Source dependent variation in phenolics, antioxidant and antimicrobial activity of Paeonia emodi in west Himalaya, India

Abstract

Paeonia emodi is one of the ethno therapeutically important Himalayan plants used to cure various diseases. However, a systematic investigation of the effect of altitude on phytochemical, antioxidant, and antimicrobial activity has not been reported so far. The present study assessed the variation in the bioactive compounds, antioxidant and antimicrobial activity of the leaf, and rhizome of P. emodi collected from different altitudes. Phytochemicals such as phenols, flavonoids, flavanol, tannins, emodin, and paeoniflorin were found in all the sampled populations, but the quantity varied significantly across the altitude. In leaf, phenolics, flavonoids, and tannins content positively correlated with altitude (p < 0.01), but flavanol did not show any connection. Similarly, in the rhizome, positive relation with altitude (p < 0.01) was observed in phenol, flavonoids, and paeoniflorin. Antioxidant activity measured by 1, 1-diphenyl- 2 picrylhydrazyl (DPPH) and nitric oxide assays showed a positive correlation (p < 0.05) with altitude. 2, 2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid), ferric reducing antioxidant power, and hydroxyl ion assays did not show any relation with altitude. Antimicrobial activity was higher in the case of rhizome for the minimum inhibitory concentration and positively correlated with phenolics, flavonoids, and flavanol (p < 0.05). The present study further revealed that the secondary metabolites in the leaf and rhizome extracts of P. emodi are an excellent source of antioxidant and antimicrobial activity, thus validating the species' therapeutic potential.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-022-01242-z.

Introduction

The trends of using natural products are growing rapidly, considering their lesser side effect and cost-effectiveness. Plant extracts are being tested for new drug discoveries as they are reported to be a source of various phytochemicals, natural antioxidants, and antimicrobials (Ouerghemmi et al. 2017; Khameneh et al. 2019). The growing concern of microbial contamination and side effects of synthetic antioxidants (Singh et al. 2017) has further accelerated the use of natural sources of antioxidants and antimicrobials. In this context, medicinal plants are viewed as potential agents for combating such problems (Senguttuvan et al. 2014; Awika and Rooney 2004).

Plants have been used for thousands of years in different systems of medicine like Ayurveda, Siddha, and Homeopathy. It has been reported that one fourth of all commercially available medicines are directly or indirectly obtained from plants, which necessitate studying the impact of environmental factors, i.e., abiotic stresses (light incidence, temperature, humidity, water and nutrients availability, heavy metals) and biotic stresses (bacteria, virus, fungi, parasites, insects) on plant secondary metabolites and related medicinal properties (Verma and Shukla 2015; Jugran et al. 2016a; Nebehaj et al. 2017).

It has been reported that a change in an individual factor may alter the content of secondary metabolites (SMs) even if other factors remain constant (Yang et al. 2018). An individual factor can generally interact with other factors. For instance, high irradiation indicates increased temperature and water deficiency. It influences the production of different groups of secondary metabolites (phenolics, flavonoids, terpenoids, alkaloids, etc.) by regulating the growth and development of plants (Verma and Shukla 2015). Nutrient availability stimulates plant growth and influences the content of SMs. Both biotic and abiotic stresses may trigger oxidative stress due to the high production of reactive oxygen species (ROS) and modify plant metabolism. Thus, plants generate many SMs to detoxify ROS (Yang et al. 2018).

Paeonia emodi Wallich ex Royle (Paeoniaceae), commonly known as 'Chandra' or 'Dhandru', is a near-endemic and vulnerable to Indian Himalayan Region (IHR) (Dhar and Samant 1993; Samant et al. 1998; Ahmad et al. 2018). The species is widely distributed in north Pakistan, North West India, west Nepal, and China (De-yuan 2004). In the IHR, the genus represents only one species Paeonia emodi; which is found from Kashmir to Uttarakhand at an altitude of 1800 to 3000 m asl (Joshi et al. 2018). In West Himalaya (Uttarakhand), P. emodi is found in places such as Triyuginarayan, Pootiwasa, Randhaar, Gwaldum, Talwar, and Chamoli area of Garhwal Himalaya (Verma et al. 2015). The species is known for its various ethnomedicinal uses (Ahmad et al. 2018). For instance, the leaf is used for atopic eczema, cardiovascular disease, diabetes, anticoagulant, anti-inflammatory, analgesic, and sedative purposes. Tea is prepared from dried petals of peony for antitussive purposes. The species is also used as an anticoagulant in treating vomiting, cholera, tuberculosis, and eye diseases (Bibi et al. 2017). Nutrition and antioxidant properties of the species are also reported (Ahmad et al. 2018; Jugran et al. 2016b).

Environmental conditions are known to influence the secondary metabolites in plants. For instance, altitude indicates a decrease or increase in temperature, radiation, atmospheric pressure, and other environmental processes (Campbell et al. 2019). Altitudinal variations also lead to variation in environmental factors such as soil nutrients and precipitation, which directly or indirectly affects the secondary metabolite content of the plants (Suyal et al. 2019). Therefore, a study on altitude facilitates the identification of conducive geographical locations for the cultivation of this threatened medicinal plant. Despite the high demand and popularity of P. emodi, no report on the effect of altitude on phytochemical, antioxidant, and antimicrobial activity is reported. This is important because the species grows at different altitudinal ranges and under wide habitat conditions. Therefore, the present study aims to study the effect of various environmental factors on phytochemicals, antioxidants, and antimicrobial properties of P. emodi in the western Himalayan region.

