Materials, microorganisms, and chemicals

Five wild plants, namely, (bitter leaf), (betel), (Imlay), (kaduk), and (stinkvine), were studied, and their botanical names, common names, families, and geographical origins are presented in Table 1. After collection, the leaves were washed to remove dirt and impurities and then air-dried at 60°C to a constant weight. The dried leaves were ground using a commercial blender (model BJY-CB2L60-A, Berjaya Steel Product Sdn Bhd, Kuala Lumpur, Malaysia) and stored in PE bags at –4°C for further use.

Pathogenic microorganisms, including seven gram-negative bacteria ( ATCC 9290, ATCC 8739, ATCC 8090, ATCC 6539, ATCC 17802, ATCC 25933, ATCC 33291), three gram-positive bacteria ( ATCC 6538, ATCC 11778, ATCC 13932), and one yeast strain ( ATCC 10231), were kept frozen in Mueller–Hinton broth (MHB) medium containing 15% v/v glycerol.

Gallic acid, DPPH, TPTZ, ABTS, and Trolox were obtained from Sigma‒Aldrich (Singapore). Folin–Ciocalteu reagent (2 N) was prepared from solid sodium tungstate, sodium molybdate, and lithium sulfate. Ampicillin and Mueller–Hinton media were obtained from Hi-Media Laboratory (Mumbai, India).

Methanol, n-hexan, ethyl acetate, hydrochloric acid, potassium chloride, aluminum chloride monohydrate, sodium hydroxide, ferric chloride hexahydrate, ferrous sulfate, potassium dihydrogen phosphate, potassium ferricyanide, and other chemicals were of analytical grade.

Preparation of plant fractions

The dried leaf material (10 g) was macerated with 250 mL of 80% v/v methanol at room temperature for 3 days. After maceration, the mixture was filtered through Whatman No. 2 filter paper to remove insoluble components. The filtrate was acquired and evaporated under vacuum in a Hei-VAP Value rotary vacuum evaporator (Heidolph Instruments, Schwabach, Germany) at 55°C to remove solvent. The concentrate was then diluted to 100 mL with distilled water and fractionated with 50 mL of different solvents in order of increasing polarity, including n-hexane and ethyl acetate, using a separating funnel to obtain three fractions: the n-hexane fraction (HE), the ethyl acetate fraction (EA), and the residual aqueous fraction (W). These fractions were also dried to calculate the dry weight of each fraction.

Antioxidant activities

To prepare the analytical solutions for HE and EA, 1 mL of each fraction was transferred to a Petri dish where the solvent (n-hexane and ethyl acetate) had evaporated spontaneously. The residues were then redissolved and diluted to 10 mL using distilled water, while the W fractions were used directly as analytical solutions.

Total phenolic content (TPC)

The total phenolic content was determined according to the Folin–Ciocalteu method described in ISO 14502–1:2005 34 based on the reaction of antioxidants with Folin–Ciocalteu reagent in an alkaline medium to form a blue chromophore with maximum absorption at 765 nm. The phenolic content was calculated based on the gallic acid standard curve and is expressed in mg gallic acid equivalent per liter of extract (mg GAE/L).

DPPH free radical scavenging activity

Antioxidant activity was evaluated through DPPH free radical scavenging capacity based on the change in the purple color of the DPPH solution (0.6 mM) measured at 515 nm upon reaction with antioxidants 35. The antioxidant activity of DPPH was calculated against the Trolox calibration curve and expressed in mg Trolox equivalent per liter of extract (mg TE/L).

ABTS cation radical scavenging activity

ABTS free radical scavenging activity was determined based on the discoloration of ABTS (7.4 mM) solution measured at 734 nm upon reaction with the antioxidant 36. The ABTS cationic radical scavenging activity was calculated against the Trolox calibration curve and expressed in mg Trolox equivalent per liter of extract (mg TE/L).

Ferric reducing antioxidant power

Ferric reducing antioxidant power (FRAP) was determined according to37 based on the chromophores formed between the working reagents (a mixture of 0.3 M acetate buffer at pH 3.6, 0.01 M TPTZ prepared in 0.04 M HCl, and 0.02 M FeCl.6HO solution at a volumetric ratio of 10:1:1) and antioxidants. Ferric reducing antioxidant activity was calculated against the Trolox calibration curve and expressed in mg Trolox equivalent per liter of extract (mg TE/L).

Antibacterial activity – Agar well diffusion test

The antibacterial activities of the leaf fractions were determined by the agar well diffusion method as described in the literature38. The bacterial pathogens were grown in liquid media for 20 h for a final microorganism concentration of 10 CFU/mL. Subsequently, 100 mL of the test strains was spread over the surface of the agar disk. The sterilized filter paper discs were loaded with 50 mL of leaf fractions, and ampicillin (0.2 mg/mL) was used as a positive control before they were incubated at 37°C for 18 h. Finally, the inhibition zone diameter (mm), which represents the extent of bacterial inhibition of the extracts compared with that of the control samples, was measured.

