Current Medicinal Chemistry, 2010, 17, 2419-2430 2419 Lentinus edodes: A Macrofungus with Pharmacological Activities P.S. Bisen*,1,2,3, R.K. Baghel2, B.S. Sanodiya2,3, G.S. Thakur2 and G.B.K.S. Prasad1

1School of Studies in Biotechnology, Jiwaji University, Gwalior-474011 (M.P.) India 2Research and Development Centre, Bisen Biotech and Biopharma Pvt. Ltd., Biotech Research Park, M-7, Laxmipuram, Transport Nagar, Gwalior-474010 (M.P.) India 3Tropilite Foods Pvt. Ltd., Davar Campus, Tansen Road, Gwalior-474002 (M.P.) India Abstract: Lentinus edodes is the first medicinal macrofungus to enter the realm of modern biotechnology. It is the second most popular in the global market which is attributed not only to its nutritional value but also to possible potential for therapeutic applications. Lentinus edodes is used medicinally for diseases involving depressed immune func- tion (including AIDS), cancer, environmental allergies, fungal infection, frequent flu and colds, bronchial inflammation, heart disease, hyperlipidemia (including high blood cholesterol), hypertension, infectious disease, diabetes, hepatitis and regulating urinary inconsistancies. It is the source of several well-studied preparations with proven pharmacological prop- erties, especially the lentinan, eritadenine, mushroom mycelium, and culture media extracts (LEM, LAP and KS-2). Antibiotic, anti-carcinogenic and antiviral compounds have been isolated intracellularly (fruiting body and mycelia) and extracellularly (culture media). Some of these substances were lentinan, lectins and eritadenine. The aim of this review is to discuss the therapeutic applications of this macrofungus. The potential of this macrofungus is unquestionable in the most important areas of applied biotechnology. Keywords: Anti-cancer, eritadenine, immunomodulation, lentinan, Lentinus edodes, polysaccharide.

1. INTRODUCTION It is a good source of , protein and essential amino acids. It is low in fat, have a high percentage of poly- Synthetic medicines (allopathic) after the Second World unsaturated fatty acids and also contain many vitamins and War completely outclassed the use of herbal folk medicine minerals. The protein in L. edodes are composed of 18 types but during the last 20 years herbal products have re-emerged of amino acids, including all essential amino acids in ratios as a major competitor of synthetic products, being used in similar to that ideal for human nutrition [10]. L. edodes con- the form of herbal preparations, herbal teas, confectionery tains significant quantities of vitamins C, B1, B2, B12, nia- and non-alcoholic beverages [1]. Historically, higher fungi cin, and (the highest content of vitamin D of any have long been used for medicinal purposes. Various impor- plant food) [11]. L. edodes mushroom-derived polysaccha- tant pharmaceutical products with proven medicinal applica- rides, immunomodulating and anticancer compounds are tions have been derived from higher Basidiomycetes fungi used in clinical applications as adjuvant to standard chemo- [2]. Lentinus edodes (shiitake) is one of the world’s second therapy. Another potential use for L. edodes is as foodstuff, largest cultivated medicinal and edible mushroom used as consumed whole or in concentrated extract or as dietary sup- ‘Functional foods’ has a long history in oriental folklore for plement [12]. There are several types of dietary supplements treatment of tumors, flu, heart diseases, high blood pressure, and medicinal formulations derived from L. edodes: dried obesity, problems related to sexual dysfunction and ageing, and pulverized fruiting bodies, hot water and alcohol extracts diabetes, liver ailments, respiratory diseases, exhaustion and of fruiting bodies, biomass or extracts of mycelia, or broth weakness. L. edodes is among the most valuable medicinal harvested from submerged liquid cultures. Commercial mushroom [3]. preparations are available as tablets, capsules, or elixirs, and It is a source of two well-studied and widely approved are available in most Asian countries and are increasingly polysaccharide medicines: LEM (an acronym for L. edodes available in USA, New Zealand, Australia, and Europe. mycelia), a protein-bound polysaccharide derived only from the mycelium [4], and lentinan – a cell-wall branched – -D- 2. THE MACROFUNGUS extracted from both the fruiting body and mycelium [5]. Both compounds are immune system enhancers that 2.1. Habit and Habitat demonstrated anticancer activity. Additionally, L. edodes L. edodes belongs to the kingdom-Fungi, Phylum- contains other compounds that inhibit blood aggregation; Basidiomycota, Class-Basidiomycetes, order-Agaricales, reduce cholesterol levels, and exhibit antibacterial and anti- family-Agaricaceae, genus-Lentinus and species-edodes viral effect [6, 7]. A lignan-rich compound derived from L. [13]. It produces sexual basidiospores, which are the repro- edodes mycelium holds a promising factor for treating both ductive units of this . The fruit bodies (basidiocarps) hepatitis-B and AIDS [8]. Another anti-tumour active poly- contain the basidiospore-producing structures, basidia [14]. saccharide, KS-2, has been isolated from L. edodes mycelia Each basidium carries four basidiospores, and each basidio- (LEM) [9]. spore in turn contains one haploid nucleus. Once basidio- spores have been released and the conditions are favourable

*Address correspondence to this author at the School of Studies in Biotech- they germinate to form hypha, a thread like fungal cell, fur- nology, Jiwaji University, Gwalior-474011 (M.P.) India; Tel: +91- ther divided into hyphal compartments. The hypha is sur- 9425122399; Fax: +91-751-4061276; E-mail: [email protected], rounded by a cell wall mainly composed of and glu- [email protected] cans (polymers of ) having a single nucleus in each

