Fractionation, identification of chemical constituents, and biological properties of cashew (Anacardium occidentale L.) leaf extracts

Abstract The current study aimed to identify the chemical constituents and bioactivities of the crude ethanolic extract (CEE) and its fractions (ethyl acetate (EAF), hexane (HEF), and aqueous (AEF)) from leaves of cashew (Anacardium occidentale L.) grown in Vietnam. A total of 31 compounds which belong to alkanes, hydrocarbons, iodine, terpenoids, phenolics, and flavonoids were determined by a gas chromatography–mass spectrometry (GC–MS) analysis, with bis(2‐ethylhexyl) phthalate being the most prevailing compound. The highest total phenolic and flavonoid contents were obtained in the EAF, followed by HEF, CEE, and AQF. All samples showed promising in vitro antibacterial activity, enzyme inhibition, and anticancer activity. Among the samples tested, the EAF exhibited the highest enzyme inhibition activity against α‐amylase and α‐glucosidase (IC50 values of 51.24 μg/mL and 99.29 μg/mL, respectively), cytotoxicity activity against HeLa cells (IC50 value of 79.49 μg/mL), and antibacterial activity against Bacillus subtilis and Escherichia coli with MIC values of 5 mg/mL and 2.5 mg/mL, respectively. These findings suggest that the leaves of A. occidentale cultivated in Vietnam are a promising source of bioactive components and that EAF is a promising bioactive material warranting further pharmaceutical investigation.

A sesquiterpene lactone zoapatanolide A was isolated from the ethanol extract of A. occidentale cultivated in Nigeria and exhibited potent cytotoxicity against HeLa cells, with an IC 50 value of 36.2 μM (Taiwo et al., 2017).Cefali et al. (2021) reported the potential use of extracts of cashew cultivated in Brazil for anti-acne treatment and the prevention of premature skin aging.Costa et al. (2021) noted that the ethanolic extract of the bark of Brazilian cashew trees could be considered an excellent candidate for drug development against cancer and fungal infection.Therefore, A. occidentale is considered a potential source of natural bioactive compounds for drug development.
Extraction and separation are two important steps in the isolation of bioactive compounds from organic materials.Different extraction methods, such as Soxhlet extraction, supercritical CO 2 , and maceration, have been used to obtain an extract from different parts of A. occidentale (Baptista et al., 2018;Tan et al., 2014).To achieve a bioactive compound-rich extract and consequently enhance its biological activity, further fractionation or purification steps are required.Among the many separation techniques, liquid-liquid extraction (LLE) can be used to enrich bioactive compounds from an initial extract (Ismail & Chua, 2020;Truong et al., 2021).In this method, the selection of solvents and design of the fractionation process are crucial to obtain the desired compounds.This is because each bioactive compound has a different solubility in different solvents (Truong et al., 2019).Barbosa-Filho et al. (2015) used solvent partitioning to obtain methanol and ethyl acetate fractions from the ethanol extract of Anacardium microcarpum stem bark and these fractions demonstrated potent antibacterial activity against a range of bacteria (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus).In a previous study, we also successfully obtained a terpenoid-rich fraction with enhanced biological activities from Serevenia buxifolia bark extract (Truong et al., 2021).Therefore, LLE is promising for concentrating different groups of bioactive compounds.Although A. occidentale has been studied for its phytochemical constituents and bioactivities (Kossouoh et al., 2008;Osman et al., 2019), the fractionation and identification of specific bioactive components from A. occidentale leaves are limited.Particularly, there is no study on the chemical profiles and bioactivities of Vietnamese cashew trees, which are an important economic plant in Vietnamthe largest producer of cashew nuts (Peng et al., 2014).Considering all the above facts, the aim of the present study was to achieve the bioactive-rich extract from A. occidentale leaves by using LLE.The chemical constituents of the extract and fractions were characterized using gas chromatography-mass spectrometry (GC-MS) analysis.The detailed scientific validation of in vitro antibacterial (B.subtilis and E. coli), antidiabetic (Type 2 diabetes, α-glucosidase and α-amylase) and anticancer (Hela cancer cell line) activities of A. occidentale leaf extract and fractions was investigated.
The findings of this study could provide new insights into the application of the Vietnamese A. occidentale leaf extract in the nutraceutical and pharmaceutical industries.

