INTRODUCTION:

Ethnopharmacological studies play asignificant role in drug discovery as a number of therapeutic products have been developed based on knowledge from ethnobotany. Phytochemicals from medicinal plants are extensively investigated due to the fact that most of the studied medicinal plants possess a number of bioactive compounds with health benefits¹. These phytochemicals are usually the secondary metabolites of the plants which support several physiological processes, including means of self-defense. Phytochemicals that are involved in defending plants from pathogens and predators are often toxic to the human body. Hence, most traditional medicinal plants are being investigated for the presence of both beneficial and toxic metabolites to address their safe usage for specific diseases.

The bioactive properties of plants from the Glochidiongenus have been studied by several groups, with many of the described Glochidion species possessing promising medicinal properties². More than 250 species have been found to be rich with flavonoids and triterpenoid saponins³.

Among other health benefits, phytochemicals are particularly known for their efficacy in preventing oxidative stress-related diseases. As metabolic byproducts, reactive oxygen species (ROS) and many free radicals are regularly created in the human body, which are then neutralized or removed from the body by endogenous enzymatic processes or dietary micronutrients. In humans, dietary vegetables and fruits are known to play the leading role in fighting against oxidative stress-inducing agents. Since natural products are safer compared to their synthetic alternatives, nutraceutical industries often use phytochemicals to formulate health supplements. Due to changes in lifestyle and environmental factors, oxidative stress and free radicals are affecting most of the population around the globe. A larger consumption of fruits and vegetables is generally recommended to combat this scenario since it is related with a lower risk of cardiovascular disease, cancer, and death, as well as improved outcomes of chronic health disorders.^6–11. In this study, we have analyzed the phytochemical contents of Glochidionlittorale Blume (Fig. 1), one of the native tropical wild shrubs found in Southeast Asia. The name of the plant is probably based on their mostly seashore habitat, and is derived from the Greek words ‘glochin’ and ‘litorolis’, which means a point and shore, respectively. Like other Glochidion species, G. littorale is being used in traditional folk medicine all over Borneo Island. However, there are limited studies on the pharmacological properties of G. littorale. Hence, this study set forth to characterize the total phenolic and flavonoid contents of G. littorale fruits, and to investigate free radical scavenging and enzyme inhibitory properties of G. littoraleextracts.

MATERIALS AND METHODS:

Plant material:

For this investigation, only mature ripe G. littorale fruits were employed (Fig. 1). Samples were taken from places that were free of pollution and non-native materials. The plant samples were washed and dried in a 30°C oven.

Soxhlet extraction:

For Soxhlet extraction, ethanol and ethyl-acetate (Merck) were utilized as solvents. The sample powder (20g) was resuspended in either ethanol or ethyl acetate (250ml of each solvent). The extraction procedure was repeated multiple times throughout the course of 24 hours. The extracts were filtered using Whatman filter paper (grade 1), and the solvents were extracted with a rotatory evaporator (Buchi Rotary Evaporator, R-205, Switzerland). The dried concentrated final extracts were kept at -20°C till further analysis.

Preliminary phytochemical analysis:

Trease and Evans (1989) standard procedures were used to determine the presence of the following preliminary phytochemicals.

Test for saponins (frothing test):

Equal volumes of the extract (1mg/ml) and distilled water (1:1 ratio) were vigorously mixed in a screw cap test tube. Samples containing saponins developed a foamy appearance.

Test for tannins (ferric chloride test):

The extracts (1mg/ml) were diluted double distilled water (1ml of each sample in 9ml distilled water) and boiled in a water bath for five minutes. The mixture was filtered after cooling at room temperature for 20 minutes to remove precipitates. The presence of tannin was indicated by the development of a brownish green color after adding 1ml of 0.1% ferric chloride (FeCl3).

Test for terpenoids (Salkowski test):

The extracts (1mg/ml) were carefully mixed with chloroform in a 1:2 ratio, followed by an equal volume of concentrated sulfuric acid (H₂SO₄ Millipore). The presence of terpenoids was indicated by the appearance of a reddish-brown color at the interface.

Test for phenols:

1ml of each extract (1mg/ml) was diluted in 4ml of distilled water, along with a few drops of 10% FeCl3 (Merck) solution. The presence of phenols was indicated by the presence of a dark green solution.

Test for flavonoids:

After adding 5ml of dilute ammonia solution to 1ml of the plant extract (1mg/ml), a further addition of 1ml of Millipore concentrated H₂SO₄ produced a temporary yellow color in flavonoid-containing samples. After that, the presence of flavonoid was confirmed by adding two drops of 1% aluminum chloride solution, which produced a yellow color.

