Current and future perspective on antimicrobial and anti-parasitic activities of Ganoderma sp.: an update

ABSTRACT Medicinal mushroom Ganoderma sp. is considered to be a key source for the production of therapeutic agents. Our current review indicates that a limited number (<19%; 79 out of >430) of isolated compounds have been tested and known to be active against several microorganisms and parasites. In this review, we aim to summarise all the antimicrobial and anti-parasitic works on Ganoderma sp. displayed on web of science, google scholar and endnote X7 from 1932 to August 2016. We further present and discuss the structure of active compounds against microorganisms and parasites. In addition, we also discuss the possible further research to identify lead compounds from Ganoderma sp. as a novel strategy to combat the potential global emergence of bad bugs and parasites.


Introduction
Ganoderma sp. is a medicinal mushroom producing a group of frequently studied bioactive compounds. They belong to the kingdom of Fungi, division of Basidiomycota, class of Agaricomycetes, order Polyporales, family of Ganodermataceae and genus of Ganoderma. A search for "Ganoderma" in the database Index Fungorum displayed 409 species records, including synonyms (http://www.speciesfun gorum.org). Ganoderma sp., especially G. lucidum, G. tsugae and G. applanatum, are well studied and have been in use in East Asian countries since the ancient times for the treatment of various diseases (Ofodile et al. 2005;Paterson 2006;Ferreira et al. 2015). Triterpenes and polysaccharides are considered key constituents isolated from fruiting bodies, gills, spores and mycelia for their bioactivities (Xia et al. 2014).
Literature reviews suggest, besides its antimicrobial activities, Ganoderma sp. components exhibit a variety of bioactivities, including anti-tumour, immune-modulatory, antioxidant, antihypertensive and anti-androgenic. Moreover, Ganoderma sp. is widely used for the remedy of various chronic diseases such as cancers, diabetes, hypertension and hepatitis (Ofodile et al. 2005;Zhang et al. 2015). To date, most of the reviews on Ganoderma sp. have been focused on its anticancer and antioxidant activities and immune modulation (Sanodiya et al. 2009). Therefore, our basic aim is to provide a glimpse on the antimicrobial and anti-parasitic activities of Ganoderma sp. In addition, we also provide possible future prospect for research on Ganoderma sp. and its compounds.
In this review, we have performed literature searches in English (ISI Web of Science and Google Scholar) and Endnote X7 (online search, Pub Med) to find publications that described Ganoderma sp. for antimicrobial activities. We have used the keywords "Ganoderma" and "Antimicrobial". Finally, we filtered individual references to determine the relevancy to our study. The inclusion criterion was the study that provided data or results or discussion on the antimicrobial activities of Ganoderma sp.
(SMs) produced by Ganoderma sp. In Ganoderma sp., more than 316 terpenes have been reported, with the majority of compounds from G. lucidium (Xia et al. 2014).
Chemical analysis of numerous Ganoderma sp. has showed Ganoderma Triterpenes (GTs) are mainly lanostanoid-type triterpene (Zhang et al. 2015). Among them, majority contain 30 or 27 carbon atoms, and some occasionally may contain 24 carbon atoms. These compounds possess the same parent skeleton, namely a trans-configuration of rings A/B, B/C, C/D and 10β, 13β, 14α, 17β substituent. In addition, the substituents are always found at the C-3, 7, 11, 12, 15, 22, 23, 24 and 25 positions of the parent nucleus (Xia et al. 2014). Thirty carbon terpenoids are usually formed by the fusion of two smaller terpenoids precursors, each containing 15 carbons sesquiterpene. Head-to-tail fashions linking of isoprene units to form linear chains and various cyclisations and rearrangements is the core mechanism to give cyclic terpenoids (Mothana et al. 2000;Hill & Connolly 2013). The parent carbon skeleton of antimicrobial and anti-parasitic GTs is shown in Figure 1, from which it can be concluded that GTs are the most common antimicrobial and anti-parasitic compounds reported from Ganoderma sp.
Farnesyl quinone, a polyketide type, is the second most common antimicrobial and anti-parasitic compound from Ganoderma sp. Quinones are known to be oxidised derivatives of aromatic compounds and are often readily made from reactive aromatic compounds with electron-donating substituent such as catechols and phenols. Besides GTs, polypeptides, small peptides such as ganodermin, polysaccharide such as sacchachitin, and chitosan also possess antimicrobial and anti-parasitic properties (Mothana et al. 2000;Wang & Ng 2006;Sanodiya et al. 2009;Chuang et al. 2013). Structures of antimicrobial and anti-parasitic compounds from Ganoderma sp. are shown in Figure 2.

Isolation of antimicrobial and anti-parasitic bioactive compounds
Extracts from fruiting bodies, both wild and cultivated, and mycelia from fermentation broth (Tables 1-4) are used for the isolation of antimicrobial and anti-parasitic bioactive compounds. Literatures divulge that most commonly ethanol (EtoAc) (Tables 1-4) is used to prepare crude extract; sometimes some researchers preferred other solvents such as chloroform (CHCl 3 ), EtOH, and acetone (Isaka et al. 2016). In addition, our review reveals that hexane and ether are poorly used for the preparation of extract from Ganoderma sp. Moreover, some techniques such as microwave, ultrasound and enzyme treatments can facilitate the breakdown of the cell wall (Ferreira et al. 2015). Solvents like MeOH, EtOH, CH 2 Cl 2 , CHCl 3 and aqueousboth cold and hotare used for further purifications and isolation. Techniques such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and column chromatography (CC) are used to facilitate the purification and isolation process (Huie & Di 2004). The general procedures of the isolation of antimicrobial and anti-parasitic compounds are shown in Figure 3. In addition, this outline can be used for other chemical investigations from Ganoderma sp.

