The α-glucosidase inhibitory activity of avicularin and 4-O-methyl gallic acid isolated from Syzygium myrtifolium leaves

Abstract

Diabetes Mellitus is the main cause of death on a global scale. In 2019, there were 463 million people with diabetes, and WHO predicts that by 2030, there will be 578 million. As an antidiabetic agent, α-glucosidase inhibitors are one of the methods employed to reduce the prevalence of diabetes. Diabetes is traditionally treated with Syzygium as a primary material, medicine, fruit, ornamental plant, and source of carpentry. This investigation aimed to examine the inhibitory effect of seven species of Syzygium against α-glucosidase enzyme using an in vitro assay and isolate active substances and ascertain their concentrations in each sample. As a solvent, ethanol was used in maceration to extract the substance. Afterward, the extract underwent a series of fractionation techniques, including liquid–liquid extraction, vacuum liquid chromatography, column chromatography, and preparative Thin Layer Chromatography (TLC) for purification and isolation. The compound's structures were elucidated using TLC, UV–Visible spectrophotometry, and nuclear magnetic resonance (NMR) spectroscopy. Based on concentrations of 100 and 200 µg/mL, Syzygium myrtifolium exhibited the most significant inhibitory effect, followed by other species of Syzygium. The proportion of ethyl acetate had the strongest activity (IC₅₀ 0.40 ± 0.02 µg/mL) contrasted to positive control acarbose (IC₅₀ 55.39 ± 0.67 g/mL) and quercitrin (IC₅₀ 6.47 ± 0.40 µg/mL). Avicularin and 4-O-methyl gallic acid were discovered in the ethyl acetate fraction of Syzygium myrtifolium with IC₅₀ values of 17.05 ± 0.75 µg/mL and 25.19 ± 0.21 µg/mL, respectively. As α-glucosidase inhibitory, the results of this study indicate Syzygium myrtifolium can be used as a dietary supplement to manage hyperglycemia.

1. Introduction

Diabetes is a metabolic condition distinguished by untreated high blood sugar (World Health Organization, 2019). High glucose levels in the body, known as hyperglycemia, can interact non-enzymatically with plasma proteins. This interaction can generate glycated hemoglobin and glycated albumin. The accumulation of progressed glycation final products in various body parts, including the kidneys and retina, can contribute to diabetes-related complications (Priya et al., 2018). World Health Organization (WHO) projects that there will be 578 million individuals with diabetes by 2030, up from 463 million in 2019. Prevalence increases by 51% between 2019 and 2045 (Saeedi et al., 2019).

Inhibiting carbohydrate hydrolysis enzymes (α-glucosidase found in the digestive organs) and lower postprandial glucose levels are critical for managing type 2 diabetes mellitus (Li et al., 2005). These medications include acarbose, miglitol, and voglibose (Kumar et al., 2011). Numerous hypoglycemic agents have undesirable side effects, for instance, bloating, nausea, diarrhea, and flatulence. Therefore, the development of natural α-glucosidase inhibitors is widespread because of fewer adverse effects (P et al., 2011).

Syzygium can be used as fruit trees, ornamental plants, and carpentry sources for nutritional and medicinal purposes (Rabeta et al., 2013). Syzygium is a Myrtaceae family member with between 1200 and 1800 species found in Africa, southern Asia, and the Pacific (Rocchetti et al., 2019). Syzygium is a plant with fruit numerous chemical constituents and bioactivity. Fruits may enhance the human diet's amount of vitamins, minerals, and bioactive compounds (Vasco et al., 2008). Several Syzygium species have been investigated for their chemical constituents and bioactivity. They were reported for antioxidant, antiviral, antidiabetic, and hepatoprotective properties (Sobeh et al., 2018b).

