Introduction

Valeriana jatamansi Jones (English name: Indian Valerian; Family: Caprifoliaceae, earlier known as family Valerianaceae) is an important medicinal herb of the Himalayan region (Jugran et al. 2020) and is used as tranquilliser, sedative, stimulant, antispasmodic, carminative, diuretic, sleep enhancing, analeptic, analgesic, nervine and to cure hysteria, urine complaints, constipation, insomnia, epilepsy, neurosis and anxiety disorder (Subhan et al. 2007; Jugran et al. 2019). The biological importance of Valeriana is attributed to valerenic acid (a sesquiterpenoid), its derivatives and valepotriates, which are mainly reported to be present in root and rhizome part of the plant (Gao and Björk 2000). Valerenic acid is the principle bioactive constituent for which this species is much valued. The species is one among the 178 traded medicinal plants which have been traded/consumed in high volumes over 100 MT/year (Rawat and Vashistha 2011) and its annual trade for the year 2014–2015 was estimated to be 1000–1500 MT (Goraya and Ved 2017). The demand of this species for pharmaceutically important compounds has been continuously increasing leading to excessive collection and continuous depletion. The changing climatic conditions has further reduced its availability for modern and traditional system of medicine, necessitating the need for its sustainable cultivation and harvesting.

Identification of suitable growing conditions for better plant growth and production and, harvesting in optimum season is essential for large scale cultivation of species. Light intensity is an important environmental factor affecting structural and functional properties of plants. V. jatamansi plants respond to high light intensity by developing thick leaf, high wax content, high photosynthesis (Vats et al. 2002), less leaf wetness, more stomata and higher capacity to retain water droplets (Pandey and Nagar 2002). V. jatamansi plants acclimatize to high photon load by altering their leaf anatomy and chloroplast arrangement and can grow well in open field conditions (Pandey and Kushwaha 2005). High or low light intensity also influence the biosynthesis of secondary metabolites. Low irradiance causes limited carbon gain due to reduced gas exchange, photosynthesis, nutrient availability, etc. and cause reduction in synthesis of secondary metabolites (Bryant 1987; Hou et al. 2010). In response to high light radiation, plants adapt by accumulating various secondary metabolites including terpenoids, phenolics and alkaloid compounds, and many of them, have high economic value due to the well-known antioxidant properties (Yang et al. 2018). These antioxidant phytochemicals play protective role in human body by reducing the level of reactive oxygen species (ROS) which in high concentration cause oxidative damage to cellular biomolecules such as lipids, proteins and deoxyribonucleic acid. These phytochemicals are also used to treat various ailments like cancer, cardiovascular disease, obesity, diabetes etc., and the consumption of antioxidant rich food and medicinal plants is proven to increase the antioxidant capacity of blood (Zhang et al. 2015). Polyphenols and carotenoids are the two main phytochemicals which contribute the most to the antioxidant in foods and plants. They possess free radical scavenging abilities through substitution of hydroxyl groups in the aromatic rings of phenolics and confer various health benefits (Zhang et al. 2015).

Various studies (Katalinic et al. 2006; Surveswaran et al. 2007; Dudonne et al. 2009; Bechlaghem et al. 2019) have been done to determine the antioxidant potential and phenolic content of many medicinal plants including V. jatamansi (Jugran et al. 2020) so as to identify the rich sources of natural antioxidants, however, studies on seasonal variations in these phytochemicals are limited specifically on the genetically uniform material (in vitro raised plants). The phytochemical composition of medicinal plants is largely affected during different seasons, suggesting that seasonal fluctuations influence therapeutic efficacy of plants (Ahmad et al. 2011; Szakiel et al. 2011). Identification of seasonal variation in bioactive compounds will determine the optimum harvesting season w.r.t commercially or pharmaceutically significant constituents (Soni et al. 2015). Systematic investigations on suitable harvesting season and light requirements of V. jatamansi have not yet been reported. Therefore, the present study was designed to determine the growth response and phytochemical content of V. jatamansi under two light conditions i.e. full sunlight and 50% shade during four different seasons in order to harness high quality medicinal herb.

