Total phenolic and flavonoid contents, antioxidant, antidiabetic and antiplasmodial activities of Garcinia forbesii King: A correlation study

2.1 Plant material

The stem bark of G. forbesii King was collected from the Somaetek forest in North Halmahera, Indonesia (1°25′22 “LU 127°46′59″ E). This plant was identified and the specimen (VII.G.237a) was stored in the Bogor Botanical Gardens.

2.2 Preparation of extracts

The bark of G. forbesii King was dried at 50 °C and crushed into powder (60–100 mesh) using a milling machine. A total of 15 g of the powder was weighed and extracted by maceration (150 mL) with four solvents: n-hexane, dichloromethane, ethyl acetate and methanol. The obtained extracts were then filtered and evaporated under reduced pressure (40–60 °C) to give dry extracts.

2.3 Total phenolic and flavonoid contents

The Folin-Ciocalteu method was employed for total phenolic content determination following previously reported paper (Badrulhisham et al., 2020) with slight modifications using gallic acid as standard (0–200 mg/L). Briefly, 0.5 mL of the extracts in methanol (1000 ppm) was mixed with 2.5 mL of 10% aqueous Folin-Ciocalteu solution, stirred and left for 5 min. A 2.0 mL of 5% aqueous Na₂CO₃ solution was then added. The mixture was further incubated at 40 °C for one hour and the absorbance was measured at a wavelength of 765 nm by using a UV–Vis spectrophotometer (Genesys UV–Vis Spectrophotometer, Thermo Fisher Scientific, Madison, WI, USA) with methanol as the blank.

The aluminum chloride method was used for total flavonoid content (Armania et al., 2013) evaluation using quercetin as standard (0–50 mg/L) in methanol. A total of 0.5 mL of each extract (100–1000 ppm) was mixed with 0.5 mL of 2% AlCl₃ (in methanol) followed by incubation for one hour and the absorbance was measured at 415 nm using UV–Vis spectrophotometer (Genesys UV–Vis Spectrophotometer, Thermo Fisher Scientific, Madison, WI, USA) with methanol as the blank.

2.4 DPPH radical scavenging assay

The DPPH antioxidant activity of extracts was determined following the reported papers (Brand-Williams et al., 1995; Dudonné et al., 2009). A 3.0 mL of 6 × 10⁻⁵ M DPPH was added to 100 µL of extracts (maximum dissolved concentration) in methanol. After 20 min of incubation at 37 °C, the absorbance of the mixture was assessed at a wavelength of 517 nm (As). The absorbance of the blank was also measured at the same wavelength (Ab). This experiment was carried out in triplo using quercetin and gallic acid as controls. The radical inhibitory activity was calculated using the equation [(Ab-As) / Ab] × 100%.

2.5 ABTS radical scavenging assay

The ABTS radical scavenging activity was evaluated based on the previous papers (Armania et al., 2013; Arteaga-Crespo et al., 2020). The ABTS solution was prepared from 5.0 mL of 7 mM ABTS and 88 mL of potassium peroxydisulfate (K₂S₂O₆). The mixture was left to stand for 12–16 h at room temperature and a dark blue solution was formed. A 250 mL of 99.5% ethanol was then added to the solution, which provided an absorbance of 0.7 ± 0.02 at 734 nm. This was referred as working solution. A total of 1.0 mL of working solution and 10 μL of the extracts (maximum dissolved concentration) in methanol was shaken for 10 s and incubated at 30 °C for 4 min. The absorbance of the mixture (As) was measured at a wavelength of 734 nm with ethanol as a blank (Ab). Quercetin and gallic acid were used as controls. The ABTS inhibition was calculated by [(Ab-As) / Ab] x100.

