Alkaloid from Phoebe declinata Nees Leaves

Introduction: Genus Phoebe have been reported to produce isoquinoline alkaloids as aporphines, noraporphines, and benzylisoquinolines. Many of these isolates exhibit diversified biological activities, including cytotoxic activity. Objective: The objective of this study is to determine cytotoxic activity of compound isolated from Phoebe declinata againts MCF-7 (breast cancer cell line). Methods: Extraction was done by reflux using n-hexane, antioxidant activity measured by DPPH method and reducing power method, cytotoxic activity measured by MTT assay using MCF-7 cell line, struture eucidation was confirmed by NMR. Results: The antioxidant activity measured using DPPH method for 1 and 2 showed IC50 value of 6.42 and 11.80 μg/mL respectively and using reducing power method for 1 and 2 showed IC50 value of 7.02 and 13.74 μg/mL respectively. Compound (1) and (2) exhibited cytotoxic activity against MCF-7 cells with an IC50 value of 82.978 and 93.179 μg/mL. Conclusion: Compound (1) and (2) exhibited antioxidant activity and cytotoxic activity against MCF-7.


INTRODUCTION
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. 1,25][6]7 Previous paper, we reported the isolation of alkaloid declinine from stem bark of Phoebe declinata. 8In 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).

Plant Material
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 NH 4 OH, and exhaustively extracted with dichloromethane by reflux again.The residue was continue extracted with methanol.The hexane, CH 2 CL 2 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). 9A 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 37 o C 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% CO 2 at 37 o C. 100 µL cell suspension with 1.5 X 10 4 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 37 o C 2% CO 2 , 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 37 o C 2% CO 2 .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. 10,11The percentage of cell viability and cell death of samples on MCF-7 cell line was calculated for each assay by using the formula: ODs ODm % viability cell 100% ODc ODm *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 IC 50 value were calculates by using curve in linier equations.Compound 1 and 2 were considered as good antioxidant agent with IC 50 value 6.42 and 11.80 µg/mL respectively which is compared to boldine as alkaloid standard with IC 50 5.80 µg/mL by DPPH methode and by reducing power assay for 1 and 2 with IC 50 value 7.02 and 13.74 µg/mL respectively which is compared to boldine with IC 50 5.95 µg/mL.Table 1.

RESULTS AND DISCUSSION
Based on the result of Table 2 shows that compound 1 and 2 non-cytotoxic because IC 50 value is very high.

CONCLUSION
Compound (1) and ( 2) exhibited antioxidant activity with IC 50 6.42 and 11.80 µg/mL by DPPH and by reducing power assay method with IC 50 7.02 and 13.74 µg/mL recpectively.Both compounds are non-cytotoxic because IC 50 value is very high (above the NCI reference).

Figure 2 :
Figure 2: Partial Structures of A, B and C and 1H, 13C-Chemical shift data of Compound 1.