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Phytochemical profiles and inhibitory effects of Tiger Milk mushroom (Lignosus rhinocerus) extract on ovalbumin-induced airway inflammation in a rodent model of asthma
© The Author(s). 2016
Received: 31 October 2015
Accepted: 25 May 2016
Published: 3 June 2016
Lignosus rhinocerus (L. rhinocerus), which is known locally as Tiger Milk mushroom, is traditionally used in the treatment of asthma by indigenous communities in Malaysia. However, to date, its efficacy on asthma has not been confirmed by scientific studies and there is also sparse information available on its active constituents. In this study, the volatile constituent of L. rhinocerus hot water extract was investigated using gas chromatography mass spectrometry (GC-MS). The potential effects of L. rhinocerus extract for anti-asthmatic activity was further investigated on ovalbumin (OVA)-sensitized asthmatic Sprague Dawley rats.
Sequential extraction using five solvents (petroleum ether, diethyl ether, hexane, ethyl acetate and methanol) was conducted prior to GC-MS analysis. Male Sprague Dawley rats were divided into the following four groups of five animals each: 1) normal rats, 2) sensitization plus OVA-challenged rats 3) sensitization plus OVA-challenged with L. rhinocerus treatment and 4) sensitization plus OVA-challenged with dexamethasone treatment. The levels of immunoglobulin E (IgE) in the serum and T-helper 2 cytokines, including interleukin (IL)-4, IL-5 and IL-13, in bronchoalveolar lavage fluid (BALF), as well as eosinophil infiltration in the lungs, were investigated.
GC-MS analysis revealed the presence of five main groups (alkane, fatty acids, benzene, phenol and dicarboxylic acid) with a total of 18 constituents. Linoleic acid (21.35 %), octadecane (11.82 %) and 2,3-dihydroxypropyl elaidate (10.47 %) were present in high amounts. The extract significantly ameliorated the increase in total IgE in serum and IL-4, IL-5 and IL-13 levels in BALF and also effectively suppressed eosinophils numbers in BALF while attenuating eosinophil infiltrations in the lungs.
L. rhinocerus hot water extract has the potential to be used as an alternative for the treatment of acute asthma.
Asthma affects 300 million people worldwide and is a result of a complex interplay between genetic and environmental factors . Allergic asthma is a chronic disease characterized by recurrent wheezing, shortness of breath, chest tightness and coughing. The airway disorder is commonly characterized by airway eosinophilia infiltration, increased serum immunoglobulin E (IgE) and T-helper 2 (Th2) cytokine levels such as interleukin (IL)-4, IL-5 and IL-13 , as well as excessive production of airway mucus.
The Th2-dominated response is initially triggered when an airway allergen is taken by the dendritic cells and presented to specific Th cells by means of major histocompatibility complex (MHC) class II, thus stimulating the immunological synapse. Activated allergen-specific Th cells then polarize into Th1 or Th2 effector populations, differentiate and expand into a Th2 subpopulation. The activated Th2 cells function in recruiting and activating cytokines, including IL-4, IL-5 and IL-13, following stimulation of eosinophils. IL-4 cytokines have been reported to cause class switching of B cells, resulting in IgE synthesis, which is involved in mast cell degranulation . The key role of IL-5 cytokines includes their involvement in the growth and differentiation of bone marrow eosinophils, as well as their release into the peripheral circulation . Additionally, IL-5 also increases the activity of eosinophil recruitment, activation and survival at the sites of inflammation . IL-13, which is known as the “central mediator of allergic asthma,” plays an important role in directing the allergic response in asthmatic patients . Various studies are therefore primarily focused on products that have the potential to reduce IgE, IL-4, IL-5, IL-13 and eosinophils as therapeutic targets for asthma treatment [7–10].
Current asthma medications are mainly based on steroids and other types of anti-inflammatory drugs . Among the available drugs, inhaled corticosteroids (ICSs) are very effective, as they suppress chronic airway inflammation in patients with asthma . ICSs are generally considered harmless, though persistent use of ICSs at higher doses often leads to various systemic and local side effects , some of which include candidiasis, osteoporosis, growth retardation, cataracts and pharyngitis . Thus, a safer alternative for the management of asthma is needed.
