Phytochemical analysis, antioxidant, antimicrobial, and toxicity studies of Schima wallichii growing in Nepal

ABSTRACT

The present study focuses on qualitative and quantitative phytochemical and biological analysis in bark and leaf extracts of Schima wallichii growing in Gulmi, Nepal. TPC and TFC were measured using the Folin-Ciocalteu reagent and the aluminum chloride colorimetric method, respectively. The DPPH assay was used for antioxidant activity, and antimicrobial activity was studied with the agar-well diffusion method. The presence of polyphenols, flavonoids, quinones, coumarins, saponins, terpenoids, and tannins was detected by qualitative phytochemical analysis. Total phenolic content in an aqueous leaf extract and methanol bark extract ranged from 199 ± 5 to 219 ± 8 mg GAE/g. Similarly, the flavonoid content found between 6.0 ± 1.7 in bark to 33 ± 2 mg QE/g in leaf extract. The antioxidant activity of IC₅₀ 36 ± 3, 39 ± 1, 52 ± 3, and 73 ± 4 µg/mL for methanolic bark extract, methanolic leaf, aqueous bark, and aqueous leaf extracts, respectively. The bark methanol extract showed the most antibacterial activity with ZOI of 10.0 ± 0.3, 8.0 ± 0.3, 14 ± 0, and 11 ± 1 mm against Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, and Escherichia coli, respectively. Plant extracts were found to be nontoxic to mildly toxic to the brine shrimp nauplii. The plant is found to be rich in phenolics, and flavonoids showing significant antioxidant and antibacterial activities, and is nontoxic against brine shrimp nauplii. The plant imparts a health-promoting effect that needs to be explored in pharmaceutical research.

KEYWORDS:

• Schima wallichii
• Phenolic
• Flavonoid
• Antioxidant
• DPPH
• Antibacterial
• Toxicity

Introduction

Plants with beneficial pharmacological effects on humans or animals are categorized under medicinal plants^[1]. Evidence from fossils suggests people have been using plants as medicine for about 60,000 years.^[2] Medicinal plants provide affordable and accessible treatments with fewer side effects. The cultivation and harvesting of medicinal plants carry significant cultural significance in many communities.^[3] Plants also hold much importance in modern medicine as phytoconstituents are used as lead compounds in the modern drug discovery process. More than 200,000 chemicals including primary and secondary metabolites are isolated and identified from higher plants around the world.^[4] Therapeutic properties of plants result from specific compounds or synergistic interaction of many compounds. The ethnobotanical approach is used during the screening of medicinal plants to obtain bioactive lead compounds that can be further developed as pharmaceutical drugs. A wide range of pharmacophores and a high degree of stereochemistry contribute to the ability of natural product collections to provide hits during screening of potential drug candidates.^[5] The Royal Botanic Garden in Kew estimates 28,187 plants are used in medicine. Among them, fewer than 16% are cited in regulatory publications.^[6] Therefore, there is a need to explore different plant species and access their medicinal properties.

Any molecular species capable of independent existence and containing unpaired electrons can be defined as free radical species.^[7] They are produced in our body during normal essential metabolic processes or exposure to X-rays, ozone, cigarette smoking, air pollution, and industrial chemicals.^[8] Accumulation of free radicals during oxidative stress can damage nucleic acids, proteins, and lipids. This can lead to various diseases such as diabetes mellitus, cancer, asthma, cataracts, and neurodegenerative and cardiovascular diseases.^[9] Antioxidant molecules scavenge free radicals and prevent cellular damage.^[10] Such antioxidants may be enzymatic or non-enzymatic, synthetic or natural. Phenolic acids and flavonoids from fruits, vegetables, and herbs contain significant antioxidant activity.^[11]

We are in the era of bacterial resistance against prevalent antibiotics. Antibiotic-resistant bacteria are associated with higher cases of mortality, morbidity, length, and cost of hospitalization.^[12] Failure to yield noble antibiotics at this time of crisis has caused a lot of concern.^[13] The genomic approach to drug discovery has met with fairly limited success,^[14] resulting in a renewed interest in natural products.^[5] Phytochemicals such as alkaloids, flavonoids, tannins, saponins, and steroids are known to contain antibacterial activity.^[15] Many plant species contain toxins and display a wide variety of adverse effects when ingested by animals or humans. Plant secondary metabolites from groups such as alkaloids, glycosides, and terpenes are reported for their toxicity.^[16] The rising awareness about the health benefits of herbal preparation has also increased the risk of herbal intoxication in people.^[17] Such risks are mainly caused by misinformation, misidentification, and overdose of herbal remedies. Hence, the evaluation of plant extracts as herbal or traditional medicine needs to be combined with their toxicological study. Brine shrimp lethality assay is used for preliminary assessment of the toxicity of S. wallichii. It is a simple, quick, and reliable technique. There are reported correlations between the response in brine shrimp assay and in vivo and in vitro acute toxicity tests in Swiss albino rats.^[18]

