Effects of Bilobol from the Fruit Pulp of Ginkgo biloba on Cell Viability

Cytotoxic activity profiling of the n-hexane extract from the fruit pulp of Ginkgo biloba L. (Gingkgoaceae) led to the purification of 8 compounds. HPLC based extract profiling identified a cytotoxic alkenylresorcinol. The extract was further separated by time-based fractionation in a gradient HPLC condition, and cytotoxicity of each fraction was evaluated using 293, B16F10, BJAB, and HCT116 cancer cells in vitro. Bilobol was isolated as an active constituent by semi-preparative HPLC and showed significant cytotoxic activities against the cancer cells in a dose-dependent manner at the concentrations of 15.0 ~ 50 μg/mL. The western blotting analysis showed that this compound increased expression of active caspase-3 and active caspase-8 in HCT116 human colon cancer cells, indicating that this compound causes apoptotic cell death in a dose-dependent manner. The results suggest that bilobol is the active component from fruit pulp of Ginkgo Biloba L. that can induce apoptotic cell death in various cancer cells.


Introduction
Cancer is the second cause of death all over the world that reached 9.6 million deaths in 2018 (World Health Organization, 2019).The incidence and mortality rate of the ten most common cancers (the cancer types include lung, stomach, liver, colorectum, esophagus etc) will increase by the year 2030 (Sankaranarayanan et al., 2014).The conventional cancer treatments such as surgery, chemotherapy and radiotherapy may offer toxicity, and multi-drug resistance and other side effects thus are often supplemented by other alternative therapies (Bell, 2010).Natural active substances from many herbs have begun to draw increasing scientific attention.Natural compounds have been used in the treatment of various cancers for a long period of time and their anticancer properties have been scientifically evaluated both in vitro and in vivo (Bonham et al., 2002;Gong et al., 2005).Many studies reported extracts from some herbal medicines that can inhibit cancer cell proliferation in vitro and in vivo (Itokawa et al., 1989).
Ginkgo biloba, also known as Salisburia adiantifolia, Salisburia macrophylla, and Pterophylla salisburiensis, is among the most sold medicinal plants.It belongs to the plant division Ginkgophyta whose all other species have extinct (Jacobs & Browner, 2000) The ginko tree has survived unchanged in the forests for over 200 million and hence it is also referred as a "living fossil" (Kozubek & Tyman, 1999;Gong et al., 2008).This plant has been used as herbal remedy for centuries in China and Korea, and now their extracts are currently used extensively in therapy for various diseases like arteriosclerosis or rheumatism.Many bioactive compounds, including flavonol and flavones glycosides, terpenoids and ginkgolides (Mahadevan & Park, 2008;Ihl, 2013) has been isolated from the plant displaying several physiological actions and attributed to these compounds, including anoxia and ischemia of brain, heart and eye, arteriosclerosis, rheumatism, inhibition of platelet activating factor and neuro-transmitter modulation.G. biloba is one of the most studied medicinal plants (Matsumoto et al., 1990) However, the chemical constituents in Ginkgo biloba that are responsible for anti-cancerous activities in vitro remain to be explored.
In the course of investigating anti-cancerous natural products, we studied Ginkgo Biloba, and significant and dose dependent in vitro apoptotic activity of isolated constituent has been found.HPLC-based profiling was applied to an n-hexane extract from the fruit pulp of the plant.The extract was separated by time-based fractionation in a gradient HPLC condition, and cytotoxicity of each fraction was evaluated using 293, B16F10, BJAB and HCT116 cancer cells in vitro.Bilobol was isolated as an active constituent by semi-preparative HPLC.The isolates were structurally identified by spectral studies of NMR.The western blotting and cytotoxicity assay were used to describe apoptotic activities of the constituent.

Extraction and isolation
The ripe fleshy seed coat of G. biloba (250 g) was exhaustively extracted three times with 2 L of MeOH.The filtered MeOH layer was concentrated at reduced pressure to obtain an extract.This MeOH extract (100.0 g) was partitioned with 400 mL of n-hexane and 300 mL of H 2 O to afford an n-hexane extract (53.84 g).An aliquot of this extract (25 mg per injection, total 9.8 g) was repeatedly separated by HPLC with a linear gradient solvent system [H 2 O containing 0.1% formic acid-MeOH (10:90) to MeOH (100%) for 70 min, flow rate 4.0 mL/min] using semiprep Atlantis™ column under 280 nm.The active compound bilobol (1) (t R 12.19 min, 31.0 mg) was isolated as yellow oil.

Cell culture
CT26, B16F10 and HCT116 cells were purchased from the Korean Cell Line Bank (Seoul, Korea), and BJAB cells were a generous gift from Genentech, Inc. (South San Francisco, USA).CT26 and B16F10 cells was cultured in DMEM, BJAB in RPMI 1640, and HCT116 in McCoy's 5A (all media contained 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified 5% CO 2 atmosphere.