Lignosus rhinocerus (L. rhinocerus) from the Polyporaceae family is a wild mushroom exclusive to Malaysia. Locally known as “cendawan susu rimau” or “Tiger milk mushroom”, this unique mushroom is one of the reported 38 available types of edible mushrooms in Malaysia used for medicinal purposes by rural and indigenous communities . The tuber is purported to contribute the most medicinal value, and the indigenous populations in Peninsular Malaysia utilise it to treat diseases such as asthma, fever, breast cancer, stomach cancer and food poisoning, as well as to heal wounds [15, 16]. Previous studies have demonstrated anti-proliferative activities  and immunomodulatory properties  of this mushroom sclerotial. A recent study by Lee et al.  reported anti-acute inflammatory properties of sclerotial powder of L. rhinocerus using carrageenan-induced paw oedema model in rats. In addition, the study also demonstrated a potent inhibition of TNF-α production by the high-molecular-weight fractions of the sclerotial powder of L. rhinocerus. However, to date, no studies have been reported on the anti-asthmatic properties of L. rhinocerus in airway inflammation models. Thus this study reported anti-asthmatic effects of L. rhinocerus sclerotial extract in ovalbumin-induced airway inflammation of the rodent model and the profile of volatile constituents of the mushroom extract by GCMS analysis.
Preparation of L. rhinocerus hot water extract
Sclerotia of L. rhinocerus cultivar TM02 was obtained in dried powdered form from Ligno Biotech Sdn. Bhd. (Selangor, Malaysia). The powder was subjected to hot water extraction using a soxhlet for 24 h and was further concentrated using a rotary evaporator (Unimax1010, Heidolph, Germany) before being freeze-dried in a freeze drier (Ilshin BioBase, Gyeonggi-do, Korea).
A sequential liquid-liquid extraction was performed using 1) petroleum ether, 2) diethyl ether 3) hexane, 4) ethyl acetate and 5) methanol. The selected solvents ranged in polarity starting from non-polar (petroleum ether, diethyl ether and hexane) to more polar solvents (ethyl acetate and methanol) to take advantage of their different properties.
Briefly, 1 ml of petroleum ether was added to the capped glass tube containing 1 g of L. rhinocerus extract. The mixture was then vortexed for 1 min using a vortex mixer (Westbury, New York, USA), followed by centrifugation (Centrifuge Universal 32R, HettichZentrifugen, Germany) at 700 × g for 5 min. The supernatant was aspirated before being transferred (100 μl) to a new auto-sampler vial for GC-MS injection. The residue was used for subsequent extraction using diethyl ether followed by hexane, ethyl acetate and finally, methanol, as previously described for petroleum ether. Following extraction by each solvent type, the samples were individually injected into the GC-MS system in duplicate. Each sample was analyzed against a blank organic solvent containing a similar type of organic solvent used in the extraction process each time.
GC-MS analysis was performed on an HP6890 GC coupled with a HP5973 mass spectrometer (Hewlett Packard, CA, USA). The column was a HP-5MS fused-silica capillary column (50 m x 0.25 mm i.d.; 0.25-μm film thickness) with helium as the carrier gas, and it was run at a constant pressure of 9.78 psi. Injection was conducted using a splitless mode at an injector temperature of 250 °C. The oven temperature was ramped from 40 °C to 280 °C (1-min hold) at a rate of 25 °C/min. The oven temperature was held at 310 °C for 6 min for each analysis. The total run time for each sample was approximately 28 min. The GC-MS interface temperature was set to 280 °C. MS mode was used during analytical scanning from 20 to 650 atomic mass units (amu). The ion source temperature was set at 250 °C.
The blank was injected first each time, followed by the sample injection. The chromatograms obtained from the total ion current (TIC) were integrated without any correction for co-eluting peaks, and the results were expressed as total abundance. The TIC peaks and chromatograms were analyzed using an Agilent Technologies 7890A GC system with 597C VL MSD. All peaks were identified based on mass spectral matching (≥ 90 %) from both the National Institute of Standards and Technology (NIST) and Wiley libraries. Only compounds with 90 % or greater spectral matching accuracy were reported.
Ethical approval was obtained from Animal Ethics Committee Universiti Sains Malaysia. Male Sprague Dawley rats (age between 6 and 8 weeks; 200–250 g in weight) were used for this experiment. The rats (n = 20) were housed in polystyrene cages in an air-controlled room. All animals were maintained at the animal house, and tap water was allowed ad libitum. The animals were acclimatized to the experimental environment prior to commencement of the study.
