Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 18  |  Issue : 2  |  Page : 102-110

GC-MS analysis and in-vitro hypocholesterolemic, anti-rotavirus, anti-human colon carcinoma activities of the crude extract of a Japanese Ganoderma spp


1 Chemistry of Natural and Microbial Products Department, Pharmaceutical Industries Researches Division, National Research Centre, Dokki, Egypt
2 Drug Bioassay-Cell Culture Laboratory, Pharmacognosy Department, National Research Centre, Dokki, Egypt
3 Environmental Virology Laboratory, Water Pollution Research Department, Environmental Research Division, National Research Centre, Dokki, Egypt
4 Mycorrhizal Systems Ltd, Lancashire; University of Stirling, Stirling, UK
5 The Engineering Research Center of Southwest Bio-Pharmaceutical Resources, Ministry of Education, Guizhou University, Guiyang, China

Date of Submission25-Nov-2018
Date of Acceptance02-Jan-2019
Date of Web Publication05-Jul-2019

Correspondence Address:
Ghoson M Daba
Chemistry of Na tural and Microbial Products Department, National Research Centre, Dokki, Giza 12622
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/epj.epj_50_18

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  Abstract 

Background and objective Medicinal mushrooms are mines of various biologically active compounds. Therefore, chemical analysis and in-vitro evaluation of some biological activities of the Japanese originated mushroom Ganoderma spp. were conducted.
Materials and methods Extraction of the fruiting bodies of Ganoderma spp. was accomplished using 80% methanol. This extract was investigated for its in-vitro cholesterol-lowering activity, anti-rotavirus effect, and anti-human colon cancer influence. Moreover, a gas chromatography–mass spectrometry analysis for this extract was performed.
Results and conclusion The gas chromatography–mass spectrometry analysis resulted in the detection of 39 compounds, which were generally saturated and unsaturated fatty acids, and alkenes. The crude extract exhibited a promising in-vitro cholesterol-lowering activity (100±0%) after 96 h of incubation at room temperature. The same crude extract showed a moderate anti-rotavirus SA-11 strain effect with a therapeutic index of 9.3. Moreover, Ganoderma spp. extract displayed a strong activity toward HCT116 human colon carcinoma cell line, resulting in a cytotoxicity of 84.03±0.93% on HCT116 cell line monolayers. Ganoderma spp. crude extract represents a promising source of biologically active compounds that could by further investigations represent support and/or alternative to the currently used drugs.

Keywords: biological activity, Ganoderma, gas chromatography–mass spectrometry, human colon cancer, hypocholesterolemic activity, rotavirus


How to cite this article:
Elkhateeb WA, Daba GM, Sheir D, El-Dein AN, Fayad W, Elmahdy EM, Shaheen MN, Thomas PW, Wen TC. GC-MS analysis and in-vitro hypocholesterolemic, anti-rotavirus, anti-human colon carcinoma activities of the crude extract of a Japanese Ganoderma spp. Egypt Pharmaceut J 2019;18:102-10

How to cite this URL:
Elkhateeb WA, Daba GM, Sheir D, El-Dein AN, Fayad W, Elmahdy EM, Shaheen MN, Thomas PW, Wen TC. GC-MS analysis and in-vitro hypocholesterolemic, anti-rotavirus, anti-human colon carcinoma activities of the crude extract of a Japanese Ganoderma spp. Egypt Pharmaceut J [serial online] 2019 [cited 2019 Sep 17];18:102-10. Available from: http://www.epj.eg.net/text.asp?2019/18/2/102/262144


  Introduction Top


According to the world health organization (WHO), about 17.7 million people die annually from cardiovascular diseases (CVDs), which represents about 31% of mortalities worldwide [1],[2]. CVD is associated with hypercholesterolemia, atherosclerosis, and lactate dehydrogenase oxidation. Hence, regulating or lowering the cholesterol level is the key factor in the treatment and prevention of CVD.

Lovastatin and its analogs are famous cholesterol-lowering agents, commonly referred to as statins, which act as inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase [3]. Despite their widespread use within the population, they are not without risk. It is broadly accepted that contraindications and interactions with certain foods exist, but further there are many side effects reported from statin use and these may be severe enough to require immediate dose reduction or cessation of medication. These statin-associated symptoms include diabetes mellitus, statin-associated muscle symptoms, and central nervous system complaints [4]. Such serious side effects, along with contraindications and interactions, present the need to identify and develop novel cholesterol-lowering compounds other than statins.

