Antioxidant activity and chemical constituents of some

Research Article

Antioxidant activity and chemical constituents of some Indonesian fruit peels

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Antioxidant activity and chemical constituents of some Indonesian fruit peels Muhtadi1, Haryoto1, Tanti Azizah Sujono1, Peni Indrayudha1, Andi Suhendi1 and Khong Heng Yen 2 1Faculty of Pharmacy, Muhammadiyah University of Surakarta, Jl. Achmad Yani Tromol Pos 1, Pabelan Kartasura, Surakarta 57102, Indonesia 2School of Chemistry nd Environtmental Studies, Faculty of Applied Sciences, University Teknologi MARA, 94300 Kota Samarahan, Sarawak, Malaysia

ABSTRACT The ethanolic extracts and their fractions of some Indonesian fruit peels were screened for their free radical scavenging properties using vitamin-E as standard antioxidant. Free radical scavenging activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. The strongest antioxidant activity was shown by the Rambutan (Nephelium lappaceum Linn.), followed by Kelengkeng (Euphoria longan Lour. Steud) and Durian (Durio zibethinus Murr.) with IC50 values of 7.74, 11.85, and 28.83 µg/mL, respectively comparable to vitaminE (IC50 = 8.48 ± 0.1 µg/mL). The ethyl acetate fraction of these fruit peels extracts demonstrated the most active antioxidant activity compared with hexane, chloroform and methanol fractions. The chemical constituents of Rambutan and Durian were successfully isolated using vacuum liquid chromatography and radial chromatography technique, and these structures were characterizated based on the proton (1H) and carbon (13C) NMR spectra. The isolated compounds were ethyl gallate (1) from Rambutan (Nephelium lappaceum Linn.) rind, 4,4-dimethylporiferasta-18(19)-en-3-ol (2) and 3α-E-ferulyloxy-lup-20(29)-en-28-oic acid (3) from Durian (Durio zibethinus Mur r.) peels. The results showed that prospective antioxidant contents in Rambutan’s fruit peel extract was higher than Kelengkeng and Durian fruit peel extracts. [Medicinal Plants 2014; 6(1) : 00-00] Keywords : Antioxidant activity, fruit peel, Rambutan (Nephelium lappaceum Linn.), Kelengkeng (Euphoria longan Lour. Steud), Durian (Durio zibethinus Murr.)

INTRODUCTION Indonesia is one of the country in southeast Asia which is rich with tropical fruits, besides Malaysia, Myanmar and Thailand (Chomchalow et al., 2008; UN ESCAP, 2007). Rambutan ( Nephelium lappaceum Linn), Kelengkeng (Euphoria longan Lour. Steud) and Durian (Durio zibethinus Murr.) are the main fruits commonly found in Indonesia. Unfortunately, not all the parts of

Corresponding author : Muhtadi e-mail : [email protected] Received : ; Accepted : doi: 10.5958/j.0975-6892.5.2.009

Medicinal Plants, 6(1) March 2014

these fruits are used as the valued materials, instead their peels are still regarded as waste. Previous chemical studies showed that these fruit peels exhibit bioactive compounds. For instance, the Mangosteen peel was proven rich in a-mangostin and xanthone derivatives, in which antioxidant potential are identif ied (Jung et al., 2006; Jose et al, 2008). Furthermore, our previous study has also shown that the extract of Euphoria longan peels contains a potent substance as antioxidant and antibacterial activity (Muhtadi et al., 2013). The present study has been undertaken to assess the antioxidant activity and chemical constituents of three Indonesian fruit peels, viz., Rambutan (Nephelium lappaceum Linn), Kelengkeng (Euphoria longan Lour. Steud) and Durian (Durio zibethinus Murr.).

