View of ANTI-CHOLESTEROL ACTIVITY TEST OF N-HEXANE, METHANOL AND ETHANOL EXTRACTS ETHANOL EXTRACT OF LEMON PEEL WITH UV-VIS SPECTROPHOOMETRY METHOD

(1)

VOLUME 10 NO 01 JUNI 2023 JURNAL MEDIKA CENDIKIA

54

JURNAL MEDIKA CENDIKIA

Volume 10 Nomor 01 Tahun 2023

ISSN Cetak: 2355-827X / ISSN Online : 2442-4412 STIKes Karsa Husada Garut

ANTI-CHOLESTEROL ACTIVITY TEST OF N-HEXANE, METHANOL AND ETHANOL EXTRACTS ETHANOL EXTRACT OF LEMON PEEL

WITH UV-VIS SPECTROPHOOMETRY METHOD

Titin Supriatin¹, Mila², N. Ai Erlinawati³

123Diploma Health Analyst Study Program, Karsa Husada College of Health Sciences Garut Correspondence Email: ¹[email protected], ²[email protected] ³[email protected]

ABSTRACT

Cholestrol is a fatty substance that is in and produced by the liver and is needed by the body.However, if cholesterol in the blood is too high it can cause blockages in blood vessels that can trigger various other types of more serious diseases such as heart disease, stroke and high blood pressure (inflammation of the pancreas organ), diabetes mellitus, thyrod disorders, etc. Lemon peel can reduce cholesterol levels in the body. Because lemon peel contains flavonoid polyphenols, which are very active in lowering cholesterol. The purpose of this study was to determine the anti-cholesterol activity and determine the EC50 value of N-Hexane partition, methanol and ethanol extracts of lemon peel (Citrus limon) invitro. This type of research is descriptive quantitative analysis method using UV- Vis spectrophotometry at a wavelength of 665.0 nm with a concentration series of test samples 150 ppm, 300 ppm, 450 ppm, 600 ppm, 750 ppm. The research data showed that the level of cholesterol reduction was directly proportional to the increase in concentration in the sample. The EC50 value of n-hexane partition was 7.6 ppm, methanol partition was 108.53 ppm and ethanol extract was 194.9231 ppm. Conclusion among the three partitions, n-hexane extract is the most effective to reduce cholesterol.

Keywords: Cholestrol, Lemon Peel, Partitioning

INTRODUCTION

Cholestrol is the most abundant sterol in the body, in its form as free cholesterol or bound to fatty acids, the main source of cholesterol from foods such as meat, fish and milk (Maulida et al., 2018).

Normal cholesterol levels are < 200 mg/dl while sometimes abnormal cholesterol is >

250 mg/dl (Maulida et al., 2018). Lack of cholesterol will disrupt functions in the

body and can even lead to death. High cholesterol can also be a triggering factor for coronary heart disease because high cholesterol causes blockages in peripheral blood vessels that reduce blood supply to the heart (Maratu Soleha, 2012).

Currently, many medicinal plants are developed by the pharmaceutical industry into traditional medicines. One of the potential plants utilized for traditional

(2)

55 medicine is lemon (Citrus limon (L.).

Lemon (Citrus limon) is one of the plants that has potential as a traditional medicine, as well as an excellent source of vitamin C and calcium. Lemon peel serves to prevent cancer (Rika P, et al, 2019). Lemon fruit (Citrus lemon) is one of the fruits that produce antioxidant compounds that can ward off free radicals. Testing the antioxidant potential of local and imported lemon fruit is taken from skin extracts and fruit pulp juice (Alfian H, et al, 2017).

Some research on Citrus sp states that all citrus groups contain flavonoids.

This flavonoid content is able to lower cholesterol because the plafonoid compound from lemon peel extract is able to bind to the methoxylation ring of lemon peel extract (Citrus lemon) (Hartanto et al., 2019).