Rotavirus is a highly contagious infectious agent causing high rates of mortalities in developing countries, especially among newborns, infants, and young children [5],[6]. According to the WHO reports, each year about 450 000 children under 5 years of age die because of diarrhea caused by rotavirus [7]. Till now, no drugs are available to treat rotavirus nor to prevent the diarrhea resulting from it [8]. The widespread existence and frequent epidemics of this dangerous virus encourage a rapid search for natural, effective, and safe compounds that exhibit a therapeutic effect toward rotavirus.

Worldwide, colorectal cancer (also termed colon cancer) is the third most commonly diagnosed cancer, after lung and breast cancers. Also, it represents the second biggest cause of cancer deaths, resulting in about 862 000 deaths annually, according to the WHO report [9]. Therefore, there is a critical need to identify further compounds that may provide effective activity against such lethal diseases.

Medicinal and edible mushrooms are natural sources of various compounds, and are used in Asian traditional medicine from the millennia as a medicinal supplementary food to treat and prevent numerous diseases [10]. Many studies have investigated the pharmaceutical characteristics of certain fungal species including their activities such as antimicrobial, antiviral, anticancer, anti-inflammatory, immunomodulating, hypocholesterolemic, hypoglycemic, antiatherogenic, and hepatoprotective agents [11],[12],[13],[14],[15],[16].

Ganoderma is a genus that includes about 80 species, and belongs to the family Ganodermataceae [17]. Ganoderma has been used from centuries in traditional oriental medicine and specifically in Japan, China, and Korea [18]. Currently, Ganoderma is available worldwide as a food supplement. Whole Ganoderma or their crude extracts have been intensively investigated for their anti-inflammatory effect [19].

In this study, a gas chromatography–mass spectrometry (GC–MS) analysis of the 80% methanolic extract of the fruiting bodies of a Japanese originated Ganoderma spp. was performed. Moreover, different concentrations of this extract were investigated for their in-vitro cholesterol-reducing activity (CRA) after different incubation times. The antiviral effect of Ganoderma spp. extract was also investigated toward rotavirus SA-11 strain. Finally, the same extract was examined for its anticancer activity against HCT116 human colon carcinoma cell line.


  Materials and methods Top


Collection and identification of the mushroom

The mature mushroom fruiting bodies were found growing in the wild, on the decaying wood of a Japanese cherry (sakura) tree (Prunus serrulata) within a park in Chihaya, Higashi ward, Fukuoka Prefecture, Japan. The fruiting body was removed and identified as belonging to the Ganoderma genus according to the classification criteria described in the comprehensive guide of the mushroom identification book [20].

Extraction of the metabolites from the mushroom

Approximately 250 g of Ganoderma spp. fruiting bodies were washed with distilled water, air dried, and then cut into small pieces and placed in an Erlenmeyer flask containing 80% methanol at room temperature and kept overnight before filtering. The resulting filtered extract was concentrated at 37°C using a rotary evaporator. The obtained extract was stored at 4°C in a clean closed container until further use.

GC–MS analysis

The analysis of the Ganoderma spp. crude extract was performed using a GC–MS instrument (TRACE GC Ultra Gas Chromatographs; THERMO Scientific Corp., Waltham, Massachusetts, USA), coupled with a THERMO mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer, Thermo Scientific, San Jose, California, USA). The GC–MS system was equipped with a TG-WAX MS column (30 m×0.25 mm daily, 0.25-µm film thickness). Analysis was carried out using helium as carrier gas at a flow rate of 1.0 ml/min and a split ratio of 1 : 10 using the following temperature program: 60°C for 1 min; rising at 3.0°C/min to 240°C and held for 1 min. The injector and the detector were held at 240°C. Diluted samples (1 : 10 chloroform, v/v) of 0.2 µl of the mixtures were always injected automatically in the splitless mode. Mass spectra were obtained by electron ionization at 70 eV, using a spectral range of m/z 40–450. Most of the compounds were identified using the analytical method: mass spectra (authentic chemicals, Wiley spectral library collection and NSIT library).The quantification of the components was based on the metabolites as detected by the mass spectrometer. Identification of the constituents was carried out by comparison of their retention times and fragmentation pattern of mass with those of published data [21] and/or with those of the Wiley 9 and NIST08 mass spectral libraries.

