Secondary metabolites of oil palm isolates of Ganoderma zonatum Murill . from Cameroon and their cytotoxicity against five human tumour cell lines

Three lanostane-type triterpenoids [lanosta-7,9(11),24-trien-3-one 15,26-dihydroxy, lanosta-7,9(11),24trien-26-oic,3-hydroxy and ganoderic acid y], four steroids[ (22E,24R)-ergosta-7,22-dien-3β,5α.6β-triol, 5α,8α-epidiory (22E,24R-ergosta-6,22-dien-3β-ol, ergosta-5,7,22-trien-3β-ol,7 (ergosterol) and ergosta7,22-dien-3β-ol,6] and a benzene derivative (dimethyl phthalate) were isolated from ethyl acetate crude extract of Ganoderma zonatum Murill. of oil palm from Cameroon. Their structures were elucidated by nuclear magnetic resonance (NMR), electron impact ionization mass spectrum experiments (EI-MS) and by comparing with the data reported in literature. The highly oxygenated lanostane triterpenoid ganoderic acid yshowed moderate cytotoxicity against two human tumour cell lines, SMMC-7721 (liver cancer) and A549 (lung cancer) with IC50 values of 33.5 and 29.9 μM, respectively and no activity on HL-60, MCF-7 and SW480, while lanosta-7,9(11),24-trien-3-one,15;26-dihydroxy and lanosta7,9(11),24-trien-26-oic,3-hydroxy, showed no activity. The three lanostane triterpeniods had the same molecular formula, molecular weight, behaviour on the TLC plates, with the same retention factor value of 3 (when petroleum ether-acetone was used in the ratio of 3:1), but with slightly different structures. These compounds have not been reported occurring together from any other species of Ganoderma. Their simultaneous occurrence might thus, serve as a diagnostic chemotaxonomic character for G. zonatum, The 1 H NMR data is provided for the first time for lanosta-7,9(11),24-trien-26-oic,3-hydroxy.


INTRODUCTION
Ganoderma species belong to the division Basidiomycota, class Homobasidiomycetes, order Aphyllophorales and family Polyporaceae (Alexopoulos et al., 1996;Wasser and Weis, 1999a).Ganoderma species are not listed among the group of edible mushrooms because the fruiting bodies are always thick, corky and tough and do not have the fleshy texture characteristics of true edible fungi (Jong and Birmingham, 1991).Although species of Ganoderma cannot be eaten directly, they have attracted great attention all over the world because of their wide range of pharmacological values (Yoon et al., 1994;Wasser and Weis, 1999b).The bioactive compounds from them have been implicated for their high antioxidant, immune-regulatory and hypoglycemic activities.They have been known for their antihydrogenic, antitumor, antihepatotoxic, antinocieptic, immunodulatory, cardiovascular, antibacterial and antiviral values (Chang and Buswell, 1996;Chang and Mshigeni, 2001).
Basidiomycetes, also called macromycetes, are among the many diverse organisms and are a major source of biologically active natural products (Liu, 2004).They provide a rich variety of active secondary metabolites (Turner, 1971).The number of different mushroom species on earth is estimated at 140000, of which only about 10% are known and from those, approximately 700 species are known to possess significant pharmacological properties (Lull et al., 2005;Chang, 1996).Medicinal basidiomycetes represent an unlimited source of phytochemicals such as primary and secondary metabolites (Roja and Rao, 1998).Quite a wide range of substances from higher basidiomycetes belonging to different classes of chemical compounds have been described and their biological properties evaluated (Lorenzen and Anke, 1998;Mizuno, 1999).
Over the past three decades, scientists all over the world have isolated more than 150 triterpenes and 50 pharmacologically active polysaccharides from different species of Ganoderma (Kim and Kim, 2002;Lin and Chou, 1984;Jong and Birmingham, 1992).Some of those bioactive compounds obtained from different basidiomycetes are a variety of terpenes, sesquiterpenes, steroids and polysaccharides (alone or forming complexes with proteins, like PSK), among others (Lindequist et al., 2005).Most of their main pharmacological properties like their antitumoral activity have been attributed to their ability to modulate the immune system.Most of them are immunostimulants, but others are immunosuppressive.
Basidiomycetes mushrooms have been used in folk medicine throughout the world since ancient times (Arisawa et al., 1986;Wasser, 1999;Stamets, 2000).Medicinal mush-rooms useful against cancer are known in China, Russia, Japan, Korea, as well as the USA and Canada, but less known in most African countries.Members of the genus Ganoderma have also been used in traditional medicine for centuries in, Japan and Korea.In 1997, the worldwide production of Ganoderma was approximately 4,300 tons, of which China contributed 3,000 tons (Chiu et al., 2000).Traditionally in China, Ganoderma is highly regarded as a herbal treatment and is claimed to alleviate or cure virtually all diseases.
The medicinal value of the mushrooms is largely a function of their secondary metabolites (Hajjaj et al., 2005).A large number of mushroom-derived compounds, both cellular components and secondary metabolites, have been shown to affect the immune system and could be used to treat a variety of disease states.Compounds that appear to enhance or potentiate host resistance are being sought for the treatment of cancer, immunodeficiency diseases or generalized immune suppression after drug treatment (Hajjaj et al., 2005).More than 140 highly oxygenated lanostane-type triterpenoids have been isolated from the fruiting bodies, mycelia and spores of Ganoderma lucidium, some of them exhibiting a very broad spectrum of biological activities and pharmacological functions.Some of them have been shown to inhibit histamine release from rats mast cells (Komoda et al., 1989), angiotensin-converting enzyme and hypocholesterol activities (Hajjaj et al., 2005).
Ganoderic acids and other triterpenoids have received considerable attention due to their conspicuous pharmacological activities.Despite the valuable dietary and therapeutic benefits of G. lucidum and related species, phytochemical evaluation of the active components have been performed predominantly in China, Korea, Japan and the United states (Paterson, 2006).
Herbalists usually consider Ganoderma as natural regulator, suppressing the immune system if it is Kinge and Mih 8441 overactive and boosting it if it is underactive such that traditional healers among the Yoruba people of southwestern Nigeria have used species of Ganoderma in the treatment of skin disorder, high blood pressure and intestinal disorder (Gao and Yang, 1991).They usually regard G. lucidum as immune booster especially when combined with other medicinal ingredients.
In Cameroon and other countries such as Malaysia and Indonesia, species of Ganoderma are highly prevalent in oil palm plantations, where they are considered pathogenic (Turner, 1981;Utomo et al., 2005) and several control measures have been considered which are aimed at killing or reducing the basidiocarp development.Ethnomycological studies in Cameroon revealed that a species of Ganoderma identified as G. lucidium (W.Curt.Fr.)P. Karst is used in the treatment of skin infections, boils, abscesses, tumours and also as a component in other medicinal preparations (Yongabi et al., 2004).There is however, paucity of information on the chemical constituents of species of Ganoderma and other macro-fungi in general in Cameroon as is the case with other Sub-Sahara African countries.The aim of this study was to identify the chemical constituents of the Cameroonian oil palm isolate of Ganoderma zonatum and subsequently, to screen some of the pure compounds against cancer cell lines in vitro as a rationale for the conservation of this medicinal fungus.