Total phenolic content:

We employed the method described by¹³ with slight modification to measure total phenolics content (TPC). 1ml of G. littorale extract (1mg/ml), 5ml of 10% Folin’s reagent (Merck), and 5ml of 7.5% NaHCO3 were mixed together and incubated at 25°C for 30 minutes. Using a spectrophotometer (BioTek), the absorbance was recorded at 750nm. To quantify the standard, gallic acid was used with values expressed as mg gallic acid equivalents (GAE)/g of extract.

Total flavonoid content:

The method described by Jia et al. (1999) was used to assess the total flavonoid content (TFC) in G. littoraleextracts. 1ml of G. littorale extract (1mg/ml), 3ml of methanol (Millipore), 200µl of 10% aluminium chloride (Merck) AlCl3, 200µl of 1M sodium acetate (Sigma) NaCH3COO, and 5.6mL of distilled water were mixed together and incubated in darkness at 30°C for 30 minutes. Absorbance was measured using a spectrophotometer at 420nm, and TFC was quantified using quercetin as the standard. The values were expressed as mg quercetin equivalents (QCE)/g of extract.

DPPH assay:

The extracts' free radical scavenging abilities were evaluated using the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) test (Sigma). A new solution of 1M DPPH in methanol (Merck) was produced, and the initial absorbance at 517nm was measured. 3ml of experimental extracts (1mg/ml) were mixed with 1ml of DPPH solution and incubated at room temperature for 30 minutes. At 517nm, the absorbance of these reaction mixtures was measured again. The percentage inhibition was computed as follows: % inhibition = (A-B)/A x 100%, where A represents the starting absorbance of pure DPPH in oxidized state and B represents the absorbance of the sample response. A graph of percentage inhibition was plotted against concentration and the IC₅₀value which is the concentration of the extract providing 50% of the radical scavenging was calculated form the analysis of linear regression.

In vitro a-amylase inhibitory assay:

G. littorale extracts of several concentrations (0.5, 1.0, 2.0, and 3.0mg/ml) were mixed in 1ml solutions with 2 units of -amylase (MP Biomedicals) and mixed with an equivalent quantity of 1% potato starch. For 30 minutes, the mixture was incubated at 37^oC to break down starch into maltose. 1ml of DNSA reagent (3,5-dinitrosalicylic acid, sodium hydroxide, and sodium potassium tartrate tetrahydrate) (Sigma) was added to each reaction mixture and heated for 5 minutes in a boiling water bath before cooling to room temperature. The quantity of maltose produced by enzyme-induced starch hydrolysis was calculated using the absorbance value at 540nm. To measure the level of released disaccharides from the experimental reactions, a standard curve was built using maltose as the standard. The percentage inhibition was computed as follows: % inhibition = (A-B)/A x 100%, where A is the starting absorbance of maltose and B is the sample response absorbance.

Statistical analysis:

“Results are presented as mean averages ± standard deviation. Statistical analysis was conducted using IBM SPSS Statistics Version 23, with statistical significance determined at P<0.05”.

RESULTS:

Phytochemical constituent in G. littorale extracts:

The presence of the five typically dispersed phytochemicals (saponins, tannins, terpenoids, phenols, and flavonoids) was found in two experimental G. littorale fruit extracts, ethanol- and ethyl acetate-derived extracts (Fig.1). The ethanol-derived extract included all five preliminary phytochemicals, whereas the ethyl acetate-derived extract contained all but the tannins of the examined phytochemicals (Table 1). These five phytochemicals are frequently connected with the bioactive qualities of plant extracts, particularly when the herbs are utilized for antibacterial, anti-oxidative, and skin protection purposes.We concentrated on the phenolic and flavonoid content of G. littorale fruit extracts in this investigation. Surprisingly, we found an inverse relationship between the amounts of TPC and TFC in the two solvent-derived extracts. The ethanolic extract had a mean average TPC of 17.146mg GAE/g, whereas the ethyl acetate extract had a mean average TPC of 7.323mg GAE/g. In contrast, the mean average TFC in ethanolic extract was 9.409mg QCE/g, whereas it was 19.140 mg QCE/g in ethyl-acetate extract (Table 2). TPC and TFC differences in ethanol and ethyl acetate extracts are statistically significant (P<0.05, ANOVA).