Materials and methods

Study site and sample collection

Plant leaf and rhizomes (five-leaf and rhizomes from five different plants) were collected in March 2018 from 4 locations viz., Triyuginarayan, Pootiwasa, Randhaar, and Gwaldum, Uttarakhand (western Himalaya), India, representing different habitats and altitudes. The plant material was identified from the Botanical Survey of India (BSI), Dehradun, Uttarakhand, with the accession number of BSI/NRC-Tech/Herb/Ident./2017–18/plant accession no. 118052. Details of habitats and meteorological data are given in Table 1. All the plant samples were put into air-tight bags and brought to the laboratory. Samples were air dried at room temperature (25ºC ± 1ºC) and grounded into a fine powder using a mixture grinder. Samples were stored at 4ºC for further analysis of polyphenols, antioxidants, and antimicrobial study.

Table 1.

Description of study sites with geographical location, habitat and associated plants

Code	Location	Longitude	Latitude	Altitude (m asl)	Habitat	Avg. annual air temp (°C)	Annual rainfall (mm)	Avg. annual UV wave (W/sq.m)
S1	Triyuginarayan	78° 59′ 25″ E°	30° 38′ 40″N°	2350	Sloppy moist with mixed forest	4.62	1905.62	274.21
S2	Pootiwasa	79° 09′ 50″ E°	30° 30′ 06″ N°	2200	Rocky, sloppy moist	10.95	1272.63	240.61
S3	Randhaar	78° 53′ 28″ E°	30° 27′ 52″ N°	2050	Sloppy moist with mixed forest	14.34	2190.80	234.14
S4	Gwaldum	79° 31′ 04″ E°	30° 01′ 35″ N°	1827	Rocky and Sloppy with mixed forest	16.12	1427.82	227.61

m asl = meter above sea level

S1 = Triyuginarayan, S2 = Pootiwasa, S3 = Randhaar, S4 = Gwaldum

Extract preparation

Leaf and rhizome 2 g of P. emodi extracted using methanol in a ratio of 1:5 (dry powder: solvents) and kept in a conical flask sealed with parafilm tape. Crude samples were macerated using a rotary shaker (Remi) at 160 rpm for 24 h until they became colorless. Filtrates were dried under ambient conditions, weighted (Fig. 1), diluted using 15 mL methanol, and stored at 4 °C for further analysis.

Fig. 1.

Extract yield (%) of P. emodi plant part at different altitude. S1 = Triyuginarayan, S2 = Pootiwasa, S3 = Randhaar, S4 = Gwaldum. Results are mean (n = 3) ± SD

Phytochemical estimation of P. emodi leaf and rhizome

Leaf and rhizome extracts of P. emodi were used to estimate total phenolics, flavonols, flavonoids, and tannins by standard methods (Rawat et al. 2011; Deleu et al. 2000; Kumaran and Karunakaran 2007; Bhatt et al. 2013).

Antioxidant potential of leaf and rhizome extract

The antioxidant activities were determined using ABTS (2, 2-azinobis 3-ethylbenzothiazoline-6-sulphonic acid) (Cai et al. 2004), DPPH (1, 1-diphenyl- 2 picrylhydrazyl) (Amarowicz et al. 2000), FRAP (ferric reducing antioxidant power) (Benzie and Strain 1996), NO (nitric oxide) (Garrat et al. 1964) and HO (hydroxyl ion) scavenging activity (Shimada et al. 1992). All the quantitative estimations were carried out using a UV–VIS spectrophotometer (Amersham Biosciences, Sweden) at the respective wavelengths of the target compounds. Results were expressed in ascorbic acid and Trolox milligram equivalent/gram dry weight (mg/g DW) of the sample. Radical scavenging (%) of antioxidant activities from different assays was calculated using the following equation.

$$\%Inhibition=\frac{A_{\mathit{Control}}-A_{\mathit{Sample}}}{A_{\mathit{control}}}\times 100$$

A _control = absorbance of control.

A _sample = absorbance of the extract.

The half-maximal inhibitory concentration IC₅₀ (µg/mL) value and relation (radical scavenging rate and concentration of extract) was analysed using linear (y = mx + n) or parabolic (y = ax² + bx + c) equation.

ABTS assay

Total antioxidant activity was analyzed using the ABTS method with minor modification (Cai et al. 2004). ABTS salt (7.0 µM) and potassium per sulphate (2.45 µM) were added to produce ABTS cation (ABTS⁺) and allowed to stand in the dark for 16 h at 20 °C. The ABTS⁺ solution was diluted using 80% (v/v) ethanol to obtain an absorbance of 0.70 (± 0.05) at 734 nm. A 3.90 mL diluted ABTS⁺ solution was added to 0.10 µl of extract, and the resulting mixture was mixed thoroughly and kept for 6 min in the dark at 20 °C. Absorbance was recorded at 734 nm for a blank prepared with 0.1 mL 80% (v/v) methanol. A standard curve of different ascorbic acid concentrations was prepared in 80% (v/v) methanol for the ascorbic acid equivalent quantification of antioxidant potential. Results were expressed in milligram ascorbic acid equivalent (AAE) per gram of extract dry weight (mg/g DW).

DPPH assay

The free radical-scavenging potential of crude extracts was determined using a DPPH assay (Amarowicz et al. 2000). A 0.50 µL aliquot of the extract solution was mixed thoroughly with 2.5 mL of 0.3 mM DPPH prepared in methanol for ~ 1 min and then allowed to stand at room temperature for 20 min. Absorbance was measured at 517 nm. Quantifying radical activity was determined based on a standard curve of ascorbic acid prepared in 80% methanol (v/v). Results were expressed in milligram ascorbic acid equivalent (AAE) per gram of extract dry weight (mg/g DW).

FRAP assay

The ferric reducing potential of the extracts was determined using FRAP solution (Benzie and Strain 1996). A 0.10 µL extract was mixed with 1.8 mL of TPTZ solution (acetate buffer: 10 mM/L TPTZ: 20 mM/L FeCl₃ in the ratio of 10:1:1). The mixture was kept at 35 °C for 10 min. The absorbance of the resultant mixture was measured at 593 nm. Results were expressed in milligram ascorbic acid equivalent (AAE) per gram of extract dry weight (mg/g DW).

