Chemical Profiling and Biological Activities on Nepalese Medicinal Plant Extracts and Isolation of Active Fraction of <i>Nyctanthes arbor-tristis</i>

Khadka, Anita; Budha Magar, Akash; Sharma, Khaga Raj

doi:https://doi.org/10.1155/2024/5080176

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Research Article | Open Access

Volume 2024 | Article ID 5080176 | https://doi.org/10.1155/2024/5080176

Chemical Profiling and Biological Activities on Nepalese Medicinal Plant Extracts and Isolation of Active Fraction of Nyctanthes arbor-tristis

Anita Khadka,¹Akash Budha Magar,¹and Khaga Raj Sharma¹

Academic Editor: Antonio M. Rabasco

Received20 Apr 2023

Revised21 Oct 2023

Accepted06 Mar 2024

Published14 Mar 2024

Abstract

The importance of medicinal plants for the treatment of different diseases is high from the aspects of the pharmaceutical industry and traditional healers. The present study involves nine different medicinal plants, namely, Neolamarckia cadamba, Nyctanthes arbor-tristis, Pogostemon benghalensis, Equisetum debile, Litsea monopetala, Spilanthes uliginosa, Desmostachya bipinnata, Mallotus philippensis, and Phoenix humilis, collected from Chitwan district of Nepal for biochemical analysis followed by the isolation of active plant fractions from the bioactive plant extract. The methanolic extracts of roots, barks, seeds, seed cover, and the other aerial parts of plants were used for the phytochemical analysis and biological activities. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay was adopted to evaluate the antioxidant activity. Antibacterial activity was evaluated using the agar well diffusion method. The antidiabetic activity was studied by the α-amylase enzyme inhibition assay. The highest antioxidant activity was observed in extracts of Nyctanthes arbor-tristis followed by Mallotus philippensis (seed cover), Pogostemon benghalensis, Litsea monopetala, Phoenix humilis, and Neolamarckia cadamba with IC₅₀ values of 27.38 ± 1.35, 32.08 ± 2.81, 32.75 ± 2.13, 33.82 ± 1.07, 40.14 ± 0.93, and 50.44 ± 3.75 µg/mL, respectively. The highest antidiabetic activity was observed in extracts of Phoenix humilis followed by Desmostachya bipinnata and Pogostemon benghalensis with IC₅₀ values of 95.69 ± 6.97, 99.24 ± 12.6, and 106.3 ± 12.89 µg/mL, respectively. The mild α-amylase enzyme inhibition was found in extracts of Nyctanthes arbor-tristis, Spilanthes uliginosa Swartz, Litsea monopetala, and Equisetum debile showing IC₅₀ values of 110.4 ± 7.78, 115.98 ± 10.24, 149.83 ± 8.3, and 196.45 ± 6.04 µg/mL, whereas Mallotus Philippensis (seed cover), Mallotus philippensis (seed), and Desmostachya bipinnata showed weak α-amylase inhibition with IC₅₀ values of 208.87 ± 1.76, 215.41 ± 2.09, and 238.89 ± 9.27 µg/mL, respectively. The extract of Nyctanthes arbor-tristis showed high zones of inhibition against S. aureus (ATCC 25923) and E. coli (ATCC 25922) of ZOI 26 and 22 mm, respectively. The chemical constituents isolated from the active plant Nyctanthes arbor-tristis were subjected to GCMS analysis where the major chemical compounds were 11,14,17-eicosatrienoic acid and methyl ester. These results support the partial scientific validation for the traditional uses of these medicinal plants in the treatment of diabetes and infectious diseases by the people living in different communities of Chitwan, Nepal.

