Antibacterial and toxicity studies of phytochemicals from Piper betle leaf extract

2.1 Phytochemical extraction

GP stems and PB leaves from Mueang district, Phatthalung province, and PR fruits from Chulabhorn, Nakhon Si Thammarat province were collected from cultivated area. The voucher specimens of GP, PB and PR with herbarium accession number as 01579, 01582, 01,585 were deposited at herbarium of Walailak Botanic Park. The cleaned samples were placed in hot air oven at 40 °C for 3 days before being ground into a fine powder (mesh size of 0.25 mm). The phytochemicals were extracted from the powdered samples with 95 % methanol and 95 % ethanol using a modified maceration method (Ungcharoenwiwat et al. 2023). A stock solution of the crude extract (500 mg/mL) was prepared using 95 % methanol or ethanol. The crude extract solutions were kept at −20 °C until use.

2.2 Analysis of phytochemical groups from plant extracts

Ten phytoconstituents in the crude extracts were screened using a modified protocol described by Ayoola et al. (2008). The presence of compounds was qualitatively evaluated by observing the color change, sedimentation, and foam formation.

2.3 Evaluation of antibacterial activity

The inhibitory efficacy of all extracts was evaluated against bacterial strains, including B. cereus TISTR747, S. aureus TISTR2329, E. coli TISTR527, P. aeruginosa TISTR357 and V. parahaemolyticus TISTR1596. The single colony of all strains was re-streaked on nutrient agar (NA) at 35 ± 2 °C for 24 h (supplement with 3 % NaCl for V. parahaemolyticus). A direct suspension of bacterial strains (1 × 10⁸ CFU/mL) was swabbed across three planes on Müller-Hinton agar (MHA) (with 3 % NaCl for V. parahaemolyticus). The crude extract solutions (50 µL) loaded in sterile discs (0.6 mm diameter) were placed on surface of agar in triplicate, and the kanamycin (500 µg/mL) and organic solvent were applied as a control. The activity was observed after incubation at 35 °C for 24 h. The clear zone of inhibition surrounding the paper disc was reported to be millimeters in diameter.

2.4 Evaluation of minimum inhibitory concentration (MIC) and minimum of bactericidal concentration (MBC)

The susceptibility of specific bacteria was studied as the MIC value using a broth microdilution assay modified from the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2009) and Ungcharoenwiwat et al. (2023). A two-fold serial dilution of the PB extract in a suitable solvent, ranging 500–0.24 mg/mL, was prepared into 96-well plates in triplicate and evaporated the solvent under vacuum at RT (28 ± 2 °C). An aliquot of bacterial suspension (10 µL), containing approximately 1 × 10⁶ CFU/mL, was inoculated to each well containing MHB or 3 % NaCl MHB and then incubated at 35 °C. Kanamycin (500 µg/mL) and organic solvent were a control. After 20 h of incubation, the oxygen consumption of bacteria was qualitative determined using resazurin solution (0.18 %) for 4 h of incubation. The blue well at the lowest concentration of the crude extract was considered the MIC value. For MBC assay, a 10 µL from each well was dropped onto agar plates in triplicate, and the bacterial growth were observed after incubation at 35 °C for 24 h. The absence of bacterial colonies at the lowest concentration of the crude extract was recorded as the MBC value.

2.5 Determination of total phenolic content and antioxidant activity

Phenolic compounds are important constituents with various bioactivities. The total phenolic content (TPC) of all crude extracts was assayed using the Folin-Ciocalteu assay with UV–visible spectrophotometry (modified from Muhamad et al. (2019)). The TPC expressed as gallic acid equivalents (GAE) per gram of sample was calculated from calibration curve of gallic acid.

The antioxidant property acting as radical scavenger of the crude extracts was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity according to the protocols described by Muhamad et al. (2019). All analyses were carried out in triplicates. Free radical-scavenging activity (%) was calculated using the following equation: $$\%\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}=\frac{\left(\mathrm{A}\mathrm{b}\mathrm{s}\left[\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right]-\mathrm{A}\mathrm{b}\mathrm{s}[\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}]\right)}{\mathrm{A}\mathrm{b}\mathrm{s}[\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}]}\times 100$$