Sensitization, airway challenge and treatment
The rats were divided into the following four groups of five animals each: (1) normal rats, (2) sensitization plus 1 % OVA challenge or “untreated group,” (3) sensitization plus 1 % OVA challenge with oral L. rhinocerus treatment (500 mg/kg per body weight) and (4) sensitization plus 1 % OVA challenge with intraperitoneal (ip) dexamethasone treatment (3 mg/kg per body weight). Sensitization on days 1 and 14 was conducted with a 2 × 1 ml i.p. injection of 10 mg/ml OVA (Nacalai Tesque, Kyoto, Japan) + 100 mg/ml alum (aluminium hydroxide) (Sigma, MO, USA) in phosphate buffered saline (PBS) (based on the modified method of Makhlouf and Hafez, ). An additional 50 ng/ml of Bordetella pertussis (Sigma, MO, USA) was also prepared with the solution as adjuvant. On day 23, the rats were challenged with 1 % OVA aerosol for 20 min/day for 7 days using an ultrasonic nebulizer (Mabist mist, Illinois, USA) and orally administered by gavage with 500 mg/kg per body weight of L. rhinocerus extract for 7 days. On day 30, 24 h after the final challenge, the rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), and blood was collected by cardiac puncture.
Eosinophils and inflammatory cell count
Following sacrifice, bronchoalveolar lavage fluid (BALF) was obtained using an endotracheal tube by instilling and aspirating the trachea with PBS solution. BALF was centrifuged at 500 × g for 5 min, and the pellet was re-suspended with PBS. The total inflammatory cell number was quantified by counting the number of cells from at least five squares of the haemocytometer after excluding dead cells stained with trypan blue.
Measurement of total IgE in BALF
The total IgE was measured with a specific rat ELISA kit (Abnova, Heidelberg, Germany) according to the manufacturer’s instructions. IgE levels in each sample were measured from optical density readings at 450 nm followed by calculation based on a standard curve that was generated using recombinant IgE.
Measurement of Th2 cytokines in BALF
An enzyme-linked immunosorbent assays (ELISA) was performed according to the manufacturer’s instructions. IL-4, IL-5 and IL-13 in BALF were measured using specific rat IL-4, IL-5 and IL-13 ELISA kits (CusaBio, Hubei, China). The bound enzyme that was proportional to the respective cytokines concentration was investigated at absorbance of 450 nm. Cytokine concentrations were calculated from standard curves that were generated using respective recombinant interleukins.
Lung tissue histology
0: no inflammation
1: mild inflammation
2: moderate inflammation
3: severe inflammation
4: extreme inflammation
For each rat, randomly distributed airway sections in the lung were analysed, and their mean scores were calculated. The inflammation present was evaluated with a double-blinded scoring method. Quantitative analysis of the lung inflammation score was performed using a Mirax Image viewer (Carl Zeiss, Jenna, Germany).
Data are expressed as the mean ± standard deviation (SD). Statistical significance (p < 0.05) was determined by a one-way ANOVA followed by Scheffe’s post hoc correction and inflammation scores were analysed with Wilcoxon-Mann Whitney test using Statistical Program for Social Science (SPSS) version 22.0 (New York, USA).
GC-MS analysis of the volatile constituent of L. rhinocerus
Volatile compositions of L. rhinocerus following extraction using various solvents and their respective retention times, RT (min)
Benzene, 1,3-bis (3-phenoxyphenoxy)
Chemical structures of the major volatiles present in L. rhinocerus
Inhibition effects of L. rhinocerus on airway inflammation
A preliminary study was conducted prior to the present investigation to optimize L. rhinocerus dosages and the dose used in the sensitization protocol that was suitable for the anti-asthmatic study. Based on this finding, a dose of 500 mg/kg of L. rhinocerus is more effective in reducing asthma-related parameters when compared to low (125 mg/kg) and medium (250 mg/kg) dosages (unpublished data) and was therefore selected for the study. A previous toxicity study indicated that 1000 mg/kg of L. rhinocerus extract fed orally to rats did not cause any adverse effects, and it is therefore considered to be safe . The sensitization method was modified from a previous study  based on similar preliminary optimization study, hence i.p. sensitization with the additional adjuvant of Bordetella pertussis and 7 days of challenge or treatment was suitable to establish the expected allergic model.