Materials and methods

Plant material and experimental conditions

V. jatamansi plants were grown in vitro as described by Purohit et al. (2015), transplanted into plastic bags containing a mixture of soil and vermiculite (1:1 v/v) and maintained in a greenhouse of the G. B. Pant National Institute of Himalayan Environment (GBP-NIHE), Kosi-Katarmal, Almora. Eighteen months old genetically identical plants of V. jatamansi were transferred (during mid August) to two different light conditions i.e. full sunlight (open field) and under shade net of 50% transmittance in the experimental field of the Institute. After six months of acclimatization under different conditions, seasonal changes in various morphological, physiological and phytochemical parameters were recorded for four seasons i.e. winter (February), spring (May), summer (August) and autumn (November). During this period, average maximum and minimum temperatures were: winter (20.1/1.4 °C), spring (20.8/9.6 °C), summer (30.8/20.4 °C) and autumn (21.5/2.1 °C); and photosynthetic photon flux density (PPFD; between 10 am to 4 pm) varied among the season i.e. winter (79–1193 µmol m−2 s−1), spring (287–1829 µmol m−2 s−1), summer (122–1906 µmol m−2 s−1) and autumn (73–1449 µmol m−2 s−1).

Morpho-physiological analysis

Various parameters like plant height, leaf number, leaf area, fresh and dry weight of different plant parts, relative water content (RWC) and photosynthetic pigments (chlorophyll and carotenoid content) were determined. Leaf area was estimated by using following allometric equation given by Walia and Kumar (2017):

$${\text{Leaf area }} = 0.{487} + 0.{\text{644 LW}}$$

where, L and W denotes length and width of leaf.

RWC was determined in fully expanded leaf from top following Dhopte and Manuel (2002). Leaf was collected and immediately fresh weight was recorded, followed by immersing the leaves in clean petri-plates filled with distilled water for 24 h. Subsequently, their weights were recorded as turgid weights after gentle surface blotting. Dry weight of leaves was recorded after 3–4 days of drying at 65 °C and RWC was calculated as:

$${\text{RWC }}\left( \% \right) = \left[ {\left( {{\text{FW}}{-}{\text{DW}}} \right)/\left( {{\text{TW}}{-}{\text{DW}}} \right)} \right] \times {1}00$$

where, FW, DW and TW denotes fresh weight, dry weight and turgid weight.

Chlorophyll and carotenoid content in leaf was estimated by DMSO method. Briefly, 10 mL of dimethyl sulfoxide (DMSO) was added in test tubes containing 50 mg of finely chopped leaves and then incubated in an oven (65 °C for 3 h). After incubation, absorbance was measured at 665, 648 and 480 nm using a spectrophotometer against pure DMSO as blank. The chlorophyll (Barnes et al. 1992) and carotenoid (Sumanta et al. 2014) content was then calculated as follows:

$${\text{Chlorophyll}}\,\,{\text{a} = 14}{.85 \times \text{A}}_{{{665}}} { - 5}{.14 \times \text{A}}_{{{648}}}$$
$${\text{Chlorophyll}}\,\,{\text{b} = 25}{.48 \times \text{A}}_{{{648}}} { - 7}{.36 \times \text{A}}_{{{665}}}$$
$${\text{Total}}\,{\text{chlorophyll} = 7}{.49 \times \text{A}}_{{{665}}} { + 20}{.34 \times \text{A}}_{{{648}}}$$
$${\text{Carotenoid}}\,{\text{content} = }\left( {{\text{1000A}}_{{{480}}} { - 1}{\text{.29C}}_{{\text{a}}} { - 53}{\text{.78C}}_{{\text{b}}} } \right){/220}$$

where, A665, A648 and A480 are absorbances at 665, 648 and 480 nm, respectively. Ca and Cb denotes chlorophyll a and chlorophyll b content.

Phytochemical analysis

Various phytochemicals (total phenolics, flavonoid and tannin content) and antioxidant activities (ABTS, DPPH and FRAP assay) were determined in different plant parts (leaf, stem, root, rhizome) and compared among light and shade grown plants.

Extract preparation

Dried plant samples (1 g) of leaf, stem, root and rhizome were added in a conical flask containing 50 mL methanol (80% v/v), stirred gently for 12 h and sonicated (22 °C, 10 min; Professional Ultrasonic Cleaner, Guang Dong GT Ultrasonic Co., Ltd, China). The extract was then centrifuged (10,000 rpm, 15 min) and supernatant was collected, filtered, and stored at 4 °C prior to analysis (Jugran et al. 2016).