Statistical analysis

All the statistical techniques, including the normality test, homoscedasticity of variances, one-way ANOVA, and post hoc Tukey test, were performed at the 5% significance level by using R version 4.1.2.

Total phenolic content

Phenolic compounds are major antioxidant components that are involved in many biological and functional activities for human health 39, 40. The total phenolic contents of different fractions, such as n-hexane (HE), ethyl acetate (EA) and water (W), from bitter leaves, betel, Imlay, kaduk, and stinkvine are shown in Table 2. According to the data obtained, the total phenolic content of the five leaves extracted from the three fractions decreased in the following order: betel (2095.09 mg GAE/L) > kaduk (1439.40 mg GAE/L) > stinkvine (1301.99 mg GAE/L) > bitter leaf (1149.36 mg GAE/L) > Imlay (715.00 mg GAE/L). Among the fractions, the EA fraction of betel leaves had the highest phenolic content (815.99 mg GAE/L), while the HE fraction of Imlay had the lowest phenolic content (27.87 mg GAE/L) compared with those of the HE, EA, W and other leaf extracts. Due to the difference in the extraction capacities of the solvents, it was found that the types of polyphenol compounds used were significantly different among the leaf extracts depending on the polarity of the solvent41. Similar results were also reported in the studies of Fasakin . 42 on the use of different solvents (methanol, ethanol, acetone, and ethyl acetate) on betel leaves, implying that methanolic and ethanolic extracts (90%, v/v) had the maximum phenolic content (205.2 and 202.9 mg GAE/g, respectively). In conclusion, the findings showed that the extraction solvent had an impact on the TPC extracted from each leaf. Water is the effective solvent for accessing bitter leaves, Imlay, and stinkvine, whereas the TPC was greater in betel and kaduk leaves extracted with EA and HE.

The color of the extract of each leaf was different for each fraction, and the changes in color of the different fractions, such as n-hexane (HE), ethyl acetate (EA) and water (W), from bitter leaf, betel, Imlay, kaduk, and stinkvine are shown in Figure 1. In the HE fraction, the color of the extracts was mostly green with a yellowish tint. However, the betel leaf extract had a different gray color than the other leaf extracts because the color level increased or decreased depending on the leaf type and the solvent polarity. In the EA fraction, the color of the leaf extract that had begun to darken and turn black clearly changed; specifically, the Imlay leaf extract had the darkest black color. In the W fraction, the Imlay extract had the darkest brown color compared to the other leaf extracts. Differences in the color of leaf extracts from other fractions are due to differences in plant species, chlorophyll content and polarity of the solvent used43.

DPPH and ABTS free radical scavenging activities

DPPH is a free radical widely used for evaluating antioxidant potential through its free radical scavenging activity44. The DPPH free scavenging activities of different fractions, such as n-hexane (HE), ethyl acetate (EA) and water (W), from bitter leaves, betel, Imlay, kaduk, and stinkvine are shown in Table 2. The antioxidant activity of DPPH in the five types of leaves ranged from 821.85 mg TE/L to 2505.35 mg TE/L and decreased in the following order: betel (25005.35 mg TE/L) > bitter leaf (4126.15 mg TE/L) > kaduk (2351.26 mg TE/L) > amloday (1873.30 mg TE/L) > stinkvine (821.85 mg TE/L). In general, the DPPH radical scavenging activities in the W fraction of bitter leaves, Imlay leaves, Kaduk leaves, and Stinkvine leaves were all greater than those in the HE and EA fractions, while in the EA extract of betel leaves, the DPPH free radical scavenging activity was also significantly greater (2270.81 mg TE/L) than that in the other fractions. The antioxidant activity of betel leaf extract was also reported in a study by Swapna .45, who demonstrated that the presence of phenols (chavicol, chavibetol, chavibetol acetate and eugenol) in betel leaves may be responsible for its antioxidant activity.

In addition, the ABTS free radical scavenging method is a more sensitive and stable method used in media with different pH values and is often used to evaluate the antioxidant capacity of polyphenol compounds46. Table 2 shows the antioxidant activity based on the ABTS free radical scavenging capacity of five leaves, the values of which ranged from 1921.35 to 30160.66 mg TE/L and were arranged in descending order: betel leaf (30160.66 mg TE/L) > bitter leaf (4712.16 mg TE/L) > kaduk (3213.77 mg TE/L) > stinkvine (2100.50 mg TE/L) > Imlay (1921.35 mg TE/L). The best leaf had the highest ABTS free radical scavenging activity, 15.7 times greater than that of Imlay. Notably, the ABTS and DPPH activities exhibited the same patterns. Specifically, for bitter leaves, the Imlay, kaduk, stinkvine, and W fractions had higher ABTS values than did the HE and EA fractions, while the EA fraction of betel leaves was superior to the other fractions. Similar results were reported in the study of Egharevba . 47 for the determination of the activities of different fractions, such as n-hexane (HE) and ethyl acetate (EA), from leaves, which showed that the EA fraction is a strong inhibitor of α-glucosidase, actively scavenging DPPH and ABTS free radicals. The different results of the fractions may be due to the presence of a high phenolic content in EA since phenolic compounds play an important role as antioxidants48.