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MATERIAL AND METHODS Plant Material The fruit peel samples of Nephelium lappaceum , Euphoria longan and Durio zibethinus were collected from local area of Surakarta, Central of Java, Indonesia. Herbarium voucher specimens were prepared and deposited in the Herbarium of Pharmacy Biology at Faculty of Phar macy, Muhammadiyah University of Surakarta, Indonesia. Spectral Studies Infrared spectra were measured using the universal attenuated total reflection (UATR) technique on a Perkin-Elmer 100 series. NMR spectra were obtained using JEOL 300MHz FT NMR spectrometer using tetramethylsilane (TMS) as internal standard. Ultra violet spectra were recorded in acetone on a Shimadzu UV-160°, UV-Visible Recording Spectrophotometer. Extraction and isolation The samples were air-dried and ground to powder and extracted with ethanol for 3 X 24 hours at room temperature. The extracts were fractionated with the par tition method, using n-hexane, chlorofor m, ethyl acetate, and methanol-water solvents. The extracts and their fractions were dried under reduced pressure and stored at 4ºC until used. The ethanol extract of Durian peels (350.0 mg) was fractionated and purif ied using a stepwise gradient system (SiO 2, hexane/chloroform, started 3.5:6.5) on radial chromatography technique to give 23 fractions. Fractions 7-10 were combined and characterized 3α-E-ferulyloxy-lup-20(29)-en-28-oic acid (3) (6 mg), and from fractions 17-19 were combined and characterized 4,4-dimethyl-poriferasta-18(19)-en-3ol (2) (2 mg). On other hand, the ethanol extract of Ramb utan peels (422.0 mg) over a silica radial chromatography (chloroform/hexane, chloroform/ethyl acetate and chloroform/methanol gradient) provided 22 fractions. Fractions 14-19 were combined and characterized as ethyl gallate (1) (30 mg). The details of fractions having characterstics of ethyl gallate, 4,4-dimethyl-poriferasta-18(19)-en-3-ol and 3α-E- ferulyloxy-lup-20(29)-en-28-oic acid are listed below: The Ethyl gallate (1) White amor phous powder UV λ max 215, 273 nm (methanol); 1H-NMR (300 MHz; aceton-d 6): δ 7.13 (s, 2H, H-2/6), 4.26 (q, J = 7.1 Hz, 2H, H-8), 1.32 (t, J =

7.1 Hz, 8H, H-9); 13C NMR (75 MHz; acetone): δ 165.8 (C-7), 145.16 (C-5), 145.11 (C-3), 137.8 (C-4), 121.2 (C-1), 108.8 (C-2/C-6), 60.0 (C-8), 13.7 (C-9). 4,4-dimethyl-poriferasta-18(19)-en-3-ol (2) White amor phous powder UV λ max 215, 273 nm (methanol); 1H-NMR (300 MHz; aceton-d6): δ 5.24 (m, 1H, H-19), 1.14 (s, 3H, H-28), 1.00 (s, 3H, H-25), 0.97 (d, J = 7.5 Hz, 3H, H-29), 0.96 (s, 3H, H-24), 0.91 (t, J = 7.5 Hz, 3H, H-23), 0.85 (s, 3H, C-27), 0.79 (s, 3H, H26). 13C NMR (75 MHz; acetone): δ 135.9 (C-18), 126.2 (C-19), 78.6 (C-3), 57.0 (C-5), 56.2 (C-21), 53.9 (C-9),48.5 (C-14),46.8 (C-13),42.9 (C-10), 39.9 (C-17), 39.8 (C-8), 39.5 (C-20), 39.3 (C-4), 37.6 (C-12), 34.0 (C-1), 31.4 (C-11),29.2 (C-15), 28.8 (C-2), 28.7(C-25), 28.1 (C-16), 25.0 (C-7), 24.1 (C-22), 24.0 (C-28), 21.5 (C-29), 19.1 (C-6), 17.7 (C-27), 17.5 (C-23), 16.4 (C24), 15.9 (C-26). 3α-E-ferulyloxy-lup-20(29)-en-28-oic acid (3) White amor phous powder UV λ max 215, 273 nm (methanol); 1H-NMR (300 MHz; aceton-d 6): δ 8.23 (s, 1H), 7.60 (d, J = 15.8 Hz, 1H. H-2’), 7.34 (d, J = 1.8 Hz, 1H, H-9’), 7.18 (dd, J = 8.2, 1.9 Hz, 1H, H-5’), 6.88 (d, J = 8.2 Hz, 1H, H-8’), 6.71 (d, J = 15.8 Hz, 1H, H-3’), 4.89 (m, 1H, H-3), 4.71 (s, 1H, H-29a), 4.59 (s, 1H, H29b), 3.92 (s, 3H, OCH3), 1.70 (s, 3H, H-30), 1.28 (s, 3H, H-27), 1.01 (s, 3H, H-26), 0.95 (s, 3H, H-23), 0.85 (s, 3H, H-24), 0.75 (s, 3H, H-25). 13C NMR (75 MHz; acetone): δ 184.5 (C-28), 177.6 (C-1’), 151.6 (C-20/C7’), 150.1 (C-6’), 141.4 (C-3’), 128.1 (C-4’), 123.8 (C9’), 122.2 (C-8’), 116.3 (C-2’), 111.6 (C-5’), 110.0 (C29), 78.6 (OCH3), 56.8 (C-17), 56.3 (C-9), 51.4 (C-19), 49.9 (C-18), 47.9 (C-18), 43.2 (C-14), 41.5 (C-8), 39.60 (C-4), 39.56 (C-1), 39.0 (C-13), 38.0 (C-10), 37.5 (C22), 35.2 (C-16), 32.8 (C-15), 31.3 (C-21), 30.4 (C-2), 28.6 (C-23), 28.2 (C-12), 26.4 (C-7), 21.7 (C-11), 19.5 (C-30), 19.1 (C-6), 16.62 (C-24), 16.53 (C-25), 16.1 (C26), 15.0 (C-27). Antioxidant Activity The extracts and their fractions were evaluated for the antioxidant activity using the 1,1-diphenyl-2picrylhydrazyl (DPPH) as described by Loo et al., (2008), with minor modif ications. Each sample of stock solution (1.0 mg/mL) was diluted to a f inal concentration of 1000, 500, 250, 125, 62.5, 31.3, 15.6, and 7.8 µg/mL. Then, a total of 3.8 mL of 50 µM DPPH methanolic solution (1 mg/50 mL) was added to 0.2 mL of each sample solution and allowed to react at room temperature for 30 min. The absobance of the