0929-8673/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd. 2420 Current Medicinal Chemistry, 2010 Vol. 17, No. 22 Bisen et al. compartment (monokaryon). When the hyphae of two differ- gions of Bhutan, Nepal, and India [16, 17]. Samgina [18] ent mating compatibility groups fuse with one another a di- reported that shiitake occurs in Kazakhstan, where the mush- caryotic hyphae are produced, containing two nuclei in each rooms were said to have been growing on conifer wood. hyphal compartment (one from each compatibility group). However, shiitake is typically known only to occur on logs The hyphae continue to grow by extension at the hyphal tip of broadleaf trees, especially Fagaceae [19, 20], which sug- and branches repeatedly to form hyphal filaments in a com- gests the fungi in Kazakhstan may have been misidentified. plex network, mycelium, which is the vegetative part of the The distribution of shiitake in the Pacific islands north and fungus. The mycelium of the fungus is responsible for the east of Papua New Guinea, such as New Caledonia, is also capturing of nutrients for growth and is often hidden under- poorly documented. ground or in the decaying organic material. Pegler [16] correlated morphological differences with When the surrounding conditions are right, mycelia start geographic distribution to recognize three species of shiitake: to form a fruit body completing one life-cycle of the mush- Lentinus edodes in northeast Asia, Lentinus lateritia in room (Fig. 1). All the tissues of the fruit body are composed Southeast Asia and Australasia, and Lentinus no- of dikaryotic hyphae, and within each basidium the two nu- vaezelandieae in New Zealand. Other mycologists have clei are fused to produce a diploid nucleus [14]. The subse- questioned whether Pegler's species are in fact morphologi- quent meiosis produces haploid nuclei, which then migrate cally distinct [17]. Pegler's species concept have also been into the developing basidiospores, one in each of the four challenged on the basis of mating criteria. Isolates of shiitake basidiospores. The basidiospores reside in the basidium until from Japan, Papua New Guinea, Borneo, Nepal, and New release and the beginning of a new life cycle of the mush- Zealand have shown to be mating compatible in laboratory room. crosses [17], which has led some mycologists to recognize only one species of shiitake Lentinus edodes. Lentinus edodes is a wood-decaying basidiomycetes, gregarious on fallen wood of a wide variety of deciduous trees, especially shii, oak, chestnut, beech, maple, sweet 3. CHEMICAL CONSTITUENTS AND BIOACTIVE gum, poplar (aspen, cottonwood), alder, hornbeam, iron- COMPONENTS OF L. EDODES wood, chinquapin, mulberry in a warm, moist climate [15]. The geographic distribution of shiitake in nature extends Scientific investigations have led to isolation of many beyond northeast Asia, but the exact limits are uncertain. The compounds from L. edodes having health promotion activi- minimum range, based on reliable, well documented reports, ties [15, 21, 22]. Fruit bodies of L. edodes contains 88–92% extends from northern Japan as far south and east as Tasma- water, protein, lipids, as well as vitamins and nia and New Zealand and as far west as the Himalayan re- minerals (Table 1) [23]. The mushroom is a good source of vitamins (Table 2), especially provitamin D2 (ergosterol),

Fig. (1). Different Growth stages of Lentinus edodes during artificial cultivation; A, Culture plate (Growing in Potato Dextrose Agar Me- dium); B, Spawn (Prepared in wheat grain); C, Mycelial colonization on solid substrate; D, Bump and primordia formation; E, Maturation of fruitbody; F, Mature fruit body. Lentinus edodes: A Macrofungus with Pharmacological Activities Current Medicinal Chemistry, 2010 Vol. 17, No. 22 2421

Table 1. Proximate and Mineral Composition of Lentinus Table 3. Fatty Acid Composition of Lentinus edodes (Values edodes (per 100 gm Sample) [23] Are Percent of Total Fat) [23]

S. No. Components Concentration S. No. Fatty acid Concentration in %

1 Moisture 4.7gm 1 C 16.0 Palmitic 19.2 2 Protein 22.8gm 2 C 18.0 Stearic 2.7 3 Fat 2.1gm 3 C 20.0 Arachidic 0.4 4 Ash 6.0gm 4 C 18.1 Oleic 8.3 5 Carbohydrate and Fibre 64.4gm 5 C 18.2 Linoleic 68.8 6 Energy (Kcal) 411 6 C 18.0 Linolenic 0.6 7 Phosphorus 439mg 7 Total Saturates 22.3 8 Magnesium 200mg 8 Total Unsaturates 77.7 9 Calcium 127mg 10 Iron 20.1mg Table 4. Amino Acid Composition of Lentinus edodes [23]

11 Zinc 4.3mg S. No. Amino acid Concentration (g16gN) 12 Manganese 5.1mg 1 Threonine 3.2 13 Copper 0.9mg 2 Valine 6.7 14 Chromium 140 g 3 Cysteine 1.4 Values are expressed on dry weight basis. 4 Methionine 0.8 Table 2. Vitamin Composition of Lentinus edodes (Quantities 5 Isoleucine 4.9 Per 100g) [25, 26] 6 Leucine 7.3 S. No. Vitamins Concentration 7 Tyrosine 3.3 fresh dry 8 Phenylalanine 4.2