Syzygium aromaticum is traditionally used to treat dental infection, toothaches, burn, and wounds (Batiha et al., 2020). Syzygium cumini seeds and leaves are used in India and Brazil to manage diabetes (Ayyanar and Subash-babu, 2012). Syzygium aqueum leaves have alleviated childbirth pains (Manaharan et al., 2014). The intake of Syzygium polyanthum leaves is linked to treating diabetes, hypertension, ulcers, diarrhea, skin disease, and infection (Ismail and Wan Ahmad, 2019). Syzygium myrtifolium addresses stomach aches (Memon et al., 2015). In India, the fruit tonic of Syzygium jambos is known to enhance the health of the brain and liver, whereas the fruit infusion exhibits diuretic properties (Ochieng et al., 2022). Syzygium malaccense has been traditionally used to treat mount ulcers and irregular menstruation (Uddin et al., 2022).

This investigation aims to analyze the potential of Syzygium species as α-glucosidase inhibitors, including Syzygium aqueum, Syzygium aromaticum, Syzygium cumini, Syzygium jambos, Syzygium malaccense, Syzygium myrtifolium, and Syzygium polyanthum leaf extracts. Most prospective species were determined by NMR analysis following the isolation of active compounds.

2. Material and method

2.1. Chemical and reagents

α-glucosidase (Saccharomyces cerevisiae) G5003-100UN Cas number 56180-94-0, and 4-nitrophenyl-α-D-glucopyranoside (pNPG) N1377-1G Cas number 3767-28-0 were acquired from Sigma-Aldrich (St. Louis, MO, USA). Acarbose hydrate was obtained from TCI (Tokyo, Japan). Quercitrin was purchased from Rotichrom. Citroborate and 10% H₂SO₄ in MeOH were specific spray reagents to TLC.

2.2. Sample and ethanolic extract preparation

The leaves of seven Syzygium species were collected in West Java, Indonesia. They were harvested and gathered between October and December 2019. The leaves were cleansed with running water. The items were then desiccated in a drying cabinet. The leaves, which were dried, were pulverized into a powder using a grinder. The pulverized leaves were mixed with a 1:10 (w/v) 96% ethanol concentration (redistilled). Macerate was conducted at ambient temperature for 3x24 hours. The collected filtrate was filtered through Whatman filter paper. The filtrate of ethanol was condensed using a rotary evaporator maintained at a temperature of 50 °C using a rotary evaporator.

2.3. Isolation and identification of active compounds from Syzygium myrtifolium leaves

The specimens with specimen B-149/2021 were authenticated at the Indonesian Institute of Sciences' Research Center for Biology in Bogor, West Java, Indonesia. The extraction was conducted at Bandung Institute of Technology's Department of Biology Pharmacy in Bandung, Indonesia. The juvenile red leaves were separated from the other leaves. Two thousand grams of powdered Syzygium myrtifolium leaves macerate with ethanol 96% at a proportion of 1:10 (w/v) (3x24 hours). Then, a 500 g portion of the extract underwent fractionation by employing a liquid–liquid extraction technique using solvents of escalating polarity, including n-hexane, ethyl acetate, and methanol.

Further purification was performed on the ethyl acetate fraction using vacuum liquid chromatography. A gradient elution system comprising n-hexane, ethyl acetate, and methanol was employed for this purpose. It produced 21 fractions. The fraction with identical chromatogram and promising activity were combined and rechromatographed using column chromatography with isocratic elution, chloroform/methanol (8:1.5). The same chromatogram fraction was merged and rechromatographed using TLC preparative with isocratic elution using chloroform/methanol (8:1.5) to yield 37.2 mg of compound IN1 and 17.5 mg of compound IN2. Compound IN1, amorphous yellow powder. Compound IN2, needle-shaped white crystal.