2.6 Ferric reducing-antioxidant power (FRAP) assay

The iron reduction power of G. forbesii King extracts was evaluated by a method of Dudonné et al. (2009) with a slight modification. This assay was conducted based on the reduction of a colorless iron complex of Fe³⁺-tripyridyltriazine to a blue complex of Fe²⁺-tripyridyltriazine. The FRAP reagent was mixed with freshly prepared 300 mM acetate buffer, 40 mM HCl, 10 mM TPTZ and 20 mM FeCl₃·6H2O at a ratio of 10:1:1 (acetate buffer:TPTZ:FeCl₃·6H₂O). The standard curves were made using various concentrations of FeSO4·.7H₂O and all solutions were prepared in a fresh condition. About 100 μL of sample solution, 900 μL of distilled water, and 2 mL of FRAP reagent were mixed and incubated at 37 °C for 30 min and the absorbance was measured at 593 nm. The blank sample measurement was carried out using acetate buffer. The difference between the absorbance of the samples and the absorbance of the blank was calculated and utilized to obtain the FRAP value and extracts’ reduction capacity. The FRAP value was expressed as µM Fe²⁺/g ((FRAP Value of sample (µM)) = abs (sample) × FRAP value of std (µM) / abs (std)). Ascorbic acid was used as control and all measurements were carried out in triplicate.

2.7 Rat intestinal α-glucosidase inhibitory activity

The inhibitory activity of rat intestinal α-glucosidase was determined according to a modified method (Worawalai et al., 2012). This technique was carried out by grouping four mixtures into four different categories: (1) blank enzyme reaction, (2) enzyme reaction, (3) blank samples reaction, (4) samples reaction. The mixture of (1) contained DMSO (10 µL), sodium phosphate buffer (50 µL), glucose kit (80 µL). The enzyme reaction group (2) consisted of mixture (1) with the addition of maltose or sucrose substrates (20 µL). The positive control of acarbose was similarly prepared as (2). The group (3) was loaded with identical mixture of (1) with samples (10 µL) replacing the DMSO. The last mixture of (4) composed of (3) and substrates maltose or sucrose (20 µL). The well plates were then incubated for 10 min (maltose) or 40 min (sucrose). A microplate reader was used to measure the absorbance at 520 nm (BioTek ELx800TM, BioTek Instruments, Inc., Winooski, VT, USA).

2.8 α-Amylase inhibitory activity

The α-amylase activity was evaluated using a method modified by Unuofin et al., 2018. A 5.0 mg of α-amylase (Porcine pancreatic α-amylase) in 1.0 mL 0,1 M phosphate buffer pH 6,9 was added to a solution of 10 mg samples in 1.0 mL DMSO. The samples (20 µL) were mixed with freshly prepared potato substrate (50 µL) and 30 µL of phosphate buffer (pH 6.9). The substrate was obtained by heating potato starch (100 mg) in 5.0 mL 0.1 M phosphate buffer for 5 min and cooled at room temperature. After a 5-minute pre-incubation period at room temperature, 20 µL of α-amylase was then added. Following that, the solution was incubated at 37 °C for 15 min and a color change was noticed by adding 50 µL of 1 M HCl and 50 µL of 10% iodine solution. A UV–Vis spectrophotometer (Genesys UV–Vis Spectrophotometer, Thermo Fisher Scientific, Madison, WI, USA) was used for absorbance measurement at a wavelength of 650 nm.

2.9 Antiplasmodial activity

The antiplasmodial activity of the extracts was judged by a method of Budimulja et al. (1997) using chloroquine sensitive Plasmodium falciparum strain 3D7. Samples were made at concentrations of 1000, 100, 10, 1 and 0.1 µg/mL. The parasites used in this test were synchronous (Ring stage) with ± 1% parasitemia. A 2.0 µL of the test solution with various concentrations was pipetted to 96 wells followed by 198 µL of parasites. The well was further placed in a chamber with O₂ 5%, CO₂ 5%, and N₂ 90% atmosphere. The chamber was then incubated at 37 °C for 48 h and subsequently the culture was harvested, and a thin blood layer was made with 20% Giemsa staining. Data analysis was carried on the blood smear that has been made by counting the number of infected erythrocytes in every 1000 normal erythrocytes under a microscope. The data was further used to determine the percent growth and inhibition.