1 Thiamin 0.05mg + 9 Lysine 6.4 2 Ribolflavin 0.15mg + 10 Histidine 2.3 3 Niacin 2.6mg + 11 Arginine 8.0 4 Ascorbic acid 2.1mg - 12 Aspartate 9.9 5 Vitamin K - - 13 Serine 5.3 6 Pantothenic acid - - 14 Glutamate 12.6 7 Biotin - - 15 Proline 8.0 8 Pyridoxin - - 16 Glycine 5.1 9 Folic acid 0.03mg - 17 Alanine 7.8 10 Vitamin D - 4000 i.u. 18 Total essential amino acids 38.2 11 Pro-Vitamin D Ergosterol 679g 19 Total amino acids 97.2 20 % Essential amino acids 39 which under ultraviolet (UV) light and heat yields calciferol 21 Chemical score 29 (vitamin D2). It also contains B vitamins, including B1 (thiamine), B2 (riboflavin) and B12 (niacin) [21, 22, 24-26]. The fatty acids account for 3.38% (Table 3) of the total lipids as -glucan and protein and water-insoluble substances ex- [21,27] with an appreciable amount of amino acids (Table 4) tractable only with salts, acids, and alkalies such as [23]. polyuronide (acidic polysaccharide), , -glucan with heterosaccharide chains, , and chitin present as In addition to -like , (1-4)-, (1- cell wall constituents. The aroma components include alco- 6)--D- and antitumor polysaccharides, lentinan, (1- hols, ketones, sulfides, alkanes, fatty acids, etc [21]. The 3)-, (1-6)--bonded heteroglucans, heterogalactans, hetero- major volatile flavor contributors are matsutakeol (octen-1- , xyloglucans, etc., have been identified. Among the ol-3) and ethyl-n-amyl ketone. The characteristic aroma of free present are , glycerol, mannitol, arabitol, shiitake mushrooms was identified as 1, 2, 3, 5, 6- , and [15, 21, 22, 24]. In shiitake mush- pentathiepane. According to Mizuno [21], the components rooms, consists of water-soluble materials such responsible for the delicious flavor are monosodium gluta- 2422 Current Medicinal Chemistry, 2010 Vol. 17, No. 22 Bisen et al. mate, 50-nucleotides, free amino acids, lower molecular It is an  -mannan peptide containing the amino acids serine, weight peptides, organic acids, and sugars. Their relative threonine, alanine, and proline (as well as residual amounts ratios are responsible for the variation in flavor naturally of the other amino acids). The polysaccharide is shown to be seen in this mushroom. Organic acids contributing to the effective on Sarcoma 180 and Ehrlich’s carcinoma, either i.p. flavor of shiitake mushroom include malic acid, fumaric or p.o., and to act via -inducing activity [9]. acid, -keto-glutaric acid, oxalic acid, lactic acid, acetic acid, formic acid, and glycolic acid. 3.4. Eritadenine

3.1. Lentinan Eritadenine was isolated from L. edodes mushroom for the first time and identified as the active cholesterol lowering Lentinan is a high molecular weight (5x105 Da); [] D component [30]. Eritadenine, 2(R), 3(R)-dihydroxy-4-(9- +20–22° (NaOH) polysaccharide [(C6H10O5)n] extracted adenyl) - butyric acid, was formerly designated as lentinacin from cell wall of fruiting body in a triple helix structure con- [30] and lentysine [31, 32] by the research groups who indi- taining only glucose molecules with mostly -(1-3)-glucose vidually isolated and structurally determined this compound linkages in the regularly branched backbone, and -(1-6)- (Fig. 3). Eritadenine is a secondary metabolite produced glucose side chains (Fig. 2) [15, 21, 28]. The configuration mainly by L. edodes. The ability of eritadenine is to lower of the glucose molecule in a helix structure is considered to the blood cholesterol level. It has been reported that erita- be important for the biological and pharmacological activity denine was present in L. edodes at levels of 400-700 mg/kg [29]. Lentinan is completely free of any nitrogen (and thus dry matter [33]. protein), phosphorus, sulphur and any other atoms except carbon, oxygen and hydrogen. It is water soluble, heat stable and alkali labile.

Fig. (3). Chemical structure of eritadenine.

4. PHARMACOLOGICAL ASPECTS OF L. EDODES

L. edodes is one of the well-known macrofungus used in several therapeutic applications. It is the source of several well-studied preparations with proven pharmacological Fig. (2). Chemical structure of lentinan. properties. The medicinal properties of L. edodes (Fig. 4) have been studied since Ming Dynasty (1369-1644) [34]. 3.2. LEM and LAP (EP3) The elders from Japanese Empire considered shiitake as the “elixir of the life” increasing vigor and energy [35]. Antibi- LEM and LAP extracts are derived from L. edodes mush- otic, anti-carcinogenic, anticancer, antifungal, antibacterial room mycelium and culture media respectively. Both are and antiviral, antidiabetic, hypolipidemic compounds have glycoproteins containing glucose, , , arabi- been isolated intracellularly (fruiting body and mycelia) and nose, mannose and . LEM also contains nucleic acid extracellularly (culture media) from L. edodes. Some of derivatives, vitamin B compounds and ergosterol. LEM and these substances were lentinan, lectin and eritadenine [30, LAP both demonstrated strong antitumour activity by i.p., 36-40]. The shiitake mushroom is used medicinally for dis- and p.o. in animals and humans. LEM is prepared from a hot eases involving depressed immune function (including water extract of powdered mycelia, incubated for 50-60 h at AIDS), cancer, diabetes, environmental allergies, fungal in- 40-50oC and partially hydrolysed by endogenous enzymes. fection, frequent flu and colds, bronchial inflammation, and The residue was extracted with water, 60oC, and the filtrate regulating urinary incontinence. A summary of the therapeu- freeze dried. The final light brown powder was LEM. An tic effects and bioactive compounds of L. edodes which is immunoactive substance EP3 has been obtained by further reported in the literature till 2009 has been represented in fractionation of LEM. [4, 22]. Table 5. Major therapeutic effects of this wonderful mush- room is being elaborated below. 3.3. KS-2-- Mannan Peptide 4.1. Antitumor and Anticarcinogenic Activity KS-2 is a peptide–polysaccharide complex (MW 6– 4 o 9.5x10 [] D +62 ; C = 0.5, water) which was obtained by The antitumor polysaccharide ‘Lentinan’ was first iso- extraction of cultured mycelia of L. edodes mushroom with lated and studied by Chihara et al. [41] who demonstrated hot water, followed by precipitation with ethanol [21, 27, 9]. that its anti-tumor effects were greater than other mushroom Lentinus edodes: A Macrofungus with Pharmacological Activities Current Medicinal Chemistry, 2010 Vol. 17, No. 22 2423

