Deepti Yadav

Longevity-promoting effects of 4-hydroxy-E-globularinin in Caenorhabditis elegans.

In modern times, there has been a major increase in the use of plants or herbal constituents for the prevention of age-related disorders. 4-Hydroxy-E-globularinin (4-HEG) is an iridoid and a major component of Premna integrifolia. This investigation represents a breakthrough in geriatrics by showing the longevity-promoting activity of 4-HEG in the animal model Caenorhabditis elegans. 4-HEG (20μM) enhanced the mean life span of worms by over 18.8% under normal culture conditions and also enhanced their survival under oxidative stress. The longevity-promoting activity was associated with reduced reactive oxygen species (ROS) levels and fat accumulation in the worms. Gene-specific mutant studies verified the role of ROS detoxification pathways and simultaneous nuclear translocation of DAF-16 in the 4-HEG-mediated effects. Quantitative real-time PCR estimations and observations of transcriptional reporters indicated that 4-HEG was able to upregulate stress-inducible genes, viz., hsp-16.2 and sod-3. Thus, 4-HEG may serve as a lead compound of plant origin for the development of important nutraceuticals superseding the aging process.

Antifilarial diarylheptanoids from Alnus nepalensis leaves growing in high altitude areas of Uttarakhand, India

Lymphatic filariasis continues to be a major health problem in tropical and subtropical countries. A macrofilaricidal agent capable of eliminating adult filarial parasites is urgently needed. Platyphyllenone (A), alusenone (B), hirustenone (C) and hirsutanonol (D) are important biologically active diarylheptanoids present in Alnus nepalensis. In the present study, we report the antifilarial activity in diarylheptanoids isolated from the leaves of A. nepalensis. Out of four compounds (A-D) tested in vitro one has shown promising anti-filarial activity both in vitro and in vivo studies. This is the first ever report on antifilarial efficacy of a compound of the plant and warrants further studies around this scaffold. In addition, a sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for the above four biomarker compounds. The separation was performed on silica gel 60F(254) high-performance thin layer chromatography plates using chloroform:methanol (9:1, v/v) as mobile phase. The quantitation of marker compounds was carried out using densitometric reflection/absorption mode at 600 nm after post-chromatographic derivatization using vanillin-sulfuric acid reagent. 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.

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 1β,3α,8β-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.

Diarylheptanoid compounds from Alnus nepalensis express in vitro and in vivo antifilarial activity.

A large number of medicinal plants remain to be explored for antifilarial compounds. In the present study a crude methanolic extract of leaves of Alnus nepalensis, chloroform- and n-butanol-partitioned fractions from the crude extract and 6 bioactivity-guided isolated compounds including two new diarylheptanoid from the fractions were assayed for microfilaricidal, macrofilaricidal and female worm sterilizing activity using the lymphatic filariid Brugia malayi in in vitro and in vivo systems. In vitro, the crude methanolic extract exerted better microfilaricidal (LC100: 15.63μg/ml, IC50: 6.00μg/ml) than macrofilaricidal (LC100: >250; IC50: 88μg/ml) activity whereas chloroform and n-butanol fractions were more macrofilaricidal (LC100: 125 and 31.25μg/ml; IC50: 13.14 and 11.84, respectively) than microfilaricidal (LC100: 250-500μg/ml, IC50: 44.16μg/ml). In addition, n-butanol fraction also caused 74% inhibition in MTT reduction potential of the adult worms. In vivo (doses: crude: 100-200mg/kg; fractions: 100mg/kg, i.p.×5 days) the chloroform fraction exerted >50% macrofilaricidal activity whereas methanolic extract and n-butanol fraction produced 38-40% macrofilaricidal action along with some female sterilizing efficacy. Of the 5 diarylheptanoid compounds isolated, alnus dimer, and (5S)-5-hydroxy-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-3-heptanone were found to show the most potent with both macrofilaricidal (LC100: 15.63μg/ml, IC50: 6.57-10.31μg/ml) and microfilaricidal (LC100: 31.25-62.5μg/ml, IC50: 11.05-22.10μg/ml) activity in vitro. These findings indicate that the active diarylheptanoid compounds may provide valuable lead for design and development of new antifilarial agent(s).

Diarylheptanoids Rich Fraction of Alnus nepalensis Attenuates Malaria Pathogenesis: In‐vitro and In‐vivo Study

Diarylheptanoids from Alnus nepalensis leaves have been reported for promising activity against filariasis, a mosquito‐borne disease, and this has prompted us to investigate its anti‐malarial and safety profile using in‐vitro and in‐vivo bioassays. A. nepalensis leaf extracts were tested in‐vitro against chloroquine‐sensitive Plasmodium falciparum NF54 by measuring the parasite specific lactate dehydrogenase activity. Among all, the chloroform extract (ANC) has shown promising anti‐plasmodial activity (IC50 8.06 ± 0.26 µg/mL). HPLC analysis of ANC showed the presence of diarylheptanoids. Efficacy and safety of ANC were further validated in in‐vivo system using Plasmodium berghei‐induced malaria model and acute oral toxicity in mice. Malaria was induced by intra‐peritoneal injection of P. berghei infected red blood cells to the female Balb/c mice. ANC was administered orally at doses of 100 and 300 mg/kg/day following Peters 4 day suppression test. Oral administration of ANC showed significant reduction of parasitaemia and increase in mean survival time. It also attributed to inhibition of the parasite induced pro‐inflammatory cytokines as well as afford to significant increase in the blood glucose and haemoglobin level when compared with vehicle‐treated infected mice. In‐vivo safety evaluation study revealed that ANC is non‐toxic at higher concentration. Copyright

Diarylheptanoids from Alnus nepalensis attenuates LPS-induced inflammation in macrophages and endotoxic shock in mice.

