Guttiferone F from the fruit of Garcinia multiflora and its anti-hepatocellular carcinoma activity

Biomedicine & Preventive Nutrition 3 (2013) 247–252

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Original article

Guttiferone F from the fruit of Garcinia multiflora and its anti-hepatocellular carcinoma activity Lain-Tze Lee a,∗ , Hui-Ping Tsai a , Chun-Chung Wang a , Chia-Ni Chang a , Wan-Chun Liu a , Hui-Chun Hsu a , Cheng-Ta Hsieh a , Yen-Chun Chen a , Hsiang-Wen Tseng a , Rung-Jiun Gau a , Szu-Hsiu Liu a , I-Sen Chen b , Munekazu Iinuma c a

Herbal Medicinal Product Technology Division, Pharmacognosy Laboratory, Industrial Technology Research Institute, Hsinchu 30011, Taiwan School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan c Laboratory of Pharmacognosy, Gifu Pharmaceutical University, Gifu 501-1196, Japan b

a r t i c l e

i n f o

Article history: Received 19 September 2012 Accepted 23 October 2012 Keywords: Garcinia multiflora Guttiferone F Xenograft Anti-hepatocellular Carcinoma Apoptosis

a b s t r a c t Garcinia multiflora is an evergreen arbor, which belongs to the family of Guttiferae. It grows mainly at the lower altitudes of the mountains in the Hengchun Peninsula, which is in southern Taiwan. In this study, bioactivity-guided screening was conducted to evaluate the anti-hepatocellular carcinoma bioactivity of the fruit’s methanol extract of G. multiflora. The methanol extract residue was partitioned with various solvents. It was then separated and purified using flash chromatography. The active component was confirmed to be guttiferone F. In our present study, cytotoxicity assay revealed that the IC50 of guttiferone F was 14.15 ± 2.36 ␮M in PLC/PRF/5 cells, 10.17 ± 0.51 ␮M in HepG2 cells and 9.71 ± 1.28 ␮M in Hep3B cells. In the in vitro anticancer study, we found guttiferone F induced apoptosis by up-regulating cleaved pro-caspase-3 and PARP expressions in PLC/PRF/5 cells. The antitumor potency is similar to sorafenib, a therapeutic drug for liver cancer. Further in vivo study showed that guttiferone F inhibits minor tumor growth on s.c. xenograft tumor of human liver cancer cell line in female SCID mice. Although antitumor efficiency of guttiferone F is not superior to sorafenib, the dose (i.p., 25 mg/kg × 5 days) is proved safe to animal. According to our studies, guttiferone F can be a promising lead for liver cancer therapy and become a new liberator for liver cancer patients after modification of the drug. © 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction Garcinia is a plant genus of the family Clusiaceae and native to Asia, Australia, tropical and southern Africa, and Polynesia. Habitat destruction threatens many species of the Garcinia genus. Many species of Garcinia have fruit with edible arils, yet are mostly eaten locally. The best-known species, the purple mangosteen (Garcinia mangostana), is commonly referred to as the “queen of fruits” and is cultivated in the tropical rainforests of some Southeast Asian countries. In those countries, the pericarp (peel, rind, hull or ripe) of mangosteen is used as a traditional medicine to treat abdominal pain, diarrhea, dysentery, infected wound, suppuration, and chronic ulcer. Most species in the Garcinia genus are characterized by their gum resin, brownish-yellow color from xanthonoids such as mangostin, its use as a purgative or cathartic and, most frequently, its use as a traditional pigment. The color term “gamboge” refers to the gambooge, whose obsolete scientific name is G. cambogia.

