Evaluation of phytochemical profile, and antioxidant, antidiabetic activities of indigenous Thai fruits

Chemicals and reagents

The following chemicals were used in the experiments: HPLC grade water containing 0.1% H₂PO₄ and methanol containing 0.1% H₂PO₄ (Phosphoric acid) were used for the HPLC analysis and purchased from Merck (Darmstadt, Germany). Standard HPLC grade, including catechin, ellagic acid, epicatechin, epicatechin gallate, gallic acid, and kaempferol were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Folin-Ciocalteu phenol reagent was purchased from Merck (Darmstadt, Germany). Methanol A.R. was purchased from RCL Labscan (Ireland). 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid, acarbose, α-amylase (procaine pancreas), Dinitrosalicilic acid (DNS), and starch azure were purchased from Sigma-Aldrich (St. Louis, MO, USA). TPTZ (2,4,6-tripyridyl-S-triazine), Iron (III) chloride (FeCl₃), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2-thiobarbituric acid, 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH), Trolox (6-hydroxy-2,5,7,8-tetramethyl chromane 2-carboxylic acid), potassium persulfate (K₂S₂O₈), and sodium carbonate (Na₂CO₃) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The α-glucosidase (Saccharomyces cerevisiae) and p-nitrophenyl- α-D-glucopyranoside (pNPG) were obtained from Sisco Research Laboratories Pvt. Ltd. (India).

Samples collection and preparation

In this study, the indigenous Thai fruits were purchased in the ripe stage. A total of 5 kg of each sample was collected from local markets as shown in Table 1. The samples were dried in an oven at 45 °C for 48 hr and afterward finely ground using a mixer until they reached a powdered consistency. The samples were extracted in triplicate using the following method 6 g of the dried samples were extracted with 80% methanol (50 ml) and sonicated for 30 min at 35 °C using an ultrasonicator (GT SONIC-R3, China). The extracts were filtered through Whatman No. 4 filter paper and the extraction was concentrated using a rotary evaporator model Büchi Rotavapor® R-210 (Mumbai, India) at 45 °C under a vacuum of 100 mbar. The concentrated extracts were then stored at −20  °C. The extracts were dissolved with 80% methanol for further HPLC and bioactivity analysis.

Table 1:

Family, scientific name, Thai name and picture of indigenous Thai fruits (the scales of the pictures are unequal).

No.	Family	Scientific name	Thai name	Picture
1	Euphorbiaceae	Antidesma puncticulatum	Mao luang
2	Dilleniaceae	Dillenia indica	Ma tat
3	Ebenaceae	Diospyros decandra	Chan
4	Elaeagnaceae	Elaeagnus latifolia	Ma lot
5	Salicaceae	Flacourtia indica	Ta khop pa
6	Clusiaceae	Garcinia dulcis	Ma phut
7	Sapindaceae	Lepisanthes fruticosa	Chamma liang
8	Sapotaceae	Mimusops elengi	Phikun
9	Muntingiaceae	Muntingia calabura	Ta khop farang
10	Euphorbiaceae	Phyllanthus reticulatus	Kang pla khruea
11	Moraceae	Streblus asper	Khoi
12	Myrtaceae	Syzygium cumini	Wa
13	Myrtaceae	Syzygium malaccense	Chompu Mameaw
14	Sapindaceae	Schleichera oleosa	Ta khro
15	Apocynaceae	Willughbeia edulis	Katang ka tio

DOI: 10.7717/peerj.17681/table-1

Phytochemical evaluation

Total phenolic content (TPC) was measured in triplicate using the Folin-Ciocalteu (F-C) method. Briefly, 30 µL of each extract was mixed with 150 µL of Folin-Ciocalteu reagent (25%, v/v) in a 96-well plate and incubated for 5 min. Then, 120 µL of 10% sodium carbonate was added to the mixture. The mixture was incubated for 60 min at 25 °C in the dark and the absorbance was recorded at 765 nm using the microplate reader model Spark™ 10M (TECAN, Switzerland). The results were given as milligrams of gallic acid equivalents per gram of sample (mg GAE/g) (Blainski, Lopes & De Mello, 2013).

