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Isolation of Phenolic Compounds of Patikan Kebo Leaves (Euphorbia Hirta. L)

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Isolation of Phenolic Compounds of Patikan

Kebo Leaves (Euphorbia Hirta. L)

Wisda Noviyanti Utami Lamek Marpaung

PG Student Lecturer

Department of Mathematics and Natural Science Department of Mathematics and Natural Science

Universitas Sumatera Utara Universitas Sumatera Utara

Sovia Lenny

Lecturer

Department of Mathematics and Natural Science Universitas Sumatera Utara

Abstract

Isolation and characterization of phenolic compound from methanol extract of Euphorbia hirta. L. has been conducted. The phenolic compound from methanol extracts isolated by several processes, such as TLC, column chromatography, and preparative TLC. The isolated phenolic compound is identified by UV-Vis spectrophotometer, FT-IR, 1H and 13C-NMR. The obtained phenolic compound is continued to the preparative TLC using chloroform: ethyl acetate (90:10, v/v) as eluent. The UV-Vis spectrum showed two peaks of maximum absorbance at 220 and 275 nm. Then, the FT-IR spectrum showed several peaks that confirmed the presence of functional group of derivative of phenolic compound, i.e. 3464, 1693, 1616, 1251, and 869 cm-1. 1H and 13C-NMR spectrum confirmed the isolated compound is derivative of phenolic compound, with data 3,81 (H-8, -OCH3) and 7,04 ppm (H-2, H-4 and H-6); 169 (-COO, C7), 146 (C3 and C5), 121 (C1), 110 (C2, C4, C6) and 52 ppm (-OCH3 C8), respectively. The E. hirta resulted in the isolation of the phenolic compound structure approached to derivative of gallic acid.

Keywords: Euphorbia hirta. L, Phenolic Compounds, Isolation, Spectroscopy

________________________________________________________________________________________________________

I. INTRODUCTION

Patikan kebo (E. hirta) is one of traditional medicinal plants from Indonesia. E. hirta is an herb vines which lives in tropical regions. E. hirta can be used as antiseptic, anti-inflammatory, antifungal, and antibacterial. Those activities are related to the secondary metabolite components, such as tannin, flavonoid (quercitrin and myricitrin), and triterpenoid (taraxerone and 11α, 12 α – oxidotaraxterol). In addition, there were also other active compounds such as alkaloids and polyphenols.

Ethanol extract of E. hirta has antibacterial activity against S. aureus, E. coli, P. aeruginosa, S. typhi, B. subtilis [7]. Other research also confirmed that activity against S. aureus and P. aeruginosawith MIC 2 mg/mL [6]. E. hirta component, triterpen, showed antibacterial and anticancer activities [8]. The ethyl acetate fraction of methanolic extract from E. hirta on fractionation afforded two known flavonol glycosides namely quercetrin and dimethoxyquercetrin, along with two new prenylated flavonosides designated as hirtacoumaroflavonoside and hirtaflavonoside-B [9]. The chemical constituents of the aerial part of E. hirta and the results nine compounds were isolated and identified as scopoletin, scoparone, isoscopoletin, quercetin, isorhamnetin, pinocembrin, kaempferol, luteolin, and gallic acid [10]. The research that focused on isolation of phenolic compound from E. hirta.

Phenolic compound are the most widely distributed secondary metabolites, ubiquitously present in the plant kingdom, even if the type of compound present varies according to the phylum under consideration. Phenolic are uncommon in vareis bacteris, fungi, and algae. Bryophytes are regular producers of polyphenols including flavonoids, but it is in the vascular plants that the full range of polyphenol is found [4].

Phenolic subtances, or polyphenols, contain numerous varieties of compounds: simple flavonoids, phenolic acids, complex flavonoid and colored anthocyanins. These phenolic compound are usually related to defense responses in the plant. However, phenolic metabolites play an important part in other processes, for instance incorporating attractive substance to accelerate pollination, coloring for camouflage and defense againt herbivores, as well as antibacterial and atifungal activities [3].