∗ Corresponding author. E-mail address: [email protected] (L.-T. Lee). 2210-5239/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2012.10.007

The Garcinia multiflora, a species of the Garcinia genus, is evergreen trees and shrubs. This species grows mainly at lower altitudes in the southern Taiwan mountains of the Hengchun Peninsula. From the G. multiflora, some isolated biflavonoids are amentoflavone, agathisflavone, robustaflavone, hinokiflavone, volkensiflavone, rhusflavanone, and succedaneflavanone. A previous study has evaluated G. multiflora’s methyls ether (volkensiflavone hexamethyl ether) and acetates (rhusflavanone hexaacetate, succedaneflavanone hexaacetate) for their antiviral activities against respiratory and herpes viruses [1]. Shih-Chang Chiena et al. investigating G. multiflora’s roots and leaves isolated 45 components, which are classified as apocartenoids (3), aromatics (13), chlorophyll (2), lignoid (1), others (7), steroids (8), triterpenoids (10), and xanthone (1). That study also discovered new compounds of polyprenylated phloroglucinol garcinialone [2]. From the stems of G. multiflora, Chiang identified two new xanthone derivatives (garcinianones A and B), two new benzophenone derivatives (4,6,4’trihydroxy-2,3’-dimethoxy-3-prenylbenzophenone and 4,6,3’,4’tetrahydroxy-2-methoxybenzophenone), and a new inseparable mixture ((1E,22Z)-1,22-diferuloyloxydocosane/(1E,24Z)-1,24diferuloyloxyteracosane), a previously known 3,8-dihydroxy-2,

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4,6-trimethoxyxanthone, 6,3’-dihydroxy-2,4-dimethoxybenzophenone, maclurin, 2,4,6,3’-tetrahydroxybenzophenone, and naringenin. That study also evaluated compounds in the brine shrimp lethality test and in the DPPH antioxidant assay [3]. From the acetone extract of the twigs of G. multiflora, a previous study isolated five new polyprenylated benzoylphloroglucinol derivatives (i.e., garcimultiflorone D, 18-hydroxygarcimultiflorone D, garcimultiflorone E, garcimultiflorone F, and isogarcimultiflorone F) and five known compounds (i.e. guttiferone E, guttiferone F, aristophenone A, isoxanthochymol, and morelloflavone). The compounds were also evaluated for their apoptotic effects against HeLa-C3 cells [4]. From the fruits of G. multiflora, a previous study isolated a novel benzoylphloroglucinol derivative, garcimultiflorone D, an unusually adamantyl-caged skeleton together with four known compounds. In that study, garcimultiflorone D exhibited inhibitory activities with IC50 values of 7.21 ± 1.07 and 6.01 ± 0.37 ␮g/mL against fMet-Leu-Phe/cytochalasin B (fMLP/CB) induced superoxide anion generation and elastase release, respectively [5]. Another study examined the constituents with their anti-inflammatory activity from the fruits of the G. multiflora, including five new benzophenone derivatives of 13,14-didehydoxyisogarcinol, garcimultiflorone A, garcimultiflorone B, 13-hydroxygarcimultiflorone B, and garcimultiflorone C. The structures of these new compounds were determined through spectroscopic and MS analyses. 13,14Didehydoxyisogarcinol, garcimultiflorone A, garcimultiflorone B, and 13-hydroxygarcimultiflorone B exhibited inhibition activity with an IC50 range of 0.11–5.58 ␮M on superoxide anion generation and elastase release by human neutrophils in response to fMetLeu-Phe/cytochalasin B (fMLP/CB) [6]. To establish the correlations between the anti-hepatocellular carcinoma activity of the extract of fruit and some of its chemical components, this study investigates the anti-hepatocellular carcinoma activity-guided fractionation of the active extracts and the anti-hepatocellular carcinoma activity of isolated compounds similar to those observed previously for a crude methanol extract.

2. Materials and methods 2.1. General 1H (500 MHz) and 13C NMR (126 MHz) spectra were measured on a Varian 500 instrument, and chemical shifts were expressed in ␦ (ppm) values. Electron spray ionization (ESI)-MS was performed on a Micromass Quattro LC, Waters 2690 mass spectrometer with the direct injection of MeOH solution of a sample. Reversed-phase high-performance liquid chromatography (HPLC) was then performed on Waters PDA996. Next, the flash chromatography was conducted on Combi flash Rf. All other reagents and chemicals were of analytical grade.

2.2. Chemicals Methanol (MeOH), alcohol (EtOH), acetone, n-hexane (n-H), ethyl ether, ethyl acetate were purchased from Echo Chemical Co.