Extraction is used to separate secondary metabolite compounds that are either polar or non-polar. In the extraction of secondary metabolites, a solvent that matches the level of polarity is needed.

Because the compounds contained in

secondary metabolites will dissolve in polar or non-polar solvents. In this case, the extraction uses two types of solvents, in a separating funnel two layers occur, the top layer is of course a solvent with a smaller density and the bottom layer is a solvent with a larger density. This extraction will result in partitioning of the extract according to the level of polarity and density.

Previous research conducted by Lesyi Lusfiyadi, et al at the Faculty of Pharmacy, Muhammadiyah University (Lesyi, et al, 2021) utilized lime peel (Citrus aurantiifolia) as an anti-cholesterol activity test. The results showed cholesterol-lowering activity by showing the EC₅₀ value of n-hexane partition as much as 448.76 ppm, methanol partition as much as 448.98 ppm and ethanol extract as much as 450.18 ppm.

Based on the above exposure, the author wants to conduct research on the anti cholesterol activity test of n-hexane partition, methanol and ethanol extracts on lemon peel (Citrus Limon) invitro using the

(3)

56 UV-VIS spectrophotometric method.

RESEARCH METHOD

This research is a quantitative descriptive research with experimental research design. In this study, the anti- colesterol activity test of N-Hexane partition, methanol and ethanol extracts of lemon peel (Citrus Limon) was conducted invitro.

Tools and Materials

The tools used in this research are analytical scales (Ohaus), baking sheet, sieve 40, oven (Memmet), blender (Cosmos®), UVVIS spectrophotometer (Shimadzu), rotary evaporator, micropipette, centrifugation, measuring pipette (Pyrex), volumetric flask (Pyrex), glass beaker (Pyrex), test tube (Pyrex), vial, vortex, water bath, water bath. The materials used in this research are lemon peel extract, 96% ethanol, n-hexane, methanol, chloroform, NaOH, concentrated H₂SO₄, anhydrous CH₃COOH, HCl, and pure cholesterol powder (Lesyi, et al, 2021).

Work Procedure

1. Preparation of Simplisia and Extract The lemon (Citrus limon) plant was washed, peeled, the lemon (Citrus limon) peel was cut, dried, blended and sieved using a 40 mesh number sieve. (Citrus limon) was macerated using 96% ethanol (1:5) for 5 days. Remaceration was done for 2 days. The maceration filtrate and remaceration filtrate were combined and then evaporated using a rotary evaporator at 40℃ to obtain a thick extract of lemon peel.

lemon peel.

2. Partitioning Preparation

Weigh 25 grams of thick ethanol extract of lemon peel and add 95 ml each of n-hexane and methanol solution into a separating funnel, shaken vigorously. Let stand for 24 hours. The n-hexane phase and methanol phase were separated and then each partition result was collected. The results of the partition were evaporated using a waterbath to obtain a thick extract.

3. Preparation of Cholesterol Standard Solution.

(4)

57 Pure cholesterol powder weighed as much as 25 mg was dissolved into chloroform in a 50 ml volumetric flask to a solution of 500 ppm. The standard solution was made concentration series of 200 ppm, 400 ppm, 600 ppm, 800 ppm, and 1000 ppm. Each concentration was put in a 50 ml volumetric flask and dissolved with chloroform until the limit mark.4.

Determination of Maximum Wavelength In determining the maximum wavelength in UV-Vis spectrophotometry by scanning the wavelength of cholesterol standard solution using concentrations of 200 ppm, 400 ppm, 600 ppm, 800 ppm, and 1000 ppm taken from 500 ppm parent stock solution. Cholesterol solution of each concentration was taken as much as 2.0 ml and added 1.5 ml of anhydrous acetic acid and as much sulfuric acid. 0.1 ml. Left for 15 minutes. Measurements were taken using a UV-Vis spectrophotometer at a wavelength of 400-800 nm.

