Variation In Secondary Metabolites Research Articles

ПРОСТРАНСТВЕННОЕ ВАРЬИРОВАНИЕ СОДЕРЖАНИЯ ВТОРИЧНЫХ МЕТАБОЛИТОВ, УГЛЕРОДА И АЗОТА В ПОДСТИЛКАХ СЕВЕРОТАЕЖНЫХ ЕЛЬНИКОВ*, "Лесоведение"

ПРОСТРАНСТВЕННОЕ ВАРЬИРОВАНИЕ СОДЕРЖАНИЯ ВТОРИЧНЫХ МЕТАБОЛИТОВ, УГЛЕРОДА И АЗОТА В ПОДСТИЛКАХ СЕВЕРОТАЕЖНЫХ ЕЛЬНИКОВ*, "Лесоведение"

Drivers of genetic diversity in secondary metabolic gene clusters within a fungal species.

Filamentous fungi produce a diverse array of secondary metabolites (SMs) critical for defense, virulence, and communication. The metabolic pathways that produce SMs are found in contiguous gene clusters in fungal genomes, an atypical arrangement for metabolic pathways in other eukaryotes. Comparative studies of filamentous fungal species have shown that SM gene clusters are often either highly divergent or uniquely present in one or a handful of species, hampering efforts to determine the genetic basis and evolutionary drivers of SM gene cluster divergence. Here, we examined SM variation in 66 cosmopolitan strains of a single species, the opportunistic human pathogen Aspergillus fumigatus. Investigation of genome-wide within-species variation revealed 5 general types of variation in SM gene clusters: nonfunctional gene polymorphisms; gene gain and loss polymorphisms; whole cluster gain and loss polymorphisms; allelic polymorphisms, in which different alleles corresponded to distinct, nonhomologous clusters; and location polymorphisms, in which a cluster was found to differ in its genomic location across strains. These polymorphisms affect the function of representative A. fumigatus SM gene clusters, such as those involved in the production of gliotoxin, fumigaclavine, and helvolic acid as well as the function of clusters with undefined products. In addition to enabling the identification of polymorphisms, the detection of which requires extensive genome-wide synteny conservation (e.g., mobile gene clusters and nonhomologous cluster alleles), our approach also implicated multiple underlying genetic drivers, including point mutations, recombination, and genomic deletion and insertion events as well as horizontal gene transfer from distant fungi. Finally, most of the variants that we uncover within A. fumigatus have been previously hypothesized to contribute to SM gene cluster diversity across entire fungal classes and phyla. We suggest that the drivers of genetic diversity operating within a fungal species shown here are sufficient to explain SM cluster macroevolutionary patterns.

Allotetraploid cryptic species in Asplenium normale in the Japanese Archipelago, detected by chemotaxonomic and multi-locus genotype approaches.

Delimitation of cryptic species provides an understanding of biodiversity and opportunities to elucidate speciation processes. Extensive flavonoid variation has been reported in the tetraploid cytotype of the fern, Asplenium normale, although related species have no intraspecific variations in flavonoid composition. We hypothesized that Japanese A. normale still harbors multiple cryptic species with different flavonoid compositions, and tested this hypothesis using chemotaxonomic and multilocus genotyping approaches. We determined the multilocus genotypes (MLGs) of 230 samples from 37 populations for one chloroplast DNA region and three nuclear genes. MLGs were used to delimit reproductively isolated lineages by population-genetic approaches. We also tested the correspondence between genetically recognized groups and flavonoid compositions. To identify the origins of putative cryptic species, we conducted phylogenetic analysis of the DNA markers used in genotyping. The genetic clusters and flavonoid compositions showed clear correspondence. We recognized three putative cryptic species in tetraploid Asplenium normale in Japan. Phylogenetic analyses revealed that cryptic species I and III originated from allopolyploidization between a diploid A. normale and an unknown diploid of A. boreale, and cryptic species II originated from allopolyploidization between a diploid A. normale and A. oligophlebium. Our study demonstrated that intraspecific variation of secondary metabolites can be a good indicator of cryptic species in ferns. The presence of the two cryptic species having the same progenitor diploid pair suggests that speciation between allopolyploid lineages of independent origin may be more common than previously considered.

