Radical Scavenging Activity and Quercetin Content of Muntingia calabura L. Leaves Extracted by Various Ethanol Concentration

Muntingia calabura is broadly cultivated and has become a common roadside tree in Indonesia that is known as "Kersen," or cherry. The current study aimed to determine the activities of the phytochemical constituent (i.e., phenolic and flavonoid total contents) with various concentrations of ethanolic extract of M. calabura leaves (EEMC) to determine the best one and the in vitro antioxidant activities and quercetin content using TLC Densitometry. The extraction was carried out by maceration with various concentrations of ethanol (96% v/v; 70% v/v and 50% v/v) to obtain the ethanolic extract of Muntingia calabura, L leaves. The determination of Quercetin was conducted by Thin Layer Chromatography (TLC) densitometry, and the total phenolic was analyzed with reagent Folin-Ciocalteu while the flavonoid content we identified by applying the colorimetric method. The antioxidant capacity was measured using the radical scavenging assay of 1,2-diphenyl-2picrylhydrazyl (DPPH). The phytochemical study showed that phenolics, saponins, tannins, and flavonoids were present in all EEMC. The extract obtained by 96% ethanol showed the highest total antioxidant activity, reducing power and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity. The same extract also exhibited the highest flavonoid content. However, the extract obtained by 70% ethanol showed the phenolic content, and the highest quercetin content was obtained by using 50% ethanol. The result from the EEMC analysis indicated that Muntingia calabura leaf extract contains essential bioactive compounds and shows potential as a source of antioxidants in M. calabura which uses 96% ethanol solvent and has the highest quercetin content in M calabura using 50% ethanol solvent.


INTRODUCTION
Medicinal plants are resources of a therapeutic device for lighting human ailments. About 80% of people in developing countries in the world rely on traditional therapy for their primary health care, and about 85% of traditional medicine involves the extracts of plants. Using the increase in the awakening of the health danger and toxicities associated with the abuse of synthetic drugs, the interest in the use of plants and plant-based drugs has faced an increase around the world. However, many medicinal plants have not been researched to find out their pharmacological effects. A plant that has recently obtained therapeutic status is Muntingia calabura L [1].
M. calabura is widely conserved and therefore, has become a common roadside tree in Indonesia that is known as "Kersen,". M. calabura (Elaeocarpaceae), commonly known as cherry which contains phenol is a species in the genus Muntingia [2]. Meanwhile, in Indonesia, the use of M. calabura is still restricted, but it has been used to treat various diseases in the overseas. The roots of M. calabura have been consumed as an abortifacient in Malaysia and as an emmenagogue in Vietnam. In another country, Colombia, the flowers are infused and consumed as a tonic and tranquillizer. The methanol extract of M. calabura fruit has exposed potent DPPH quenching capacity [3].
Furthermore, phytochemical studies of various constituents of the plant have identified many bioactive flavonoids, sesquiterpenes, chalcones, and phenolic compounds [2]. The secondary metabolite classes present in the crude extracts of M. calabura, generally the flavonoid class, are known to be reliable for the plant's different bioactivities [4]. Past studies have proved flavones, flavanones, flavans, and biflavans to be the primary ingredients of this M. calabura, some of which have shown anti-platelet aggregation and cytotoxic activities [5]. However, the above research is mostly done in other countries than in Indonesia. Different sites for the growing of M. calabura plants will cause differences in the content of phenols and flavonoids. Besides, this research also aims to find the highest quercetin, phenols and flavonoids content from various variations of solvent concentration.
The current study is mainly focusing on the flavonoid and phenolic content of this plant.
Flavonoids are the largest group of plant phenolics, for over half of the eight thousand naturally occurring phenolic compounds [6]. The main strength of dietary flavonoids is its antioxidant properties. Flavonoids are a group of natural antioxidants which are usually found in plants, fruits and vegetables. They are known to be the excellent scavengers of oxygen free radicals [7]. Phenolic and polyphenolic compounds comprise the main class of natural antioxidants present in plants, foods, and beverages [8]. Phytochemicals such as phenolics, carotenoids and flavonoids, have been exposed to possess functional properties such as antimicrobial and free radical scavenging activity.
The phytoconstituents acts as a natural antioxidant and prevent free radical formation [9].
Flavonoids have continued beyond one hundred years and possess a broad spectrum of biological activities that might be able to influence processes which are not regulated in disease.
Quercetin is a plant pigment that a potent antioxidant flavonoid and especially a flavonol. It is a flexible antioxidant known to possess protective abilities against damage tissue caused by several drug toxicities [10].
The determination of the total flavonoid and phenolic content of ethanol extracts from Muntingia calabura leaves (EEMC) with various concentrations and the examination on their potential antioxidant activities is essential to supply more scientific evidence for the research and development of the wild M. calabura tree. The main objective of this study is to find the best bioactive constituents of plant Muntingia calabura, the best quercetin content using TLC densitometry, the best antiradical activity using various concentrations of ethanol which could serve as a right candidate for the development of new antiradical agents.