Fig. (4). Medicinal importance of Lentinus edodes.

Table 5. Therapeutic Effects and Bioactive Compounds of L. edodes Reported in the Literature Until 2009

Therapeutic effects Bioactive compounds References

Antitumor Lentinan ( -D-glucans), KS-2--mannan-peptide, LEM,LAP (heteroglucan-protein), EP3 [2,5,9,21,27] Immunomodulation Mannoglucan, polysaccharide [5,22,41,49,53,54] protein complex, glucan, Lentinan, polysaccharide L-II, (1-3)--D-glucan Antimicrobial Lentinamicin [55] Antiviral Lentinan, LEM, JLS-18, EP3, EPS4 [2,62-64] Antibacterial LEM, Lenthionine , chloroform and ethylacetate extracts [63,64,66] Antifungal Lentin [69] Cardiovascular and Hypolipidemic Eritadenine, lentinacin, lentysine [3,30-32,70,72] Hepatoprotective Lentinan, LEM, hot-water extraction and ethanol extraction [75,76] Hemagglutinating Lectin [77-79] Antioxidant methanol and water extracts, [80,82,83] polyphenolic compounds polysaccharides and was active for some types of tumors cells in a dose-dependent manner when administrated orally [42]. The antitumor effect of lentinan was confirmed by us- [46]. ing Sarcoma-180 transplanted in CD-1/ICD mice [5]. Later, The carcinostatic effect of lentinan results from the acti- it showed prominent antitumor activity not only against allo- vation of the host’s immune system. ß-D-glucan binds to genic tumors, such as Sarcoma-180, but also against various lymphocyte surfaces or serum-specific proteins, which acti- synergic and autochthonous tumors, and it prevented chemi- vate macrophage, T-helper cells, natural killer (NK) cells, cal and viral oncogenesis [43]. The tumor inhibitory effect of and other effector cells. All these increase the production of lentinan was highly striking. In 1-5 mg/kg x 10 doses, the antibodies as well as (IL-1, IL-2) and interferon inhibition ratio was 95 to 97.5%, and in dosages of 0.2 (IFN-) released upon activation of effector cells [47]. The mg/kg x 10, the tumors underwent complete regression in 6 antitumor studies conducted with L. edodes thus far are very out of 10 mice [41]. Combination treatment of L. edodes interesting and do show a potential for providing therapeutic mycelium extracts with 5-Fluorouracil represent a novel control of cancer. It is, however, difficult to say whether L. chemotherapeutic strategy in colon cancers and that p53, edodes could have preventive effects against cancer when p21/Cip1 and p27/Kip1 may play some important roles for consumed as part of the diet. the involvement in antitumor activity [44]. Four antitumor (1/3)--D-glucans coded as L-I1, L-I2, L-I3 and L-I4 with 4.2. Immunomodulating Effects high molecular weight (1.47x106–1.67x106) were isolated from four kinds of fruiting bodies of Lentinus edodes [45]. Lentinus edodes has attracted a lot of attention owing to Exo-biopolymer from rice bran cultured with L. edodes [rice its immunomodulatory effects. Lentinan is well known as a bran exo-biopolymer (RBEP)] induced the activation of NK type of biological response modifier (BRM). Augmentations 2424 Current Medicinal Chemistry, 2010 Vol. 17, No. 22 Bisen et al.

