Isolation Compound Anti-Obesity from the Bark Ara (Ficus Racemosa) of Aceh

Introduction

Obesity (overweight) is one disease that often occurs among children, adolescents and adults. According to the International Obesity Task Force (IOTF) that more than 1.1 billion adults worldwide are overweight and 312 million are obese. Obesity is very dangerous because it can cause a wide range of other diseases such as diabetes, heart disease, stroke and certain types of cancer [1].

One of the plants that have the potential in the medical field is F. racemosa of the family Moraceae, which is used in the system of traditional medicine for diarrhea, dysentery, hemorrhoids, wound healing, skin disorders, diabetes, ulcers, impaired bile, anti bleeding, colds, antioxidants and as well as agents anticancer[2]. The results reported, suggested that F.racemosa compounds containing tannins, saponinsgluanol acetate, betasitosterol, lupeol, lupeol acetate and a-amyrinacetate[3]. Leaf extract of Ficusracemosa Linn. active as an anti-bacterial of Escherichia coli, Basilluspumilis, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus[4].

Material and Methods

Plant Material

The bark  ofF. racemosa was collected in February 2015  in Aceh, Indonesia. The plant was identified at Department of Biology, University of North Sumatera, Medanense, Medan.

Animal

All experiment were carried out using breeding 4-6 week old male Swiss Webster mice chosen from animal colony  of central animal research facility, University of North Sumatera, Medan. The colony was maintained  under controlled condition soft temperature.

Spectroscopic Investigation

IR spectrum was found using a Perkin Elmer Spectrum One FT-IR spectrophotometers. Mass spectra were measured using a Shimadzu GC-MS QP 2010 Ultra.  The ¹HNMR (400 MHz), Spectrum  wererecordedon a JEOL in CD₃Cl.

Testing Phytochemicals

The method used for testing of phytochemicals can be found in: Phytochemical methods, Simplified Determination Method to Analyze plant [5]

Extraction And Isolation Of β-Amiryn Acetate From The Bark Of F. Racemosa

The air dried of  bark of F. racemosa(3 kg) was extracted with hexane  for two times in percolator and filtered. The filtrate was evaporated in vacuo to give the dark brown residue, and the yield was 68.50  g.  Then the sample was also extracted  withethylacetate solvent, and  the yield was  50.52 g (extract of ethylacetate). All of these extract were collected separately and preserved for bioassay test and analysis. Antiobesity test results from both of the  extracts, the ethyl acetate extract   slightly more active  from  hexane extract, so the isolation and testing activities was directed at the  ethyl acetate extract.

Ethylacetate Extract Fractionation

The  ethyl acetate extract (30 g) was separated by column chromatography with  silica gel (70-230 mesh, 150 g, Merck) as stationary phase and eluted with n-hexane gradually with ethylacetate acetate (100: 0;  98: 2;  95: 5;  90:10;  80:20),  to efford 86 fractions by TLC profile.

Fractions (1-16, A)  as much as 7.01g, contained terpenoid, fractions  (17-34, B)  as much as 9.8 g, contained steroid, and  fractions (35-86, C)  as much as 1.55 g, contained terpenoids. Fraction 1-16  (3 g) was further fractionated using column chromatography (70-230 mesh, 100 g, Merck) eluting with eluent system of n-hexane: ethyl acetate (98: 2;  95: 5;  90:10)  to obtain a yellowish white solids were 250 mg (A4). This solid was further fractionated using column chromatography (70-230 mesh, 15 g, Merck) using a system of eluent n-hexane: ethyl acetate (98: 2;  95: 5;  90:10),to obtained a white solid (A4₄). Furthermore, this pure isolate  was  tested  its antiobesity  activity and characterized using ¹H-NMR and FT-IR.

Antiobesity activity test

Before use, the mice were acclimatized for 7 days in laboratory conditions as well as getting enough food and drinks. After 7 days, selected mice were healthy, characterized by weight stable or increased and did not show any abnormal behavior. Having fasted for 20-24 hours , the weight of mice were weighed, then mice were divided into 5 groups, each of the groups contain three of mice,  group I:  obesity control  (negative control is given food (pellets) without medication, no given extract  or  xenical), group II: treated with  standard drug, xenical,  was given orally at dose of  1500 mg/kg bw of mice), group III, IV, and V: treated with extract of F. racemosa, with  dosis 1500 mg/kg bw of mice. Weight measurement is done by weighing the mice every day in five days [7].

Results and Discussion

Phytochemical Test Results

Phytochemical test results of fresh bark  F. racemosa and the ethyl acetate extract of F. racemosa showed a class of secondary metabolites, respectively, steroids, terpenoids and saponins.