2.6 Study of thermal stability and storage time

The lifetime of the phenolic contents and antibacterial stability of the crude PB extracts are of concern for quality control. The extracts were incubated at various temperatures at 28 (RT), 37 °C, and 65 °C for different durations (3, 6, and 12 h) and 121 °C for 15 min (autoclave condition). The temperature that resulted in the highest remaining total phenolic content was selected to study the effect of storage time (1, 7, 14, 30, and 90 days). All treatments were done in triplicate. The TPC, MIC, and MBC of the extracts were assessed as described above. The stability value of TPC (%) was reported using the following formular: $$\%\mathrm{S}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{\mathrm{P}\mathrm{h}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}}{\mathrm{P}\mathrm{h}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{t}0\mathrm{h}}\times 100$$

2.7 Analysis of phytochemical metabolites by LC-QTOF MS

Liquid Chromatograph-Quadrupole Time-of-Flight Mass Spectrometer (1290 Infinity II LC-6545 Quadrupole-TOF, Agilent Technologies, USA) was used for chemical components analysis. The substances from PBM were separated on Zorbax Eclipse Plus C18 Rapid Resolution HD (150 mm length x 2.1 mm inner-diameter, particle size 1.8 μm, Agilent, USA). The mobile phase A was 0.1 % Formic acid and 0.5 mM ammnonium formate in water, and B was 0.1 % Formic acid and 0.5 mM ammnonium formate in acetonitrile, at a flow rate of 200 µL/min. The program was specified as follows, 90 % B (15 min), 90 % B (24 min), 5 % B (26 min) and 5 % B (30 min). The UPLC condition was positive and negative electrospray mode for the MS analysis using data-dependent automatic MS/MS acquisition modes (AutoMSMS).

2.8 Hemolytic activity against human erythrocytes

Hemolytic activity refers to the ability of a phytochemicals in PB extracts to damage or lyse red blood cells, leading to the leakage of hemoglobin from the cells. This phenomenon can be used as an indicator of cellular toxicity, indicating the potential of a substance to harm the cells. This method was described by Madakka et al. (2021). Briefly, the PB extract was mixed with 10 % human erythrocytes at a ratio 1:2 and then incubated at 37 °C for 1 h (water bath). The final volume (1 mL) in each tube was adjusted by PBS solution, and the supernatant was separated at 1000 × g for 5 min. Triton X100 (1.0 %), PBS solution, and organic solvent were used as controls. Hemoglobin released into the supernatant was absorbed at 540 nm using a spectrophotometer, and hemolysis (%) was calculated using the following equation: $$\mathrm{H}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}(\%)=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\left[\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right]-\mathrm{A}\mathrm{b}\mathrm{s}[\mathrm{N}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}]}{\mathrm{A}\mathrm{b}\mathrm{s}\left[\mathrm{P}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right]-\mathrm{A}\mathrm{b}\mathrm{s}[\mathrm{N}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}]}\times 100$$

The hemolyticity was considered by comparing percentage of hemolyticity with negative control according to ASTM F756-00, if % hemolysis was 0 to 2.0 = non-hemolytic, 2.1 to 5.0 = slightly hemolytic, and more than 5.0 = hemolytic.

2.9 Determination of toxicity with Artemia salina model

The brine shrimp lethality assay, a modified method of Ramos et al. (2009) provides a preliminary evaluation of the toxicity of the crude extract. PB extract solution (2.4–153.6 mg/mL, 500 µL) were added to vials, and the organic solvent was evaporated with vacuum desiccator until dry. Five milliliters of 2.5 % artificial seawater were added to the vials to prepare a final concentration of 0.24–15.36 mg/mL PB extracts. Ten nauplii (24-h old) were transferred to each vial, and all vials were placed at room temperature (28 ± 2 °C) for 24 h. Evaporated solvent vials containing 2.5 % artificial seawater were used as negative controls. The toxicity was considered as LC₅₀ < 100 µg/mL=high toxicity, 100 µg/mL>LC₅₀ < 500 µg/mL=moderate toxicity, and LC₅₀ > 1000 µg/mL=low toxicity.