General experimental procedures
Optical rotations were measured on a Horiba SEPA-300 polarimeter.UV spectra were measured in Shimadzu UV-2401 PC spectrophotometer.IR spectra were obtained on a Tensor 27 with KBr pellets.NMR spectra were recorded on Bruker AV-400 and Bruker DRX-500 spectrometer.Chemical shifts (δ) were expressed in ppm with reference to the solvent signals.FAB-MS were recorded with a VG Autospec-3000 spectrometer.EI-MS were recorded with a VG Autospec-3000 spectrometer.ESI-MS and HRESI-MS were recorded with an API QSTAR Pulsar 1 spectrometer.Preparative HPLC was performed on an Agilent 1100 series with a Zorbax SB-C18 (5 µm, 9.4 × 150 mm) column.Preparative MPLC was performed on Büchi apparatus equipped with Büchi fraction collector C-660, Büchi pump module C-605 and manager C-615.Silica gel (200 to 300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), RP-18 gel (40 to 75 µm, Fuji Silysia Chemical Ltd., Aichi, Japan) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) were used for column chromatography.Fractions were monitored by TLC and spots were visualized under visible and UV light (254 and 365 nm).Spots were also visualized by heating silica gel plates sprayed with 10% H2SO4 in ethanol.All solvents used were of analytical reagent grade.

Collection and identification of fungus
Ganoderma species were collected from naturally infected oil palm plants of the PAMOL Plantations Ltd, Lobe, South West Region, Cameroon and were identified using gross morphology and microscopy (Ryvarden, 1984).The gross morphology is exemplified in Figure 1.

Extraction and isolation of secondary metabolites
Fruiting bodies of G. zonatum (1.4 kg) were oven dried at 70°C for 48 h.The dried samples were powdered and then extracted three times with 8 L of chloroform/methanol in 1:1 ratio (v/v) at room temperature.The combined extract was evaporated in a rotator evaporator and concentrated in vacuo and the residue was suspended in water and partitioned with petroleum ether, ethylacetate and n-hexane.The ethylacetate extract (240 g) was subjected to MPLC and then TLC and like fractions were combined to give three fractions.

