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CHEMICAL CONSTITUENTS AND ANTIOXIDANT ACTIVITY OF Salix tetrasperma ROXB

Six (1-6) and two (7-8) compounds were obtained from ethyl acetate extract of S. tetrasperma Roxb bark and leaves, respectively. Structures of isolated compounds were determined by spectroscopic data, including H NMR, C NMR, and two dimensional NMR as β-sitosterol glucoside (1), 3,4-dihydroxybenzoic acid (2), 2(hydroxymethyl)phenol (3), 1,3-dihydroxyphenol (4), 3-(hydroxymethyl)phenol (5), 4-methylbenzaldehyde (6), 1,2dihydroxybenzene (7) and 3’,4’,5,7-tetrahydroxyflavone (8). Interestingly, compounds 2, 4, 5, 6, and 8 were reported for the first time isolated from this plant. The antioxidant activities of all crude extracts were evaluated by the diphenylpicrilhydrazyl (DPPH) method. Among all extracts, methanol extract of the bark has the highest antioxidant activity with an IC50 value of 6.85 μg/mL. The activity of ethyl acetate extract of the root (IC50 19.53 μg/mL) was stronger than the bark (IC50 22.69 μg/mL).


INTRODUCTION
Salix tetrasperma Roxb belongs to the Salicaceae family. This plant is commonly called "dalu-dalu" in West Sumatera, Indonesia and has been used as a traditional medicine to treat coughs and skin diseases as well as to reduce fever. The pharmacological activities of this plant showed that the bark extract of this plant possessed hypoglycemic activity 1 , while the leaf extract showed antimicrobial 2 , antipyretic 3 , analgesic, and anti-inflammatory activities. 4 The previous phytochemical work on this genus revealed that most species contain numerous phenolic compounds. According to our previous study, the polyphenolic compounds showed good biological activities. [5][6] Salicin glycosides from S. glandulosa showed antineuroinflammatory activity. 7 Recently the salicin derivatives from S. acmophylla were reported as cytotoxic and anti-inflammatory. 8 Simple phenolic compounds from S. capensis bark had the antibacterial activity to P. aerugino. 9 Only a few phytochemical studies have been reported on S. tetrasperma Roxb. 4,[10][11] The phytochemical composition of Salix from different regions may vary, due to the environment of the growing plant. However, the information on chemical constituents from S. tetrasperma Roxb growing in Indonesia is still limited. Due to a large number of phenolic compounds that were reported from this genus, the authors are also interested to evaluate the antioxidant activity from various parts of this plant. Hence, this study aims to isolate secondary metabolite compounds from ethyl acetate extract of S. tetrasperma Roxb bark and leaves and to investigate the antioxidant activity of nhexane, ethyl acetate, and methanol extracts various parts of S. tetrasperma Roxb.

EXPERIMENTAL Material and Methods
All solvents were purchased from the suppliers with further purification before usage. Silica gel for column chromatography was performed on silica gel 60 (Merck). TLC plates pre-coated with silica gel 70 F 254 (60-200 mesh) were purchased from Wako Pure Chemical Industries, Japan. TLC spots were detected under the UV lamp (254 and 365 nm). 1 H, 13 C, HMQC, HMBC, and 1 H-1 H COSY NMR spectra were recorded with JEOL JNM-ECS 400, 500, and 600 spectrometers using TMS as an internal standard. Twodimensional 1 H-1 H COSY, HMQC, and HMBC correlations were made by using standard pulse sequences.

Plant Material
Leaves, root, and bark of S. tetrasperma Roxb were collected from the surrounding of Singkarak Lake, West Sumatera Province, Indonesia in May 2015. The plant was identified by a taxonomist at Herbarium of Andalas University (ANDA), Padang, Indonesia.

Antioxidant Activity
The antioxidant activities of various S. tetrasperma Roxb extracts were determined by using DPPH radical scavenged method 12 with slight modification. 3 mL of each solution [in various concentrations] of tested samples (n-hexane, EtOAc, and MeOH extracts of the leaves, roots, and bark) was poured into 1 mL of DPPH solution [0.1 mmol/L]. The mixture was incubated for 30 minutes at room temperature in the dark place and then recorded by UV-spectrophotometer at 517 nm. As a comparison, the ascorbic acid and methanol were used as a standard and a blank, respectively. The antioxidant activity was calculated based on the percentage of DPPH radical scavenged (% Inhibition) using the following equation: Where A control is the absorbance of control containing all reagents that exclude the sample, and A sample is the absorbance of the sample. The measurements were taken in triplicate. The antioxidant results were expressed as the IC 50 value ± standard deviation (SD).

