Abstract Sauauria vulcani serves as a rich source of secondary metabolites, including triterpenoids, steroids, tannins, and flavonoids. The present study aimed to assess the biological activities of the S. vulcani leaves, which is an indigenous plant found in West Indonesia. Dried S. vulcani leaves were macerated with hexane to obtain HPE, ethyl acetate to obtain EPE, and methanol to obtain MPE. The TPC was assessed using the Folin-Ciocalteu method, and the TFC was determined utilizing the aluminum chloride assay. Antioxidant activity was evaluated through the DPPH and ABTS assays. The α-amylase inhibition activity of HPE, EPE, and MPE was assessed using the DNS method. The study identified various secondary metabolites in the extracts, including phenolics, flavonoids, triterpenoids, steroids, tannins, and saponins. MPE exhibited the highest TPC and TFC with values of 38.501 ± 0.891 mg GAE/g and 31.436 ± 0.797 mg QAE/g, respectively. In the DPPH and ABTS assays, MPE showed the highest antioxidant capacity, with IC₅₀ values of 54.74 µg/mL and 61.10 µg/mL, respectively. EPE and MPE of S. vulcani displayed increased levels of polyphenolic content, which contributed to their antioxidant effects. The α-amylase inhibitory studies showed that the extracts of S. vulcani (EPE, MPE) had significant inhibitory effects. This study suggests that the EPE and MPE extracts of S. vulcani have the potential as natural sources of bioactives for drug development.

Keywords: Antioxidant, ABTS, Anti-diabetic, DPPH, Sauauria vulcani

Funding: The authors are grateful for the research funding provided by the Universitas Negeri Gorontalo, Indonesia.

Citation: JA Musa, W., Bialangi, N., Kilo, A. K., and Situmeang, B. 2025. Evaluation of polyphenolic content, antioxidant and anti-diabetic activity of different solvent extracts of Sauauria vulcani Korth. leaves. Natural and Life Sciences Communications. 24(2) e2025022.

INTRODUCTION

The development of natural resources is becoming a more significant objective in numerous countries, particularly Indonesia, which has a wealth of herbal plants (Hatami et al., 2014). Herbal plants are commonly utilized in beverages and to address infections, with both the public and private sectors frequently investing in research to discover new beneficial phytochemicals from medicinal plants. Sauauria vulcani Korth. commonly known as pirdot in North Sumatra province, Indonesia, is a tropical tree that can be found in Western Indonesia (Lubis et al., 2022). Part of the Actinidiaceae family, the leaves of this plant are utilized by the community to create herbal green tea (Hutahaean et al., 2018). Sauauria vulcani serves as a rich source of secondary metabolites, including triterpenoids, steroids, tannins, and flavonoids (Sinaga et al., 2020).

Indonesia is one of the top ten countries with the highest number of adults (20–79 years) living with diabetes. In 2021, the International Diabetes Federation (IDF) estimated that 19.5 million Indonesians had diabetes, equivalent to 10.6% of the adult population. According to the IDF Diabetes, 537 million adults (20–79 years) globally live with diabetes, approximately 10.5% of the world's adult population (Bialangi et al., 2024).

In previous research, our group isolated and characterized two triterpenoid acids compounds from the ethyl acetate extract of S. vulcani (Musa et al., 2019). These isolated compounds exhibited significant cholesterol-lowering activity. Despite limited research on this plant, its biological screening has revealed intriguing pharmacological properties, including anti-diabetic and antioxidant effects. Oxidative stress contributes to the progression of diabetic complications such as neuropathy, nephropathy, retinopathy, and cardiovascular diseases. It damages cellular structures, including lipids, proteins, and DNA. Phenolics are potent antioxidants due to their ability to donate hydrogen atoms or electrons to neutralize free radicals. In diabetic patients, phenolic compounds can reduce oxidative stress by scavenging excess free radicals (Ratnasari et al., 2022). The choice of solvent in extracting bioactive compounds significantly impacts the antioxidant and antidiabetic activities of S. vulcani. Methanol is ideal for antioxidant and antidiabetic research focusing on phenolics and flavonoids, while hexane and ethyl acetate can uncover less-polar bioactives.