Schima wallichii (DC.) Korth is an evergreen tree belonging to Theaceae family. It is commonly found in northern India, southern China, and Southeast Asia.^[19,20] It reaches up to 47 m in height and is found between 900 and 2100 m in altitude.^[21] It has white, fragrant flowers with yellow ovaries and leathery, lanceolate leaves with 8–12 pairs of secondary veins. The leaves and bark of S. wallichii pose various medicinal properties. Its bark is used to treat fever and pain by the local people of Sikkim and boiled leaves are used as a tonic in Northern Thailand.^[22,23] The bark is also reported to be used as an antiseptic, anthelmintic, vermicide, and cure for gonorrhea.^[24] Corolla of the plant is used to treat uterine disorders and hysteria by Malaysian people.^[20] A survey conducted by Seikh et al., (2015) reported the use of plant extract for extensive treatment of hypertension in diabetic patients in Northeast India. The same study reported a significant α-glucosidase enzyme inhibitory activity in the plant.^[25] Its antioxidant, antimicrobial, and anticoagulant properties are reported in literatures^[22,26]. The country of Nepal is rich in indigenous cultures and traditions. S. wallichii is used as traditional medicine in various parts of the country. In the Nuwakot district of Nepal, a decoction of root is used for diarrhea and dysentery, whereas juice is used for gastric problems.^[27] Local people of Rashuwa, Nepal, use bark powder to treat cuts and burns.^[28] Indigenous Gurung people use the crushed roots for scorpion bites and the Satar tribe uses bark for fever, pain, and bone fracture.^[29,30] The leaves of the plant are also used as fodder and bedding. The present study focuses on the quantification of total phenolic and flavonoid content, antioxidant, antifungal, antimicrobial, and toxicity of S. wallichii methanol and aqueous extract. To the best of our knowledge, a comparative study of phytochemicals, biological activity, and toxicity in leaves and barks of the plant using methanol and aqueous solvent is the first attempt in this study. The photographs of the plant samples collected from the study site are shown in Figure 1.

Figure 1. Photographs of the leaves and barks of the plant from the collection site and the herbarium sample.

Materials and methods

Collection and identification of plant sample

Leaves and barks of S. wallichii were collected from Tamghas 7, Gulmi, Nepal, in November 2021. The coordinates of the collection site are 28° 03’ 41” N and 83° 14’ 48” E and the elevation is 1610 m. A herbarium sample was sent to the Central Department of Botany, Tribhuvan University, Nepal, for identification (voucher no. TUCH-21051). The harvested plant materials were cleaned and air-dried to a constant weight.

Preparation of methanol and aqueous extracts

The 300 g of each plant powder was weighed and equally divided into two conical flasks. Five hundred millilitres of methanol was added to one conical flask, whereas the same amount of distilled water was added to another conical flask as solvent. After 24 h, the contents of the conical flask with distilled water were filtered using a clean muslin cloth. The residue was resuspended in the same conical flask with distilled water. The filtrate was again filtered using Whatman filter paper 1. The resultant filtrate was concentrated using a rotavapor (Buchi RE111) at 40 ^○C. The process was repeated three times. A similar process was carried out for methanol solvent at intervals of 72, 48, and 24 h.

Phytochemical analysis

The qualitative preliminary phytochemical analysis of plant extract was performed according to the protocol reported.^[31,32]

Alkaloids

Five millilitres of plant extract in a test tube was concentrated to yield a residue. Thus, the obtained residue was dissolved by adding 1.5 mL of 2% (v/v) HCl followed by adding three drops of Meyer’s reagent (0.679 g of HgCl₂ and 2.5 g of KI in 50 mL distilled water). The formation of white precipitates indicates the presence of alkaloids.

Phenols

The plant extract was mixed with 2 mL of 2% FeCl₃ solution and observed for the formation of blue, green, or black coloration.