Mapping the Arabidopsis Metabolic Landscape by Untargeted Metabolomics at Different Environmental Conditions

Metabolic genome-wide association studies (mGWAS), whereupon metabolite levels are regarded as traits, can help unravel the genetic basis of metabolic networks. A total of 309 Arabidopsis accessions were grown under two independent environmental conditions (control and stress) and subjected to untargeted LC–MS-based metabolomic profiling; levels of the obtained hydrophilic metabolites were used in GWAS. Our two-condition-based GWAS for more than 3000 semi-polar metabolites resulted in the detection of 123 highly resolved metabolite quantitative trait loci (p ≤ 1.0E-08), 24.39% of which were environment-specific. Interestingly, differently from natural variation in Arabidopsis primary metabolites, which tends to be controlled by a large number of small-effect loci, we found several major large-effect loci alongside a vast number of small-effect loci controlling variation of secondary metabolites. The two-condition-based GWAS was followed by integration with network-derived metabolite–transcript correlations using a time-course stress experiment. Through this integrative approach, we selected 70 key candidate associations between structural genes and metabolites, and experimentally validated eight novel associations, two of them showing differential genetic regulation in the two environments studied. We demonstrate the power of combining large-scale untargeted metabolomics-based GWAS with time-course-derived networks both performed under different abiotic environments for identifying metabolite–gene associations, providing novel global insights into the metabolic landscape of Arabidopsis.

Transcriptome analysis of terpene chemotypes of Melaleuca alternifolia across different tissues.

Plant chemotypes or chemical polymorphisms are defined by discrete variation in secondary metabolites within a species. This variation can have consequences for ecological interactions or the human use of plants. Understanding the molecular basis of chemotypic variation can help to explain how variation of plant secondary metabolites is controlled. We explored the transcriptomes of the 3 cardinal terpene chemotypes of Melaleuca alternifolia in young leaves, mature leaves, and stem and compared transcript abundance to variation in the constitutive profile of terpenes. Leaves from chemotype 1 plants (dominated by terpinen-4-ol) show a similar pattern of gene expression when compared to chemotype 5 plants (dominated by 1,8-cineole). Only terpene synthases in young leaves were differentially expressed between these chemotypes, supporting the idea that terpenes are mainly synthetized in young tissue. Chemotype 2 plants (dominated by terpinolene) show a greater degree of differential gene expression compared to the other chemotypes, which might be related to the isolation of plant populations that exhibit this chemotype and the possibility that the terpinolene synthase gene in M. alternifolia was derived by introgression from a closely related species, Melaleuca trichostachya. By using multivariate analyses, we were able to associate terpenes with candidate terpene synthases.

Utility of the Colorimetric Folin-Ciocalteu and Aluminum Complexation Assays for Quantifying Secondary Metabolite Variation among Wild Sunflowers

AbstractSecondary metabolites serve multiple functions in plants, and play a key role in many ecological processes. Accordingly, the quantification of such compounds is central to addressing many questions in plant science. Alongside precision analytical methods like gas and liquid chromatography-mass spectrometry, there exists a substantial niche for inexpensive and rapid spectrophotometric approaches if their usefulness in a system can be demonstrated. This study seeks to examine the utility of two commonly used colorimetric methods – the Folin-Ciocalteu assay and the aluminum complexation assay – for quantifying variation in leaf phenolic and flavonoid content among members of the genus Helianthus, the sunflowers. Among species known a priori to vary substantially in both the diversity and relative concentrations of secondary metabolites, both assays detect substantial variation among species. Moreover, total phenolic content as assessed by the Folin-Ciocalteu assay correlates positively with concentrations of multiple individual phenolic compounds as quantified by high performance liquid chromatography-mass spectrometry, indicating that the Folin-Ciocalteu describes variation in sunflower phenolic content. Additionally, the diversity of flavonoids known from Helianthus include a number of those known to be sensitive to the aluminum complexation assay, indicating that this assay may also be a useful descriptor of relative variation in sunflower flavonoid content. In total, both the Folin-Ciocalteu and aluminum complexation assays appear to capture useful, if coarse, variation in secondary metabolites among Helianthus species, and seem useful as rapid low-cost methods for exploratory research, preliminary analyses, and potentially useful for high-throughput phenotyping within wild or cultivated sunflower with proper calibration.

