Introduction

Changing environmental conditions (Fokum et al. 2017), living style (McKillop and Schrum 2005) and food habit (Shin et al. 2016; Singh et al. 2019) of people caused several health complications. Several phytoconstituents available in medicinal plants contribute major role in scavenging reactive oxygen species (ROS), which provides protection against damages caused by the xenobiotics. Several factors viz; genetical set up of the plant, physiological factors, environmental conditions, and geographical variations influence the phytochemical constituent of plant (Yosr et al. 2018). Seasonal variations have a major influence on the phytochemical composition of a species (Lopez-Lázaro 2009). Premna integrifolia L. commonly known as Agnimantha is a member of family Lamiaceae (Group 2009). Traditionally, it is used as anticoagulant (Gopal and Purushothaman 1984), cardioprotective (Bose et al. 2012), anti-inflammatory and anti-arthritic (Rajendran and Krishnakumar 2010), antidiabetic (Majumder et al. 2014), and anti-hyperlipidaemic (Patel and Patel 2012). The present study reports the comparative analysis of seasonal influence on polyphenolic constituent and antioxidant activity of different plant parts of P. integrifolia. Stems, roots and leaves of P. integrifolia were extracted in different solvents like ethanol, methanol, ethyl acetate and water in different season and subjected to analysis. Among different extracts, ethyl acetate extract of leaves was showing maximum antioxidant activity with high polyphenolic constituent. Therefore, this extract was characterized by UHPLC-Q-TOF-MS/MS and GC–MS analysis to know the active ingredient present in the extract. Previous reports validates the antioxidant property of P. integrifolia root (Gokani et al. 2011), stem bark (Majumder et al. 2014), wood (Muthukumaran et al. 2013; Rajendran et al. 2009) and leaves (Selvam et al. 2012), but there is no report of the comparative study of different plant parts influenced by season of collection for their total phenol, flavonoid content and antioxidant activity. In previous study, we reported the hepatoprotective activity of P. integrifolia (Singh et al. 2018) but we could not find the bioactive constituent responsible for hepatoprotective activity. Therefore, in present study we have performed in silico analysis of hepatoprotective property of major compounds of P. integrifolia identified by GC–MS and LC–MS as ligand by using an important liver enzyme alanine amino transaminase. This is the first report of the in-silico docking analysis in P. integrifolia for hepatoprotective biomarkers from UHPLC-Q-TOF–MS/MS and GC–MS identified compounds.

Materials and methods

Chemicals 0.1,1-Diphenyl,2-picryl hydrazyl (DPPH), riboflavin, nitroblue tetrazolium (NBT), L-methionin, thio barbutiric acid (TBA), ethylenediaminetetraacetic acid (EDTA) gallic acid, rutin, ascorbic acid, potassium ferricynide [K3Fe(CN)6], trichloro acetic acid (TCA),and ferric chloride (FeCl3) were purchased from Hi- media Ltd. All reagents were of analytical grade.

Plant collection and extract preparation

Plant parts namely leaves, stem bark and root of P. integrifolia were collected from the ayurvedic garden of Banaras Hindu University, Varanasi during month of December for winter, May for summer and August for rainy season (Fig. 1). The plant was taxonomically identified by Prof. V.K. Joshi, Department of Dravyaguna, Faculty of Ayurveda, IMS, Banaras Hindu University, Varanasi, India. A voucher specimen BSI/CRC/2016–17 was deposited in the Botanical Survey of India. Collected plant sample leaves, stem bark and root were washed thoroughly under running tap water in order to avoid dust, and shade dried at room temperature and then powdered in a mechanical grinder. All extracts were prepared using protocol of Upadhyay et al. (2014).

Fig. 1
figure 1

Premna integrifolia a whole plant, b stem, c leaf, and d root

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DPPH radical scavenging activity

The free radical scavenging capacity of the extracts were estimated using modified protocol of Upadhyay et al. (2014). Different concentrations of plant extracts were mixed with 5 ml of a 0.005% methanolic solution of DPPH and incubated for 15 min at room temperature. Absorbance was recorded at 517 nm.

Lipid peroxidation assay

Lipid peroxidation was measured using modified protocol of Upadhyay et al. (2014). 50 μl of each extract and egg homogenate (250 μl, 10% in distilled water, v/v) were mixed in a flask, and the final volume was made up to 500 ml by adding distilled water. Finally, 20 μl FeSO4 (0.07 M) was added to the above mixture and incubated for 40 min, to induce lipid peroxidation. This mixture was mixed with 750 μl of 20% acetic acid (pH 3.5) and 750 μl of 0.8% TBA (w/v) (prepared in 1.1% sodium dodecyl sulphate) and 25 μl 20% TCA and then heated in a boiling water bath for 60 min. After cooling, 3.0 ml of 1-butanol was added to each tube and centrifuged at 3000 rpm for 10 min. The absorbance of the organic upper layer was measured against 3 ml butanol at 532 nm. Distilled water was used as blank in place of the extract.