4. Cholesterol Standard Curve

Cholesterol standard solution of each concentration series was taken as

much as 2.0 ml which was then added to 1.5 ml anhydrous acetic acid solution and 0.1 ml concentrated sulfuric acid and then incubated at room temperature then the sample was measured in the 15th minute.

The absorbance was measured using UV- Vis Spectrophotometry.

5. Preparation of test solution

Preparation of 1000 ppm test solution. The test solution of n-hexane partition, methanol partition and ethanol extract of lemon peel were each made a concentration series of 150 ppm, 300 ppm, 450 ppm, 600 ppm, and 750 ppm.

6. Cholesterol Level Measurement

Test solution of n-hexane partition, methanol partition and ethanol extract of lemon peel lemon peel ethanol extract of each concentration was taken in an amount of 2.0 ml which was added to 600ppm cholesterol solution in an amount of 2.0 ml put into a test tube, the mouth of the test tube was given aluminum foil. Each test solution was vortexed. The solution was centrifuged at 3000 rpm for 10 minutes.

The supernatant was taken as much as 3.0

(5)

58 ml and also added 1.5 ml of acetic acid and 0.1 ml of concentrated sulfuric acid, let stand at room temperature for 15 minutes.

The absorbance of the solution was carried out using a UV light spectrophometer with a wavelength of 665.0 nm.

Data Analysis

The data obtained in this research is quantitative. Quantitative data through the calculation of Y = mX + C and the formula A = 1-Bx100%, where A = percent decrease in coelstrol, 1 = constant, B = final absorbance value. The EC₅₀ value of N- Hexane partition, methanol and ethanol extracts of Lemon peel (CitrusLimon) was obtained.

RESULTS AND DISCUSSION RESULTS

Table 1. Yield results of Lemon Peel Extract (Citrus Limon)

Weight of simplified powder

(gr)

Extract Weight (gr)

Extract Yield

(%)

160 30 18,75

Table 1 shows the weight of lemon peel simplisia powder (Citrus limon) 160 grams, the weight of lemon peel extract (Citrus Limon) 30

grams, and the yield of lemon peel extract (Citrus Limon) 18.75%.

Table 2. Results of lemon peel extract partition yields

Pelarut partisi

Bobot Ekstrak (gram)

Bobot partisi (gram)

Randemen (%) N-

Heksana

30 0,5 1,6

Metanol 30 8 26,6

Table 2 shows the weight of the extract is 30 g, the solvent used is 100 ml of N-Hexane and 100ml of methanol. The weight of n-hexane partition was 0.5 g and methanol partition was 8 g. The yield of n-hexane partition was 1.6% and methanol partition was 26.6%.

Table 2 Shows the weight of the extract 30 g, the solvent used is 100 ml N- Hexane and 100ml methanol. The weight of n-hexane partition is 0.5 g and methanol partition is 8 g. The yield of n-hexane partition is 1.6% and methanol partition is 26.6%.

Figure 1. partition of ethanol extract of lemon peel.

(6)

59 Figure 2. Methanol extral partitioning

Figure 3. n hexane partition

Figure 4. standardized curve for cholesterol

Figure 5: EC50 values of N-Hexane, Methanol, and Ethanol Extracts of lemon peel (Citrus lemon).

DISCUSSION

1. Simplisia Preparation and Extraction In the lemon peel (Citrus limon) made dry simplisia. Lemon peel simplisia powder (Citru limon) is sieved, with the aim of getting the uniformity of the size of lemon peel simplisia powder (Citrus limon).