2.10 Digestive enzyme inhibitory activity

Glucoamylase activity was determined by the DNS assay with 1 % soluble starch as the substrate. The crude extract solutions at 1.95 mg/mL were mixed with citrate buffer (pH 5.5) at ratio 1:1 to final volume at 500 µL. A glucoamylase solution from Aspergillus niger (5 U/mL in citrate buffer, pH 5.5, 500 µL) was mixed into test tube at 37 °C for 15 min. Enzyme activity was measured according to Sharma et al. (2014). The percentage inhibitory activity was calculated using the following equation: $$\%\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\frac{\left(\mathrm{A}\mathrm{b}\mathrm{s}\left[\mathrm{e}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e}\right]-\mathrm{A}\mathrm{b}\mathrm{s}[\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}]\right)}{\mathrm{A}\mathrm{b}\mathrm{s}[\mathrm{e}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e}]}\times 100$$

Bovine pancreatic lipase activity was assayed using p-nitrophenyl phosphate (pNPP) as a substrate. The PB extract solutions at 1.95 mg/mL were mixed with Tris-HCl buffer (pH 8.5) (1:1 v/v) to final volume 200 µL. A lipase solution from the bovine pancreas (25 mg/mL, pH 8.5) was added to the test tubes and placed at 37 °C for 15 min. The enzyme activity was determined using method of Kanwar et al. (2005) and the inhibitory activity (%) was calculated using the above equation.

Bovine pancreatic trypsin activity was measured using azocasein as a substrate. The crude extract solutions at 1.95 mg/mL were mixed with Tris-HCl (pH 8.5) at ratio 1:1 to final volume 200 µL. Trypsin solution from bovine pancreas (1 U/mL, 50 µL) was mixed into each tube and incubated at 37 °C for 15 min. The enzyme reaction was analyzed using a method of Spelbrink et al. (2011) and the inhibitory activity (%) was calculated using a previously described equation.

2.11 Statistical analysis

All assays were conducted in triplicate and standard deviation, graph plots, and linear regression were generated using Microsoft Excel 2020. Statistical analyses were performed using one-way analysis of variance (ANOVA) and Tukey’s multiple tests using Minitab19. Statistical significance was set at p < 0.05.

3.1 Phytochemical screening from medicinal plants

Plant extractions were performed for PB, PR and GP using 95 % methanol and ethanol. The extracts from GP yielded low amounts (3.1-4.2%) of brownish-orange paste. Crude PR extracts showed the highest yield of a brownish paste (PRE 15.23% and PRM 6.86%). For PB, the methanolic crude extract (PBM) had a higher yield (14.14%) than the ethanolic crude extract (PBE) (8.13%). Both the extracts appeared as dark greenish pastes. Ten phytochemical groups were screened in the crude extracts (Table 1). There were slightly different phytochemical groups in the two solvents except for GP. Detectable phytochemical groups were divided into five primary compounds, namely terpenoids, saponins, flavonoids, coumarins and tannins. Phenolic compounds are the large group and have high solubility in polar organic solvents, especially alcohols and often combine with numerous compounds in plant extracts.

4.1 Determination of MIC and MBC of plant extracts

Both alcoholic extracts of PB had potential inhibition against five pathogenic bacterial strains (Table 3). The MIC and MBC values of PBM and PBE ranged from 0.24 to 3.91 mg/mL and 0.98 to 3.91 mg/mL. The lowest MIC was found in V. parahaemolyticus (0.24 mg/mL, PBM), followed by B. cereus (0.98 mg/mL, PBE), P. aeruginosa and E. coli (1.95 mg/mL, PBM). For MBC values, there were one or two-folds increasing from MIC values that were found in treatments of B. cereus (3.91 mg/mL, PBM and PBE) and V. parahaemolyticus (1.95 mg/mL, PBE).

These results were correspondence with the investigation of Chan et al. (2009), who reported that methanol-extracted bioactive substances, especially phenolic compounds, were more abundant than those extracted from ethanol because of their higher polarity. Phenolic compounds promote the cell membrane permeability in K⁺ and H⁺ transfer using carboxyl groups in aromatic hydrocarbons, which inhibit ATP synthesis by consuming a proton motive force (Nouri et al., 2014). Hydroxychavicol, eugenol, and chavibetol as main phenolic compounds in PB extracts, can precipitate DNA or penetrate bacterial cell walls (Jesonbabu et al., 2011).

4.2 Total phenolic content and antioxidant activity

The TPC analysis in extract was investigated to determine the redox properties acting as antioxidants. The methanolic crude extracts generally exhibited higher TPC than the ethanolic crude extracts, except for the crude PB extracts (Table 4). TPC values ranged 44.32–130.07 mg of GAE/g for methanolic extracts and 34.38–147.69 mg of GAE/g for ethanolic extracts. The highest TPC was observed in PBE at 147.69 ± 0.03 mg of GAE/g, followed by PBM at 130 ± 4.46 mg of GAE/g.