Flavonoids

The plant extract was mixed with a few pieces of magnesium followed by a dropwise addition of conc. HCl. The formation of a pink scarlet color after a few minutes indicates the presence of flavonoids.

Tannins

Two millilitres of 5% FeCl₃ was added to 2 mL of plant extract and observed for the formation of yellow or brown precipitates.

Reducing sugars

A total of 2.5 mL of Benedict’s solution (17 g of trisodium citrate dihydrate, 10 g of Na₂CO₃, and 1.74 g of copper sulfate pentahydrate in 100 mL of distilled water) was taken in a test tube followed by adding 0.5 g of plant extract and then warmed in a hot water bath for about 5 min. The formation of green/red or yellow coloration indicates the presence of reducing sugar.

Saponins

The plant extract was mixed with 5 mL of distilled water and then shaken vigorously. The formation of stable foam indicates the presence of saponins.

Coumarin

A single pellet of KOH was dissolved in 1 mL of ethanol. Then, 1 mL of extract solution was added. The formation of precipitates indicates the presence of coumarins.

Terpenoids

A small amount of plant extract was dissolved in chloroform and an equal volume of conc. H₂SO₄ was added. Reddish-brown coloration at the junction of two liquids indicates the presence of terpenoids.

Quinones

One millilitre of conc. H₂SO₄ was added to 1 mL of plant extract solution. The formation of red coloration indicates the presence of quinones.

Sterols

Two millilitres of plant extract was mixed with chloroform. Overall, 1–2 mL of acetic anhydride was added followed by adding one or two drops of conc. H₂SO₄ from the side of the test tube. An array of red, blue, and green colors indicates the presence of sterols.

Glycosides

Two millilitres of glacial acetic acid, one drop each of 5% FeCl₃ and conc. H₂SO₄ was added to 5 mL of plant extract. The appearance of a brown ring indicates the presence of glycosides.

Proteins

Plant extract is mixed with 2 mL of Millon's reagent and observed for a white precipitate that turns red on gentle heating.

Total phenolic content (TPC)

The total phenolic content of plant extracts was measured by using the Folin-Ciocalteu phenol reagent method with slight modifications.^[33] A total of 20 µL each of Gallic acid solutions (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µg/mL in methanol) and plant extract solutions (500 µg/mL in 50% DMSO) were added in triplicates to different bores of a 96-well plate. One hundred microlitres of 1:1 v/v FCR diluted with distilled water and 80 µL of 1 M Na₂CO₃ were added to each bore. The reaction mixture was placed in a dark place for about 25 min and then absorbance was measured at 760 nm using a microplate reader (SYNERGYILX, BioTek Instruments Inc., USA). TPC was calculated using the regression equation, y = 0.023× (R² = 0.982) obtained from the standard calibration curve of gallic acid and expressed in terms of milligrams of gallic acid equivalent per gram dry weight of plant extract (mg of GAE/g).

Total flavonoid content (TFC)

The total flavonoid content of the plant extracted was estimated by using the aluminum chloride colorimetric method with slight changes.^[34] A total of 130 µL each of quercetin solutions (15.4, 30.8, 46.2, 61.6, 77, 92.4, 107.8, 123.2, 138.6, and 154 µg/mL in methanol), 60 µL of ethanol, 5 µL of AlCl₃ and 5 µL of CH₃COOK were added in triplicates to different bores of a 96-well plate. Similarly, 20 µL of plant extract solutions (500 µg/mL in 50% DMSO), 110 µL of distilled water, 60 µL of ethanol, 5 µL of AlCl_3, and 5 µL of CH₃COOK were added in triplicates to the remaining bores of the 96-well plate. The reaction mixture was placed in the dark for about 30 min and then absorbance was measured at 415 nm using a microplate reader. TFC was calculated by using the regression equation, y = 0.024× (R² = 0.993) obtained from the standard quercetin curve and expressed in terms of milligrams of quercetin equivalent per gram dry weight of plant extract (mg of QE/g)

Brine shrimp toxicity

Brine shrimp toxicity assay was performed using the methods described.^[35] Two millilitres each of plant extracts (10, 100, 1000 µg/mL in methanol) were added to different test tubes in triplicates, and the solvent was evaporated to dryness using a water bath. An equal volume of methanol was taken as the negative control. The leftover residue in the test tubes was dissolved with 5 µL of artificial seawater, and 10 healthy Brine shrimp nauplii were added to each test tube. The number of surviving nauplii was counted after 24 h. The concentration of plant extract lethal to half of the test organism (LC₅₀) was calculated using the percentage mortality versus concentration curve.