Comparative functional characterization of eugenol synthase from four different Ocimum species: Implications on eugenol accumulation

Isoprenoids and phenylpropanoids are the major secondary metabolite constituents in Ocimum genus. Though enzymes from phenylpropanoid pathway have been characterized from few plants, limited information exists on how they modulate levels of secondary metabolites. Here, we performed phenylpropanoid profiling in different tissues from five Ocimum species, which revealed significant variations in secondary metabolites including eugenol, eugenol methyl ether, estragole and methyl cinnamate levels. Expression analysis of eugenol synthase (EGS) gene showed higher transcript levels especially in young leaves and inflorescence; and were positively correlated with eugenol contents. Additionally, transcript levels of coniferyl alcohol acyl transferase, a key enzyme diverting pool of substrate to phenylpropanoids, were in accordance with their abundance in respective species. In particular, eugenol methyl transferase expression positively correlated with higher levels of eugenol methyl ether in Ocimum tenuiflorum. Further, EGSs were functionally characterized from four Ocimum species varying in their eugenol contents. Kinetic and expression analyses indicated, higher enzyme turnover and transcripts levels, in species accumulating more eugenol. Moreover, biochemical and bioinformatics studies demonstrated that coniferyl acetate was the preferred substrate over coumaryl acetate when used, individually or together, in the enzyme assay. Overall, this study revealed the preliminary evidence for varied accumulation of eugenol and its abundance over chavicol in these Ocimum species. Current findings could potentially provide novel insights for metabolic modulations in medicinal and aromatic plants.

Intra-Population Variation of Secondary Metabolites in Cistus ladanifer L.

In previous studies, secondary metabolites in the leaf exudate of Cistus ladanifer, specifically aglycone flavonoids and diterpenes, were demonstrated to play an ecophysiological role. They protect against ultraviolet radiation, have antiherbivore activity, and are allelopathic agents. Their synthesis in the plant was also found to vary quantitatively and qualitatively in response to various environmental factors. In view of these findings, the present work was designed to clarify whether within a single population there are differences among individuals subject to the same environmental conditions. To this end, we analyzed the leaves of 100 individuals of C. ladanifer. The results showed the existence of intrapopulational variation, since, although all the individuals had the same composition of secondary chemistry, the amounts were different. The individuals of a given population of C. ladanifer differ from each other even when growing under similar conditions. According to the ammount of flavonoids and diterpenes observed in each individual, it was possible to distinguish four different groups of individuals. Most individuals, evenly distributed within the population, had low concentrations of the studied compounds, whilst other individuals synthesized greater amounts and were randomly distributed among the former. Given the functions of flavonoids and diterpenes in this species, the quantified intra-population variation may involve greater plasticity for the species in the face of environmental changes.

Intraspecific phytochemical variation shapes community and population structure for specialist caterpillars.

Summary Chemically mediated plant–herbivore interactions contribute to the diversity of terrestrial communities and the diversification of plants and insects. While our understanding of the processes affecting community structure and evolutionary diversification has grown, few studies have investigated how trait variation shapes genetic and species diversity simultaneously in a tropical ecosystem.We investigated secondary metabolite variation among subpopulations of a single plant species, Piper kelleyi (Piperaceae), using high‐performance liquid chromatography (HPLC), to understand associations between plant phytochemistry and host‐specialized caterpillars in the genus Eois (Geometridae: Larentiinae) and associated parasitoid wasps and flies. In addition, we used a genotyping‐by‐sequencing approach to examine the genetic structure of one abundant caterpillar species, Eois encina, in relation to host phytochemical variation.We found substantive concentration differences among three major secondary metabolites, and these differences in chemistry predicted caterpillar and parasitoid community structure among host plant populations. Furthermore, E. encina populations located at high elevations were genetically different from other populations. They fed on plants containing high concentrations of prenylated benzoic acid.Thus, phytochemistry potentially shapes caterpillar and wasp community composition and geographic variation in species interactions, both of which can contribute to diversification of plants and insects.

Variation in Lichen Species Assemblages and Secondary Metabolites SurroundingStereocaulonSpecies in the Boreal Forest of Northwestern Manitoba

Abstract. Lichen assemblages and their secondary metabolites may be influenced by the broader habitat. More specifically, the function of the thallus structure within a species may influence the production of some secondary metabolites. The objectives of this study were to examine thallus variation in secondary metabolites for one genus, Stereocaulon, and to compare the diversity of lichen assemblages and their secondary metabolite distributions in the boreal forest of northwestern Manitoba. Five sites in each of three study regions were selected (Sherridon, North Star, and Athapap), around the presence of a Stereocaulon thallus. All ground lichen species were collected from five quadrats within each site totaling 75 quadrats. Lichen species were identified and thin-layer chromatography was performed to identify secondary metabolites. A total of 59 ground lichen species and 20 secondary metabolites were identified. This study showed that norstictic acid and constictic acid were absent from the pseudopodet...

