INTRODUCTION:

Indonesia is a country that has a variety of plant species because it has around 4000 plant species, no less than 40% of which are species that only grow in the Indonesian region and are not found anywhere else in the world¹. Plants carry out metabolism to produce metabolites. Plants produce two metabolites namely primary and secondary metabolites. Types of secondary metabolite compounds that act as biological activities include flavonoids, alkaloids, tannins, saponins, quinones, steroids and terpenoids¹. Based on the Ministry of Health (2007), out of 9,600 plants, only 300 have been used as traditional medicine, one of which is gotu kola².

Gotu kola (Centella asiatica (L.) Urban) has been used as a traditional medicine both in fresh and dry forms or in the form of concoctions³.

Degenerative diseases are still a concern for every country in the world and are the main driver of death⁴. One of the things that causes this degenerative disease is free⁵. Antioxidant compounds are needed to neutralize, reduce and inhibit the formation of free radicals in the body by acting as electron donors for free radicals, causing them to turn into free electrons in free radicals by pairing and stopping damage in the body. Atoms or molecules with an unpaired electron are known as free radicals, and this property makes them very reactive. Free radicals are created when food is broken down by our bodies or when we are exposed to radiation or smoke in the environment. The oxidative stress is brought on by free radicals like hydrogen peroxide, hydroxyl, superoxide anions, peroxynitrite, and nitric oxide. Damage to biological macromolecules including DNA, lipids, and proteins is caused by oxidative stress. Many illnesses, including anemia, arthritis, inflammation, diabetes, neurological disorders, atherosclerosis, and carcinogenesis, are brought on by cellular damage⁶. This prevents the accumulation of free radicals which can lead to the development of degenerative diseases⁷. The solution to keeping the body protected from free radicals is the use of compounds to neutralize free radicals such as antioxidants².

One of the plants that can be used for traditional medicine for various diseases is Gotu Kola (C.asiatica (L.) Urban). Gotu kola (C.asiatica (L.) Urban) is known to have pharmacological effects as an antibacterial⁸, antioxidant, anti-leprosy, and anti-diabetic^9,10, anti-aging¹¹, anti-cancer¹², anti-inflamation¹³, etc. Centella asiatica leaves are used as fresh vegetables, drinks, skincare and traditional medicine¹⁴. Herbal medicines have been used for the management of certain ailments for centuries¹⁵. Based on empirical data and research, gotu kola has been shown to offer several therapeutic benefits for treating various diseases, including canker sores, skin diseases, fever, urine damage, hypertension, anemia, diarrhea and being able to improve memory¹⁶. The part of gotu kola that is commonly used for traditional medicine is the leaves³. Compounds that have an active role as antioxidants in gotu kola are spolyphenols, flavonoids, carotene, tannins, vitamin C, kaemferol, quercetin, glycosides and asiaticoside^10,17. Centella asiatica contains triterpene glycosides such as centellasaponin, asiaticoside, sceffoleoside and madecassoside, also asiatic acid and madecassic acid¹⁸.

Several studies have been conducted regarding the pharmacological effects of Centella asiatica, namely testing the antioxidant activity of the ethanol extract of Centella asiatica herb using the phosphomolybdate method¹⁹. Dewi & Maryani (2015) have also succeeded in isolating secondary metabolites from the ethyl acetate fraction with the compounds obtained, namely kaempferol and quercetin. Both of these compounds have a role in antioxidant activity and inhibition of α-glucosidase (IC₅₀ each of the two compounds, namely 16.50 and 21.61μg/mL). Besides the ethyl acetate fraction, the n-hexane and n-butanol fractions are also often isolated^8,20,21. Then research on gotu kola from the ethyl acetate fraction is still rarely used, so further research is needed. to isolate, identify, and test the free radical scavenging activity of DPPH from the active ethyl acetate fraction of Centella asiatica herb (C.asiatica (L.) Urban).

MATERIALS AND METHODS:

Materials:

C. asiatica (L.) Urban were collected from BALLITRO, Bogor, West Java, Indonesia in June 2020. The identification of the plant was determined by the staff of Research Center for Biology-LIPI (BRIN).