In-vitro cholesterol reduction assay

Overall, 0.4 g of the methanolic extract of Ganoderma spp. was dissolved in 5 ml distilled water; then different dilutions of this mixture were prepared as illustrated in [Table 1]. After that, mixtures were supplemented with 1 ml of soluble cholesterol to bring the total volume to 5 ml. These different mixtures were incubated at room temperatures for 24, 48, 72, and 96 h. Cholesterol assay was then performed using the cholesterol assay kit (Biodiagnostic, Cairo, Egypt) to determine the residual amount of cholesterol in the spent broth. A measure of 4 ml of distilled water supplemented with 1 ml of soluble cholesterol was used as a control. Finally, the percentage of cholesterol-reducing activity (CRA) was calculated as described previously [22] as follows:
Table 1 Preparation of different concentrations of Ganoderma spp. crude extract mixture for cholesterol-reducing activity (CRA) assay

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where A0 is the absorbance of the control (500 nm) and A is the absorbance of the sample (500 nm). Tests were carried out in triplicate.

XXXAntiviral activity of crude extract against rotavirus SA-11

Cell lines and virus titration

The Rhesus monkey kidney cell line (MA 104) was used in this study for culturing of the simian rotavirus SA-11 strain. MA 104 cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM). The media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 µg/ml streptomycin and 100 units/ml penicillin, and 1% HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid). The cell culture was then incubated under humidified 5% CO2 atmosphere in CO2 incubator. The medium used for both cytotoxicity and antiviral assays was containing only 2% of FBS. RV SA-11 for antiviral experiments was activated by 10 mg/ml trypsin for 30 min at 37°C. RV SA-11 stock was titrated using MA 104 in 96-well microtiter plates as described previously by Shaheen et al. [23]. The viral titers were calculated as TCID50/0.1 ml (50% tissue culture infectious doses/0.1 ml) according to Spearman–Kärber formula [24]. RV SA-11 stock was kept in small aliquots at −80°C until use.

Cytotoxicity assay

Different concentrations from the Ganoderma spp. methanolic crude extract (7.8, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 µg/ml) were prepared in DMEM (containing 2% FBS and 2% antibiotics). The cytotoxic activities of the tested extract was examined onto MA 104 by using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [25]. Briefly, the cell lines (5×103 and 5×104 cells/well) were seeded in 96-well microtiter plates. After 24 h in 5% CO2 incubator at 37°C, the cell monolayers were treated with various concentrations of the extract (each dilution in triplicate). Cell control was included using only the medium. The treated or nontreated cells were incubated for 2 days at 37°C in a 5% CO2 incubator with checking the cell morphology under inverted microscope daily. After the previous incubation period, the culture medium was discarded and replaced by 100 µl of MTT solution (5 mg/ml) for 4 h at 37°C in a CO2 incubator. After that, MTT solution was removed and replaced by 50 µl DMSO/well. After 30 min at 37°C, the optical densities were measured using an enzyme-linked immunosorbent assay reader at 540 nm. The percentage of cytotoxic effects was calculated as [(CTC)×100], where C and T refer to the optical densities of cell control and treated cells, respectively.

XXXAntiviral activity of crude extract on RV SA-11 by the MTT method

MA 104 cells at a concentration of 5×104 cells/well were cultured for 24 h in a CO2 incubator at 37°C in 96-well microtiter plates. After removing the culture media, three nontoxic concentrations of the crude extract were tested against viral infections. A measure of 50 µl of 106 TCID50 virus suspensions was incubated with 50 µl of culture media (with or without the test compound) in humidified 5% CO2 atmosphere for 1 h at 37°C and then the mixed solution was added to cell monolayers. After 1 h in CO2 incubator, the mixed solution was removed. The cell lines were washed two times with a culture medium and then 200 µl of infectious medium (FBS free DMEM containing 2 µl of trypsin) was added to the cells. Virus controls, containing the virus suspension, and cell controls, containing culture medium, were included in the assay. All plates were incubated for 3 days at 37°C in a CO2 incubator and the cytopathic effect of the virus was monitored daily and then measured by the MTT as described above. The percentage protection was calculated as [(TV)/(CV)×100], where T, V, and C are the absorbance readings of the extract with virus, virus control, and cell control, respectively. Therapeutic index (TI) of the tested extract was calculated as ratio CC50 over IC50.