Antibacterial activity

Antibacterial assay of plant extract was performed by using the agar well diffusion method described.^[36] American-type culture collection of gram-positive (Staphylococcus aureus, ATCC 25,923; Bacillus subtilis, ATCC 35,021) and gram-negative (Klebsiella pneumoniae, ATCC 700,603; Escherichia coli, ATCC 25,922) bacteria were used as test organisms. Broth cultures of test bacteria were prepared, and the concentration of test organism was maintained at 0.5 McFarland standard (10^6–8 CFU/mL). One hundred microlitres of inoculum was spread on MHA plates. Twenty microlitres of plant extract solutions (25 mg/mL in DMSO) were added in triplicates to the wells (7 mm diameter) of MHA plates and incubated at 37 ^○C. After 24 h, the zone of inhibition (ZOI) was measured using a ruler. Ampicillin (1 mg/mL) was used as standard and DMSO was used as blank.

Antifungal activity

The antifungal assay was performed by using the agar well diffusion method following the standard protocol.^[36] Fusarium solani was used as a test organism. Overnight incubated broth culture of the fungus was prepared in potato dextrose agar (PDA). One hundred microlitres of microbial pathogen (0.5 McFarland) was spread on PDA plates and wells of 7 mm diameter were bored. Overall, 20 µg/mL of plant extracts (25 mg/mL in DMSO) were loaded into each well and incubated at 37 ^○C. The ZOI was measured after 48 h. DMSO was used as a control and cycloheximide (20 mg/mL) was used as standard.

Antioxidant activity

2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to determine the free radical scavenging activity of plant extracts.^[37] One hundred microlitres of DPPH solution (0.1 mM in ethanol) and 100 µL of plant extract solutions of different concentrations (7.81, 15.62, 31.25, 62.5, 125, 250, 500, and 1000 µg/mL in 50% DMSO) were added in triplicates to the bores of a 96-well plate. The reaction mixture was incubated in the dark for 30 min and then, absorbance was measured at 517 nm by a microplate reader. Fifty percent DMSO was used as blank and quercetin was used as standard.

$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$

Half maximal inhibitory concentration (IC₅₀) was calculated from percentage scavenging at different concentrations of plant extract using GraphPad Prism 9 software.

Statistical analysis

All the tests were performed in triplicates, and the values are expressed as mean ± standard error (n = 3). The results were compared with one-way ANOVA followed by Tukey’s test using SPSS version 29 software. The values with p < .05 were considered statistically different.

RESULTS and DISCUSSION

Phytochemical analysis

The results of the preliminary qualitative phytochemical analysis are shown in Table 2. The identification number, site of plant collection, parts used and the traditional medicinal applications are shown in Table 1.

Table 1. Voucher number, the plant used, and traditional medical practices.

Name of the plant	Identification no.	Altitude/plant growing	Plant parts	Traditional uses
Schima wallichii	TUCH-21051	altitude 1610 m, coordinates 28° 03’ 41” N 83° 14’ 48” E	Bark	uterine disorder and hysteria^[Citation20] fever and pain^[Citation22]
			Leaf	tonic^[Citation23]
				antiseptic, anthelmintic, vermicide, and, treatment of gonorrhea^[Citation24] Hypertension^[Citation25] diarrhea, dysentery, and gastric problems^[Citation27] cuts and burns^[Citation28] Scorpion bites^[Citation29] bone fracture^[Citation30]

Bark and leaf extracts of S. wallichii were found to be rich in primary and secondary metabolites. Polyphenols, flavonoids, quinones, coumarins, saponins, terpenoids, and tannins were detected in all extracts of S. wallichii while alkaloids and sterols were completely absent. Glycosides and sugar were detected in methanol extracts and proteins were found in aqueous extract (Table 2). The presence of saponin, terpenoid, flavonoid, tannins, and phenolic compounds in ethanol and aqueous extract of the plant was also reported in the literature.^[26] Phytochemicals are an important source of bioactive compounds. Many phytochemicals play a significant role in their bioactivity in the modern drug discovery process. It has been reported that the compound Kaempferol-3-O-rhamnoside isolated from the leaves of S. wallichii displays significant anticancer activity.^[38] The presence of important phytochemicals in S. wallichii makes it an important medicinal plant.