1. Introduction

Ayurveda is considered to be the most ancient medicinal system that had developed as a fully fledged medical system during 1000 BC [1]. Many drugs used in modern medicinal practices are derived from the herbal remedies in the history of traditional therapies including opium, aspirin, digitalis, and quinine. In recent years, the use and search for potent natural compounds as drug candidates and dietary supplements have derived from plants. It has been reported that plant-derived chemical compounds have potential against simple to life-threatening diseases, and at least 7,000 medical compounds in modern pharmacopeia are derived from plants [2]. The World Health Organization estimated that about 80% of the world population relies on plant extracts or their active constituents as folk medicine in traditional therapies. However, due to the inaccessibility of modern medicinal practices, a large number of Nepalese people depend upon a wide range of natural medicines for their primary healthcare based on ethnobotanical knowledge [3]. Nepal is rich in natural biodiversity which reflects the floral and faunal variations. This biodiversity supports the livelihood of people (80–90%) regarding the utilization of plants as medicine. Medicinal plants have assumed greater importance in recent days due to the tremendous potential they offer in developing new drugs against many diseases and illnesses that affect human beings. Herbal plants constitute the major constituents of most indigenous medicines, and a large number of medical preparations contain one or more ingredients of plant origin [4]. Plant-derived pharmaceuticals have high demand in the world market as fragrances, flavors, and color ingredients [5].

People of different communities in the world have been using medicinal plants for many years in the treatment of different diseases like cancer, Parkinson, Alzheimer, diabetes, and several infectious diseases [6, 7]. These plants are used as a source of anticancer, antidiabetic, antioxidant, and antimicrobial agents. Antioxidants are compounds that are present at low concentrations but effectively impede or retard the oxidation of substrates [8]. They protect the cell from oxidative damage caused by the reactive oxygen and nitrogen species. An imbalance between antioxidants and reactive oxygen species is the underlying basis of oxidative stress, leading to cellular damage [2, 9].

Compounds of natural, synthetic, and semisynthetic origin that kill or inhibit the growth of microorganisms without little or no damage to the host cell are antimicrobial agents. Traditionally, people of different communities have been using medicinal plant extracts in the crude form as an herbal medicine against several infectious diseases [10]. The chemical compounds that act as the most important weapons against microbial infections in humans are antibiotics. Many commonly used antibiotics have become less and less effective against certain illnesses due to the emergence of drug-resistant bacteria [11]. The scope of antimicrobials from natural sources is in high demand because of the increasing rate of multidrug-resistant strains as well as newly developed strains with reduced susceptibility to available antimicrobial agents [12]. The systematic screening of antimicrobial plant extracts is a continuous effort to find new drug candidates with high activity against multidrug-resistant microorganisms [13].

Diabetes is a life-threatening disease that arises due to the insufficient production of insulin by the pancreas (type 1 diabetes) or the human body’s failure to properly use produced insulin (type 2) [14]. Type 2 diabetes has received considerable attention due to its prevalence among the population. One major therapeutic target for antidiabetic drugs is the α-amylase and α-glucosidase enzymes. α-Amylase is a group of endoglucanases secreted by the salivary glands and pancreas. They initiate carbohydrate digestion by hydrolyzing α-1,4-glycosidic bonds in carbohydrates to produce oligosaccharides [15, 16]. Oligosaccharides and disaccharides thus produced are cleaved into glucose by α-glucosidase enzymes produced near the brush border enterocyte region of the jejunum [17]. Inhibition of α-amylase and α-glucosidase significantly delays the digestion of carbohydrates and subsequent absorption of glucose [18]. Thus, both α-amylase and α-glucosidase inhibitors are effective in controlling post prandial hyperglycemia [19]. Compounds bearing more than one type of therapeutic action are found to be effective in the treatment of diabetes [20].

The present study focused on the collection of nine selected medicinal plants from the Chitwan district of Nepal based on their traditional uses (Figure 1). Biochemical profiling of the medicinal plants was established based on phytochemical analysis, antioxidant, antidiabetic, and antibacterial activity giving partial scientific support for using such medicinal plants as antioxidants and antidiabetic and antibacterial agents.

2. Materials and Methods

2.1. Chemicals and Equipment

The chemicals and reagents used in the experimental section of this research were of analytical grade from Merck, T. Fisher Scientific, and Qualigens chemical companies, India. The major equipment used was Buchi RE111 rotavapor, Thermo Scientific Spectronic 20+ spectrophotometer, grinder, digital weighing machine (GT 210), column (Fortuna W.G.C, Optifix, Germany), micropipettes (Erba Biohit), and water bath (Physio Lab Scientific Industries, Ambala Cantt, India).