DPPH scavenging activity, the activity increased in dose dependent manner, with IC₅₀ values ranging 0.03–3.36 mg/mL. The highest IC₅₀ values were observed for PRM and PRE, whereas the lowest IC₅₀ values were observed in PBE and PBM. These suggested that the bioactive constituents possessing the antibacterial activity of PB extracts had also associated with their TPC and antioxidant activity. Moreover, the PB extract contained high amount of phenolic compound. This was supported by Altemimi et al. (2017), who reported that phenolic compounds transmitted electrons to other compounds, thereby exhibiting antioxidant activity. Antioxidant activity held significance for various applications, including reduction of inflammation, prevention of blood clots, and inhibition of enzymes.

4.3 Thermal stability and storage time

Temperature significantly affected the stability of both the TPC and the inhibitory potency of the PBM extracts, as shown in Fig. 1. TPC remained stable at temperatures between 28–35 °C and significantly decreased (p < 0.05) at higher temperatures. However, only 37.2 % of TPC remained after 12 h at 65 °C, and a similar decrease was observed under autoclave condition (33.21 %). The remaining antibacterial activity was stable at more than 50 % from MIC (1.95 mg/mL). A long-term storage study revealed a significant decrease in TPC (p < 0.05), with 85.9–22.3 % remaining after 7–90 days. Moreover, MBC decreased 2 folds of the MIC (3.81 mg/mL) for 90 days. According to Cheng et al. (2017), the temperature and storage time affected the structure of phenolic compounds by modifying their hydroxy groups. Moreover, the loss of TPC might be due to thermal degradation or heat-labile nature or degradative enzymes (Benjamin et al., 2022). On the other hand, microwave drying treatment could increase more the TPC than fresh sample (Benjamin et al., 2022), and high temperature showed the increasing of flavonoid content in ginger powder (Chan et al., 2009). The complex of polysaccharide and flavonoids had reported in thermostability improvement (Fernandes et al., 2020). These supported that crude PB extracts contained complex forms of phytochemical groups that might be released or change their forms leading to affect on thermal resistance and antibacterial activity. The stability of other active compounds and their functional group transformation should be intensive studied in the future.

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Fig. 1

Stability of TPC and antibacterial activities against E. coli of PBM at different temperatures and autoclave condition (A) and at room temperature (28 °C) for 90 days (B).

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4.4 Phytochemical metabolites by LC-QTOF/MS

The phytocomponents from PBM analyzed by LC-QTOF/MS with positive and negative electron spray ionization modes (ESI−/ESI+) revealed the retention time, mass, matching score and area sum (%) (Fig. 2 and Table 5.). The main components in extract from positive mode were steroids as mometasone furoate (12.3 %), alkaloids as erysopine (6.64 %) and quinazoline (4.31 %), quinones as heptaprenyl diphosphate (4.79 %), lipids as diglycerides (6.22 %), gingerglycolipid A (3.81 %) and stearidonic acid methyl ester (3.33 %). For the negative mode, the major components were flavonoids as demethoxyegonol (15.34 %), and phenolics that composed of 4-hydroxycinnamaldehyde (6.40 %), 2,5-dihydroxybenzal-dehyde (5.09 %), 1-(4-hydroxy-3-methoxyphenyl)-5-(4-hydroxyphenyl)-1,4-pentadien-3-one (4.76 %) and isopulegone caffeate (2.72 %). Moreover, 3-hydroxychavicol 1-glucoside had also found in small area. The most of phenolics were detected by negative mode and other metabolites were determined by positive mode. The prevalent components in PBM extract had reported their biological properties including anti-bacteria of mometasone furoate (Neher et al., 2008), 2,5-dihydroxybenzaldehyde (Schabauer et al., 2018), 2,5-dihydroxybenzal-dehyde (Wong et al., 2008); anxiolytic activity of erysopine, antimicrobial and anti-complement activities of egonol and derivatives (Emirdağ-Öztürk et al., 2011); anti-inflammatory and antioxidant effects of 4-hydroxycinnamaldehyde (Lee et al., 2022). The chemical components of PBM varied based on several influences including the plant type, developmental phase, collecting time, habitat as well as extraction method.