The aluminum chloride method analyzed the flavonoid content (TFC) in triplicate using the aluminum chloride method. Briefly, 90 µL of the extract was mixed with 90 µL of a 2% aluminum chloride solution in a 96-well plate. The mixture was incubated for 15 min at 25 °C, and the absorbance was recorded at 440 nm. The results were presented in milligrams of quercetin equivalents per gram of sample (mg QE/g) (Molole, Gure & Abdissa, 2022).

Identification and quantitative analysis of phenolic compound

The method was modified by Soto et al. (2022). Ultra-high pressure liquid chromatography (UHPLC) was performed on an Agilent 1290 Infinity II LC system (Agilent, Santa Clara, CA, USA), which includes a quaternary solvent pump, an automatic injector, and a column oven. A diode array detector (DAD) was used for analysis. The extracts were separated using a Raptor ARC-18 column (150 mm × 4.6 mm, 2.7 µm particle size; Restek, Centre County, PA, USA). The injection volume was 10 µL and the column was maintained at 40 °C. The mobile phase consisted of a gradient mixture of solvent A (water containing 0.1% H₂PO₄) and solvent B (methanol containing 0.1% H₂PO₄) with a 0.5 ml/min flow rate. The gradient was started with 90.0% solvent A and 10.0% solvent B and was adjusted to 82.8% A and 17.2% B at 3 min, 77.0% A and 23.0% B at 6.5 min, 68.7% A and 31.3% B at 8.5 min, 54.0% A and 46.0% B at 10 min, 45.0% A and 55.0% B at 11.5 min, 0.0% A and 100.0% B at 13 min, and 90.0% A and 10.0% B at 17 min. The DAD was used at 286 nm. Data acquisition and processing were performed using the Agilent HPLC OpenLAB CDS 2.X software.

In vitro antioxidant assays of extracts

2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay was used to determine the antioxidant activity of the extracts in triplicate. Briefly, 90 µL of the extract was added to 90 µL of methanolic DPPH dye and 90 µL of methanol in 96-well plates, and the reactants of control were prepared by adding 90 µL of methanol to 90 µL of methanolic DPPH dye and 90 µL of methanol in 96-well plates. The mixtures were incubated for 30 min at 25 °C in the dark and the absorbance was measured at 520 nm (Molyneux, 2003).

A ferric-reducing antioxidant power (FRAP) assay was used to determine the antioxidant activity of the extracts. Briefly, the FRAP reagent was prepared by mixing a solution of 10 mM TPTZ in 40 mM HCl, acetate buffer (300 mM, pH 3.6), and 20 mM FeCl₃ at 10:1:1 (v/v/v). The reactants were prepared by adding 285 µL FRAP reagent to the 15 µL extracts and Trolox (used as a standard) and then incubated for 30 min in the dark at 25 °C. The absorbance was measured at 593 nm. The results were expressed as Trolox equivalents (mm TE). The samples were determined in triplicate (Fernandes et al., 2016).

2,2′-azinobis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assay was used to determine the antioxidant activity of the extracts. The samples were determined in triplicate. Briefly, the ABTS⁺ radical was prepared by mixing 2.45 mM K₂S₂O₈ and 7 mM ABTS at a 1:1 (v/v) ratio. Then, the mixture was incubated at 25 °C and was kept in the dark for 16 hr. The reactants of the sample were prepared by adding 20 µL of the sample and 180 µL of the ABTS⁺ radical into 96-well plates; the reactants of control were prepared by adding 20 µL of the methanol and 180 µL of the ABTS⁺ radical into 96-well plates; The reactants were incubated for 15 min at 25 °C and the absorbance was measured at 734 nm (Dong et al., 2015).

