PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL POTENCY OF JERUK KUNCI FRUIT WASTE (Citrus x microcarpa Bunge) EXTRACT AGAINST Propionibacterium acnes

The growth of acne bacteria can be treated using antibacterial compounds, a plant that has the potential to be developed as an acne antibacterial Citrus x microcarpa Bunge (jeruk kunci). Based on the literature review, the genus Citrus contains steroids, flavonoids, terpenoids, tannins, and saponins with bioactivity as antioxidants and antibacterials. This study aims to determine the secondary metabolite content and antibacterial activity of jeruk kunci fruit waste extract against Propionibacterium acnes. In this study, the extraction used the maceration method with ethanol as a solvent. The determination of the phytochemical test was carried out qualitatively. The antibacterial test was conducted by the disc diffusion method. The results of the phytochemical test contained secondary metabolites, such as flavonoids, tannins, terpenoids, and steroids. Based on the data from the antibacterial test of the Jeruk Kunci fruit waste extract, it was found that the antibacterial power of the extract against P. acnes was relatively strong. In conclusion, the extract of the Jeruk Kunci fruit waste can be developed as an antibacterial agent.


Extraction
The 250 grams of simplicia were macerated using 2.5 L of ethanol for 3 x 24 hours. They were stirring every 1 x 24 hours to obtain a concentrated extract. The filtrate obtained was concentrated with a vacuum rotary evaporator [5].

Phytochemical Testing
Qualitative testing of the secondary metabolites of jeruk kunci fruit waste extract by using several reagents. Qualitative secondary metabolic testing work procedures are as follows [6]:

Alkaloids Test
Tested with Wagner, Dragendroff, Mayer reagents by adding 1 mL H2SO4 2 N was shaken vigorously. The formation of sediment indicates the presence of alkaloid compounds. The positive results of Mayer's reagent were white deposits, Dragendroff was red-orange with residue, and Wagner produced brown deposits.

Saponin Test
Saponins were detected with hot water with the addition of 2 drops of 2 N HCl. The sample is positive if it contains a lot of foam and is consistent for 10 minutes.

Tannin / phenolhydroquinonetest
A total of 1 mL of sample ethanol extract was added with 1% FeCl3 as much as 2-3 drops. A positive sample contains phenol if it produces a blackish-blue color.

Flavonoid Test (HCl + Mg)
Sample 1 mL of ethanol extract, add 2 drops of concentrated HCl while shaking firmly, add Mg powder, and shake vigorously. The presence of flavonoids is indicated by change the color of the solution to orange or red color.

Terpenoid and Steroid Test
A total of 3 drops of the sample extract in the test tube were added 3 drops of glacial acetic acid. The mixture was then added with 5 drops of concentrated H2SO4 through the tube wall. Samples containing terpenoids are indicated by the presence of a purple or red solution and blue or green if they have steroids.

Antibacterial Testing
Propionibacterium acnes bacteria were inoculated on nutrient agar, 6 mm disc paper is placed on the media surface total of 50 μL was taken from each test solution with various concentrations of 20%, 40%, 60%, 80%, and 100% (w/v) as well as positive control and negative control solutions. The positive control used clindamycin, and the negative control used distilled water. They then entered into the concentration variations that have been made. Incubated for 24 hours at 37 o C [7]. To see the zone of inhibition formed from the test using a caliper [8]. Measurement of the clear zone formed as an inhibition against bacterial growth. The category of antibacterial inhibition according to Davis and Stout (1971), the inhibition zone diameter <5 mm is classified as weak, 5-10 mm is moderate, and 10-20 mm is classified as strong, and> 20 mm is classified as very strong.

RESULTS AND DISCUSSION Phytochemical testing
Phytochemical testing serves to identify the active compounds contained in the ethanol extract of the sample. Several reagents are used to identify phytochemical compounds such as alkaloids, saponins, tannins/phenols hydroquinone, flavonoids, terpenoids, and steroids. The results of phytochemical testing are presented in (Table 1). Table 1 shows the phytochemical test. The ethanol extract of the sample contains bioactive compounds in the form of terpenoids, phenol hydroquinone/tannins, flavonoids, and steroids. In the alkaloid test of the ethanol extract of the sample, there was no precipitate because it did not react with the reagents. It was stated that the extract did not contain alkaloids. In contrast, alkaloid compounds have the characteristic feature of having a nitrogen atom. The nitrogen atom makes the alkaloid be base.
Flavonoid identification. The results of phytochemical testing of the ethanol extract of the sample indicated flavonoids marked by a change in color to orange or red. Generally, flavonoids dissolve in ethanol because they are polar. Ethanol is used to free flavonoids from their salt form. Addition of concentrated chloride to protonate flavonoids to form flavonoid salts. After the addition of magnesium powder, the formation of orange or red color in the presence of flavonoids. It due to reduction by HCL and magnesium [9].
Saponins identification. Saponins are active substances that contain foam. Phytochemical test results show negative results because no stable foam is formed. Saponins have a steroid group as a nonpolar group and a glycosyl group as a polar group. The two groups will produce micelles when shaken with water. In the micellar structure, the polar groups face outwards while the nonpolar groups point inward, which produces foam [10]. Green + Phenol hydroquinone/tannin identification testing. The tannin content of the sample ethanol extract showed positive results with a marked blackish-blue color change because FeCl3 reacts with the aromatic -OH group [11].
Terpenoid and steroid identification. The lieberman-Burchard reagent is used to identify steroids and terpenoids. The reagents used were H2SO4 and CH3COOH [10]. The phytochemical test of the ethanol extract of the sample showed a positive result of the formation of a brownish-red color for the terpenoid test and blue for the steroid test. Sulfuric acid with glacial acetic acid solvent functions to protonate the hydroxy groups on steroids or terpenoids, forming a greenish-blue color [12].
It is similar to what was expressed in previous studies. The FT-IR spectrum shows the presence of OH groups overlapping with the aromatic signal at 3159-3050 cm-1 which is supported by the C = C signal. The research states that the extract of the Jeruk Kunci may contain phenolic compounds such as phenolic acids and flavonoids [13]. Other studies claim that the fruit peel extract C microcarpa phenolic compounds and flavonoids, which phenolic levels higher than the Merdeka lime and a higher level of flavonoids than C. hystrix [14]. Based on the research of Lou and Ho (2016), HPLC analysis of C. x microcarpa extract contains many major glucosylated flavonoids. The compound 3', 5' -di-C-β-glucopyranosylphloretin and Apigenin-6,8-di-C glucoside (vicenin-2) is a compound that is extracted major falvonoid with respective levels of 2335 ± 22 and 637 ± 21 mg / 100 gr dry [15]. The structure of the two compounds is presented in Figure 1. Based on phytochemical analysis and literature review, it shows that the dominant active compounds in Jeruk Kunci are phenolic and flavonoids.