Superoxide radical scavenging property

A modified protocol of Upadhyay et al. (2018) was used for superoxide radical scavenging activity. In this assay, reaction mixture of 5 ml was made by adding 0.01 M a phosphate buffer solution (PBS) (pH 7.8), 130 mM methionine, 60 μM riboflavin, 0.5 mM EDTA, 0.75 mM NBT and 0.5 ml of test sample solution. The reaction mixture was kept in front of fluorescent light for 5 min, and absorbance was recorded at 560 nm. For control, identical tubes containing the reaction mixture was kept in the dark. The percentage inhibition was measured by comparing the absorbance of the control and the test sample. The blank was 0.01 M PBS.

Reducing power assay (RP). The reducing power of extracts was determined by using modified protocol of Mishra et al. (2016). Different concentrations (50–1000 µg ml−1) of extracts were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mixture was incubated at 37 °C for 15 min and after that 2.5 ml of trichloroacetic acid (TCA, 10%) was added to the mixture and centrifuged at 1000 rpm for 115 min. The upper organic layer of solution (2.5 ml) was taken and mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and then absorbance was recorded at 700 nm. High absorbance indicated high reducing power. Ascorbic acid was used as the standard in this assay.

Determination of total phenol content (TPC)

The TPC was determined by Folin-Ciocalteu method described by McDonald et al. (2001) with some modification. Briefly, 1 ml of distilled water, 0.1 ml of 1 mg ml−1 sample, and 0.3 ml of Folin-Ciocalteu reagent with distilled water (1:1) were mixed in a test tube, and kept for 5–8 min at room temperature. Then, 2 ml of 7% sodium carbonate solution was added, and reaction volume was maintained at 3 ml. Solutions were incubated for 1 h at room temperature, the absorbance was recorded at 750 nm. All determination was carried out in triplicate. Various concentrations of gallic acid used for the preparation of the standard curve and expressed as microgram per milligram of gallic acid equivalents (GAE).

Measurement of total flavonoid content (TFC)

Determination of TFC was done by AlCl3 colorimetric method with slight modification in protocol (Chang et al. 2002). Different extracts (0.1 ml of 1 mg ml−1) in ethanol were mixed with 0.1 ml of 2% AlCl3, 0.1 ml of 1 M potassium acetate, and 2.7 ml of ethanol. The reaction mixture was kept at room temperature for 30 min and absorbance was recorded at 415 nm. TF content was calculated using rutin as the standard and expressed as micrograms per milligram of rutin equivalents (RE).

Profiling of bioactive compounds using UHPLC-Q-TOF-MS/MS

Ethyl acetate extract of leaves collected from rainy season (EAEPI) was characterized using modified protocol of (Singh et al. 2018). The separation of EAEPI was done on an Acquity UPLC system (Waters, Milford, MA, USA) equipped with a BEH C18 column (100 mm × 2.1 mm, 1.7 μm). The column temperature was set at 25 °C. The mobile phase consisted of acetonitrile (B) and methanol (C) acidified with 0.1% formic acid (A). The flow rate of the mobile phase was set at 300 μl/min while 5 μl injection volumes were used. Analytes were eluted by using a gradient elution programme as follows: The initial composition of B:C was 90:10% and increased to 80:20% in 2 min, 50:50% for 1–3 min, 30:70% for 3–6 min, then 10:90% for 1 min and finally, decreased quickly to 90:10% for 7–10 min. The sample was analyzed by mass spectroscopic method (positive and negative ion modes equipped with an electrospray ionization (ESI) source) for the presence of any bioactive compounds. The following mass spectroscopy parameters were applied: The cone and desolation gas flows were 52 l/h and 647 l/h, respectively; the source and desolation temperatures were 40 °C and 450 °C, respectively. The capillary and cone voltage were set at 2.72 kV and 40 eV, respectively. The Q-TOF mass spectrometer was conducted in MSE mode with a low collision energy set at 6 eV in the first function and a collision energy ramp from 20 to 40 eV in the second function. Centroid mode data was collected over the m/z range 100–1000 in both functions, and the scan time was 1 s with an interscan delay of 0.024 s. The accurate mass and molecular formula denomination were acquired with the Mass Lynx 4.1 software (Waters MS Technologies).

