Variability in morphology, phytochemicals and antioxidant activity in Bistorta amplexicaulis (D. Don) Greene populations under variable habitats and altitudes

Abstract Phytochemicals have become significantly important for scientific research since these possess incredibly remarkable health benefits, especially antioxidant potential to scavenge free radicals and combat the harmful effects of oxidative stress caused by adverse environmental factors. The efficacy and quantity of these phytochemicals relies upon numerous factors including the extraction method, solvent polarity and the habitat features in which the plant is growing. In this study we emphasized on phytochemical analysis and antioxidant activity of Bistorta amplexicaulis, an important medicinal plant species from Kashmir Himalaya. We evaluated antioxidant activity using different assays from all the selected sites to enumerate the impact of habitat. The sites were selected based on varying habitat features and altitude. Our results revealed that Ethyl acetate is the potent solvent for the extraction of phytochemicals. Below ground parts exhibited better scavenging activity than the above ground parts. Amongst the sites, we found the maximum antioxidant potential at Site I. A positive correlation was found between antioxidant activity and altitude while soil attributes (OC, OM, N, P, and K) and most of the morphological traits showed a negative correlation. Overall, our study identified the elite populations that could be utilized for mass propagation and harness the ultimate antioxidant potential of B. amplexicaulis. Graphic Abstract


Introduction
The plant kingdom consists of a vast number of species with a huge diversity of bioactive metabolites with different chemical scaffolds (Mir et al. 2019).It is estimated that only 15% and 6% of medicinal plants have been systematically investigated for phytochemical and pharmacological potentials, respectively (Choudhary 2001).Ethnopharmacology, a source of knowledge-driven drug discovery is playing a significant role in drug discovery from plants, animals, and fungi based on local or traditional knowledge of its pharmacological or toxicological properties in local population (Heinrich 2010).Currently, 119 drugs that have been licenced for clinical usage come from medicinal plants.Among them, 74% were found by chemical analysis of the components that humans utilize as medicines.These 119 plant-based medications are made in large quantities from about 90 different plant species.Given that there are more than 25,000 species in the world, thorough analysis of these species can result in the creation of more effective medications to treat variety of disorders (Farnsworth 2007).Natural products possess qualities that are inherently optimized for a variety of biological processes since they are produced by living things.In comparison to synthetic compounds, natural compounds own selective biological properties, have better chemical diversity and biosynthetic complexity making them more suitable for drug discoveries (Atanasov et al. 2015).All biological systems produce free radicals and different reactive oxygen species during cellular metabolism, which causes oxidative stress in human beings.If not regulated properly, oxidative stress can induce a variety of chronic and degenerative diseases as well as the aging process and some acute pathologies (trauma, stroke).Oxidative stress has been investigated in neurological diseases including Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), memory loss, depression (Halliwell 2001;Singh et al. 2004) Therefore some sort of defensive system is required to lessen this damage, and in fact, oxidative stress is managed by using a variety of natural and synthetic antioxidants, particularly plant-derived secondary metabolites.Recently, numerous medicinal plants have been thoroughly examined for their antioxidant and radical scavenging activity for the treatment of several diseases (Kumar et al. 2017;Nweze et al. 2017;Mushtaq et al. 2018;Javed et al. 2019;Torma et al.2019;Rani et al. 2020;Chaturvedi et al. 2021).
Plants, due to their sedentary nature are unable to avoid the vulnerabilities of the environment in which they grow.The competence of plants to transform their morphology and physiology in order to adapt to fluctuating environmental settings is known as phenotypic plasticity (Badyaev 2009).Various environmental stress, such as high temperatures and UV rays, frequently have a direct impact on plants and are known to change their morphological and structural characteristics.Additionally, it has been suggested that factors such as climate, nutrient availability, plant-to-plant interactions, and genetic makeup have an impact on plant secondary metabolites in addition to their morphology (Suyal et al. 2019).One significant environmental factor that has been linked to changes in plant secondary metabolism includes altitude (Zlatev et al. 2012).A plant's overall structural and functional qualities are both impacted by oxidative stress, which is caused by adverse environmental conditions that promote the production of free radicals.Plants produce distinct phytochemicals such as polyphenolic compounds, terpenes, alkaloids, and carotenoids as part of their defensive mechanism to deal with the harmful implications of oxidative stress caused by various environmental factors.
Bistorta amplexicaulis (D.Don) Greene (Syn: Polygonum amplexicaule D. Don), locally known as 'machran chai' belongs to the family Polygonaceae and grows in hilly regions of Kashmir as a medium-sized herb (Dar et al. 2002).B. amplexicaulis is medicinally significant and has applicability in the treatment of many diseases such as maintaining proper blood circulation, reducing stomach pain, muscle injuries, fractures, rheumatism, osteoporosis, and inflammation of the tongue and mouth (Xie 2008;Xiang et al. 2011).In this regard, we have conducted the phytochemical analysis using HR-LCMS, of this particular species and address the following questions: (i) Impact of habitat variability and altitude on the antioxidant potential, morphology, and soil parameters across the study sites, (ii) Effect of solvent polarity on the antioxidant potential of B. amplexicaulis, (iii) Variation in antioxidant activity with respect to the plant part used for extraction and (iv) the correlation between antioxidant potential, morphology and soil parameters of B. amplexicaulis.