Phytochemical Analysis
Chemical tests were carried out to the extract to screen for and identify bioactive chemical constituents in the medicinal plants, such as tannins, flavonoids, alkaloids, and saponins, using the standard procedures as described by Harborne [11] with slight modifications.

The Determination of Total Phenolic Contents
As suggested by Orak [12] that using gallic acid is the standard phenolic compound, the total phenolic content was determined by using the Folin-Ciocalteu reagent. Concentration was calculated using Gallic acid, as standard and the results were expressed as mg gallic acid equivalent/100 g wet weight. Gallic acid, in varying concentrations, were used to prepare a standard curve. This curve was used to relate the absorbance of the unknown samples to Gallic acid equivalents (GAE). Results were expressed as mg GAE in 1 g of dried sample (mg GAE/g). In short, 1 ml of extract solution was mixed with 45 ml of distilled water, then 1 ml of Folin-Ciocalteu reagent was added, and the contents of the flask were mixed thoroughly. After three minutes, 3 ml of Na2CO3 was added, and the mixture was allowed to stand for two hours. The absorbance was measured at 750-760 nm.

The Determination of Total Flavonoid Contents
The flavonoid total content was determined using a colorimetric method. The aluminum chloride colorimetric method was modified from the procedure suggested by Chang et al [13]. The total flavonoid content was determined using a standard curve with quercetin as the standard.
Several milligrams of quercetin were dissolved in 80% ethanol and then diluted to 25, 50 and 100 μg/mL. The diluted standard solutions (0.5 mL) were separately mixed with 1.5 mL of 95% ethanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water.
After incubated at room temperature for 30 min, the absorbance of the mixture was measured at 425 nm. The quantity of 10% aluminum chloride was reversed by the same amount of distilled water in blank. Similarly, 0.5 mL of ethanol extracts or 15 mL standard flavonoid solutions (100 µg/mL) were reacted with aluminum chloride for the determination of flavonoid content as described above.

Thin Layer Chromatography Study for Quantification of Quercetin
The standard and the sample were dissolved in ethanol and were filtered using Whatman Filter paper no. 41 before spotting on thin layer chromatography (TLC) plate. Each standard solution and samples were spotted on a 60 F254 silica gel plate and then eluted using a solvent system of chloroform, acetone, and formic acid (10:2:1) with track distance 0.50 cm and the migration distance was 90 mm. Detection was done under ultraviolet at 254 and 366 nm [14].

DPPH Antiradical Scavenging Assay
Assay of the antioxidant activity of extracts was carried out by spectrophotometric methods   [16]. Natural plant products are mainly divided into three major compounds including phenolic, terpenoids, and alkaloids. Accordingly, the study on preparation of plant extracts would be useful in the bioassay of the bioactive compounds. The extract's yield should depend on the polarity of solvents. Additionally, the solubility of the natural products and the solvent could also determine the yield. For example, polar solvents including methanol, acetone and ethanol are most commonly used to extract some flavonols, alkaloids, polyphenols and saponins [17].
The use of ethanol solvent with the different concentrations was intended to attract polar and non-polar chemical components in M. calabura leaves powder. The filtrate was concentrated using a rotary evaporator with a temperature of 80°C based on the boiling point of ethanol which is at 78C [18]. The quantitative determination of phenolic compounds by using Folin-Ciocalteu (F-C) reactive is a broad method [19]. The quantitative determination of phenolic compounds result that was measured with a maximum wavelength of 762 nm for all variant concentrations solvent (96% v/v; 70% v/v and 50% v/v) were identified.
The standard curve for gallic acid was y = 0.0110x + 0.0003 (R² = 0.9992) where y is the peak area (mAU) and x is the concentration (mg mL-1). The content of each compound was expressed as mg/100 g of fresh leaves (  (table 3). In contrast, the phenolic total content of EEMC 70% was higher than EEMC 96% and 50%. They should depend on the polarity of solvents. Besides, the level of phenolics in plant sources depends on factors such as cultivation techniques, cultivars, growing conditions, maturation processes, and processing and storage conditions among others [20].
The basic concept of the aluminum chloride colorimetric method is that aluminum chloride  (Table 3). It indicated that the highest flavonoid total content was the EEMC 96%.    ( Table 4). The highest of quercetin content was EEMC 50%.    In the present study, the percentage of inhibition was measured to determine the antioxidant activity of the extracts, which was able to inhibit free radicals. DPPH assay was considered to be a more valid, more comfortable and more economical method to evaluate the radical scavenging activity of antioxidants since the radical compound was stable and needed not to be generated. DPPH is nitrogen-centered free radical having a free electron which gives a strong absorption at 517 nm.
Purple DPPH changed to yellow when its odd electron paired with the radical scavenger to reduce the DPPH-H. The decrease in the absorbance of DPPH free radical at 517 nm was because of the radical scavenger donated hydrogen [23]. Linearity equations and IC50 of reference ascorbic acid and EEMC are shown in Table 5.

CONCLUSION
Muntingia calabura leaf extract contains essential bioactive compounds and shows potential as a source of antioxidants in M. calabura which uses 96% ethanol solvent and has the highest quercetin content in M. calabura using 50% ethanol.