2.4. α-Glucosidase inhibitory activity

The α-Glucosidase inhibition investigation adopted an adapted method (Vonia et al., 2022). In 96 well microplates, 30 μL of the sample, 36 μL of 0.1 M phosphate buffer solution (pH 6.8), and 17 µL of 6 mM p-nitrophenyl-α-D-glucopyranoside substrate were added, and preincubated at 37 °C for 5 min. After a 5 min preincubation at 37 °C, 17 μL (0.2 units/ml) of the α-glucosidase enzyme were combined, followed by 15 min of incubation at 37 °C to complete the reaction. The chemical response was ended by combining 100 μL of 200 mM Na₂CO₃ in every single well. The absorbance of the chemical process was assessed using a microplate reader at 400 nm (Tecan Infinite 200 pro). Each measurement was conducted in triplicate. Positive control was acarbose, and negative control was the absence of any inhibitor. Calculating the percentage of inhibition:

$$\%\text{Inhibition}=\frac{\mathrm{B}1-\mathrm{B}2}{\mathrm{B}1}\times 100\%$$

• B1 = Blank absorbance * – control of blank absorbance **.

• B2 = Sample absorbance – control of sample absorbance ***.

• * Blank comprises PBS + pNPG + enzyme + Na₂CO₃.

• ** Control of blank comprises PBS + Na₂CO₃, excluding enzyme.

• *** Control of sample comprises extract + PBS + Na₂CO₃, excluding enzyme.

The IC₅₀ was determined by employing a linear regression equation (y = a + bx), where the x-axis corresponds to the concentration of the samples and the y-axis represents the percentage of inhibition. The IC₅₀ value represents the model's strength to inhibit 50% of α-glucosidase activity below the experimental conditions.

2.5. Qualitative analysis of active compound

The analysis employed 2D TLC, TLC with three distinct eluents, and HPLC. 2D TLC using two distinct eluents: chloroform/methanol/formic acid (8:2:0.25) and ethyl acetate/methanol/water (8:2:1.5). TLC with three different eluents using chloroform/methanol/formic acid (8:1.5:0.25), ethyl acetate/methanol/water (8:2:1.5), and methanol/acid formic (10:0.25). HPLC analysis was performed on the Shimadzu LC-20AD with stationary phase using Phenomenex HPLC Column Kromasil 5u 100A C18 with an injection number of 20 µL volume and mobile phase using 0.02% H₃PO₄ in water and methanol. The elution system was gradient and isocratic, 0.01 to 3.00 min using a gradient from 40 to 60% methanol, 3.01 to 7.00 min using isocratic 60% methanol, 7.01 to 12.00 min using isocratic 70% methanol, 12.01 to 15.00 min using isocratic 40% methanol and stop at 15.01 min. HPLC Detector utilized UV 210 and 360 nm.

2.6. NMR analysis

NMR spectrums were obtained using an Agilent 500 MHz with a DD2 console system operating at 500 MHz for ¹H and 125 MHz for ¹³C. Compound IN1 was extracted with solvent CD₃OD and analyzed for ¹H, ¹³C, COSY, HMBC, and HSQC. Compound IN2 was analyzed for ¹H and ¹³C using solvent DMSO-d₆.

2.7. Statistical analysis

The data on α-glucosidase inhibitory activity belonging to extracts, fractions, and isolated compounds were expressed as the mean value and the standard deviation (SD). Statistical analysis of the data was performed using SPSS version 26.0. The independent-sample t-test and one-way analysis of variance (ANOVA) with Tukey's posthoc test were conducted to evaluate the significance. A value of p < 0.05 was considered statistically significant.

3. Result

3.1. Phytochemical screening

The standard phytochemical analysis detected flavonoid, phenol, tannin, quinone, saponin, and steroid/triterpenoid in the crude drug and ethanolic extract of Syzygium myrtifolium (Harborne, 1984).