2.4. Preparation of extracts and anti-hepatocellular carcinoma-guided fractionation The dried fruit of G. multiflora (3 kg) was pulverized and extracted with MeOH (3 × 10 L) for 3 days. The MeOH extracts were concentrated under reduced pressure at 35 ◦ C to obtain the residue (330 g). The reddish-brown methanol extra residue (8.5 g) was suspended in 50 mL MeOH/H2O (10:90, v/v) and partitioned successively with 50 mL × 3 n-hexane, ether, ethyl acetate respectively. Next, the extracts and the remaining aqueous solution were concentrated in a rotary evaporator to remove the solvents, which produced the n-hexane layer (S-01) 6.144 g, ether layer (S02) 0.3572 g, ethyl acetate layer (S-03) 0.214 g and aqueous layer (S-04) 0.5066 g. Finally, those extracts were submitted to antihepatocellular carcinoma evaluation. 2.5. Flash chromatography The active fraction (n-hexane layer, S-01) was subjected to flash chromatography on 120 g Slica gel. The sample (S-01, 5.4265 g) was dissolved in n-hexane. Mobile phase: n-hexane/ethylacetate gradient: initial 3 min n-hexane 100%, 60 min 100%→0%, in which the flow rate was set at 85 mL/min. Each of the 162 fractions collected was 25 mL. Those fractions were analyzed by thin layer chromatography and combined into 18 new fractions. Those fractions were then submitted to anti-hepatocellular carcinoma evaluation. Fraction 11 was recrystallized and was confirmed to be guttiferone F, based on LC-MS and NMR analyses as well as literature data. 2.6. Culture of human hepatocellular carcinoma cells Human hepatocellular carcinoma HepG2 cells, Huh-7 cells, SKHep1, and J-5 were purchased from the Bioresource Collection and Research Center (Taiwan). All cells were grown in the DMEM medium with 10% FBS. PLC/PRF/5 (BCRC 60223), Huh-7 (JCRB-0403), DMEM media, and fetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, NY, USA). 2.7. Cytotoxic effect on cancer cells with MTT-based colorimetric assay All cancer cell lines were seeded onto 96-well plate (Corning Incorporation COSTAR, 3599) at a concentration of 5 × 103 ∼ 1 × 104 Huh-7 cells/well and were grown in DMEM medium with 10% FBS. After 24 h, the test samples were applied to culture wells and then incubated at 37 ◦ C with a humidified atmosphere containing 5% CO2, for 72 hours. Next, 20 ␮L of MTT solution (5 mg/mL in PBS, pH 7,2) was added to each well, and the plates are incubated for 2∼4 h at 37 ◦ C. After incubation, the medium was aspirated, and 100 ␮L of DMSO was added to the wells to solubilize the formazan dye. Absorbance was read at 540 nm by ELISA reader, and the surviving cell fraction was calculated. Finally, inhibition of cell viability was calculated through means of the following formula: % inhibition = (1 − absorbancy of treated cells/ absorbancy of untreated cells) × 100

2.3. Plant material The fruit of G. multiflora was collected from Mudan of Pingtung County in southwestern Taiwan, in September 2007 and identified by Dr. I.S. Chen. A voucher specimen (Chen 6061) was deposited in the Faculty of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.

2.8. Animals All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC No.: ITRI-IACUC-2012010M, Industrial Technology Research Institute of Taiwan, HsinChu,

