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Proceeding of The 5thAnnual Basic Science International Conference

Received, , Accepted, , Published online, .

Isolation of Secondary Metabolites Compound and Antioxidant

of Neem Root (Azadiractha indica) From Poteran-Madura Island

* Prima Agusti Lukis1

Taslim Ersam2

1,2 Department of Chemistry, Faculty of Mathematic and Science, Institut

Teknologi Sepuluh Nopember, Surabaya, Indonesia

* Corresponding authors: [[email protected]]

Abstract Numerous studies on medicinal plants reported that it contain large

amount of antioxidants. Antioxidant effects mainly due to the secondary

metabolites compounds. In this study, carried out the isolation of secondary

metabolites compound of the neem roots (Azadirachta indica) from Poteran-

Madura Island and quantitatively antioxidant test using DPPH (2,2-diphenyl-1-

picrylhydrazyl) method. Isolation method used was macerated with methanol andthen fractionated by vacuum liquid chromatography using the eluent n-hexane,

dichloromethane, ethyl acetate and methanol based on increasing polarity. The

resulting fraction is then purified by recrystallization using the solvent n-hexane.

Pure compound were obtained to test the melting point, solubility test, and

antioxidant test with DPPH method. Compounds obtained white needle-shaped

crystals with a melting point of 133-134C. Bases on GC-MS analysis of this

compound obtained molecular formula is C29H48O and that supported the data1H-

NMR and 13C-NMR. Antioxidant test using DPPH method showed that this

compound having IC50of more than 600 ppm. This indicates that these compound

inactive antioxidant.

1. INTRODUCTION

Antioxidants are compounds electron donor or reductant. This compound has a small

molecular weight, but is able to inactivate the development of the oxidation reaction with

radical binding. Related oxidation reactions in the body, antioxidant status is an important

parameter to monitor the health of a person. Antioxidant has gained importance in the current

scenario as it has an ability to trap free radicals which are produced during the degenerative

diseases. The human body has a system of antioxidants to counteract the reactivity of free

radicals, which are continuously formed by the body [1].

Several new compounds have been isolated from medicinal plants such as alkaloids,

terpenoids, flavonoids and xanthones which have potential antioxidant activity. One island in

Indonesia, Poteran island has some endemic plants are considered potential sources of

phenolic compounds as antioxidants. Several potentially productive plants as medicine are

Moringa (Moringa oleifera), Neem (Azadirachta indica), Saga (Adenanthera pavonima),

Breadfruit (Artocarpus altilis), Chilli herbs (Piper retrofactum, Vahl), Kesambi (Schleichera

aleosa), Pulai (Alstonia scholaris), and Pigeon pea (Cajanus cajan). In this research, selected a

plant that has potential as an antioxidant that Neem (A. indica). Its have been used

medicinally to treat various diseases such as fever, abdominal pain, itching, and a reduction in

blood sugar levels. Aerial parts of neem has been isolated more than 140 chemical compounds.

The compounds contained in the neem plant through research bioactivity in vitro and in vivostudies have demonstrated activity as an anti-inflammatory, anti-histamine, antipyretic, and

anti-fungal, but it is still rare to find compounds from neem potential as antioxidants [2].


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Research on neem root has not been done, so in this study will be conducted to determine the

activity of compounds the neem roots from Poteran-Madura Island. The secondary metabolites

of neem potentially containing compounds are expected to be potentially as an antioxidant

compound.

2. METHODS

2.1 Materials.

Plant Materials. Roots of A. indica were collected from Poteran Island-Madura,

Indonesia in March, 2014.

Instrumens and Chemicals. Melting points were determined on Fisher John-melting

point Apparatus. IR spectra were measured with FT-IR PRESTIGE 21 (SHIMADZU)

spectrophotometers, respectively. GC-MS spectra were measured with GC/MS Autosampler

Agilent Technologies, 5973 inert, Mass Selective Detector. 1H and 13C-NMR spectra were

recorded with JEOL-Nuclear Magnetic Resonance (JNM ECS-400), operating at 400.0 MHz

using standards from residual and deuterated solvent peaks, respectively. VLC was carried outusing Merck Si gel 60 GF254and TLC analysis on precoated Si gel plates (Merck Kieselgel 60

F254, 0.25 mm).

2.2 Procedures

Extraction and Isolation

The dried powdered of neem roots (5 kg) was macerated in MeOH and the MeOH extract (30

g) was sequentially fractionated by VLC Si-gel using n-hexane, dichloromethane (CH2Cl2),

ethyl acetate (EtOAc), and MeOH based on increasing polarity to give 7 fractions (I-O).