Qualitative phytochemical analysis
The phytochemical analysis of various parts of B. amplexicaulis in different solvents confirmed the presence of numerous bioactive substances (Supplementary material, Table S1).

Total flavonoid (TFC) and phenolic content (TPC)
TFC and TPC varied significantly depending on the plant part, the solvent used and the habitat selected (Supplementary material, Tables S2 and S3).In general, higher TPC and TFC were recorded in ethyl acetate extracts (149.129± 1.299 mg GAE/g and 170.474 ± 5.762 mg rutin/g, respectively) in comparison to other solvents used (Figure 1).TPC and TFC increased as follows P.E > Aqueous > Methanol > Ethyl acetate extracts.TPC and TFC also varied depending on the plant parts, as maximum TPC was found in the below-ground parts (Rhizome) in comparison to the above-ground parts (Stem, leaf and inflorescence).However, TFC was maximum in above-ground parts and ranged from 170.474 ± 5.762 mg rutin/g at Site I to 15.084 ± 0.476 mg rutin/g at Site IV.Amongst different study sites, high altitude sites at 2730 and 2657 m a.s.l had maximum phenolic and flavonoid content.Above-ground parts had relatively lower total phenols (109.195± 0.605 mg GAE/g to 7.743 ± 0.198 mg GAE/g) but shows a similar trend with altitude and solvent used.
The efficacy of phytochemical extraction relies upon numerous factors including the type of phytochemical, extraction method, sample particle size, extraction time, pH, temperature, solute-solvent ratio and polarity of the solvent (Do et al. 2014).Proper selection of solvent systems is important for the efficient extraction of polyphenols and other phytochemicals from the samples (Iloki-Assanga et al. 2015).Flavonoids and other plant-derived polyphenols possess multiple biological properties; thus, it is important to assess their presence in various plant parts extracted using diverse organic solvents (Apetrei et al. 2011;Azadi Gonbad et al. 2015).

Phytochemical composition
Using the HR-LCMS approach, the phytoconstituents in ethyl acetate extract of B. amplexicaulis (below ground and aboveground parts) were analysed and further identified (Supplementary material, Tables S4 and S5).The major phenolic and flavonoid compounds were found to be Catechin, Quercetin, 3-Hydroxycoumarin, and Paradol.In the current study, the antioxidant activity of different extracts can be associated with Catechin, Quercetin and 3-Hydroxycoumarin identified by LC-MS.Ethyl acetate extracts of both belowground and aboveground parts revealed 15 and 20 important peaks in ESI + ve and ESI − ve modes using HR-LCMS, respectively (Figure 2) This study provided the first report of phytochemical analysis of B. amplexicaulis using HR-LCMS/MS.The role of Catechin, Quercetin and 3-hydroxycoumarin as antioxidants has already been reported by several workers (Jomová et al. 2019;Pheomphun et al. 2019;Zaplatic et al. 2019).Consequently, these compounds can work in synergy with other phytochemicals to enhance antioxidant activity (Okur et al. 2021).

Reducing power assay
The reducing power of different extracts was dependent on the concentration of the extract and the solvent used.The absorbance value increases with the increase in reducing power activity of the sample.Ethyl acetate extracts of aboveground parts had the strongest reducing power ability in comparison to the other extracts, with the highest values of 0.514 ± 0.0005 followed by methanol (0.461 ± 0.0005), aqueous (0.393 ± 0.0005) and P.E (0.37 ± 0.001) extracts (Supplementary material, Table S6) Reducing power ability of below-ground parts (rhizome) was comparatively less and showed a significant difference (p ≤ 0.05) when compared to the above-ground parts (Figure 3).Among the study sites, Site I showed the highest antioxidant potential, and the lowest was shown by Site IV (0.135 ± 0.004).Paired comparison plots revealed that the differences are significant (p ≤ 0.05) for most of the study sites (Figure 4).