Effect of crude extract on HCT116 human colon carcinoma cell lines

Cell culture

HCT116 colon carcinoma human tumor cell lines were cultured in 95% humidity, 5% CO2 at 37°C<AQ: Pls check whether the change retains the intended meaning>. The cell line was maintained in McCoy’s 5 A medium supplemented with 10% FBS.

Cytotoxicity assay

The acid phosphatase assay was used to assess cytotoxicity according to the method described by Yang et al. [26]. Overall, 10 000 cells were seeded per well in 96-well plates, left to attach overnight, and then treated with samples for 3 days. For one plate, a substrate solution was prepared where 20 mg tablet of p-nitrophenyl phosphate (cat. no. N2765; Sigma, Darmstadt, Germany) was dissolved in 10 ml buffer solution (0.1 mol/l sodium acetate, 0.1% triton X-100, pH=5). Cell monolayers were washed with 250 µl PBS. One hundred microliter of pNPP substrate solution was added per well, then the plates were incubated for 4 h at 37°C. Ten microliter of 1 N sodium hydroxide stop solution was added per well. Absorbance was measured directly at a wavelength of 405 nm. All samples were tested in triplicates, and 0.5% DMSO was used as negative control and 50 µmol/l cisplatin was used as positive control. The sample was tested at serial dilutions with a final concentration of 400, 200, 100, 50, 25, 12.5, and 6.25 µg/ml. Percent cytotoxicity was calculated by the formula



where D and S denote the optical density of drug-treated and solvent-treated wells, respectively.


  Results Top


GC/MS analysis

As shown in the chromatogram in [Figure 1], GC–MS analysis of the crude extract of Ganoderma spp. showed the presence of about 60% oxygenated compounds and 40% nonoxygenated compounds. Most of the compounds (listed in [Table 2]) were alkenes, saturated and unsaturated fatty acids, such as pentadecane; hexadecane; octadecane; eicosane; tricosane; decosane; pentacosane; heneicosane; 11-(1-ethylpropyl); methyl-18-methylnonadecanoate; 17-pentatriacontene; (Z)-9-octadecenamide; tetratetracontane; docosanoic acid methyl ester; 3-nitro-1,2-benzenedicarboxylic acid;, 1-heptatriacotanol), tricosanoic acid, methyl ester, decosane; 2, 6, 10, 14, 18, 22-tertacosahexaene; 2, 6, 10, 15, 19, 23-hexamethyl; and others.
Figure 1 Gas chromatography–mass spectrometry chromatogram for the methanolic extract of Ganoderma spp. fruiting bodies.

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Table 2 List of compounds identified from the methanolic extract of Ganoderma spp. by GC–MS analysis

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Hypocholesterolemic activities of the Ganoderma spp. methanolic crude extract

The results shown in [Table 3] showed that the methanolic extract of Ganoderma spp. exhibited high cholesterol reduction activity in vitro with results ranging from 35.1±1.51 to 63.5±1.06% after 24 h, from 54.2±0.95 to 77.3±0.60% after 48 h, from 72.6±1.85 to 90.5±1.05% after 72 h, and from 83.4±1.93 to 100%±0 after 96 h depending on the concentration of the extract. The highest cholesterol-reducing activity of Ganoderma spp. was achieved after 96 h of incubation by using concentration 5, which is equivalent to using 32 mg/ml of the methanolic crude extract.
Table 3 In-vitro cholesterol-reducing activity of the methanolic extract of Ganoderma spp.

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The anti-rotavirus SA-11 activity of Ganoderma spp. extract

The cytotoxicity of the methanolic crude extract of Ganoderma spp. was investigated on MA 104 cells by the help of the MTT colorimetric assay. As shown in [Table 4], Ganoderma spp. exerted toxic effects on MA 104 cells with CC50 of 650±0.80 μg/ml. This result indicated that this methanolic extract exhibited a promising anti-rotavirus activity with a TI of 9.3.
Table 4 Results of cytotoxicity and antiviral activity of the methanolic extract of Ganoderma spp. on MA 104 cells using the MTT method

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The anti-HCT116 human colon carcinoma activities of the methanolic crude extract

The cytotoxic effect of the methanolic crude extract was evaluated against HCT116 human colon carcinoma cell line. Results represented in [Figure 2] suggested that this extract had a promising cytotoxic effect, and that the sensitivity of the treated colon cells was concentration dependent. Treatment with Ganoderma spp. in concentration of 100 μg/ml resulted in a cytotoxicity of 84.03±0.93% whereas the positive control (cisplatin) in concentration of 50 μmol/l caused only 70.18±4.46% cytotoxicity.
Figure 2 Cytotoxicity % of Ganoderma spp. methanolic extracts on HCT116 cell line monolayers. Error bars represent the SD of three independent experiments.