2.2. Collection and Identification of Plants

Plant materials were collected from the Bharatpur and Tandi areas of Chitwan, Nepal. The collected plants were identified at the Central Department of Botany, Tribhuvan University. The plant samples were washed under tap water to remove contaminants such as dust, soil, eggs, and larvae of insects. The clean plant samples were dried in the shade, ground to make powder, and stored in clean and labeled plastic bags. The list of plants collected, parts used, common names, and therapeutic uses are shown in Table 1. The voucher identification number is indicated as AK₁-AK₁₀.

2.3. Extraction of Plant Metabolites

The 100 g powder of each plant sample was extracted by cold percolation in methanol (300 mL) at 25°C for 48 hours with frequent agitation. The mixture was filtered through clean cotton. This process was repeated for the complete extraction, and the filtrate was concentrated in a rotary evaporator under reduced pressure at 55°C. The extracts were kept in a beaker, and the percentage yields of the dried plant extracts were calculated. The extracts were stored at 4°C until the biological and chemical analyses were carried out.

2.4. Phytochemical Analysis

The preliminary qualitative phytochemical analysis of plant extracts was performed by color differentiation reactions adopting the protocol [30].

2.5. DPPH Radical Scavenging Activity

The use of 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a rapid, simple, and inexpensive method to measure the radical scavenging activity by adopting the standard protocol which was previously described by Jamuna et al. [31]. The percentage of DPPH radical scavenging was calculated by using the equation:where is the absorbance of the control DPPH solution and methanol and is the absorbance of the test sample. IC₅₀ (50% inhibitory concentration) is the effective concentration of the sample that is required to scavenge 50% of the DPPH free radicals. IC₅₀ values were calculated using the linear regression equation graphically with GraphPad Prism 9 software.

2.6. In Vitro α-Amylase Inhibitory Studies

The α-amylase inhibition assay was performed by adopting the standard protocol of Kusano et al. with slight modifications [32]. The excess starch due to α-amylase inhibition was measured at 630 nm as a blue, starch-iodine complex. 200 mg starch was dissolved in 25 mL of NaOH (0.4 M) by heating at 100°C for 5 minutes to prepare the substrate. After cooling, the pH was adjusted to 7.0, and the final volume was made to 100 mL by adding distilled water. Acarbose was used as a positive control. 400 μL of test solution was preincubated at 37°C for 5 minutes with 200 μl of acarbose or plant extract with varying concentrations (40, 80, 160, 320, and 640 μg/mL), followed by 200 μL of 50 μg/mL α-amylase (20 mM phosphate buffer with 6.7 mM NaCl, pH 6.9), and incubated at 37°C for 15 min. Terminations of the reaction was carried out by adding 800 μL of HCl (0.1 M). After that, 1000 μL of 2.5 mM iodine reagent was added to the mixture, and the absorbance was measured at 630 nm. The triplicate reading was measured by using a spectrophotometer. The α-amylase enzyme inhibition was expressed as percent inhibition calculated using the equation given below: The % α-amylase inhibition was plotted against the extract concentration, and the IC₅₀ values were calculated graphically:

Abs₁ is the absorbance of the incubated mixture containing plant sample, starch, and α-amylase, Abs₂ is the absorbance of the incubated mixture of sample and starch, Abs₃ is the absorbance of the incubated mixture of starch and α-amylase, and Abs₄ is the absorbance of the incubated solution containing starch.

2.7. Antibacterial Activity Agar Well Diffusion Method

Antibacterial activity of the plant extracts was performed using the agar well diffusion method. The antimicrobial activity was evaluated by measuring the zone of inhibition (ZOI) [11]. The bacterial strains were obtained from the Central Department of Microbiology, Tribhuvan University. The strains included a Gram-positive bacterium (Staphylococcus aureus ATCC 25923) and a Gram-negative bacterium (Escherichia coli ATCC 25922). The organisms to be tested were aseptically touched with the help of an inoculating loop from the primary culture plate. Then, it was transferred into a test tube with 10 mL of sterile liquid media of nutrient broth and incubated overnight at 37°C. The media used in the study were prepared according to the manufacturer’s recommendation [33].