XTT assay
Cells were cultured in 96 well plates (2 x 10 5 /well or 1 x 10 4 / well) overnight and treated with indicated amounts of bilobol for 24 hours.Cell viabilities were assessed using XTT-based colorimetric assay kit, according to the manufacturer's instructions (Promega, WI, USA) and absorbances at 493 nm were monitored by TECAN Infinite M200 monochromator (Tecan, Switzerland).Data are representative of three independent experiments and are reported as means ± SDs.

Isolation and identification of active compound
To identify the anti-cancerous molecules from G. biloba, we extracted n-hexane soluble substances from the fruit pulp of sarcotesta of G. biloba.The n-hexane extract was subjected to partition and subsequent repeated HPLC column chromatography.The fractions were obtained and further purified by HPLC using reverse phase C18 column.Methanol: water solvent system was found to be the best solvent to extract fractions from the G. biloba.We isolated 8 fractions from the fruit pulp of G. biloba through bioassay-guided separation of the fractionated Methanol: water extract using repeated column chromatography (Figure 1).A linear gradient solvent condition applied to HPLC separation was H 2 O containing 0.1% formic acid-MeOH (10:90) to MeOH 100% for 70 min, with flow rate 4.0 mL/min, at 280 nm.Of eight fractions evaluated, fraction b exhibited significant cytotoxic activity, and bilobol (1, tR 12.19 min) was identified as a major component of this fraction.

Cancer cell growth inhibition assay
To evaluate the cytotoxic effects of isolated compound on tumor cells, we treated CT26 cells with different concentrations of bilobol (3.125 µg/mL, 6.25 µg/mL, 12.5 µg/mL, 25 µg/mL, 50 µg/mL and 100 µg/mL) for 24 hours.It showed dose dependent cytotoxic effects of the compound suppressing more than 50% of cell viability with highest concentration compared to that of control group (Figure 3).
The cytotoxic effects of bilobol on other tumor cells were further determined by XTT based colourimetric assay using 293, B16F10, BJAB, HCT116.As shown in Figure 4, bilobol exhibited considerable inhibition of cancer cell growth in a dose dependent manner.293 cells showed the highest sensitivity against bilobol (Figure 4).These results support the previous anti-tumor studies of bilobol where administration of bilobol in a dosage of 40 mg/kg per day for 4 days after injection of tumor cells in mouse caused inhibition of the tumor cell growth (Risberg et al., 1998).Our results suggested that bilobol may have inhibiting effects on different human cancers and may be used in future tumor treatments.

Western blot assay
We evaluated the mode of cell death by measuring expression level of caspase-3 and caspase-8 in HCT116 cells.Activation of caspase-3 and caspase-8 are considered to be key events for induction of apoptotic cell death (Schwartsmann et al., 2002) HCT116 cells were treated with 50 µg/mL bilobol for 0, 2, and 4 hrs and activation of caspases were determined by western blotting.Bilobol significantly augmented caspase-3 activation as reflected by production of cleaved caspase-3 in a dose-dependent manner (Figure 5).Also, caspase-8 activation by bilobol was observed.
Detailed molecular mechanism by which bilobol causes caspase activation is not clear.Bilobol could directly damage the mitochondria to release cytochrome c or possibly changes gene expression levels of death-inducing machinery proteins as indicated by Gohil et al. (2000) and Papadopoulos et al. (2000), showing that Ginkgo leaf extract changes the transcriptional level of genes involved in cell proliferation, cell differentiation and apoptosis in breast and cancer cell lines (Tanaka et al., 2011;van Beek & Montoro, 2009).bilobol (1) with a single injection of the n-hexane extract of the fruit pulp of Ginkgo biloba L. (Gingkgoaceace) without subsequent isolation procedures.We determined cytotoxic profiles of the fractions obtained from the n-hexane extract of Ginkgo biloba L. (Gingkgoaceace) using XTT assay.The activity-guided isolation of compounds using HPLC provided a known compound, bilobol from the active fraction b, exhibiting dose-dependent cytotoxic effects on human colon cancer cells (CT26, B16F10, BJAB and HCTT116 cells).However, further studies would be desirable to elucidate the signaling pathway for bilobol induced apoptosis as well as therapeutic potential to cancer using in vivo tumor model.
However, we hypothesize that bilobol may directly act on caspase activation through mitochondrial damage or activation of apoptotic pathways rather than through transcriptional gene expression, because it can activate casapse-3 and -8 within 4 hours after treatment.

Conclusions
In this study, the fast and effective structural screening was successfully accomplishes by employing HPLC based extract profiling for the identification of a known alkenylresorcinol,

Figure 1 .
Figure 1.HPLC profile and peak-based fractions (a-h) of n-hexane extract from ginkgo fleshy seed coat.A linear gradient solvent condition was applied to HPLC separation; H 2 O containing 0.1% formic acid-MeOH 10:90 to MeOH 100% for 70 min, flow rate 4.0 mL/min, 280 nm.Of eight fractions evaluated, fraction b exhibited significant biological activity, and bilobol (1, t R 12.19 min) was identified as a major component in this fraction.

Figure 4 .
Figure 4. 293 cells, B16F10 cells, BJAB cells or HCT116 cells were treated with indicated amounts of bilobol for 24 hours.XTT assays were carried out to measure viabilities.