Standardization Study of Simplicia and Extract of Calamondin (Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and Antibacterial Assay

Calamondin (Citrus microcarpa Bunge) is a commodity that is widely grown in Indonesia, even promoted by the One Village One Product (OVOP) of calamondin in Bengkulu1. However, wastewater from calamondins such as peel, pulp, seeds, and extracted water has not been processed into products which has a high sale value2. Calamondin contains vitamin C 7.3 gr, vitamin A 57.4 mg IU, calcium 8.4 mg, water 15.5 gr, potassium 37 mg, and fiber 1.2 gr. Calamondin peel contains essential oils, 0.15% ascorbic acid, and 5.5% citric acid3. Calamondin peels and leaves contain about 1.2% and 0.93% volatile oil4. The study results of Cheong et al., dichloromethane and hexane extracts from calamondin, identified 79 compounds representing 98% of compounds that evaporate with N-methylantranylate were the most detected compounds. Methanol extract contains a lot of phenolic acids, especially p-Coumaric and ferulic acid5. The study results of Bhat et al., stated that calamondin contains flavonoids around 1.41 mg / 100 mL with antioxidant capacity around 777 mg / 100 mL and ascorbic acid around 40.2 mg / 100 mL6. The main flavonoid found in calamondin is hesperidin7. Hesperidin has been shown to have antioxidant, anti-cancer, and anti-inflammatory activity8. One of the potentials of hesperidin can be developed as a chemotherapy drug because it has been known to have a cytotoxic effect. The research of Adam et al., showed that hesperidin can increase the cytotoxic effect of doxorubicin9. The combination of hesperidin and vitamin C can overcome blood flow disorders and accelerate the healing of bruises10. Hesperidin has anti-hyperglycemic potential and improves heart function in mice with type two diabetes mellitus11, an antibacterial activity, such as against Helicobacter pylori12.


INTRODUCTION
Calamondin (Citrus microcarpa Bunge) is a commodity that is widely grown in Indonesia, even promoted by the One Village One Product (OVOP) of calamondin in Bengkulu 1 . However, wastewater from calamondins such as peel, pulp, seeds, and extracted water has not been processed into products which has a high sale value 2 . Calamondin contains vitamin C 7.3 gr, vitamin A 57.4 mg IU, calcium 8.4 mg, water 15.5 gr, potassium 37 mg, and fiber 1.2 gr. Calamondin peel contains essential oils, 0.15% ascorbic acid, and 5.5% citric acid 3 . Calamondin peels and leaves contain about 1.2% and 0.93% volatile oil 4 . The study results of Cheong et al., dichloromethane and hexane extracts from calamondin, identified 79 compounds representing 98% of compounds that evaporate with N-methylantranylate were the most detected compounds. Methanol extract contains a lot of phenolic acids, especially p-Coumaric and ferulic acid 5 . The study results of Bhat et al., stated that calamondin contains flavonoids around 1.41 mg / 100 mL with antioxidant capacity around 777 mg / 100 mL and ascorbic acid around 40.2 mg / 100 mL 6 . The main flavonoid found in calamondin is hesperidin 7 . Hesperidin has been shown to have antioxidant, anti-cancer, and anti-inflammatory activity 8 . One of the potentials of hesperidin can be developed as a chemotherapy drug because it has been known to have a cytotoxic effect. The research of Adam et al., showed that hesperidin can increase the cytotoxic effect of doxorubicin 9 . The combination of hesperidin and vitamin C can overcome blood flow disorders and accelerate the healing of bruises 10 . Hesperidin has anti-hyperglycemic potential and improves heart function in mice with type two diabetes mellitus 11 , an antibacterial activity, such as against Helicobacter pylori 12 .
The community widely likes this plant as a flavoring and scented food and drink in Indonesia 13 . Almost all parts of the calamondin has the potential as a medicine. The methanol extract of the leaves has efficacy as an anti-inflammatory and inhibits the growth of E.coli, Citrobacter freundii, Aeromonas hydrophila, P.aeruginosa, S.agalatiae, and Yersinia enterocolitica 14 , an antioxidant activity and has anti-inflammatory activity 15 . Calamondin juice can stimulate appetite, improve digestive disorders, reduce blood pressure and cholesterol 16,17 .