Profiling of bioactive compounds using GC–MS analysis

The EAEPI was used for the GC–MS analysis using modified protocol of Tiwari et al. (2016). The bioactive constituents were identified by comparison of their retention indices (RI) relative to homologous alkane series (purchased from Sigma, St. Louis, USA) and by comparison of their mass spectral fragmentation patterns with those data provided in WILEY8.LIB and NIST11.LIB. Identification was assumed when a good match of mass spectrum and RI was achieved. EAEPI was subjected to GC–MS analysis on a GCMS-QP2010 Plus (Shimadzu, Kyoto, Japan) system with headspace sampler (AOC-20 s) and auto-injector (AOC-20i), equipped with the mass selective detector, having ion source temperature of 230 °C, interface temperature of 270 °C, a solvent cut time of 3.50 min threshold of 1000 eV and mass range of 40 to 650 m/z. Compounds were separated using a Rtx 5 MS capillary column (Restek Company, Bellefonte, USA: cross bond 5% diphenyl/95% dimethyl polysiloxane) having dimensions 30 m (length) × 0.25 mm (diameter) × 0.25 μm (film thickness). The split mode at a ratio of 10:1 was used. The temperature of the injector was initialized to 260 °C, having a split injection mode. The temperature was programmed from 80 °C (2 min), then further increased to 250 °C at a ramp rate of 10 °C/min (5 min hold) and finely increased to 280 °C at a ramp rate of 15 °C/min (24 min hold). Helium (> 99.999%) was used as the carrier gas at a linear flow velocity of 40.5 cm/s. The debit of gas (helium) vector with a total flow of 1.21 mL/min was fixed to 16.3 mL/min. The volume of the injected sample was 2 μL of methanol extract. Total MS running time was 46 min.

Proteins and ligands for in silico docking

The major compounds identified in UPLC-Q-TOF-MS/MS and GCMS compounds and a standard hepatoprotective drug known as silibinin were used as ligand for molecular docking analysis. The three-dimensional (3d) structure of the compounds was downloaded from Pubchem database (https://pubchem.ncbi.nlm.nih.gov) in sdf file then converted into Pdb file through Open Babel Gui. The protein structure (3d) of the enzyme alanine aminotransaminase with pdb id 3IHJ was downloaded from protein data bank (https://www.rcsb.org/) in pdb format.

Molecular docking analysis

The molecular docking studies of all selected compounds and proteins were performed with Auto doc tools 1.5.6 (Morris 2009). The interaction between ligand and protein were assessed on the basis of binding energies.

Evaluation of drug likeness and ADMET properties

The drug likeness properties of the compounds were evaluated by Lipinski’s rule of five (http://scfbio-iitd.res.in/software/drugdesign/lipinski.jsp#anchortag) (Lipinski et al. 1997). ADMET properties of compounds viz; adsorption, distribution, metabolism, excretion, and toxicity were evaluated using ADMETlab2 https://admetmesh.scbdd.com/service/evaluation/cal to know the pharmacologically potent hepatoprotective lead molecule (Xiong et al. 2021). For ADMET properties evaluation, different parameters viz; blood–brain absorption (BBB), human intestinal absorption (HIA), AMES toxicity, and carcinogenicity were used for scoring.

Statistical analysis

All experiments were performed in triplicate and data were expressed as means ± SE. Statistical comparisons were made by means of one-way ANOVA test followed by post hoc analysis with Dunnett's test by using SPSS (version 16). P < 0.05, P < 0.01 was considered a significant difference, while P > 0.05 was considered to be non-significant. EC50 values were calculated from linear regression analysis.

Results

DPPH radical scavenging assay

All three extracts of leaf, stem and root have significant free radical scavenging activity (Table 1). Lowest EC50 value was observed in leaf extract of ethyl acetate of rainy season (301.23 ± 1.25 µg/ml) compared to other extracts in their respective season. Antioxidant activity of rainy season ethyl acetate extract was higher compared to summer and winter extracts. Leaf sample of summer and winter season showed EC50 values 345.46 ± 1.35 and 369.12 ± 1.23 µg/ml respectively. EC50value of leaf sample extracted in ethanol during rainy season was 485.04 ± 1.32 µg/ml, while for summer and winter season extracts EC50 values was 498.14 ± 1.23 and 505.13 ± 1.23 µg/ml respectively. In case of methanol and aqueous extract of leaf, EC50 value of different seasonal extracts was in this order rainy < summer < winter. If we compare the EC50value of DPPH free radical scavenging activity of different extracts of stem, the trend was in this order ethyl acetate < ethanol < methanol < aqueous extract. On the basis of comparison of EC50 values of different root extracts the range was as follows: ethanol extract (EC50 = 489.16–490.16) < ethyl acetate (EC50 = 502.56–507.29) < methanol (EC50 = 998.12–999.89) < aqueous extract (EC50 = 1603.28–1608.19). Antioxidant activity of rainy season ethanol extract of root was higher (EC50 = 489.16 ± 0.29) compared to summer (EC50 = 490.18 ± 0.18) and winter (EC50 = 490.10 ± 1.67) extracts.