2.10 Statistical analysis

The statistical analysis was carried out with IBM 23 statistical software to determine total phenolic and flavonoid contents, antioxidant activities and antidiabetic activities. This study was carried out in thrice to determine the mean value (mean SD). A linear regression equation was used to determine the percentage of α-glucosidase and α-amylase inhibition of each extract's concentration. Meanwhile, probity analysis was used to determine the proportion of growth inhibition on P. falciparum.

3.1 Total phenolics and total flavonoids contents

In this study, the total phenolic and flavonoid contents of n-hexane, dichloromethane, ethyl acetate and methanol extracts from the bark of G. forbesii King were tested. The results of total phenolic content were expressed as gallic acid equivalents using the equation of a standard curve y = 0.0042x + 0.0234, R² = 0.9901, as shown in Fig. 1a. The n-hexane extract showed the highest phenolic content (187.37 ± 0.06 mg GAE/g dry extract). Ramirez and co-workers (2019) found the same profile of n-hexane extract with high phenolics from the epicarp and leaves of G. madruno. In contrast, methanol extract, as shown in Table 1, had the lowest phenolic contents (73.40 ± 0.11 mg GAE/g dry extract). This result indicated that phenolic compounds of G. forbesii King were accumulated in nonpolar and semipolar extracts, and less found in polar extract. This finding was supported by the isolation of five xanthones from dichloromethane extract, including lichexanthone, subelliptenone H, 12b-hydroxy-des-D-garcigerrin A, garciniaxanthone B and garcigerin A (Wairata et al., 2021). The flavonoid contents were expressed as quercetin equivalent using the equation y = 0.0337x: R² = 0.9973, as shown in Fig. 1b. The methanol extract displayed the highest flavonoids (35.97 ± 0.02 mg EQ/g dry extract) while the n-hexane extract was the lowest (6.60 ± 0.14 mg EQ/g dry extract). It was noticed that flavonoid compounds were accumulated in polar solvents and lower in nonpolar solvents (Arteaga-Crespo et al., 2020; Barbouchi et al., 2020).

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Fig. 1

Standard curves for gallic acid (a) and quercetin (b).

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3.2 Antioxidant activity

3.3 α-Glucosidase and α-amylase inhibitory activities

The enzyme inhibitory activity for all extracts was assessed using α-glucosidase and α-amylase. The α-glucosidase and α-amylase are key enzymes involved in carbohydrate metabolism. α-Amylase degrades complex carbohydrates into oligosaccharides and disaccharides which further degrade into monosaccharides by α-glucosidase. The liberated glucose is then absorbed by the intestine and causes postprandial hyperglycemia. The inhibition of this process reduces the postprandial glucose levels by slowing the hydrolysis and absorption of carbohydrates in the blood which is useful in the treatment of type 2 diabetes (Shinde et al., 2008).

The α-glucosidase inhibitory activity of extracts from G. forbesii King was observed using rat intestinal enzymes with sucrose and maltose substrates. α-Glucosidase breaks sucrose into glucose and fructose while maltose becomes two glucoses (Kadouh et al., 2016; Unuofin et al., 2018). Table 3 showed that the inhibition percentage and IC₅₀ value of dichloromethane extract using sucrose substrate were 86.68 ± 0.05% and 35.13 ± 2.01 µg/mL at a concentration of 312.5 µg/mL. This value was the highest compared to other extracts. The n-hexane extract produced lowest inhibition in sucrose with 63.14 ± 0.02% of inhibition and IC₅₀ of 60.87 ± 1.06 µg/mL. In contrast, the n-hexane extract displayed the highest activity using maltose with a percent inhibition and IC₅₀ value of 86.32 ± 0.02% and 38.94 ± 1.45 µg/mL. The methanol extract was the least active extract in maltose with 64.14 ± 0.01% of inhibition and IC₅₀ of 29.89 ± 1.54 µg/mL. These data indicated that nonpolar extracts were very active for α-glucosidase inhibition compared to others.

