SPECIFIC AND NON-SPECIFIC PARAMETERS BASED ON SIMPLICIA AND ETHYL ACETATE FRACTIONS OF STAR FRUIT LEAVES (AVERRHOA CARAMBOLA L.)

Objective: Star fruit (Averrhoa carambola L.) is a plant with numerous benefits, including anti-skin inflammation properties. The aim of the presentstudy was to evaluate simplicia and ethyl acetate fractions of star fruit leaves collected from three regions in Indonesia, including Depok, Subang, andSukabumi, and to characterize specific and non-specific parameters.Methods: Extraction was performed through maceration. Next, liquid partitioning was performed to obtain ethyl acetate fractions. Chromatogramsfor simplicia and ethyl acetate fractions for samples from three regions were obtained using chloroform: methanol:water (8:2:0.5) with apigenin asthe standard.Results: The yield of the ethyl acetate fraction from the star fruit leaves was 4.2–6.2%. Water content, total ash content, and acid-soluble ash contentswere 4.79%, 1.55%, and 0.064%, respectively, and total flavonoid levels were 14.63–22.14 mg QE/g of a fraction. The water-soluble and ethanol-solubleextracts were 14.32% and 9.69%, respectively, and total flavonoid contents were 0.12–0.18 mg QE/g simplicia. In addition, drying losses, ash content,and acid-soluble ash content were 9.70%, 7.14%, and 0.31%, respectively. The results of solvent residues and heavy metal contaminant (Hg, Pb, Cd,and As) tests indicated that the ethyl acetate fraction did not contain solvent residue or heavy metal contamination.Conclusion: The specific and non-specific parameters based on simplicia and ethyl acetate fractions from star fruit leaves meet the requirement.


INTRODUCTION
Standardization is necessary to obtain uniform raw materials which would guarantee consistent pharmacological effects of medicinal plants [1,11]. In addition, the efficacy of a medicinal plant depends on the chemical constituents, which is under the influence of numerous factors, such as the place of growth, climate, and rainfall, in addition to harvesting practices [4,7,10]. A key challenge, therefore, is how to determine the consistency of the quality of crude drugs and extracts of a plant cultivated across regions with different altitudes, soil conditions, and weather [9].
Quality requirements for extracts and botanicals consist of specific predefined parameters and various general standards (nonspecific) [2,3]. Specific parameters include the identity of the extract, the organoleptic extract, and the concentrations of substances to be dissolved in certain solvents, while the non-specific parameters include drying shrinkage, moisture content, total ash content, residual solvents, pesticide residues, and contamination with heavy metals, in addition to microbial contamination [12].
Averrhoa carambola L., which is commonly known in Indonesian society as star fruit, is a plant used extensively in traditional medicine [5,8]. Star fruit leaves have numerous health benefits, for example, they have antiulcer, anthelmintics, and antitumor properties, in addition to being used to manage hypotension [15]. In addition, studies have shown that the ethyl acetate fraction of star fruit leaves contains high levels of apigenin most and exhibits the highest lipoxygenase enzyme inhibitory activity [13,14].
Considering the potential pharmacological applications of extracts from star fruit leaves, the manufacture of ethyl acetate fractions, in addition to their standardization, with the aim of maintaining consistency with regard to quality, and guaranteeing safety and efficacy, need to be explored. In addition, the present study aimed to establish key specific and non-specific parameters for crude drugs and obtain   Subang Dried round leaves, short stems, pointed edges, blunt or rounded bases, flat-leaf edges, pinnate leaf bones, and brownish-green 3.
Sukabumi Dried round leaves, short stems, pointed edges, blunt or rounded bases, flat-leaf edges, pinnate leaf bones, and brownish color

Materials
A. carambola leaves were obtained from Depok, Subang, and Sukabumi regions in Indonesia.