Table 1 Comparative DPPH free radical scavenging activity of leaf, stem, and root extracts of P. integrifolia in different seasons
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Superoxide radical scavenging assay

Results revealed that leaf, stem and root exhibited potent scavenging activity for superoxide radicals in a concentration dependent manner (Table 2). Superoxide scavenging activity of leaf extract was higher compared to stem and root extracts as evident from EC50 values. Among leaf extracts, ethyl acetate extract has a lower range of EC50values (349.12 ± 1.26–383.19 ± 1.67) compared to ethanol (477.15 ± 0.12–495.18 ± 0.12), methanol (677.02 ± 1.26–697.25 ± 0.27) and aqueous (1096.12 ± 2.10–1112.45 ± 0.67) extracts. In conclusion, among all extracts, ethyl acetate extract of leaf of rainy season exhibited (EC50 = 349.12 ± 1.26) highest potential for superoxide radical scavenging activity compared to stem (EC50 = 707.12 ± 0.27) and root (EC50 = 733.91 ± 1.21) extracts.

Table 2 Comparative superoxide radical scavenging activity of leaf, stem, and root extracts of P. integrifolia in different seasons
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Lipid peroxidation assay

If we compare the EC50 values of lipid peroxidation of different extracts derived from different parts of plant, we can say that leaf extracts showed lower range of EC50 values (339.12 ± 0.23–599.19 ± 1.18) compared to stem (EC50 = 648.24 ± 0.38–1367.99 ± 0.54) and root (EC50 = 485.12 ± 0.67– 991.88 ± 0.65) extracts. It means leaf extract showed better inhibition of lipid peroxidation compared to stem and root extracts. Among all extracts, leaf ethyl acetate extracts exhibited lower range of EC50 value (339.12 ± 0.23–354.00 ± 0.13) compared to ethanol (503.15 ± 0.19–531.37 ± 1.12), methanol (579.56 ± 0.76–589.14 ± 0.56) and aqueous (585.23 ± 0.45–599.19 ± 1.18) extracts. EC50 values of ethyl acetate extracts obtained in different seasons are in the order rainy (339.12 ± 0.23) < summer (348.43 ± 0.25) < winter (354.00 ± 0.13). So, we can conclude that ethyl acetate extract of leaves derived during rainy season has highest lipid peroxidation inhibition (Table 3).

Table 3 Comparative lipid peroxidation activity of leaf, stem, and root extracts of P. integrifolia in different seasons
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Total phenolic content (TPC)

The phenolic compounds may contribute directly to antioxidant action (Awika et al. 2003). Total phenolic content was determined spectrophotometrically by Folin-ciocalteu method and reported as GAE in reference to standard curve (y = 0.003x–0.041, R2 = 0.945). In ethyl acetate extract of leaf, TPC was higher in rainy season which was 74.33 ± 2.26 mg g−1 followed by summer and winter having phenolic content 70.13 ± 0.26 mg g−1, 65.10 ± 1.26 mg g−1, respectively. In ethanolic extract of leaf the TPC was higher in rainy season which was 68.33 ± 0.56 mg g−1 while summer and winter had 64.3 ± 1.26 mg g−1, 62.32 ± 0.23 mg g−1, respectively. In methanol extract of leaf, TPC was higher in rainy season which was 64.43 ± 0.76 mg g−1 while by summer and winter had 60.3 ± 1.06 mg g−1, 54.06 ± 0.23 mg g−1, respectively. TPC in aqueous extract of leaf of rainy season was 49.0.35 ± 0.35 mg g−1 while by summer and winter season had 41.09 ± 0.20 mg g−1 and 39.00 ± 0.26 mg g−1. TPC in ethyl acetate extract of stem bark of rainy season was 50.33 ± 1.23 mg g−1 while summer and winter season had 42.09 ± 0.25 mg g−1 and 39.00 ± 0.36 mg g−1. TPC in ethanol extract of stem bark of rainy season was 48.66 ± 0.35 mg g−1 while summer and winter season had 39.09 ± 0.27 mg g−1 and 37.65 ± 0.44 mg g−1, respectively. TPC in methanolic extract of stem bark of rainy season was 46.33 ± 1.73 mg g−1 while summer and winter season had 35.19 ± 0.20 mg g−1 and 32.01 ± 0.26 mg g−1. TPC in aqueous extract of stem bark of rainy season was 41.66 ± 0.25 mg g−1 while summer and winter season had 31.29 ± 0.20 mg g−1 and 27.66 ± 0.46 mg g−1. Root extract, TPC was higher in ethyl acetate extract of summer season which was 46.35 ± 2.12 mg g−1 while rainy and winter season had 46.33 ± 1.32 mg g−1 and 45.84 ± 0.81 mg g−1, respectively, which indicates almost same value (Fig. 2).