MATERIAL AND METHODS

Materials

Sauauria volcani leaves as raw material, AR grade ethyl acetate, AR grade methanol, AR grade n-hexane, Folin Ciocalteu reagent, 7.5 % sodium carbonate (Merck D1435), gallic acid (Sigma Aldrich), quercetin (Sigma Aldrich), 3,5-dinitrosalicylic acid (DNS), 2,2-diphenyl-1-picryl-hydrazine-hydrate (DPPH) (Sigma Aldrich), 2.2 Azinobis (3-Ethylbenzotiazolin) 6 Sulfonat (ABTS) (Sigma Aldrich), alfa amylase (Sigma Aldrich).

Plant preparation

In June 2022, fresh S. vulcani leaves were gathered from Sungkean village, Nainggolan Subdistrict, Samosir Island District, Sumatera Utara Province, Indonesia. A botanical specialist analyzed and classified the plant samples, identifying the specimen using available literature. The plant was identified by the staff of LIPI Cibinong, Indonesia with a voucher specimen Number FR-SP 0101. At the herbarium, a specimen was deposited. The leaves were cleaned, cut into approximately 0.5 x 0.5 cm pieces, and dried at room temperature (27°C) for 14 days. The materials were pulverized into coarse powder and macerated in n-hexane, ethyl acetate, and methanol, with occasional agitation at room temperature for three days each. After three days, the extracts were filtered with filter paper (Whatman No. 1), Subsequently, a rotary evaporator used to concentrate the extracts. The crude concentrated extracts were then refrigerated until utilized. The extract is stored in a refrigerator using an airtight container.

Qualitative phytochemical screening

Standardized phytochemical tests (flavonoids, phenolics, triterpenoids, steroids, tannins, saponins, and alkaloids) were conducted on Saurauia vulcani extracts to assess the presence of various bioactive constituents (Nurhasnawati et al., 2019). The results were determined by observing colour changes or the formation of a precipitate occurred upon the addition of specific reagents for 10-15 minutes.

Total phenolic content (TPC)

A 750 μL of freshly diluted Folin-Ciocalteu reagent (1:10 ratio with distilled water) was mixed with 100 μL aliquot of each HPE, EPE, and MPE solution.  After a period of 10 minutes, 750 μL of 6% b/v (Na₂CO₃ solution was added to the mixture. A spectrophotometer used to determine the absorbance at 725 nm. The TPC was established using a standard calibration curve constructed with gallic acid (2.00 to 10.00 µg/mL). The TPC was quantified as milligrams of gallic acid equivalent per gram of sample (Tavanappanavar et al., 2024).

Total flavonoid content (TFC)

The AlCl₃ used to evaluated TFC of the tested HPE, EPE, and MPE solutions (Samid et al., 2023). A 200 μL aliquot of appropriately concentrated crude extracts was blended with 2.3 mL of MeOH solution, then added of 100 μL each of 0.5 M NaNO₂ and 0.3 M AlCl₃.  500 μL NaOH 1M was added after the mixture was vortexed thoroughly. Subsequently, a UV-spectrophotometer utilized to determine the absorbance at 506 nm with a blank (the identical mixture lacking AlCl3). The TFC was determined utilizing a quercetin curve of calibration (0.25-4.00 mg/mL) and presented  as milli grams of quercetin equivalent per 100 grams of sample (Samid et al., 2023).

DPPH radical scavenging assay

The DPPH assay was used to measure the in vitro antioxidant activity of HPE, EPE, and MPE (Aldayel, 2023). In a methanol solution, 600 μL of DPPH was combined with 2400 μL of samples at various concentrations (ranging from 20.00 to 100 µg/mL). The mixtures were then incubated in darkness at room temperature for 30 minutes. A wavelength of 517 nanometers used to measure the absorbance of the mixture. Ascorbic acid and Trolox served as positive controls, and the percentage of DPPH scavenging activity was utilized to assess the antioxidant activity of the extract (Ludwaba et al., 2024). The IC₅₀ values, were determined through graphical analysis (Chaima et al., 2023).