Monographs on calamondin peels are still minimal. Therefore, it is necessary to standardize specific and non-specific parameters and determine levels of hesperidin which is the identity compound of calamondin. Specific parameters are aspects of qualitative chemical content and quantitative aspects of the levels of chemical compounds that are directly responsible for certain pharmacological activities, while non-specific parameters are useful for maintaining compound content, safety, and stability of extracts to have a safety and efficacy consistency in consumers 18 . Because hesperidin has been shown to have antibacterial activity, so researchers are interested in testing the antibacterial activity of the calamondin peel extract against the bacteria S.aureus, E.coli, E.faecalis, and P.auruginosa. P. auruginosa and E. coli represent Gram-negative bacteria whereas E. faecalis and S. aureus represent Gram-positive bacteria

Place and time
The research conducted in two months in April-September 2019. The preparation and extraction of calamondin peel to determinating the standardization from calamondin peel's extract were conducted in three weeks at Sumatra Biota Laboratory Andalas University.
2. Microscopic characterizations: Identifying the simplicia powder identification fragment using aqua dest and chloral hydrate reagents.
3. Loss on drying: Simplicia powder of 1-2 grams is put into a weighing bottle that has been measured. Smooth over the simplicia powder in the weighing bottle. Then dry it at 105 ° C in an open container using an oven until the weight remains.
4. Total Ash: The simplicia powder or extract of 2-3 grams is inserted into the curs that has been measured, then incandesce sample until the charcoal is used up. Ash content is expressed in % w / w. 5. Acid-insoluble ash: Ash obtained from determination of total ash content was boiled with 25 mL of 2N HCl for 5 minutes. Collect insoluble acid parts, filter using ash-free filter paper, wash with hot water, incandesce ash until the weight remains. Acid insoluble ash content is expressed in% w/w.
6. Alcohol-soluble extractive: Simplicia powder of about 5 grams that have been dried in the air. Put in the clogged pumpkin, add 100 mL of ethanol-water, shake it repeatedly for the first 6 hours and leave it for 18 hours. Then strain and evaporate 20 mL of the filtrate to dry in a vaporizer cup that has been incubated at 105° C until the weight remains.
7. Water-soluble extractive: Simplicia powder of about 5 grams that have been dried in the air. Put in the clogged pumpkin, add 100 mL of saturated chloroform water, repeatedly shake for the first 6 hours and leave it for 18 hours. Then strain and evaporate 20 mL of the filtrate to dry in a vaporizer cup that has been incubated at 105 ° C until the weight remains.
8. Water content: Weigh the extract which is estimated to contain 1-4 mL of water put in a dry flask. For substances that can cause sudden fluctuation when boiling add a boiling stone. Add about 200 mL of water-saturated toluene to the flask, heat the flask carefully for 15 minutes. After the toluene boils, set the distillation to about two drops per second until most of the water is distilled and increase the distillation speed to 4 drops per second. After all the water has been distilled, the inside of the condenser is washed with water-saturated toluene while cleaning with a tube brush connected to a copper wire and soaked with water-saturated toluene. Continue distillation for 5 minutes. Cool the receiver tube to room temperature. If there are drops of water attached, rub the coolant tube and receiver tube with rubber that is attached to a copper wire and moistened with water-saturated toluene until the water drops drop. Read the volume of water after the water and toluene separate. Water content is calculated in% v / w.
9. Chromatographic analysis: The material used in this work is the 60 F254 silica gel plate as the stationary phase and ethyl acetate: formic acid: water (100: 15: 17) as the mobile phase. The test solution is an extract with a concentration of 5% in 2 ml of aqua dest and sufficient methanol up to 10 ml. The comparison used was hesperidin with a concentration of 1000 µg / ml in a suitable solvent. Then, 5 µl of the test solution and 5 µl of the comparison were made spots on the TLC plate using capillary tubes. The TLC plate is placed on a buffer rack until the making spots station is located below and put into the vessel until it reaches the bottom absorbent layer where the spots is not submerged. The vessel is closed and left until the mobile phase propagates to the limit at the creepage distance. Then, the TLC plate is removed from the vessel and dried. Blemish detection under UV light with a wavelength of 365 nm.
Quantification of hesperidin by TLC-densitometry method 21 1. Determination of the maximum wavelength of absorption of hesperidin: 10 mg/ml hesperidin solution was determined for its maximum absorption wavelength using a UV-Vis spectrophotometer.
2. Preparation of calibration curve: Perform TLC on the comparison made in concentrations of 50, 100, 150, 200, 250, 500, and 1000 (µg / mL) make spots with a volume of 5 μl each. Then, it is scanned using a densitometer with a maximum absorption wavelength. The curve between concentration and AUC that is formed must be linear (R2> 0.99) and determine the regression equation.