Fig. (5). Early phase of the mechanism of action of Lentinan and possible pathway for inflammatory and immune reactions. IL-1, IL-2, IL-3, and IL-6: -1, -2, -3, and –6. APPIF: acute phase protein-inducing factor VDHIF: vascular dilation and hemorrhage-inducing fac- tor; CSF: colony-stimulating factor; MIF: migration inhibition factor;’ TNF: ; VPF: vascular permeability factor; NKF: natural killer cell-activating factor; MAF: macrophage-activating factor; CTL: cytotoxic T lymphocyte; LAK: lymphokineactivated killer cell G. [52]. of NK (Natural Killer), CTL (Cytotoxic T Lymphocyte), spleen cells of infected mice revealed that the Th1 immune LAK (Lymphokine Activated Killer) activities and DTH response was stimulated against malaria infection. In vitro (Delayed Type Hypersensitivity) responses against tumor and in vivo, lentinan enhanced the expression of MHC II, antigen were observed after administration of Lentinan [48]. CD80/CD86, and Toll-like receptors (TLR2/TLR4), and These activities are responsible for the antitumor effects of increased the production of IL-12 in spleen dendritic cells lentinan. Antitumor polysaccharide L-II was isolated and (DCs) co-cultured with parasitized red blood cells (pRBCs). purified from the fruiting body of L. edodes [49]. The anti- It was concluded that lentinan has prophylactic potential for tumor activity of the polysaccharide L-II on mice- the treatment of malaria [53]. The immunomodulation ef- transplanted sarcoma 180 was mediated by immunomodula- fects of lentinan through oral administration were investi- tion by inducing T-cells and macrophage-dependent immune gated [54] and the results obtained proved its efficacy, that system responses. Kupfahl et al. [50] evaluated   prefeeding of the mice for 7 days at an optimum dose of 3                mg/mouse was most effective against tumor induction,   Listeria monocytogenes   The results achieving a tumor inhibition rate (TIR) of 94.44%. Possible showed that the lentinan enhances the protective CD8 T-cell immune system regulating actions of lentinan were summa- response against L. monocytogenes probably by a mecha- rized [43] and are depicted in Fig. (6). nism that involves the IL-12-mediated augmentation of the specific antilisterial CD8 T-cell response Fruit body and 4.3. Antimicrobial Activity mycelial extracts of L. edodes are able to enhance the prolif- eration of rat thymocytes directly and act as co-stimulators in Antimicrobial activity has been found in liquid cultures, the presence of the T-mitogen PHA [51]. Many interesting chloroform, ethyl acetate, water and dried fruit body extracts mechanisms of action of Lentinan and possible pathways for of L. edodes [55-57]. These extracts are active against gram inflammatory and immune reactions have been represented positive and gram-negative bacteria, yeasts and mycelial in Fig. (5) [52]. fungi, including dermatophytes and phytopathogens [58]. Mycelial-free culture of L. edodes exhibited greater antimi- Pre-treatment of mice with lentinan significantly de- crobial effect against grampositive than gram-negative bacte- creased the parasitemia and increased their survival after ria with Bacillus subtilis and Staphylococcus aureus among infection by Plasmodium yoelii 17XL (P.y17XL). Enhanced the most highly inhibited [56]. Antimicrobial compounds IL-12, IFN- and NO production induced by lentinan in isolated from L. edodes liquid cultures include lentinamicin Lentinus edodes: A Macrofungus with Pharmacological Activities Current Medicinal Chemistry, 2010 Vol. 17, No. 22 2425

Fig. (6). Mechanisms of antitumor and immunomodulating activity of lentinan as a -D-glucan [43, 60]. Mac, macrophages; TL(H), T- lymphocyte (helper); NK, natural killer cells; IL-1, -2 and -13, interleukin-1, -2, -13; CSF, colony-stimulating factor; MAF, macrophage- activating factor; PC-TL, precytolytic T lymphocyte; CTL, cytolytic (cytotoxic) T lymphocyte; BL, B lymphocyte.