ABTS radical scavenging assay

The antioxidant capacity of HPE, EPE, and MPE was assessed through the ABTS radical cation (ABTS^•+) decolorization assay to determine their free radical scavenging activity. The ABTS assay is a colourimetric method that depends on the generation of the ABTS^•+, which is subsequently diminished with antioxidants, causing a colour transition from pale greenish to colourless. In brief, 10 mL ABTS 7 mM was combined with 5 mL K₂S₂O₈ 2.4 mM. The generation of ABTS^•+ was facilitated by the mixture for 16 hours at dark room temperature (Idowu et al., 2023). The ideal absorbance range for the prepared ABTS^•+ standard solution was found to be 0.600-0.700. The absorbance measured at 734 nm after the samples were dissolved in methanol at concentrations about 20 µg/mL to 100 µg/mL. Trolox and ascorbic acid were utilized as standards within a concentration range of 2.0 mM to 10.0 mM. The percentage of ABTS inhibition (ABTS inhibition %) was determined using the formula [(Aa – Ab) / Aa] × 100. Aa represents the absorbance of a blank, while Ab denotes the absorbance of the sample (Indira et al., 2023).

In vitro anti-diabetic activity

The α-amylase Inhibitory activities of HPE, EPE and MPE were evaluated using a standardized procedure, which included incubating 500 µL of acarbose and plant extracts with 500 µL of α-amylase (1 IU/mL in 0.02 M Na₃PO₄ buffer, pH 6.9) at room temperature for 10 minutes (Wickramaratne et al., 2016). After the 10-minute incubation, the mixture was mixed with a 1% (w/v) starch solution and incubated at 37°C for 20 minutes. The reaction was halted by adding 1 mL of 3,5-dinitrosalicylic acid (DNS) reagent, followed by boiling the mixture for 5 minutes (Tinrat and Jiraprasertwong, 2023). Subsequently, 10 mL of distilled water was added to the mixture, which was then cooled to room temperature before measuring the absorbance at 540 nm. A blank control was prepared using the same procedure as described above, omitting the extract sample. The α-amylase inhibition percentages were calculated using the formula below:

α-amylase inhibition (%) = [(Ab - As) / Ab] × 100,

where Ab denotes the absorbance of the control, while As denotes the absorbance of the samples (HPE, EPE, MPE).

Statistical analysis

The results were reported as the mean ± standard deviation. Variations in TPC, TPC, DPPH and ABTS radical scavenging were assessed using one-way ANOVA in triplicate. Statistical analysis was conducted using Microsoft Excel and GraphPad Prism 10.0.0 with significance set at P < 0.05.

RESULTS

Qualitative phytochemical screening

Table 1 presents several of the phytochemical screening results of n-hexane (HPE), ethyl acetate (EPE), and methanol (MPE) extracts of S. vulcani. The result revealed the absence of alkaloids in all extracts, saponins in the n-hexane extract, and tannins in both the n-hexane and ethyl acetate extracts.

Table 1.  Phytochemical screening of whole Sauauria vulcani Korth. leaves extracts.

Note: +: Presence; -: Absence

Total phenolic and total flavonoid content

Table 2 presents the TPC and TFC of all extracts. According to the quantitative analysis, MPE exhibited the highest total phenolic content (38.501 ± 0.891) and flavonoid content (31.436 ± 0.797). Conversely, the lowest levels of TPC and TFC were observed in HPE.

Table 2.  Polyphenolic content of S. vulcani extracts.     

Note: The results show the average values of three replicates with standard deviation (SD).

Antioxidant activity result

The scavenging capabilities against free radicals were assessed using the DPPH and ABTS methods. The DPPH radical scavenging activities of HPE, EPE, and MPE are detailed in Table 3, while Table 4 presents the ABTS radical scavenging activities. IC₅₀ values were determined through linear regression analysis of DPPH and ABTS scavenging activities against sample concentration, representing the concentration needed to neutralize 50% of the free radicals for each method. Different IC₅₀ values for DPPH and ABTS were observed for the whole extracts. The highest antioxidant activity in DPPH and ABTS are shown in MPE with IC₅₀ value of 54.74 and 61.10, respectively. The % inhibition figures of HPE, EPE, and MPE in DPPH are presented in Figure 1 and in ABTS shows in Figure 2.

Table 3. DPPH free radical-scavenging of S. vulcani extracts.

Table 4.  Free radical-scavenging activity of S. vulcani extract in ABTS.

Figure 1. DPPH radical capacity of the extracts of Sauauria vulcani Korth. leaves. Data are mean ± SD (n = 3).

Figure 2. ABTS radical scavenging capacity of the extracts of Sauauria vulcani Korth. leaves. Data are mean ± SD (n = 3).