Extraction and Fractination:

The dried aerial parts of C. asiatica was extracted by 70% ethanol to provide the crude extracts and continued to fractionate of the extracts. Fractionation is carried out by partitioning using a separatory funnel. In a separatory funnel, 400 g of dry extract was previously dissolved in distilled water, then n-hexane was added. The mixture was homogenized and allowed to form two layers, namely the water layer and the n-hexane layer. The n-hexane layers were separated and collected, then concentrated using a rotary vacuum evaporator (BUCHI Rotavapor R-100®) at a temperature range of 40-50°C. The aqueous layer is mixed with ethyl acetate and then carried out in the same way as the partition process with n-hexane solvent. After partitioning with ethyl acetate, the water layer obtained was partitioned with n-butanol solvent²².

Separation:

The viscous extract of the ethyl acetate fraction was separated by Vacuum Liquid Chromatography (VLC) using silica gel stationary phase and a gradient mobile phase system with steps from n-hexane : ethyl acetate (10:0, 9:1, 8:2, 7:3, 6 :4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10) then followed by ethyl acetate : methanol (10:0, 9:1, 8:2, 7: 3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10). The results of each fraction are collected in vials and then evaporated using a rotary vacuum evaporator at a temperature range of 40-50 °C. The fractions obtained from VLC were tested by TLC and the fractions that had the same TLC pattern were combined. Furthermore, the combined fractions were tested again with TLC and the fractions that had not too many spots or stains and indicated the presence of the intended compound were separated again using gravity column chromatography using silica gel as a stationary phase with the same mobile phase mixture as VLC²⁰. Each subfraction obtained from the column is dried in an oven. The purity test of each fraction obtained from VLC and the subfraction obtained from gravity column chromatography was carried out by Thin Layer Chromatography (TLC) using 3 different eluent systems (n-hexane, ethyl acetate and methanol). Further separation was carried out using a sephadex column with dichloromethane:methanol (1:1) solvent²³.

Purification and Purity Test:

Compounds that have been obtained from separation are purified by recrystallization. The recrystallization technique used was recrystallization with multiple solvents. Isolate was dissolved in chloroform solvent then methanol was added slowly. Compounds and impurities will dissolve in the solvent and precipitate, the main compounds will remain in the solution²⁴.

Antioxidant Test:

a) Antioxidant Activity of Gotu Kola Extract and Fractions:

Extract and fraction solutions were prepared at a concentration of 1000 ppm and DPPH at a concentration of 1mM. Furthermore, the extract and fraction solutions were diluted and prepared with four different concentrations (the calculation of each fraction and extra is in the appendix). 2 mL of each extract or fraction solution with 0.5 mL of DPPH was mixed, then shaken and allowed to stand for 30 minutes. The resulting absorption was measured at a wavelength of 517nm by spectrophotometer UV-Vis (Carry 60 ®). The positive control used as a comparison was quercetin with a concentration of 1, 5, 10 and 20 ppm. This test duplo⁹. Interpretation of the data on these results using IC₅₀value. The following formula was used to calculate the percentage of DPPH radical scavenger activities that were inhibited: A0 was the absorbance of the control reaction, and As was the absorbance in the presence of the sample. %Inhibition is calculated as [1-(As/A0)] x 100²⁵. Using logarithmic regression, the IC₅₀ values were determined from the mean inhibitory values.

b) Antioxidant Activity of Pure Isolates and Sephadex Chromatography Fractions:

Antioxidant activity test was carried out using microplate reader (Thermo Scientific®) with the DPPH method. Antioxidant measurements were carried out in the dark at a wavelength of 517 nm. The initial sample was made with a concentration of 1000 ppm. Then the sample solution was diluted and prepared with four different concentrations (the calculation for each sample is in the attachment). A total of 100 µL of sample solution with 100 µL of 0.1mM DPPH was mixed and shaken then incubated for 30 minutes. The positive control used as a comparison was quercetin with a concentration of 1, 5, 10 and 20ppm. This test is duplo²⁶. Interpretation of the data on these results using IC₅₀value_.The following formula was used to calculate the percentage of DPPH radical scavenger activities that were inhibited: A0 was the absorbance of the control reaction, and As was the absorbance in the presence of the sample. % Inhibition is calculated as [1-(As/A0)] x 100²⁵. Using logarithmic regression, the IC₅₀ values were determined from the mean inhibitory values.

c) Antioxidant Acitivity of VLC Fraction and Gravity Column Chromatography:

Each sample was made at a concentration of 1000 ppm in methanol and then diluted to 100 ppm. A total of 100 µL of sample solution (concentration of 100 ppm) was put in microplate reader (Thermo Scientific®). After that, 100 µL of 0.1 mM DPPH was added, then homogenized and left at room temperature in the dark for 30minutes. The absorbance value was measured using microplate reader (Thermo Scientific®) at a wavelength of 517nm.