Characterization of Isolated Compounds
After separation by chromatography technique, six (l-6) and two (7-8) compounds were isolated from EtOAc extract of S. tetrasperma Roxb bark and leaves, respectively. The structures of isolated compounds were displayed in Fig.-  Based on H and C NMR spectral data, and compared to related literature, compound 1 was identified as β-sitosterol glucoside. [13][14][15] Compound 2 was obtained as a yellow crystal (8 mg ) and it also had orthocoupling with the proton at δ H 6.77 ppm (J = 8.2 Hz), that assigned trisubstitution at 1, 3 and 4 positions in the aromatic ring. A broad singlet signal in the most downfield region (δ H 12.28 ppm) assigned the occurrence of carboxylic acid bound to the aromatic ring of 2. By H and C NMR analyses and comparison with the previously reported data, the chemical structure of 2 was determined as 3,4dihydroxybenzoic acid or protocatechuic acid. [16][17][18] Compound 3 was isolated as an orange crystal (6 mg). The H NMR of 3 displayed four aromatic protons and an oxidized methylene proton (δ H 4.71 ppm). The most downfield proton at δ H 7.21 ppm revealed ortho-coupling with the proton at δ H 6.78 ppm (J = 7.32 Hz), while the proton at δ H 7.07 ppm as a triplet also showed ortho-coupling with the protons at δ H 6.78 and 6.77 ppm (J = 8.24 Hz). The C NMR indicated seven carbon signals including six aromatic carbons at δ C 155.36 (C-1), 127.99 (C-3), 127.53 (C-5), 127.45 (C-2), 119.32 (C-4), 115.17 ppm (C-6) and one oxidized methylene carbon (δ C 61.02 ppm) bound to aromatic carbon, respectively. Based on the comparison of H and C NMR data with the literature, the structure of compound 3 was assigned as 2-(hydroxymethyl)phenol or salicyl alcohol. 19 Compound 4 was acquired as a white pale crystal (4 mg). The H NMR spectrum showed three aromatic protons. While, four signal aromatic carbons (δ C 135.9, 130.45, 119, and 117 ppm) appeared in the C NMR spectrum. The proton at δ H 7.51 ppm (td, 1H) had ortho-coupled with two protons at δ H 6.91 ppm (dd, J = 8 Hz) and also had para-coupling with a proton at δ H 7.87 ppm (dd, J = 1.84 Hz). It is predicted that the aromatic ring had two hydroxyl groups at 1, 3-position. Based on the comparing spectral data of 4 with literature, it was identified as 1, 3-dihydroxyphenol or resorcinol. 20 Compound 5 was obtained as an orange crystal (10 mg . This compound assigned two substituents bound to the aromatic ring. It was suggested that the hydroxyl and hydroxymethyl groups were separately attached to carbon at δ C 156.14 (C-1) and δ C 129.61 (C-3). Compared to the spectral data of 5 with the literature, the chemical structure of 5 was determined as 3-(hydroxymethyl) phenol. [20][21] Compound 6 was isolated as a brownish crystal (3 mg showed long-range correlations to C-2 (δ C 164.65 ppm), C-4' (δ C 149.64 ppm), C-3' (δ C 145.70 ppm), and C-1' (δ C 122.33 ppm) suggested that two hydroxyl groups are attached at C-3' and C-4'. A remaining proton as a singlet at δ H 6.53 ppm exhibited long-range correlations to C-10 (δ C 103.96 ppm), and C-1' (δ C 122.33 ppm), indicated that proton is attached to the C-3 position of C ring. According to the HMBC spectrum, a hydroxyl group was suggested bound at C-5, C-7, C-3', and C-4' positions of 8. In comparison, the NMR data of 8 with the related literature data, [24][25] compound 8 was confirmed as 3',4',5,7-tetrahydroxyflavone or luteolin.

Antioxidant Activity
The DPPH radical scavenging results of S. tetrasperma Roxb barks, root, and leaves extracts were assayed as described in Materials and Methods. The highest antioxidant scavenging among the nine different extracts was in the MeOH extract of the bark, while the minimum was found in the n-hexane extract of the root (  The number of electrons taken up is very important for antioxidant scavenging activity. This is due to the ability of hydrogen donors from polyphenol compounds and also the correlation with the total flavonoid content contained in the extract. [26][27] Our phytochemical investigation showed that the presence of phenolic and flavonoid in MeOH and EtOAc extract of the bark and leaves of S. tetrasperma Roxb might contribute to the high antioxidant activity. Thus, this plant can be considered as a good natural antioxidant source.