Plant Materials

Based on the ethnomedicinal uses, six plants chosen for this study were collected from different areas of Tanahu and Kaski district of Nepal. The dried specimens were mounted on authentic standard herbarium sheets, labelled correctly, and stored in the Laboratory of Pharmacognosy, School of Health and Allied Sciences, Pokhara University, Nepal. The herbarium was then sent to National Herbarium and Botanical Laboratories, Nepal for identification purpose. Table 1 contains information about the plant samples that were collected for this study.

Extraction of Plant Materials

The plants were extracted by using double maceration method with 70% methanol in the ratio of 1:10 w/v (dried plant material: solvent). Then the obtained extracts were filtered through cotton and were dried in a rotatory evaporator (40°C, 70-80 rpm). The obtained concentrated extracts were then kept in a vacuum desiccator (50-60 mbar pressure) until complete drying. The weight of each extract was measured daily until a constant weight was obtained.

Phytochemical Screening

All six plant extracts were screened for different classes of secondary metabolites using analytical test methods as mentioned in different literatures (Chen et al., 2022; Shaikh & Patil, 2020). The stock solution of concentration 1 mg/ml was prepared by dissolving the plant extract in methanol and water to prepare methanolic and aqueous extract solutions, respectively. Table 2 illustrates the methods employed for phytochemical screening of plant extracts.

Antioxidant Activity

Antibacterial Activity

Chemical Isolation

Dried extract of Dischidia bengalensis (whole plant) (550 g) was used for chemical isolation. Extraction in 70% methanol yielded 76.2 g of the crude extract, which was dissolved in sufficient amounts of distilled water and then the water-soluble extract (DNW) (66.19 g) was introduced into MCI (Mitsubishi Chemical Corporation) GEL^TM column. The column was eluted with water (1500 ml), 40% MeOH (1500 ml), 60% MeOH (1500 ml), 80% MeOH (1000 ml), MeOH (1000 ml) and chloroform (500 ml) respectively. TLC (Thin layer chromatography) patterns of alternate fractions were observed on solvent system CHCl₃: MeOH: H₂O (6:4:1) for fractions 1-35 and with CHCl₃: MeOH: H₂O (7:3:0.5) for fractions 36-74. The developed TLC chromatograms were then visualized under UV (long UV-365 and short UV-254) followed by H₂SO₄ (10% v/v) spray with heat for 2 min and FeCl₃ (10% w/v) spray. Fractions with similar spots on TLC were mixed to give 12 sub-fractions (DNW-1 to DNW-12) and were evaporated in rotary evaporator. Of the 12 subfractions, DNW-6 and DNW-9 were further subjected to column chromatography to obtain compound 1 (DNW-6-4-2-4) and compound 2 (DNW-9-4-2-3).

Phytochemical Screening

Around the world, there has been a lot of interest in studying the chemical constituents of medicinal plant as they possess variety of biological properties, including medicinal properties, and are often used in traditional medicines (Chhetri & Khatri, 2017). The phytochemical screening of 70% methanolic extract of six plant samples revealed either presence or absence of the different secondary metabolites i.e., alkaloids, tannins, saponins, phenols, carbohydrates, flavonoids, terpenoids, and quinones. The presence or absence of phytochemicals was confirmed by visual observation of the change in color or turbidity. In the present study, Gynura nepalensis (GSL) leaves lacked flavonoids but the study conducted by (Chakrabarty et al., 2022) revealed the presence of flavonoids. Similarly, the extract of Angiopteris helferiana and Mikania spp. showed no response to FeCl₃ phenol test indicating the absence of phenol. However, a study reported high phenol content in rhizome of A. helferiana (Lamichhane et al., 2019). The extract of Curcuma caesia revealed the presence of alkaloid, saponin, quinone, phenol, flavonoid, carbohydrate, and tannin, but lacked terpenoid. Interestingly, a study reported the presence of terpenoids in this plant (Pakkirisamy, Kalakandan, & Ravichandran, 2017). This variation in phytochemicals may be due to difference in plant collection time, difference in solvent system used for extraction, parts of the plant used, age and various ecological and climatic conditions. Moreover, the number of phytochemical substances varies greatly from species to species and even from plant to plant (Shaikh & Patil, 2020). The result of the phytochemical screening has been shown in Table 3.

Figure 1

Percentage DPPH free radical scavenging activity of selected plant samples with reference to standard ascorbic acid at 517nm

Figure 2

Zoneof inhibition of A. helferiana (2), D. bengalensis (4), and D. coronans (5) along with standard Azithromycin (a) and Gentamycin (b) against S. aureus and E. coli at three different concentrations, i.e. 100 mg/ml, 50 mg/ml, and 25 mg/ml.