The prepared sterile Muller–Hinton Agar (MHA) plates were dried to remove the excess moisture from the surface of the media. The agar plates were prepared by labeling them with the name of the bacteria and the name code of the disc. The inocula of bacteria were transferred into a Petri dish containing solid nutrient media of agar using a sterile swab. The sterile cotton swab was dipped into the prepared inocula and swabbed carefully all over the plates. The plate was rotated through an angle of 60° after each swabbing. Finally, the swab was passed around the edges of the agar surface. The inoculated plates were left to dry for minutes at room temperature with a lid closed. For each incubated media plate, wells were made of 6 mm diameter with the help of sterile cork borer no. 6 and labeled properly. Then, 50 µL of the working solution of the plant extract, DMSO as a negative control, and chloramphenicol as a positive control at the same time in the separate well were loaded. The plates were kept for half an hour with a closed lid so that the extract is completely diffused into the media. The plates were kept in an incubator overnight at 37°C. After 24 hours of incubation, the plates were observed for the presence of inhibition of bacterial growth indicated by a clear zone around the wells. The size of the zone of inhibition was measured, and the antibacterial activity was expressed in terms of the average diameter of the zone of inhibition in millimeters. The absence of the zone of inhibition was interpreted as the absence of bacterial activity. The ZOI was measured with the help of a millimeter ruler, and the mean was calculated [34].

2.8. Extraction and Isolation of Compounds

The extract of Nyctanthes arbor-tristis bark with promising antioxidant and antimicrobial activity was selected for the isolation of chemical constituents. The shade-dried and powder bark of N. arbor-tristis (300 g) was extracted exhaustively with methanol by cold percolation. 30 g of methanol extract was fractionated with hexane, ethyl acetate, and dichloromethane of increasing polarity successively. The ethyl acetate soluble fraction showed good separation with a large number of spots in TLC and was subjected to column chromatography to isolate the pure chemical compounds. A slurry was made by mixing 1.8 g of the ethyl acetate fraction with silica gel and loaded onto a silica gel (200 g, E-Merck, 60–120 mesh) packed column having an internal diameter of 4.5 cm with an adsorbent height of 60 cm. The column was eluted with gradients of ethyl acetate in hexane and MeOH in EtOAc to obtain several fractions. The polarity of the eluent was changed after the TLC report of each fraction. The number of spots observed in TLC was visualized under UV-visible light and an iodine chamber.

2.9. Analytic Conditions for FTIR

FTIR is an analytical device used to detect functional groups in the organic compounds providing the covalent bonding information (IRTracer-100 Shimadzu). In FTIR, infrared radiation of about 400 to 4000 cm⁻¹ is sent through a sample in which some radiation is absorbed and passed through the sample. The radiation absorbed by the molecule is converted into rotational/vibrational energy that produces a signal at the detector in the form of a spectrum typically from 400 cm⁻¹ to 4000 cm⁻¹ representing a molecular fingerprint of the sample. The 21 scans were used for the better resolution of the IR spectra. Each chemical compound will produce a unique spectral fingerprint, making FTIR analysis a great tool for chemical identification.

2.10. Analytic Conditions for GC

GC analysis was performed on the gas chromatography-mass spectrometer GCMS-QP 2010 under the conditions: injection volume 1 µL with a slit ratio of 1 : 90, helium as a carrier gas with an Rtx-5MS column of dimension 30 m × 0.25 mm × 0.25 µm, and temperature programmed at 50, 150, and 250°C with a hold time of 0.0 and 4.0 min identification was accomplished by comparison of MS with those reported in NIST 08 and FFNSCI.3 libraries [35, 36].

2.11. Statistical Analysis

Data were recorded as a mean of three determinations of absorbance for each concentration, from which the linear correlation coefficient () value was calculated. The data were measured as a mean ± standard deviation (SD). The regression equation is given as Y = mx + c, where Y is the absorbance of extract, m is the slope from the calibration curve, x is the concentration of extract, and c is the intercept. The regression equation was used to calculate the concentration of extracts. All the determinations were conducted at least 3 times (n = 3); the statistical mean was calculated with ±SD using Microsoft Excel 2013. Means were compared using one-way ANOVA followed by the Tukey test with SPSS version 29 software. Values with were considered significantly different.