The DPPH and ABTS assay results in this study were reported as half-maximal inhibitory concentration values (IC₅₀), the concentration of a substance that can inhibit 50% of biological function. The IC₅₀ value was calculated in Eqs. (1) and (2). The inhibitory concentration (IC) was calculated by Eq. (1), where the absorbance control represents the absorbance value obtained from the control sample. The absorbance sample represents the absorbance value obtained from the tested sample. (1)${\displaystyle \text{\%}\text{IC}=\left(\frac{{\text{Absorbance}}_{\text{control}}-{\text{Absorbance}}_{\text{sample}}}{{\text{Absorbance}}_{\text{control}}}\right)\times 100\text{\%}.}$ Then, to determine the IC₅₀, a graph of the relationship between the inhibitory concentration and percent inhibition was made. The regression equation was derived from the graph and the IC₅₀ value was calculated by the following equation “a” represents the slope of the dose–response curve, while “b” represents the y-intercept of the dose–response curve. (2)${\displaystyle {\text{IC}}_{50}=\left(\frac{50-b}{a}\right).}$

In vitro antidiabetic assay of extracts

The anti-diabetic activity was quantified using the α-glucosidase inhibition assay as follows: 100 µL of potassium phosphate buffer (10 mM, pH 6.8), 20 µL of α-glucosidase (1U/ml), and 40 µL of extract were mixed into 96-well plates, and afterward incubated for 15 min at 37 °C. Then, 40 µL of 2mM pNPG were added and incubated for 15 min at 37 °C. After the incubation, 100 µL of 0.1 M Na₂CO₃ were added and the absorbance was measured at 405 nm. A mixture without the extract was used as a blank, while a mixture without the extract and enzyme was taken as a control. Acarbose was used in the assay as a positive control (Lordan et al., 2013).

The α-amylase inhibition was determined by the following method: 40 µL of the extract and 40 µL of 1% starch solution were added into a microcentrifuge tube (1.5 ml, Thermo Fisher), incubated for 10 min at 25 °C. Then, 40 µL of α-amylase solution (0.5 mg/ml) was added and incubated for 10 min at 25 °C. Afterward, 80 µL of DNS was added and incubated for 5 min at 100 °C. The reactants were cooled at 0 °C for 5 min. Next, 50 µL of the reactant’s solution and 200 µL of DI water were added to 96-well plates. The absorbance was measured at 540 nm. A mixture without the extract was used as a blank, while a mixture without the extract and enzyme was taken as a control. Acarbose was used in the assay as a positive control (Figueiredo-Gonzalez et al., 2016).

The inhibition of α-glucosidase and α-amylase was determined using Eq. (1). After determining IC, the IC₅₀ values were calculated using Eq. (2).

Statistical analysis

A two-way analysis of variance was performed in the Jamovi Program (The Jamovi Project, 2019) version 0.9.5.12 to determine the effect of indigenous Thai fruit extracts on chemical constituents and bioactivities. Post-hoc comparisons between the extracts were performed with Tukey’s HSD test. The p-values <0.05 were considered statistically significant.

Phytochemical evaluation

Methanolic extracts were used for phytochemical evaluation in this study. The total phenolic content was reported as gallic acid equivalents per gram of methanolic extract (mg GAE/g). As shown in Table 2, the highest amounts of total phenolic contents were found in S. cumini (980.42 ± 0.89 mg GAE/g), followed by S. malaccense (235.98 ± 0.41 mg GAE/g), and L. fruticose (188.19 ± 0.95 mg GAE/g), respectively. Furthermore, this study compared the quantity of flavonoids in 15 fruits. As shown in Table 2, the total flavonoid content was reported as quercetin equivalents per gram of methanolic extract (mg QE/g). The highest amounts of flavonoid content were found in S. cumini (3.55 ± 0.02 mg QE/g), followed by E. latifolia (1.06 ± 0.08 mg QE/g), D. indica (0.94 ± 0.14 mg QE/g), and L. fruticosa (0.77 ± 0.05 mg QE/g), respectively. These findings indicate that S. cumini could be a good source of phenolic and flavonoid supplements compared to all of the fruits in this study. The variation in total phenolic and flavonoid content among samples may be due to genetic factors and ecological conditions. The high phenolic and flavonoid content in S. cumini is consistent with previous studies demonstrating its potential health benefits. S. cumini, therefore, offers antioxidant, anti-inflammatory, and anti-diabetic properties (Priya, Prakasan & Purushothaman, 2017).