Antioxidant Activity

Antioxidant activity was measured using DPPH free radicals scavenging assay with ascorbic acid as the standard. A graph of concentration versus percentage of free radical scavenging activity was generated, as well as the IC₅₀ value of each plant was calculated. The antioxidants in the DPPH method react with the stable free radical, 2,2-diphenyl-1-picrylhydrazyl (deep violet color), and convert it to 2,2-diphenyl-1-picrylhydrazine with discoloration, indicating the sample's scavenging potential (Gurung, Kaundinnyayanna, & Parajuli, 2019). It is a pink-colored nitrogen-containing radical that loses its pink color when it accepts an electron or a hydroxyl group from antioxidants. Color loss is proportional to the number of electrons accepted and can be quantified using light absorption changes at 517 nm. The IC₅₀ value in DPPH free radical scavenging method is the sample concentration that can scavenge 50% of the DPPH free radical, evaluated by recording the change in absorbance due to DPPH reduction, which is inversely related to the sample's antioxidant property (Giri et al., 2020). In the current study, all plant extract showed a dose-dependent increase in activity from concentrations of 0.1 µg/ml to 100 µg/ml. Comparatively, the extract of Dischidia bengalensis and Curcuma caesia showed potent antioxidant activity (IC₅₀= 4.60 μg/ml and 5.81 μg/mL respectively), as compared to the positive control, ascorbic acid (IC₅₀ = 2.76 μg/mL), and this may be due to presence of phytochemicals like flavonoids and phenols. According to studies, phenolic compounds with higher concentrations have increased antioxidant properties. The antioxidant property of phenolic compounds is mainly due to their ability to scavenge injurious free radicals such as superoxide and hydroxyl radicals (Meng et al., 2021). Also, the current investigation found the IC₅₀ value of Angiopteris helferiana extract to be 30.19 μg/ml, which is lower than the value reported by (Lamichhane et al., 2019). Comparatively, Mikania spp. stem extract showed less scavenging activity (IC₅₀ = 59.17 μg/ml) than other plant extracts, which may be correlated with the absence of phenols in it (Ekram et al., 2017). The IC₅₀ value of Drynaria coronans and Gynura nepalensis were found to be 9.46 μg/ml and 7.61 μg/ml, respectively. The results of DPPH free radical scavenging assay are depicted in Table 4 and Figure 1.

Antibacterial Activity

Antibacterial activity of 70% methanolic extract of Angiopteris helferiana, Curcuma caesia, Dischidia bengalensis, Drynaria coronans, Gynura nepalensis, and Mikania spp. were screened by the agar well diffusion method. The diameter of the zone of inhibition of plant samples indicated sensitivity or resistance against Escherichia coli and Staphylococcus aureus as shown in Table 5 and Figure 1.

In the present study, A. helferiana extract modestly inhibit the growth of E. coli at concentrations of 50 and 100 mg/ml (zone of inhibition = 3.00±1.41 and 5.50±2.12 mm, respectively) but at the conc. of 50 mg/ml, A. helferiana extract exhibited the maximum zone of inhibition (8.00±1.42 mm) against S. aureus. However, in the study conducted by (Bhattacharjee et al., 2017). A. helferiana ethyl acetate extract exhibited antibacterial activity against S. aureus only but not against E. coli and Pseudomonas aureus. Although the extract of Curcuma caesia rhizome and Gynura nepalensis leaves in the current investigation failed to demonstrate any antibacterial activity against E. coli and S. aureus. G. nepalensis essential oil and methanolic extract exhibited antibacterial against S. aureus, P. aeruginosa, and E. coli in the study performed by (Meng et al., 2021). Also, (Rajkumari & Sanatombi, 2017) reported that C. caesia has potential antibacterial properties. This variance in antimicrobial activities may be brought on by the different concentration of extract used throughout the study, difference in the solvent used for extraction, variations in geographic location, climatic conditions, and plant growing season (Rasheed, Hamudi, & Kreem, 2010). In the current investigation, the methanolic extract of Dischidia bengalensis exerted antibacterial activity against E. coli (zone of inhibition 5.00±0.00 mm) at a concentration of 100 mg/ml but failed to do so against S. aureus. The methanolic extract of Drynaria coronans was found to inhibit the growth of both S. aureus and E. coli, where the highest zone of inhibition was measured against E. coli at a concentration of 100 mg/ml (zone of inhibition 8.00±1.42 mm). The value of zone of inhibition discovered in the present study, however, is significantly lower than what the study reported (Khanal, Sharma, Pokharel, & Kalauni, 2020). Similarly, Mikania spp. methanolic stem extract exhibited antibacterial action against E. coli only. This may be due to the presence of flavonoids and tannins, as these phytochemicals are responsible for antibacterial action against many of the bacterial strains that humans encounter (Havsteen, 2002). Furthermore, the antibacterial activity of Angiopteris helferiana, Dischidia bengalensis, and Drynaria coronans may be due to presence of these phytochemicals as well.