Determination of Antibacterial Activity
An antibacterial test was used to determine the inhibitory strength of the ethanol extract of the sample against the growth of P. acnes bacteria using the disc diffusion method. Disc diffusion is used to determine whether there is an effect of ethanol extract samples on P. acnes. The medium used for bacterial growth is nutrient agar. The use of agar nutrients is a common medium for bacterial growth. Aquadest as a negative control and clidamycin as a positive control. Antibacterial activity the extract by looking at the formation of a clear zone around the disc paper. The clear zone that is formed is called the zone of inhibition. Measurement of the developed zone of inhibition uses a caliper. Based on the results of the research, the inhibition test carried out on the ethanol extract of the sample obtained the results in (Table 2): Figure 1. Structure of the Jeruk Kunci flavonoid compound [15] Description: + Positive control -Negative control Table 2 shows that the mean inhibition zone formed from variations in the concentration of ethanol extract samples at concentrations of 20%, 40%, 60%, 80%, and 100% has antibacterial activity against P. acne bacteria in the strong category ( Figure 2). Figure 2 shows that the higher the ethanol extract concentration of the sample, the higher the average inhibition zone formed. Positive control has potent bacterial inhibition; negative control has no bacterial inhibition. It is because distilled water does not have antibacterial properties against P. acnes. Then the variation in pure concentration of the ethanol extract of the sample is not influenced by the solvent. The ethanol extract of the sample was able to inhibit the growth of P. acne because it had antibacterial properties. The inhibitory power of the sample ethanol extract against P. acnes of low concentrations has been shown great inhibition. The extract can be used as an invention that can be developed in the future to be used as an antibacterial agent. Inhibitory power as antibacterial of the ethanol extract samples is the possibility of a synergistic collaboration between the compounds contained in the sample ethanol extracts such as flavonoids, phenols, terpenoids, steroids, and tannins.  The mechanism of flavonoid inhibition as an antibacterial is by inhibiting energy metabolism. It inhibits the use of oxygen through the movement of bacteria that play a role in microbial activity and extracellular protein and prevents the formation of cytoplasmic energy. Inhibits nucleic acid synthesis where the A and B rings of flavonoids play an essential role in the hydrogen bonding process by accumulating nucleic acid bases to be inhibited in the formation of DNA and RNA. Limiting the function of the cell membrane through the formation of complex compounds from extracellular proteins on flavonoids, causing the cell membrane to break out of the intracellular compounds [16]. The phenolic mechanism in inhibiting bacteria by increasing the cytoplasmic membrane permeability causes cytoplasmic coagulation and leakage of intracellular components resulting in lysis or death [17]. The mechanism of action of terpenoid as antibacterials is as follows: steroid compounds are able to interact with cell phospholipid membranes, resulting in decreased membrane integrity and changes in cell morphology so that cells become brittle and undergo lysis [18]. Inhibition of terpenoid compounds is by reacting with porin (transmembrane protein) on the outer membrane of the bacterial cell wall. Then the formation of strong polymer bonds that destroy porin [19]. The tannin/phenol hydroquinone as an antibacterial is by inhibiting the transcriptase enzyme so that DNA topoisomerase is not formed. Other tannins' mechanism as antibacterials is by activating cell adhesin, activating enzymes, and disrupting protein transport. Disruption in bacterial cell protein synthesis due to the presence of tannin compounds will have fatal consequences, which lead to bacterial cell death [20].

CONCLUSION
Based on the our finding it can be concluded that jeruk kunci fruit waste contains secondary metabolites terpenoids, phenolhydroquinone/ tannins, flavonoids, and steroids. Jeruk kunci fruit waste has antibacterial activity against the Propionibacterium acnes bacteria with strong category.