Scavenging activity of nitric oxide (NO)

Nitric oxide was generated from sodium nitroprusside (Garrat et al. 1964) and 0.80 mL of extract was diluted using 0.50 µL of sodium nitroprusside followed by incubation for 150 min under a light source (24-W compact fluorescent light). After incubation, 0.60 µL of a mixture from the stock was transferred in tubes containing 0.60 µL Griess reagent, then again incubated for 10 min in the dark. Absorbance was taken at 546 nm. Control experiments were performed with an equal amount of distilled water instead of extract solution. Results were expressed in milligram ascorbic acid equivalent (AAE) per gram of extract dry weight (mg/g DW).

Scavenging activity of hydroxyl ion (HO)

Deoxyribose assay was used to determine the hydroxyl radical scavenging activity in an aqueous medium (Shimada et al. 1992). A 0.20 µL of extract was mixed with Fenton reaction mixture containing (0.2 mL of FeSO₄ (10 mM), 0.2 mL of EDTA (10 mM), 0.2 mL of 2-deoxy- D-ribose (10 mM), 0.2 mL of 10 mM H₂O₂ and 1.2 mL of phosphate buffer 0.1 M (7.4 pH). The mixture was incubated for 4 h at 37ºC and then heated at 95ºC in the water bath for 10 min, followed by adding 1 mL each of 2.8% of trichloroacetic acid (TCA) and thiobarbituric acid (TBA). Finally, the reaction mixture was cooled in an ice bath and centrifuged at 5000 rpm for 15 min. The absorbance of the supernatant was measured at 532 nm. Fenton reaction mixture and water were taken as a blank. Results were expressed in milligram ascorbic acid equivalent (AAE) per gram of extract dry weight (mg/g DW).

Physico-chemical properties of soil

To estimate physicochemical parameters like pH, total moisture, total carbon, total nitrogen, and total phosphorus were tested using standard methods (Walkley and Black 1934; Parkinson and Allen 1975).

Antimicrobial activity

The test microorganisms, the microbial culture collection established in the microbiology lab of the institute (GBPNIHE, Almora, Uttarakhand) was used. The microbial cultures were represented by Gram-positive and Gram-negative bacteria and fungi. The bacterial cultures were maintained in tryptone yeast extract (TYE) agar slants at 4 °C and glycerol stock at -20 °C. In contrast, the fungal cultures were maintained at potato dextrose (PD) agar slants at 4 °C. Leaf and rhizome extracts were individually tested against one Gram-positive bacteria (Bacillus subtilis- NRRLB-30408), one Gram-negative bacteria (Escherichia coli- GBPI_125), and two fungi (Aspergillus niger- ITCC2546, Fusarium oxysporum- ITCC4219). Antimicrobial activities of extracts were tested following the disk diffusion method, and the minimum inhibitory concentration (MIC) of needle extracts was determined (Adhikari et al. 2018). Streptomycin and Chloramphenicol were used as positive control.

HPLC analysis of active constituents

Active metabolite gallic acid, tannic acid, quercetin, ascorbic acid, emodin, and paeoniflorin were quantified in the P. emodi leaf and rhizome extracts and analyzed using reverse-phase High-Performance Liquid Chromatography (RP-HPLC) (Alliance Waters e2695, Waters, Milford, USA) equipped with photodiode array detector (PDA, Waters 2998) detector and SPHERISORB C18 reverse phase column (5 µm particle size, 4.6 × 250 mm i.d.).

Gallic acid, tannic acid, quercetin, and ascorbic acid were measured at 275 nm (wavelength). Briefly, a 20 µL sample was injected and run with a mobile phase of methanol: 1% glacial acetic acid in mili Q water (75:25 v/v) with a flow rate of 1 mL/min for 10 min. Emodin quantification was performed at 254 nm with methanol: Mili Q water (70:30 v/v) containing 0.5% acetic acid with the flow rate of 0.8/mL for 8 min; however, in the case of paeoniflorin mobile phase of methanol: acetonitrile: Mili Q water (10:10:80 v/v/v) containing 0.4% formic acid were used and quantification was done at 230 nm with flow rate of 1 mL/min for 8 min. Results for each compound were expressed in mg/g DW of P. emodi leaf and rhizome. Experiments were carried out in triplicate.

GC–MS analysis of P. emodi

P. emodi leaf and rhizome collected from Triyuginarayan were analyzed for volatile secondary metabolite using GC/MS-QP 2010 ultra (Shimadzu, Japan). Helium was used as a carrier gas, and the parent ions' electron impact (EI) mass spectrum was recorded. The initial temperature of the column was kept at 50 °C for 2 min, then held for 3 min at 250 °C and finally at 280 °C for 18 min. Helium was used as carrier gas with a flow rate of 1.21 mL/min. Mass range setting was considered 50–1000 Da. The chemical constituents were identified using NIST and Willey library spectra provided by the software of the GC/MS system.

Statistical analysis

Estimating phytochemical compounds (total phenolics, tannins, flavonols, and flavonoids), antimicrobial and antioxidant activities by 2, 2- azinobis 3-ethylbenzothiazoline-6-sulphonic acid, 1,1-diphenyl- 2 picrylhydrazyl, ferric reducing antioxidant power assays were conducted in triplicates. The data expressed as the means ± standard errors (SE) from experiments performed in triplicate. Statistical significance and mean between groups were tested using Student's t-test and two-way ANOVA. The p-value < 0.05 was considered significant. Homogenizing grouping for all the values was done separately using DMRT (Duncan multiple range test) in SPSS version 20. Pearsons's correlation was calculated among different parameters used in the present study using R software (R-3.4.1).