DPPH radical scavenging activity
The results of the DPPH radical scavenging test showed that the scavenging capacities of various extracts varied significantly.The findings showed that different extracts possessed different scavenging capacities that depended on the solvent and concentration of the plant extracts.The strongest scavenging action was demonstrated by Ethyl acetate extracts of (below ground part) rhizome, which had the highest inhibition values of 70.415 ± 0.373%, followed by methanol, aqueous and P.E extracts (67.24 ± 0.574, 56.0081 ± 0.313 and 52.5 ± 0.208%, respectively) (Supplementary material, Table S7).Aboveground parts had relatively lesser scavenging activity and showed significant differences (p ≤ 0.05) when compared to the scavenging activity of belowground parts and followed a similar trend in terms of the solvent used for extraction (Figure 3).% Inhibition values decreased from ethyl acetate, methanol, aqueous to P.E extracts (42.0537 ± 0.373, 36.9080 ± 1.149, 35.863 ± 0.318 and 34.863 ± 0.203%, respectively.Amongst the sites, Site I showed the maximum percent inhibition with the lowest IC 50 values ranging from 348.59 ± 4.11 to 935.123 ± 1.87 µg/mL followed by Site II, Site III and IV sites.

Nitric oxide (NO) radical scavenging assay
NO scavenging assay of B. amplexicaulis extract and fractions varied considerably (Supplementary material, Table S8).Extracts scavenge NO radicals in a concentration-dependent manner.Maximum scavenging activity was shown by ethyl acetate fraction of rhizomes followed by methanolic extracts with the IC 50 value of 162.72 ± 3.43 and 244.786 ± 3.21, respectively as compared with the IC 50 value of ascorbic acid 105.952 ± 3.34 µg/ml.The order of reactivity for all other extracts was methanol > Aqueous > P.E.Aboveground parts had lower scavenging activity and showed significant differences (p < = 0.05) when compared to the below-ground parts (Figure 3).

Hydroxyl (OH − ) radical scavenging activity
Hydroxyl Radical Scavenging (OH − ) Activity and IC 50 values showed considerable variations concerning the solvent and plant part used for extraction.The maximum percentage reduction of OH − radical was demonstrated by ethyl acetate extracts of belowground parts (83.300 ± 0.175) followed by methanol, aqueous, and petroleum ether extracts (74.885 ± 0.466, 63.089 ± 0.260 and 58.606 ± 0.277%, respectively) (Figure 3).The IC 50 values of ethyl acetate, methanol aqueous and petroleum ether extracts rhizome extract were 127.57± 2.22, 280.59 ± 3.43, 426.718 ± 4.22 and 510.98 ± 3.22 µg/ mL, respectively).Site I had the highest OH -radical reduction and lowest IC 50 value followed by Site II, Site III and Site IV (Supplementary material, Table S9).Antioxidant activity of the plant extracts carried out using different assays shows a positive correlation with the total phenolic and flavonoid content (Figure 5).As a result of their redox capabilities, phenolics are the most important plant secondary metabolite having antioxidant properties (Jimoh et al. 2011;).Flavonoids are among the most widely distributed class of compounds found in plants, having health-enhancing effects.As shown in the results significant differences in antioxidant potential amongst different parts (belowground and aboveground) of B. amplexicaulis could be due to the difference in the quantity of phenolics as they possess a strong correlation.Also, the effect of solvent on the extractability of various phytochemicals (Phenolics/ Flavonoids) can be attributed to the fact that the polarity of a solvent affects their solubility and hence the antioxidant activity as supported by Gulcin (2020) and Maisuthisakul et al. (2007).The wide range of chemical properties and polarity of phytoconstituents also leads to their varied solubilities in different solvents (Sultana et al. 2009).A strong correlation was also found between antioxidant activity and altitude.Populations of higher altitudes possess better scavenging activities than populations at lower altitudes.Amongst various environmental factors, altitude has a significant impact on the geology, topography, rainfall, soil moisture, texture, and species composition of an area (Suyal et al. 2019).This correlation of antioxidant activity with altitude is indicative of the higher level of stress in the populations of B. amplexicaulis present at higher altitudes.In a similar study carried out by Cirak et al. (2017), a positive association between antioxidant activity and altitude suggests that stress at higher altitudes is mainly caused by low temperature and UV-B radiation.Plants accumulate more phenolics and additional phytochemicals for defense in open, exposed settings, due to direct sunlight and UV radiation in open and exposed sites.These phytochemicals function as antioxidants and can take a variety of forms to mitigate potentially harmful UV radiation before they can damage the chromophores (Ruhland et al. 2007).In exposed and open habitats, UV and other ionizing radiations can damage DNA directly by generating reactive oxygen through an indirect photo-sensitization reaction.