| Plant material
The mature leaves of A. occidentale L. (red cashew tree) were collected from Dong Nai province, Vietnam.The taxonomical identification of the plant was conducted by the VNM Herbarium, Institute of Tropical Biology, Vietnam.The leaves of A. occidentale were collected during the flowering-fruiting stage.The leaves were cut, washed carefully with water to remove debris, dried at 45°C for 48 h, and then ground to a powder.The sample was subsequently macerated with 80% ethanol for 24 h at 40°C and the extraction was repeated three times.After extraction and filtration, the ethanol was completely evaporated using a rotary vacuum evaporator, and the sample was subsequently freeze-dried to obtain the crude ethanolic extract (CEE).The CEE was kept in the dark at 4°C for further experiments.

| Fractionation of crude extract by LLE
The A. occidentale leaf extract was fractionated by LLE (Figure 1).Briefly, the CEE was dissolved in distilled water (1:10, w/v), transferred into a separating funnel, equilibrated, and successively fractionated with n-hexane and ethyl acetate to yield different fractions, respectively called hexane fraction (HEF), ethyl acetate fraction (EAF).The residue was generated, namely aqueous fraction (AQF).
The resulting fractions (HEF, EAF, and AQF) were then concentrated using a rotary evaporator.All fractions were then freeze-dried and kept in the dark at 4°C for further experiments.The fractionation process was repeated three times.

| Determination of total flavonoid content and total phenolic content
The total phenolic content (TPC) of the A. occidentale leaf extract and its fractions was measured using a modified Folin-Ciocalteu method (Encarnação et al., 2016) with some minor modifications.Briefly, 1 mL of the extract was mixed with 7 mL of diluted Folin-Ciocalteu reagent (1:10, v/v, in water) and 2 mL of 7.5% sodium carbonate.The reaction mixture was then incubated at room temperature for 1 h before measuring the absorbance at 765 nm using a spectrometer (V-730 UV-Vis Spectrophotometer, Jasco).Gallic acid (50-250 μg/ mL) was used to establish a standard curve (y = 0.003x -0.0134; R 2 = .9982)for the calculation of the TPC.The TPC was expressed as milligrams of gallic acid equivalents per gram of dry matter (mg GAE/g DM).
The total flavonoid content (TFC) of the crude extract and its fractions was estimated using a modified spectrophotometric method (Medina-Medrano et al., 2019).Briefly, the extracts (2 mL) were mixed with 5% AlCl 3 (0.5 mL) and 1 M potassium acetate solution (0.5 mL).The mixture was then incubated at room temperature for 15 min before measuring the absorbance at 415 nm using a V-730 UV-Vis spectrophotometer (Jasco).Quercetin (20-100 μg/mL) was used as a standard to establish a calibration curve (y = 0.01x + 0.017; R 2 = .9967)for the calculation of the TFC.The TFC was shown as milligrams of quercetin equivalents per gram of dry matter (mg QE/g DM).

| Phytoconstituents identified by GC-MS
The phytoconstituents were analyzed using a GC/MS system (Agilent 7890B/7000C) based on the method of Haron et al. (2019), with minor changes.The dried A. occidentale crude extract and its fractions were dissolved in acetonitrile (Sigma) (1:20, w/v).The samples were filtered using a 0.45 μm PTFE filter (Millipore, Sigma-Aldrich) before injection into GC-MS.The separation was performed with an Agilent DB-5MS capillary column (40 m × 250 μm × 0.25 μm).The injector was set at 325°C (split mode with the ratio adjusted to 25:1 and an injection volume of 1 μL).The oven temperature started at 70°C for 1 min, increased to 280°C, and was then held for 5 min at 15°C ramp/min.The flow rate of the carrier gas (helium) was maintained at 2.25 mL/min.The mass spectrometer was operated with ionization in the electron impact mode using ionization energy MS 2.0.The proportion of each detected component was calculated as the ratio between their area and the total area of all detected compositions from each A. occidentale extract or fractions.