Results and discussion

Altitude is an overall reflection of multiple ecological factors, such as temperature, humidity, and solar radiation, which vary across altitudes (Liu et al. 2016). The results of the present study demonstrated that the samples (leaf and rhizome) of P. emodi collected from varying altitudes (1700–2250 m above sea level) under different forest types represented dominant tree species like Alnus nepalensis, Quercus semicarpifolia, Q. floribunda and Aesculus indica showed variation in active metabolites, antimicrobial and antioxidant activities. The details of the results have described in the following sections:

Phytochemical estimation through spectrophotometric methods

The highest phenolics (108.94 ± 0.82 mg GAE /g DW, 59.75 ± 1.21 mg GAE /g DW) and flavonoids (89.94 ± 1.10 mg QE/g DW, 37.47 ± 0.92 mg QE/g dw) content were obtained in leaf and rhizome, respectively of P. emodi from S1 site (Triyuginarayan), followed by S2, S3, and S4 sites. Total flavanol (16.84 ± 0.57 mg QE /g DW) and tannin content (36.38 ± 0.86 mg TAE /g DW) of the leaf were higher in the S2 site (Pootiwasa), followed by S1, S4, and S3, respectively. Flavanol (41.89 ± 1.57 mg QE/g dw) and tannin (39.59 ± 0.85 mg TAE/g dw) content of rhizome was higher in S1 and S3 sites, respectively (Fig. 2). Furthermore, total phenolics and total flavonoid content of P. emodi leaf leaf (r = 0.963; r = p < 0.977 respectively) and rhizome (r = p < 0.961; r = 0.871 respectively) were significantly correlated with altitude. Flavanol and tannin content did not show any trend with the altitude, and no significant difference was observed among the studied sites.

Fig. 2.

Phytochemical constituent of P. emodi. S1 = Triyuginarayan, S2 = Pootiwasa, S3 = Randhaar, S4 = Gwaldum, phenol = gallic acid equivalent, flavanol and flavonoid = quercetin equivalent, tannin = tannic acid equivalent. Results are mean (n = 3) ± SD. Homogenize grouping were done using Duncan test, alphabet a-e denotes their significance (p ≤  0.05)

This type of relationship was also observed elsewhere. For instance, the total phenols, proline, and proteins were found to increase with the increase in altitude of desert plants (Sharaf et al. 2013). It might be because plants at higher altitudes face higher UV-B radiations, which have pleiotropic effects on plant development, morphology, and physiology; the most effective protection mechanism stimulated under this light regime is the biosynthesis of flavonoids and phenols (Frohnmeyer and Staiger 2003; Ruhland et al. 2007). This could be the reason of higher phenol and flavonoid content in P. emodi plants at higher elevation as compared to lower elevation; however, same trends were reported from some other Himalayan plants e.g., Coleus forskohlii (Rana et al. 2020), Hedychium spicatum (Rawat et al. 2011), Gaultheria trichophylla (Bahukhandi et al. 2017), Thalictrum foliolosum (Pandey et al. 2018) and Valeriana jatamansi (Jugran et al. 2013).

While comparing between rhizome and leaf of Paeonia, the total phenol and flavonoid contents were considerably higher in leaf as compared to rhizome (Fig. 2). Similar pattern was also recorded earlier in two species of Paeoniaceae viz., Paeonia arietina and P. kesrounansis (Sut et al. 2019) where the aerial parts showed maximum TPC (total phenol content) and TFC (total flavonoid content) than the rhizome extracts. This may be due to the use of solvent and extraction procedure, as they used all non-polar solvents, i.e., ethyl acetate, hexane, and dichloromethane. However, methanolic extracts (polar solvent) were used in the present study.

Antioxidant activity

Different methods have been used for analyzing the antioxidant activity of P. emodi. Antioxidant activity depends on various factors such as the type of radicals used, sample matrix, solvent composition, pH, and sensitivity of the compounds present in the target samples (Nebehaj et al. 2017). In the present study, five assays (ABTS, FRAP, DPPH, NO, and HO) have been used to estimate the antioxidant activity of the leaf and rhizome extract of P. emodi (Table 2); after that, leaf and rhizome samples were further evaluated for their radical scavenging rate (%) and 50% inhibitory concentration (IC₅₀) (Table 3).

Table 2.

Effect of altitude on antioxidant activity of P. emodi

Antioxidant activity [ascorbic acid mg/g (DW)]
Study site	Plant parts	ABTS	DPP	FRAP	NO	HO
S1	Leaf	19.38 ± 1.05d	28.15 ± 2.04a	08.07 ± 0.98d	29.75 ± 2.15d	29.02 ± 1.67c
	Rhizome	49.45 ± 2.65a	20.38 ± 2.65b	11.51 ± 0.98bc	44.19 ± 3.15a	07.32 ± 0.68f
S2	Leaf	37.31 ± 1.32c	22.25 ± 2.65b	09.01 ± 0.57d	22.35 ± 1.75e	25.53 ± 1.29d
	Rhizome	36.68 ± 2.54c	21.38 ± 1.98b	16.84 ± 0.68a	35.85 ± 2.09c	05.69 ± 1.98f
S3	Leaf	35.34 ± 3.42c	19.29 ± 2.25b	11.22 ± 1.09bc	18.65 ± 0.98f	39.22 ± 3.68a
	Rhizome	43.01 ± 4.21b	12.68 ± 1.35d	13.27 ± 1.11b	42.39 ± 3.18ab	06.42 ± 2.37f
S4	Leaf	36.45 ± 2.23c	17.78 ± 2.54bc	13.19 ± 1.35b	16.89 ± 0.98f	32.27 ± 5.98 b
	Rhizome	38.10 ± 2.43c	06.66 ± 0.35e	06.79 ± 1.65de	11.85 ± 0.85 g	09.52 ± 0.68e

Results are mean (n = 3) ± SE. Homogenize grouping was done (each antioxidant activity separately) using Duncan test, alphabet a-g denote their significance (p ≤ 0.05). S1 = Triyuginarayan, S2 = Pootiwasa, S3 = Randhaar, S4 = Gwaldum

Table 3.