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Taiwan). SCID (CB17/Icr-Prkdcscid/CrlBltw) mice were purchased from BioLASCO Ltd (Taipei, Taiwan). Female SCID mice (6 to 8 weeks old) were implanted subcutaneously (s.c.) in the right flank with PLC/PRF/5 hepatocellular carcinoma cells (3 × 106 cells per mice) in 100 ␮L mix (equal volumes of PBS and Matrigel). Tumor size was then measured with calipers and tumor volumes (mm3 ), as calculated by using the formula: V = LS2 /2 (where L denotes the longest diameter and S represents the shortest diameter). PLC/PRF/5 tumors were allowed to grow to 100–200 mm3 . Tumor-bearing mice were administrated intraperitoneally (i.p.) of guttiferone F (25 mg/kg, n = 5) or a vehicle control (n = 5) once daily for 14 days. The formula of vehicle is 10% NMP (M6762, Sigma-Aldrich, St. Louise, MO), 20% Cremophor EL (C5135, SigmaAldrich, St. Louise, MO), and 70% saline. Tumor volumes and body weight of animal were determined twice weekly. Next, weights and tumor sizes were determined and clinical observations were made throughout the study until the animal was removed from the study. The antitumor activity was illustrated by percentage tumor growth inhibition (TGI). TGI was calculated as [1 − (tumor volume final − tumor volume initial for the treated group)/(tumor volume final − tumor volume initial for the vehicle group)] × 100. 2.9. Western blot analysis Human hepatoma PLC/PRF/5 cells were plated at 2 × 105 cells per well in six-well plates (Orange Scientific, Belgium) and cultured for 24 h, then incubated in a serum-free medium overnight before (preparing the OR preparation of) cell lysates. Cells were treated with guttiferone F for 1, 2, 6 and 24 h or different concentrations of guttiferone F for 24 h. Next, lysates were prepared by adding the cell lysis buffer (Cell Signaling Technology, USA); the lysates were clarified by centrifugation (11,400 × g, 15 min, 4 ◦ C). Additionally, lysate protein content was quantified using the Bio-Rad protein assay according to the manufacturer’s instructions with bovine serum albumin used as the reference standard. Proteins were separated by electrophoresis on 10% and 15% SDS-polyacrylamide gels and electroblotted onto NC membranes (Hybond-C Extra, Amersham Biosciences, USA). The membrane was blocked for 1 h in Tris-buffered saline containing 0.1% Tween 20 and 5% powdered milk and, then, incubated 48 h at 4 ◦ C with primary antibody antiPARP antibody (Cell Signaling Technology, USA) or anti-caspase-3

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Methanol extract (S-00 , 8.5g with MeOH 10%) n-Hexane 50ml extract 3 times

Ether 50ml extract 3 times

n-Hexane layer (S-01, 6.14g)

EA 50ml extract 3 times Ether layer (S-02, 0.35g)

EA layer (S-03 ,0.21g)

H2O layer (S-04, 0.51g)

Fig. 1. Partitioning of the methanol extract of Garcinia multiflora fruit.

(8G10) (Cell Signaling Technology, USA) or ␤-actin (GeneTex). The membrane was then washed and incubated for 1 h at room temperature with horseradish peroxidase–labeled secondary antibody (Cell Signaling Technology, USA). Moreover, western blots were visualized by enhanced chemiluminescence with horseradish peroxidase–labeled secondary antibodies and the SuperSignal® HRP substrate (Pierce, USA). Finally, the image was quantified using Bio-Imaging Analysis System (FloGel (FGIS-2) Fluorescent Gel Image System). 3. Results 3.1. Bioassay-guided fractionation of the extract of G. multiflora’s fruits Bioassay-guided fractionation of the dried air-dried powdered fruits of G. multiflora was performed to determine the active constituents responsible for anti-hepatocellular carcinoma activity in the crude extract. The MeOH solution of crude extract was partitioned sequentially with hexane, ether, and ethyl acetate (Fig. 1). Each layer and the crude extract were tested for activity (Fig. 2).

Fig. 2. Anti-hepatoma effects of extracts on hepatoma cells. Evaluation of anti-hepatocellular carcinoma activity of each layer and the crude extract revealed that the n-hexane layer is the major portion and shows antitumor activity.

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Fig. 3. Effects of subfractions obtained from flash chromatography fractionation on anti-HCC activity. n-Hexane layer (S-01) was fractionated by flash chromatography to produce 17 fractions, in which fractions 10–14 had anti-hepatocellular carcinoma activity, Fraction 11 (22.4% of n-hexane layer) had the highest ability.