This test is duplo. The positive control used was quercetin with the same concentration as the sample. The antioxidant capacity of the VLC yield fraction is expressed in absorbance values²⁶. Interpretation of data on this fraction using percent inhibition is calculated as [1-(As/A0)] x 100²⁵.

RESULT AND DISCUSSION:

Fractination:

A total of 400grams of 70% ethanol extract of gotu kola was partitioned with three solvents with different polarities namely n-hexane, ethyl acetate, and n-butanol. The results of the three partitions were concentrated with a vacuum rotary evaporator. The result of the water layer from the partition of n-butanol is maintained, so that the following fractions are obtained

Table 1: Data on the yield of 70% Ethanol Extract of Gotu kola

Name of Fraction	Mass (g)	Fraction Yield (%)
n-Hexanes	15.24	3.81
Ethyl Acetate	26.85	6.70
n-Butanol	25.06	6.27

The purpose of the partition of this research is to maximize the separation process based on its polarity. Solvent n-hexane which is non-polar to separate compounds that are non-polar, then with ethyl acetate which is semi-polar in nature to separate compounds that are semi-polar and finally uses a solvent n-butanol which is polar to attract polar compounds. After that, the antioxidant activity test was carried out on the 70% ethanol extract of gotu kola and all the fractions in table 1. The method used in this study was the DPPH method and the standard used was quercetin. The use of DPPH is because this method has advantages such as easy, fast analysis, and makes it possible to visually determine the presence of compounds that act as antioxidants. This test is characterized by a color change from purple to yellow which occurs when the sample donates electrons to the DPPH radical (1,2-diphenyl-1-Picrylhydrazyl) and converts it into a more stable molecule, resulting in a color change from purple to yellow. The parameter that is also used to measure the antioxidant activity of the extract formulation samples is the IC₅₀ value (sample concentration in ppm (µg/mL) which can inhibit 50% of DPPH free radical activity). The standard used in this test is quercetin. Quercetin is a flavonoid compound in the flavonol class which is active as an antioxidant ²⁷. When the flavonols in quercetin react with free radicals (DPPH), quercetin will donate its proton to become a radical compound. The resulting unpaired electrons are delocalized into the aromatic system so that the quercetin radical compound has very low energy and is relatively less reactive, therefore this compound is very good at inhibiting DPPH free radicals. Therefore this compound is good for use as a standard for testing natural antioxidant²⁸. The results of the antioxidant activity test of extracts and fractions of gotu kola are in table 2.

Table 2: Results of Antioxidant Activity Test Analysis of Extracts and Centella asiatica Fractions

Name	IC₅₀value ±STD
Quercetin	4.71 ± 0.07
70% Ethanol Extract	42.21± 0.75
n-Hexanes Fraction	1289.06 ± 18.79
Ethyl Acetate Fraction	37.82 ± 0.76
n-Butanol Fraction	13..33 ± 0.92

Based on the table above, it can be seen that the n-butanol fraction (IC₅₀ = 13.33 ppm) has a higher activity compared to other extracts and fractions. A compound is categorized as active as an antioxidant if the IC₅₀ value which is owned ≤ 100ppm, is said to be potentially moderate when the IC₅₀ value ranges from 100-200ppm and is declared inactive as an antioxidant if the IC₅₀ value >200 ppm. Then for the category that antioxidants are very active when the IC₅₀ value < 50 ppm⁵. Due to the fact that the yield of the ethyl acetate fraction is the highest and has a strong antioxidant category, the ethyl acetate fraction is used for further separation. The n-butanol fraction and 70% ethanol extract are not used because a lot of research has been done^20,21,29. Then there are several cases in the journal Rumyati, et al (2014) where the IC₅₀result the antioxidant activity of the fraction obtained from the partition results may have a greater value than the extract. This is because the compounds contained in the extract still contain metabolite compounds which are greater than the fractions, and partitioning affects the content of compounds drawn by the solvent so that there is the potential for differences in antioxidant activity.