IC₅₀ value of P. emodi leaf and rhizome

Study site	Plant parts	IC 50 (µg/mL)
		ABTS	DPPH	FRAP	NO	HO
S1	Leaf	476.43 ± 4.25b	447.87 ± 7.24bc	505.24 ± 7.21bc	474.98 ± 5.98 b	324.98 ± 8.21d
	Rhizome	316.98 ± 4.87d	387.67 ± 4.87d	476.56 ± 5.27d	225.98 ± 4.00f	298.56 ± 4.25e
S2	Leaf	596.13 ± 2.54a	498.78 ± 6.24b	511.23 ± 4.28bc	587.37 ± 7.25a	387.89 ± 5.24bc
	Rhizome	247.98 ± 2.87e	467.78 ± 4.55bc	412.34 ± 3.54de	297.78 ± 4.09e	265.98 ± 2.54f
S3	Leaf	485.54 ± 5.24b	507.56 ± 8.14b	545.87 ± 4.78ab	434.76 ± 4.21c	434.76 ± 6.24a
	Rhizome	393.98 ± 4.98c	498.45 ± 6.92b	576.87 ± 3.48a	307.34 ± 4.32e	296.34 ± 6.21e
S4	Leaf	585.45 ± 5.87a	576.37 ± 4.35a	526.65 ± 8.21ab	576.67 ± 3.78a	413.76 ± 5.24ab
	Rhizome	309.98 ± 4.35d	598.98 ± 4.47a	587.34 ± 4.87a	398.45 ± 3.87d	287.45 ± 3.75e

IC ₅₀ = inhibitory concentration at which the inhibition percentage reaches 50%

S1 = Triyuginarayan, S2 = Pootiwasa, S3 = Randhaar, S4 = Gwaldum, Results are mean (n = 3) ± SE, homogenize grouping were done (eachbioassay separately) using Duncan test, alphabet a-f denote their significance (p ≤ 0.05)

Results of NO and DPPH assay indicated that radical scavenging activity of extracts increased significantly (p < 0.05) with an increase in altitude (Table 3), and similar patterns was observed in Hedychium spicatum and Ginkgo biloba (Rawat et al. 2011; Sati et al. 2013). It has been reported that the plant's habitat can affect the arrangement of secondary active substances in the plant (Zargoosh et al. 2019). Relative humidity and temperature of different sites may affect the increase in antioxidant activity. The reduced temperature and increased exposure to UV radiation at high altitudes on different plant parts are also previously reported for synthesizing antioxidant compounds, thereby inducing variation in antioxidant activity (Zargoosh et al. 2019).

Plant secondary metabolites act as antioxidants and reducing agents by the hydrogen-donating property of their hydroxyl groups. Results of the ABTS and DPPH assays for plant extracts were highly correlated (p < 0.001) (Table 1). This is inconsistent with earlier studies where ABTS and DPPH assay share a similar mechanistic basis, viz., transfer of electrons from the antioxidant to reduce an oxidant (Huang et al. 2005). A similar pattern was reported earlier for ABTS and DPPH assays in P. emodi aerial parts (p < 0.05) (Jugran et al. 2016b) and in Meconopsis aculeate rhizomes (p < 0.05) (Bahukhandi et al. 2018).

In leaf extract, the antioxidant activity was obtained higher in HO scavenging assay followed by NO, ABTS, FRAP and DPPH; however, in rhizome extract, the pattern was slightly different with higher ABTS scavenging assay followed by NO, FRAP, HO and DPPH (Table 2). Genetic changes, environmental conditions, and physiological factors can significantly affect the bio-accessibility and bio-availability of phytochemicals (Li et al. 2012). A similar finding has also been reported for the species Larrea tridentate (Skouta et al. 2018). On the contrary, Meconopsis aculeate demonstrated a different pattern with higher activity in ABTS assay followed by DPPH, FRAP, OH, and NO ion assay (Bahukhandi et al. 2018). Present results indicate that high NO, FRAP, and ABTS activities were present in P. emodi plant parts (leaf and rhizome). Similar results were also recorded from the study conducted in aerial and root portions of Valeriana jatamansi and rhizomes of Polygonatum verticilatum collected from different altitudinal ranges; however, maximum activity was obtained in ABTS assay (Jagran et al. 2016a; Suyal et al. 2019).

The concentration of the extracted sample at which the inhibition percentage was reached 50% known as its IC₅₀ value. The IC₅₀ value is negatively related to antioxidant activity as it expresses the amount required to decrease its radical concentration by 50%; the lower the IC₅₀ value, the higher the antioxidant activity (Chanda et al. 2011). The effect of altitude on IC₅₀ value of this plant was negatively correlated as the altitude increased, the IC₅₀ decreased and the antioxidant property increased. Value determined from plant extracts (leaf and rhizome) of the different sites are given in Table 3. Similar results were reported from various medicinal plants, i.e., Peltophorum ferrugineum, Celastrus peniculatus, Ocimum gratissimum, and Larrea tridentate (Chanda et al. 2011; Skouta et al. 2018). Lower IC₅₀ values were observed with better antioxidant potential in rhizome extracts compared to leaf extract of P. emodi.

Significant relationships were observed between the different antioxidant methods for leaf and rhizome extracts. This may be attributed to the fact that tested antioxidant assays are significantly related and follow similar mechanisms (electron transfer-based assays such as DPPH, ABTS, and FRAP and hydrogen atom transfer-based assays such as HO and NO) (Miguel 2010). Hence, the significantly related methods could be equally useful for assessing the antioxidant activities of P. emodi. Although these methods are significantly correlated, the responses (in terms of antioxidant equivalent activity) are not the same, so the use of different assays can increase the overall assessment of the antioxidant capacity of the plant extracts (Tachakittirungrod et al. 2007). Such information is useful to determine compounds and types of compounds responsible for the antioxidant potential of the plant extracts. Therefore, the total polyphenol contents of the extracts were used and compared with their respective antioxidant capacity values.