Structure Elucidation

Compound separation from the various fractions of Dischidia bengalensis entire plant was performed using adsorption chromatography based on TLC profiling in the current study, and structural elucidation of the isolated compounds was done using ¹H NMR, ¹³C NMR, and DEPT 135° (Distortion less Enhancement by Polarization Transfer) spectra. To get pure chemicals, a 70% MeOH extract of Dischidia bengalensis was fractionated using MCI GEL^TM, Sephadex® LH-20, ODS gel column, and Silica gel column chromatography, in a sequential manner. TLC profiling of each fraction and sub-fractions were performed based on the solvent system ratios CHCl₃: MeOH: H₂O (7:3:0.5 and 6:4:1) followed by detection using 10% aq. FeCl₃ and 10% H₂SO₄ as spraying agent. Comparable solvent ratios and spraying agents in similar concentrations were employed in reference with various literature addressing chemical isolation and TLC profiling (Joshi, Devkota, Watanabe, & Yahara, 2014; Sai, Devkota, Thapa, Poudel, & Joshi, 2020).

Figure 3

Structure of 2"-O-rhamnosylvitexin (copmpound-1) isolated from Dischidia bengalensis.

Figure 4

Structure of isovitexin (compound-2) isolated from Dischidia bengalensis.

The entire assignment of the proton and carbon atoms, their location and linkage with anomeric protons were determined and compared with chemical shift values of related similar compounds on the literature (Joshi, Devkota, & Yahara, 2013; Thoa et al., 2019; Wen et al., 2017). Two compounds were isolated; compound 1 as 2"-O-rhamnosylvitexin (Figure 3) and compound 2 as isovitexin (Figure 4). The ¹³C NMR spectrum of compound 1 showed signals equivalent to total 27 carbons, in with 12 carbon signals were assignable to a 2”-O- substituted rhamnosyl moiety. The detection of 15 carbon signal through ¹³C NMR spectra for Kaempferol moiety confirmed the compound carbon skeleton structure of flavonoid. Similarly for compound 2, ¹³C NMR spectrum showed signals equivalent to total 21 carbons, in which 15 carbon signals were assignable to kaempferol moiety.

Similarly, in DEPT-135° ¹³C NMR spectroscopy, negative signals at around 61.0 ppm indicates the presence of CH₂ group in both the isolated compounds. Likewise, CH₃ group present in the rhamnose moiety was confirmed by the appearance of signals at 17.67 ppm in compound 1 (Joshi et al., 2013). All quaternary carbon signals were absent in the given spectrum.

In the study conducted by (Wei et al., 2014), 2"-O-rhamnosylvitexin significantly reduced H₂O₂-induced late and early apoptosis/necrosis in human adipose-derived stem cells (hADSCs) compared to the corresponding H₂O₂-treated group, indicating that 2"-O-rhamnosylvitexin protects hADSCs by inhibiting apoptosis. Similarly, isovitexin had effective free radical scavenging capacities of 91.48%, indicating that it has anti-oxidant properties, possibly because it can act as both proton donor or metal ion chelators (Azubuike-Osu, Ohanenye, Jacob, Ejike, & Udenigwe, 2021; Lee et al., 2019). Prevention in reduction of antioxidant defense i.e. free radical scavenging activity, ferric reducing ability, and Glutathione levels in inflammatory foci resulted in analgesic and anti-inflammatory response. Thus, indicating that potent free-radical scavenging activity leads to increased anti-inflammatory response and pain relief (Babaei et al., 2020; Borghi et al., 2013).

A study found that isovitexin, when pretreated with acute lung injury (ALI) mice, can have anti-inflammatory properties. It showed a dramatical decline in tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6) production, reactive oxygen species (ROS) generation, myeloperoxidase (MPO) and malondialdehyde (MDA) content. This in turn, effectively inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, which protects against LPS (Lipopolysaccharide) induced inflammation and oxidative stress in ALI mice (Lv et al., 2016). ​Since, this plant is traditionally used to treat bone fracture and muscle pain, and have potent antioxidant activity, these compounds might be responsible for this activity. However, a detail investigation is needed to fully understand the role of these compounds on traditional uses of this plant.