3. Determination of Hesperidin Levels in Extracts: Scan the TLC plate that has eluted the sample with a concentration of 5% and comparison The missing compound is a volatile compound such as water, essential oils, and others. It is intended that the simplicia is not easy to moldy so it does not affect the chemical content of the simplicia. In addition, loss on drying parameters can be used to ensure the drying process of plants has been carried out perfectly. Loss on drying value of simplicia calamondin peel obtained no more than 10.76%. The next parameter is the determination of water-soluble and ethanol extracts content. Determination of the extract content in certain solvents provides an initial picture of the amount of compounds present in the sample. The water used is chloroform saturated water to avoid microbial growth in the water. Ethanol used is 96% ethanol.

RESULTS AND DISCUSSION
In this study, samples were used in the form of calamondin peel from three different regions based on administrative differences (at least districts) in West Sumatera randomly selected, namely Bukittinggi and Padang, and Pesisir Selatan District. The sample is dried in an oven at 45 ° C for several days until the sample is dry. The use of an oven with this temperature to keep the simplicia from impurity and guarantee the quality 22 . Dry samples are then mashed using a grinder so that the simplicia powder is obtained. Furthermore, standardization is performed on simplicia (Figure 1).
The first parameter determined is macroscopic simplicia. This examination is carried out by directly observing the physical form of the simplicia and extract of the calamondin peel, aiming to determine the characteristics of the calamondin peel. The next parameter that is carried out is microscopic observation, which is an examination to identify the identification fragment of the simplicia powder using a microscope to prevent the falsification of the simplicia 19 . The observation is carried out using a microscope at 400 times magnification with a chloral hydrate reagent. The addition of chloral hydrate aims to eliminate the contents of cells such as starch and protein so that other cells can be seen clearly under a microscope. The simplicia calamondin peel had brownish outer surface, and the inside is yellow, slightly smelly and sour taste ( Figure 2). Microscopic examination obtained identifiers of calcium oxalate crystal fragments, fibers, parenchyma with oil cells, ladder-shaped transport tissue. Microscopic examination can be seen in Figure 3.
The next step is to determine the simplicia standardization parameters, namely loss on drying. The purpose of the loss on drying parameter is to give a range of magnitude of the lost compound to the drying process 23 .   (1) (2) (3)

(4) (5)
found more in water solvents than ethanol solvents. So calamondin simplicia contains a higher polar compound than semipolar-nonpolar compounds. The result of all parameter simplicia can be seen in Table 1.
The extract is made by maceration of simplicia powder by using ethanol solvent because it is able to dissolve almost all substances, both polar, semi-polar and non-polar in nature and can precipitate proteins and inhibit enzymes work so that they can avoid the process of hydrolysis and oxidation 24 . Ethanol used is ethanol 70% which is commonly used for the extraction of dry samples. Maserat is evaporated under vacuum so that air pressure on the surface is reduced so that the boiling point drops. This condition can reduce the possibility of decomposing the compounds contained in the sample at high temperatures. After evaporating, the extract was obtained not less than 25.33% ± 1.3%. The results of giving calamondin peel extract in the form of thick extract, special odor, black color and bitter taste. The standardization of extracts, the water content was determined. The water content in the extract also determines the stability of the extract, because high water content results in the ease of bacteria, mold, and yeast to multiply so that there will be changes to the extract 25,26 . Determination of extract water content is done by distillation. One of the chemicals that can be used in determining the extract water content is toluene. The use of toluene as a distillation-carrying chemical liquid because toluene has a boiling point higher than water that is 110 ° C, and toluene cannot mix with water because toluene is non-polar while water is more polar and toluene has a lower specific gravity than water that is 0.867 gr / mL. In addition, the use of toluene is intended so that fats do not evaporate when distilled because non-polar fats will dissolve in toluene which is also a non-polar solvent 27 . Water content obtained from the extract of the calamondin peel is not more than 17.47% ± 0.82%. The result of all parameter extract can be seen in Table 1.
The next parameter in extract standardization is TLC. The TLC profile is a qualitative analysis to show the presence of certain marker compounds present in the sample and to describe the composition of the chemical compounds contained in the sample. The TLC profile of the ethanol extract of calamondin peel used the comparative compound hesperidin. It is because hesperidin is a compound that is mostly contained in oranges 28 . In detecting patches of hesperidin, citroborate stain reagents are used, assisted with heating and detected by UV with wavelength 365 nm. Citroborate is used as a stain-viewer to identify flavonoid compounds 29 . The results of TLC calamondin peel extract was Rf1: 0.34; Rf2:0.43; Rf3: 0.58; Rf: 0.68 and Rf5: 0.78 while hesperidin (comparison) Rf was 0.68. From this pattern, it can be concluded that hesperidin is found in the calamondin peel extract ( Figure 3).
