Densitometric method for assessment of six specialized metabolites in four Sida sp. and its congener Abutilon indicum: Targeted metabolomics, greenness assessment, and chemometrics analysis.

Sida is one of the most diverse genera, with about 200 species distributed in tropical and subtropical regions of the world. Among 18 species distributed in India, Sida acuta, Sida cordifolia, Sida rhombifolia, and Sida cordata are used in traditional medicines along with its possible adulterant Abutilon indicum for several therapeutic uses. The non-availability of marker-based validated methods for the identification and classification of these species leads to adulteration. Indoloquinoline and quinazoline are the major bioactive alkaloids distributed in Sida spp. First time, a simple, economical and high throughput method was developed and validated for the simultaneous determination of 20-hydroxyecdysone (1), vasicine (2), vasicinone (3), cryptolepine (4), quindolinone (5), and cryptolepinone (6) using HPTLC-UV densitometry. The method was validated to meet globally accepted ICH guidelines. The method was sensitive with LOD and LOQ ranging from 0.38-0.63 and 1.57-2.12 µg/band. The samples were spiked at 3 different concentrations, the recovery values were 93.49-98.88%. In addition, the greenness index of the HPTLC method was estimated using four different greenness assessment techniques. Targeted HPTLC analysis indicated the distribution of specialized metabolites in Sida spp. and A. indicum. However, the occurrence of cryptolepine in A. indicum was not reported in the literature, so this was further confirmed by liquid chromatographic studies of the samples from different locations. The chromatographic data was statistically evaluated by principal component analysis (PCA) and hierarchical clustering (HCA). HPTLC-based targeted metabolite quantitation explains the adulteration/substitution in Sida raw material and derived herbal preparations.

Simultaneous estimation of five biomarkers of neuroprotective herb Ashwagandha NMITLI-118R AF1 in rat plasma and brain using LC-ESI-MS/MS: Application to its pharmacokinetic and stability studies.

Withania Somnifera (WS) is a popular nutritional supplement in the USA, Europe, and Asia, known for its pharmacological effects on neurological disorders. However, the bioanalytical method development, validation, and pharmacokinetics of WS NMITLI-118R AF1 biomarkers Withanolide A (WLD A), Withanone (WNONE), Withanolide B (WLD B), Withaferin A (WF A), and 12 Deoxywithastramonolide (12 DEOXY) in rats have not been comprehensively explored. This study aimed to develop and validate a sensitive and selective LC-ESI-MS/MS method for these biomarkers in male Sprague Dawley rats plasma and brain matrix. Rats were divided into eight groups, each containing five rats. A plant extract of NMITLI-118R AF1 at 50 mg/kg was orally administered to the rats for in-vivo pharmacokinetic investigation. All the analytes had a linear calibration curve (r2 > 0.999), and intra-day and inter-day precision (%) were found in the range of 2.46 - 13.71% and accuracy were within the acceptable range (±15%). The biomarkers of NMITLI-118R AF1 were found stable in in-vitro plasma and simulated gastro-intestinal fluids. The observed (Cmax) and (Tmax) values for the biomarkers in the systemic circulation were WLD A (5.59 ± 0.34 ng/mL, Tmax 1.00 ± 0.00 h), WNONE (6.28 ± 0.41 ng/mL, Tmax 0.95 ± 0.11 h), WLD B (6.45 ± 2.87 ng/mL, Tmax 0.95 ± 0.11 h), WF A (6.50 ± 0.27 ng/mL, Tmax 1.00 ± 0.00 h), and 12 DEOXY (5.68 ± 0.39 ng/mL, Tmax 1.00 ± 0.00 h). In contrast to the old method, our approach exhibits a lower limit of quantification (LLOQ), shorter run time (less than10 min), and enables the detection of WF A and WNONE in fresh rat plasma by other quantitative analysis of mass spectrometry (m/z) [M]+. Shows high sample volumes for both, larger plasma volumes, costlier sample collection techniques dried blood spot (DBS), more expensive solid phase extraction techniques (SPE) and longer analysis time 14 min. Moreover, our method requires a smaller sample volume 10 µL, offers faster analysis time 4 min, and achieves a higher sensitivity 1 ng/mL. This is the first report of a comprehensive study on in-vitro and in-vivo pharmacokinetics of NMITLI-118R AF1 biomarkers, which may aid in further pre-clinical and clinical trial investigations.

Stimulation of glucose uptake by glycosides from Alectra parasitica subsp. chitrakutensis (M.A. Rau) K.K. Khanna & An. Kumar: An in-vitro study of TNF-α induced insulin resistance in L6 myoblasts.