Antioxidant activity and chemical constituents of some Indonesian fruit peels

mixtures was measured at 517 nm. A control was prepared without sample or standard and measured immediately at 0 min. The precent inhibition (I %) of DPPH radical were calculated as follows: I % =

Ablank – Asample A blank

× 100

where Ablank is the absorbance value of the control reaction (containing all reagents except the test sample) and Asample is the absorbance value of the test samples. The sample concentration provide 50% inhibition (IC50) was calculated by plotting inhibition percentages against concentrations of the sample. All tests were carried out in triplicate and IC50 value were reported as means ± SD of triplicates. RESULTS AND DISCUSSION Antioxidant activity The ethanolic extract and their fractions of fruit preels of Rambutan, Kelengkeng and Durian were evaluated for their antioxidant properties by DPPH radical scavenging (Table 1). It has been reported that free

Table 1. Antioxidant activity of some Indonesian fruit peelsa Sample

DPPH IC 50 (mg/mL)

Ethanol extract of Kelengkeng fruit peels

11.85 ± 0.96

Hexane fraction of Kelengkeng fruit peels

374.81 ± 9.73

Ethyl acetate fraction of Kelengkeng fruit peels

The isolated chemical constituents One chemical constituent namely ethyl gallate (1) had been isolated from Rambutan and two compounds from Durian, namely 4,4-dimethyl-poriferasta-18(19)-en-3-ol (2) and 3α-E-ferulyloxy-lup-20(29)-en-28-oic acid (3). These compounds (Fig. 1) were elucidated based on their proton and carbon NMR spectra, and the structure of the ethyl gallate (1) was conf irmed with reference spectral data (Mahajan and Pai, 2010), 4,4-dimethylporiferasta-18(19)-en-3-ol (2) with the Ersoz’s NMR spectral data (Ersoz et al., 2002) and 3α-E-ferulyloxylup-20(29)-en-28-oic acid (3) with Khumar and Sharma’s spectral data (Khumar and Sharma, 2006). The ethyl gallate (1) has been reported to exhibit good antioxidant properties (Romero et al., 2010; Kubo et al., 2010; Bonacorsi et al., 2011), antiviral (Juliana, 2006), and induces apoptosis of HL-60 Cells (Kim et