This table revealed that the n-hexane layer is the major portion and shows antitumor activity. The active fractions (n-hexane layer) were subjected to flash chromatography on Slica gel column and then eluted with n-hexane/ethylacetate to produce 17 fractions, in which fractions 10–14 had anti-hepatocellular carcinoma activity (Fig. 3). Fraction 11 (22.4% of n-hexane layer) had the highest ability to test with the anti-hepatocellular carcinoma. LC-MS and NMR analyses as well as literature data confirmed that the pure compound of Fraction 11 was guttiferone F (Fig. 4). 3.2. Growth inhibition of guttiferone F on hepatocellular carcinoma cells Cells were seeded onto 96-well plate at 5 × 103 ∼ 1 × 104 cells/well and treated with guttiferone F, nexavar as a positive control at various concentrations. The percentage of cell viability was determined by MTT assay after 3 days of treatment. A dose-dependent growth inhibition was observed at concentrations ranging from 3 to 100 ␮M. Cytotoxicity assay revealed that the IC50 values (half maximal inhibitory concentration for 72 h) of guttiferone F were 14.15 ± 2.36 ␮M in PLC/PRF/5, 10.17 ± 0.51 ␮M in HepG2 cells and 9.71 ± 1.28 ␮M l in Hep3B cells. In comparison, the IC50 values of Nexava were 8.35 ± 3.63 ␮M in PLC/PRF/5, 5.46 ± 0.72 ␮M in HepG2 cells and 7.81 ± 0.71 ␮M l in Hep3B cells (Table 1 and Fig. 5). Results of our in vitro anticancer study found that guttiferone F induced apoptosis by up-regulating cleaved

OH HO

O

O

Table 1 The IC50 of guttiferone F on hepatocelluar carcinoma (HCC) cell lines.

Guttiferone F Sorafenib

PLC/PRF/5

HepG2

Hep3B

14.15 ± 2.36 8.35 ± 3.63

10.17 ± 0.51 5.46 ± 0.72

9.71 ± 1.28 7.81 ± 0.71

pro-caspase-3 and PARP expresstion in PLC/PRF/5 cells. To further study the apoptotic mechanisms underlying the cytotoxic effects of guttiferone F, the effects of guttiferone F on the cleavage of pro-caspases-3 and poly (ADP-ribose) polymerase (PARP) were investigated after 24 h treatment. Uttiferone F at 10 ␮M induced the cleavage of pro-caspase-3 into its two active fragments (17 and 19 kDa). Guttiferone F also stimulated the cleavage of PARP, a DNA repair enzyme that is one of the cleavage targets of activated caspase-3. The treatment of guttiferone F after 2, 6, 8 and 24 h in PLC/PRF/5 cells, the result showed guttiferone F induced the cleavage of pro-caspase-3 and PARP at 2 hours (Fig. 6). 3.3. Inhibition of guttiferone F in the PLC/PRF/5 tumor growth xenograft model Further in vivo study indicated that guttiferone F inhibits tumor growth on s.c. xenograft tumor of a human liver cancer cell line PLC/PRF/5 in female SCID mice. Intraperitoneal injection (i.p.) with guttiferone F (25 mg/kg/day) for 14 days (day 7 to day 21) displayed an inhibitory effect on the tumor growth. At the end of the experiment, the mean tumor volume of the control mice was 1598 ± 242 mm3 , while those of the mice treated with guttiferone F were 1045 ± 216 mm3 . The tumor growth inhibition (TGI) was 35 ± 14%. During guttiferone F treatment, no evidence of toxicity responses was identified and the body weight did not significantly change in comparison with the vehicle group (Fig. 7). 4. Discussion

O

OH

Fig. 4. Graphic formula of guttiferone F.

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide. The incidence of HCC is more than 560,000 cases, and the 5-year survival rate is below 9%. Despite scientific advances and the implementation of measures for early detection of HCC in patients at risk of this disease, survival of patients has not improved

L.-T. Lee et al. / Biomedicine & Preventive Nutrition 3 (2013) 247–252

A

sorafenib

120

Guttiferone F

100

Viability

251

80 60 40 20 0 1

10

concentration (uM)

B

sorafenib

120

Guttiferone F

Viability

100 80 60 40 20 0 1

10

concentration (uM)

C

sorafenib

120

Guttiferone F

Fig. 6. Guttiferone F induced apoptosis by the cleavage of pro-caspase-3 and PARP in PLC/PRF/5 cells. A. The effects of guttiferone F on the cleavage of pro-caspases-3 and PARP were investigated after 24 h treatment. Guttiferone F at 10 ␮M induced the cleavage of pro-caspase-3 into its two active fragments (17 and 19 kDa) and stimulated the cleavage of PARP. B. The treatment of guttiferone F after 2, 6, 8 and 24 h in PLC/PRF/5 cells.