Identification and quantitative analysis of phenolic compound

The methanolic crude extract was analyzed for phenolic compounds through ultra-high-performance liquid chromatography (UHPLC) at a wavelength of 286 nm. The study identified six phenolic compounds (catechin, epicatechin, epicatechin gallate, gallic acid, kaempferol, and ellagic acid). The results, presented in Table 3, showed that catechin has the highest concentration in S. cumini (2048.83 ± 0.98 µg/mg), followed by S. oleosa (728.26 ± 0.69 µg/mg), and L. fruticose (473.79 ± 0.58 µg/mg). Epicatechin had the high concentration in S. cumini (5397.40 ± 0.03 µg/mg), and M. calabura (1101.8 ± 0.16 µg/mg). Epicatechin gallate was abundant in S. cumini (3843.07 ± 1.93 µg/mg). Ellagic acid was found in high concentrations in S. cumini (172.45 ± 0.16 µg/mg), and M. calabura (89.91 ± 0.63 µg/ml). Kaempferol was detected in low concentrations in G. dulcis (24.45 ± 0.95 µg/ml), F. indica (16.75 ± 0.11 µg/mg), and D. indica (13.85 ± 0.99 µg/mg). Gallic acid was found in most of the samples, with the highest concentration in M. calabura (2118.55 ± 0.44 µg/mg), followed by M.s elengi (689.26 ± 0.49 µg/mg), and G. dulcis (552.51 ± 0.99 µg/mg).

Antioxidant capacities of crude extracts

The methanolic extracts were determined for antioxidant activities using three assays: DPPH, FRAP, and ABTS, shown in Table 4. The DPPH assay is a colorimetric reaction that is widely used and easy to perform. The results are expressed as IC₅₀ value and indicate that S. cumini (IC₅₀ value of 3.00 ± 0.01 µg/ml) had the highest antioxidant potential among the compounds tested, followed by D. decandra (IC₅₀ value of 110 ± 0.04 µg/ml), and G. dulcis (IC₅₀ value of 120 ± 0.01 µg/ml). The ABTS assay measures the ability of antioxidants to scavenge ABTS radicals generated in aqueous phase. The results are expressed as mg of Trolox and show that S. cumini (IC₅₀ value of 40 ± 0.01 µg/ml) had the highest antioxidant potential, followed by S. malaccense (IC₅₀ value of 430 ± 0.02 µg/ml) and L. fruticose (IC₅₀ value of 500 ± 0.06 µg/ml). The FRAP assay measures the antioxidant capacity by reducing ferric ions to ferrous ions, and the results are expressed as Fe²⁺ equivalents or FRAP values. The results revealed that S. cumini (898.63 ± 0.02 mg TE/ml) had the highest antioxidant potential, followed by S. malaccense (484.75 ± 0.66 mg TE/ml), and A. puncticulatum (169.41 ± 0.69 mg TE/ml). Overall, the results indicate that S. cumini and S. malaccense are excellent sources of antioxidant compounds.

Antidiabetic activities of crude extracts

The antidiabetic capacity of methanolic extracts was determined using two key enzyme assays. Including α-glucosidase inhibition and α-amylase inhibition. The α-glucosidase inhibition assay measures the potential of antidiabetic activity and the results are expressed as IC₅₀. The results, shown in Table 5, reveal that S. cumini (IC₅₀ value of 0.13 ± 0.01 mg/ml) had the highest potential of α-glucosidase inhibition among the samples tested, followed by M. calabura (IC₅₀ value of 3.27 ± 0.82 mg/ml), and D. decandra (IC₅₀ value of 3.96 ± 0.19 mg/ml). Additionally, Acarbose was included as a positive control, exhibiting an IC₅₀ value of 0.86 ± 0.01 mg/ml. The α-amylase inhibition assay is also used to measure the potential of antidiabetic activity and the results are expressed as IC₅₀values. The results, shown in Table 5, emphasize that S. cumini (IC₅₀ value of 3.91 ± 0.05 mg/ml) had the highest ability of α-amylase inhibition, followed by L. fruticosa (IC₅₀ value of 4.14 ± 0.04 mg/ml), and W. edulis (IC₅₀ value of 4.88 ± 0.02 mg/ml). Acarbose, the positive control, exhibited an IC₅₀ value of 0.39 ± 0.05 mg/ml. Overall, the results indicate that S. cumini has the highest potential for antidiabetic activity among the samples tested.