HO O

42.89 ± 1.91

Ethanol extract of Durian fruit peels

28.83 ± 0.48

Chloroform fraction of Durian fruit peels

32.81 ± 2.64

Ethyl acetate fraction of Durian fruit peels

14.91 ± 3.23

HO OCH2CH3

HO

HO

Ethanol extract of Rambutan fruit peels

7.74 ± 0.76

Chloroform fraction of Rambutan fruit peels

6.64 ± 1.14

Ethyl acetate fraction of Rambutan fruit peels

4.29 ± 0.72

Methanol-water fraction of Rambutan fruit peels

8.34 ± 0.59

Vitamin E as standard

8.48 ± 0.77

Ethyl gallate (1)

4,4-dimethyl-poriferasta-18(19)-en-3-ol (2)

O

O

OH

O

HO OCH3

represent meand ± standard deviation of three independent experiment. Medicinal Plants, 6(1) March 2014

radical scavenging activity is greatly influenced by the phenolic component of samples (Cheung et al., 2003). In the present study, ethanolic extract and their fractions of Rambutan fruit peels demonstrated more activity than vitamin-E and other extract and fractions. This indicated abundance of phenolic compounds and prospective antioxidant ingredients in the Rambutan fruit peels compared to Durian and Kelengkeng. All of the ethyl acetate fractions were more active than the crude extract and their fraction, which showed that the phenolic compounds were readily soluble in ethyl acetate solvent.

9.23 ± 0.15

Methanol-water fraction of Kelengkeng fruit peels

aData

3

3-E-ferulyloxy-lup-20(29)-en-28-oic acid (3)

Fig. 1. The chemical structure of the isolated compounds from Rambutan and Durian fruit peels

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al., 2012). In addition, Shabana et al. (2013) also reported that the sterol glycosides from fruit peels of Solanum melongena L. have a promising anticancer activity against hepatocellular carcinoma. The sterol glycosides has similar skeleton as that of 4, 4-dimethylporiferasta-18(19)-en-3-ol (2) and therefore, compound 2 is expected to have cytotoxic effect against cancer cell lines whereas 3α-E-ferulyloxy-lup-20(29)-en-28-oic acid (3), is one of the alkyl ferulate derivative which is expected to be antioxidative (Kikuzaki et al., 2002). CONCLUSION The bioassay studies showed that the peels of Rambutan (Nephelium lappaceum Linn), Kelengkeng (Euphoria longan Lour. Steud) and Durian (Durio zibethinus Murr.) exhibited very potent anti-oxidative properties. Rambutan ( Nephelium lappaceum Linn) peels demonstrated the highest antioxidant acitivity, comparable with vitamin-E. In addition, the ethyl gallate was succesfully isolated from Rambutan fruit peels extract whereas 4,4-dimethyl-poriferasta-18(19)en-3-ol and 3α-E-ferulyloxy-lup-20(29)-en-28-oic acid were isolated from Durian fruit peels extract. ACKNOWLEDGEMENT The authors wish to thank Muhammadiyah University of Surakarta and Ministry of Education and Cultural Republic, Indonesia for Excellent Research Grant Scheme for f inancial support. The authors also wish to thank the staff of Phar macy Biology Laborator y, Universitas Muhammadiyah Surakarta for their assistance in the determination of the samples and Universiti Teknologi MARA Sarawak for laboratory facilities. REFERENCES Bonacorsi C, Fonseca LM, Raddi MSG, Kitagawa RR, Sannomiya M and Vilegas W (2011). Relative antioxidant activity of Brazilian medicinal plants for gastrointestinal diseases. Journal of Medicinal Plants Research, 5(18): 4511-4518. Chaver ri JP, Rodríguez NC, Ibarra MO and Rojas JMP (2008). Review Medicinal properties of mangosteen (Garcinia mangostana), Food and Chemical Toxicology, 46: 32273239.

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