Morphology
The phenotypic traits of B. amplexicaulis studied during the field survey vary substantially, signifying remarkable phenotypic variation between populations across different altitudes (Supplementary material, Table S10).The plant height is highest in Site IV (92.54 ± 2.12 cm) and lowest in Doodhpathri (34.93 ± 2.54 cm).Similarly, Rhizome length and breadth were maximum in Site IV (17.48 ± 2.12 and 2.11 ± 0.33 cm) and minimum in Site I (8.87 ± 0.48 and 1.28 ± 0.36 cm).Leaf number per plant ranged from 3.2 ± 0.60 (Site I) to 4.50 ± 0.41 cm (Site IV).The mean apical and basal leaf length per plant is 4.74 ± 1.32 cm (Site I) to 9.02 ± 1.63 cm (Site IV) and 5.877 ± 2.30 (Site I) to 11.84 ± 2.47 cm (Site IV), respectively.The mean apical and basal leaf breadth per plant ranged from 2.24 ± 0.56 (Site I) to 4.41 ± 1.10 cm (Site IV) and 2.514 ± 2.31 (Site I) to 5.21 ± 1.64 cm (Site IV), respectively.Inflorescence length also varied considerably from 3.54 ± 0.61 cm (Site I) to 7.42 ± 0.46 cm (Site IV) and the number of flowers ranged from 42.50 ± 2.32 (Site I) to 97.4 ± 1.58(Site IV), respectively.The decrease in plant height with increasing altitude can be a survival mechanism to endure harsh climatic conditions such as strong winds, also as leaves remain close to the warmer soil, photosynthetic conditions are amended.At higher altitudes plants intensify super cooling capability by diminishing intercellular spaces and cell size which ultimately results in an overall decrease in plant size (Gulzar et al. 2017).This reverse relationship between plant height and increasing altitude, as an adaptation has already been reported by various studies (Baret et al. 2004;Magray et al. 2022;Qadir et al. 2022).Apart from the severe conditions, plants growing at high altitudes have a shorter growing season due to which they are less tall in comparison to plants at lower altitudes where the growth period is relatively longer (Gulzar et al. 2017).The difference in plant height may also be due to the difference in age at different sites (Zotz et al. 2001).At low altitudes, light is a critical limiting resource for plant survival and growth (Zhang et al. 2012).Plants growing in low light intensities have limited photosynthetic capabilities per unit leaf area (Niinemets 2007;Poorter et al. 2008) therefore they would develop to allocate the most biomass to laminas.Due to their wider foliar display, large leaves may intercept a substantial amount of light at low light intensities (Poorter et al. 2008).

Soil analysis
Soil analysis at various sites revealed that the soil is slightly acidic to slightly basic with average pH ranging from 6.183 ± 0.153 (Site1) to 8.05 ± 0.29 (Site IV).The average OC and OM content ranges between 8.16 ± 0.192% to 9.113 ± 0.023% and 14.079 ± 0.332% to 15.746 ± 0.040%.The value of N, P, and K ranged from 269.21 ± 8.13 (Site I) to 532.17 ± 7.57 ppm (Site IV), 40.766 ± 8.71 (Site I) to 66.96 ± 8.18 ppm (Site IV) and 214.56 ± 4.58 (Site I) to 341.50 ± 7.5 ppm (Site IV), respectively (Supplementary material, Table S11).As the results depict, with the increase in altitude the OC, and OM decrease from Site IV to Site I. Site IV shows the highest fertility as the habitat is shady with the accumulation of litter.The main source of OM is litter and its quantity and quality are determined by the plant species which is dominant (Quan et al. 2005).The continuing supply of nutrients is ensured by the decomposition of OM.OM is the main source of soil C that governs soil characteristics.According to Mishra et al. (2017) the quantity of OC and OM positively correlates with the available N and K.In forests, the OM content of the soil completely determines the amount of N (Mishra et al. 2017).Generally, it has been found that high-altitude soils are invariably rich in organic matter with favorable pH.With the increase in altitude temperature decreases, rainfall increases low temperature reduces the rate of mineralization of organic matter, thereby leading to the accumulation of the maximum amount of organic matter.However, in the present study, we found that the selected sites of higher altitudes range from partial rocky to fully rocky habitats.As rocky environments are generally less fertile (Fitzsimons and Michael 2017) so despite being at higher altitudes their fertility remains low.A similar trend of decreasing OC and OM with altitude has also been reported by Saeed et al. (2014).A significant relationship was found between the nutrient level of soil and the quantity of secondary metabolites (phenolics and flavonoids) (Figure 5).Our study has revealed, as the soil becomes enriched in various nutrients the quantity of secondary metabolites decreases.A strong negative correlation was found between nitrogen and TPC (r = −0.967)and TFC (r = −0.931).A decrease in the number of secondary metabolites with increasing nutrients (particularly N) is backed up by the carbon/nutrient balance (CNB) hypothesis.According to this hypothesis (Bryant et al. 1983), nitrogen fertilization increases growth at the cost of the production of carbon-based secondary metabolites (such as terpenoids, phenolics and other compounds with only C, H and O in their primary structure), potentially increasing concentrations of alkaloids and other nitrogen-based compounds.This hypothesis proposes that the production of carbon and nitrogen-based allelochemicals is greatly affected by variations in the availability of carbon, light and nitrogen (resources required for plant growth).Increased quantities of non-nitrogenous metabolites synthesized from the shikimic acid pathway are typically due to nutrient deficiency.Previous studies carried out by Duarte et al. (2012) also support this relationship between secondary metabolites and nitrogen availability.