Physico-chemical properties of soil

Results of soil analysis indicated that maximum pH was recorded from the S3 site (6.18 ± 0.12), followed by S1 (5.82 ± 0.19) and S2 (5.47 ± 0.13). However, maximum moisture content was present in the S2 site (23.19 ± 0.60%), followed by S1 (22.88 ± 0.50%) and S4 (20.32 ± 0.30%). Soil minerals like total nitrogen (1.81 ± 0.60%) and total phosphorus (0.107 ± 0.08%) were higher in the S1 site, while the S2 site had the maximum amount of organic carbon (4.88 ± 0.22%) (Table S1).

Antimicrobial potential

Antimicrobial activities of the species were tested against two groups of microorganisms, bacteria (Gram-positive and negative) and fungi (Table 4; Fig. 3). Results showed that altitude does not affect antimicrobial activity; it varied among the tested microorganisms. The inhibition pattern was different for different tested microorganisms, with particular reference to the altitude. In leaf antibacterial activity was higher in the S1 site against Bacillus subtilis (12.00 ± 0.10 mm), followed by S4 (11.00 ± 0.06 mm), S3 and S2. For Escherichia coli zone of inhibition was higher in S1 (14.29 ± 0.16 mm), followed by S3 (10.25 ± 0.56 mm) and S2 (10.03 ± 0.19 mm), respectively. In the rhizome, the zone of inhibition did not show any difference between altitudes for both bacteria (Table 4; Fig. 3).

Table 4.

Effect of altitude on antimicrobial activity of P. emodi leaf and rhizome

Microorganism		Zone of inhibition (mm)								Positive control (antibiotic)
		S1		S2		S3		S4
		Leaf	Rhizome	Leaf	Rhizome	Leaf	Rhizome	Leaf	Rhizome	Streptomycin	Chloramphenicol
Bacteria	B. subtilis	12.00 ± 0.10a	5.21 ± 0.03d	5.49 ± 0.03d	5.33 ± 0.06d	7.27 ± 0.12c	4.00 ± 0.06e	11.00 ± 0.06b	4.31 ± 0.03e	14.25 ± 2.14	15.87 ± 0.03
	E. coli	14.29 ± 0.16a	2.67 ± 0.03e	10.03 ± 0.19b	3.33 ± 0.03d	10.25 ± 0.56b	3.33 ± 0.03d	6.26 ± 0.12c	2.54 ± 0.06e	18.19 ± 1.28	18.24 ± 0.06
Fungi	F. oxysporum	3.00 ± 0.12d	6.33 ± 0.12b	2.93 ± 0.09d	NA	9.56 ± 0.15 a	5.97 ± 0.19c	6.67 ± 0.12c	NA	9.18 ± 0.98	8.98 ± 0.03
	A. niger	7.62 ± 0.12d	12.69 ± 0.09a	NA	NA	10.61 ± 0.09b	8.00 ± 0.06c	6.52 ± 0.09e	NA	8.37 ± 1.24	8.54 ± 0.98

Microorganism		Minimum Inhibitory Concentration (µg/mL)
Bacteria	B. subtilis	300	700	500	650	400	650	400	650	100	100
	E. coli	150	650	100	600	250	600	350	600	100	100
Fungi	F. oxysporum	550	550	500	NA	450	650	550	NA	200	200
	A. niger	500	600	NA	NA	400	650	500	NA	200	200

NA = activity not detected. Results are mean (n = 3) ± SE. Homogenize grouping were done (each microorganism separately) using Duncan test, alphabet a-e denote their significance (p ≤ 0.05).S1 = Triyuginarayan, S2 = Pootiwasa, S3 = Randhaar, S4 = Gwaldum

Fig. 3.

Antibacterial and antifungal activity of P. emodi leaf and rhizome. LE = leaf extracts, RE = rhizome extract, 1 = E. coli, 2 = B. subtilis, 3 = A. niger, 4 = F. oxysporum, S1 = Triyuginarayan, S2 = Pootiwasa, S3= Randhaar, S4= Gwaldum

Antifungal activity was observed to be higher in rhizomes as compared to leaves. Leaf demonstrated antifungal activity in all the studied sites, but rhizome showed only in two sites (S1 and S3). Antifungal activity was not observed against Aspergillus niger in S2 and S4 sites, but the S1 and S3 sites showed high activity in the rhizome and leaf, respectively (Table 4). Quantitative estimation of antimicrobial activity using MIC (minimum inhibitory concentration) value revealed that Gram positive and Gram negative bacteria ranged from 300 to 700 and 150 to 600 µg/mL, respectively, and for fungi, 400 to 650 µg/mL (Table 4). As compared with the plant part, antimicrobial activity was higher in the rhizome.

Results revealed that altitude did not influence the antimicrobial activity of P. emodi leaf and rhizome extracts. However, a plant's antimicrobial properties depend on the kind of microorganism against the antimicrobial activity. Various conditions responsible for antimicrobial activity are microbial population size, composition, the effectiveness of an antimicrobial agent, the concentration of an antimicrobial agent, etc. (Redondo 2014). Antimicrobial activities do not influence by altitude; rather, it depends on the microorganism against which activity was tested. While plate assays are qualitative in nature, they reveal the presence or absence of antimicrobial activity. To understand the antimicrobial potential of any plant, the measurement of MIC is considered a relatively authentic parameter (Lambert and Pearson 2000).