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  Discussion Top


Species within the Ganoderma genus are proving to be promising sources of compounds with important biological activities. Many studies have previously reported numerous pharmacological properties of species within the Ganoderma genus [18],[27],[28],[29],[30],[31],[32],[33].

The GC–MS analysis of the methanolic extract of Ganoderma spp. fruiting bodies resulted in the detection of 39 compounds. Majority of those compounds were alkenes, saturated and unsaturated fatty acids, which came in accordance with the GC–MS profile of some Ganoderma spp. that showed the presence of similar compounds [34],[35].

The tested methanolic extract of Ganoderma spp. showed a remarkable cholesterol-reducing activity in vitro, indicating that Ganoderma spp. represents a promising source of biologically active compounds having hypocholesterolemic effects. Many studies have described the cholesterol-lowering activity of Ganoderma extracts and here we have quantified the impact in detail [36],[37],[38]. Previously, in the species Ganoderma lucidum the presence of some oxygenated lanosterol compounds were identified, and these work through inhibiting cholesterol synthesis in T9A4 hepatocytes to reduce total cholesterol and high-density lipoprotein % in tested hamsters to 9.8 and 11.2%, respectively [37]. However, α-glucans and β-glucans have also been nominated as compounds responsible for the cholesterol-lowering behavior of G. lucidum in mice [38]. On the other hand, many reports have pointed out polyunsaturated fatty acids as food constituents that reduce serum cholesterol [39],[40],[41]. In the current study, many unsaturated fatty acids have been detected in the extract of Ganoderma spp., such as octadecadienoic acid and oleic acid which may contribute in the hypocholesterolemic activity exerted by the extract.

Replication in viruses includes many steps such as attachment of the virus to the host, penetration of the host cell, replication of the virus within the host cell, assembly, and departure of the virus from the infected cells. Targeting these various steps can be used in the evaluation of the antiviral activities of different compounds [42]. In the current study, the effect of the methanolic extract of Ganoderma spp. on the attachment and penetration steps was investigated. As shown in [Table 3], treatment with this extract resulted in an in vitro anti-RV SA-11 activity of TI 11, which indicated the capability of this extract to attach to viral capsids, and hence stopping them from binding to cell receptors. Therefore, penetration and entry processes into host cells were prevented. Different compounds were previously identified from Ganoderma applanatum extracts and were nominated as antiviral agents [43].A promising in-vitro anti-human colon cancer activity was observed from treatment with Ganoderma spp. extract. This may be due to the presence of many unsaturated fatty acids such as oleic acid and octadecadienoic acid. Unsaturated fatty acids such as oleic acid have been nominated for their anticancer activities [44],[45],[46]. The mechanism of this action includes activating GPR40 and inducing oxidant stress and mitochondrial dysfunction in cancer cell lines [44]. It was also reported that free fatty acids can selectively inhibit the growth of tumor cells [47]. Moreover, a study conducted on the fatty acids from G. lucidum spores had proven its ability to inhibit tumor cell proliferation [46]. Octadecenes was also detected in the extract of Ganoderma spp. and it was reported for its anticancer activities [35],[48]. On the other hand, reports for the anticancer activities of Ganoderma extracts explained this effect by the presence of many compounds such as applanoxidic acid C, D, F, G; nujiangexanthone B; heptemerone D; trichiol C; camphoratin E, xylariacin B, sphaeropsidin D, 7-methoxy-2, 3, 6-trimethylchromone, applanatumin A, and berkedrimane B [43],[49],[50],[51].


  Conclusion Top


Exploring the miraculous mushroom, Ganoderma, for biological activities is always resulting in promising outcomes. Results of this study highlighted the GC–MS analysis, in addition to the promising in-vitro capabilities of the methanolic crude extract of a Japanese Ganoderma spp. fruiting bodies. This extract exhibited hypocholesterolemic, anti-rotavirus, and a promising anti-human colon carcinoma activities. Investigating Ganoderma extracts and studying their potential therapeutic effects may contribute in the future in the identification of alternatives to the currently used drugs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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