Relationship between antioxidant potential, morphology and soil parameters
The current study revealed antioxidant potential shows a negative correlation with various morphological traits (stem height, root and leaf dimensions) and soil parameters (Figure 5).The study sites at different elevations had a significant impact on the morphological and phytochemical composition of B. amplexicaulis.This variation in morphology is predicted because different habitats result in diverse phenotypic expressions.Other Himalayan medicinal plants have also shown this kind of variation between populations (Suyal et al. 2019).The positive correlation between various morphological and soil parameters occurs because plants grow luxuriously in nutrient-rich soils in comparison to nutrient-deficient soils (Inagaki et al. 2011).The growth of plants is often limited by the availability of nitrogen (Kant et al. 2011), hence plant growth is restricted under nutrient-deficient soils (Site I).A negative correlation between morphology and antioxidant potential occurs due to higher levels of stress at higher altitudes.This in turn leads to an increased concentration of secondary metabolites (hence the antioxidant activity) (Cirak et al. 2017) and a reduction in the overall growth of the plant (Qadir et al. 2022).The study has revealed that Site I is favorable for harnessing antioxidant potential while for most of the morphological traits Site IV is suitable as depicted by the PCA plot (Figure 6).

Survey and selection of study sites
Extensive field surveys were carried out during 2020-2021 in varied habitats of Kashmir Himalaya to recognize specific areas across diverse geographical conditions.Four sites were selected at high, mid, and low altitudes differing in habitat features, temperature, and precipitation based on the ease of access, habitat dynamics, and abundance of the selected plant population.Three natural populations Doodhpathri Site I (2730 m a.s.l),Gulmarg Site II (2657 m a.s.l),Tangmarg Site III (2158 m a.s.l), and one population at Kashmir University Botanical Garden Site IV -1588 m a.s.l (KUBG), as shown in the map (Supplementary material, Table S11).

Soxhlet extraction
To conduct the phytochemical analysis, 20-30 mature flowering individuals were randomly selected from the specified populations.Plants were shade dried for 15-20 days and then subjected to grinding using an electric grinder until a fine powder was formed.About 50 grams of dried powder of belowground (Rhizome) and aboveground (stem, leaf and flower) parts from each site were subjected to hot successive extraction in a soxhlet apparatus using 500 mL of various solvents (Petroleum ether, Ethyl acetate, Methanol and Aqueous) (Bimakr et al. 2011).The dried extracts were stored at 4 °C in air-tight glass vials until further use.

Morphological characterization
To observe various morphological parameters, twenty mature flowering individuals were randomly selected from the selected sites (Qadir et al. 2022).The plants from each site were recognized in Kashmir University Herbarium (KASH) with voucher specimens No. 2964, 3749, 4318 and 4319.The plants were further assessed for various morphological characteristics.

Quantitative analysis of phytochemicals present in the extracts of B. amplexicaulis
Qualitative analysis of phytochemicals was carried out using the standard method (Wallis 1990;Harborne 1998;Kokate et al. 2005;Sadashivan and Manickam 2005).