Alectra parasitica subsp. chitrakutensis (M.A. Rau) K.K. Khanna & An. Kumar (Orobanchaceae) is a parasitic plant indigenous to India. Locally, the plant is known as 'Midaki and Nirgundikand'. It is used to treat fever, piles, cardiovascular disorders, and blood-borne non-infectious diseases by ethnic communities. The phytochemical investigation of A. parasitica subsp. chitrakutensis rhizome led to the isolation of azafrin (1), rehmaionoside-C (2), and mussaenoside (3). Compounds (2) and (3) are being reported for the first time from this plant. Compounds were evaluated for their intercellular glucose uptake activity in basal and insulin-TNF-α-stimulated L6 muscle cells. In particular, rehmaionoside C exhibited activity comparative to metformin, increasing uptake by basal- and insulin-TNF-α-stimulated cells by 4.88- and 3.90-fold and 5.04- and 4.04-fold. While azafrin and mussaenoside have produced 3.03- and 2.36-fold; 4.03- and 3.22-fold increase in intercellular glucose uptake. Compounds did not show toxicities in rat L6 myoblast cells. The study suggests that rehmaionoside-C from A. parasitica subsp. chitrakutensis might activate glucose uptake by insulin mimics and could be a nontoxic anti-diabetes lead for drug discovery.

SlDREB3, a negative regulator of ABA responses, controls seed germination, fruit size and the onset of ripening in tomato.

SlDREB3 was identified as a ripening up-regulated gene of the AP2/ERF-domain family of transcription factors. Its manipulation affects processes primarily governed by ABA. It negatively regulates ABA responses in tomato by altering ABA levels/signaling and is, in turn, negatively regulated by ABA. SlDREB3 over-expression lines show higher transcript levels of the ABA metabolism genes CYP707A3 and UGT75C1 and an 85% reduction in ABA levels leading to early seed germination. In contrast, suppression lines show decreased CYP707A3/UGT75C1 expression, 3-fold higher ABA levels and delayed germination. The expression of other ABA signaling and response genes is also affected. Suppression of SlDREB3 accelerates the onset of ripening by 4-5 days while its over-expression delays it and also reduces final fruit size. SlDREB3 manipulation effects large scale changes in the fruit transcriptome with suppression lines showing early increase in ABA levels and activation of most ripening pathway genes that govern ethylene, carotenoids and softening. Strikingly, key transcription factors like CNR, NOR, RIN, FUL1, governing ethylene-dependent and ethylene-independent aspects of ripening, are activated early upon SlDREB3 suppression suggesting their control by ABA. The studies identify SlDREB3 as a negative regulator of ABA responses across tissues and a key ripening regulator controlling ethylene-dependent and ethylene-independent aspects.

Validated HPTLC method for the simultaneous quantification of diterpenoids in Vitex trifolia L.

Vitex trifolia L. is an important Indian medicinal plant with diverse pharmacological properties. In a recent study, we reported the isolation and antitubercular activity evaluation of three new diterpenoids from its leaves; here we have developed a validated rapid, simple, precise, and accurate high-performance TLC method for the simultaneous quantification of isolated diterpenoids in V. trifolia. Diterpenoids, 6α,7α-diacetoxy-13-hydroxy-8(9),14-labdadien (A), 13-hydroxy-5(10),14-halimadien-6-one (B), and 9-hydroxy-13(14)-labden-16,15-olide (C) were separated on silica gel 60F254 high-performance TLC plates using chloroform/acetone (98:2, v/v) as mobile phase. The quantitation of diterpenoids was carried out using densitometric reflection/absorption mode at 610 nm after postchromatographic derivatization using a vanillin/sulfuric acid reagent. A precise and accurate quantification can be performed for compounds A and B in the linear working concentration range of 333-1000 ng/band and for C in the range of 670-2000 ng/band with good correlations (r = 0.9984, 0.9991, and 0.9994, respectively). The method was validated for peak purity, precision, accuracy, robustness, LOD, and LOQ, as per the ICH guidelines. The method reported here is simple, reproducible and may be applied for the quantitative analysis of the above diterpenoids in the leaves of V. trifolia.

Antitubercular diterpenoids from Vitex trifolia.

A new halimane diterpenoid, 13-hydroxy-5(10),14-halimadien-6-one (1) and two new labdane diterpenoids, 6α,7α-diacetoxy-13-hydroxy-8(9),14-labdadien (2) and 9-hydroxy-13(14)-labden-15,16-olide (3), were isolated for the first time, along with fifteen known compounds, from the hexane soluble fraction of methanolic extract of Vitex trifolia leaves. The structures of these new diterpenoids were elucidated by spectral analysis. Their relative configurations were established using analysis of NOESY correlations and coupling constants observed in (1)H NMR. Compounds 2, 3 and another known diterpenoid, isoambreinolide (4) were evaluated for antitubercular activity. 3 and 4 exhibited antitubercular activity (MIC=100 and 25 µg/ml) against Mycobacterium tuberculosis H37Rv in BACTEC-460 assay.