Weighing as much as 160 grams of lemon peel powder was macerated with 96%

ethanol (1: 5) for 5 days and remaseation for 2 days. Lemon peel extract results in table 2 obtained ekstrak kental kulit jeruk lemon dengan nilai rendemen 18,75%. Extract yield is the active substance compound that can be taken from the simplisianya which is the ratio between the total extract obtained

y = 0.0013x - 0.1084 R² = 0.9878

0 0.5 1

0 500 1000

Absorban

Ekstrak etanol kulit jeruk Lemon

Y

Linear (Y)

y = 0.0015x - 0.0928 R² = 0.9188

0 0.2 0.4 0.6 0.8 1 1.2

0 500 1000

Absorban

Ektrak metanol kulit jeruk Lemon

Y

Y Linear (Y)

y = 0.0012x - 0.0375…

0 0.5 1

0 1000

absorbansi

uji seri N heksana

y

y Linear (y)

y = 0.0003x + 0.0936 R² = 0.9208

0 0.1 0.2 0.3 0.4

0 500 1000 1500

Absorban

Baku kolesterol

Y

Y Linear (Y)

(7)

60 and the weight of the simplisia powder used for extraction. The extract yield states that the chemical compounds contained in the lemon peel simplisia powder are extracted by the solvent. The more the yield value of the resulting extract, the more chemical compounds are produced. (Lesyi, et al, 2021).

2. Partitioning of thick extract

Partitioned as much as 25 g by adding 95% ml methanol solvent and 95%

ml n-hexane which is put into a separatory funnel then the separatory funnel is placed on a statip to let the solution stand for 24 hours partition extraction process.

The results of the lemon peel extract partition in table 2 obtained the n-hexane partition yield value of 1.6% and the methanol partition yield value of 26.6%.

The results obtained from the solvent separation process used by partition extraction on ethanol extract of lemon peel were more in the methanol partition than the n-hexane partition.

According to (Harborne, 1996) a compound will dissolve in a solvent that has

the same polarity. In this case there are several types of plafonoid compounds. Each plaphonoid has a different level of polarity depending on the number and position of hydroxyl groups of each type of plavonoid, so that it will affect the solubility of plaphonoid in lemon (Citrus lemon).

In the partition process there are chemical compounds that are not attracted by n- hexane solvent as much as 1.6% but dissolve in methanol as much as 26.6%.

This happens because the nature of the solvent N- hexane is non-polar while the lemon peel is polar, while methanol is quite polar solvent so it can dissolve some of the lemon peel.

According to (Sigarlaki &

Tjiptaningrum, 2016) n-Hexane is a good solvent for extracting compounds that are nonpolar. In the standard state this compound is a colorless liquid that does not dissolve in water. According to research (Richmond, 2008) the sample extract produced bio active in the form of chlorophyll a in N-Hexan extract of 10.88 ml/gr sample, higher than the methanol

(8)

61 extract of 8.47ml/gr sample. Chlorophyll is a type of non-polar pigment that dissolves in non-polar solvents such as N-hexanes.

Therefore, in the study of lemon peel in N- Hexan solvent produced less mass due to the polar nature of lemon peel and non-polar nature of N-Hexan.

According to Gritter et.al,. 1991 extraction with solvents is based on the nature of polarity in the solvent during extraction. Polar compounds will only dissolve in polar solvents, such as methanol, ethanol, butanol and water. Non-polar compounds will only dissolve in non-polar solvents, such as chlorophyll and hexane.

3. Maximum Wavelength

The wavelength obtained in this study is in accordance with the orientation results, namely 665.0 nm with a concentration of 600 ppm in the cholesterol standard solution reacted with 1.5 ml of anhydrous acetic acid and 0.1 ml of concentrated sulfuric acid. This is because the peak forms the optimal absorption (Lesyi, et al, 2021).

4. Cholestrol Standard Curve

The correlation coefficient (R) on the cholesterol standard curve in this study is R = 0.9208 with a value of a = 0.0003x and a value of b = 0.0936 so that a linear regression equation y = 0.0003x + 0.0936 is obtained. The correlation coefficient is used to determine the linearity of an analysis, where the value of a is the intersection of the curve on the y-axis, while the value of b is the slope of the curve (Lesyi, et al, 2021).