Antibacterial activities of compounds and extracts of Ganoderma sp
Currently bioassay-guided antibiotics identification, TLC and chromatography bio-autography are used to track antibacterial ingredients from the extract (Huie & Di 2004). Minimum inhibitory concentration (MIC) and 50% inhibitory concentration (IC 50 ) values are used to determine the potency of antibacterial agents. Our literatures review showed that MeOH and EtOH are good solvents for the extraction of antibacterial compounds of interest rather than other organic solvents; however, the parts of Ganoderma sp. used and the tested bacterial strains may be the limiting factors in choosing the solvent. Most studies that use alcoholic solvents for extraction showed very low MIC (Li et al. 2012;Shang et al. 2013;Cilerdzic et al. 2016). Several studies on the fruiting bodies of Ganoderma sp. reveal that the compounds have the inhibitory ability to the different types of Gram positive bacteria (GPB), Gram negative bacteria (GNB) including the mycobacteria (Al-Fatimi et al. 2005;Isaka et al. 2016).
Literatures reveal most of the antibacterial tests are performed on crude extract with significant effective results rather than pure compounds (Sa-Ard et al. 2015;Zengin et al. 2015;Cilerdzic et al. 2016). In addition, scanty information is available on the in vivo model test of effective compounds; we noticed only compounds (27)

Antifungal activities of compounds and extracts of Ganoderma sp
An antifungal proteinganoderminisolated from the fruiting bodies of G. lucidium inhibits the growth of Botrytis cinerea, Fusarium oxysporum and Physalo   ) sporapiricola with an IC 50 value of 15.2 mM, 12.4 mM and 18.1 mM, respectively (Wang & Ng 2006). Terpeneoids like applanoxidic acids A (1), C (2) and F (3) isolated from G. annulare inhibit the growth of the fungi Microsporum cannis and Trichophyton mentagrophytes at concentrations of 500-1000 µg/ml (Smania et al. 2003).
In another study, researchers synthesised the complexes of polysaccharide with different rare earth metal (RE-CGAP (RE: La, Eu and Yb)) and evaluated their efficacy against fungi and reported that rare earth carboxymethylated G. Applanatum polysaccharide (RE-CGAP) complexes with antifungal activities with EC 50 value of 1.01-28.48 mg/ml (>100 mg/ml not included) . The details of the antifungal action of Ganoderma sp. are demonstrated in Table 2.

Anti-parasitic activities of compounds and extracts from Ganoderma sp
Nortriterpenes-ganoboninketals A-C (15-17) obtained from the biochemical analysis of the fruiting bodies of  Table 3. Illustration of antiviral activities of Ganoderma sp. parts, products and compounds.

Conclusion and future perspective
Ganoderma sp. has been used for treatment in various diseases over a long period (Paterson 2006). Our review clearly showed that compounds from Ganoderma sp., under the extensive in vivo and pharmacological research, can be used in various microorganisms and parasitic diseases. However, the in vivo experiment and pharmacological research of the identified compounds are very limited. Therefore, future work should be focused on in vivo and pharmacological assays of known compounds, especially Ganoderma terpenes that have antimicrobial and anti-parasitic properties. A better understanding of the antimicrobial and anti-parasitic compounds from Ganoderma sp. is crucial for identifying the potential side effects and trace out the new host target and molecular mechanisms, which will provide evidence to further clinical applications of these compounds. Although extensive researches have been carried out on Ganoderma sp., most of the studies were concentrated on few species, G. lucidum for instance. Researchers must need to pay more attention to closely related species based on the phylogenic analysis though numerous challenges including genetic analysis, biosynthetic metabolism, separation, isolation and identification may be encountered. In addition, due to the rapid emergence of drug resistance in microorganisms and parasites, fewer options have been left for the treatment of diseases caused by microorganism and parasites. To fight back this problem, further research should be focused on this field for all the identified compounds and the unidentified compounds, which are on the way to be identified. Our review revealed numerous extracts of Ganoderma sp. exhibit the inhibition to microorganisms including parasites, indicating that Ganoderma sp. in particular still seem to possess opportunities for new drug lead compounds.
Scanty literatures are found on the assay of identified compounds for animal and plants pathogens including parasites, indicating that this area of research for the Ganoderma sp. compounds is overlooked. Also, our current experience on a literatures review of Ganoderma sp. compounds, more than 430 compounds identified (Baby et al. 2015;Rai et al. 2015), most of the compounds have not been performed on the antimicrobial and anti-parasite assay. Therefore, further studies need to be carried out in order to explore this concealed area.
No doubt, it is evident that Ganoderma sp. is going to serve as one of the potential sources of novel antibiotics and anti-parasitic drugs in the near future. To reach the apex and specificity of effective antimicrobial and anti-parasite activity, cooperative investigations need to be carried out in the areas of genomic, bioinformatics, chemistry and pharmacology. Moreover, strategies to evoke the sleeping gene clusters linked for the production of bioactive compounds and its regulation need to be adopted.