(octa-2, 3-diene-5, 7 diyne-1-ol) [55], -ethyl phenyl alcohol Candida albicans and Saccharomyces cerevisiae; (f) in- [55]. creased host resistance to infections with potentially lethal Listeria monocytogenes [2]. 4.3.1. Antiviral Activity LEM and a new lignan-rich compound ‘JLS-18’ derived L. edodes had been believed to cure the common cold for hundreds of years. More recently, some scientific evidences from LEM, blocked the release of infectious Herpes simplex virus in animals [62] and it has been suggested because of its have been obtained to support this belief. L. edodes showed high activity that JLS-18 could be of value in the treatment an activity (expressed as the percentage decrease in lung of hepatitis B and AIDS patients [63]. Water-soluble lesion score compared with the control) of 46%, which was from EP3 and EPS4 from shiitake mushroom mycelium have of the same magnitude as for amantadine hydrochloride, a shown antiviral effects [64]. common drug against influenza (40%). A watery extract from L. edodes was also reported to prevent the multiplica- 4.3.2. Antibacterial Activity tion of polio virus [59]. Lentinan enhanced the host resis- Anti-bacterial activity is an exciting result, with increas- tance against infections with bacteria, fungi, parasites, and ing bacterial resistance to antibiotics, improving host immu- viruses, including the agents of AIDS [60]. Lentinan reduced nity may be the way forward in fighting bacterial infection. the toxicity of azidothymidine AZT (a drug commonly used The antibacterial activity of L. edodes against Bacillus sub- for treating HIV carriers and AIDS patients). Prevention of tilis was evaluated in cell-free filtrates obtained after growth the onset of AIDS symptoms through potentiation of host in 14 different culture media [65]. Lenthionine, a sulphur defense is now being actively investigated both experimen- containing peptide from shiitake has antibacterial and anti- tally and clinically [61]. In addition to lentinan, other sub- fungal activity and bis [(methylsulfonyl) methyl] disulphide stances from L. edodes have also been shown to have antivi- [66], a derivative of lenthionine, has strong inhibitory effects ral activity. The mechanism of their effect is in most cases against Staphylococcus aureus, Bacillus subtilis and Es- via induction of interferon [3, 21]. cherichia coli [67]. The chloroform and ethylacetate extracts Lentinan has also shown: (a) antiviral activity in mice of the dried shiitake mushroom have bactericidal activity against VSV (vesicular stomatis virus), encephalitis virus, against both growing and resting Streptococcus mutans and Abelson virus, an adenovirus type 12; (b) stimulated non- Prevotella intermedia [68]. specific resistance against respiratory viral infection in mice; (c) conferred complete protection against an LD75 challenge 4.3.3. Antifungal Activity dose of virulent mouse influenza A/SW15; (d) increased A novel protein designated lentin with potent antifungal resistance to the protozoal parasites Schistosoma japanicum, activity was isolated by Ngai and Ng [69] from the fruiting Sch. mansoni; (e) exhibited activity against Mycobacterium bodies of the L. edodes. Lentin, which had a molecular mass tuberculosis bacilli resistant to antituberculosis drugs, Bacil- of 27.5 kDa, inhibited mycelial growth in a variety of fungal lus subtilis, Staphylococcus aureus, Micrococcus lenteus, species including Physalospora piricola, Botrytis cinerea 2426 Current Medicinal Chemistry, 2010 Vol. 17, No. 22 Bisen et al. and Mycosphaerella arachidicola. Lentin also exerted an cemic activity in streptozotocin (STZ)-induced diabetic rats inhibitory activity on HIV-1 reverse transcriptase and prolif- [74]. In dose-dependent study, orally administered L. strigo- eration of leukemia cells. sus EPS, at the dose of 150 mg/kg, exhibited a considerable hypoglycemic effect in STZ-induced diabetic rats. Plasma 4.4. Cardiovascular and Hypolipidemic Activity insulin levels of STZ-induced diabetic rats decreased as compared to control group rats. Although insulin levels Cardiovascular diseases are among the main causes of slightly increased in the EPS-treated groups. The hypogly- death in our society and there is a strong correlation between cemic potential of the EPS was further supported by his- enhanced blood cholesterol levels and the development of tological observations of pancreatic islets of Langerhans. such diseases. The popular macrofungus L. edodes, has been shown to produce blood cholesterol lowering compound 4.6. Hepatoprotective Activity designated eritadenine [2(R), 3(R)-dihydroxy-4-(9-adenyl)- butyric acid] [30]. The hypocholesterolemic action of this A polysaccharide fraction from L. edodes showed liver compound has been quite extensively examined in rats. Eri- protective action in animals together with improved liver tadenine is suggested to accelerate the removal of blood cho- function and an enhanced production of antibodies to hepati- lesterol either by stimulating tissue uptake or by inhibiting tis B [21]. Lentinan and LEM have given favourable results tissue release [70]. It was demonstrated that when rats were in treating chronic persistent hepatitis and viral hepatitis B fed a diet supplemented with 5% (dry weight) of L. edodes patients [75]. Lentinan improved serum glutamic pyruvic fruiting bodies for 10 weeks the plasma cholesterol levels of transaminase (SGPT) and completely restored GPT levels in the animals decreased significantly [71]. Eritadenine works the livers of mice with toxic hepatitis. Crude extracts of shii- by lowering levels of all lipoprotein types, i.e., high-density take mushroom cultures have demonstrated liver-protecting as well as low-density lipoproteins [3]. actions [2, 22, 27]. In addition to animal tests, the effectiveness of L. edodes The hot-water extraction and ethanol extraction of the in lowering blood serum cholesterol was also tested in hu- mycelia of L. edodes were examined for their hepatoprotec- man subjects. A daily intake of 90 g of fresh shiitake, 9 g of tive effect on dimethylnitrosamine-injured mice [76]. Both dried shiitake, and 9 g of UV-irradiated dried shiitake for 7 fractions decreased the blood aspartate aminotransferase and days lowered the mean serum cholesterol levels in young alanine aminotransferase levels, partially inhibited the over women by 12, 7 and 6%, respectively [7]. All three diets accumulation of collagen fibrils, and suppressed the over decreased serum cholesterol levels of older persons (60 years expression of genes for alpha-smooth muscle actin and/or of age) by 9% over 7 days. Eritadenine