Cytotoxicity assay
following human tumour cell lines were used: breast cancer MCF-7, hepatocellular carcinoma SMMC7721, human myeloid leukaemia HL-60, colonic cancer W480 and lung cancer A549.These are cell lines maintained at the Kunming Institute of Botany of the Chinese Academy of Sciences, Kunming, China.All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% foetal bovine serum (Hyclone, USA) at 37°C in a humidified atmosphere with 5% CO2.Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), purchased from sigma, USA.Briefly, 100 µl adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with initial density of 1 × 105 cells/ml in 100 µl medium.Each tumour cell line was exposed to the test compound at various concentrations (0.064, 0.32, 1.6, 8 and 40 µM) in triplicates for 48 h, with cisplatin and taxol (sigma, USA) as positive control.After the incubation, MTT (100 µg) was added to each well and the incubation continued for 4 h at 37°C.The cells were lysed with 100 µl 20% SDS-50% DMF after of 100 µl medium.The optical density of the lysate was measured at 595 nm in a 96-well microtitre plate reader (Bio-Rad 680, USA).The concentration of inhibitors that result in 50% inhibition of enzyme activity (IC50) was calculated from the leastsquares regression line of the logarithmic concentrations plotted against the remaining activity.

RESULTS
Following purification of the various fractions by preparative TLC methods, the spectral analysis revealed the following data for the eight compounds:
of the fungus, their simultaneous occurrence in G. zonatum is reported here for the first time.This simultaneous occurrence could be used as a diagnostic feature for the species and could serve as a chemotaxonomic character.
The anti-tumour effects of Ganoderma are apparently mediated by numerous biologically active compounds such as polysaccharides, triterpenes and immunomodulatory proteins (Silva et al., 2002).Due to differences in climate, composition of extracts and culturing methods, individual species of Ganoderma may differ in their bioactivity (Chen et al., 2004).
For the active compound ganoderic acid Y, the percentage cell viability of liver and lung cancer cell lines was less than 50% at 40 µM concentration, while for HL-60 (leukaemia), MCF-7 (breast cancer) and SW480 (colon cancer) it was greater or equal to 50% at the same concentration.The reasons for these differences are unclear and there is need for further investigation on the underlying mechanism of the action of ganoderic acid Y on liver and lung cancer cell lines.
Ganoderic acid Y (113-b), an oxygenated lanostane type triterpenoid, has been shown to have biological activity.For example, Haijaji et al. (2005) showed that it inhibits cholesterol biosynthesis.Also, Berger et al. (2004) found that organic fractions of G. lucidum containing oxygenated lanosterol derivatives inhibited cholesterol synthesis in T9A4 hepatocytes.Toth et al. (1983) also isolated ganoderic acid Y from Ganoderma lucidum and found that it showed cytotoxic activity in vitro on hepatoma cells, while Huie and Di (2004) showed that ganoderic acid Y from the fruiting body of G. lucidum possess antihypertensive properties and ACE (Angiotensin-converting enzymes).In this study, it was found to have moderate antiproliferative activity against two cancer cell lines; liver and lung cancer cell lines, respectively.This affirms the claims of the use of this fungus for the treatment of tumours by traditional medical practitioners in Cameroon (Yongabi et al., 2004).This shows that ganoderic acid Y (113-b) has pharmaceutical potentials and G. zonatum could serve as a source of this compound.

Conclusion
Based on the literature available to us, this is the first report on the isolation, structural elucidation and bioactivity of compounds from the fruiting bodies of G. zonatum.It serves as a baseline study for further research on this medicinal mushroom species.The three highly oxygenated lanostane triterpenoids isolated from G. zonatum, can be used as a taxonomic marker for this species since they possess the same molecular weight, molecular formula and retention factor value, but with slightly different structures.Our recent surveys revealed that G. zonatum, a member of the G. lucidum complex, was among the most prevalent species on oil palm in south western Cameroon.These could serve as a source of secondary metabolites.There is need for further research in vivo to ascertain the efficacy of ganoderic acid Y on lung (A-549) and liver (SMMC-7721) cancer cell lines, since they are natural products.There is also the need for the research on different species of Ganoderma and other macro fungi from Cameroon and Africa for their secondary metabolites and subsequently, their bioactivity against different cell lines.

Figure 1 .
Figure 1.Percentage cell viability of five tumor cell lines against different concentrations (µM) of ganoderic acid Y.

Table 1 .
Structure, molecular formula and weight of eight secondary metabolites from a Cameroonian oil palm isolate of G.zonatum Murill.