Separation:

One method for isolating and identifying the bioactive chemical from plant extracts is column chromatography, which uses a stationary phase like silica gel or alumina³⁰. Separation was carried out by vacuum liquid column chromatography (VLC) and gravity column chromatography (GCC). Then the results of the fractions were combined based on the results of the same chromatogram from thin layer chromatography (TLC). The first separation of the ethyl acetate fraction was carried out by vacuum liquid column chromatography (VLC) with a gradient system as the mobile phase. The total fractions obtained from this separation are 69 fractions and a combined total of 14 fractions. The fourteen fractions that have been obtained were tested again for their antioxidant activity microplate reader with a concentration of 100 ppm. The highest inhibition value was found in F8 with an inhibition value of 89.99 %±0.09. The second separation was carried out by gravity column chromatography (GCC) with silica gel 60 stationary phase (0.063 – 0.2 mm) and the same mobile phase as VLC. The total fraction obtained from this second separation using GCC was 348 fractions and 20 combined fractions were obtained from the Rf value of thin layer chromatography (TLC). After that, the antioxidant activity test was carried out again with microplate reader with a concentration of 100ppm. The highest inhibition value was obtained, namely F10 with an inhibition value of 97.6% ± 0.06. The third separation was carried out by GCC with sephadex LH-20 as the stationary phase and dichloromethane:methanol (1:1) as the mobile phase. The fractions obtained from this separation were 45 fractions with a combined total of 11 fractions. From this separation it was found that FS-5 contained white crystalline compounds (pure isolates).

In Fadhli, et al (2020) showed that the principle of using the sephadex LH-20 adsorbent will produce eluate without residue and separate components more selectively so that this adsorbent is used to obtain pure isolates. Dichloromethane (DCM): methanol (1:1) was used to determine the solvent due to the characteristics of the Sephadex LH-20 adsorbent using methanol as a solvent. This was also supported by previous studies which also used the solvent as a re-separation step in order to obtain pure isolates²⁰. The fractions obtained from this separation were 45 with a combined total of 11. From this separation it was found that FS-5 contained white crystalline compounds (pure isolates).

Purification and Purity Test:

Purification of FS-5 or the isolate is carried out by recrystallization. The recrystallization technique used was recrystallization with multiple solvents. FS-5 was dissolved in chloroform solvent then added methanol slowly. Compounds and impurities will dissolve in the solvent and precipitate, the main compound will remain in solution. The mass of pure isolate produced was ±5.5 mg. After that, the purity test was carried out with TLC. A compound is said to be pure if it has a single spot after testing. In this test, the TLC plate was cut and eluted with chloroform: methanol (9:1) as the mobile phase. The spots were observed under UV light (254 and 366 nm wavelength). Then the TLC plate was sprayed with an H₂SO₄ 10% and heated. The results of the TLC showed that FS-5 was pure and was suspected of being a terpenoid compound because terpenoid compounds would form a pink to purple or violet color after being sprayed with H₂SO₄ 10% and heated^8,31. Because many plants employ their terpenes as a deterrent to draw insects, microbes, and animals that use those plants as food, terpenes play a crucial function as phytohormones compounds of the plant³².

Pure Isolate Antioxidant Acitivity Test:

The antioxidant activity test of pure isolates from sephadex chromatographic separation was carried out using an microplate reader with the DPPH method and quercetin standards. This test was repeated in duplicate. Variation of concentration used in FS-5 is 31,25; 125; 250; and 500ppm. Antioxidant test results are expressed ^{in IC}50 ^{value. The test results for the
antioxidant activity}of quercetin and FS-5 were 2.82 ± 0.01 and 106.99 ± 1.76, respectively.