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Fig. 2

Percentage of antiplasmodial inhibition activities of G. forbesii extracts.

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α-Amylase is an enzyme responsible for starch processing into glucose (Venkatachalam et al., 2020). Therefore, the inhibitory activity of the extracts from G. forbesii King was also tested against this enzyme. The dichloromethane extract showed a strong inhibition activity against α-amylase with IC₅₀ value of 4.83 ± 0.20 μg/mL at a concentration of 99.01 μg/mL ( Table 3). The lowest activity was shown by methanol extract with IC₅₀ of 16.47 ± 0.51 μg/mL. The standard acarbose had an IC₅₀ value of 2.61 ± 0.32 μg/mL. The α-amylase activity evaluation confirmed the profile of nonpolar and semipolar extracts in G. forbesii King.

3.4 Antiplasmodial activity

The in vitro antiplasmodial assay of G. forbesii King stem bark extracts were performed against P. falciparum strain 3D7. The results, as presented in Table 4 and Fig. 2, revealed that n-hexane, ethyl acetate, and methanol extracts showed promising activity at a 100 μg/mL concentration compared to chloroquine standard. The methanol extract produced the highest activity at a concentration of 10 μg/mL compared to other extracts and chloroquine. Furthermore, at a concentration of 1 μg/mL, the inhibition percentage of n-hexane extract was 63.32%. The IC₅₀ values ​​of the n-hexane and methanol extracts were of 0.23 ± 0.01 μg/mL and 0.73 ± 0.01 μg/mL close to the chloroquine standard (IC₅₀ 0.002 ± 0.01 μg/mL). The IC₅₀ values ​​in the range of 10 to ≤ 10 μg/ mL were expressed as very active. Therefore, the extracts of n-hexane and methanol were active (IC₅₀ value ≥ 10 μg/mL) (Kainama et al., 2019). The results indicated that the extracts of G. forbesii King are potential therapeutics against P. falciparum 3D7 strain (Lulan et al., 2020).

3.5 Correlation analysis

This study correlates the total phenolic and flavonoid contents, antioxidant, antidiabetics, and antiplasmodial activities of G. forbesii Kings extracts. The relationship was calculated by using Kendall's Tau correlation. Hunter and co-workers (2021) reported the use of Kendall’s Tau calculation in the correlation of Manuka-type honeys with their bioactivities. The total phenolics negatively correlated with total flavonoids (r = -1,000, p < 0.01) means that the higher the total phenolic content, the lower the total flavonoid content. In general, total phenolics and flavonoids play an important role in the antioxidant activity acting as a chain breaker, free radical scavenger and electron donor (Kainama et al., 2020). Table 5 shows a strong positive correlation between DPPH vs total phenolics and DPPH vs total flavonoids with r = 0.718 (p < 0.005). A weak correlation was observed between ABTS vs total phenolics and ABTS vs total flavonoids with r = 0.174 (p < 0.05), but ABTS had a strong correlation with DPPH (r = 0.718, p < 0.05). The total phenolics and FRAP were negatively fierce (r = -1,000, p < 0.01) which shows an inverse relationship between phenolics and Fe³⁺ reduction activity. The higher the phenolic, the lower the reduction activity. In contrast, a positive correlation was occurred for total flavonoids vs FRAP (r = 1.000, p < 0.01). This indicated the flavonoid compounds contained in the extracts were directly proportional to the reduction power. Furthermore, the ABTS was strongly correlated with α-glucosidase inhibitory activity (sucrose r = 1.000, p 0.05; maltose r = 1.000, p < 0.01). In addition, the α-amylase inhibitory activity was also showed a relationship with ABTS (r = 1.000, p < 0.05). This data concluded that there is a correlation between antidiabetic and antioxidant activities. The lipophilic and hydrophobic compounds are active in ABTS and as antidiabetics (Huang et al., 2020).