| Sensitivity test
The bacteria strains (B. subtilis ATCC 8012 and E. coli ATCC 25922) were collected from stock of culture collections, maintained on trypticase soy agar (TSA), and sub-cultured monthly.The appropriate bacteria strain was prepared according to the Clinical and Laboratory Standards Institute (CLSI) recommendation, where the OD 600 value was adjusted to the equivalent of 10 8 colony-forming units (CFU)/mL.The disc diffusion method described by Razmavar et al. (2014) was employed to examine the antibacterial capabilities of A. occidentale crude extract and fractions against E. coli ATCC 25922 and B. subtilis ATCC 8012.A serial concentration (20, 40, 80, and 100 mg/mL) of the crude extract and fractions (HEF, EAF, or AQF) was prepared in 10% of dimethylsulfoxide (DMSO).Subsequently, sterile filter paper disks (6 mm in diameter) were impregnated with 30 μL of each sample (1 mg of tested sample/disk) and allowed to dry at room temperature.Streptomycin (1 mg/mL) and 10% of DMSO were used as the respective standard antibiotic (positive control) and negative control.The tested bacterial suspensions (100 μL) were uniformly spread in petri plates containing TSA medium.The paper disks impregnated with the samples were then placed on the surface of the agar.Finally, the plates were incubated aerobically for 24 h at 37°C, and the antibacterial activity was evaluated by measuring the diameter of the inhibition zone (IZ, mm) (Abew et al., 2014).

| Determination of minimum inhibitory concentration and minimum microbicidal concentration values
The minimum inhibitory concentration (MIC) values of the crude extract and fractions were determined using a broth microdilution method with resazurin (Sarker et al., 2007).Resazurin (0.01%) was prepared in sterile distilled water, vortexed well, filter sterilized (0. where A c and A s are the absorbance of the control (DMSO) and the samples, respectively.The results were then shown in terms of the IC 50 values, which are the concentration of the tested sample required to inhibit 50% enzyme activity.

| In vitro α-glucosidase inhibition
The α-glucosidase assay was carried out using the method of Damsud et al. (2013), with some changes.The sample (100 μL) and 100 μL of α-glucosidase (1.0 U/mL) in DMSO were incubated at 25°C for 15 min in the dark before adding 100 μL of 10 mM pNPG solution in DMSO.
The reaction solution was then incubated at 25°C for 30 min before adding 300 μL of Na 2 CO 3 (100 mM).The absorbance was measured at 405 nm using a spectrophotometer.The percentage of α-glucosidase inhibition was estimated in the same manner as in the α-amylase assay.Acarbose was also used as a standard (positive control).

| Sulforhodamine B assay
The antiproliferative activity of the extracts against HeLa cells was evaluated using the Sulforhodamine B (SRB) assay (Haron et al., 2019).Briefly, the cells were prepared at a density of 5000 cells per well in 96-well plates at 37°C with 5% CO 2 .The cells were then treated with various concentrations of tested samples (1-100 μg/mL) for 24, 48, and 72 h.Treated cells were fixed with 50% cold trichloroacetic acid (50 μL) for 1 h at room temperature and then washed gently with tap water (5 times) and dried.The cells were subsequently stained with 100 μL of 0.2% (w/v) SRB in 1% acetic acid for 20 min.After five washes with 1% acetic acid, the plate was dried, added with 100 μL of 10 mM Tris base solution, and shaken for 5 min.Finally, the relative cell viability was determined by measuring the absorbance at 540 nm on a 96-well microtiter plate reader (Synergy HT, Biotek Instruments).Camptothecin (Calbiochem) (1 μg/mL) was used as a positive control.The cytotoxicity was determined as follows (Nguyen et al., 2016): where OD t and OD c are the absorbances of the tested sample and the untreated sample, respectively.The concentration of the sample that is able to inhibit cell proliferation by 50% (IC 50 ) was estimated from the dose-response curve.