Diarylheptanoids, a group of plant secondary metabolites are increasingly recognized as potential therapeutic agents. The aim of study was to ascertain the anti-inflammatory profile of diarylheptanoids from Alnus nepalensis against lipopolysaccharide (LPS)-induced inflammation in macrophages and endotoxic shock in mice. Extracts prepared from dried leaves of A. nepalensis using standard solvents were tested against LPS-induced inflammation in macrophages. Among all, butanol extract (ANB) has shown most significant inhibition of pro-inflammatory cytokines without any cytotoxicity. HPLC analysis of ANB showed the presence of diarylheptanoids. The diarylheptanoids were further isolated and tested in-vitro for anti-inflammatory activity. Treatment of isolated diarylheptanoids (HOG, ORE and PLS) was able to reduce the production and mRNA level of pro-inflammatory cytokines (TNF-α and IL-6). Furthermore, we demonstrated that it inhibited the expression of NF-kB protein in LPS-induced inflammation in macrophages. In-vivo efficacy and safety profile of ANB revealed that oral treatment of ANB was able to improve the survival rate, and inhibited the production of pro-inflammatory cytokines in serum, attenuated vital organ injury in a dose dependent manner without any toxic effect at higher dose in mice. The results suggest that diarylheptanoids from A. nepalensis can be considered as potential therapeutic candidates for the management of inflammation related diseases.

Simultaneous Quantification of Diarylheptanoids in Alnus nepalensis Using a Validated HPTLC Method

Platyphylloside, oregonin and hirsutanonol 5-O-β-D-glucopyranoside are known bioactive metabolites in Alnus nepalensis. In this article, the aforementioned three markers were isolated and simultaneously quantified by the thin-layer chromatography densitometric method. A sensitive, selective and robust qualitative and quantitative densitometric high-performance thin-layer chromatographic method was developed and validated for the determination of above markers in the leaves of A. nepalensis. The separation was performed on silica gel 60F254 high-performance thin-layer chromatography plates using chloroform:methanol:formic acid (75:25:2, v/v) as mobile phase. The quantitation of diarylheptanoids was carried out using the densitometric reflection/absorption mode at 610 nm after post-chromatographic derivatization with vanillin-sulfuric acid reagent. A precise and accurate quantification can be performed in the linear working concentration range of 333-3330 ng/spot with good correlation (r(2) = 0.999). The method was validated for peak purities, precision, robustness, limit of detection and quantitation, etc., as per ICH guidelines. Specificity of quantitation was confirmed using retention factor and spectra correlation of markers in standard and sample tracks.

Acacetin 7-O-α-l-rhamnopyranosyl (1–2) β-D-xylopyranoside Elicits Life-span Extension and Stress Resistance in Caenorhabditis elegans

The advancements in the field of gerontology have unraveled the signaling pathways that regulate life span, suggesting that it might be feasible to modulate aging. To this end, we isolated a novel phytomolecule Acacetin 7-O-α-l-rhamnopyranosyl (1-2) β-D-xylopyranoside (ARX) from Premna integrifolia and evaluated its antiaging effects in Caenorhabditis elegans The spectral data analysis revealed the occurrence of a new compound ARX. Out of the three tested pharmacological doses of ARX, viz. 5, 25, and 50 µM, the 25-µM dose was able to extend life span in C. elegans by more than 39%. The present study suggests that ARX affects bacterial metabolism, which in turn leads to dietary restriction (DR)-like effects in the worms. The effect of ARX on worms with mutations (mev-1, eat-2, sir-2.1, skn-1, daf-16, and hsf-1) indicates that ARX-mediated life-span extension involves mechanisms associated with DR and maintenance of cellular redox homeostasis. This study is the first time report on longevity-promoting activity of ARX in C. elegans mediated by stress and DR-regulating genes. This novel phytomolecule can contribute in designing therapeutics for managing aging and age-related diseases.

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes

Abstract Purpose: To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. Materials and methods: Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. Results: The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). Conclusion: DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.

Isolation and HPTLC analysis of iridoids in Premna integrifolia, an important ingredient of Ayurvedic drug Dashmool

Premna integrifolia is an important constituent of the famous herbal formulation “Dashmool” of the Indian Ayurvedic system of medicines. The plant is traditionally used in the treatment of skin diseases, arthritis, gonorrhea, rheumatism, anorexia, and jaundice. 10-O-trans-p-coumaroylcatalpol (A), 4″-hydroxy-E-globularinin (B), and premnosidic acid (C) are major iridoid glycosides (chemical markers) present in P. integrifolia. In the present study, an attempt has been made to develop a high-performance thin-layer chromatography (HPTLC) method for quantitative estimation of iridoid glycosides in the stem bark of P. integrifolia. Separation was performed on silica gel 60F254 high-performance thin-layer chromatographic plates. The solvent system consisted of ethyl acetate-methanol-H2 O-acetic acid (80:12:6:2 v/v). Densitometric analysis of iridoids was carried out in the absorbance mode at 510 nm after post-chromatographic derivatization using vanillin-sulphuric acid reagent. 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. This HPTLC method was found to be reproducible, accurate, and can detect iridoids at a nanogram level. The developed HPTLC method would be an important tool in the quality control method for polyherbal formulations.