reduces blood serum heat-shock protein 47 in the mice. Both fractions also inhib- cholesterol in mice, not by the inhibition of cholesterol bio- ited the morphologic change and proliferation of isolated rat synthesis, but by the acceleration of the excretion of ingested hepatic stellate cells (HSCs), which play a central role in cholesterol and its metabolic decomposition. It has been liver fibrosis, in a dose-dependent manner and without cyto- shown to lower blood levels of cholesterol and lipids in ani- toxicity. The direct interaction between the extracts and mals. When added to the diet of rats, eritadenine (0.005%) HSCs appears to be important for the hepatoprotective activ- caused a 25% decrease in total cholesterol in as little as one ity. contained in both fractions are considered to week. The cholesterol-lowering activity of this substance is be potential candidates for expressing the hepatoprotective more pronounced in rats fed a high fat diet than in those on a effects. low-fat diet. Although feeding studies with humans have indicated a similar effect [6, 22, 24]. The amount of choles- 4.7. Hemagglutinating Activity terol reducing agent (eritadenine) in L. edodes, in search of a potential natural medicine against blood cholesterol was The agglutinating activity of different morphogenetic quantified [70, 72]. structures of L. edodes F-249, including mycelium, brown mycelial mat, primordia, and fruiting bodies was studied. 4.5. Antidiabetic Activity Data showed that mycelial mat was found to possess the maximum hemagglutinating activity, which can be explained The hypoglycemic effect of an exo-polymer produced by the possible involvement of agglutinins in the formation from a submerged mycelium culture of L. edodes was inves- of mycelial mat, which is composed of glued hyphae. The tigated in streptozotocin- induced diabetic rats [6], the ad- changes of the hemagglutinating activity of intracellular lect- ministration of the exo-polymer (200 mg/kg BW) reduced ins of the basidiomycete L. edodes at various stages of its the plasma glucose level by as much as 21.5%, and increased morphogenetic development depending on erythrocyte type, plasma insulin by 22.1% as compared to the control group. It growth medium, and lectin purification degree was studied also lowered the plasma total cholesterol and triglyceride [77-79]. levels by 25.1 and 44.5%, respectively. The hypoglycemic effect of L. edodes has been also demonstrated and proved its 4.8. Antioxidant Activity potential in lowering the blood glucose and triglyceride (TG) levels in the serum of rats [73]. It also lowered the plasma An antioxidant is a molecule capable of slowing or total cholesterol and triglyceride levels by 25.1 and 44.5%, preventing the oxidation of other molecules. Oxidation is a respectively. chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free Exopolysaccharide (EPS) produced from submerged my- radicals, which start chain reactions that damage cells. celial culture of Lentinus species was evaluated for hypogly- Antioxidants terminate these chain reactions by removing Lentinus edodes: A Macrofungus with Pharmacological Activities Current Medicinal Chemistry, 2010 Vol. 17, No. 22 2427 free radical intermediates, and inhibit other oxidation toneal and/or pleural effusions of a group of 16 patients with reactions by being oxidized themselves. advanced cancer [86]. Eighty percent of the lesions showed probable clinical responses, with an improvement in per- Pharmacological effect of polysaccharides from L. edo- formance demonstrated in seven subjects. The survival time des on serum oxidative status in high-fat-diet rats were in- vestigated [80]. The rats were fed a standard diet and had for those who responded immunologically to the treatment was 129 days and 45 days for those who did not respond. free access to water. The administration of polysaccharides from L. edodes significantly reduced serum total cholesterol Mycelium extract of L. edodes was used to boost the im- (TC), triglyceride (TG), low density lipoprotein cholesterol mune response in AIDS patients [15, 22]. When it was used (LDL-c) and enhance serum antioxidant enzyme activity and to treat HIV-positive patients with AIDS symptoms, the T- thymus and liver index in high-fat rats. In addition, the ad- cell count rose from a baseline of 1250/mm3 after 30 days up ministration of polysaccharides from L. edodes significantly to 2550/mm3 after 60 days. Forty patients with chronic viral decreased the increased expression level of VCAM-1mRNA. hepatitis B and seropositive for Hbe antigenemia were given In conclusion, the data suggested that the administration of 6 g of LEM daily (orally) for 4 months. The study focused polysaccharides from L. edodes could decrease the increased on the number of patients seroconverting from Hbe antigen oxidative stress induced by high-fat diet in rats. positive to antiHbe positive, which was 25% after LEM ther- The antioxidant activity of L. edodes extracts obtained by apy, and was higher in patients with chronic active hepatitis (36.8%). In addition, 17 patients (43%) became seronegative organic solvents and supercritical fluids using different ex- for Hbe antigen. Liver function tests improved even for pa- traction techniques; high-pressure operations and low- tients who remained seropositive, and they had raised plasma pressure methods were investigated [81]. The mushroom albumin, and adjusted protein metabolism. extract was mixed with a 0.3 mM 2, 2-diphenyl-1-picryl- hydrazyl-hydrate (DPPH) ethanol solution, to give final con- Dried shiitake mushroom (9 g/day) decreased 7–10% se- centrations of 5, 10, 25, 50 and 100 g of extract per mL of rum cholesterol in patients suffering with hypercholes- DPPH solution. Fractions obtained with CO2 using ethanol terolemia. For many patients 60 years of age or older with as co-solvent, at 40ºC, 20 MPa and 15% EtOH and for di- hyperlipidemia, consuming fresh shiitake mushroom (90 chloromethane in low-pressure technique had antioxidant g/day in 7 days) led to a decrease in total cholesterol blood activities. level by 9–12% and triglyceride level by 6–7% [22, 6]. The effect of heat treatment on the changes in the overall antioxidant activity and polyphenolic compounds of L. edo- 6. TOXICITY AND SIDE EFFECTS OF L. EDODES des was investigated by Choi et al. [82]. The heat treatment significantly enhanced the overall antioxidant activities of Lentinus edodes is an edible mushroom, but some indi- shiitake mushroom. The low molecular weight (LMW) viduals may experience minor side effects or allergic reac- subfraction of the water