| TPC and TFC of A. occidentale leaf extract and fractions
In this study, the LLE was applied to yield three fractions, namely HEF, EAF, and AQF, from the CEE of A. occidentale leaves (Figure 1).
The TFC and TPC of the crude extract and its fractions were examined and the results are illustrated in Table 1.Significant variations in the TFC and TPC of the A. occidentale crude extract and its fractions were observed in this study (p < .05;Table 1).The TFC and TPC showed the following order: EAF > HEF > CEE > AQF.The highest TPC and TFC were obtained in the EAF (233.47 mg GAE/g DM and 43.97 mg QE/g DM, respectively), whereas the recovery of phenolic and flavonoid components was lowest in the AQF (79.47 mg GAE/g DM and 2.0 mg QE/g DM, respectively).

| Phytoconstituents of A. occidentale leaf extract and fractions
Table 2 shows the constituents identified from GC-MS chromatography analyses of A. occidentale extract and its fractions.A total of 31 chemical components belonging to alkanes, hydrocarbons, iodine, terpenoids, phenolics, and flavonoids were found in the CEE, HEF, EAF, and AQF of the A. occidentale leaves (Figure S1).
The HEF obtained from the A. occidentale leaf CEE led to the identification of 12 organic compounds, accounting for 38.71% of identified compounds in the total extract and fractions (Figure 2;  2).
The EAF prepared from the A. occidentale leaf extract and analyzed using the same GC-MS conditions led to the identification of 15 chemical constituents, representing 48.39% of phytochemicals in the total extract and fractions (Figure 2; Table S1).were found as minor components in the EAF (Table 2).

| Antibacterial activity
This study evaluated the antibacterial activity of the extracts against 1 Gram-positive (B.subtilis ATCC 8012) and 1 Gram-negative (E. coli the samples examined (CEE, HEF, EAF, and AQF), the EAF exhibited the greatest inhibitory effects against two tested bacteria, followed by HEF, CEE, and AQF (Table 3, Figures S2 and S3).
The resazurin reduction test is recommended to examine the antibacterial potential of natural extracts and to determine the MIC and MMC values (Sarker et al., 2007).This method was therefore applied in the present study to estimate the MIC and MMC values of the crude extract and fractions of A. occidentale leaves.In this work, the fractions possess better inhibitory effects against the tested bacteria, E. coli and B. subtilis, than the crude extract.Among the fractions examined, the EAF showed the highest inhibitory activity with MIC values of 2.5 mg/ mL against E. coli and 5 mg/mL against B. subtilis, and MMC values of 5 mg/mL and 10 mg/mL against E. coli and B. subtilis, respectively.The MIC values of the CEE and AQF were 20 mg/mL against E. coli and >10 mg/mL against B. subtilis, while the MMC values of these samples were > 20 mg/mL against E. coli and > 10 mg/mL against B. subtilis.The MIC of the HEF was observed to be 5 mg/mL against E. coli and 10 mg/ mL against B. subtilis, while the MMC of this fraction was 10 mg/mL against E. coli and >10 mg/mL against B. subtilis (Table 3).
The inhibitory effect of the extracts on α-glucosidase and αamylase were also assessed using the IC 50 values.Among the tested samples, the EAF revealed the highest inhibitory capacity against α-amylase and α-glucosidase with IC 50 values of 51.24 μg/mL and 99.29 μg/mL, respectively.These values were slightly higher than those of a standard antidiabetic drug, acarbose (IC 50 values of 48.13 μg/mL and 90.77 μg/mL against α-amylase and α-glucosidase, respectively), indicating that EAF is a promising antidiabetic agent.
However, the IC 50 values for α-glucosidase and α-amylase inhibition by other tested samples were significantly higher than that of EAF and acarbose (Table 4).respectively.Notably, its antiproliferative activity against HeLa cells was comparable to camptothecin (no significant difference at all treatment times), indicating that EAF is a promising anticancer agent for further application.However, the HEF, AQF, and CEE showed significantly lower activity than the EAF.Hence, treatment of HeLa cells for 48 h with the EAF was chosen for further analysis.