A below-ground herbivore shapes root defensive chemistry in natural plant populations.

Plants display extensive intraspecific variation in secondary metabolites. However, the selective forces shaping this diversity remain often unknown, especially below ground. Using Taraxacum officinale and its major native insect root herbivore Melolontha melolontha, we tested whether below-ground herbivores drive intraspecific variation in root secondary metabolites. We found that high M. melolontha infestation levels over recent decades are associated with high concentrations of major root latex secondary metabolites across 21 central European T. officinale field populations. By cultivating offspring of these populations, we show that both heritable variation and phenotypic plasticity contribute to the observed differences. Furthermore, we demonstrate that the production of the sesquiterpene lactone taraxinic acid β-D-glucopyranosyl ester (TA-G) is costly in the absence, but beneficial in the presence of M. melolontha, resulting in divergent selection of TA-G. Our results highlight the role of soil-dwelling insects for the evolution of plant defences in nature.

Effect of Genotype and Environment on Salvia miltiorrhiza Roots Using LC/MS-Based Metabolomics.

Salvia miltiorrhiza (S. miltiorrhiza) Bunge is broadly used as herbal medicine for the clinical treatments of cardiovascular and cerebrovascular diseases. Despite its commercial and medicinal values, few systematic studies on the metabolome of S. miltiorrhiza roots have been carried out so far. We systematically described the metabolic profiles of S. miltiorrhiza using high pressure liquid chromatography mass spectrometry (LC/MS) in conjunction with multivariate statistical analyses, aimed at monitoring their biological variations of secondary metabolites related to three locations and four S. miltiorrhiza genotypes. A total of 40 bioactive constituents were putatively annotated in S. miltiorrhiza root samples. This study found that both the same S. miltiorrhiza genotype growing at three different locations and four S. miltiorrhiza genotypes growing at the same location had significant metabonomic differences identified by the principal component analysis (PCA) approach. By using orthogonal projection to latent structure with discriminant analysis (OPLS-DA), 16 and 14 secondary metabolites can be used as potential location-specific and genotype-specific markers in S. miltiorrhiza, respectively. The specificity of LC/MS profiles offered a powerful tool to discriminate S. miltiorrhiza samples according to genotypes or locations.

Metabolic Profiling and Antioxidant Assay of Metabolites from Three Radish Cultivars (Raphanus sativus).

A total of 13 anthocyanins and 33 metabolites; including organic acids, phenolic acids, amino acids, organic compounds, sugar acids, sugar alcohols, and sugars, were profiled in three radish cultivars by using high-performance liquid chromatography (HPLC) and gas chromatography time-of-flight mass spectrometry (GC-TOFMS)-based metabolite profiling. Total phenolics and flavonoids and their in vitro antioxidant activities were assessed. Pelargonidins were found to be the major anthocyanin in the cultivars studied. The cultivar Man Tang Hong showed the highest level of anthocyanins (1.89 ± 0.07 mg/g), phenolics (0.0664 ± 0.0033 mg/g) and flavonoids (0.0096 ± 0.0004 mg/g). Here; the variation of secondary metabolites in the radishes is described, as well as their association with primary metabolites. The low-molecular-weight hydrophilic metabolite profiles were subjected to principal component analysis (PCA), hierarchical clustering analysis (HCA), Pearson’s correlation analysis. PCA fully distinguished the three radish cultivars tested. The polar metabolites were strongly correlated between metabolites that participate in the TCA cycle. The chemometrics results revealed that TCA cycle intermediates and free phenolic acids as well as anthocyanins were higher in the cultivar Man Tang Hong than in the others. Furthermore; superoxide radical scavenging activities and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging were investigated to elucidate the antioxidant activity of secondary metabolites in the cultivars. Man Tang Hong showed the highest superoxide radical scavenging activity (68.87%) at 1000 μg/mL, and DPPH activity (20.78%), followed by Seo Ho and then Hong Feng No. 1. The results demonstrate that GC-TOFMS-based metabolite profiling, integrated with chemometrics, is an applicable method for distinguishing phenotypic variation and determining biochemical reactions connecting primary and secondary metabolism. Therefore; this study might provide information on the relationship between primary and secondary metabolites and a synergistic antioxidant ability derived from the secondary metabolites in the radish cultivars.