5. Reduction of cholesterol in extracts Preparation of 1000 ppm test solution was carried out. The concentration series of test solutions are 150 μg/ml; 300 μg/ml; 450 μg/ml; 600 μg/ml; and 750 μg/ml, each of which is put into a 10ml volumetric flask and added with chlorophyllipt until the limit mark. The higher the concentration, the more concentrated the solution. Chloroform is used as a cholesterol solvent because it adjusts the treatment in the manufacture of cholesterol standard stock solution. In each concentration series made as much as 10 ml, taken for testing each as much as 2.0 ml is

(9)

62 put into a centrifuge tube that has been coated with aluminum foil.

The purpose of test tubes and centrifuge tubes coated using aluminim foil is to protect the test solution from light because if exposed to light, the compounds contained will be damaged. Addition of cholesterol solution with a concentration of 600 μg/ml as much as 2.0 ml. Furthermore, centrifugation was carried out for 10 minutes at 3000 rpm which had previously been vortexed for 2 minutes to homogenize the sample solution with cholesterol solution.

The purpose of centrifugation is to separate the liquid from the particles against the density of the screen. The next stage is the addition of 1.5 ml of anhydrous acetic acid and 0.1 ml of concentrated sulfuric acid. The purpose of adding anhydrous acetic acid is to extract and the reaction will form acetyl derivatives of steroids while the addition of sulfuric acid with the aim of forming a complex color reaction of green solution. The green solution is due to the presence of steroid content in it which

includes cholesterol. The type of steroid in plants is phytosterol with a molecular structure similar to cholesterol (Lesyi, et al, 2021).

There is a change in color from a clear yellow color in cholesterol solution, then after the addition of concentrated sulfuric acid, there is a change in color to green in the chemical structure of cholesterol, there is an addition of conjugated bonds. In this reaction must be free from water content in it because it can affect the unstable reaction process.

Furthermore, the solution was allowed to stand for 15 minutes in a dark place, because it has unstable photodegradation properties when exposed to light. The purpose of standing for 15 minutes, to optimize the reaction to the maximum so that the green color complex terbantuk. The compound formed was measured on a UV-Vis spectrophotometer with a wavelength of 665.0 nm (Lesyi, et al, 2021).

The relationship between the concentration of the sample test solution (x value) and the average percentage of

(10)

63 cholesterol level reduction (y value). Based on the results of curve calculations on ethanol extracts of lemon peel and cholesterol listed in Figure 6, the results obtained Y = 0.0013X-0.1084 and based on the results of calculations with the formula A = 1-Bx100% and the results of the calculation of Y = bX + a, the results of cholesterol reduction or EC50 obtained a value of 194.9231 ppm.

Based on the results of curve calculations on methanol extracts of lemon peel and cholesterol listed in Figure 7.

obtained the results Y = 0.0015X-0.0928 and based on the calculation results with the formula A = 1-BX100% and the calculation results Y = bX + a, obtained the results of cholesterol reduction or EC50 obtained value X = 108.53 ppm.

Based on the results of curve calculations on N- Hexane extract of lemon peel and cholesterol listed in graph 8, the results obtained y = 0.0012x - 0.0375 and based on the calculation results with the formula A = 1-BX100% and the calculation results Y = bX + a, the result of cholesterol

reduction or EC50 obtained a value of 7.6 ppm. So that from the three partitions a bar chart is made with the name EC50 value as in Figure 9. above. From these results, it shows that the ethanol extract of lemon peel, methanol and N-hexan experienced a significant decrease in cholesterol levels.

This is due to the order of polarity of ethanol

> methanol > N-Hexan. While cholesterol is a compound that is nonpolar.