3. Results

3.1. Phytochemical Analysis

The results of the preliminary qualitative phytochemical analysis are shown in Table 2.

The phytochemical analysis showed positive results for glycosides, alkaloids, and terpenoids. The result shows that terpenoids were present in all the extracts, and reducing sugars and glycosides were present in most of the plant extracts. Flavonoids were present only in Neolamarckia cadamba, Desmostachya bipinnata, Nyctanthes arbor-tristis, and Phoenix dactylifera. Coumarin was present only in Phoenix humilis.

3.2. Antioxidant Property

In this study, the effect on the free radical scavenging ability was determined through the DPPH assay because it is one of the most effective, reactive, reliable, and simple in vitro methods. The results of percentage radical scavenging at a different concentration of plant extract with standard ascorbic acid are shown in Figure 2.

The scavenging effects of both plant extracts and the standard on the DPPH radical were expressed as half-maximal inhibitory concentration (IC₅₀) values, and the results are reported in Table 3. The linear regression of the percentage of radical scavenging versus concentration was used for the calculation of the concentration of each plant extract required for 50% inhibition of DPPH activity (IC₅₀) Table 3. The antioxidant potential has an inverse relation with the IC₅₀ value; the lower value of IC₅₀ indicates high antioxidant potential. Antioxidants are tremendously important substances that possess the ability to protect the body from damage caused by free radical-induced oxidative stress.

According to the results obtained, Nyctanthes arbor-tristis, Mallotus philippensis (seed cover), Pogostemon benghalensis, and Litsea monopetala extract showed promising antioxidant activity with the IC₅₀ values of 27.38 ± 1.35, 32.08 ± 2.81, 32.75 ± 2.13, and 33.82 ± 1.07 µg/mL. The values were comparable to the IC₅₀ values of 24.86 ± 4.6 µg/mL for ascorbic acid used as a positive standard. The plant extract of Phoenix humilis, Neolamarckia cadamba, Equisetum debile, and Desmostachya bipinnata showed moderate antioxidant activity with IC₅₀ values of 40.14 ± 0.93, 78.15, 50.44 ± 3.75, 80.14 ± 4.49, and 81.49 ± 1.59 µg/ml, whereas the plant extracts of Spilanthes uliginosa Swartz and Mallotus philippensis (seed) impart the poor antioxidant activity with IC₅₀ values of 93.41 ± 10.04 and 122.98 ± 6.29 µg/mL, respectively.

3.3. Antidiabetic Activity

The graphical relationship between the α-amylase enzyme inhibition and the concentration of plant extracts is shown in Figure 3.

The α-amylase enzyme inhibition assay showed that some of the plant extracts had significant inhibitory concentration and found promising α-amylase inhibition activity, while the remaining extracts showed mild to poor. The inhibitory concentration (IC₅₀) is shown in Table 4.

Many plant extracts have been reported for their antidiabetic activities and are currently being used in ayurveda for the treatment of diabetes. However, there is a lack of scientific validation for using these medicinal plants against diabetes and infectious diseases. In the present study, ten indigenous medicinal plants used against diabetes were screened for their α-amylase enzyme inhibition activity. The plant extracts of Phoenix humilis and Desmostachya bipinnata showed promising α-amylase enzyme inhibition activity with IC₅₀ values of 95.69 ± 6.97 and 99.24 ± 12.698.54 µg/mL, respectively. The plant extracts of Pogostemon benghalensis, Nyctanthes arbor-tristis, Spilanthes uliginosa Swartz, Litsea monopetala, and Equisetum debile exhibited moderate α-amylase inhibition, whereas Mallotus philippensis (seed cover), Mallotus philippensis (seed), and Neolamarckia cadamba exhibited poor α-amylase enzyme inhibition. Acarbose used as a positive standard displayed an IC₅₀ value of 28.88 ± 1.4 µg/mL.

3.4. Antibacterial Activity

In the present study, the antibacterial activity on ten medicinal plant extracts was recorded against E. coli (ATCC 25922) and S. aureus (ATCC 25923). The effects shown by the plant extracts against the bacterial strain as zones of inhibition (mm) are listed in Table 5.