3.2. In vitro α-Glucosidase inhibitory activity

Leaves of seven species of Syzygium were assessed with in vitro α-glucosidase inhibitory using pNPG as substrate (Table 1). α-glucosidase enzyme inhibition percentages were displayed for each sample. The sample extract with a significant inhibitory effect will be forwarded to the IC₅₀ values and then isolated for the active compound. Syzygium myrtifolium exhibited the most significant inhibitory effect, followed by Syzygium jambos, Syzygium polyanthum, Syzygium aromaticum, Syzygium malaccence, Syzygium aqueum, and Syzygium cumini, respectively based on concentration 100 and 200 µg/mL.

Table 1.

The percentage α-glucosidase inhibitory activity of Syzygium leaf extract.

Samples (leaf extract)	Inhibition (%)
	100 µg/mL	200 µg/mL
Syzygium aqueum	26.51 ± 3.90a,d	48.58 ± 5.23
Syzygium aromaticum	51.69 ± 2.92b	81.93 ± 1.29a
Syzygium cumini	23.13 ± 5.83a	34.55 ± 4.15
Syzygium jambos	83.75 ± 1.37c	92.27 ± 1.13a,b
Syzygium malaccense	37.64 ± 4.37d	62.23 ± 3.30
Syzygium myrtifolium	90.88 ± 1.21c	99.70 ± 1.26b
Syzygium polyanthum	59.45 ± 4.49b	91.22 ± 3.83a,b
Acarbose	50.73 ± 1.27	66.03 ± 1.11*

Note: Data are the mean of three replicates (n = 3) ± standard deviation. At p < 0.05, the means in the vertical column with a similar alphabet are not significantly different. Values are the final concentration. * (Vonia et al., 2022).

The most inhibitory activity of α-glucosidase was fractionated from Syzygium myrtifolium by liquid–liquid extraction from non-polar to polar solvents (Table 2). The IC₅₀ values of 0.40 ± 0.02 g/mL and 0.44 ± 0.03 g/mL for ethyl acetate and water fraction indicate a significant inhibitory effect. The Ethyl acetate fraction was sent to isolate the active compound, yielding IN1 and IN2. IC₅₀ values are 17.05 ± 0.75 µg/mL and 25.19 ± 0.21 µg/mL, respectively.

Table 2.

The IC₅₀ values for α-glucosidase inhibitory activity of extract, fraction, and active compound of Syzygium myrtifolium.

Samples	IC50 (µg/mL)
Ethanol extract	0.42 ± 0.65a
n-hexane fraction	11.61 ± 0.94
Ethyl Acetate fraction	0.40 ± 0.02a
Water fraction	0.44 ± 0.03a
IN1	17.05 ± 0.75
IN2	25.19 ± 0.21
Quercitrin	6.47 ± 0.40
Acarbose	55.39 ± 0.67

Note: Data are the mean of three replicates (n = 3) ± standard deviation. At p < 0.05, the means in the vertical column with a similar alphabet are not significantly different.

3.3. Isolated compound

IN1 was obtained as a yellow amorphous powder. The compound was eluted with Chloroform/Methanol/Formic acid (8:1.5:0.25) on TLC silica gel GF₂₅₄ and reacted with citroborat to produce a green-to-yellow spot, which was then transformed into a yellow dot with 10% H₂SO₄. Analysis of IN1 using 2D TLC and TLC with three distinct eluents revealed one spot. This compound's UV peaks at 298 and 357 nm indicated the existence of flavonoids, mainly the flavonol group (Harborne et al., 1975). The HPLC chromatogram showed one peak at a retention time of 5.7 min with mobile phase Methanol/Water (4:6) using UV 210 and 360 nm detectors. The ¹H NMR spectrum of IN1 (Supplementary Fig. 1s) detected two single protons at flavonoid's A ring with meta-coupled at positions 6 and 8 at δ 6.19 and 6.38. Chemical shifts at 7.75, 6.86, and 7.57 indicated three single aromatic proton signals at position 2′, 5′, and 6′ and indicated 3′,4′-disubtituted flavonoid's B ring. Sugar is present at δ 3.45–5.15 as arabinofuranose. The ¹³C NMR spectrum of IN1 (Supplementary Fig. 2s) showed 20 carbon. Several correlations with heteronuclear single quantum coherence (HSQC) (Supplementary Fig. 3s). 2D NMR, heteronuclear multiple bond connectivity (HMBC) (Supplementary Fig. 4s), and correlation spectroscopy (COSY) (Supplementary Fig. 5s) spectra exhibited a correlation between ¹H→¹³C and ¹H↔¹H. The result of the NMR spectrum has been confirmed as avicularin and compared with previously published data (Ahn et al., 2019, Da Silva Sa et al., 2017).