Antioxidant activity of G. littorale extracts:

To assess the antioxidant properties of the two G. littorale fruit extracts, we employed the conventional DPPH free radical method. As the IC50 value of the ethanol extract is lower than that of the ethyl acetate extract (183.11µg/ml and 197.61µg/ml, respectively), this means the DPPH free radical scavenging property was slightly higher in the ethanol extract compared to the ethyl acetate extract. The IC50 values suggest that the experimental extracts are less efficient in scavenging DPPH free radical molecules than the control ascorbic acid (Table 2).

Fig 1. Fruits of Glochidionlittorale Blume at different stages: (A) white when unripe; (B) pinkish white in early ripening stage; (C) and darker pink at full ripening.

Table 1. Screening of the selected phytochemicals in G. littoralefruit extracts

Chemical constituent	Ethanol extract	Ethyl-acetate extract
Saponins	+	+
Tannins	+	–
Terpenoids	+	+
Phenols	+	+
Flavonoids	+	+
(+) Present; (–) Absent

Table 2. Total phenolic, flavonoid and antioxidant contents of G. littorale fruit extracts

Sample/Extract	TPC (mg GAE/g)	TFC (mg QCE/g)	DPPH IC50 (µg/ml)
Ethanol^a	17.146 ± 0.051b	9.409 ± 0.002b	183.11 ±0.053b,c
Ethyl acetate^b	7.323 ± 0.086a	19.140 ± 0.006a	197.61 ±0.037a,c
Ascorbic acid^c	ND	ND	168.89 ± 0.045a,b

Each value is expressed as the mean ± standard deviation (n=3). (a-c) represents a significant difference (P<0.05; ANOVA, Tukey’s post hoc test) between mean values within the specific assay. (ND = not determined)

Inhibition of α-amylase by G. littorale extracts:

G. littorale fruits are known to be edible, and there has yet been reported side effects. Hence, we also investigated the potential of our fruit extracts in inhibiting α-amylase, the enzyme is frequently used to reduce glucose absorption by decreasing the availability of dextrin oligosaccharides in the digestive system. Medicinal herbs with such α-amylase inhibitory effects have been shown to reduce blood sugar levels. In our study, we discovered that both ethanolic and ethyl acetate extracts inhibit α-amylase and starch hydrolysis in vitro. Both extracts inhibited α-amylase throughout all experimental units (0.5, 1.0, 2.0, and 3.0mg/ml), with the degree of inhibition increasing as fruit extract concentrations increased (Fig. 2). Across all four concentrations tested, the ethanolic extract inhibited α-amylase more effectively than the ethyl acetate extract. (8.13% vs 6.7% for 0.5mg/ml; 44.16% vs 20.09% for 1.0mg/ml; 64.37% vs 28.65% for 2.0mg/ml; 89.62% vs 49.54% for 3.0mg/ml). According to statistical analysis, the level of α-amylase inhibition was considerably higher in the presence of 1.0, 2.0, and 3.0mg/ml of ethanolic extract than in the presence of equivalent quantities of ethyl acetate extract. (P< 0.05, t-test).

Fig 2.Efficacy of α-amylase inhibition by G. littorale fruit extracts. Ethanolic extract (solid line) shows significantly higher α-amylase inhibitory property compared to ethyl acetate extracts (dotted line) at concentrations 1.0, 2.0 and 3.0mg/ml.

DISCUSSION:

Existing information suggests that phytochemicals in G. littorale and G. glaucum were first investigated by¹⁵ who reported the presence of triterpenoids and hydrogen cyanide by using several basic approaches such as comparing the amount of precipitates, or observing the changes of colour reactions by eye, or by smelling the reaction mixtures. Though such crude methods are questionable and not free from artefact, this was the first known study on the phytochemicals of Glochidionspecies in Borneo. In recent years, several researchers have reported the phytochemical profiles of several Glochidionspecies, but that of the experimental plant G. littorale remain largely unexplored. Wiart (2006) reported this plant as a potential herb with beneficial pharmacological properties based on evidences of its usage in traditional medicine in Malaysia in treating dysentery. Although G. littoraleis being used in traditional medicine in several parts of Southeast Asia, the phytochemicals present in this species are not well documented.