In vitro anti di-abetic activities

The study examined the potential antidiabetic activity of S. vulcani leaves extracts through their inhibition of α-amylase activity. Table 5 presents the IC₅₀ values for these anti-α-amylase activities. The highest anti-α-amylase activities were indicated in MPE and the lowest anti-α-amylase activities showed in HPE.

Table 5. The IC₅₀ value of anti-α-amylase activities of S. vulcani extract.

DISCUSSION

In this research, n-hexane was chosen for extraction due to its effectiveness in dissolving non-polar secondary metabolite groups, being an alkane derivative. Ethyl acetate was selected for extraction as it is an ester derivative known for its ability to effectively dissolve semi-polar secondary metabolite groups. Methanol was employed in the extraction process for its capability as an alcohol derivative to dissolve both polar and non-polar groups of secondary metabolites (Elish et al., 2023). Moreover, methanol's non-hydrolyzing nature, low volatility at a low boiling point, and compatibility with thermolabile compounds.

Table 1 presents the outcomes of the phytochemical screening conducted on the HPE, EPE, and MPE extracts. The leaf extracts of S. vulcani were found to contain various phytochemical components, including flavonoids, tannins, phenolics, steroids, and saponins. The data indicates that HPF contains only flavonoids, tannins, and saponins, while EPE and MPE contain almost all components except alkaloids. Flavonoids and phenolics were detected in all leaf extracts, as these compounds are natural polyphenols found in various fruits, vegetables, nuts, seeds, flowers, leaves, and barks.

Table 2 presents the quantitative determination of TFC in the HPE, EPE, and MPE extracts using the colorimetric method with aluminum chloride. The TFC values ranged from 2.875 to 31.436 mg QE/g. Statistical analysis indicated notable variances among the three types of solvents (P < 0.05). The MPE exhibited the highest TFC content among the S. vulcani extracts, while HPE had the lowest. Moreover, MPE not only had the highest TFC but also the highest TPC content in S. vulcani leaves, with HPE showing the lowest TFC and TPC content contrary to our observations, MPE exhibited greater TFC compared to HPE and EPE. Although MPE displayed the highest flavonoid content, it also produced a notable quantity of phenolics. This observation is unsurprising, as flavonoids have been known to be extractable using solvents with high polarity (Alkowni et al., 2023; Warinthip et al., 2023).

Phenolic compounds are essential secondary metabolites known for their antioxidant properties (Mishra et al., 2022; Sirisa-Ard et al., 2023). The hydroxyl groups (-OH) are responsible for scavenging free radicals (Kabré et al., 2023). Table 2 shows the TPC measured using the Folin–Ciocalteu reagent. Statistical analysis indicated significant differences in TPC among the three S. vulcani extracts (P < 0.05). The MPE exhibited the highest TPC, while the HPE had the lowest. This suggests that methanol was the most effective solvent for extracting TPC in this study. This preference for polar solvents is due to their superior ability to extract phenolic compounds compared to nonpolar and semi-polar solvents. We also found significant differences in the TPC among the three extracts. The study found that the redox properties of S. vulcani leaves, which acted as singlet oxygen quenchers, reducing compounds, and hydrogen donors, were the main source of the antioxidant activity of the leaves.

In this study, the IC₅₀ values for radical scavenging in S. vulcani extracts were determined using the DPPH and ABTS assays (Tables 3 and 4). The DPPH free radical has been used to test antioxidant activity. It measures the change in violet color caused by electron transfer. At room temperature, it is a stable free radical that forms a diamagnetic molecule by accepting electrons or other free radicals, with the highest absorbance at 517 nm due to the presence of an odd electron. As antioxidant molecules quench the DPPH free radicals and lower the absorbance, the freshly generated DPPH solution transitions from deep blue to colorless (Mishra et al., 2022). DPPH and ABTS radical scavenging assays are simple and widely used techniques to investigate the antioxidant capacity of various plant extracts.

The strength of the antioxidant activity was determined based on IC₅₀ value, referring to Musa et al. (2023) where the value of < 50 ppm is considered as very strong, 50-100 ppm as strong, 100-250 ppm as moderate, 250-500 ppm as weak, and > 500 ppm as inactive. The extracts were ranked from strongest to weakest antioxidant activity as MPE, EPE, and HPE, respectively. Results are presented as both percentage inhibition and IC₅₀ values.