| Preliminary cytotoxicity effect
The viability of the HeLa cells also changed in a concentrationdependent manner (Figure 4).Increasing the EAF concentration led to a significantly decreased viability of HeLa cells after 48 h of incubation (p < .05).The EAF showed the most potent activity against HeLa cells with an IC 50 of 79.49 μg/mL, whereas the IC 50 values of the other tested extracts were > 100 μg/mL.

| DISCUSS ION
LLE is an established method to achieve the separation of compounds with different polarities (e.g., phenolics and flavonoids) (Alara et al., 2021).In this work, LLE was applied to fractionate the ethanolic extract of A. occidentale leaves using different solvents.Consequently, HEF, EAF, and AQF were obtained from the CEE.These extracts revealed the presence of different phytoconstituents, including alkanes, hydrocarbons, iodine, terpenoids, phenolics, and flavonoids.Phenolics and flavonoids are known as important bioactive compounds in cashew plants and exhibit potent bioactivities (Salehi et al., 2020).Therefore, the content of these compounds in the crude extract and its fractions (HEF, EAF, The numbers correspond to the phytoconstituents identified by GC-MS and their names are shown in Table 2.

TA B L E 3
In vitro antibacterial activity of the A. occidentale leaf extract and fractions.31 phytochemical compounds, which most likely contribute to the bioactivities of the extracts (Salehi et al., 2020;Sassi et al., 2022).
The highest number of components (15) were found in the EAF, followed by the HEF (12), AQF (12), and CEE (8) (Table S1).Therefore, the highest antibacterial, antidiabetic, and anticancer activities of the EAF could be correlated to the occurrence of more bioactive components in this fraction.Cell death of a cancerous tumor due to effect of EAF from herbal plants was reported in the study of Idris et al., 2017.Cancer cells could be dying by secondary necrotic under the induction of EAF after 24 h of treatment.The release of secondary necrotic is advantageous to allow the efficient activation of the immune system that relates to the clearance of tumor cells from the tumor microenvironment.Besides, cells may also be died by apoptosis at earlier time-post-treatment and switch to secondary necrosis over time (Idris et al., 2017;Vakkila & Lotze, 2004).

(
70 eV) to obtain the mass spectra in the range from 40 to 700 m/z.The chemical profiles were identified based on retention times and by examination of their main spectra against the spectral databases of Wiley and the National Institute Standard and Technology (NIST) 22 μm filter), and stored at 4°C for further analysis.The crude extract and the fractions were dissolved in DMSO.Serial two-fold dilutions of the samples (20 mg/mL -0.078 mg/mL) were then prepared in sterile 96-well plates containing Mueller-Hinton broth (MHB).Subsequently, 10 μL of bacteria suspension (final concentration of 5 × 10 5 CFU/mL) and resazurin indicator (10 μL) was added to each well.The plates were then incubated at 37°C for 24 h and the color change was assessed visually.The MIC value was defined as the lowest concentration of the tested samples that prevented the resazurin color change from blue to pink.Each test contained a positive growth (streptomycin) and a negative control (10% DMSO).All experiments were established in triplicate and performed until MICs were constant.The minimum microbicidal concentration (MMC) value was estimated by plating 10 μL of the test material from the wells where no indicator color change was observed onto the MHB medium.The MMC value was then determined as the lowest concentration without growth (no colony) at the end of the incubation period.2.7 | Determination of antidiabetic activity2.7.1 | In vitro α-amylase inhibitionThe α-amylase inhibition assay was adapted fromThaidi et al. (2020), with minor modifications.Briefly, the samples (crude extract and fractions) were serially diluted in DMSO to obtain different concentrations (25-400 μg/mL).α-Amylase (3100 U/mg, Sigma) was dissolved in a phosphate buffer solution (pH 6.9) to reach a concentration of 2 U/mL.Soluble starch in water (1%, w/v) was used as the substrate solution.The reaction mixture containing 200 μL α-amylase solution and 200 μL of each sample was incubated at 37°C for 10 min.Subsequently, 200 μL of the starch solution was added to the reaction mixture and incubated for 10 min at 37°C.The enzyme reaction was then terminated by adding 500 μL of DNS reagent solution (96 mM DNS and 5.31 M sodium potassium tartrate in 2 M NaOH) and placing into boiling water for 10 min.Finally, the mixture was cooled and diluted with 5 mL of distilled water.Acarbose and DMSO were used as the reference standard and control, respectively.The absorbance of the reaction mixture was then measured at 540 nm.The percentage of α-amylase inhibition (I) was estimated as follows:

Figure 3
Figure 3 presents the viability of HeLa cells treated with different extracts (CEE, HEF, EAF, and AQF) at a concentration of 100 μg/mL and an anticancer drug (camptothecin).All tested samples exhibited significant antiproliferative activity against HeLa cells in a timedependent manner (p < .05).Among the samples tested, EAF was the most active fraction, resulting in the lowest viability of HeLa cells of 53.56%, 43.61%, and 35.54% for 24, 48, and 72 h of treatment,

F
Venn diagram of chemical constituents in the crude ethanolic extract and different fractions of A. occidentale leaves.
and AQF) was evaluated.The results revealed that the TPC and TFC showed statistically significant differences among the extract and fractions (p < .05).The EAF showed the highest TPC (233.47 mg GAE/g DM) and TFC (43.97 mg QE/g DM), followed by the HEF, CEE, and AQF.This could be due to the chemical components of the phenolic and flavonoid groups being more semi-polar to nonpolar.Consequently, these compounds were present at high levels in the EAF.The solvent is an important factor affecting the success of extraction and fractionation(Nur et al., 2019).Solvent selection is based on the principle of "like dissolves like," where a component can dissolve in a solvent with similar polarity(Amaro et al., 2015).The findings obtained in this work are similar to that ofNur et al. (2019) who obtained the highest content of phenolics and flavonoids in the ethyl acetate fraction from the crude extract of paku atai tuber.The biological activities of an extract are attributed to its bioactive constituents and their concentration.In this study, EAF showed the highest antibacterial activity against two tested bacteria (B.subtilis and E. coli), inhibitory capacity against α-amylase and α-glucosidase, and cytotoxicity against HeLa cells.This could be because the EAF contained the highest level of phenolics and flavonoids.These compounds are known to possess potent bioactivities(Salehi et al., 2020).To provide more information about the chemical constituents and biological activities of A. occidentale extracts and fractions, GC-MS analysis was applied and showed the presence of TA B L E 4 α-glucosidase and α-amylase inhibitory activities of the crude ethanolic extract and its fractions from A. occidentale leaves.

E 1
Fractionation of the crude ethanolic extract of A. occidentale leaves by liquid-liquid extraction.
The diameter of ZI of streptomycin (1 mg/mL) for E. coli ATCC 25922 and Bacillus subtilis ATCC 8012 was 20 mm and 17 mm, respectively.No ZI was observed in the negative control (10% DMSO). a Viability of HeLa cells treated with different concentrations of ethyl acetate fraction (1-100 μg/mL).Different letters indicate statistical significance at p < .05.
F I G U R E 3The cytotoxicity of the crude ethanolic extract and fractions (100 μg/mL) from A. occidentale leaves at different treatment times (24, 48, and 72 h).F I G U R E 4