Validated high performance thin layer chromatographic method for simultaneous quantification of major iridoids in Vitex trifolia and their antioxidant studies.

Negundoside (1), agnuside (2) and 6'-p-hydroxy benzoyl mussaenosidic acid (3) are known bioactive metabolites in Vitex trifolia. In the present study a simple precise and reproducible method was developed for simultaneous quantitation of NS (1), AS (2) and HMA (3) and the antioxidant capacity of above markers has also been determined. Marker compounds have been resolved using silica gel 60 F(254) plates, petroleum ether (60-80)/toluene/acetone/water (10:10:80:2 v/v/v/v) as the mobile phases. The method does not employ any derivatisation procedure and can be used as a quality control tool for routine analysis of drugs V. trifolia and V. negundo together with their commercial extracts. NS (1), AS (2) and HMA (3) showed significant activity in DPPH and NO radical scavenging assays.

Simultaneous quantification of diterpenoids in Premna integrifolia using a validated HPTLC method.

A sensitive, selective and robust densitometric high-performance thin layer chromatographic method was developed and validated for the determination of diterpenoids in the root bark of Premna integrifolia. Diterpenoids 1ß,3α,8ß-trihydroxy-pimara-15-ene (A), 6α,11,12,16-tetrahydroxy-7-oxo-abieta-8,11,13-triene (B) and 2α,19-dihydroxy-pimara-7,15-diene (C) were used as chemical markers for the standardization of P. integrifolia plant extracts. The separation was performed on silica gel 60F(254) high-performance thin layer chromatography plates using hexane/acetone/ethylacetate (60:20:20 v/v) as mobile phase. The quantitation of diterpenoids was carried out using densitometric reflection/absorption mode at 475 nm after post-chromatographic derivatization using vanillin-sulfuric acid reagent. A precise and accurate quantification can be performed for compounds A, B and C in the linear working concentration range of 1-10 µg/spot with good correlations (r(2) =0.9985, 0.9996 and 0.9992, respectively). The method was validated for peak purity, precision, robustness, limit of detection (LOD) and quantitation (LOQ) etc., as per the International Conference on Harmonization (ICH) guidelines. Specificity of quantitation was confirmed using retention factor (R(f)) and spectra correlation of markers in standard and sample tracks. The method reported here is simple and reproducible which may be applied for quantitative analysis of above diterpenoids in the root bark of P. integrifolia.

Two new lignans from Phyllanthus amarus.

Two new lignans, 3-(3,4-dimethoxy-benzyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl-methyl)-dihydrofuran-2-one (1) and 4-(3,4-dimethoxy-phenyl)-1-(7-methoxy-benzo[1,3]dioxol-5-yl)-2,3-bis-methoxymethyl-butan-1-ol (2), were isolated from the leaves of Phyllanthus amarus and their structures were established by spectral analysis. Additionally, eight known lignans were also isolated and characterized.

Simultaneous quantification of withanolides in Withania somnifera by a validated high-performance thin-layer chromatographic method.

This paper describes a sensitive, selective, specific, robust, and validated densitometric high-performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of 3 key withanolides, namely, withaferin-A, 12-deoxywithastramonolide, and withanolide-A, in Ashwagandha (Withania somnifera) plant samples. The separation was performed on aluminum-backed silica gel 60F254 HPTLC plates using dichloromethane-methanol-acetone-diethyl ether (15 + 1 + 1 + 1, v/v/v/v) as the mobile phase. The withanolides were quantified by densitometry in the reflection/absorption mode at 230 nm. Precise and accurate quantification could be performed in the linear working concentration range of 66-330 ng/band with good correlation (r2 = 0.997, 0.999, and 0.996, respectively). The method was validated for recovery, precision, accuracy, robustness, limit of detection, limit of quantitation, and specificity according to International Conference on Harmonization guidelines. Specificity of quantification was confirmed using retention factor (Rf) values, UV-Vis spectral correlation, and electrospray ionization mass spectra of marker compounds in sample tracks.

Iridoid glycosides from Gmelina arborea.

Three iridoid glycosides 6-O-(3''-O-benzoyl)-alpha-L-rhamnopyranosylcatalpol (1a), 6-O-(3''-O-trans-cinnamoyl)-alpha-L-rhamnopyranosylcatalpol (2a) and 6-O-(3''-O-cis-cinnamoyl)-alpha-L-rhamnopyranosylcatalpol (3a) were isolated from aerial parts of Gmelina arborea and structures were elucidated by spectral analysis. Additionally a known iridoid 6-O-(3'', 4''-O-dibenzoyl)-alpha-L-rhamnopyranosylcatalpol (4) was also isolated and identified.