Viability

100 80 60 40 20 0 0.1

1

10

concentration (uM) Fig. 5. The cytotoxicity of guttiferone F on hepatocelluar carcinoma (HCC) cells. A. PLC/PRF/5. B. HepG2. C. Hep3B. Cytotoxicity assay revealed that the IC50 value (half maximal inhibitory concentration for 72 h) of guttiferone F was 14.15 ± 2.36 ␮M in PLC/PRF/5, 10.17 ± 0.51 ␮M in HepG2 cells and 9.71 ± 1.28 ␮M in Hep3B cells. In comparison, the IC50 value of Nexava was 8.35 ± 3.63 ␮M in PLC/PRF/5, 5.46 ± 0.72 ␮M in HepG2 cells and 7.81 ± 0.71 ␮M l in Hep3B cells.

significantly in recent decades [7]. Although sorafenib has shown a modest survival benefit for patients with early disease, further research is warranted to improve the clinical outcomes of patients with advanced and treatment-resistant HCC [8]. Previous studies have examined the anticancer activity of polyisoprenylated benzophenone and xanthon compounds isolated from Garcinia species such as garcinol [9–11], gambogic acid [12–28], mangostins [29–32], guttiferone K [33], oliganthin [34], and cluvenone [35]. This study screened approximately 300 plant extracts for in vitro anticancer activity against HepG2 and Huh-7 cell lines. According to our results, the fruit’s methanol extract of G. multiflora exhibited anti-hepatocellular carcinoma bioactivity.

Fig. 7. Role of guttiferone F in decreasing PLC/PRF/5 tumor growth in sc. Xenograft the in vivo model. A. PLC/PRF/5 cells were subcutaneously injected into SCID mice at 3 × 106 cells/mice. Guttiferone F was intraperitoneal (i.p.) injection into SCID mice daily after initial tumor injection (25 mg/kg). B. Body weight change effect of guttiferone F treatment in human PLC/PRF/5 bearing SCID mouse.

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The conventional approaches for studying the bioactivity components of natural products are direct isolation and purification of the metabolites of crude extracts followed by an evaluation of the bioactivity of every component. Although these direct approaches have led to the findings of bioactivity ingredients, bioassay-guided fractionation and purification would be a preferred means of linking the activity of crude extract directly to its active components. In this study, the methanol extract residue was partitioned with various solvents. The active portion was then subjected to flash chromatography to perform the following separation procedure. The active fraction was purified into a pure compound. LC-MS and NMR analysis as well as literature data confirmed that this compound is guttiferone F. Guttiferone F, the first prenylated benzophenone from Allanblackia stuhlmannii, has been found to possess anti-HIV activity [36]. Biological evaluation revealed that polyisoprenyl benzophenonoids from Garcinia subelliptica exhibited cytotoxic activity against several tumor cell lines, yet had not been studied in hepatocellular carcinoma. Our results further indicated that the bioactivity of guttiferone F is similar to that of nexavar, a therapeutic drug for liver cancer. Moreover, guttiferone F inhibited the proliferation activity of liver cancer cell lines and induced apoptosis by the cleavage of pro-caspase-3 and PARP in PLC/PRF/5. Another in vivo study was performed. According to those results, guttiferone F inhibited tumor growth on s.c. xenograft tumor of human liver cancer cell line (PLC/PRF/5) in female SCID mice. The tumor growth inhibition was 35 ± 14% for 14 days treatment. Consequently, guttiferone F is a promising lead for liver cancer therapeutic development. 5. Conclusions This study demonstrates that guttiferone F is the active constituent responsible for anti-hepatocellular carcinoma activity in the crude extract. Importantly, guttiferone F is a promising lead for liver cancer therapy. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements The authors would like to thank the Department of Industrial Technology (MOEA) of the Republic of China, Taiwan for financially supporting this research under Contract No. 101-EC-17-A-02-040317. References [1] Lin YM, et al. Antiviral activities of biflavonoids. Planta Med 1999;65:120–5. [2] Chiena SC, et al. A novel polyprenylated phloroglucinol, garcinialone, from the roots of Garcinia multiflora. Tetrahedron Lett 2008;49:5276–8. [3] Chiang YM, Kuo YH, Oota S, Fukuyama Y. Xanthones and benzophenones from the stems of Garcinia multiflora. J Nat Prod 2003;66:1070–3. [4] Liu X, Yu T, et al. Apoptotic effects of polyprenylated benzoylphloroglucinol derivatives from the twigs of Garcinia multiflora. J Nat Prod 2010;73:1355–9. [5] Ting CW, et al. A new benzoylphloroglucinol derivative with an adamantyl skeleton and other constituents from Garcinia multiflora: effects on neutrophil pro-inflammatory responses. Chem Biodivers 2012;9:99–105. [6] Chen JJ, Ting CW, Hwang TL, Chen IS. Benzophenone derivatives from the fruits of Garcinia multiflora and their anti-inflammatory activity. J Nat Prod 2009;72:253–8. [7] Spangenberg HC, Thimme R, Blum HE. Targeted therapy for hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 2009;6:423–32. [8] Finn RS. Development of molecularly targeted therapies in hepatocellular carcinoma: where do we go now? Clin Cancer Res 2010;16:390–7.