Of the three partitions seen in the final value, the n-hexane partition has an EC50 value of only 7.6, this shows that cholesterol is a non-polar sample and n- hexane is a non-polar solvent. So between non-polar substances with non-polar solvents will dissolve each other, this is in line with research from (Sigarlaki &

Tjiptaningrum, 2016), that n-Hexane is one of the good solvents for extracting compounds that are nonpolar. In the standard state this compound is a colorless liquid that does not dissolve in water. The value of cholesterol reduction in methanol extract, has an EC50 of 108.53 ppm higher than n-hexane. This is because cholesterol is

(11)

64 non-polar and methanol is polar, so they do not dissolve each other. This is in accordance with research (Andini et al, 2019) that methanol is a polar solvent whose polarity is higher than ethanol because it has a smaller number of c atoms, so the compounds bound by the two solvents have different levels of polarity. In Figure 4, the EC50 results of cholesterol solubility in ethanol extract showed a cholesterol reduction value of 194.9231 ppm. This value is the highest among the three partitions. This is due to the polarity of ethanol below the polarity of methanol, so that only a little cholesterol dissolves in ethanol extract of lemon peel. This is in line with the opinion (Hartanto et al., 2019). That ethanol compounds are semi-polar and able to dissolve polar or non-polar compounds including lemon peel.

CONCLUSION

Based on the results of the study, it can be concluded that ethanol partition, methanol partition, and n-hexane extract of lemon peel (Citrus limon) have cholesterol- lowering activity by showing the EC₅₀

value. Among the three partitions, the lowest cholesterol-lowering value was the n-hexane lemon peel extract partition. So among the three partitions, the most effective in lowering cholesterol is n-Hexan.

REFERENCE

Alfian HK, 2017, POTENSI

ANTIOKSIDAN EKSTRAK KULIT DAN PERASAN DAGING BUAH LEMON (Citrus Lemon) LOKAL DAN IMPOR, Jurnal UMJ.

Andini, et al, 2019, Uji Aktifitas antioksidan dan toksisitas ekstraks methanol beberapa jenis daun benalu, Volume 2

Hartanto,et al, 2019, "Potensi ekstrak etanol kulit jeruk lemon (Citrus limon L.) sebagai obat alternatif hiperkolesterolemia pada tikus wistar hiperglikemik." Kartika: Jurnal Ilmiah Farmasi.

Harborne, 1996, Analisis Flavonoid, alkoloid, kuinon dan terpenoid pada tanaman herbarium. laut, Jurnal Biologi Covid 19.

Gritter, 1991, Metode Ekstraksi, Jurnal Biologi Covid 19.

Lutfiyati, Iesyi, et al, 2019 "Uji Aktivitas Antikolesterol Partisi N-Heksana, Metanol Dan Ekstrak Etanol Kulit Jeruk Nipis (Citrus Aurantiifolia)

(12)

65 Secara In Vitro." Prosiding Seminar Nasional Kesehatan. Vol. 1. 2021.

Richmond, 2004, Pengaruh pencahayaan terhadap Kandungan Pigmen Tetraselmis chuii sebagai Sumber Antioksidan Alami. Nada Kristiani Ginting, Sri Sedjati*, Endang Supriyantini, Ali Ridl. Departemen Ilmu Kelautan dan Perikanan.

Maulida, et al, 2018, "Pengaruh Rasio Kolesterol Total terhadap High Density Lipop rotein (HDL) pada Kejad ian stroke Iskemik.

Soleha, Maratu, 2012, "Kadar kolesterol tinggi dan faktor-faktor yang

berpengaruh terhadap kadar kolesterol darah." Jurnal Biotek Medisiana Indonesia 1.2 (2012): 85- 92.

Rika P, et al, 2019), KARAKTERISASI SIMPLISIA DAN SKRINING FITOKIMIA SERTA ANALISIS SECARA KLT (Kromatografi Lapis Tipis) DAUN DAN KULIT BUAH JERUK LEMON (Citrus limon (L.) Burm.f.), Farmasi Stikes Imelda.

Sigarlaki,et al, 2016),"Pengaruh pemberian buah naga merah (Hylocereus polyrhizus) terhadap kadar kolesterol total." Jurnal Majority 5.5.