Our early phytochemical examination of G. littorale fruits indicated the presence of secondary metabolites known to be free radical scavengers, such as saponins, tannins, terpenoids, phenols, and flavonoids. The occurrence of these phytochemicals in different therapeutic plants has been ascribed to their ability to act as anti-aging, anti-carcinogenic, anti-inflammatory and anti-microbial agents. It is also apparent that almost all of the medicinal plants that are used in traditional medicine possess these phytochemicals albeit with varying amounts^16,17. Furthermore, tocharacterize the phytochemical constituents of G. littorale fruit extracts, we proceeded with quantifying the phytochemical contents, followed by assessing the antioxidant activity of the fruit extracts using DPPH scavenging activity assay.Among the two extracts investigated in this study, G. littorale ethanolic extracts had greater total phenolic contents than ethyl acetate extracts. (P<0.05). This might be related to the fact that ethanol dissolves both hydrophilic and hydrophobic substances.Because of their numerous hydroxyl groups that efficiently quench free radicals, phenolic acids are the principal category of antioxidants. Our findings imply that for future studies on the antioxidative activities of G. littorale, an ethanolic extract may give more polyphenolic components than an ethyl acetate-derived extraction. Total flavonoid contents were found to be higher in the ethyl acetate extract than in the ethanol one (P<0.05). This may indicate that the flavonoids present in ethyl acetate extracts of G. littorale fruits are semi-polar or non-polar in nature. Hence, the difference in the flavonoid contents between these extracts might be due to differences in solvent polarity. This is in line with previous studies on different plant extracts which suggest that “ethyl acetate is the most efficient solvent in extracting flavonoids”¹⁸. The DPPH scavenging technique was used to calculate the IC50 value, which is inversely related to antioxidative activity - the lower the IC50 value, the greater the efficacy of free radical scavenging. The ethanolic extract has a lower IC50 value than the ethyl acetate extract. The existence of polyphenolic chemicals was confirmed by a significant association between antioxidant activity and total phenolic content of the extracts, as predicted. Apart from polyphenols, terpenoids are produced for several purposes such as protein glycosylation, photosynthesis, and plant growth and development. The terpenoids is one of the largest group of phytochemicals that are also involved in defending hosts from pathogens and predators¹⁹. Terpenoids are receiving increased attention in nutraceuticals due to their role in regulating lipid metabolism by targeting several key molecule markers such as AMPK and SREBP²⁰. Similar to the terpenoids, flavonoids are also involved in regulating lipid metabolism by altering a number of key lipid metabolizing proteins such as AMPK (Tung et al., 2016), PPARα²¹, PPARγ²², and SREBP1c. The presence of saponin in our experimental extracts is another interesting feature. This natural product possesses detergent-like properties and is mostly amphipathic in nature. Saponins from plant extracts have been shown to inhibit cholesterol absorption in the gut which eventually leads to amelioration of total cholesterol level in the body²³. In future work, it is important to identify the types of saponins in G. littorale as certain saponins are toxic to the human body. This would be crucial since both ethanol and ethyl acetate extracts of G. littorale can be utilized for exploring their potential in reducing cholesterol absorption across the intestinal wall. After from that potential diet benefit, our findings also suggest that theG. littorale fruit might have hypoglycemic property as we observed the ability in our extracts to inhibit α-amylase enzyme. Herbal extracts with such property can be used to reduce postprandial blood glucose level as α-amylase is the first enzyme to hydrolyze dietary starch during digestion. Taken together, it would be promising to proceed with further investigations surrounding bioactive properties of G. littorale using in vitro and in vivo models.

CONCLUSION:

The presence of bioactive compounds with therapeutic properties in G. littorale is evident by its usage in traditional folk medicine. The described phytochemicals in this study are universally studied for most of the known medicinal plants; pharmaceutical industries often utilize sophisticated techniques to isolate and characterize these individual chemical compounds. Hence, our findings denote just an addition to the growing pool of information gathered for preliminary phytochemical analysis in ethnopharmacology. However, to our knowledge, this is the first attempt to systematically screen phytochemicals in G. littorale fruits using two commonly used solvents of different polarity. The total phenolic and flavonoid contents in both ethanolic and ethyl acetate extracts indicate that the G. littorale species is a potential source of natural antioxidants. This may open up their avenue into the functional food sector, and further biochemical analyses in future will reveal their therapeutic potential in the areas of anti-aging, anticancer, anti-inflammatory, antimicrobial, anthelmintic, skin protection, and wound healing activities.

ACKNOWLEDGEMENT:

The authors wish to thank the technical staff of theEnvironmental and Life Sciencesat the Faculty of Science, Universiti Brunei Darussalam for laboratory support.

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Received on 15.08.2022 Modified on 30.09.2022

Research J. Pharm. and Tech 2023; 16(1):339-344.

DOI: 10.52711/0974-360X.2023.00060