Phoebe declinata Nees belongs to Lauraceae family which commonly grows in Indonesia.1 The plant is a multy years plant (perennial) of moderate size (about 30-40 feet). This plant is called in Indonesia as huruhejo or bedagai, and grows commonly at Sumatera and Java.1,2 Genus Phoebe have been reported to produce isoquinoline alkaloids as aporphines, noraporphines, and benzylisoquinolines.3-5 Many of these isolates exhibit diversified biological activities, including anti-diabetes, anti-inflammation, cytotoxic, antibacterial, antifungal activities and antioxidant properties.3-6,7 Previous paper, we reported the isolation of alkaloid declinine from stem bark of Phoebe declinata.8 In our present research, a new alkaloid declinatine (1) was obtained from the hexane extract of the plant and a known alkaloid declinine (2) from diclormetana extract (Figure 1).
MATERIALS AND METHODS
The 1H-NMR and 13C-NMR were recorded in deuterated chloroform on JEOL 500 MHz instrument. Silica gel 60, 70-230 mesh ASTM (Merck 7734) was used for column chromatography, Mayer’s reagent was used for alkaloid screening, TLC aluminum sheets (20 × 20 cm Silica gel 60 F254), were used in the TLC analysis. The TLC spots were visualized under UV light (254 and 366 nm) followed by spraying with Dragenderff ’s reagent for an alkaloid detection.
The leaves of Phoebe declinata (Lauraceae) collected from Bogor, west Java, Indonesia in June 2012, was Identified by Dr. Joko Ridho Witono. A voucher specimen (PD 1065) has been deposited in the Faculty of Pharmacy, University of Indonesia.
Extraction and Isolation
The air-dried leaves P. declinata (500g) were reflux in hexane. The plant residue was moistened with 54% of NH4OH, and exhaustively extracted with dichloromethane by reflux again. The residue was continue extracted with methanol. The hexane, CH2CL2 and methanol extracts were evaporated. The hexane extracts (10 g) were subjected to column chromatography using silica gel as stationary phase and n-hexane-ethyl acetate and ethyl acetate-methanol systems, gradually polarity affording 15 fractions. The seven fractions were chromatographed using silica gel and purified to give 1 (40 mg). The dichloromethane extracts (10 g) were subjected to column chromatography using silica gel as stationary phase and ethyl acetate-methanol systems, gradually polarity affording 10 fractions. Fraction 4 was chromatographed using silica gel and purified to give 2 (20 mg).
Free radical scavenging ability using DPPH radical
The antioxidant activity of isolate was assessed by measuring their scavenging potency against stable free radical 1,1 Diphenyl -2-picryl-hydrazyl
(DPPH).9 A total of 1 mL of DPPH (100 µg/mL/ solution and 1 mL sample at various concentrations (20, 40, 60 and 80 µg/mL or boldine as the alkaloid standard solution (5,6,7,8,9 and 10 µg/mL were added into mixed solution at the separated place. The reaction mixture was incubation the dark at temperature 37oC for 30 min. Optical density of each solution was measured at 517 nm using methanol as blank. DPPH scavenging activity of samples represented as value of inhibition concentration 50 % was calculated using the following equation:
Free radical scavenging ability using reducing power
The reducing power of the isolate was determined by the method described by Chang et al. Different concentrations of the extracts (0.06-1 mg/mL) were mixed with phosphate buffer (0.2 mM, pH 6.5), ferric chloride solution (2 mM) and potassium ferricyanide (4 mM). To this, 100 mg/mL trichloroacetic acid was added to the reaction mixture and was made up to 1 mL with water and incubated at 37°C for 10 min. The absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.
Assay of Cytotoxic activity
The MCF-7 cell line was cultures stock in DMEM with 10 % FBS, 100 µg/mL streptomycin and penicillin (100 IU/mL) and 2 mm glutamine. Cell were incubated in humidified atmosphere of 5% CO2 at 37oC. 100 µL cell suspension with 1.5 X 104 cells included in microplate 96 well. The Samples with concentration 3.125; 6.25; 12.5; 25, 50 and 100 µg/mL with triple replications each cell controls and medium controls. Microplate incubated for 24h at 37oC 2% CO2, the culture medium removed and washed with PBS. Into each well plate added 10 µL of MTT solution (1 mL MTT in 10 mL culture medium) and microplate incubated at 37oC 2% CO2. After 4h of stopper reagent added 100 mL of 10 % SDS in 0.1 N HCL into each well (to dissolve the purple formazan crystals). Absorbance is read using an ELISA reader at wavelength of 550 nm.10,11 The percentage of cell viability and cell death of samples on MCF-7 cell line was calculated for each assay by using the formula:
*Where ODc = optical density cell with samples, ODc = optical density cell without sample, ODm = optical density media without cell.