Table 2. Qualitative phytochemical analysis of plant extracts.

Group of compounds	SBM	SBA	SLM	SLA
Alkaloids	-	-	-	-
Coumarins	+	+	+	+
Flavonoids	+	+	+	+
Glycosides	+	-	+	-
Polyphenols	+	+	+	+
Quinones	+	+	+	+
Reducing Sugars	+	-	+	-
Saponins	+	+	+	+
Terpenoids	+	+	+	+
Tannins	+	+	+	+
Proteins	-	+	-	+
Sterols	-	-	-	-

SBM, Bark methanol extract; SBA, Bark aqueous extract; SLM, Leaf methanol extract; SLA, Leaf aqueous extract; +, present; -, absent.

Total phenolic and flavonoid content

Polyphenols are an important and diverse group of plant secondary metabolites with more than 8000 phenolic structures currently known. They are involved in growth, reproduction, pigmentation, protection from ultraviolet radiation, and pathogens in plants.^[39] Phenolic compounds are responsible for most of the antioxidant activity of plants. The biological activities of phenolic compounds such as subduing oxidative stress, protecting from neurodegenerative disease, and reducing the risk of cardiovascular disease can be attributed to their antioxidant activities.^[40] Flavonoids are the most common and diverse group of phenolic compounds. They display a variety of pharmacological and health-promoting effects depending upon their structure. Antibacterial, hepatoprotective, anticancer, antiviral, and anti-inflammatory activity of flavonoids has been reported.^[41] Extracts of S. wallichii were found to be rich in phenolic and flavonoid contents. The highest concentration of phenolics was found in SBM (219.42 ± 8.44 mg GAE/g), followed by SBA (211 ± 4.01 mg GAE/g), SLM (204 ± 9.45 mg GAE/g), and SLA (199 ± 4.10 mg GAE/g) (Table 3). TFC was found to be highest in SLM (33.45 ± 1.1). SLA, SBA, and SBM contained a TFC of 28.05 ± 2.6, 6.22 ± 0.5, and 6 ± 1.7 mg QE/g, respectively (Figure 2). The bark extracts display higher concentrations of phenolic content, whereas leaf extract contains comparatively more flavonoids as high concentrations of flavonoids are located in the mesophyll cells of leaves. A similar trend of plants displaying higher phenolic compounds in barks and higher flavonoid content in leaves was also reported for Berberis baluchistanica.^[42] Phenolic content was also reported in ethyl acetate and chloroform fraction of S. wallichii bark as 163.4 ± 2.22 µg and 90.35 ± 3.11 µg per mg of the fraction.^[43] The same study found the flavonoid content to be 57.32 ± 2.31 µg and 28.27 ± 1.1 µg per mg of the ethyl acetate and chloroform extract. The estimated amount of the phenolic and flavonoid contents may vary according to temperature, rainfall, soil composition, and rainfall.^[44] Nevertheless, the extracts of S. wallichii display significant phenolic and flavonoid contents that may explain its antioxidant and antibacterial activity observed in the present study and its prevalence in traditional medicine.

Figure 2. TPC (mg GAE/g) and TFC (mg QE/g) of plant extracts.

Table 3. TPC, TFC, and DPPH free radical scavenging activity (IC₅₀) of different extracts of S. wallichii.

Plant extracts	TPC (mg GAE/g)	TFC (mg QE/g)	IC50 (µg/mL)
SBM	219 ± 8	6 ± 2^b, c	36 ± 3^a, b, d
SBA	211 ± 4	6.2 ± 0.5^b, c	52 ± 3^a, b
SLM	204 ± 9	33 ± 1	39 ± 1^a, b
SLA	199 ± 4	28 ± 3	73 ± 4^a
*Quercetin	#	#	3.4 ± 0.2

Values are mean ±SE (n = 3); * positive control; #, values not measured; ^{a, b, c, d} p < .05 vs. quercetin, SLA, SLM, and SBA.