Fig. 2
figure 2

Total phenol content in leaf, stem and root extracts of P. integrifolia in different seasons. Data are presented as mean ± SD. *P < 0.05; **P < 0.01 significantly different from the control

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Total flavonoid content (TFC)

Total flavonoid content was determined by AlCl3 colorimetric method and rutin was used as standard for making curve (y = 0.006x–0.104, R2 = 0.966). Flavonoid content in different extracts of different parts during change in the season was represented in Fig. 3. Flavonoid content was higher in ethyl acetate extract of leaf of rainy season (98.83 ± 0.26 µg/mg) compared to summer (86.43 ± 0.43 µg/mg) and winter season (76.33 ± 0.23 µg/mg). In ethanol extract of leaf, flavonoid content in rainy season was observed higher (68.5 ± 0.19 µg/mg) compared to summer (65.5 ± 1.23 µg/mg) and winter (51.81 ± 0.82 µg/mg). In methanol extract of leaves of rainy season, flavonoid content was higher (50.12 ± 2.13 µg/mg) compared to summer (45.23 ± 0.56 µg/mg) and winter (42.12 ± 1.23 µg/mg). The flavonoid content in aqueous extracts of different seasons was in this order: rainy (37.16 ± 0.82 µg/mg) > summer (30.12 ± 2.31 µg/mg) > winter (29.43 ± µg/mg). Flavonoid content in different extracts of stem during rainy season was as follows: ethyl acetate (70.12 ± 0.56 µg/mg) > ethanol (51.81 ± 2.13 µg/mg) > methanol (49.16 ± 0.32 µg/mg) > aqueous extract (35.00 ± 0.32 µg/mg). Flavonoid content in ethanol, methanol, ethyl acetate and aqueous extracts of stem in summer season was 45.12 ± 0.12 µg/mg, 45.83 ± 0.53 µg/mg, 62.12 ± 0.73 µg/mg and 31.32 ± 1.32 µg/mg, respectively. Flavonoid content in ethanol extracts of roots of all three season was comparable. In rainy season, it was 64.33 ± 0.86 µg/mg, while in summer and winter flavonoid content was 65.12 ± 0.82 and 65.09 ± 0.82 µg/mg, respectively. TFC in methanol extract of rainy, winter and summer season was 45.66 ± 0.82, 45.45 ± 0.82, and 45.83 ± 1.21 µg/mg, respectively. Aqueous extract of winter contained lower flavonoid content (32.12 ± 0.39 µg/mg) compared to rainy (37.16 ± 0. 56 µg/mg) and summer season (32.15 ± 1.32 µg/mg). There was no significant difference in TFC of summer (65.12 ± 1.2 µg/mg) and winter (65.09.12 ± 0.13 µg/mg) extracts, while in rainy season extract it was 64.33 ± 0.43 µg/mg. TFC in ethyl acetate extract of root in rainy season it was 53.66 ± 1.13 µg/mg, while in summer and winter season it was 54.12 ± 0.41 and 55.01 ± 0.13 µg/mg, respectively. There was no significant difference in flavonoid content in methanol extract of root in rainy (45.66 ± 1.42 µg/mg), summer (45.45 ± 1.02 µg/mg) and winter (45.83 ± 0.73 µg/mg) seasons. Similarly, flavonoid content in aqueous extract of roots of all three season was almost similar and it was 32.16 ± 0.41, 32.15 ± 0.19 and 32.12 ± 0.57 µg/mg in rainy, winter and summer season, respectively (Fig. 3).