Charecterization of Pure Isolate FS-5:

a) UV-Vis Spectroscopy:

Figure 1: UV-Vis spectroscopy 11-hydroxy-9-(hydroxymethyl)-1, 2, 6a, 6b, 9, 12a-hexamethyl-10-((3,4,5-trihydroxytetrahydrofuran-2-yl)oxy )-1, 2, 3, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 11, 12, 12a, 12b,13,14b-icosahydropicene-4a-carboxylic acid

Analysis of isolates by UV spectrophotometry aims to determine whether there are chromophore groups in the isolates being analyzed. The detection of chromophore groups in isolates can be seen from the observation of the isolate's UV spectrum at 2. In the UV spectrum of pure isolates, there are 2 spectral peaks. This could be due to the possibility of the presence of a C=C group in the isolate resulting in a transition from π → π* resulting in absorption at a maximum wavelength of around 200 nm³³.

b) IR Spectroscopy:

Figure 2.IR spectroscopy 11-hydroxy-9-(hydroxymethyl)-1, 2, 6a, 6b, 9, 12a-hexamethyl-10-((3,4,5-trihydroxytetrahydrofuran-2-yl)oxy )-1, 2, 3, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 11, 12, 12a,12b,13,14b-icosahydropicene-4a-carboxylic acid

Isolate analysis by IR spectrophotometry aims to determine the functional groups in the pure isolate being analyzed. Functional group detection in isolates can be known from observations on the IR spectrum of isolates based on the wave number in Figure 2. In the IR spectrum of pure isolates, the OH group was detected which was marked with a wide band at wave number 3375.02 cm^-1. The aliphatic CH groups are detected because there is a band at each wave number of 2929.66 cm^-1and 2866.84 cm^-1. Number of waves 2929.66 cm^-1possibly indicating CH₃ stretching, while CH₂stretching is shown at a wave number of 2866.84 cm^-1. Other potential aliphatic groups are marked on the band with a wave number of 1584.66cm^-1indicating the presence of a C=C group. At wave numbers 1456.85, 1411.12, and 1372 cm^-1shows an aliphatic CH group³³.

c) LC-MS/MS Spectroscopy:

One of the advantages of using the LC-MS/MS analysis technique is that it can identify the molecular mass at the absorption peak resulting from a sample separation process and confirm the purity of the compound. Therefore, to support the data from the UV spectrophotometer and IR spectrophotometer, a molecular mass analysis was carried out at a retention time of 12.51 minutes. The results of the LC/MS-MS examination show value m/z 511.3383 for [M-ribose+Na]⁺ fragment from 11-hydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamethyl-10-((3,4,5-trihydroxytetrahydrofuran-2-yl)oxy)-1, 2, 3, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 11, 12, 12a, 12b, 13, 14b-icosahydropicene-4a-carboxylic acid.

Figure 3: LC-MS/MS spectroscopy 11-hydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamethyl-10-((3,4,5-trihydroxytetrahydrofuran-2-yl)oxy )-1, 2, 3, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 11, 12, 12a, 12b, 13, 14b-icosahydropicene-4a-carboxylic acid

d) NMR Spectroscopy:

The NMR spectrophotometer uses data ¹H, ¹³C, HMQC, HMBC, and H-H COSY. The results of isolate FS-5 were compared with the results of asiatic acid obtained from the literature, these results can be seen in tables 3 and 4.

Table 3: Results of Chemical Shift Data For Compound 1H (500 MHz), 13C (125 MHz), and HMBC Isolate 11-hydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamethyl-10-((3,4,5-trihydroxytetrahydrofuran-2-yl)oxy )-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylic acid

Position	¹H-NMR	¹³C-NMR	HMBC (δ_C, ppm)	H-H COSY (δ_H, ppm)
Position	(δ_H, ppm), multiplicity, J (Hz))	(δ_C, ppm)	HMBC (δ_C, ppm)	H-H COSY (δ_H, ppm)
1	2.32 (m) 2.39 (m)	38.7	-	3.51
2	3.51 (m)	79.2		2.32 dan 2.19
3	3.19 (m)	73.5	101.1 dan 56.4
4		45.8
5	0.98 (m)	56.4
6	1.92 (m)	39.7
7	0.96 (s)	19.803	39.4
8		51.2
9	1.11 (m)	39.4
10		36.7
11	1.81 (m)	37.2
12	5.29 (br,s)	122.2	31.8 dan 37.2
13		138.4
14		42.2	11.95
15	0.97 (m)	37
16	1.17 (m)	29.7	37.0
17		42.3
18	1.45 (m)	31.8		0.91
19	0.91 (m)	24.9	42.3	1.45
20	1.45 (m)	24.3
21	1.35 (m)	31.9		1.53
22	1.53 (d.10)	31.9	179.3	1.35
23	3.68 (dd,5) 3.77 (dd.5)	61.8	45.8
24	0.93 (s)	19.32
25	0.84 (d,7)	18.98	36.7 dan 39.4
26	0.75 (s)	21.05	51.2
27	0.61 (d,10)	11.95
28		179.3
29	0.72 (s)	12.23	24.9
30	0.73 (s)	18.95	31.95
31	4.33 (d,0.82)	101.1
32	3.22 (d,12.5)	75.7		3.37
33	3.37 (d,2.5)	70.1		3.39 dan 3.22
34	3.39 (d,2)	76.3		3.37