IN2 was obtained as a white needle crystal. The compound was eluted with Chloroform/Methanol/Formic acid (8:1.5:0.25) on TLC silica gel GF₂₅₄ and did not react when sprayed with citroborat. IN2 did exhibit a dark spot when treated with 10% H₂SO₄. Analysis of IN2 using 2D TLC and TLC with three distinct eluents revealed one spot. This compound showed that UV maximum of 289 nm. The HPLC chromatogram exhibited a single peak at the retention time of 5.1 min with mobile phase Methanol/Water (4:6) and detector UV 210. The proton NMR spectrum (Supplementary Fig. 6s) detected three peaks. The broad single peak at δ 9.21 indicated three substituted OH at positions 2, 5, and 7. The proton signal at δ 6.93 was identified single proton at positions 2 and 6 in aromatic substitution. The peak at δ 3.74 indicates the presence of CH₃ at position 8. Six peaks with different intensities were detected in the ¹³C NMR for the remaining signals (Supplementary Fig. 7s). The spectrum at δ 108.5 and 145.6 exhibits higher intensity peaks than the others, indicating two C for each peak. These data agree with previous reports on 4-O-methyl gallic acid (Abhishek et al., 2019, Farag et al., 2015).

4. Discussion

Hyperglycemia characterizes a group of metabolic disorders known as diabetes. Long-term specific effects cause additional secondary conditions, including retinopathy, nephropathy, and neuropathy, and increase the risk of developing other diseases (World Health Organization, 2019). Inhibiting α-glucosidase found in the digestive organs delayed glucose absorption. That is essential to type 2 diabetes management (Li et al., 2005).

By inhibiting the work of the α-glucosidase enzyme, it is possible to prevent diabetes mellitus, primarily type 2 diabetes mellitus, thereby reducing diabetes-related mortality. The α-glucosidase enzymes (maltase, isomaltase, glucomaltase, and sucrase) function in the small intestine wall to hydrolyze oligosaccharides and disaccharides. This enzyme can be inhibited to effectively decrease the digestion and absorption of complex carbohydrates. The condition helps minimize the postprandial glucose spike in people with diabetes (Shinde et al., 2008). Many gastrointestinal adverse effects are associated with α-glucosidase inhibitors, such as bloating, nausea, diarrhea, and flatulence. The α-glucosidase inhibitors from naturally derived can be used to treat postprandial hyperglycemia, and they have lesser adverse effects and are less expensive in contrast to synthetic antihyperglycemic drugs (P et al., 2011).

Most species of Syzygium were determined to have hypoglycemic effects in vivo and in vitro. On Syzygium species, the following in vitro hypoglycemic effects were examined: α-glucosidase inhibitory activity, α-amylase inhibitory activity, DPP-IV inhibitory activity, and antiglycation assay (Priya et al., 2018). A previous in vitro study of multiple Syzygium revealed their potential as α-glucosidase inhibitors. Other species include Syzygium aqueum (Manaharan et al., 2012), Syzygium aromaticum (Adefegha and Oboh, 2012, Oboh et al., 2015), Syzygium cumini (Ajiboye et al., 2020, Franco et al., 2020, Perera et al., 2018, Liu et al., 2018, Omar et al., 2012, Priya et al., 2018, Trinh et al., 2016), Syzygium malaccence (Arumugam et al., 2014, Arumugam et al., 2016); Syzygium polyanthum (Abdulrahman et al., 2019, Dewijanti et al., 2020, Elya et al., 2015, Rahayu et al., 2019).