Seasonal variation in secondary metabolites of edible shoots of Buck’s beard [Aruncus dioicus (Walter) Fernald (Rosaceae)

Aruncus dioicus (Walter) Fernald is an herbaceous plant belonging to the Rosaceae family. In the northern regions of Italy young shoots of A. dioicus are collected and used as vegetable. No studies on the chemical composition of young shoots of A. dioicus have been reported so far. Thus, the aim of the present paper was to develop and validate a comprehensive chromatographic method allowing the identification and quantification of chemical compounds occurring in A. dioicus shoots harvested at different developmental stages. The results showed that shoots contain mainly polyphenols. Twenty-four compounds were detected. Caffeoylglucose derivatives were recognized as major compounds. It was also confirmed that shoots contain the cyanogenic compound prunasin at all investigated vegetation stages. It was shown that the prunasin content significantly increases during plant development, thus justifying the belief that after the development of the first green leaves, shoots should not be used for culinary purposes any longer.

UPLC-QTOFMS based metabolomics followed by stepwise partial least square-discriminant analysis (PLS-DA) explore the possible relation between the variations in secondary metabolites and the phylogenetic divergences of the genus Panax.

Phylogenetic and metabolomic approaches have long been employed to study evolutionary relationships among plants. Nonetheless, few studies have examined the difference in metabolites within a clade and between clades of the phylogenetic tree. We attempted to relate phylogenetic studies to metabolomics using stepwise partial least squares-discriminant analysis (PLS-DA) for the genus Panax. Samples were analyzed by ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QTOFMS) to obtain metabolite profiles. Initially, conventional principal component analysis was subsequently applied to the metabolomic data to show the limitations in relating the expression of metabolites to divisions in the phylogenetic tree. Thereafter, we introduced stepwise PLS-DA with optimized scaling methods, which were properly applied according to the branches of the phylogenetic tree of the four species. Our approach highlighted metabolites of interest by elucidating the directions and degrees of metabolic alterations in each clade of the phylogenetic tree. The results revealed the relationship between metabolic changes in the genus Panax and its species' evolutionary adaptations to different climates. We believe our method will be useful to help understand the metabolite-evolution relationship.

A metabolomic and phytochemical based study of Rhodiola species sourced from Asia and Europe

Rhodiola rosea L. has a long history of use in Europe as an economically valuable medicinal plant. It is used for the treatment of stress-induced fatigue, anxiety and high-altitude sickness [1]. With the increasing demand and over-collection of R. rosea, some adulterated products have been found. According to our investigations, approximately 25% of products claiming to be R. rosea on the European market are adulterated. Adulteration may occur at different stages along the value chain. Therefore the authenticity and quality of raw materials of R. rosea is an important area for investigation. The aim of the project is to investigate the metabolite differences of Rhodiola spp. collected from China and Europe and assess their phytochemical variation. Approximately 45 crude root dried plant specimens were sourced from different suppliers. HPTLC and 1H-NMR analysis was used to evaluate the samples phytochemical variation and metabolites differences. R. crenulata was found to contain no rosavin (a marker compound for R. rosea) but another marker compound, particular to R. crenulata, was identified. Approximately 30% of R. rosea and 27% of R. sachalinesis crude root dried specimens were adulterated with R. crenulata on account of containing no rosavin. R. sachalinesis contains a low concentration of rosavin and salidroside compared with R. rosea. Variations in secondary metabolites of Rhodiola spp. were identified and can be used to distinguish different species of Rhodiola. A marker compound was identified for R. crenulata. Adulteration among these species appears to be commonplace. The method of combining HPTLC and 1H-NMR is shown to be a robust quality control method.

Oviposition Behavior of the Pollen Beetle (Meligethes aeneus): A Functional Study

The recognition by female phytophagous insects of a plant as a ‘good’ or ‘bad’ host for egg laying is based on a variety of cues (either visual, physical or chemical). Specific cues are often looked for during stereotypic oviposition behaviors, composed of several phases having their own function(s). In this study the oviposition behavior of the pollen beetle Meligethes aeneus, a pest which lays eggs in flower buds of only some brassicaceous plants, was described in detail on five oilseed rape (Brassica napus) genotypes. In parallel, setae borne by the ovipositor were characterized by scanning electron microscopy. Observations showed that the stereotypic oviposition sequence is functionally divided into three independent phases: external inspection, internal inspection and egg laying. The ovipositor plays a role in all phases by gaining information about external and internal bud parts. This role appears to be only physical since all the setae it bears are mechanoreceptors. Despite the fact that the pollen beetle is a specialist for oviposition, important variations in secondary metabolites that are typical of its host plant family (i.e., glucosinolates) on the bud did not influence clutch size. The crucial phase in the oviposition sequence seems to be the external inspection, during which poor and high-quality host plants are probably discriminated. Chemical information on bud surface is likely to be determinant in this process.