Our study targeted serum IgE detection, while Th2 cytokine detection was conducted in BALF. As in asthmatic models, airway obstructions and difficulties in breathing are assumed to result from the responses of immunoglobulins and cytokine levels, which lead to worsening of asthma symptoms. Eosinophils are also found in large numbers within the submucosa and epithelium in the allergic model. Most allergic mediators are stimulated by IgE and are released into the blood stream in response to inflammatory reactions . In addition, BALF biofluid is widely accepted as a reliable sample with which to determine the composition of secreted pulmonary proteins and the products of activated cells. Therefore, in this study we aimed to investigate OVA-induced IgE detection in serum, while Th2 cytokines were detected in BALF.
Our study is the first to elucidate the composition of hot water extract of L. rhinocerus widely used in the traditional treatment of asthma. Alkanes were the major group found to be present, while linoleic acid was the major constituent detected. To our knowledge, our study is also the first to confirm the airway inhibition effects of L. rhinocerus.
The GC-MS analysis was conducted following a sequential extraction method that employs both polar and non-polar properties of solvents in separating compounds based on polarity. In this study, a simple liquid-liquid extraction method is beneficial because it does not involve a heating process, which minimizes damage to the volatiles and can prevent the formation of other artefacts [24, 25]. This sequential extraction conserves the amount of volatiles as well as selectively improves the process and the recovery of different types of extracts from analogous materials. The compounds were selected based on spectral library matching to the NIST and Wiley libraries.
Most of the compounds detected were from the alkane group. In comparison, a study of other types of medicinal mushrooms such as Termitomyces sp. and Termitomyces microcarpus prepared from petroleum ether crude extracts reported the presence of small amounts of octadecane (0.64 %) and heptadecane (1.01 %) , which are alkanes. Another researcher reported the presence of octadecane (0.15 %) in Trichodermaharzianum following GC-MS analysis . However, our study detected the presence of higher amounts of octadecane (10.47 %) and heptadecane (9.29 %) in L. rhinocerus. Similar to our study, a study on Agaricus bisporus comparing different strains of mushrooms (strains 310 and 342) reported high amounts of non-polar compounds such as octadecane (13.8 %) and nonadecane (14.4 %) in addition to eicosane and heptadecane . In addition, several studies [28–30] have reported fatty acids to be the predominant constituent in mushrooms. Specifically, linoleic acid has been detected to be the major constituent (~65 %) of Schizophyllum commune and Lentinus edodes , which is also reported in our study as the most common compound (21.35 %). Similar to our study, the fatty acid profile of several Tricholoma species, such as T. portentosum and T. terreum, also revealed the presence of linoleic acid (∼28 %) .
Linoleic acid is a known precursor of 1-octen-3-ol (alcohol of fungi), which functions as the principal aromatic compound in most fungi [33–35]. A study on L. rhinocerotis comparing mycelium of shaken cultures and static cultures revealed the presence of 2.3-dihydroxypropyl elaidate compound (0.27–0.28 %) . The presence of a similar compound was also detected in the spore lipids of Ganoderma lucidum (0.07 %) . In our study, 2,3-dihydroxypropyl elaidate was reported to be present at approximately 11.82 % and constituted the second most common compound detected. In comparison, linoleic acid was compared in different parts of A. bisporus mushroom and was reported to be present in 12.2 % of the volume of the whole fresh sample, in 1.5 % of the stalks and in 0.8 % of the gills .
Linoleic acid was present in varying concentrations in different types of mushrooms, and unique properties were observed. In fact, such variations are key to understanding the aromatic and other medicinal properties. Nonetheless, GC-MS analysis of L. rhinocerus revealed compounds that were detectable and comparable with other mushrooms types and plants [29, 39, 40], making them both similar and different to plants of numerous species and genus.
This study is the first to report on the inhibition effects of L. rhinocerus on airway inflammation in asthmatic model. Asthmatic model of Sprague Dawley rat was used in the study and dexamethasone was used as a positive control as this compound is commonly used as a potent inhibitor of airway inflammation and remodelling. The administration of corticosteroids has been demonstrated to inhibit structural changes associated with airway fibrosis in animal models and is therefore widely utilized as a control in asthma studies . In the context of the experimental design, all rats in this study (except for the normal group) underwent sensitization on days 1 (primary sensitization) and 14 (secondary sensitization). The purpose of an additional sensitization step on day 14 is to ensure that the levels of airway eosinophils do not revert back to the baseline, as well as to avoid the development of tolerance. In addition, these sensitizations were coupled with OVA along with alum and Bordetella pertussis to enhance Th2-dominated responses .