Fig. 3
figure 3

Total flavonoid content in leaf, stem and root extracts of P. integrifolia in different seasons. Data are presented as mean ± SD. *P < 0.05; **P < 0.01;***P < 0.001 significantly different from the control

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Reducing power assay

The reducing power of all the three extracts of leaf, stem and root in three different seasons like rainy, winter and summer, increased in concentration dependent manner as depicted in Figs. 3 and 4. Ethanol extract of leaf showed higher reducing potential in rainy season compared to summer and winter (Fig. 5a–c). Methanol extract of leaves of rainy season exhibited higher reducing potential compared to summer and winter season (Fig. 6a–c). Ethyl acetate extract of leaves of rainy season showed higher reducing potential compared to stem and root. Absorbance of rainy season's leaves extract was highest (OD = 1.21) (Fig. 6a–c). Aqueous extract of leaf showed higher reducing potential in rainy season compared to winter and summer season (Fig. 7a–c). On the basis of above results, it was observed that ethyl acetate extract of leaves during rainy season exhibited highest reducing potential.

Fig. 4
figure 4

Reducing power of ethyl acetate extract of a leaf, b stem and c root of P. integrifolia in different season

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Fig. 5
figure 5

Reducing power of ethanol extract a leaf, b stem and c root of P. integrifolia in different season

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Fig. 6
figure 6

Reducing power of methanol extract of a leaf, b stem and c root of P. integrifolia in different season

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Fig. 7
figure 7

Reducing power of aqueous extract of a leaf, b stem and c root of P. integrifolia in different season

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Correlation between total antioxidant activity and total polyphenol content

Ethyl acetate extracts of different parts of the plant during rainy season contained highest polyphenol content, so a correlation between the total phenol, flavonoid content and antioxidant activity in rainy season extracts was presented in Fig. 8. A positive correlation was observed between phenol and flavonoid content as well as total antioxidant activity in the ethyl acetate extract of leaf (R2 value ranges from 0.975 to 0.999). Similarly, the strong positive correlation was observed between antioxidant activity and phenol and flavonoid content in ethyl acetate extract of stem (R2 value ranges from 0.921 to 0.927) and root (R2 value ranges from 0.981 to 0.947), which is clear from the correlation coefficient (Fig. 8). A significant positive correlation was observed between ferric reducing antioxidant power (FRAP) and total antioxidant activity (R2 = 0.975) of rainy season leaf extract. Similarly, the ethyl acetate extract of stem (R2 = 0.948) and root (R2 = 0.792) of rainy season showed positive correlation with total antioxidant activity and ferric reducing power (Fig. 9). These results indicate that there was strong relationship between polyphenol content in leaf, stem and root with antioxidant activity. Therefore, in conclusion it was observed that presence of flavonoid and phenol in extracts contribute significant role for their antioxidant potential.

Fig. 8
figure 8

Correlation between total antioxidant activity and polyphenol content of rainy season ethyl acetate extract of leaf (a), stem (b) and root (c)

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Fig. 9
figure 9

Correlation between total antioxidant activities and reducing power of rainy season ethyl acetate extract of leaf (a), stem (b) and root (c)

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GC–MS analysis of EAEPI

The result obtained from GC–MS analysis of rainy season ethyl acetate extract of leaves of P.integrifolia showed the presence of important phytoconstituents. The GC–MS chromatogram (Fig. 10) showed the presence of several peaks. It indicates about presence of bioactive compounds. The major compounds identified based on their retention time (RT), molecular formula, molecular weight, peak area percentage and bio activities as presented in Table 4. Among these identified compounds some major compounds with their percentage pick area (%) and retention times (min) were as follows: citronellol (29.1%, 30.67 min), phytol acetate (16%, 32.48 min), campesterol (12.5%, 50.83 min), phytol acetate (5.83%, 59.74 min), squalene (4.78%, 41.77%), stigmasterol (4.76%, 49.16 min) and hexadecanoic acid (4.52%, 28.12 min).

Fig. 10
figure 10

GC–MS total ion chromatogram of rainy season ethyl acetate extract of leaves of P. integrifolia

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Table 4 Bioactive compounds identified in ethyl acetate extract of leaves of P.integrifolia by GC–MS
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UPLC-Q-TOF–MS/MS analysis of EAEPI

EAEPI was profiled for the presence of bioactive compounds by their mass through UPLC–Q-TOF–MS/MS (Fig. 10). The identified compounds with their biological activities were listed in Table 5. These compounds were tentatively identified based on their retention time, ESI [M-H-], MS/MS and m/z base ions. The compounds were identified by using the combination of mass spectrometry data and established database (http://spectra.psc.riken.jp/). Data revealed that EAEPI contained 8 polyphenolic compounds. These compounds with their m/z values were as follows: quercetin-3-Dxyloside (256), kaempferol-3,7-O-bis-alphaL-rhamnoside (284), isorhamnetin-3-O-glucoside (317), luteolin-3′,7-di-O-glucoside (149), eriodictyol-7-O-glucoside (872), syringetin-3-O-galactoside (359), petunidin-3-O-beta- glucopyranoside (329) and vitexin-2′′-O-rhamnoside (425). These compounds possess different pharmacological activities like antioxidant, hepatoprotective, antitumor, anticancer and antidiabetic, which is summarized in Table 5.