* the data has been determined based on the correlation of HMQC and HMBC

Table 4. Results of Chemical Shift Data For Compound 1H (500 MHz), 13C (125 MHz), and HMBC Asiatic Acid ³⁴

Position	¹H-NMR	¹³C-NMR
Position	(δc, ppm), multiplicity, J (Hz))	(δc, ppm)
1		47.9
2	4.23 (d,10)	68.8
3	4.23 (d,10)	78.2
4		43.6
5		47.9
6		18.5
7		33.1
8		40.0
9		48.1
10		38.3
11		23.8
12	5.45 (t,4)	125.6
13		139.3
14		42.6
15		28.6
16		24.9
17		48.0
18		53.5
19		39.4
20		39.4
21		31.0
22		37.4
23	3.72 (d, 12) 4.21 (d,12)	66.5
24	1.05 (s)	14.4
25	1.18 (s)	17.5
26	1.05 (s)	17.5
27	1.05 (s)	23.9
28		179.9
29	0.90 (d, 6.4)	17.5
30	0.92 (d, 6.4)	21.4

The results of the analysis using¹H NMR provides general information that a chemical shift < δ 3ppm indicates the presence of a proton from the sp₃ carbon bond in the terpenoid framework and proton signals in the downfield (δ 5 – 7) are the proton signals of alkenes (doublebond). Most of the methyl groups appear near δ 1 ppm if the methyl group is attached to the sp₃ carbons another. The methylene group protons, -CH2- (bonded to the sp₃) appears at a larger chemical shift (near δ 1.2 – 1.4 ppm) than the methyl group protons. The tertiary methine protons appear at a greater chemical shift than the secondary protons. The protons that are close enough to the alkene will have little effect on the symptoms.deshielding from pi electrons (π) to absorption occurs around the downfield of alkyl protons in general, namely at δ > 2 ppm³⁵.

Absorption at a shift around δc 179 is characteristic of the presence of carboxylic acids at C-22 from asiatic acid compounds produced from gotu kola plants. Whereas a shift around δc 120-140 ppm indicates the presence of carbon double bond (C=C) in the terpenoid framework present in C-12 and C-13. At C-31 it is suspected that there is a ribose sugar that binds to the main framework of asiatic acid (terpenoids).

The correlation image of the FS-5 isolate can be seen above.

Based on UV/Vis, FTIR, LC-MS/MS, and NMR spectrophotometer data, it can be concluded that isolate FS-5 is an asiatic acid derivative compound shown in Figure 4. The results of a literature search via Pubchem®, Chem Spider®, and The Human Metabolosme Database® show that This compound is a new compound with the IUPAC name, namely 11-hydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamethyl-10-((3,4,5-trihydroxytetrahydrofuran-2-yl)oxy )-1, 2, 3, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 11, 12, 12a, 12b, 13,14b-icosahydropicene-4a-carboxylic acid.

CONCLUSION:

Isolation of the active ethyl acetate fraction of Centella asiatica (L.) Urban to obtain a new compound derived from asiatic acid with the IUPAC name 11-hydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a- hexamethyl-10-((3,4,5-trihydroxytetrahydrofuran-2-yl)oxy)-1, 2, 3, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 11, 12, 12a,12b,13,14b-icosahydropicene-4a-carboxylic acid. The antioxidant activity of this compound is based on the IC₅₀value with the DPPH test of 106.99±1.76ppm.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This research was supported and facilitated by the National Research and Innovation Agency (BRIN). We thank to Mr. Azhar Darlan from Puslabfor for LC-MS/MS measurement. Bunga Nur Annisa, Vriezka Mierza, and Sofa Fajriah are main author for this research.

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Received on 27.02.2023 Modified on 07.04.2023

Research J. Pharm. and Tech 2024; 17(1):51-58.

DOI: 10.52711/0974-360X.2024.00009