Generally, the activation, growth and differentiation of eosinophils are conducted by Th2-dominated responses . It is reported that cytokines secreted by Th2 cells can help in the recruitment and activation of eosinophils in the nasal region. Eosinophil counts performed on BALF in this study revealed that treatment with hot water extract of L. rhinocerus has reduced the number of eosinophils to 6.54 × 104 cells in BALF when compared to the untreated group, which contained 8.34 × 104 cells; this could indicate attenuated severity and possible withdrawal in eosinophil infiltration along the submucosa and epithelium regions. Groups of rats receiving hot water extract of L. rhinocerus and dexamethasone treatment had also significantly decreased the numbers of total inflammatory cells in BALF. These decreases are correlated with the level of cellular infiltration. Furthermore, the deterioration of inflammatory cell numbers in BALF was confirmed by lung tissue histology, thus possibly be valuable in controlling the inflammatory processes underlying exacerbation of allergic asthma. This result is in accordance with the previous study that demonstrated the reduction of eosinophils in OVA-treated animals after treatments with herbal extract .
An early finding indicated that in sensitized individuals, the nasal mucosa contains high level of IgE-binding mast cells . Upon sensitization, increasing numbers of IgE-coated mast cells traverse the epithelium. During the phase of allergen exposure or challenge, the IgE-coated mast cells recognize the allergens deposited at mucosal regions, and the mast cells then degranulate. This leads to the release of mediators such as histamines and leukotrienes, which are responsible for blood vessel leakage to stimulate the sensory nerves leading to congestion, airway inflammation and obstruction as well as systemic reflexes such as sneezing . Thus, reducing IgE levels has been the major focus in combating asthma. Accordingly, animals receiving hot water extract of L. rhinocerus had significantly reduced IgE levels (14.88 ng/ml) when compared to the untreated group (16.29 ng/ml). A recent study also reported that another type of mushroom polysaccharide from A. camphorata has similar anti-asthmatic effects shown by reducing IgE levels in an animal model of asthma .
In our study, it was noted that hot water extract of L. rhinocerus significantly reduced IL-4 levels in BALF. This is supported by a study using murine allergen challenged models where intranasal chitin polysaccharide components of fungal cell decreased IL-4 (16.22 pg/ml) production compared to untreated group (23.07 pg/ml) in vivo [47, 48]. Moreover, Th2 cytokine concentration levels of IL-5 (38.82 ng/ml) and IL-13 (80.54 pg/ml) were also reduced when hot water extract L. rhinocerus was used to treat sensitized rats in comparison with untreated groups (42.79 ng/ml and 83.69 pg/ml), respectively. Similarly, both IgE levels in the serum and Th2 cytokines in BALF were also reduced by dexamethasone. However, the reduction observed in the IL-13 levels due to L. rhinocerus administration was not significant, which may illustrate the varying effects of L. rhinocerus extract on IL-13 regulation. Interestingly, IL-13 has been reported to potentially have independent roles from eosinophils, IgE, IL-4 and IL-5 . It has been reported that the production and high activity of eosinophils, the hallmark of asthma, was not affected despite inhibition of IL-13 . However, the potential independent role of IL-13 in prompting the complete allergic asthmatic phenotype has been underlined. In a study on IL-13-dependent airway hyper responsiveness (AHR), it was revealed that the mechanisms are independent of IgE and eosinophils in mice .
The lung histopathology study further confirmed how structural changes or responses involving lung tissues further contribute to asthma. Indications of such as high leukocytes or eosinophil infiltration at regions around the peribronchial and perivascular space in the bronchioles are important keys that point to such a severe condition . Consistent with previous findings regarding immunoglobulin and Th2 cytokines, both the hot water extract of L. rhinocerus and dexamethasone were noted to reduce the number of eosinophil cells infiltrating into lungs tissue. A similar histological study on A. Camphorata also reported attenuation of lung inflammatory leukocytes in OVA-sensitized animals . Relief of bronchoalveolar inflammation in a murine model of airway sensitivity has also been reported in a histology study that used another type of mushroom i.e., Ganoderma tsugae (G. tsugae) as a possible treatment for allergic asthma .