FRAP

The FRAP free scavenging activities of different fractions, such as n-hexane (HE), ethyl acetate (EA) and water (W), from bitter leaves, betel, Imlay, kaduk, and stinkvine are shown in Table 2. The FRAP values of the five leaves varied from 1114.26 g TE/L to 3588.88 g TE/L and were in descending order: betel (3588.88 g TE/L) > kaduk (2110.46 g TE/L) > bitter leaf (1925.39 g TE/L) > stinkvine (1598.21 g TE/L) > Imlay (1114.26 g TE/L). It is evident that the FRAP values of betel leaves were outstanding and were the highest for the EA fraction, which is consistent with the findings of Mohammed 49

In addition, the results also showed the variation in FRAP values among the different fractions. The FRAP values of three of the five leaf types (bitter leaf, Imlay, and stinkvine) were greater for the W fraction than for the other two fractions, ranging from 629.20 to 1184.19 g TE/L. In contrast, betel and kaduk extracted by EA (1384.40 mg TE/L) and HE (1191.48 g TE/L) solvents exhibited higher FRAP activity than did those extracted by W. Similar results were also reported in the studies of Guleria . 50 on the fractions of fruit and Park .51 on the fractions of cultivated with .

Correlation

Correlations between total phenolic content (TPC) and antioxidant capacities (FRAP, DPPH and ABTS free radical scavenging activity) of different fractions, such as n-hexane (HE), ethyl acetate (EA) and water (W), from bitter leaves, betel, Imlay, kaduk, and stinkvine are shown in Table 3. The correlation between antioxidant activities and phenol content was also statistically significant (p ≤ 0.05). In general, the correlation coefficients for the relationship between ABTS and DPPH radical scavenging activity (0.996) and between TPC and FRAP (0.963) were the highest. The above result implied that TPC is responsible for FRAP activity, whereby higher phenolic contents result in stronger antioxidant activity. This result is in agreement with the findings of Zheng . 52, who reported a strong correlation between the total phenolic content and FRAP assay results for selected herbs. Interestingly, the total phenolic content in the present study did not correlate with DPPH or ABTS activity, which is similar to the findings of Rajurkar53 for several traditional Indian medicinal plants.

Antibacterial activity

Infectious diseases caused by drug-resistant bacteria are a worldwide concern, and plants are a natural source of many biological compounds with potential antibacterial properties54, 55. The antibacterial activities of different fractions, such as n-hexane (HE), ethyl acetate (EA) and water (W), from bitter leaves, betel, Imlay, kaduk, and stinkvine are shown in Table 4. According to the results, betel leaf has the best antibacterial properties among the five leaf types. All three fractions of betel (especially the EA and HE fractions) were resistant to most of the gram-positive and gram-negative bacteria included in the study. Although it did not have outstanding antibacterial activity like betel leaves, the EA fraction of four leaf types (bitter leaf, Imlay, kaduk, and stinkvine) had greater antibacterial activity than the HE and W fractions. In the EA fraction, the diameter of the inhibition zone ranged from 12–33 mm and was particularly sensitive to and . The W fraction showed weak antibacterial ability, and the diameter of the inhibition zone was only approximately 8–16 mm, particularly for bitter leaves, which inhibited only among the bacteria tested. In contrast to the antibacterial ability of the EA fraction, the HE fraction of the four leaf samples was mostly resistant to 2-3 bacterial strains, with less sensitive inhibition zones ranging from 11–17 mm. The results showed that the betel leaf extract had the greatest antibacterial ability against most bacteria.

This may be because betel leaves contain antibacterial compounds, even those against multidrug-resistant bacteria, such as hydroxychavicol, stearic acid, and palmitic acid 56. According to Muruganandam 57, high contents of phenols and flavonoids can impart high inhibitory effects on microorganisms. However, the biological activity of these compounds is strongly dependent on the chemical nature and polarity of the extraction solvent. Haminiuk 58 demonstrated that phenolic and flavonoid contents are significantly lower when these compounds are extracted with hexane. Therefore, the antibacterial ability of hexane extracts is also more limited than that of extracts from other polar solvents, such as water, methanol, ethyl acetate and ether, from betel leaves. These results are similar to those of the study by Armansyah . 59 on the antibacterial activity of the EA fraction from red betel leaves, which revealed that the EA fraction has a broad spectrum of antibacterial activity against all tested microorganisms (, and ).