[9] Ahmad A, Sarkar SH, Bitar B, Ali S, Aboukameel A, Sethi S, et al. Garcinol regulates EMT and Wnt signaling pathways in vitro and in vivo leading to anticancer activity against breast cancer cells. Mol Cancer Ther 2012. [10] Saadat N, Gupta SV. Potential role of garcinol as an anticancer agent. J Oncol 2012. [11] Ahmad A, Wang Z, Wojewoda C, Ali R, Kong D, Maitah MY, et al. Garcinolinduced apoptosis in prostate and pancreatic cancer cells is mediated by NFkappaB signaling. Front Biosci (Elite Edition) 2011;3:1483–92. [12] Lupescu A, Jilani K, Zelenak C, Zbidah M, Shaik N, Lang F. Induction of programmed erythrocyte death by gambogic acid. Cell Physiol Biochem 2012;30:428–38. [13] Yang LJ, Chen Y, He J, Yi S, Wen L, Zhao S, et al. Effects of gambogic acid on the activation of caspase-3 and downregulation of SIRT1 in RPMI-8226 multiple myeloma cells via the accumulation of ROS. Oncol Lett 2012;3:1159–65. [14] Cheng H, Su JJ, Peng JY, Wang M, Wang XC, Yan FG, et al. Gambogenic acid inhibits proliferation of A549 cells through apoptosis inducing through upregulation of the p38 MAPK cascade. J Asian Nat Prod Res 2011;13:993–1002. [15] Wang K, Tang Y, Sun M, Lu B, Zhu H, Ji O, et al. The mechanism of neogambogic acid-induced apoptosis in human MCF-7 cells. Acta Biochim Biophys Sin (Shanghai) 2011;43:698–702. [16] Ren Y, Yuan C, Chai HB, Ding Y, Li XC, Ferreira D, et al. Absolute configuration of (-)-gambogic acid, an antitumor agent. J Nat Prod 2010;74:460–3. [17] Liu Y, Li W, Ye C, Lin Y, Cheang TY, Wang M, et al. Gambogic acid induces G0/G1 cell cycle arrest and cell migration inhibition via suppressing PDGF receptor ␤ tyrosine phosphorylation and Rac1 activity in rat aortic smooth muscle cells. J Atheroscler Thromb 2010;17:901–13. [18] Li Q, Cheng H, Zhu G, Yang L, Zhou A, Wang X, et al. Gambogenic acid inhibits proliferation of A549 cells through apoptosis-inducing and cell cycle arresting. Biol Pharm Bull 2010;33:415–20. [19] Yang J, Ding L, Jin S, Liu X, Liu W, Wang Z. Identification and quantitative determination of a major circulating metabolite of gambogic acid in human. J Chromatogr B Analyt Technol Biomed Life Sci 2010;878:659–66. [20] Nie F, Zhang X, Qi Q, Yang L, Yang Y, Liu W, et al. Reactive oxygen species accumulation contributes to gambogic acid-induced apoptosis in human hepatoma SMMC-7721 cells. Toxicology 2009;260:60–7. [21] Chen J, Gu HY, Lu N, Yang Y, Liu W, Qi Q, et al. Microtubule depolymerization and phosphorylation of c-Jun N-terminal kinase-1 and p38 were involved in gambogic acid induced cell cycle arrest and apoptosis in human breast carcinoma MCF-7 cells. Life Sci 2008;83:103–9. [22] Qi Q, You Q, Gu H, Zhao L, Liu W, Lu N, et al. Studies on the toxicity of gambogic acid in rats. J Ethnopharmacol 