Several findings further confirm that immune disorders are attributable to the collapse of the system that controls the balance between levels of Th1 to Th2 cells. In fact, diseases such as leprosy, allergic asthma and multiple sclerosis are associated with aberrant Th1 and Th2 polarizations. Th2 cells are also strongly implicated in atopy and allergic inflammation. A. camphorata polysaccharides, for instance, are potential inducers of Th1-type cytokines such as tumour necrosis factor and interferon but not of Th2 cytokines, which have successfully been proven to reduce asthmatic conditions . The importance in discovering and identifying new drugs that are safe and effective and have minimal side effects has made the medicinal properties of plants more valued and thereby more widely explored. Our study demonstrated that treatment of OVA-induced airway inflammation among Sprague Dawley rats treated with hot water extract of L. rhinocerus significantly reduced allergic asthma parameters i.e., IgE level, Th2 cytokines, eosinophil count and eosinophil infiltration in the lungs.
Various speculations denote the potential anti-asthmatic effects of L. rhinocerus. Investigating the roles of fatty acids in reducing asthma symptoms has been gaining high attention in studies of animal models as well as children [51, 52]. Interestingly, a previous chemical study on L. rhinocerotis revealed that major compounds such as fatty acids are present .
Polyunsaturated fatty acids (PUFA) have been found to have potential anti-inflammatory properties with fewer side effects, and they are claimed to be mildly beneficial for the treatment of allergic conditions such as asthma and atopic dermatitis . In fact, previous studies [53, 54] have proven that PUFAs are closely associated with enhanced lung function and reduced asthma symptoms. It has been shown that n-3 PUFAs suppress a major mediator of asthma known as leukotriene synthesis . The role of such fatty acids is also associated with the modulation of prostaglandin metabolism, which is the primary cause of exacerbated conditions of allergic asthma. It has been proven that the anti–asthmatic potential of Zanthoxylum bungeanum is due to the presence of some phytochemical compounds, specifically those rich in linoleic acids . Therefore, it is plausible that the presence of high concentrations of linoleic acid in L. rhinocerus which is also one of the main key constituents, contributes to the anti-asthmatic effect. Nevertheless, this should be confirmed in a further study.
It should be noted that there may be differences in the type of detectable volatile compounds present in fresh L. rhinocerus in contrast to the cultivated sample used. In addition, the spectral library matching performed in this study during the detection of volatile compounds provides preliminary presumptive data; therefore, future studies that contribute to the utilization of authentic standards should be conducted to further confirm our findings. Confirming the presence of compounds with long-chain fatty acids can be further supported by additional methylation steps as is also recommended by Mendez et al. . Moreover, this study only focused on volatile compounds of L. rhinocerus as these compounds are reported to be the intermediate or end products of diverse metabolic pathways of various classes . Identification of non-volatile and semi-volatile organics, which are more suitable for investigation using liquid chromatography/mass spectrometry, was not performed at this point of the study.
Alkanes were the major group present in hot water extract of L. rhinocerus, while linoleic acid was the major constituent detected. L. rhinocerus showed anti-asthmatic effects in vivo as confirmed by the decreased levels of IgE and Th2 cytokines responsible for eosinophils recruitment, as well as the attenuation of eosinophil infiltration in the lungs. Thus, L. rhinocerus has the potential to be a promising alternative or adjuvant to the current drugs used for the management of allergic asthma.
BALF, bronchoalveolar lavage fluid; GC-MS, Gas chromatography mass spectrum; Ig, immunoglobulin; IL, interleukin; OVA, ovalbumin; TIC, total ion current.
We would like to thank LignoBiotech™Sdn. Bhd for contributing sclerotial powder L. rhinocerus (Tiger Milk mushroom) cultivar TM02, the staff of the Animal Research and Service Centre (ARASC) and the staff of the Craniofacial Science Lab (USMKK).
This study was financially supported by the Research University Grant (RU) from Universiti Sains Malaysia (1001/PPSK/813065).
Availability of data and materials
All data related to the study are presented in the paper.
MJ involved in the experimental works and drafted the manuscript. AHF assisted in the pathological analysis. AAN, SHG and MFWE involved in the planning of the research, data collection and analyses and manuscript writing. All authors have read and give approval to the manuscript to be published.
The authors declare that they have no competing interests.
Consent for publication
Ethic approval and consent
The experimental protocols and animal care were approved by the Animal Ethics Committee, Universiti Sains Malaysia, ethical approval no (AEC/2010/(59) (208).
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