EXPERIMENTAL METHOD 1 Plant material and the explant

Bogor, 21-22 October 2015 385 isorhamnetin, triterpenes e.g. sterols, organic acids, polyprenols including ginkgolic acid, and tannic acid Zimmerman et al. 2002. The ginkgolide, a unique C 20 cage molecule, is a naturally occurring platelet-activating factor PAF antagonist Braquet 1987; Nunez et al. 1986. PAF is a potent mediator of anaphylaxis and inflammation and is also implicated in shock, graft rejection, renal diseases, ovoimplantation, and certain disorders of the central nervous system Kuster et al. 1986; Dive et al. 1989; Singh et al. 2013. Different types of PAF antagonists are available in present time, such as synthetic PAF antagonists and natural ones. Natural compounds have less toxic effects compare to synthetic one as more and higher dose of natural compounds shows safer therapeutic margins Singh et al. 2013. Indeed, the growing importance of PAF as a mediator of diverse pathologies increases the possible medicinal benefits that may be derived from ginkgolide, specific PAF antagonist Liu and Xia, 2006. However, only trace amounts of ginkgolides are contained in the ginkgo leaves Nakanishi 1967; Teng 1988. In addition, location, climate, and seasonal variations have been affecting the ginkgolides content. Thus the constant and continuous commercial-scale supply of the ginkgolides from the field-grown leaves appears to be quite uneconomically and questionable. Tissue culture of the ginkgo leaves was conducted as an alternative way to solve the problem. This study was carried out to investigate the effect of growth hormone on biomass and production of bilobalide, ginkgolides A and B in callus and cell suspension cultures from G. biloba leaves. 2. EXPERIMENTAL METHOD 2.1 Plant material and the explant G. biloba leaves were collected from the education garden of Faculty of Agriculture, Ehime University, Japan, on September 2012 fall season. The plant material was washed using neutral detergent two times and rinsing six to seven times with sterile distilled water. After surface sterilization with 70 ethanol EtOH for 1 min followed by a solution of 1 sodium hypochlorite for 15 min, and cleanings in sterilized distilled-water for 10 min. Thus, Leaves have been already using as the explant material. 2.2 Optimization of culture condition Calluses were generated from leaves of G. biloba. The explant of leaves after pretreatment describe above section was cut and transferred to Murashige and Skoog MS medium, containing sucrose 30 gL and gellan gum 2 gL, with pH 5.6. The growth calluses were sub- culture every month and incubated at 24˚C in the dark. Callus growth was expressed as fresh weight gram and dry weight gram. Callus with 0.5-0.6 g of fresh weight was transferred to MS medium supplemented with 1-naphthaleneacetic acid NAA, 2,4- dichlorophenoxyacetic acid 2,4-D and kinetin in certain combination, then the weight was determined after 30, 60 and 90 days. Table 1: The design combination of growth hormone treatment in cell suspension cultures of G. biloba Kinetin mgL NAA mgL 2,4-D mgL 1.0

2.0 1.0

2.0 0.1 TR1 TR2 TR3 TR4 1.0 TR5 TR6 TR7 TR8 To induce cell suspension cultures, 2 g fresh weight FW of friable callus was transferred into 250 mL Erlenmeyer flask containing 40 mL MS liquid medium. In order to optimize cell growth, cells were cultured in different combination of growth regulators auxin and Bogor, 21-22 October 2015 386 cytokinin. For auxin optimization, the cells were cultured on 2,4-D concentrations of 1.0 and 2.0 mgL, and NAA at concentrations of 1.0 and 2.0 mgL. The design combination of growth hormone in this experiment is shown on Table 1. The cell suspension cultures were incubated in shaken at 100 rpm in the dark with temperature 24°C. 2.3 Measurement of growth The biomass of cell suspension cultures was measured for 30, 60 and 90 days using packed- cell volume PCV. The biomass in PCV was calculated using the equation: [V p V t ] x 100 where V p is precipitate volume, and V t is total volume. 2.4 Determination of bilobalide and ginkgolides Fresh callus from leaves were harvested by filtering through filter paper 70 mm, Advantech. The filtrate consisting of fresh weight cells was freezed in a refrigerator -30°C overnight and then freeze-dried for three days. The dried cells were ground with a mortar and pestle. Ten mL n-hexane was added, sonicated and centrifuged. The supernatant upper layer was removed and precipitin was extracted with 10 ml ethyl acetate EtOAc for 2 h in a sonicator. Supernatant ethyl acetate solution obtained by centrifugation was concentrated using a rotary vacuum evaporator. The concentrate of EtOAc was separated using preparative high performance liquid chromatogram HPLC to obtain biobalide, ginkgolides A and B, and each collected sample was confirmed by gas chromatography-mass spectrometry GC-MS. HPLC Analysis was performed on a reverse phase column Sh imadzu VP Shimpak ODS RP column 250 x 4.6 mm i.d., 5 m in a Waters equipped with a UV ultraviolet detector wavelength 220 nm by isocratic elution with methanol-water- isopropanol 17.5-72.5-10 with a flow rate of 1.0 mLmin. The amount of biobalide, ginkgolides A and B was calculated through a standard curve, the regression linier with coefficient R 2 , 0.99 for each standard. The GC-MS analysis was performed to confirm the molecular ion [M + ] of biobalide, ginkgolides A and B. After trimethylsilylation, the sample was subjected to a Shimadzu GC-MS QP2010 Shimadzu, Japan equipped with a TC-5 capillary column 30 m, id: 0.24 mm. The carrier gas was Helium delivered at a constant rate of 1.5 mL min -1 , with a column pressure of 100 Kpa and interface t emperature of 280˚C. The temperature program was 60˚C, 10˚C min -1 to 300˚C, and 300˚C for 18 min to allow the late eluting peak to exit the column. The injection volume was 1 L and the injector temperature was maintained at 280˚C. The GC-MS conditions consisted of 40-700 mass ranges. Comparison of mass spectrum of each collected sample with that of each standard sample bilobalide, ginkgolides A and B was conducted.

3. RESULT AND DISCUSSION