2008;117:433–8. [23] Qiang L, Yang Y, You QD, Ma YJ, Yang L, Nie FF, et al. Inhibition of glioblastoma growth and angiogenesis by gambogic acid: an in vitro and in vivo study. Biochem Pharmacol 2007;75:1083–92. [24] Lu N, Yang Y, You QD, Ling Y, Gao Y, Gu HY, et al. Gambogic acid inhibits angiogenesis through suppressing vascular endothelial growth factor-induced tyrosine phosphorylation of KDR/Flk-1. Cancer Lett 2007;258:80–9. [25] Yang Y, Yang L, You QD, Nie FF, Gu HY, Zhao L, et al. Differential apoptotic induction of gambogic acid, a novel anticancer natural product, on hepatoma cells and normal hepatocytes. Cancer Lett 2007;256:259–66. [26] Han Q, Yang L, Liu Y, Wang Y, Qiao C, Song J, et al. Gambogic acid and epigambogic acid, C-2 epimers with novel anticancer effects from Garcinia hanburyi. Planta Med 2006;72:281–4. [27] Guo QL, Lin SS, You QD, Gu HY, Yu J, Zhao L, et al. Inhibition of human telomerase reverse transcriptase gene expression by gambogic acid in human hepatoma SMMC-7721 cells. Life Sci 2006;78:1238–45. [28] Zhang HZ, Kasibhatla S, Wang Y, Herich J, Guastella J, Tseng B, et al. Discovery, characterization and SAR of gambogic acid as a potent apoptosis inducer by a HTS assay. Bioorg Med Chem 2004;12:309–17. [29] Chang HF, Yang LL. Gamma-mangostin, a micronutrient of mangosteen fruit, induces apoptosis in human colon cancer cells. Molecules 2012;17:8010–21. [30] Kosem N, Ichikawa K, Utsumi H, Moongkarndi P. In vivo toxicity and antitumor activity of mangosteen extract. J Nat Med 2012. [31] Johnson JJ, Petiwala SM, Syed DN, Rasmussen JT, Adhami VM, Siddiqui IA, et al. ␣-Mangostin, a xanthone from mangosteen fruit, promotes cell cycle arrest in prostate cancer and decreases xenograft tumor growth. Carcinogenesis 2012;33:413–9. [32] Krajarng A, Nakamura Y, Suksamrarn S, Watanapokasin R. ␣-Mangostin induces apoptosis in human chondrosarcoma cells through downregulation of ERK/JNK and Akt signaling pathway. J Agric Food Chem 2011;59:5746–54. [33] Kan WL, Yin C, Xu HX, Xu G, To KK, Cho CH, et al. Antitumor effects of novel compound, guttiferone K, on colon cancer by p21Waf1/Cip1-mediated G(0)/G(1) cell cycle arrest and apoptosis. Int J Cancer 2012. [34] Gao XM, Yu T, Cui MZ, Pu JX, Du X, Han QB, et al. Identification and evaluation of apoptotic compounds from Garcinia oligantha. Bioorg Med Chem Lett 2012;22:2350–3. [35] Batova A, Altomare D, Chantarasriwong O, Ohlsen KL, Creek KE, Lin YC, et al. The synthetic caged garcinia xanthone cluvenone induces cell stress and apoptosis and has immune modulatory activity. Mol Cancer Ther 2010;9:2869–78. [36] Fuller RW, Blunt JW, Boswell JL, Cardellina JH, Boyd MR. Guttiferone F, the first prenylated benzophenone from Allanblackia stuhlmannii. J Nat Prod 1999;62:130–2.