Antioxidant potential

The results of the antioxidant assay are shown in Figure 3. The antioxidant activity of the plant extracts is measured in terms of DPPH free radical scavenging activity. Hydrogen atom donated by antioxidants reduces the odd electron of nitrogen in the DPPH molecule resulting in the loss of violet color.^[45] S. wallichii plant extracts contained significant DPPH free radical scavenging activity. Concentration-dependent increment in radical scavenging activity was observed. Half maximal inhibitory concentration for plant extracts was 36.21 ± 3.06 µg/mL (SBM) < 39.11 ± 1.14 µg/mL (SLM) < 52.02 ± 3.19 µg/mL (SBA) < 73 ± 4.1 µg/mL (SLA). The antioxidant activity was found to be higher in bark extracts than in leaf. Although the observed IC₅₀ values were higher than that of quercetin (3.38 ± 0.23 µg/mL) used as a positive standard, all the values were less than 100 µg/mL. A loose correlation between total phenolic content and antioxidant activity was observed as SBM with the highest phenolic content displayed the most and SLA with the lowest phenolic content displayed the least antioxidant activity. However, SLM with lower phenolic content displayed higher antioxidant activity than SBA. Such results may have been caused by the presence of new chemotype antioxidants other than phenolics in SLM.^[46] The observed IC₅₀ values for plant extracts were comparatively higher than 3.38 ± 0.23 µg/mL recorded for quercetin used as a positive standard. All the values were significantly different from those of the quercetin and SBM and were significantly different from those of the SLM and SBA at p < .05. Similarly, both SBA and SLM were also significantly different from SBA. The estimated IC₅₀ value for methanol and aqueous extract of bark was lower than that of ethanol (98.7 µg/mL) and aqueous (257 µg/mL) extract of bark reported by Bhattacharjee et al., (2019)^[26] Das et al., (2012) reported IC₅₀ values of 63.30 ± 3.33 µg/mL, 18.70 ± 3 µg/mL, and 7.33 ± 3.32 µg/mL for petroleum ether, chloroform, and ethyl acetate fraction of S. wallichii bark.^[43] The present study and the results available in the literature suggest that S. wallichii extracts were found to be rich in antioxidants. Natural antioxidants have applications in food, cosmetics, pharmacology, and medicine.^[47] The high antioxidant activity of the plant holds its importance in natural product research.

Figure 3. Plot of percentage of DPPH radical scavenging against the concentration of the plant extracts and standard quercetin.

Antimicrobial activity

Plant extracts exhibited significant activity against both gram-positive and gram-negative bacteria, but significant activity was not observed against the fungal species Fusarium solani (Table 4). All four extracts were active against K. pneumoniae, B. subtilis, and E. coli, whereas S. aureus, a multidrug-resistant bacterium was only susceptible to SBM (Figure 4) Inhibition toward bacterial growth was not observed in 50 % DMSO used as the negative control. Maximum ZOI among extracts was displayed by SBM (8 ± 0.33 mm against S. aureus and 14 ± 0 mm against B. subtilis) and SLM (11 ± 0 mm against K. pneumoniae and 12 ± 0.33 mm against E. coli). The ZOI displayed by methanol extracts was higher than that of their aqueous counterpart. A similar trend was reported by Dwivedi et al., (2020) for methanol and aqueous extracts of leaves of Carica papaya.^[48] The results indicate that the presence of plant secondary metabolites with antibacterial properties is at a higher number and concentration in methanolic extract than their aqueous counterparts. ZOI of SBM (14 ± 0 mm) against B. subtilis was comparable to that of Ampicillin (15 mm) used as a positive standard. Ampicillin also displayed a ZOI of 25 mm against K. pneumoniae, 30 mm against S. aureus, and 22 mm against E. coli, whereas cycloheximide displayed a ZOI of 27 mm against F. solani. Consequently, the observed values were significantly different from each other and the positive control at p < .05. The ZOI of 14 ± 0.7 mm and 13 ± 0 against E. coli and B. subtilis for methanol extract (25 mg/mL) of leaf were similar to 20 mm and 17 mm reported for ethanol extract (50 mg/mL).^[49] ZOI of 19 mm against S. aureus was reported for ethanol extract of leaf in the same report, whereas no ZOI was observed for methanol extract of leaf in the present study. Differences in solvent and concentration of plant extracts may have caused such dissimilarities. The variation in bacterial susceptibility for different plant extracts in the present study may be due to differences in bacterial tolerance, antimicrobial resistance mechanism, types of phytochemicals, their concentration, and underlying synergistic and antagonistic interactions. The antibacterial activity displayed by the compound may be due to the presence of flavonoids, phenolics, saponins, and quinones.^[50] Further study of the plant extracts including isolation and characterization of specific secondary metabolites might provide a lead compound for novel antibacterial medicine.