Antiobesity activity

The test results antiobesityof  extract n-hexane and ethyl acetate to mice can be seen in Figure 1.

Figure 1: Curves of reduction average weight of mice with different doses of the extract n-hexane and ethyl acetate (for 5 days) Click here to View Figure

 

Based on Figure 1 shows that mice fed an  crude extract n-hexane and ethyl acetate of   with a dose of 100 mg / kg bwof mice, the weight on average increased, respectively by 10.64% and 7.50% , weight is risen because a given dose of the extract is still very low. Mice were given extracts of n-hexane and ethyl acetate at a dose of 500 mg / kg bw of mice, weight  of mice  increased relatively minor, respectively: 5.87% and 2.57%. Mice that were fed n-hexane crude extract and ethyl acetate extract  at the dose of 1000 mg / kg bwof mice,  increased weight is relatively smaller, respectively 3.68% and 0.63%. Mice that were fed n-hexane crude extract and ethyl acetate extract rough at doses 1500 mg / kg bwof mice, body weight of rats was reduced by respectively 0.33% and 1.38%. So that this dosage used as a dose of work.

Further testing anti-obesity activity, carried out against a group fraction chromatography results ethyl acetate extract (fraction groups A, B, and C).The test results antiobesity fraction (A, B, C) of the mice are shown in Table 1.

Table 1: Results of testing the activity of anti-obesity of fractions A, B, and C (%)

Fraction	The decrease /increase of weight of mice  with given  fraction A, B, C for 5 days (each 3 mice) (%)			Average total (%)
	Mice (I)	Mice (II)	Mice (III)
fraction A	3.54	-3.03	-9.19	-2.89
Fraction B	0.16	-4.78	-3.10	-2.57
Fraction C	-1.61	-0.49	0.40	-0.56
Control (+)	-0.81	-5.86	-5.76	-4.14
Control (-)	4.48	4.84	5.16	4.82

Note: (+) weight gain in mice

(-) Weight loss in mice

 

Based on the result of a decrease / increase in the average weight of mice contained in Table 1, can be seen that all fractions A, B, and C, can reduce the body weight of mice. Fraction  of A  may reduce   mice weight   (2.89%) is greater than the  fraction of  B (2.57%) and the  fraction C (0.56%). Positive control,  xenical  can lose weight of mice  by an average of 4.14%, while the negative control  increase  the mice  weight  of average  as much as 4.82%.

Test Antiobesity Pure Isolates (A4₄) On Swiss Webster Mice

A4₄ pure isolates can lose weight in mice  as much as  2.31%, while Xenical as a positive control  to lose weight as much as 4.49%, and the weight of negative control mice rose as much as 4. 31% in  the same experiment.

Determination Structure  A4₄

A4₄ compound, was obtained as a colourless amorphous powder  fromethilacetate extract.  The ¹H NMR spectrum of A4₄ compound, measured in CDCl₃, Characterization of compound A4₄ with ¹H-NMR can be seen in Figure 2.

Figure 2:1H-NMR spectrum of Compound A44   Click here to View Figure

 

¹H-NMR spectrum for proton aliphatic (CH₃, CH₂ and CH) on triterpenoid compounds shown in chemical shifts (δH) 2 ppm, which is characteristic of proton cyclic of basic framework of triterpenoids that are not separated properly [8]. Compound A4₄ showed 8 C-methyl chemical shifts (δH ppm) 0.90 (3H, s, H-23); 0.97 (3H, s, H-24); 0.86 (3H, s, H-25); 1.00 (3H, s, H-26); 1.06 (3H, s, H-27); 0.84 (3H, s, H-28); 0.86 (3H, s, H-29); 0.79 (3H, s, H-30).

Acetyl group at chemical shifts (δH ppm) 2.03 (3H, s, H-32),  one proton broad multiplet at 4.49,(m,1H) was ascribed to proton of C-3, due to the influence of the electronegativity of O atoms and COCH₃ and presence of the olefin proton at δH shift 5.11 ppm (t, 1H),  characteristics of the Δ¹² (isolated double bonds, order oleanan) [2].

Furthermore,  determination of structure of the compound A4₄ done by comparing the chemical shifts (δH) between compounds A4₄  with compounds β-Amyrin acetate standard [9]. Data comparison of chemical shifts (δH) compound A4₄ with β-Amyrinasetat compounds, can be seen in Table 2.