Other parameters in the standardization of simplicia and extracts are a determination of total ash content and acid insoluble ash content. Determination of total ash content aims to provide an overview of the internal and external mineral content and organic matter originating from the initial process until the extract is formed. Determination of total ash content is done by heating at high temperatures where the organic compounds and their derivatives are decomposed and evaporated at a temperature of 700 0 C until only mineral and inorganic elements remain 23 . Total ash content obtained from simplicia and extract of calamondin peel is not more than 4.33% ± 0.03% and 4.65% ± 0.06%. Ash obtained from the determination of total ash content is used to determine acid insoluble ash content. Determination of acid-insoluble ash content aims to evaluate the material against contamination of ingredients which contain silica like soil and sand. Acid insoluble ash content obtained from simplicia and extracts calamondin peel no more 1.01% ± 0.07% and 0.13% ± 0.04%. The result of all parameter of extract can be seen in Table 2.
The next parameters was determination of chemical content (hesperidin) in the extract. It used TLC-densitometry method with a maximum absorption wavelength of hesperidin was 284nm. The linear regression of the hesperidin comparison calibration curve for the calculation of hesperidin levels was y = 15.914x -49.778 R² = 0.996 and the heperidin level of calamondin peel extract was not less 4.78%. The spectrum λ max of hesperidin can be seen in Figure 4.
Furthermore, the antibacterial activity of the calamondin peel extract was tested using the agar diffusion method. The principle of the agar diffusion method is that the test substance is dripped on a blank disc, then placed on the agar media which has been inoculated by bacteria.
The test substance will diffuse into the agar medium, if a clear area is formed around the disc on the media indicating the presence of bactericidal activity by antibacterial agents on the surface of the agar media 30 .
The agar Nutrient Media is chosen because it has complete nutritional content for the growth of bacteria such as peptone, NaCl, yeast extract, and beef extract 31 . The antibacterial activity test of calamondin peel extract was carried out at a concentration of 20% and 15% in DMSO. DMSO is used as a test extract solvent because it can dissolve polar and nonpolar compounds 32 . The negative control used is DMSO aimed to prove that DMSO which is to dissolve the test extract does not have antibacterial activity. The positive control used in antibacterial testing is chloramphenicol at 0.3%. Chloramphenicol is used as a positive control because it belongs to a broad spectrum of antibiotics that can inhibit the growth of Gram-positive and Gram-negative bacteria 33 .
The test is carried out three times repeated for the accuracy of the test and minimize errors. The bacterial suspension was made in 0.9% NaCl solution. NaCl as a source of bacterial minerals because one of the factors that influence bacterial growth. NaCl 0.9% is used to keep bacterial cells in an isotonic state. 0.9% NaCl used must be sterile to avoid contamination of other microbes 34    The examination results of calamondin peel simplicia (Citrus microcarpa Bunge) are outer surface is brown and the inner is yellow, slightly smell and sour taste. Microscopic examination obtained identifiers of calcium oxalate crystal fragments, fibers, parenchyma with oil cells, ladder-shaped transport tissue. Water soluble extract and ethanol content is not less than 19.73% ± 0.97% and 10.26% ± 0.25%, loss on drying value is not more than 10.78% ± 0.05%, and the total ash content and acid insoluble ash is not more than 4.33% ± 0.03% and 1.01% ± 0.07%.
2. The examination results of the calamondin peel extract are thick, distinctive smell, black and bitter taste. The TLC pattern used a comparison hesperidin with an Rf value of 0.68, a hesperidin level of not less than 4.78% ± 0.09%, rendemen value of not less than 25.33% ± 1.3%, a water content of no more than 17.47% ± 0.82%, and the total ash content and acid insoluble ash are not more than 4.65% ± 0.06% and 0.13% ± 0.04%.
3. Extracts of calamondin peel from three regions namely Bukittinggi, Padang and Pesisir Selatan have antibacterial activity against bacteria Staphylococcus aureus, Eschericia coli, Enterococcus faecalis, and Pseudomonas auruginosa at a concentration of 15% with a range of inhibitory diameters of 7.65 mm ± 0.36 mm to 9.96 mm ± 0.52 mm and at a concentration of 20% with a range of inhibitory diameter of 9.26 mm ± 0.72 mm to 13.39 mm ± 0.28 mm.

SUMMARY
Research on the Standardization Study of Simplicia and Extract of Calamondin (Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and Antibacterial Assay was carried out. Calamondin had antioxidant and Antibacterial activity.