Estimation of total phenolic content

Total phenolic content in the methanolic extract was determined by Folin-Ciocalteu’s colorimetric method (Singleton and Rossi 1965). The extract (0.25 mL) was diluted with distilled water (2.25 mL) and Folin-Ciocalteu’s reagent (0.25 mL) was added. Reaction was allowed for 5 min and then 7% sodium carbonate (2.5 mL) was added. Mixture was kept in the dark and absorbance was measured at 765 nm using UV–Vis spectrometer (U-2001, Hitachi, Japan). Results were expressed in milligram gallic acid equivalent (GAE)/ gram of dry weight.

Estimation of total flavonoids

Flavonoid content was determined by method given by Chang et al. (2002). Briefly, the methanolic extract (0.5 mL) was diluted with distilled water (1.5 mL) and 10% (w/v) aluminium chloride (0.5 mL) was added to it, followed by 1 M potassium acetate (0.1 mL) and distilled water (2.8 mL). The mixture was incubated at room temperature for 30 min and absorbance was recorded at 415 nm. Total flavonoid content was calculated as milligram quercetin equivalent (QE)/ gram of dry weight.

Estimation of total tannin

Total tannin content was determined following the method of Nwinuka et al. (2005) with slight modifications. Briefly, methanolic extract (5.0 mL) was added to a volumetric flask containing Folin-Dennis reagent (0.5 mL). 7% sodium carbonate solution (1 mL) was added and the mixture was diluted to 10 mL with distilled water. The mixture was shaken vigorously, allowed to stand for 20 min at room temperature and the absorbance was measured at 700 nm. Total tannin content was expressed in milligram tannic acid equivalent (TAE)/gram of dry weight.

ABTS [2–2-azinobis (3-benzylthiazole)-6-sulphonic acid] assay

ABTS assay was performed following the method of Cai et al. (2004). Breifly, ABTS cation (ABTS+) was produced by adding ABTS (7.0 µM) and potassium persulphate (2.45 µM) in an amber colour bottle, kept in dark for 16 h and diluted with 80% ethanol till an absorbance of 0.70 ± 0.05 at 734 nm is attained. 3.9 mL of this solution was added to 0.1 mL of methanolic extract, allowed to stand for 6 min in dark at 23 °C and absorbance was recorded at 734 nm. Quantification was done using standard curve with various concentrations of ascorbic acid which were prepared in 80% methanol. Results were expressed in milligram ascorbic acid equivalent (AAE)/gram of dry weight.

DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay

DPPH assay was performed as described by Brand-William et al. (1995), with minor modification. Briefly, DPPH cation (0.1 mM) was prepared in 80% ethanol (v/v). About 3.0 mL of freshly prepared DPPH solution was mixed with 1 mL of diluted sample, shaken vigorously and kept in dark at room temperature for 20 min. Absorbance was recorded at 520 nm and results were expressed in milligram ascorbic acid equivalent (AAE)/gram of dry weight.

FRAP (Ferric reducing antioxidant power) assay

FRAP assay was performed as described by Faria et al. (2005), with minor modifications. FRAP reagent was prepared by adding 10 volume of 300 mM acetate buffer, 1 volume of 10 mM 2,4,6-Tris(2-pyridyl)-1,3,5-triazine (TPTZ) in 40 mM HCl and 1 volume of 20 mM FeCl3 and pre-warmed at 37 °C. A 3.0 mL of this reagent was mixed with 0.1 mL methanolic extract, kept at 37 °C for 8 min and absorbance was recorded at 593 nm. Results were expressed in milligram ascorbic acid equivalent (AAE)/ gram of dry weight.