Discrimination and similarity evaluation of tissue-cultured and wild Dendrobium species using Fourier transform infrared spectroscopy

The FTIR method was applied to evaluate the similarity of tissue-cultured and wild Dendrobium huoshanense C.Z. Tang et S.J. Cheng, Dendrobium officinale Kimura et Migo and Dendrobium moniliforme (Linn.) Sw and discriminate different Dendrobium species, especially D. huoshanense and its main goldbrick Dendrobium henanense J.L. Lu et L.X. Gao. Despite the general pattern of the IR spectra, different intensities, shapes and peak positions were found in the IR spectra of these samples, especially in the range of 1800–600cm−1, which could be used to discriminate them. The methanol, aqueous extracting procedure and the second derivative transformation obviously enlarged the tiny spectral differences among these samples. The similarity evaluation based on the IR spectra and the second derivative IR spectrum revealed that the similarity of the methanol extracts between tissue-cultured and wild Dendrobiums might be lower than that between different Dendrobium species. The similarities of the powders and aqueous extracts between tissue-cultured and wild Dendrobiums were higher than those between different Dendrobium species. The further principal component analysis showed that the first three components explained 99.7%, 87.7% and 85.1% of data variance for powder, methanol extract and aqueous extract, respectively, demonstrating a good discrimination between samples. Our research suggested that the variations of secondary metabolites between different origins of the investigated Dendrobiums might be higher than what we had supposed. Tissue culture techniques were widely used in the conversation of rare and endangered medicinal amedica, however, our study suggested that the chemical constituents of tissue-cultured plants might be quite different from their wild correspondences.

Essential Oil Secondary Metabolites Variation of Salvia palaestina Leaves Growing wild from Different Locations in Palestine

Article history: Received on: 05/10/2015 Revised on: 20/10/2015 Accepted on: 09/11/2015 Available online: 27/11/2015 Herbal medicine is widely practiced in Palestine. Salvia palaestina (Lamiaceae) in particular is heavily used because of its acquired traditional reputation over the years rather than scientific basis. S. palaestina essential oils contain secondary metabolites whose production is influenced by many factors that determine their composition and yield. Wild leaves of S. palaestina were collected from eight different locations in Palestine. Air dried leaves were subjected to steam distillation (SD) and the composition of essential oils was determined for the first time by GC-MS in the electron impact mode. Twenty volatile and semi-volatile components were identified. The major components in all samples were eucalyptol and the percentages ranged from 51.9% to 63.06%. Other components were found but to a lesser extent mainly β-Thujene, -Myrcene, (±)-Camphor, α-Terpineol, and βCaryophyllene.

HPTLC Method for Quantitative Evaluation of Seasonal Variation of Stigmasterol in Rauvolfia serpentina (L). Benth. ex Kurz.

Rauvolfia serpentina (L). Benth. ex Kurz. (Apocynaceae) occupies a prominent position in the ethno-therapeutics practiced in the Indian subcontinent. The plant is known to contain a number of bioactive secondary metabolites including indole alkaloids. The objective of our present study is to estimate the seasonal variation of stigmasterol content in the roots of the plant. Seasonal modulation in stigmasterol content in R. serpentina roots was analyzed by High Performance Thin Layer Chromatography (HPTLC) performed on Silica gel 60 F254 TLC plates with benzene-acetone 86:14 (v/v) as mobile phase. Densitometric analysis was carried out at λ=366 nm after derivatization with vanillin-10 % (v/v) sulphuric acid alcohol. In the root samples stigmasterol was present at Rf value of 0.44. Stigmasterol content was found to be the highest in roots harvested during summer and the lowest in winter and there was a gradual decrease from summer to winter through monsoon and autumn and from spring the hike was again found. Modulation in stigmasterol content during different seasons was noted which can be implicated to the popular notion of variation in secondary metabolites depending on temperature, ultraviolet radiation and other factors related to seasonal changes. Popular use of the plant in Indian ethnomedicine may be attributed to the presence of stigmasterol as one of the important constituents of the plant.