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(IJSTE/ Volume 4 / Issue 09 / 030)

II. MATERIALS AND METHODS

General

All the solvents and reagents used in this study were of analytical grade and were used without further purification. TLC was performed using silica gel 60 F254 (E. Merck). The remaining mentioned chemicals and solvents used in this experiment were obtained from Sigma–Aldrich. Uv-Vis spectroscopy Varian Cary 100 Conc was used to measure λmax, FT-IR Shimidzu Prestige (Japan) was used to determine the presence of functional group of phenolic compound.1H NMR and 13C NMR spectra were recorded on a Agilent (500 MHz and 125 MHz) instrument in CD3OD (purity 99.99%) with TMS (99.999%) as an internal standard (chemical shifts in δ ppm)

Plant Materials

The samples was collected from Nagan Raya Aceh, during the month of Maret 2017. The taxonomic identification of the plan was conducted at the Herbarium Madanense, University of Sumatera Utara.

Preparation of Crude Extract

The collected samples was washed thoroughly with water and cut into small pieces. The wet sample of E. hirta (300 g) were extracted for 24 h with methanol (4 L). The methanol was evaporated from the methanol crude extract using a rotary evaporator at 60oC under reduced pressure to get the crude extract. The methanol free crude extract (70 g) was suspended in ethyl acetate to remove tannin from extract. The ethyl acetate was evaporated using a rotary evaporator at 60 oC. The crude extract (10 g) was fractionated with n-hexane and methanol. The phenolic compound was assumed to be present in the methanol fraction (5 g).

Isolation of Pure Compound

Gravitational elution of methanol extract in silica gel column with chloroform: ethyl acetate in increasing amounts of methanol gave two fractions; fractions 2-1-1 and 2-1-2. Fraction 2-1-1 was continued for further purification using chloroform:ethyl acetate (90:10 v/v) in preparative TLC and giving greenish pictorial paste (2-1-1).

III. RESULTS AND DISCUSSION

Preparative was conducted to obtain pure compounds using eluent of chloroform:ethyl acetate (90:10 v/v) and 2-1-1 as pure fractions were obtain. The other data, FT-IR spectrum of the phenolic compound (2-1-1) is shown in Figure 3. Fig. 3 showed several band that indicated the presence of phenolic compound, such as at 3464 cm-1 ascribed for the appearance of –OH group. The other peak is observed at 1693 dan 1251 that indicated the present of ester group (C=O dan C-O). The confirmation of phenolic compound is declared by the presence of C=C aromatic band at 1616, 1514 and 869 cm-1. The results of FT-IR of phenolic compound (2-1-1) is shown similar result to gallic acid spectra [1].

Fig. 1: The FT-IR Spectrum results for phenolic compound 500 750 1000 1250 1500 1750 2000 2500 3000 3500 4000 4500 1/cm 20 30 40 50 60 70 80 90 100 %T 34 64 .1 5 29 54 .9 5 29 20 .2 3 28 50 .7 9 16 93 .5

0 1616.3

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UV-Vis spectrum of the phenolic compound that diluted in methanol is shown in Figure 2.

There are two peak that was observed, 220 and 275 nm, in Figure 2. Those maximum absorptions described the presence of n→σ* and n→π* transition, respectively. The trasition of n→σ* is ascribed to the appearance of C-O group in the phenolic compound and C=O group for n→π* transition. The results of UV-Vis of phenolic compound (2-1-1) is shown similar result to gallic acid spectra [2].

Fig. 2: The UV-Visible Spectrum of phenolic compound

The 1H-NMR spectrum of the phenolic compound that diluted in methanol is shown in Figure 3. In the 1H-NMR spectrum, all proton signal showed one singlet. The first signal at δ 7.04 ppm indicated the presence of three aromatic proton (2, 4 and H-6). The last signal is appeared at δ 3.81 ppm ascribed the presence proton of methoxy group –OCH3 (H-8). To support the elucidation structure of phenolic compound (2-1-1) of E. hirta, the analysis of 13C-NMR was also conducted (Figure 4).