Table 5 Major bioactive compounds identified in ethyl acetate extract of rainy season leaves of P. integrifolia using UPLC-MS/MS in + ve ion mode
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In silico docking analysis

During in-silico docking analysis, three compounds stigmasterol, campesterol, and eriodictyol-7-O-glucoside were showing best binding potential with their binding energies − 8.98, − 8.67 and − 5.99 respectively (Table 6) (Fig. 11a and b). Their binding affinities were even higher than silibinin, the standard hepatoprotective compound.

Table 6 Docking of Alanine amino transferase with major compounds identified in UPLC-MS/MS and GC–MS analysis
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Fig. 11
figure 11

UPLC-MS/MS positive ion chromatogram of ethyl acetate extract of P. integrifolia leaves showing peaks of identified compounds with their m/z values. (1) Quercetin-3-Dxyloside (256), (2) Kaempferol-3,7-O-bis-alpha-L-rhamnoside (284), (3) Isorhamnetin-3-O-glucoside (317), (4) Luteolin-3′,7-di-O-glucoside (149), (5) Eriodictyol-7-O-glucoside (872), (6) Syringetin-3-O-galactoside (359), (7) Petunidin-3-O-beta-glucopyranoside (329) and (8) Vitexin-2′'-O-rhamnoside (425)

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Drug likeness and ADMET Properties All the compounds showing best binding score with enzyme were also satistfying drug likeness criteria according to Lipinski’s rule of five except eriodictyol-7-O-glucoside (Table 7). The Lipinski’s rule differentiates between drug-like and non drug-like properties of compounds by compliance with 2 or more than two of five different parameters viz; molecular mass less than 500 Dalton, Log P value (indicator of lipophilic property) less than 5, hydrogen bond donor less than 5, hydrogen bond acceptor less than 10 and molar refractivity between 40 and 130. Among three compounds stigmasterol, campesterol, and eriodictyol-7-O-glucoside only two compounds stigmasterol, and campesterol were satisfying Lipinski’s rule of five while eriodictyol-7-O-glucoside was rejected by above criteria (Table 7). Besides drug likeness, any drug should fulfill ADMET parameters viz. absorption, distribution, metabolism, excretion and toxicity. It is evident from Table 8 that both stigmasterol, campesterol were showing best intestinal absorption and distribution. These two compounds were also non-ames toxic and non-carcinogenic. While the third compound eriodictyol-7-O-glucoside was toxic and carcinogenic with poor intestinal absorption (Table 8). Thus, it could not be recommended in pharmaceutical formulations.

Table 7 Drug likeness properties of compounds
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Table 8 ADMET properties of compounds
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Discussion