HPLC analysis

HPLC analysis of phenolics

HPLC analysis of various phenolic compounds in the methanolic plant extract (prepared as described earlier under the section extract preparation) was conducted using a reverse phase chromatography system (Alliance e2695; Waters, Milford, MA, USA), equipped with a quaternary solvent delivery system, a vacuum degasser, an autosampler, and a Waters 2996 photodiode array detector. Various phenolic compounds such as m-coumaric acid, p-coumaric acid, o-coumaric acid, gallic acid, caffeic acid, vanillic acid, chlorogenic acid, catechin, phloridzin, ferulic acid, 4-hydroxybenzoic acid, 3-hydroxybenzoic acid and rutin were procured from Sigma-Aldrich (St. Louis, Missouri, United States). The separation was done on a Spherisorb C-18 column (250 mm × 4.6 mm, i.d. 5 μm particle size) at 30ºC using mobile phase of water: methanol (60:40, v/v) at a flow rate of 0.8 mL/min in an isocratic mode. Mobile phase was filtered through Millipore filtration assembly and degassed by sonication, and the column effluent was monitored over the range 190–400 nm at every 1.2 nm intervals of the absorption spectrum. Peak purity test was performed and separation of components with a peak purity angle lower than its purity threshold was accepted and considered to be a pure substance. The identification of phenolic compounds was done with respect to the retention time (RT) and absorbance spectrum of the corresponding external standard. To measure the concentrations of phenolic compounds, various standards of phenolics were prepared by dissolving in methanol, and stock solutions (20, 40, 60, 80, 100 ppm) of standards were used for calibration (Belwal et al. 2016). The results were expressed in microgram per gram of dry weight (µg/g DW).

HPLC analysis of valerenic acid (VA)

Valerenic acid content in different plant parts (leaf, stem, root and rhizome) was analysed by the method described by Bos et al. (1998). Briefly, 1 g of ground plant material (leaf, root, rhizome and stem) was extracted with three portions of 3 mL methanol for 5 min in an ultrasonic bath. The extracts were filtered, volume was then adjusted to 10 mL with methanol and used for HPLC analysis. The mobile phase consisted of 20% CH3CN (eluent A) and 80% CH3CN (eluent B) and both eluents contained 1 mM H3PO4. The following elution programme was used: first isocratic at 55% A and 45% B for 5 min, followed by a linear gradient up to 100% B in 19 min and isocratic elution at 100% B for 2 min. Subsequently, a linear gradient to 45% B in 2 min followed by isocratic at 55% A and 45% B for 5 min was used. The flow rate was 1.5 mL/min and detection of VA was carried out at 225 nm. The identification of valerenic acid was done with respect to the retention time and absorbance spectrum of the corresponding external standard i.e. pure valerenic acid (Sigma-Aldrich, St. Louis, Missouri, United States). Stock solutions (5, 10, 15, 20, 25 ppm) were prepared by dissolving valerenic acid in methanol and used for calibration. The results were expressed as microgram per gram of dry weight (µg/g DW).

Method validation

The HPLC method was validated by defining the linearity, limits of detection (LOD) and limits of quantification (LOQ) of all phytochemical compounds. The linearity was assessed by means of linear regression regarding the amounts of each standard and the area of corresponding peak on the chromatogram. Limits of detection and quantification were determined by calculation of the signal-to-noise ratio. Signal-to-noise ratios of 3:1 and 10:1 were used for estimating the detection limit and quantification limit, respectively.

Statistical analysis

All the statistical analysis was performed using the SPSS 16.0 statistical software program. A two way analysis of variance (ANOVA) was performed to assess the effects of light conditions (L) and seasons (S) on various morphological and physiological parameters. Three-way ANOVA analysis was performed for all phytochemical parameters, and the effect of light conditions (L), seasons (S), plant parts (P) and their interaction was analysed. Tukey test was performed among different seasons for all the studied variables. Data were presented as the mean ± standard error of three replicates with significance level at (p < 0.01) and (p < 0.05). Relationship between plant growth parameters and phtytochemical content under two different light conditions were evaluated using the Pearson’s correlation analysis.

Results

Morpho-physiological parameters

Morpho-physiological parameters like plant height, leaf area, leaf number and RWC were significantly (p < 0.01) affected by light conditions during all the seasons (Fig. 1). Plant height in all four seasons was 30–59% higher under shade condition, as compared to plants grown under full sunlight (open field). Two-way ANOVA analysis showed that variation in plant height was significantly (p < 0.01) influenced by light conditions (L) and seasons (S), but their interaction (L x S) was not significant (Supplementary Table S1). Reduction in leaf number (13–43%) and leaf area (48–67%) was observed in plants grown under full sunlight conditions as compared to shade conditions, during all the seasons (Fig. 1), and the reductions were significantly (p < 0.01) affected by L, S and L x S (Supplementary Table S1). The RWC showed no significant variations among seasons except in shade grown plants during winter season (Fig. 1). Based on the results of two-way ANOVA, RWC varied significantly (p < 0.05) with L, S and L x S (Supplementary Table S1).