Quantification of total flavonoids
Methodology by Kumaran and Karunakaran (2006) was followed for the quantification of flavonoids.

Quantification of total phenolics
Following the standard methodology by Hagerman et al. (2000) the total amount of phenolic content in plant extracts was measured.

Soil sampling and analysis
Soil profiles were assembled up to a depth of 15 cm (triplicates) from the study sites for soil analysis.Before the further examination, the obtained soil samples were air-dried and sieved through a 0.5 mm size sieve (Khan et al. 2017;Iqbal et al. 2021).Using a digital pH meter, the pH of the soil was determined in suspensions of soil water (1:5) (Khan et al. 2012).Walkley and Black (1934) rapid titration method was employed to measure Organic carbon (OC) in soil.Organic matter (OM) was calculated by multiplying OC with a factor of 1.724 (Magray et al. 2022).Other chemical characteristics of soil, such as available nitrogen(N), phosphorus (P), and potassium (K) were examined at each study site using the established methods of Subbaiah and Asija (1956); Olsen et al. (1954) (spectrophotometer method) and Black (1968) (flame photometric method).

Liquid chromatography coupled with high-resolution mass spectrometry
High-resolution liquid chromatography and mass spectrometry (HR-LCMS) analyses were conducted to identify the bioactive compounds.HR-LCMS analysis was operated on the ethyl acetate extract of upper ground and below ground parts.High-resolution liquid chromatography and mass spectrometry model-1290 Infinity ultra-high performance liquid chromatography (UHPLC) System, 1260 infinity Nano HPLC with Chipcube, 6550 iFunnel Q-TOFs (Agilent Technologies, Santa Clara, CA, USA) was employed to obtain a chemical fingerprint of the plant extract (Rafiq et al. 2022).

DPPH radical scavenging assay
The method described by Saeed et al. 2012 with slight modifications was followed to conduct the DPPH radical scavenging assay

Reducing power assay
The ability of plant extract to reduce Fe 3+ was assessed using a method given by Misan et al. 2011, with some minor modifications

Nitric oxide radical (NO) scavenging assay
NO activity was conducted following the methodology by Francis and Rew (2010).

Hydroxyl radical (OH − ) scavenging assay
The ability to scavenge hydroxyl radicals was assessed using the method described by Rafiq et al. (2022).

Statistical analysis
SPSS 23 (SPSS Inc., Chicago, IL, USA) was used to analyse variance (ANOVA) with Tukey's test of multiple comparisons for the antioxidant tests.R was used to create the PCA plot (version 3.2).Origin Pro 2021 was used for pairwise comparison and other correlation plots.

Conclusion
The study of medicinal plants as a source of possible herbal medicines has seen a renaissance during the last several decades.There is a need to advance research for the discovery and characterization of novel natural medications from plants and other natural sources with the help of enhanced screening methods as herbal medicine may usher in a new era of medical systems for the management of human ailments.However environmental conditions have a prevalent role, affecting the phenotype and the quality of phytoconstituents.In this regard, the current study was carried out to understand the interrelation between morphology, phytochemistry, and soil attributes among four populations of B. amplexicaulis across different habitats varying in altitude.In this study, we have been able to identify elite populations in terms of the quantity of phytochemicals and their antioxidant potential.The LC-MS analysis of B. amplexicaulis yielded bioactive compounds with the vast majority of them having therapeutic applications.The present study revealed that the rhizome is a primary source of antioxidants and ethyl acetate is probably the most suitable solvent for the efficient extraction of phytochemicals in B. amplexicaulis.A considerable increase in antioxidant activity with varying habitats and increasing altitude signifies that the species can be cultivated for harnessing the ultimate potential in these habitats.The study established that antioxidant activity was better in plants growing at Site I while soil parameters and most of the phenotypic traits were prominent at Site IV.This study will also provide insight into the potential resilience of the species to continued habitat changes.Apart from that, baseline data would be set for future studies that may further authenticate whether the changes are due to phenotypic plasticity or essentially genetic.

Figure 2 .
Figure 2. Phytochemicals identified in B. amplexicaulis ethyl acetate extract of Below ground parts (rhizome) (a -esI positive mode, B -esI negative mode) and above ground parts (leaf, stem, and flower) (c -esI positive mode, d -esI negative mode) using the hr-lcMs technique.

Figure 6 .
Figure 6.Principal component analysis (Pca) of morphological traits, quantification of phenolics/ flavonoids, and various antioxidant assays of Bistorta amplexicaulis across the different study sites.