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Fig. 2

Total ion chromatogram of PBM analyzed by LC-MS/MS on positive and negative modes.

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4.5 Biological toxicity assay

4.5.1

4.5.1 Hemolytic activity

Hemolytic activity against erythrocytes is an important property to evaluate the potential safety of crude extracts for in vivo applications. In addition, the data can provide indirect information regarding the potential general toxicity. PBE and PBM exhibited hemolytic activity in a dose-dependent manner with IC₅₀ values of 0.44 and 0.24 mg/mL, respectively (Fig. 3A). Potential mechanisms include the disruption of red blood cell membranes, ripping of the lipid bilayer and changes in the concentration of surrounding solution. According to Zohra and Fawzia (2014), the terpenoids and glycosides in betle leaf extracts could adhere to the erythrocytic membrane, eventually leading to disintegration.

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Fig. 3

Hemolytic activity against human erythrocyte (A) and toxicity evaluation using A. salina larvae (B) by crude PB extracts.

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4.5.2

4.5.2 Determination of toxicity against a. Salina

A. salina larvae lethality assay was investigated to assess the in vivo toxicity of PBM and PBE extracts (0.06–7.80 mg/mL). The morphology of A. salina larvae treated with all concentrations of extracts remained unchanged compared to that of the control group for 24 h. Abnormalities in swimming behavior and body size were observed (Table 6). The swimming or body size compared to the control group showed no significant difference (p > 0.05). At low concentrations (0.06–0.24 mg/mL), the survival rate exceeded 90 % between treatments (Fig. 3B).

Table 6 Swimming behavior and body size of A. salina treated with crude PB extracts.

Crude extracts	Concentration (mg/mL)	Abnormality characters
		Swimming (%)	Body sizes (mm)
Control	0	0	3.00 ± 0.17
PBM	0.06	76.67 ± 5.77	2.93 ± 0.11
	0.12	93.33 ± 11.54	2.93 ± 0.05
	0.24	80.00 ± 10.00	2.93 ± 0.00
	0.49	84.72 ± 6.05	3.00 ± 0.17
PBE	0.06	90.00 ± 0.00	3.00 ± 0.17
	0.12	80.00 ± 10.00	3.00 ± 0.30
	0.24	83.33 ± 5.77	3.00 ± 0.10
	0.49	86.67 ± 11.54	2.90 ± 0.10

Larval characteristics are shown in Fig. 4. After 24 h, the LC₅₀ value (lethal concentration for 50 % of the population) of A. salina treated with PBM and PBE ranged 0.49–0.98 mg/mL. Complete mortality (100 %) was exhibited at 0.98–7.80 mg/mL. Therefore, the LC₅₀ of PBM and PBE against A. salina were estimated as LC₅₀ at 0.58 and 0.61 mg/mL that could be classified as moderate toxic. The toxicity of the crude PB extracts was evaluated to be low to moderate in human erythrocyte and organism. Therefore, it may not be suitable for internal use in organisms and can potentially be applied as a disinfecting agent at specific concentrations.

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Fig. 4

Morphology of A. salina treated with crude PB extracts at 24 h for toxicity evaluation.

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4.6 Determination of digestive enzyme inhibition

Enzyme inhibition by plant extracts has potential as an alternative healthcare supplements. The results showed that PBM exhibited a low degree of alpha-glucoamylase inhibition as 10.39 %, whereas PBE activated the enzyme activity (−25.62 % inhibition). PBM and PBE showed lipase inhibition rates of 50.04 and 64.52 %, respectively. Conversely, PBM and PBE exhibited minimal inhibition of trypsin activity (5.86 and 10.03 %). Polyphenol, flavonoids, and glycoside groups in plant extracts are likely to contribute to this effect (Alias et al., 2017). Ado et al. (2013) reported that some plant extracts promoted enzyme activity by more than 20 %; moreover, phytochemical substances such as eugenol or flavonoids could delay the onset of enzyme reaction (Boskabady and Ramazani-Assari, 2001). Active inhibitors were mainly found in the phenolic and alkaloids groups that can directly bind to the catalytic or active sites of enzyme especially hydroxy groups (Ado et al., 2013). These findings suggest that PB extract has potential for applications as an alternative supplement in healthcare.