Although previous studies have investigated the α-glucosidase inhibitory activity of the genus Syzygium, neither Syzygium myrtifolium nor Syzygium jambos have been mentioned. This study compared the inhibitory activity of α-glucosidase enzyme from seven Species of Syzygium and found that Syzygium myrtifolium had the most significant inhibitory effect (Table 1), followed by Syzygium jambos, Syzygium polyanthum, Syzygium aromaticum, Syzygium malaccence, Syzygium aqueum, and Syzygium cumini, respectively. The isolation phase of Syzygium myrtifolium was continued to determine its active components. The ethyl acetate and water components of Syzygium myrtifolium demonstrated the most significant inhibitory activity. The ethyl acetate fraction was selected for the next step. Two active compounds, Avicularin (Fig. 2) and 4-O-methyl gallic acid (Fig. 3), were determined by NMR Analysis from the ethyl acetate fraction. Their purity was evaluated using HPLC (Fig. 1). Table 3, Table 4 exhibits the information obtained from NMR analysis.

Fig. 2.

(A) Structure of IN1 (Avicularin), (B) Correlation of HMBC and COSY of IN1 (Avicularin).

Fig. 3.

Structure of IN2 (4-O-methyl gallic acid).

Fig. 1.

HPLC Chromatogram of Active Compound. (A) IN1 at 360 nm, (B) IN1 at 210 nm, (C) IN2 at 210 nm.

Table 3.

1D and 2D NMR data of IN1 (Avicularin).

Position	13C	HSQC	HMBC	COSY
	δC	δH (J in Hz)	1H→13C	1H↔1H
1
2	149.9
3	135.6
4	179.4
5	162.9
6	99.8	6.19 (1H, d, 2.1)	C-5, C-7, C-8, C-10	H-6, H-8
7	165.9
8	94.7	6.38 (1H, d, 2.1)	C-6, C-7, C-9, C-10	H-8, H-6
9	158.37
10	105.6
1′	123.0
2′	117.4	7.75 (1H, d, 2.2)	C-2, C-1′, C-3′, C-4′	H-2′, H-6′
3′	145.9
4′	158.6
5′	116.1	6.86 (1H, d, 8.45)	C-2, C-3′, C-6′	H-5′, H-6′
6′	122.8	7.57 (1H, dd, 2.2, 2.2)	C-2, C-2′, C-4′	H-6′, H-5′, H-2′
1″	104.6	5.15 (1H, d, 6.6)	C-3, C-4″	H-1″, H-2″
2″	72.8	3.9 (1H, t, 7.15)	C-1″, C-3″	H-2″, H-1″, H-3″, H-5″
3″	74.1	3.65 (1H, dd, 3.25, 3)		H-3″, H-2″, H-5″
4″	66.9	3.45 (1H, dd, 3.15, 3.2)	C-1″, C-3″	H-4″, H-5″
5″	69.1	3.83 (2H, dd, 3.7, 3.75)	C-1″, C-3″	H-5″, H-2″, H-4″, H-3″

d, duplet; dd, duplet of duplet; t, triplet.

Table 4.

¹H and ¹³C data of IN2 (4-O-methyl gallic acid).

Position	13C	Type of C	1H
	δC		δH (J in Hz)
1	119.3	C—C
2	108.5	CH	6.93 (2H, s) – H
3	145.6	C—OH	9.21 (3H, brs) – OH
4	138.43	C—O
5	145.6	C—OH	9.21 (3H, brs) – OH
6	108.5	CH	6,93 (2H, s) – H
7	166.35	COOH	9.21 (3H, brs) – OH
8	51.63	CH3	3.74 (3H, s) – CH3

brs, broad singlet; s, singlet.

