chebulinic acid (CN) in the extracts prepared from the bark and fruits of four Terminalia species available in India. The separation of the five analytes was achieved on an RP-18 column (4.6 × 250 mm, 5 µm) at 25°C using a solvent mixture comprising of acetonitrile and (0.05%) trifluoroacetic acid-water in a gradient elution mode. Limit of detection was 1.0, 0.5, 1.0, 0.5 and 1.0 μg/mL for GA, CL, CB, EA and CN, respectively. Similarly, limit of quantification was 2.5, 1.0, 2.5, 1.0 and 2.5 μg/mL for GA, CL, CB, EA and CN, respectively. Good linearity (r(2) > 0.992) was observed for all the five compounds in wide concentration range. Using the developed HPLC method, the five analytes were identified and quantified in bark and fruit extracts of Terminalia chebula, Terminalia bellirica, Terminalia arjuna and Terminalia catappa. This is the first report of identification and quantification of the five tannin-related marker constituents in the bark and fruit extracts of T. chebula, T. bellirica, T. arjuna and T. catappa.

Accumulation of Three Important Bioactive Compounds in Different Plant Parts of Withania somnifera and its Determination by the LC–ESI-MS-MS (MRM) Method

A comprehensive experiment was conducted to study the accumulation pattern and determination of three important bioactive compounds namely withaferin-A (WA), 12-deoxywithastramonolide (WO) and withanolide-A (WD) and its determination by the liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) method in root, stem, fruits and leaves of Withania somnifera. A rapid and sensitive LC-ESI-MS-MS method was developed and validated for the determination of these three important bioactive compounds, having same molecular weight. The multiple reaction monitoring method was established by two transitions for each analyte and intense transition used for quantification. Separation of the three analytes was achieved within a run time of 5 min on an RP-18 column using a mobile phase consisting of acetonitrile and 0.1% acetic acid in water in an isocratic condition. The developed method was validated as per the ICH guidelines. The developed method was found to be suitable for identification and quantification of WA, WO and WD in different plant parts such as roots, stems, fruits and leaves of W. somnifera. The accumulation of WA was highest in leaves samples (8.84 ± 0.37 mg/g) and it was 2.23, 5.85 and 27.26 times higher than its concentration in fruits, stems and roots, respectively. WO and WD contents were highest (0.44 ± 0.016 and 0.72 ± 0.016 mg/g, respectively) in root.

Comparison of green extraction methods with conventional extraction method for extract yield, L-DOPA concentration and antioxidant activity of Mucuna pruriens seed

ABSTRACT Mucuna pruriens is a plant of Fabaceae family. Seed of M. pruriens is considered as a rich source of levo-3,4 dihydroxyphenylalanine (L-DOPA), a non-protein phenolic amino acid. In the present study, three different extraction methods were compared for extract yield, concentration of bioactive compounds such as total phenol, L-DOPA and antioxidant capacity. Extracts were prepared using water acidified with hydrochloric acid (0.1 N) by conventional method of refluxing as well as two green methods namely ultrasound and microwave assisted solvent extraction. A rapid and qualified high-performance liquid chromatography method was also developed for quantification of L-DOPA in different extracts. Among the three extraction methods, microwave assisted extraction provided the best results for yield and quality of M. pruriens extract in much shorter time in comparison to refluxing method of extraction.

Development and validation of a rapid high performance liquid chromatography - photodiode array detection method for estimation of a bioactive compound wedelolactone in extracts of Eclipta alba

Following optimization of extraction, separation and analytical conditions, a rapid, sensitive and simple reverse-phase high performance liquid chromatography-photo diode array (HPLC-PDA) method has been developed for the identification and quantification of wedelolactone in different extracts of Eclipta alba. The separation of wedelolactone was achieved on a C18 column using the solvent system consisting of a mixture of methanol: water: acetic acid (95: 5: 0.04) as a mobile phase in isocratic elution mode followed by photo diode array detection at 352 nm. The developed method was validated as per the guidelines of the International Conference on Harmonization (ICH). Calibration curve presented good linear regression (r2>0.998) within the test range and the maximum relative standard deviation (RSD, %) values for intra-day assay were found to be 0.15, 1.30 and 1.1 for low (5 µg/mL), medium (20 µg/mL) and high (80 µg/mL) concentrations of wedelolactone. For inter-day assay the maximum RSD (%) values were found to be 2.83, 1.51 and 2.06 for low, medium and high concentrations, respectively. Limit of detection (LOD) and limit of quantification (LOQ) were calculated to be 2 and 5 µg/mL respectively. Analytical recovery of wedelolactone was greater than 95%. Wedelolactone in different extracts of Eclipta alba was identified and quantified using the developed HPLC method. The validated HPLC method allowed precise quantitative analysis of wedelolactone in Eclipta. alba extracts.

Nanotechnology for enhanced bioactivity of bioactive phytomolecules

Nanotechnology is emerging as a very promising tool in the area of phytomedicine. It covers materials, applications, and processes that find applications on very-very small dimensions. Unique size and physical properties of nanoparticles provide an array of salient characteristics. Nanoparticles of different sizes, shapes, and functionality can be synthesized in order to meet the specific requirements of delivery system. These benefits are being potentially explored by the researchers in order to overcome both the physical and chemical limitations of drugs primarily derived from phytochemicals. Nanotechnology has been used as an aid in different aspects of drug discovery from natural sources, and plants in particular. Nanotechnological interventions could be utilized for increasing the efficiency of plant-derived medicines, as novel formulations using nanotechnology have a considerable edge over conventional formulations.

Comparison on extraction yield of sennoside A and sennoside B from senna (Cassia angustifolia) using conventional and non conventional extraction techniques and their quantification using a validated HPLC-PDA detection method

Abstract Senna is an important medicinal plant and is used in many Ayurvedic formulations. Dianthraquinone glucosides are the main bioactive phytochemicals present in leaves and pods of senna. The extraction efficiency in terms of yield and composition of the extract of senna prepared using both conventional (cold percolation at room temperature and refluxing) and non conventional (ultrasound and microwave assisted solvent extraction as well as supercritical fluid extraction) techniques were compared in the present study. Also a rapid reverse phase HPLC-PDA detection method was developed and validated for the simultaneous determination of sennoside A and sennoside B in the different extracts of senna leaves. Ultrasound and microwave assisted solvent extraction techniques were more effective in terms of yield and composition of the extracts compared to cold percolation at room temperature and refluxing methods of extraction.