Extraction and fractionation
A. carambola leaves were obtained from Depok, Subang, and Sukabumi.
Simplicia powder was extracted from the leaves using 70% ethanol using the maceration method, with a powder: solvent ratio of 1:10.
The maceration process began with soaking Simplicia for 6 h while stirring occasionally and then the mixture was allowed to stand for 18 h. The mixture was evaporated on a rotary vacuum evaporator to reduce the solvent until a sticky extract was obtained. The extract was fractionated to purify the compounds using n-hexane, ethyl acetate, and water. The extract was ground into powder, suspended in n-hexane, and homogenized using a sonicator for 10 min. The suspension was filtered using filter paper and then the filtrate was evaporated on a rotary evaporator until a viscous extract was obtained. The viscous extract was then dried using a vacuum oven at 40°C-50°C until a constant weight.  Afterward, the residue was dissolved in ethyl acetate and homogenized using sonicator for 10 min. The solution was then partitioned by adding distilled water, shaken in a separator funnel, and allowed to stand for 30-60 min until two layers were formed (ethyl acetate layer on top and distilled layer water at the bottom). The layers formed were then separated. The ethyl acetate and water layers were evaporated on a rotary evaporator until a viscous extract was obtained.

RESULTS AND DISCUSSION
Organoleptic parameters are specified using the senses, which are used to describe the properties of crude drugs based on shapes, smell, color, and taste. According to the results, the samples from the three regions exhibited similar organoleptic properties, with a distinct smell and a slightly tart flavor. However, the plants from the three regions could be distinguished based on a different color. The different color from leaves may be influenced by environmental factors, in addition to the time harvest and the drying process [6]. Organoleptic test results are listed in Table 1.
The first stage in the present study was the macroscopic observation of star fruit levels. Traits observed included leaf shape and color of the star fruit plants. The results of the macroscopic examinations of star fruit leaves from the three regions are listed in Table 2. The star fruit leaves from the three locations had star fruit leaves with similar shapes, which were circular. In addition, leaf petioles were short, with pointed tips, bases were obtuse or rounded, and the leaf edges were flat. The only factor that could be used to distinguish the leaves from the three regions was color.
Microscopic examinations were performed to determine the fragments that could be used to characterize Simplicia. Microscopic plant tissue from specific parts displays unique features so that they could be used to distinguish similar parts from different plants, based on sections referred to as identification fragments, as illustrated in Fig. 1. Microscopic examination of simplicia from star fruit leaves at a 10 × revealed identifiable fragments in the form of one-to three-celled cover hairs, parasitic stomata, and oil gland cells. We used chloroform: methanol:water mobile phase with a ratio of 8:2:0.5 to reveal chromatograms of the simplicia and ethyl acetate fractions. The eluted plate was then sprayed with 5% AlCl 3 and observed under 366 nm ultraviolet light. The chromatogram results of the simplicia and ethyl acetate fractions are presented in Figs. 2 and 3, respectively. In the simplicia results, there are identical spots between the standards and the samples; therefore, the simplicia from star fruit leaves contained apigenin flavonoids.

CONCLUSION
The simplicia of the A. carambola leaves produced a dry powder, ranging in color from green to brown and with a distinctive smell. Phytochemical screening was performed for the simplicia from the three regions. According to the results, the simplicia contained flavonoids, tannins, saponins, terpenoids, and glycosides ( Table 3). The specific and non-specific parameters of simplicia from star fruit leaves are listed in Table 4, organoleptic examination of ethyl acetate fraction from star fruit leaves from three regions are listed in Table 5, while the specific and non-specific parameters of the ethyl acetate fractions are listed in Table 6. They had a somewhat tart taste. The leaves were circular in shape and the leaf petioles had short pointed tips, and the leaves had obtuse or rounded bases, flat-leaf edges, and green-brown color. Microscopic examination of fragments revealed identifiers on the hair bulbs, with one-to three-celled hairs, parasitic type stomata, and oil gland cells, file thickening carrier with nets and with oil present, with thickening of the ladder carrier file. In addition, water-soluble compound contents were not lower than 14.23% and ethanol-soluble compound contents were not lower than 9.62%.
Phytochemical screening tests revealed that the presence of flavonoids, tannins, saponins, terpenoids, and glycosides, and total flavonoid contents was not lower than 0.12 mg QE/g of bulbs. Non-specific parameters included drying shrinkage not greater than 9.70%, total ash content not greater than 7.14%, and acid-insoluble ash content not greater than 0.31%. The organoleptic properties of the ethyl acetate fraction from star fruit leaves included a thick extract with a blackishbrown color, a distinctive smell, and a bitter taste.

CONFLICTS OF INTEREST
We declare that we have no conflicts of interest.