Avicularin is derived from quercetin, but little is known about its biological activity (Fujimori and Shibano, 2013). Avicularin exerts its effect on lipid accumulation in 3T3-L1 cells by inhibiting C/EBP-activated GLUT4-mediated glucose uptake, leading to reduced lipid accumulation (Fujimori and Shibano, 2013), as reported in previous investigations to have biological activity as antibacterial (Da Silva Sa et al., 2017). The previous study showed avicularin exhibited a significant concentration-dependent reduction in cell proliferation in Hcc. Furthermore, it demonstrated a significant inhibitory effect on the moving and invasive capabilities of Huh7 cells. Avicularin also inhibits G0/G1-phase cell population and reduces the aggregation of S-phase cells in the cell stages while inducing cell apoptosis (Wang et al., 2019). Avicularin demonstrated antioxidant activity at a concentration of 100 µg/L, showing a scavenging rate of 87.54% against OH radicals (Lee et al., 2019). Avicularin successfully decreases hepatic inflammation, endoplasmic reticulum stress (ERS), and glucose metabolism problem caused by Pb (Qiu et al., 2022).

Similarly, 4-O-Methyl gallic acid was identified as a gallic acid derivative via NMR spectroscopy (Dao et al., 2019). Compared to gallic acid, 4-O-methyl gallic acid was less effective against distinct influenza strains (Dao et al., 2019). In addition, 4-O-methyl gallic acid inhibits bacteria and yeast, particularly against Streptococcus faecalis, Cryptococcus neoformans (Abhishek et al., 2019), and R. solanacearum (Farag et al., 2015). Other investigations have also reported the efficacy of 4-O-methyl gallic acid in reducing the expression of AT1R and CD36 mRNA in Ang II-treated MDPs. This indicates a potent restraint of Ang II-triggered pro-atherogenic process involved in foam cell formation (Oliveira et al., 2004). 4-O-methyl gallic acid effectively suppressed the expression and synthesis of genes and signaling molecules related to inflammation, containing NO, PGE2 (iNOS, COX-2, and TNF-α expression), and IL-1β, in both RAW264.7 cells and primary macrophages when activated with lipopolysaccharide (LPS) (Na et al., 2006). 4-O-methyl gallic acid not only restrains VEGF creation below the hypoxic state but also suppresses the creation of ROS in endothelial cells that are activated with VEGF. The compound also effectively blocks the invasion of endothelial cells and the formation of tubes when activated with bFGF (Jeon et al., 2005). Based on previous studies, no existing literature documenting the inhibition of the α-glucosidase enzyme by avicularin and 4-O-methyl gallic acid. Therefore, this study is the initial report on the inhibitory ability of these compounds on the α-glucosidase enzyme.

Avicularin and 4-O-methyl gallic acid have not previously been reported in Syzygium myrtifolium. Active compounds that have been reported from Syzygium myrtifolium include anthocyanin, betulinic acid, dimethyl cardamonin, ursolic acid, (2S)-7-Hydroxy-5-methoxy-6,8-dimethyl flavanone, (S)-5,7-dihydroxy-6,8-dimethyl flavanone, (E)-2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone, (E)-2′,4′-dihydroxy-6′-methoxy chalcone (Aisha et al., 2013, Anggraini, 2017, Memon et al., 2015). Avicularin was isolated from Euphorbia humifusa (Ahn et al., 2019), Taxillus kaempferi (Fujimori and Shibano, 2013), Myrcia tomentosa (Da Silva Sa et al., 2017), Vaccinum vitis-idaea (Riihinen et al., 2013), whereas 4-O-methyl gallic acid was isolated from Elaocarpus tonkinensis (Dao et al., 2019), Phyllanthus polyphyllus (Abhishek et al., 2019), Acacia arabica and Punica granatum (Farag et al., 2015). Our study represents the initial documentation of the isolation of Avicularin and 4-O-methylgallic acid from the leaves of Syzygium myrtifolium.

