Identification and Bioactivity Studies of Flavonoid Compounds from Macaranga hispida (Blume) Mull.Arg

Two flavonoid compounds, 5,7,3’,4’-tetrahydroxy-6-g eranylflavonol (1) and kaempferol 7-Oβ-glucose (2) have been isolated from the leaves of Macaranga hispida (Blume), Mull.Arg. Isolation and purification were conducted by chromatography methods and chemical structure chara terization was carried out by spectroscopic method s. The 5,7,3’,4’-tetrahydrxyi-6-geranyl flavonol (1) and k aempferol 7-O-glucose (2) had moderate cytotoxic ac tivity against murine leukemia P-388 cell lines with IC50 value of 0.22 and 101.5 μg/mL, respectively. The IC 50 for antioxidant activities of (1) and (2) were 2.83 and 13.95 μg/mL, respectively. The LC 50 of (1) and (2) from BSLT were 350 and >1000 μg/mL, respectively.


Introduction
Macaranga hispida (Blume) Mull. Arg, known locally as Mahang (Indonesia) is one of approximately 300 species of the Macaranga (Euphorbiaceae) genus, It was widespread growth, from Africa and Madagascar, to the Asian tropic region, North Australia and the Pacific Islands [1]. As part of our study about natural products, drug discovery, including exploration of potential bioactivity of plants originating from Indonesia, the chemical content of the ethyl acetate and butanol fraction obtained from partitioning methanol extract of M. hispida was explored using the column chromatography method.
A Previous study reported that flavonoid and terpenoid are the major components of the Macaranga genus [2][3][4][5][6][7][8][9]. Apigenin, isolated from Portulaca. oleracea L and the bioactive compound also had potential antibacterial activity indicating that it can be used for development of antibacterial drugs for the treatment of diseases associated with these pathogenic bacteria [10]. Meanwhile, flavonoid glycoside compounds have been found in abundance in plants (flowers, fruits, vegetables, bean and tea) [11][12][13].
However, there are no publication established about chemical constituents of M. hispida plant. In this paper, we describe the structure elucidation and cytotoxic properties of flavonoid compounds isolated from the methanol extract of the leaves of M. hispida.