Fig. 1
figure 1

Morpho-physiological parameters of V. jatamansi plants grown under full sunlight and shade conditions, during different growing seasons. Bar denotes standard error and different letters (a-d) above bar denote significant (p < 0.01) difference among seasons

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Among the photosynthetic pigments, significant reduction in chlorophyll a (17% reduction in winter, 20% in spring, 3% in summer and 1% in autumn season) and total chlorophyll content (20% reduction in winter, 25% in spring, 5% in summer and 3% in autumn season) was observed under full sunlight condition as compared to shade condition, while, carotenoid content was significantly higher (4% higher in winter, 21% in spring, 23% in summer and 3% in autumn season) under full sunlight during all the four growing seasons studied (Fig. 2). Two-way ANOVA analysis revealed that variation in these parameters were significantly (p < 0.05) influenced by light conditions (L) and seasons (S), but their interaction (L x S) was not significant (Supplementary Table S1). Chlorophyll b content was significantly (p < 0.05) reduced (11–39%) under full sunlight as compared to shade conditions, while the effect S and L x S was not significant (Fig. 2; Supplementary Table S1).

Fig. 2
figure 2

Photosynthetic pigments in V. jatamansi plants grown under full sunlight and shade conditions, during different growing seasons. Bar denotes standard error and different letters (a-d) denote significant (p < 0.05) difference among seasons

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Fresh and dry biomass of aerial plant parts i.e. stem and leaf was significantly higher in plants grown under shade condition as compared to full sunlight during all four seasons (except leaf dry weight under winter and spring seasons). Similarly, fresh and dry weight of belowground plant parts (root and rhizome) was higher under shade conditions during summer and autumn seasons, while higher in full sunlight grown plants in winter and spring seasons (Fig. 3). Among different seasons, significantly higher above and below ground biomass was observed under autumn season, followed by summer season. Two-way ANOVA analysis showed that variation in biomass was significantly (p < 0.01) influenced by L, S, and L x S (Supplementary Table S1). Difference in morphology of V. jatamansi plants under different light conditions during vegetative stage (in summer) and flowering stage (in winter) is represented in Fig. 4.

Fig. 3
figure 3

Fresh and dry weight of different parts of V. jatamansi grown under full sunlight and shade conditions, during different growing seasons. Bar denotes standard error and different letters (a-c) denote significant (p < 0.05) difference among seasons

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Fig. 4
figure 4

Difference in plant morphology of Valeriana jatamansi grown under shade and full sunlight during a vegetative stage in summer and b flowering stage in winter

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Phytochemical content and antioxidant activity

Among different light conditions, significantly (p < 0.01) higher total phenolics, flavonoids, tannin content and antioxidant activities (ABTS, FRAP and DPPH) were observed under full sunlight as compared to shade condition in all plant parts during all growing seasons except autumn season where total phenolic content in leaf and rhizome, and total tannin content in stem, rhizome and root were higher in shade grown plants (Fig. 5). Among different seasons, highest phytochemical content and antioxidant activities were observed in summer season under both light conditions. Among different plant parts, leaf contains highest total phenolics (9.6 mg GAE/g DW), flavonoid (10.5 mg QE/g DW), tannin (5.4 mg TAE/g DW), ABTS activity (41 mg AAE/g DW), DPPH (35 mg AAE/g DW) and FRAP activity (23 mg AAE/g DW) as compared to other parts (root, rhizome and stem). Three-way ANOVA analysis revealed that variations in phytochemical content and antioxidant activities were significantly (p < 0.01) influenced by light conditions (L), seasons (S), plant parts (P) and their interactions (Supplementary Table S1).

Fig. 5
figure 5

Phytochemical content and antioxidant activities in different parts of V. jatamansi grown under full sunlight and shade conditions, during different growing seasons. Bar denotes standard error

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HPLC analysis of methanolic plant extract (chromatogram shown in Fig. 6a) revealed that growth season and light conditions significantly affect the amount of phenolic compounds in different plant parts (Table 1). Out of the twelve phenolic compounds measured, five compounds viz. vanillic acid, 4-hydroxy benzoic acid, rutin, phloridzin and o-coumaric acid were detected in very low amount in some season, while not detected in most of the seasons and plant parts (data not shown). Vanillic acid and 4-hydroxy benzoic acid were only detected in leaf during summer and autumn season; rutin was detected in leaf in summer season; phloridzin was detected in leaf and rhizome during summer and winter seasons and; o-coumaric acid was only detected in rhizome part in winter, spring and summer seasons. Among other phenolic compounds, it was found that chlorogenic acid was quantitatively the most abundant phenolic compound in almost all plant parts, during all the seasons. The regression equation and linearity range for each phenolic compound, together with LOD and LOQ values, are shown in Table 2.