extract of L. edodes had the highest tions. Allergic reactions to the spores of L. edodes have been antioxidant activity against lipid peroxidation of rat brain reported in workers picking mushrooms indoors. Symptoms homogenate with IC50 values of 0.109 and 1.05 mg/ml [83]. include fever, headache, congestion, coughing, sneezing, Bioprocessing was very effective in mobilizing phenolic nausea, and general malaise [87]. A water extract of the antioxidants from soybean powder. Post processing, phenolic fruiting body was found to decrease the effectiveness of content had increased 90% to 11.3 mg/g dry weight (wt) in blood platelets in initiating coagulation [88]. L. edodes my- water extract and 232% to 5.8 mg/g dry wt in ethanol extract celium has shown no evidence of being acutely toxic, even in [84]. Hence, it was clearly concluded that L. edodes had a massive doses of over 50 mg/day for 1 week, though mild power-pack antioxidant property which would be beneficial side effects such as diarrhea and skin rash may occur. for mankind. Lentinan has no known serious side effects. However, in clinical trials of patients with advanced cancer, minor side 5. CLINICAL STUDIES OF L. EDODES IN HUMANS reactions occurred such as a slight increase in glutamate- oxaloacetate transminase (GOT) and GPT liver enzymes and Lentinan was demonstrated to have antitumor activity a feeling of mild pressure on the chest. But these changes and increased the survival time of patients with inoperable disappeared after lentinan administration was stopped [89]. gastric cancer [22] and women with recurrent breast cancer following surgical therapy [15, 22]. When the polysaccharide of L. edodes was administered once or twice a week with 7. COMMERCIAL ASPECTS OF L. EDODES chemotherapy to a patient with progressive cancer but with Today, present need in every aspect has increased so fast no serious liver, kidney, or bone marrow dysfunction, it pro- that every research work with commercial outlook requires duced a statistically significant improvement in immune and major modifications to meet the upcoming challenges and anticancer activity when compared to chemotherapy alone obstructions. Similarly, the present review is an attempt to [85]. Two hundred seventy-five patients with advanced or recurrent gastric cancer were given one of two kinds of che- create an understanding and make aware about the vital im- portance of the macrofungus “Lentinus edodes”.The great motherapy (mitomycin C with 5- fluorouracil or tegafur) interest in L. edodes commercialization is due to its fla- either alone or with lentinan injections. The best results were vor/taste, and several medicinal applications. More than obtained when lentinan was administered prior to chemo- 130,000 tons of L. edodes are produced per year, out of them therapy and in patients with a basis of prolongation of life, 45% of this is sold fresh, the rest being sold dried. L. edodes regression of tumors or lesions, and the improvement of im- mune responses. Lentinan was injected into malignant peri- is commercially available in many forms. It may be injected 2428 Current Medicinal Chemistry, 2010 Vol. 17, No. 22 Bisen et al. as a solution (1 mg/vial) or ingested as a -coated tablet, [4] Suzuki, H.; Iiyama, K.; Yoshida, O.; Yamazaki, S.; Yamamoto, N.; capsule, concentrate, powdered extract, syrup, tea, wine, and Toda, S. Structural characterization of the immunoactive and anti- viral water-solubilized lignin in an extract of the culture medium of or as a medicinal dish. The therapeutic uses of L. edodes for Lentinus edodes mycelia (LEM). Agric. Biol. Chem., 1990, 54(2), the treatment of a variety of diseases, including, AIDS/HIV 479-487. support, cancer, fatigue, hepatitis, high cholesterol, hyperten- [5] Chihara, G.; Maeda, Y.Y.; Hamuro, J.; Sasaki, T.; Fukuoka, F. sion, immune support, intestinal parasites/worms, longevity, Inhibition of mucose sarcoma 180 by polysaccharides from respiratory infections speak about L. edodes's commercial Lentinus edodes (Berk.) Sing. Nature, 1969, 222, 687-688. significance. [6] Yang, B.K.; Kim, D.H.; Jeong, S.C.; Das, S.; Choi, Y.S.; Shin, J.S.; Lee, S.C.; Song, C. H. Hypoglycemic effect of a Lentinus edodes exopolymer produced from a submerged mycelial culture. Biosci. 8. CONCLUSION AND FUTURE PROSPECTS Biotechnol. Biochem., 2002, 66, 937-942. [7] Suzuki, S.; Ohshima, S. Influence of L. edodes (Lentinus edodes) The Lentinus edodes has been described in the literature on human serum cholesterol. Mushroom Sci., 1976, 9(1), 463-467. [8] Jong, S.C.; Birmingham, J.M. Medicinal and therapeutic value of as a macrofungus with great potential for therapeutic appli- the shiitake mushroom. Adv. Appl. Microbiol., 1993, 39, 153-184. cations. Medicinal value of this mushroom has become a [9] Fujii, T.; Aeda. H.; Suzuki, F.; Shida, N. Isolation and characteriza- matter of great significance particularly in preventing or tion of a new antitumor polysaccharide, KS-2 extract from culture treating serious health conditions such as cancer, acquired mycelia of Lentinus edodes. J. Antibiot., 1978, 11, 1079-1090. immune deficiency syndrome (AIDS), and hypercholes- [10] Vetter, J. Mineralstoff and Aminosauregehalte des esbaren, kulti- vilten Pilzes Shii-take (Lentinus edodes). Z. Lebensm. Unters. terolemia. The potential of this macrofungus is unquestion- Forsch., 1995, 201, 17-9. able in some of the most important areas of applied biotech- [11] Mattila, P.; Suonp, K.; Piironen, V. Functional properties of edible nology. However, there are currently no standard protocols mushrooms. Nutrition, 2000, 16, 694-696. for verifying the product quality and efficacy. From a phar- [12] Wasser, S.P.; Nevo, E.; Sokolov, D.; Reshetnikov, S.; Timor- macological point of view, safety is the primary issue and Tismenetsky, H. Dietary supplements from medicinal mushrooms: diversity of types and variety of regulations. Int. J. Med. Mushr., research in this direction is desired. L. edodes has a great 2000, 2, 1-20. potential for the production of useful bioactive metabolites [13] Boer, C.G.; Obici, L.; Souza, C.G.M.; Peralta, R.M. Decolorization and that they serve as a prolific resource for drugs. The iden- of synthetic dyes by solid state cultures of Lentinula (Lentinus) tified bioactive compounds in L. edodes of known molecular edodes producing manganese peroxidase as the main ligninolytic structures account for a wide range of beneficial biomedical enzyme. Bioresour. Technol., 2004, 56, 1384-1387. 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Received: February 09, 2010 Revised: May 06, 2010 Accepted: May 09, 2010