Materials and Methods
General. The 1 H-and 13 C-NMR spectra were recorded with a JEOL JNM-ECA 500 spectrometer instrument with CD 3 OD as a solvent and TMS as an internal Extraction and isolation. Powdered dried leaves of M. hispida (2.15 kg) were macerated in methanol. The methanol extract was evaporated to obtain 220 g of crude methanol extract. The methanol extract was partitioned with n-hexane, ethyl acetate (EtOAc), and butanol (BuOH), successively. The EtOAc soluble fraction (20 g) was chromatographed over a silica gel column and eluted successively with a gradient solvent system of n-hexane:EtOAc to obtain nine fractions (F1-F9). Compound 1 (40 mg) was crystallized from fraction 5 (F5) after it was evaporated and re-diluted with EtOAc and further purified with CHCl 3 , acetone and MeOH to obtained a pure compound. The butanol soluble fraction (20 g) was chromatographed over a silica gel column and eluted successively with a gradient solvent system of n-hexane:EtOAc to obtained five fractions (F1-F5). Compound 2 (20 mg) was purified from F4 by column chromathography with CHCl 3 , acetone and MeOH eluents to obtained a pure compound. Antioxidant activity [14]. Antioxidant analysis was conducted using "DPPH free scavenging activity" method with a slight modification. Various concentrations of the M. hispida extract/fractions in 0.8 ml methanol were mixed with 0.2 mL of methanolic solution containing 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, resulting a final concentration of the DPPH of 0.2 mM and sample concentrations up to 100 µg/mL. The mixture was shaken vigorously and left to stand for 30 min at room temperature, the absorbance was then measured using a spectrophotometer at 515 nm. The percentage of inhibition (free radical scavenging activity) was calculated by the equation: [1 -(B/A)] x 100%; whereas A is absorbance in the absence of a sample and B is absorbance in the presence of a sample. The IC 50 value denoted the concentration of the sample required to scavenge 50% of the DPPH free radicals.
BSLT activity [15,16]. Brine shrimp eggs (Artemia salina, Sanders TM Great Salt Lake, Brine Shrimp Company L.C., U.S.A.) were hatched in sea water. Two unequal compartments in the plastic chamber with several holes in the divider were used for hatching. The eggs were sprinkled into the larger compartment which was darkened, while the smaller compartment was illuminated.
After a 48 hours incubation period at room temperature, nauplii (larvae) were collected by pipette from the illuminated side, whereas their shells were left in another side.
Before adding 100 µL of sea water. Serial dilutions were made in the wells of 96-well microplates (Nunc, Denmark) in triplicate in 100 µL sea water. Control wells with DMSO were included in each experiment. A suspension of nauplii containing 10 to15 organisms (100 µL) was added to each well. The plates were covered and incubated at room temperature for 24 hours. Plates were then examined under the binocular steromicroscope and the numbers of dead (non-motile) nauplii in each well were counted. One hundred microliters of methanol were then added to each well to immobilize the nauplii and after 15 minutes the total numbers of brine shrimp in each well were counted. Analysis of the data was performed by probit analysis on a Finney computer program to determine the lethal concentration to half of the test organisms (LC 50 ).
Cytotoxic activity. Compounds 1 and 2 were tested in vitro for its cytotoxicity against the P388 cell line. The P388 cells growth inhibition by compound 1 and 2 were analyzed by the 3-(4,5-dimethylthyazole-2-yl)-2,5diphenyl-tetrazolium bromide (MTT) assay [17]. MCF-7 cells were seeded in 96 well plates and incubated with MTT (5 mg/mL) for 4 h. Cells were further solubilized by adding 100 µL of DMSO. Absorbance was read at 570 nm. Cell viability of treated cells was expressed as the amount of dye reduction compared to untreated control cells. The wells that contained only medium and 10 mL of MTT were used as blanks for the plate reader. Based on the 1 H-NMR data, compound 1 had four aromatic protons with two aromatic systems, The ABX system was at δ 7.69 (dd, J=8,48 and 1,97 Hz ), 6,99 (d, J=8,48 Hz) and 7,79 (1H, dd, J=1,97 Hz) and had a singlet proton at δ 6.40 in ring A. Two olefinic proton triplet signals at δ 5.09 and 5.30 indicated a typical isoprenyl group, with three methyl singlets at δ 1.82, 1.54 and 1.59, and three methylene signals at δ 3.35, 1.95 and 2.08. 13  Based on this NMR data, ( 1 H-and 13 C-NMR) compound 1 is a geranylated flavonoid derivative. This was supported by the HMBC experiment, which showed the geranyl group was located at C-5, with the presence of long range coupling between the methylene group at δ 3.35 (H-1") which correlated with C-5 at δ 112.32 (Figure 1). From the 1D-and 2D-NMR data, confirmed by LCMS data (m/z 439.20 [M+H] + ), compound 1 was Based on NMR data, supported by LC-MS data and reference comparisons (Figure 1.), Compound 1 was identified as the 5,7,3',4'-tetrahydroxy-6-geranyl flavonol [18]. This was the first report of 5,7,3',4'-tetrahydroxy-6geranyl flavonol content in Macaranga hispida. The 1 H-NMR data showed six aromatic protons at 8.08 (d, 9.08) (2H) / δ C 132.4 and 6.88 (d, 9.08) (2H) / δ C 116/ δ C 116.3, which indicated the presence of an A2B2 ring system. In addition, signal two singlets at δ H δ 6.2 (d, 1.95)/ δ C 100, 6.4 (d, 1.95) / δ C 94.8, were shown in rings A and B. Based on this chemical shift data, compound 2 was predicted as a kaemferol derivative. The anomeric proton (H-1") at δ H 5.15 ((d, J=7.79), which correlated to carbon at δ C 105.1, indicated that the linkage between the sugar residue and aglycone (flavone) was O-glycosidic at C-7 of the aglycon oxyigen atom. The O-glycosidic linkage was confirmed by HMBC correlations between the anomeric proton at δ H 5.14 with the proton atom at δ H 6.4 (H-8) (Figure 2).   Table 1 showed that, quercetin (positive control), compounds 1 and 2 have IC 50 values 4.39, 2.83 and 13.95 µg/ml, respectively. Due to similarities as the flavonoid, compound 1 has the highest antioxidant activity compared to quercetin (positive control) and compound 2. Both compounds had IC 50 values lower than 100 µg/mL. Thus they could be categorized as antioxidant active compounds.

Result and Disscussion
BSLT activity. The BSLT test was conducted on compounds 1 and 2. The toxicity test method was carried out because the cost was low and the results were be trustworthy. This test observed the mortality rate caused by the test compound. According to Meyer, active compounds will result in high mortality, and compounds with LC 50 ≤1000 µg/mL can be categorized as compounds that have potent toxicity. A Smaller LC 50 value means the compound was to be increasingly active/toxic. Table 2 showed no dead nauplii in the control groups, which means the sea water was in good condition. The obtained LC 50 value for compound 1 was smaller than compound 2, which means that compound 1 was more toxic than compound 2.
Cytotoxic activity. Based on the results in Table 3 above, showed that compound 1 was very active as anticancer against P388 cancer cells with IC 50 value 0.22 µg/mL, compared to compound 2 with IC 50 value 101.5 µg/mL. the different between compound 1 and 2 are in the isoprenyl and sugar group. Based on the anticancer activity result (Table 3) indicated that isoprenyl substituent bound to A ring of flavonoid increased the activity (compound 1), while sugar substituent decreased the anticancer activity (compound 2).

Conclusions
After isolation, purification and characterization of the phenolic compound isolated from ethyl acetate and butanol fraction of the methanol extract of M. hispida leaves, we concluded that there are two compound obtained of i.e compound of the 5,7,3',4'-tetrahydroxy-6geranyl flavonol (1) and kaempferol 7-O-β-glucose (2). Compound 1 has a higher activity than the compound 2 in a row with a value of 2.83 µg/mL and 13.95 µg/mL, The BSLT analysis results obtained smaller LC 50 for compound 1 (350 µg/mL) compared to compound 2, which had a value greater than 1000 µg/mL, Therefore compound 1 was more toxic than compounds 2. P388 for anticancer activity of compounds 1 and 2 respectively, 0.22 dan 101.5 µg/mL.