Fig. 6
figure 6

a HPLC chromatogram of methanolic extract of V. jatamansi, b HPLC overlay chromatogram of valerenic acid standards at 225 nm

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Table 1 Various phenolic compounds (μg/g DW) present in different parts of V. jatamansi plants grown under full sunlight and shade conditions, during different growing seasons. Values indicate mean ± standard error. nd denotes not detected
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Table 2 Regression curves, linearity, LOD and LOQ of phytochemical compounds
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Highest content of chlorogenic acid (38,160 μg/g DW) and 3-hydroxybenzoic acid (4967 μg/g DW) were found in leaf part during autumn season, followed by summer; p-coumaric acid was highest (5937 μg/g DW) during autumn season in stem; gallic acid was found highest (992 μg/g DW) during winter season in rhizome followed by summer; m-coumaric acid (15,903 μg/g DW), caffeic acid (13,711 μg/g DW) and ferulic acid (2453 μg/g DW) were found highest in summer season in leaf (Tables 1 and 3). Total phytochemicals were highest during summer season and among different light conditions, all compounds were higher under full sunlight as compared to shade condition. Among different plant parts, higher amount of phytochemicals was found in leaf compared to stem, root and rhizome, under both light conditions (Table 1). Results of three-way ANOVA revealed highly significant (p < 0.01) influence of L, S, P and their interactions on these phenolic compounds (Supplementary Table S4).

Table 3 Suitable harvesting season, plant portion and light condition for different phytochemicals  and antioxidant activities of V. jatamansi
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Valerenic acid content

HPLC overlay chromatogram of VA standard is represented in Fig. 6b. Among different seasons, highest amount of VA was observed during summer season in both light conditions (Fig. 7). Highest VA was found in leaf part during summer under both shade (162.8 µg/g DW) as well as full sunlight condition (108.7 µg/g DW), followed by rhizome. Results of three-way ANOVA showed that VA content varied significantly (p < 0.01) with L, S, P and their interactions (Supplementary Table S1).

Fig. 7
figure 7

Valerenic acid content in different parts of V. jatamansi, grown under different light intensities during different seasons. Bar denotes standard error and different letters (a–d) indicate significant (p < 0.01) difference among seasons

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Correlation Analysis

Correlation analysis among various morpho-physiological and phytochemical attributes of V. jatamansi revealed a significant positive correlation among plant height, phenolics, flavonoid, tannin content and antioxidant activities of various plant portions under both light conditions (Supplementary Tables S2 and S3). A significant positive correlation was found among leaf area, phenolics, flavonoid and DPPH activity. Under shade condition, leaf number and fresh/dry biomass of plant was significantly correlated with phenolic compounds and antioxidant activity. Under full sunlight, less or no correlation of leaf number and plant fresh/dry biomass with antioxidant compounds/activity was observed. RWC and carotenoid content did not correlate with antioxidant compounds and activity under both light conditions, while chlorophyll content was significantly but negatively correlated with most of these phytochemical compounds and antioxidant activities.

Discussion

The present study identified differential response of V. jatamansi plants during different season and under exposure to different light conditions. The negative impact of high light intensity in open field on overall plant performance is well known (Phee et al. 2004; Yasoda et al. 2018) but the patterns differ with season. Reduction in plant height, leaf number and leaf area was observed under high light or full sunlight condition as compared to shade condition (Fig. 1a,b,c). Reduced growth parameters under full sunlight condition might be due to higher evapo-transpiration which impaired plant water balance and reduced overall plant growth. In this study, reduction in leaf relative water content under full sunlight condition was also observed (Fig. 1d). Increased water loss from leaf due to high irradiation is reported by Li and Ma (2012). Similar to results of this study, high leaf water content under shading was reported by Bakhshy et al. (2013) and Hamdani et al. (2018). Better plant growth under shading may be due to the favourable micro climatic conditions such as light intensity, temperature and relative humidity in 50% shade which increased the photosynthetic activity and assimilate accumulation (Yasoda et al. 2018). Also, it is reported that V. jatamansi plants are capable to respond to high light intensity through morphological adaptations of leaves like more trichome length, more number of stomata, less leaf wetness, higher contact angle and higher capacity to retain water droplets (Pandey and Nagar 2002).