Figure 4. Antibacterial slides: (a) K. pneumoniae, (b) S. aureus, (c) B. subtilis, and (d) E. coli, (e) F. solani; PC, positive control; NC, negative control; SBM, bark methanol extract; SBA, bark aqueous extract; SLM, leaf methanol extract; SLA, leaf aqueous extract.

Table 4. Antibacterial activity (ZOI) is shown by plant extracts as compared to positive and negative control.

Plant extracts	ZOI (mm) shown by the plant extracts
	K. pneumonia	S. aureus	B. subtilis	E. coli	F. solani
SBM	10.0 ± 0.3^a	8 ± 0.33^a	14 ± 0^b	11± 1^a, d	-
SBA	9.0 ± 0.3^a, c	-	13 ± 0.9^a	7.0 ± 0.3^a, b, c	-
SLM	11 ± 0^a, b	-	13 ± 0^a	14.0 ± 0.7^a	-
SLA	9 .0± 0.3^a	-	11.0± 0.3^a	12.0 ± 0.3^a	-
Ampicillin*	25	30	15	22	#
Cycloheximide*	#	#	#	#	27
100% DMSO^&	-	-	-	-	-

Values are the mean ± SE (n=3); * positive control; ^& negative control; -, no significant ZOI; #, value not measured; ^{a, b, c, d} p < .05 vs. ampicillin, SLA, SLM, and SBA.

Toxicity

The Brine shrimp assay was employed to estimate the toxicity. Concentration-dependent increments in percentage mortality were observed in the study. The percentage mortality versus concentration curve is given in Figure 5.

Figure 5. Percentage mortality vs. concentration curve for Brine shrimp assay.

Significant toxicity was not observed in the plant extracts (Table 5). Only the methanol extract of the bark displayed an LC₅₀ value of less than 1000 µg/mL. Lower toxicity of bioactive plant extract toward eukaryotic cells is considered beneficial from a pharmaceutical point of view.^[51] The decreasing order of LC₅₀ value was found to be SLM (1556 ± 126 µg/mL) > SLA (1198.41 ± 95.48 µg/mL) > SBM (1171.33 ± 79.60 µg/mL) > SBA (574.45 ± 17.7 µg/mL) that represents increasing order of toxicity. A statistically significant difference was observed for SBA at p < .05. The bark extracts of the plant were more toxic than leaf extracts. Similarly, aqueous extracts displayed less LC₅₀ value than their methanol extract counterparts. This may be due to the difference in phytochemicals and their concentration in different parts of the plant and different extracting solvents. The distribution of secondary metabolites within a plant is not uniform.^[52] The absence of alkaloids may support lesser toxicity of plant extracts as they are known to be the largest category of plant toxins.^[16]

Table 5. Lethality dose (LC₅₀) shown by plant extract against Brine shrimp nauplii.

Plant extract	LC50 (µg/mL)
SBM	1171.33 ± 79.60^c
SBA	574.45 ± 17.7^a, b
SLM	1556 ± 126
SLA	1198.41 ± 95.48

Values are the mean ± SE (n = 3). ^{a, b, c} p < .05 vs. SLA, SLM, and SBA.

Conclusion

Methanol and aqueous extracts of S. wallichii are rich in phenolic and flavonoid content. They display significant antioxidant and antibacterial activities. Plant extracts inhibited the growth of both gram-positive and gram-negative bacteria, but significant activity was not observed against fungal species. Antibacterial activity was higher in methanol extracts than in their aqueous counterparts. The plant extract does not display significant toxicity toward Brine shrimp nauplii. Thus, the plant is nontoxic and relatively safe. The present study supports the use of S. wallichii leaves and barks in traditional medicine. It also validates the high antioxidant and antimicrobial activities of the plant extract that needs to be explored. Further work should be carried out on the isolation, purification, characterization, and standardization of bioactive compounds.

Authors’ contributions

DS: Study design and experimental analysis. KRS: Original idea presentation, study supervision, and final approval of the version to be published, ABM: prepare a manuscript draft, data analysis SP: Literature review, data analysis.

Ethical approval

All the experimental works were carried out by a valid protocol with ethical approval.

Acknowledgments

The authors would like to acknowledge the Central Department of Botany, Tribhuvan University, Kathmandu, Nepal, for the identification of the plant. The Central Department of Chemistry, Tribhuvan University is grateful for providing the laboratory facilities.

Data availability statement

Access to raw data is possible upon justifiable request.

Disclosure statement

The authors declare no conflict of interest.