The presence of aromatic ring in the phenolic compound was supported with the presence of several signal of 13C-NMR at δ 146 (C3 dan C5), δ 121 (C1) and δ 110 (C2, C4, C6). The present of methoxy proton in 1H-NMR is support with the presence of methoxy carbon signal at 52 (C8). In 13C-NMR, also showed the presence of carbonyl group in the phenolic compound at δ 169 ppm (C7). The presence of carbonyl and methoxy signal in the 13C-NMR leads to the ester group according to the structural identification of derivative of gallic acid, methyl gallate [5].

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(IJSTE/ Volume 4 / Issue 09 / 030)

Fig. 4: 13C-NMR Spectrum of the phenolic compound

According to the result of elucidation structure of phenolic compound of E. hirta from UV-Vis, FT-IR, 1H-NMR and 13C-NMR data, the phenolic compound structure approached to derivative of gallic acid structure with ester group (Figure 6).

Fig. 6: Proposed structure of gallic acid derivative structure

IV. CONCLUSION

The isolated phenolic compound (2-1-1) from E. hirta has green color (greenish pictorial paste) and its structure approached to the gallic acid derivative with ester group that obtained from the analysis of UV-Vis, FT-IR, 1H-NMR and 13C-NMR data.

REFERENCES

[1] Banday, J.A., Mi, F., Farooq, S., Qurishi, M.A., Koul, S., Razdan, T.K. (2012) “ Salicylic Acid and Methyl Gallate from the Roots of Conyza canedensis”

J&K India.

[2] Chanwitheesuk, A., Teerawutgulrag, A., Kilburn, J.D., Rakariyatham, N. (2007) “Antimicrobial Gallic Acid from Caesalpinia mimosoides Lamk” Food

Chemistry 100 : 1044-1048.

[3] Derong, L., Xiao, M., Zhao, J., Li, Z., Xing, B., Li, X., Kong, M., Li, L., Zhang, Q., Liu, Y., Chen, H., Qin, W., Wu, H., Chen, S. (2016) “Review An Overview of Plant Phenolic Compounds and Their Importance in Human Nutrition and Management of Type 2 Diabetes” Molecules 21 : 1374. Doi: 10.3390/molecules21101374.

[4] Latanzio, V. (2013) “Phenolic Cpmpound : Introduction” Springer-Verlag Berlin Heidelberg. Doi: 10.1007/978-3-642-22144-6_57.

[5] Ma, X., Wu, L., Tian, W. (2005) “Application of Preparative High-Speed Counter-Current Chromatography for Separation of Methyl Gallate from Acer

truncatum Bunge” Journal of Chromatography A : 212-215.

[6] Ngemenya, M.N., Mbah, J.A., Tane, P., Titanji, V.P.K. (2006) “Antibacterial Effects of Some Cameroonian Medicinal Plants Against Common Pathogenic

Bacteria” African Journal of Traditional, Complementary and Alternative Medicines. 3(2): 84-93. C

OH HO

1

2

3

4 5 6

7

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[7] Ogbulie, J.N., Ogueke, C.C., Okoli, I.C., Anyanwu, B. (2007) “Antibacterial Activities and Toxicological Potentials of Crude Ethanolic Extracts of Euphorbia hirta” African Journal of Biotechnology. 6(13): 1544-1548.

[8] Ragasa, C. Y., Cornelio, K. B. (2013) “Triterpenes from Euphorbia hirta and Their Cytotoxicity” Chinese Journal of Natural Medicines, 11(5) : 0528-0533.

[9] Sheliya, M. A., Rayhana, B., Ali, A., Pillai, K. K., Aeri, V., Sharma, M., Showkat, R. M. (2015) “Inhibition of α-glucosidase by New Prenylated Flavonoids

from Euphorbia hirta L. Herb” Journal of Ethnopharmacology, 176 : 1-8.

Figure

Fig. 1: The FT-IR Spectrum results for phenolic compound
Fig. 2: The UV-Visible Spectrum of phenolic compound
Fig. 4: 13C-NMR Spectrum of the phenolic compound

References

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