HPLC analysis of active metabolite

HPLC analysis of leaf and rhizome bioactive compounds (emodin, paeoniflorin, gallic acid, tannic acid, quercetin, and ascorbic acid) showed that phenolic compounds viz. tannic acid (2.43 ± 0.35 min), gallic acid (2.95 ± 0.65 min), ascorbic acid (3.38 ± 0.29 min), and quercetin (4.80 ± 0.54 min) eluted in different retention time. Paeonia leaf and rhizome showed the presence of two marker compounds, emodin, and paeoniflorin. Emodin is a polyphenol (anthraquinone) present in the whole plant and reported for its strong anti-inflammatory, antioxidant, cardiovascular, antimicrobial and laxative property (Tzeng et al. 2012); however, paeoniflorin (monoterpene glycoside) is used as a gynecologic agent for infertility associated with PCOS, hyper pro-lactinemia, endometriosis (Ong et al. 2019). Emodin and paeoniflorin eluted at 3.72 ± 0.11 min and 3.07 ± 0.15 min, respectively (Fig. 4).

Fig. 4.

Antibacterial and antifungal activity of P. emodi leaf and rhizome. LE= leaf extracts, RE = rhizome extract, 1 = E. coli, 2 = B. subtilis, 3 = A. niger, 4 = F. oxysporum, S1 = Triyuginarayan, S2 = Pootiwasa, S3 = Randhaar, S4 = Gwaldum

HPLC analysis revealed that the gallic acid, ascorbic acid, and quercetin contents in P. emodi (leaf and rhizome) increase with the increase in elevation. On the contrary tannic acid, emodin, and paeoniflorin contents did not relate to altitude. Tannic acid, gallic acid, ascorbic acid, quercetin, and emodin were higher in the aerial part, but paeoniflorin was higher in the underground part of P. emodi (Table5).

Table 5.

HPLC data for active metabolite of P. emodi leaf and rhizome

	mg/g dw
Active metabolite	Plant parts	S1	S2	S3	S4
Ascorbic acid	Leaf	24.15 ± 0.23a	21.54 ± 0.53b	18.87 ± 1.11c	17.12 ± 0.93 cd
	Rhizome	21.15 ± 0.37b	23.83 ± 0.51a	18.43 ± 0.65c	13.55 ± 0.97e
Emodin	Leaf	13.66 ± 0.21f	23.43 ± 0.34c	16.39 ± 0.56e	9.81 ± 0.76 g
	Rhizome	32.11 ± 1.65b	45.27 ± 2.43a	31.02 ± 0.93b	21.74 ± 0.53d
Gallic acid	Leaf	59.75 ± 1.06a	55.18 ± 1.13b	37.65 ± 1.22d	30.53 ± 1.15e
	Rhizome	41.23 ± 2.32c	36.15 ± 1.32d	32.43 ± 1.43e	24.45 ± 0.32f
Paeoniflorin	Leaf	27.14 ± 1.23d	24.32 ± 1.23e	24.91 ± 1.23e	14.03 ± 1.23 g
	Rhizome	37.95 ± 0.65b	21.08 ± 0.76f	32.90 ± 0.43c	61.17 ± 0.61a
Quercetin	Leaf	25.23 ± 0.65b	22.23 ± 0.76c	28.43 ± 0.86a	20.65 ± 0.54d
	Rhizome	19.27 ± 0.67d	18.18 ± 0.84de	17.98 ± 0.67ef	13.65 ± 0.87 g
Tannic acid	Leaf	13.75 ± 0.54c	15.97 ± 0.57b	18.65 ± 0.94a	14.56 ± 1.05c
	Rhizome	11.34 ± 0.76d	15.06 ± 0.76b	13.43 ± 0.62c	14.43 ± 0.46c

Results are mean (n = 3) ± SE. Homogenize grouping was done (each secondary metabolite separately) using Duncan test, alphabet a-g denote their significance (p ≤ 0.05). S1 = Triyuginarayan, S2 = Pootiwasa, S3 = Randhaar, S4 = Gwaldum

Among different populations, tannic acid in P. emodi leaf was found higher in the S3 site (18.65 ± 0.94 mg TAE/g dw), followed by the S2 site (15.97 ± 0.57 mg TAE/g dw), S4 site (14.56 ± 1.05 mg TAE/g dw) and S1 site (13.75 ± 0.54 mg TAE/g dw), respectively. Rhizome samples showed a significantly (p < 0.05) higher amount of paeoniflorin (61.17 ± 0.61 mg/g DW) in the S4 site than S1 site (37.95 ± 0.65 mg/g DW), followed by the S3 and S2 site. The amount of paeoniflorin was higher in P. emodi compared to the dry powder of P. lactiflare (8.116 mg/g) (Zhou et al. 1998). Leaf extract of site S2 contained a significantly (p < 0.05) higher amount of emodin (23.43 ± 0.34 mg/g DW) than S3 (16.39 ± 0.56 mg/g DW), followed by S1 and S4 (Table5). Emodin content in P. emodi leaf and rhizome was higher compared to an earlier study reported from roots and rhizomes of Rheum emodi (2.19 mg/30 g) (Tabin et al. 2016).

GC–MS analysis

GC/MS analysis of rhizome (Supplementary Table S2; Fig. S1) and leaf (Supplementary Table S2; Fig. S2) extract showed a wide range of 29 major compounds. GC–MS analysis of methanol extract of leaf and rhizome of Triyuginarayan revealed a mixture of organic and aromatic compounds, including phenol, aldehyde, alkanes, and their derivatives with functional groups, carbonyls, and esters. GC–MS analysis of leaf and rhizome extract also showed presence of various active metabolites i.e., Acetophenone, benzoic acid 1-pentacene, 9,12-Octadecadienoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester, n-propyl 9,12-octadecadienoate, 3-Bromo-2-propynyl palmitate, propanoic acid, trilinolein, docosanoic acid, cyclooctasiloxane, benzene methanol, 9-octadecenamide, glycidyl palmitate, octadecanoic acid, hexadecanoic acid, 8-octadecanone, ethanone, benzaldehyde, acetophenone, pyrrolidine, Cholestane, 1-pentacene, cis-15-tetracosenoic acid, γ-sitosterol, docosanoic acid, salicyl alcohol, palmitic acid, benzene-carboxylic acid and glycidyloleate etc. Similar compounds have earlier been reported from the essential oil of P. suffruticosa flower (Yin et al. 2012), seed oil of P. ostii (Liu et al. 2020), and roots of P. emodi (Verma et al. 2015).