The extracts of Nyctanthes arbor-tristis and Mallotus philippensis (seed cover) showed promising antibacterial activity with the highest ZOI of 26 and 24 mm, respectively, against S. aureus. The plant extracts of Litsea monopetala, Equisetum debile, and Phoenix humilis showed weak antibacterial activity with ZOIs of 14, 12, and 12 mm against S. aureus, respectively. The extract of Nyctanthes arbor-tristis and Mallotus philippensis (seed cover) showed high antibacterial activity against E. coli with ZOIs of 22 mm and 14 mm, respectively. The rest of the plant extracts were found to be poor or not active against these organisms.

3.5. Isolation of Active Fractions

Based on antioxidant and antimicrobial activities, the bark extract of Nyctanthes arbor-tristis was selected as the potent extract for the isolation of active constituents. Different fractions were collected, and purity was checked for each fraction by TLC. Fractions having a similar TLC report were mixed. The list of fractions collected is shown in Table 6.

Fractions 82–86 were seen as single spots in TLC, but the yield was very low (no significant yield), so the isolation of the compound was not carried out from these fractions. The isolation, characterization, and identification of the compounds are under process. The fractions that show the single spot seen in TLC were subjected to FTIR analysis. FTIR is a useful analytical tool to identify the functional groups in a molecule as each specific chemical band often has a unique energy absorption band. The FTIR spectrum of the fraction collected in 1% MeOH in EtOAc is shown in Figure 4.

The -COOH functional group of carboxylic acid showed IR spectra at 2500–3000 cm⁻¹, and the C=O group of ketones, the –CHO group of aldehydes, and the -COOR group of esters showed IR spectra at 1690–1760 cm⁻¹. Similarly, the functional group of amines (-NH₂) showed IR spectra at 1180–1360 cm⁻¹, and alcohols (-OH) and ethers (R-O-R′) showed IR spectra at 1080–1300 cm⁻¹. It shows the possibility of the presence of compounds having a functional group -COOH, -C=O, -CHO, -COOR, -NH_2, -OH, and R-O-R′ in the ethyl acetate fraction collected in 1% MeOH in EtOAc of N. arbor-tristis.

3.6. GC Analysis

The fraction collected in 1% MeOH in EtOAc was further subjected to GCMS analysis using a single spot-on TLC report. The gas chromatogram is shown in Figure 5.

The GC/MS analysis of the partially purified fraction of Nyctanthes arbor-tristis bark extract search (NIST 08 and FFNSCI.3) revealed the presence of several compounds. The number of peaks was seen in the gas chromatogram out of which 4 compounds were visualized clearly. The major constituents present in the partially purified fraction (1% methanol in ethyl acetate) of Nyctanthes arbor-tristis bark were hexadecanoic acid, methyl ester, 11,14,17-eicosatrienoic acid, methyl ester, docosanoic acid, methyl ester, and 2-chloroaniline-5-sulfonic acid. The chemical constituents are shown in Table 7.