For the DPPH assay, the IC₅₀ values for HPE, EPE, and MPE were 343.62, 79.37, and 54.74 µg/mL, respectively, while for the ABTS assay, they were 124.28, 78.72, and 61.10 µg/mL, respectively. The MPE and EPE classified as strong antioxidant, while HPF classified as weak antioxidant. Ascorbic acid, the positive control in the DPPH method, exhibited the highest IC₅₀ value compared to HPE, EPE, and MPE. Trolox, the positive control in the ABTS method, showed an IC₅₀ value of 4.96 µg/mL. It was hypothesized that MPE would exhibit the highest antioxidant activity due to its higher TPC and TFC levels (Table 2). The outcomes of the DPPH and ABTS assays validated that the methanol extract (MPE) from S. vulcani leaves exhibited the greatest antioxidant capability.

The variation in the quantity of antioxidants is influenced by several factors, with the choice of solvent for extraction being the most critical (Abdallah et al., 2024). The type of solvent used for extraction is the most important. In addition, the plants contained the mixture of polar and non-polar secondary metabolites compounds which can be dissolved in solvents with different polarities (Musa et al., 2023). The empirical rule, “like dissolves like”, means that polar compounds dissolve in polar solvents and lesspolar compounds dissolve in less-polar solvents (Hatami et al., 2014). The HPE showed the lowest antioxidant capacity in both method (DPPH and ABTS). The study reported that the compounds dissolve in n-hexane was the less-polar compunds like lipids which have weak radical scavenging capacity (Bialangi et al., 2024).

Additionally, the high positive correlation between antioxidant capacities and the total phenolic and flavonoid content reveals a substantial involvement of phenolic and flavonoid compounds in the antioxidant activity of S. vulcani extracts (Musa et al., 2023). Mwangi et al. (2024) suggested that the majority of natural antioxidants have multiple functions, and the response of each compound varies depending on the method used. Therefore, in the current study, using different assessments for antioxidant activity could have produced varying results.

The α-amylase inhibitory assays revealed significant inhibitory potential in the extracts of S. vulcani (HPE, EPE, MPE). The IC₅₀ value of MPE (21.141 ± 0.19 μg/mL) was comparable to that of acarbose (18.63 ± 1.21 μg/mL), a widely prescribed anti-diabetic medication. The IC₅₀ values of HPE (113.734 ± 0.14 μg/mL) and EPE (98.893 ± 0.15 μg/mL) significantly differ from that of acarbose. Among the extracts of S. vulcani, the methanol extract (MPE) exhibited the highest anti-α-amylase activity. Inhibiting α-amylase can potentially alleviate symptoms of type 2 diabetes by reducing starch breakdown and delaying glucose absorption in affected individuals. Phenolic compounds found in plant extracts demonstrate anti-diabetic effects by strongly inhibiting α-amylase (Tinrat and Jiraprasertwong, 2023). These findings are entirely argreement with the total phenolic content observed in the S. vulcani leaf extracts (methanol > ethyl acetate > n-hexane; Table 2). The potent α-amylase inhibitory activity in MPE is likely attributed to its polar compounds, suggesting the need for further investigation and isolation of the active compounds. Diabetic complications are anticipated to be linked with oxidative stress, which arises from the production of free radicals during glucose oxidation and the consequent oxidative breakdown of glycated proteins.

CONCLUSION

EPE and MPE of S. vulcani displayed increased levels of polyphenolic content, which contributed to their antioxidant effects. In the DPPH and ABTS assays, MPE showed the highest antioxidant capacity, with IC₅₀ values of 54.74 µg/mL and 61.10 µg/mL, respectively. The anti-diabetic studies showed that the EPE and MPE had significant inhibitory effects. The IC₅₀ value of MPE were 21.141 ± 0.19 μg/mL. This study suggests that the EPE and MPE extracts of S. vulcani have the potential as natural sources of bioactive for drug development.

ACKNOWLEDGEMENTS

The authors expressed their deepest gratitude to Universitas Negeri Gorontalo, for the funding support from the Research Budget Implementation List 2024.

AUTHOR CONTRIBUTIONS

Nurhayati Bialangi and Ahmad Kadir Kilo assisted in conducting the experiments, performed the statistical analysis and data visualization and wrote the manuscript. Weny JA Musa and Boima SItumeang designed and conducted all of the experiments and wrote the manuscript. All authors have read and approved of the final manuscript.

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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