Table 2: Comparison of the ¹H-NMR spectrum data of compound A4₄ (CDCl₃, 500 MHz) with standard of β– amyrin acetate compound

Position H	Chemical shift (δH) ppm ( J Hz)		Position H	Chemical shift (δH) ppm ( J Hz)
	A1	β– amirin asetat*		A1	β– amirin asetat*
1	1.28-1.35 (m)	1.04 -1.64 (m)	16	0.79-1.91 (m)	0.79 – 1.98 (m)
2	1.67 (m)	1.60 – 1.88 (m)	17	–	–
3	4.49 (t)	4.50 (t, 8.0 Hz)	18	1.91 (d)	1.93 (d; 4.2 Hz)
4	–	–	19	1.00-1.67 (m)	1.00 -1.66 (m)
5	0.84 (m)	0.84  (m)	20	–	–
6	1.54 (m)	1.40 – 1.53 (m)	21	1.30 (m)	1.08 – 1.32 (m)
7	1.38 (m)	1.33 – 1.52 (m)	22	–	–
8	–	–	23	0.90 (s)	0.88 (s)
9	1.54 (m)	1.58 (m)	24	0.97 (s)	0.96 (s)
10	–	–	25	0.86 (s)	0.86 (s)
11	1.62 (m)	1.63 -1.80 (m)	26	1.00 (s)	0.97 (s)
12	5.11 (t)	5.18 ( t; 3.5 Hz)	27	1.06 (s)	1.13 (s)
13	–	–	28	0.84 (s)	0.83 (s)
14	–	–	29	0.86 (s)	0.88 (s)
15	0.86 (m)	0.85 – 1.76 (m)	30	0.79 (s)	0.87 (s)
OCH3	2.03 (s)	2.05 (s)

 

Table 2 shows that the compound of  A4₄  is similiar   with  β-amyrin  acetate standard. Based on the comparison of the data of the ¹H-NMR, this compound is thought to contain the compounds β-amyrin acetate. Chemotaxonomic approach based compounds β-amyrin acetate is also present in plants A. camansi, [10], FicusretusaL.variegata[11],Dorsteniaarifolia[12], which is also a family Moraceae

The above data  is reinforced with Mass  Spectrometry (MS)  spectrum of data, which indicate the presence of β-amyrin acetate compound.

Figure 3: Mass spectrometry compound A44   Click here to View Figure

 

From  fragmentation of compound  ß-amyrin acetate  standar , showed molecular ion    [H+] m/z:468,  218 , 207, 189 and the 69. Pattern fragmentation 218 shows the breakdown C16H25 [M+] 203 ([CH₃M]+). The abundance ion at m/z 218, 203, 189, and 69 are typical for the fragmentation of ß– amyrin acetate [13].

However, the MS spectrum  of compound   A4₄ looks a molecular ion at m / z 426 which constitutes the frame of β-amirin [9, 14]. This is because the group COCH₃ (m / z 43) was not seen in the data of MS. Characteristic of β-amyrin acetate compound contained in fragment  the molecular ion m / z 218 as the base peak [13, 15]. The solving molecular ion β-amyrin compound [15]

Based on the results of data analysis and FT-IR spectrum compound of A4₄, shows the absorbance of carboxyl groups C-O (ester) at wave number 1241 cm^-1. At wave number 1366 cm^-1 showed absorbance C(CH₃)₂ (gem-dimethyl), namely the existence of two methyl groups on the same carbon. At wave number 1735 cm^-1 there is absorbance of carbonyl C = O ester (R-CO₂-R ‘) and the wave number 2851-2919 cm^-1 there is absorbance of C-H (alkyl). Based on comparative data ¹H-NMR and Mass Spectra and FT-IR of isolate A4₄, is suggested isolate A4₄ as ß-amyrin acetate, which can be seen in Figure 4.

The structure of β-amyrin acetate compound can be seen in Figure 4.

Figure 4: β-amyrin acetate Click here to View Figure

 

Conclusion

Based on the results obtained can be concluded that the test results showed F. racemosa extract contains secondary metabolites, terpenoids and steroid group. The crude extract ethyl acetate at a dose of 1500 mg / kg bw of mice, can lose weight by 1.38% .All  fractions A, B, and C, can reduce the body weight of rats.  Fraction of A  may reduce weight of mice as much as   (2.89%) is greater than the fraction of B (2. 57%) and the fraction C (0.56%), while  A4₄ pure isolates can lose weight in mice  as much as  2.31%.

Characterization of isolates A4₄ with ¹H-NMR, IR and mass spectra, allegedly fraction of A4₄ is β-amyrin acetate.

Acknowledgments

The author would like to thank the Directorate of Research and Community Service. Strengthening the Directorate General Research and development Ministry of Research, Technology and Higher Education, Indonesia.

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