Photosynthetic capacity and plant growth is largely determined by chlorophyll content of leaves (Li et al. 2018). In the present study, chlorophyll a and total chlorophyll content during all the seasons was significantly reduced in plants grown under full sunlight as compared to shade condition (Fig. 2a,c). Reduction in chlorophyll a content was 17% during winter, 20% during spring, 3% during summer and 1% during autumn season. Likewise, the reduction in total chlorophyll content was 20% in winter, 25% in spring, 5% in summer and 3% in autumn season. A decline in chlorophyll content might be due to pigment degradation or reduced synthesis under high light and temperature conditions (Incesu et al. 2014). Reduction in chlorophyll content after exposure to intense radiation was reported and it was suggested that this might be an adaptation strategy to prevent absorption of excessive energy by plant (Han et al. 2003). In this study, higher amount of carotenoid was found in plants grown under full sunlight condition (Fig. 2d). Carotenoids possess antioxidant potential and thus their increased concentration protects plants from oxidative damage (McElroy and Kopsell 2009).

Growing plants under shade nets provide suitable conditions for plant growth by decreasing temperature, high light intensity and by maintaining soil moisture (Hamdani et al. 2018). In our study, total plant biomass was several times higher in shade grown plants as compared to full sunlight (open field), in all four growing seasons, with highest (3.2 fold) increase during autumn season followed by summer season (2.2 fold) (Fig. 3). The findings are corroborated with Singh et al. (2010) who reported that dry matter accumulation in belowground parts of V. jatamansi was significantly higher in autumn (October) season. Similar to this study, shade nets of different colors were found to increase the vegetative growth of plants as compared to no shading (Zare et al. 2019).

A significantly higher level of total phenolics, flavonoids, tannin content and antioxidant activities (ABTS, DPPH, FRAP) was recorded under full sunlight condition compared to shade, summer season compared to other seasons and, in leaf part as compared to other parts of plant (Fig. 5). The results are in agreement with an earlier study where polyphenolics content and all three antioxidant assays were found significantly higher in the aboveground part than the belowground part of planted individuals of V. jatamansi (Bhatt et al. 2012). Similarly, total phenolics, tannins and antioxidant activity in first year of plant growth were found higher in aerial portion of plant (Jugran et al. 2015). Significant variation in total phenolics, flavonoids and antioxidant activity in aerial and root portions among 25 populations of V. jatamansi was also reported (Jugran et al. 2016). A six month study on antioxidant activity in essential oil of V. jatamansi revealed higher antioxidant activity during March as compared to January and May (Rawat et al. 2017). The higher phenolics in the aerial portion of V. jatamansi plants grown under full sunlight may be attributed to higher exposure of leaves to solar radiation and the accumulation of more phenolics help to prevent photo-inhibition caused by high radiation (Bhatt et al. 2012). Higher antioxidant activity during autumn and summer season might be due to high temperature and photon flux density during these seasons, which otherwise could cause accumulation of reactive oxygen species (ROS). Improved antioxidant potential helps to reduce the ROS mediated photo-oxidation and prevents oxidative damage under high light conditions (Bhandari and Sharma 2006). It is reported that growing season is responsible for variation in phenolic compounds of blueberry which might be due to difference in seasonal temperature and sunshine duration and, the concentration of phenolics was positively correlated with antioxidant activities (Dragović-Uzelac et al. 2010).

Out of the twelve phenolic compounds studied, concentration of chlorogenic acid, 3-hydroxybenzoic acid, p-coumaric acid, gallic acid, m-coumaric acid, caffeic acid and ferulic acid varied significantly among different season and light conditions (Table 1). Majority of these phenolics were higher under full sunlight condition compared to shade and, during autumn and summer season compared to other seasons (Table 3). Similar to this study, chlorogenic acid was reported to be the most abundant phenolic acid in the aerial part of Tamarix africana, a medicinal plant widely used in Algerian traditional medicine (Bechlaghem et al. 2019). Leaf had highest phenolics as compared to other plant parts. Similar to the present study, considerably higher amount of hydroxybenzoic acid, caffeic acid, chlorogenic acid, catechins, gallic acid, p-coumaric acid were observed in aerial portion of V. jatamansi than the root portion (Bhatt et al. 2012).