Graph percentage of viability cell against logarithm concentration was plottes. The IC50 value were calculates by using curve in linier equations.
RESULTS AND DISCUSSION
Compound 1 was obtained as a white crystal. The LCMS-IT-TOF revealed a pseudo molecular ion peak at m/z 372.4417 [M+H]+, thus suggesting a molecular weight and formulae are 371.449 and C22H28O5. (calc. 372.45). The 1H-NMR spectrum (Figure S1-S5) contained the presence of three protons of phenyl as ABX type (C ring) at at δ 6.60 (H-2ʹ, s), 6.77 (H-5ʹ, d, 8.0 Hz) and 6.53 (H-6ʹ, dd, 2, 8.0 Hz). Two singlet aromatic protons indicated this signal have para position (B ring), appear at δH 6.42 (H-8, s) and 7.55 (H-5, s). The other signals indicated the presence of ring A : 2 methyls (d) were shown at δ 0.98 (2-Me, d, 7.0 Hz) and 1.12 (3-Me, d, 7.0 Hz). The presence of two methine at δ 2.77 (sixtet) and 2.41 (sixtet) with 7.1 Hz constants J coupling, indicated this signals have cis orientation. Two singlet aromatic protons at δ 6.42 (H-8, s) and 7.55 (H-5, s), indicated this signal have para position in the ring B. Two signal methoxy (-OCH3) were showed at δ 3.76 (6-OMe, s) and 3.86 (7-OMe, s). That were ilustrated in partial structure A, B and C, was shown in Figure 2.
The 13C-NMR (Figure S6-S9) and HMQC spectrum (Figure S10) also supported the presence of A, B and C, with the presence of ring C signals at δ 111.8 (C-2ʹ), 110.9 (C-5ʹ) and 121.2 (C-6ʹ). Signals at δ 200.2 (C-1), 42.8 (C-2), 42.6 (C-3,), 16.11 (2-Me) and 12.10 (3-Me) support for the presence of ring A, and signals at 111.9 (C-8), 108.2 (C-5), 56.0 (6-OMe) and 56.1 (7-OMe) confirm for the presence of ring B. Base on this spectral data, indicated that the structure is alkaloid. The compound 1 also showed alkaloid spot which was visualized by Dragendorf ’s spray method in aluminum sheet. For construct this partial structure was elucidated by use HMBC (Figure S11-S21). The presence of long range coupling in the HMBC experiment between C-2 (δ, 42.6, d) and H-3 at δ 4.41 (d) and C-10 (δ 133.84, s), C-2 (δ 46.04, d) indicated ring B was connected with ring A at C-9 and C-10. For construct this partial structure was elucidated by use HMBC experiments (Figure S22).
The presence H-H correlation (COSY) (Figure S7) between H-2 dan H-3 indicated that protons is very close, and the presence of NOESY correlation between H-2 dan H-3, Constant coupling value between H-2 dan H-3 is 7.1 Hz, showed that H-2 is cis to H-2.
Compound 2 was obtained white crystal, m.p. 102-104oC, molecular formula C20H22N2O4. 1H-NMR (CDCl3, δ): 6.88 (s, CH-1), 7.07 (s, CH-3), 6.99 (s, CH-10), 3,86 (s, OCH3-2), 1.08 (m, CH3-5), 3.88 (s, OCH3-6), 3.89 (s, OCH3-7), 3.9 (s, OCH3-8), 0.67 (m, CH3-9) (Figure S23). 13C-NMR (CDCl3, δ): 118.51 (C-1), 148.99 (C-2), 110.38 (C-3), 147.90 (C-3a), 135,69 (C-3b), 147.78 (C-4a), 133.48 (C-5), 148.60 (C-6), 148,96 (C-7), 148,64 (C-8), 134.84 (C-8a), 133.26 (C-8b), 133.82 (C-9), 109.35 (C-10), 55.88 (OCH3-2), 11.89 (CH3-5), 55.87 (OCH3-6), 55.95 (OCH3-7), 55.90 (OCH3-8), 15.05 (CH3-9) (Figure S24).
Compound 1 and 2 were considered as good antioxidant agent with IC50 value 6.42 and 11.80 µg/mL respectively which is compared to boldine as alkaloid standard with IC50 5.80 µg/mL by DPPH methode and by reducing power assay for 1 and 2 with IC50 value 7.02 and 13.74 µg/mL respectively which is compared to boldine with IC50 5.95 µg/mL. Table 1. Based on the result of Table 2 shows that compound 1 and 2 non-cytotoxic because IC50 value is very high.
Compound (1) and (2) exhibited antioxidant activity with IC50 6.42 and 11.80 µg/mL by DPPH and by reducing power assay method with IC50 7.02 and 13.74 µg/mL recpectively. Both compounds are non-cytotoxic because IC50 value is very high (above the NCI reference).