There are 2 fractions (L and M) which has relatively the same Rfvalue, so that the combined

(P = 4.5680 g) and further fractionated by VLC method using n-hexane : CH2Cl2based onincreasing polarity. Results from the second fractionation combined fractions obtained 4 (P 1-

P4) are monitored by TLC. At P3 fraction of precipitation, then filtered using vacuum

filtration, purified by recrystallization using n-hexane and P3cas compound 1(0.1303g) were

obtained.

DPPH free radical scavenging activity

The DPPH method was used to evaluated the antioxidant activity of the phenolic

compounds [7], [8]. The antioxidant activity of the compound 1 was determined in terms of

hydrogen donating or radical scavenging ability, using the stable radical DPPH. Samples at

various concentrations (600; 400; 200; 100; 50; 10 g/ml) were added to 1 ml of DPPH and 2

ml methanol and allowed to stand for 30 min at 37 C. The absorbance of the sample was

measured at 517 nm. All experiments were repeated three times. Radical scavenging activity

was expressed as the inhibition percentage of free radical by the sample and was calculated

using the formula [9].

.. (1)

Compound 1: white solid, melting point133-134C, 1H-NMR (CDCl3, 400MHz) and13C-

NMR (CDCl3, 400MHz) see Table1; GC-MS (m/z): 412[M +], 394, 369, 351, 314, 300,

271, 255, 213, 159, 133, 83, 55; IRmaxKBr (cm-1) : 3429, 2958, 1654, 1463, 1379, 1054.

3. RESULTS AND DISCUSSION


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Compound 1was isolated in the form of a white solid with a melting point of 133-134

C. MS data indicate that the compound has a molecular formula C 29H48O, so that the value of

DBE = 6. Then, it was supported by the data of IR, 1H-NMR and 13C-NMR, and compared

with data from previous studies. The IR signal absorption band observed at 3429 cm -1 is

characteristic of O-H stretching. Absorption at 2958 cm-1 is typical of cyclic olefinic

HC=CH- stretching while the absorption at frequencies such as 1654 cm-1is as a result of

C=C absorption. However, this signal is weak, the absorption at 1463 cm -1was assignable tocyclic (CH2)n bending absorption frequency. While the one at 1379 cm

-1is attributable to OH

deforming absorption. The absorption at 1054 cm-1is typical of cycloalkane moieties. These

absorption frequencies were in very close agreement with the ones observed for Stigmasterol

[3]. 1H-NMR spectra of compound 1(Table 1) showed the presence of two methyl singlets at

H0.78ppm (s, 3H) and 1.01ppm (s, 3H) ; three methyl doublet that appeared at H0.80 ppm

(d, 3H,J=6.8 Hz), 0.82 ppm (d, 3H,J=6.8 Hz), and 0.91ppm (d, 3H,J=6,4 Hz) ; and a methyl

triplet at H0.84 ppm (t, 3H,J=6.8 Hz). Compound 1also showed protons at H4.99, 5.14,

and 5.34 ppm suggesting the presence of three protons corresponding to that of a

trisubstituted and a disubstituted olefinic bond. The proton multiplet signal at H3.52 ppm

showed a hydroxy proton. The signal at H1,01 ppm (s, 3H) corresponds to C-28 and C-29

protons respectively of Stigmasterol [3].

Table 1. Data Comparation of 1H-NMR and 13C-NMR Compound 1with Stigmasterol [4]

in CDCl3

Position Stigmasterol Compound 1

H(ppm) C

(ppm)

H(ppm) C

(ppm)

1 - 37.6 - 37.3

2 - 32.1 - 31.8

3 3.51 (tdd, 1H, J= 4.5,4.2, 3.8 Hz)