In this study, valerenic acid, the principle bioactive compound present in V. jatamansi, was found to be highest in leaf part during summer season and the concentration increased under shading (Fig. 7). High VA content in shade during summer season suggests that very high light intensity (PPFD) along with high temperature during summer in open field (full sunlight) reduced VA content, while shading provided suitable microenvironment which positively influenced VA production. Based on this study, summer season (August) could be suggested as optimum harvesting time since higher phytochemicals were present, especially in aerial part and this might also help to prevent the destructive harvesting of the species. High VA content during summer season can be an adaptive mechanism to protect plant against oxidative stress under high light and temperature conditions of summer.

The results are in accordance with Jugran et al. (2016) where significant variations in VA content in above and belowground plant parts were observed among different populations of V. jatamansi. Singh et al. (2010) reported higher amount of valepotriates, another major constituent of V. jatamansi, during autumn season. In another species i.e. V. officinalis, maximum content of VA and valepotriates was obtained in February and March (Bos et al. 1998). This suggests that appropriate harvesting time depends on plant species, location and the compound which is more desired. The prevailing environmental conditions, mainly temperature, light intensity and relative humidity are crucial for determining the amount of phytoactive compounds at harvesting time and should be considered when planning optimal harvesting time for a specific area.

Further, correlation analysis revealed a significant correlation among plant growth parameters and antioxidant compounds/activities under different light conditions (Supplementary Tables S2 and S3). Under shade condition, a significant positive correlation was found among plant growth parameters (leaf area, leaf number, and biomass), phytochemical content and antioxidant activities. This suggests that under optimum light conditions (shade), high plant growth supports more carbon gain and thus higher production of secondary metabolites. Leaf number and leaf area increases the light interception capacity resulting in higher carbon assimilation, which can be utilized in biomass accumulation (Irving 2015; Weraduwage et al. 2015) and production of phytochemical compounds. However, under exposure to stressful conditions, plants accumulate secondary metabolites by compensating plant growth (Cipollini et al. 2018; Isah 2019). In this study, under full sunlight condition, less significant correlations among growth parameters and antioxidant compounds/activities were found. This might be due to differential response of plants to balance plant growth and defence response under high light stress, which reduced plant growth/biomass and increased production of antioxidant compounds to prevent oxidative damage under high light intensity. Similar to this study, a significant positive relationship between the total phenolics, flavonoids and tannins content with the ABTS and DPPH radical scavenging capacity and FRAP antioxidant capacity of plants was reported (Katalinic et al. 2006; Surveswaran et al. 2007; Dudonne et al. 2009; Addai et al. 2013; Bechlaghem et al. 2019). Medicinal plants with higher amount of phenolic compounds and higher radical scavenging activity are promising source of natural antioxidants which are considered safe and efficacious for treating various human ailments.

Conclusion

The current study concludes that season and light intensity significantly affect the growth and phytochemical content of V. jatamansi. Plant growth parameters along with the principle bioactive compound (valerenic acid) increased under shade conditions, indicating shade loving nature of species. However, various antioxidant compounds/activities and phenolic compounds were higher under full sunlight in open field, indicating that the species is capable to survive high photon load by inducing metabolic changes. These antioxidant phytochemicals play important role in mitigation of oxidative damage and adaptation of plants during fluctuating environmental conditions. Higher amount of various phenolics as well as terpenoid (valerenic acid) compounds in the leaf portion of plant demonstrate great commercial implications since this can subsequently reduce the destructive harvesting of root and rhizomes.

The present study recommends cultivation of V. jatamansi under shade net and it’s harvesting during summer season as the plant biomass along with antioxidant activity and phytochemical content including principle bioactive compound was highest. Seed maturation and dispersal of V. jatamansi is also completed before summer, thereby facilitating regeneration and sustainable utilization of this species. Further, systematic study on monthly fluctuation of these phytochemicals, especially therapeutically important bioactive compounds, on different locations should be carried out to get clear insight into major environmental factors and signalling compounds affecting their synthesis.