4. Discussion

The results of the phytochemical analysis showed that plants are rich sources of secondary metabolites. The results of the present study were found to be different from the previously reported results on the phytochemical analysis of some medicinal plants (Ganjewala et al. 2013) [21]. The difference in the phytochemicals as secondary metabolites is due to the variation in altitude of plants, different environmental conditions, method and time of sample collection, extraction procedure, and also due to lab setup and chemical grades [37]. Ascorbic acid can be used as a standard antioxidant showing strong DPPH scavenging activity. Several studies have demonstrated a linear correlation between antioxidant activity and the phenolic content of plant extracts [38, 39]. The results of the present study were compared to the previously reported results of some medicinal plants and found to be almost similar in most of the cases. The antioxidant activity of N. arbor-tristis extract was found having an IC₅₀ value of 57.93 µg/ml [40]. The same trend for DPPH scavenging activity has been shown in previously reported results of the antioxidant activity (IC₅₀) for leaf, flower, root, stem, and fruits of L. camara extracts as 16.02 ± 0.94, 28.92 ± 0.19, 31.52 ± 0.74, 46.96 ± 2.51, and 90.11 ± 0.57 µg/ml, respectively. The results are comparable to the results of the present study [41]. Saeidnia et al. reported the α-amylase enzyme inhibition activity of butanolic and methanolic extract of Z. majdae with IC₅₀ values of 24.5 ± 2.1 and 22.0 ± 2.7 mg/mL as compared to acarbose 6.6 ± 3.1 mg/mL. This result was compared to the results of the present study and found that the plant extracts exhibited moderate α-amylase enzyme inhibition activity [42]. Khan et al. 2013 reported the antibacterial activity of the cold-water extract of J. officinale (leaves) with a ZOI of 16 mm against P. aeruginosa followed by B. subtilis (13 mm), E. coli (7 mm), and P. vulgaris (7 mm). Similarly, cold-water extract of Santalum album (wood) exhibited a zone of inhibition (15 mm) against P. vulgaris followed by B. subtilis (14 mm), E. coli (10 mm), and P. aeruginosa (9 mm). The results of the present study showed that the plant extracts exhibited potent antibacterial activity as compared to the previously reported results [43]. The results of quantitative phytochemical analysis and antioxidant activity in this study were found comparable to the results reported by Bhatt and Dahal in the plant Elaeocarpus ganitrus in various solvent extracts of seed from the Ilam District of Nepal [44].

5. Conclusion

The present study provides the first pharmacological insight into the antioxidant, antidiabetic, and antibacterial potential of less explored selected medicinal plants from the Chitwan district of Nepal. Phytochemical analysis showed that plant extracts are found to be rich in phenolic and flavonoid content as secondary metabolites. The extracts of Nyctanthes arbor-tristis, Mallotus philippensis (seed cover), Pogostemon benghalensis, and Litsea monopetala showed high antioxidant capacity, whereas Phoenix humilis, Neolamarckia cadamba, Equisetum debile, and Desmostachya bipinnata showed moderate antioxidant capacity. These plant extracts showed significant α-amylase enzyme inhibition as compared to the most common drug acarbose indicating that the polyphenols and flavonoids present in the extracts reduce postprandial hyperglycemia by delaying carbohydrate digestion. An IC₅₀ value of 95.69 ± 6.97 µg/mL observed for Phoenix humilis was slightly higher than 28.88 ± 1.4 µg/mL observed for acarbose. The plant extracts of Nyctanthes arbor-tristis and Mallotus philippensis (seed cover) showed promising antibacterial activity against E. coli and S. aureus. The results of the present study could be made scientifically more valid by performing the in vivo assay in the isolated pure compounds from the active plant extracts selected in this study. The findings of the recent study would be useful for future research directions on the traditionally used medicinal plants in the development of nutraceuticals and pharmaceuticals as drug candidates in the future drug discovery process.

Abbreviations

DPPH:	2,2′-Diphenyl-1-picrylhydrazyl
IC₅₀:	The half-maximal inhibitory concentration
ROS:	Reactive oxygen species
µg/mL:	Microgram per milliliter
mg:	Milligram
ZOI:	Zone of inhibition
ATCC:	American-type culture collection
PPA:	Porcine pancreatic amylase
SD:	Standard deviation
GCMS:	Gas chromatography and mass spectrometry
FTIR:	Fourier-transform infrared spectroscopy.

Data Availability

The data that support the findings of this study can be obtained from the corresponding authors on request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Khaga Raj Sharma supervised the work and wrote the manuscript as well as final approval, Anita Khadka carried out the laboratory work and data analysis, and Akash Budha Magar carried out the statistical analysis and literature review. All the authors have read and approved the final manuscript.

Acknowledgments

The authors are thankful to the Central Department of Chemistry, Tribhuvan University, for the laboratory facilities, the Central Department of Botany, Tribhuvan University, for the identification of plants, the Chinese Academy of Sciences, Nepal, for providing a master’s thesis supporting grant to Anita Khadka, the Central Department of Microbiology, Tribhuvan University, for the antibacterial test, and the Department of Food Technology and Quality Control, Babar Mahal, Kathmandu, Nepal, for GC/MS analysis. This research was partially supported by the Chinese Academy of Science.

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