The identified compounds in the leaf and rhizome are reported to possess various biological activities. Constituents like hexadecanoic acid, 1,2-benzene dicarboxylic acid, tridecanedial, phthalic acid, and eicosanoic acid are the major compounds present in the methanolic fraction of P. emodi and responsible for antimicrobial activity. These compounds have also been detected in Streptomycin sp (Lin et al. 2005). These results demonstrated that the leaf and rhizome of P. emoji are a noble source of natural bioactive compounds and can be used to treat many diseases.

Correlation among phenolics, antioxidants, and antimicrobial activity

Pearson correlation was used to determine the correlation among environmental parameters, bioactive compounds, and biological activities. In leaf, phenol, flavonoids, tannin, DPPH, NO, phosphorus and carbon are positively correlated with short waves and altitude. Temperature showed a significant positive correlation with ABTS and FRAP while negatively correlated with all other parameters. Soil nutrients like nitrogen, phosphorus, and carbon are positively related to phytochemical activity measured by phenol, flavonoids, and tannin. Total phenol contents demonstrated a significant positive relationship with antioxidant activity measured by DPPH and NO and a negative correlation with ABTS, FRAP, and OH assay (Fig. 5). While in the rhizome, phosphorus, carbon, phenol, flavonoids, flavanol, and DPPH were demonstrated significant positive correlation with altitude and short waves. Soil nutrients like phosphorus showed a significant positive correlation with phenol, flavonoids, flavanol, and DPPH; however, nitrogen and carbon establish the same relationship with phenol and DPPH. Temperature establishes a significant negative correlation with phosphorus, carbon, phenol, flavanol, flavonoids, and DPPH, while no significant positive correlation was recorded (Fig. 5).

Fig. 5.

Pearson correlation matrix among environmental parameters, altitude, polyphenolic content, the antioxidant activity of P. emodi A) leaf and B) Rhizome. T = Temperature, N = Soil Nitrogen, P = Soil Phosphorus, C = Soil Carbon. Correlation is significant at 0.05 level, gray highlighted box = p0.05

Results from the present study indicated that high antioxidant activity is associated with a high phenolic and flavonoid content. Several species e.g., Hedychium spicatum, Valeriana jatamansi, and Polygonatum verticilatum, Taxus wallichiana (Rawat et al. 2011; Jugran et al. 2016a; Suyal et al. 2019; Adhikari et al. 2022) showed the presence of high phenolic and flavonoid content and therefore, exhibited higher antioxidant capacity, where total phenolic content, flavonoid content and antioxidant activity were positively correlated with each other. UV absorbing compounds such as flavonoids and phenolics are two leading group of antioxidants. Phytochemicals play a crucial role in the protection mechanism of plants by inhibiting the production of free radicals and scavenging reactive oxygen species, thus preventing oxidative damage and lipid peroxidation (Fusco et al. 2007). They can also donate electrons and have the capacity to stabilize radical intermediate. Phenolic compounds are known to act as antioxidants, and flavonoids contain ortho-dihydroxylated structure, which exhibits the characteristics of absorbing ultraviolet and scavenging free radicals (Huyut et al. 2017).

Correlation analysis demonstrated that emodin (anthraquinone) and paeoniflorin (monoterpene) content has no relation to the change in altitude. Terpene and anthraquinone are both used by the plant for their defense mechanism. The high pressure of parasites and herbivores at lower elevations may be one of the reasons for the high concentration of this metabolite (Forbey et al. 2009; Kant et al. 2015). In the plant part, i.e., leaf and rhizome, phenol, flavanol, and flavonoid content showed a significant positive correlation with antimicrobial activity (Fig. 5). Phenol and flavonoids showed a positive relation with antibacterial activity, and flavanol was responsible for the antifungal activity. It has been known that plants synthesize various bioactive compounds in plant tissues as secondary metabolites that have an antimicrobial activity to stop or inhibit the development of mycelia growth, substrate, and membrane disruption, inactivating the enzyme and inhibiting germination (Zhang et al. 2011). Phenol and flavonoid are reported for their antibacterial activity against both the bacteria i.e., gram-positive and gram-negative. The major mode of action phenolic compounds used in bacterial cell membrane damage, inhibition of virulence features, and destruction of bacterial biofilm development (Majdanik et al. 2018). Flavonoid compounds have different mechanisms to kill bacteria. A significant role has been played by the presence of phenolic hydroxyl group in flavonoids which increases the protein binding affinity due to which microbial enzyme production is inhibited and concurrently destruct the cell wall, and consequently cell death occurs (Liu et al. 2008). Flavanols kill the fungal species by supporting the breakdown of the plasma membrane and hindering cell wall development and protein synthesis (Aboody and Mickymaray 2020).

Conclusion

This is the first report that evaluates the effects of altitude on various phytochemical contents, antioxidants, and antimicrobial activity of P. emodi leaf and rhizome extract. This study concludes that synthesizing secondary metabolites in P. emodi leaf and rhizome depend on the prevailing edaphic and environmental conditions (temperature, rainfall, and precipitation). The leaf and rhizome of the plants are sources of antioxidants and antimicrobial agents that could be used for nutritional purposes (e.g., food additives, food preservatives), pharmaceuticals, healthcare products, and for medical applications. They should be tested in the future to determine their anti-diabetic, anti-proliferative, and anti-cancerous effects. The leaves can be utilized as a source of antioxidants and antimicrobials in food and healthcare products. This will help to promote its conservation as a non-destructive harvesting method.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution statement

KJ and PA carried out the experimental work, performed the data analysis, and written the draft. IDB and VP provide supervision for carried out the study, helped in data analysis, review and editing the manuscript. All the authors have read the final manuscript and agreed for submission.

Declarations

Conflict of interest

The authors do not have any conflict of interest.