72.1 3.52 m 71.8

4 - 42.4 - 42.3

5 - 141.1 - 140.8

6 5.31 (t, 1H, J= 6.1 Hz) 121.8 5.34 (t, 1H, J= 4.8 Hz) 121.8

7 - 31.8 - 31.9

8 - 31.8 - 31.9

9 - 50.2 - 51.3

10 - 36.6 - 36.6

11 - 21.5 - 21.2

12 - 39.9 - 39.8

13 - 42.4 - 42.4

14 - 56.8 - 56.815 - 24.4 - 24.4

16 - 29.3 - 28.3

17 - 56.2 - 56.1

18 - 40.6 - 40.6

19 0.91 (d, 3H, J= 6.2 Hz) 21.7 0.91 (d, 3H, J= 6.4 Hz) 21.3

20 4.98 (m, 1H) 138.7 4.99 (m, 1H) 138.4

21 5.14 (m, 1H) 129.6 5.14 (m, 1H) 129.3

22 - 46.1 - 45.9

23 - 25.4 - 25.5

24 0.83 (t, 3H, J= 7.1 Hz) 12.1 0.84 (t, 3H, J= 6.8 Hz) 11.9

25 - 29.6 - 31.9

26 0.82 (d, 3H, J= 6.6 Hz) 20.2 0.82 (d, 3H, J= 6.8 Hz) 21.3

27 0.80 (d, 3H, J= 6.6 Hz) 19.8 0.80 (d, 3H, J= 6.8 Hz) 19.5

28 0.71 (s, 3H) 18.9 0.68 (s, 3H) 18.9

29 1.03 (s, 3H) 12.2 1.01 (s, 3H) 12.1


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Then, the data is reinforced by the data 13C-NMR that the compound 1has a minimum of

29 carbon atoms. This is consistent with the data MS that the amount of carbon as much as

29. The 13C-NMR (Table 1) shows some recognizable signals at C140.8 and 121.8 ppm

which is assignable to the double bond at C-5 and C-6 [3], signal at C138.4 and 129.3 ppm is

also assignable to the double bond at C-20 and C-21 [4]. The signal at C71.8 ppm is due to

C-3 -hydroxyl group [3]. Again the signals observed at C18.9 and 12.1 ppm correspond to

angular carbon atoms at C-28 and C-29 respectively (Table 1). It had been reported that the

lower value for C-29 is attributable to -gauche interaction that increases the screening of C-

29 thereby causing lower chemical shift [3]. Likewise the loss of proton in C-6 results in

decrease in screening of C-28 leading to an increase in 13C chemical shift to higher frequency

[3]. This explanation is also accepted C18.9 and 12.1 ppm (for C-28 and C-29 respectively).

Carbon signals at C-28 more deshielding than C-29 due to the proton at C-6 is experiencing

dehydrogenation olefinic protons, so that it is more deshielding, while C-29 only have C12.1

ppm which is a methyl carbon. Then, 13C-NMR data of compound 1were compared with data

from the literature (Table 1) that it has a structural similarity with stigmasterol [4].

HO

1

3

10

5

6

7

28

29

9

8

14

13

12

11

16

17

18

1920

21 22

23 24

27

26

2

4

15

25

Compound 1: Stigmasterol

Thus, the structure of compound 1 was assigned as the known compound stigmasterol.

The physical and spectral data are consistent to the reported literature values of stigmasterol

[3], [4]. Antioxidant test using DPPH method showed that this compound having IC50 of more

than 600 ppm. This indicates that these compound inactive antioxidant, as a compound having

IC50of less than 200 ppm showed it has potent antioxidant activity [5]. When compared with

the antioxidant activity of vitamin C that have IC50values 6.316 ppm, the antioxidant activity

of these compound far exceeds the standards.

4. CONCLUSIONS

Phytochemical study of methanol extract of Neem (A.indica), a medicinal plant from Poteran

Island-Madura resulted to the isolation of known compound stigmasterol (1). This compound

was evaluated for its in vitroantioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH)

radical-scavenging have IC50 of more than 600 ppm. This indicated that these compound

inactive antioxidant.

5. REFERENCES

1. Winarsi, H., 2007, Natural Antioxidants and Free Radicals (Potential and Its

Application in Health), Kanisius, Yogyakarta.

2. Soegihardjo, C.J., 2007,Neem (Azadirachta indica A.Juss, Tribe Meliaceae), Multi Crop

Benefits Issues to Tackle Rakyat Indonesia, SIGMA, Yogjakarta, 10, 83-102.

3. Isah, Y., Ndukwe, I., and Amupitan, J.O., 2012, Isolation Of Stigmasterol From AerialPlant Part Of Spillanthes Acmella Murr, World J Life Sci. and Medical Research, 2 (2) :

77.


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4. Chaturvedula, V.S.P, and Prakash, I., 2012, Isolation Of Stigmasterol And -Sitosterol

From The Dichloromethane Extract Of Rubus Suavissimus, International Current

Pharmaceutical Journal, 1 (9): 239-242.

5. Blois, MS., 1958, Antioxidant determination by the use of a stable free radical, Nature

181, 1199-1200.

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