Medicinal Plants are the rich sources of polyphenolic compounds. These polyphenolics act as antioxidants by scavenging free radicals and chelation of metal ions (Halliwell et al. 1995). Plant accumulates phenolic compounds under various stress conditions such as drought, heat, ultraviolet light exposure, air pollution and pathogens attack (Pasqualini et al. 2003). Changes in climatic conditions also influence the biosynthesis of polyphenol (Christie et al. 1994). The stresses enhance the production of reactive oxygen and nitrogen species in the body of the host plant. Host defends itself against ROS by escalating the biosynthesis of phenolic compounds (do Nascimento and Fett-Neto 2010). Temperature fluctuation from optimal level also cause alteration in physiology and biochemistry of several plants (Malhi et al. 1998). In present study, significant variation in total phenol and flavonoid content in different plant part extracts collected in different season have been recorded (Figs. 1 and 2). The polyphenol (TPC and TFC) content in all extracts derived from different plant parts varied with the seasonal variation. Phenolic content was highest in ethyl acetate extract leaf collected in rainy season, while it was lowest in winter season leaf extract. Since the microbial load enhances during the rainy season, so the biotic stress during rainy season might be responsible for higher polyphenol production in rainy season in P. integrifolia. Seasonal variation also affected the phenolic content in leaf exudates in Citrus species (Chaves et al. 1997). Plant parts extracted in different solvents showed steady increase in percentage inhibition by DPPH radicals with increasing concentration (Table 1). The degree of reduction in absorbance is the marker of the increased antioxidant capacity of the extract. Ethyl acetate extract of leaves and roots of rainy season depicted greater antioxidant activity compared to stem. Nearly similar variation pattern of EC50 value was obtained for stem and root sample extracted in ethanol methanol and aqueous extract. It was evident that season of collection of plant parts greatly affects the antioxidant activity towards DPPH free radical. Superoxide radical is very harmful species to cellular components because they are precursor of ROS (Buonocore et al. 2010). Current study revealed that all extracts are observed as effective scavenger of singlet oxygen generated in riboflavin-NBT-light system and their activity increased with increase in concentration of extracts (Table 2). In this assay ethyl acetate extract of leaves of rainy season has greater scavenging potential compared to other plant part extracts of different seasons. Ferric reducing antioxidant power (FRAP) is a useful assay for the measurement of antioxidant activity. FRAP was determined based on the colorimetric detection of the reaction mixture. Increase in the absorbance is the indication of the improved antioxidant activity (Mishra et al. 2016). In the present investigation, reducing potential of all extracts increased with increase in concentration, which was in close correlation with its observed antioxidant activity (Figs. 3, 4, 5, 6, 7, 8). Thus, this reducer might be responsible for antioxidant potential of extracts. Lipids are responsible for maintenance of cellular integrity. Results revealed that the rainy season ethyl acetate extract of leaves has higher inhibitory potential for lipid peroxidation compared to other plant parts (Table 3) The GC–MS analysis of ethyl acetate leaf extract of rainy season of P. integrifolia showed the presence of 26 compounds with seven major compounds (Table 4; Fig. 9). UPLC-Q-TOF–MS/MS analysis of above extract illustrated the presence of eight polyphenolic compounds whose antioxidant properties were reported earlier (Kähkönen and Heinonen 2003; Wu et al. 2012; Hu et al. 2012) (Table 5; Fig. 10).

Alanine amino transferase (ALT) test is normally used to check the hepatic damage such as hepatitis and cirrhosis. When the liver is damaged, the level of ALT rises in the liver and thereby in the bloodstream. Therefore, in present study, ALT was taken for the in-silico docking analysis against ligands. Among all compounds, three compounds stigmasterol, campesterol, and eriodictyol-7-O-glucoside were showing maximum binding energies against ALT (Table 6, Figs. 12 and 13). The residues were showing van der Walls, hydrophobic, hydrogen bond interactions which is presented in Figs. 12 and 13. Thus, it indicates that these three compounds have potential to protect hepatic damage. The two compounds stigmasterol and campesterol were satisfying the drug likeness properties according rule of the Lipinski’s five, while the eriodictyol-7-O-glucoside was rejected by Lipinski’s criteria (Table 7). Both compounds stigmasterol and campesterol were showing best absorption, distribution, and were non-toxic and non-carcinogenic (Table 8). While, eriodictyol-7-O-glucoside was showing poor intestinal absorption, and was observed as toxic and carcinogenic while ADMET scoring. This is the first report of the in-silico evaluation of hepatoprotective evaluation of stigmasterol and campesterol. There are reports supporting hepatoprotective activity of quercetin-3-D-xyloside, kaempferol-3,7-O-bis-alpha-L-rhamnoside, isorhamnetin-3-O-glucoside, vitexin-2''-O-rhamnoside, eriodictyol-7-O-glucoside (Table 5) but these compounds were not binding efficiently during in silico docking, which is evident from their binding energies (Table 6).

Fig. 12
figure 12

In silico interaction between campesterol and receptor alanine aminotransferase a docked complex, b complex showing the active site where ligand interacted, c 3D level interaction between receptor and campesterol, d 2D level interaction between receptor and campesterol

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Fig. 13
figure 13

In silico interaction between stigmasterol and receptor alanine aminotransferase a docked complex, b complex showing the active site where ligand interacted, c 3D level interaction between receptor and stigmasterol, d 2D level interaction between receptor and stigmasterol

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Conclusion

It can be concluded that polyphenol content and antioxidant activity was dependent on the season of collection of plant materials, parts of the plant used for extraction and nature of solvents used for extraction. Lipid peroxidation activity, superoxide radical scavenging activity and reducing power varied significantly based on season, solvent as well as plant parts. Results revealed that ethyl acetate extract of leaves of rainy season, contained highest total phenol and flavonoid content and showed highest antioxidant activity. UPLC-MS/MS analysis of EAEPI confirmed the presence of eight major bioactive compounds. GC–MS analysis reported the presence of twenty-six bioactive compounds with six major compounds. In silico docking analysis of major compounds against liver enzyme revealed the presence of two hepatoprotective lead compounds that can be used in hepatic drug formulations after clinical trials.