Many active compounds have been found in other Syzygium species, and some have been reported to possess antidiabetic activity. 4-hydroxybenzaldehyde; myricetin-3-O-rhamnoside; europetin-3-O-rhamnoside; phloretin; myrigalone-G; myrigalone-B were found off Syzygium aqueum, which showed inhibitory activity against α-glucosidase and α-amylase enzymes (Manaharan et al., 2012). Clove bud essential oils contained dominant volatile compounds such as α-pinene; β-pinene, and 1,8-cineole also showed α-glucosidase and α-amylase inhibitory activity (Oboh et al., 2015). Active compounds such as jamutannins; iso-oenothein C; oenothein C; cornussin B; swertisin; gallic acid; chlorogenic acid; syringic acid; coumaric acid; ellagic acid; myricetin; ferulic acid; cinnamic acid; quercetin; phloridzin; and p-coumaric acid were found in the seeds of Syzygium cumini. These compounds also demonstrated anti-hyperglycemic activity (Liu et al., 2018, Omar et al., 2012, Priya et al., 2018). Moreover, the leaves were found to contain quercetin; quercetin glycoside; gallic acid; myricetin glycoside; kaempferol glycoside; luteolin glycoside; rosmarinic acid, epicathechin-3-gallate (Franco et al., 2020). Nonanoic acid, methyl ester; Eicosanoic acid, methyl ester; 9,12-Octadecadienoic acid(Z, Z)-, methyl ester; 9-Octadecenoic acid, methyl ester; Tricosanoic acid; (-)-Caryophyllene oxide; Cyclopropane, 1-(2-methylene-3-butenyl)-1-(1-methylenepropyl)-; 1-Tridecene; Tetradecane; 1-Decene; Hexadecane; 2-Pentadecanone were found in Syzygium polyanthum and reported to have antidiabetic activity (Widjajakusuma et al., 2019). Myricitrin was identified in Syzygium malaccense and exhibited anti-hiperglycemic properties (Arumugam et al., 2014). Various compounds were identified in the leaves of Syzygium jambos, including anacardic acid analog, myricitrin, myricetin, ursolic acid, gallic acid, squalene, malic acid, citric acid, castalagin, hexahydroxydiphenoyl-hexoside, casuarinin, ellagic acid, and myricetin rhamnoside (Sharma et al., 2013, Sobeh et al., 2018a).

Although the α-glucosidase inhibitory activity of Syzygium myrtifolium and its isolates is promising, additional progress is required for comprehensive evaluation. This includes conducting toxicity assays, preclinical studies, and clinical trials. Further development is necessary to assess the potential side effects of Syzygium myrtifolium and its isolates, such as avicularin and 4-O-methyl gallic acid. Advance research on avicularin and 4-O-methyl gallic acid is also needed to determine if, in the future, these compounds can be used in the treatment as hypoglycemic agents in addition to acarbose, miglitol, and voglibose.

5. Conclusion

Syzygium myrtifolium and Syzygium jambos leaf extracts inhibited the α-glucosidase enzyme more effectively than other Syzygium species in this study. Two bioactive compounds: Avicularin (IN1) and 4-O-methyl gallic acid (IN2), were determined as the active compounds isolated from Syzygium myrtifolium. Two bioactive compounds have the potential to inhibit α-glucosidase enzyme, in contrast to the positive control acarbose. Avicularin is more potent than 4-O-Methyl gallic acid, but the extract and fraction of ethyl acetate and water are more promising to be developed. This study’s findings suggest that Syzygium myrtifolium leaf extract and its bioactive fraction can be used as a dietary supplement to manage hyperglycemia associated with type 2 diabetes mellitus with further development.

Funding

This work is supported by World Class Research program 2019 managed by the Bandung Institute of Technology.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: