HYPOGLYCEMIC ACTIVITY OF A. BILIMBI IN STREPTOZOTOCIN (STZ)-DIABETIC RATS

II. HYPOGLYCEMIC ACTIVITY OF A. BILIMBI IN STREPTOZOTOCIN (STZ)-DIABETIC RATS

The single-high-dose-STZ-induced diabetic rat is one of the animal models of human insulin-dependent diabetes mellitus (IDDM), or type I diabetes mellitus. In this model, diabetes arises from irreversible destruction of the

h -islet cells of the pancreas by streptozotocin, causing degranulation or reduction of insulin secretion. In this type I model of diabetes, the insulin is markedly depleted, but not absent (3).

The chemical structure of STZ consists of a glucose moiety (Fig. 2) with

a highly reactive nitrosourea side chain that is thought to initiate its cytotoxic action. As shown in Figure 3, the glucose moiety directs this agent to the pancreatic h cells, where it binds to a membrane receptor to cause structural damage (4). The deleterious effect of STZ results from the generation of highly

reactive carbonium ions (CH + 3 ) that cause DNA breaks by alkylating DNA bases at various positions, resulting in activation of the nuclear enzyme poly (ADP-ribose) synthetase, thereby depleting the cellular enzyme substrate (NAD + ), leading to the cessation of NAD + -dependent energy and protein metabolism. This in turn leads to reduced insulin secretion (5). It has been suggested that free-radical stress occurred during h-cell destruction mediated by mononuclear phagocytes and cytokines (6,7). Since free-radical scavenger was demonstrated to protect against the diabetogenic properties of STZ, it is likely the oxidative stress may play a role in determining STZ toxicity (8). However, some poly (ADP-ribose) synthetase inhibitors, such as nicotin- amide and 3-aminobenzamide, could prevent the onset of diabetes (9).

A. Hypoglycemic Activity of A. bilimbi in STZ-Diabetic Wistar Rats

STZ-induced diabetic male Wistar rats were given five intraperitoneal (i.p.) injections of the 80% ethanolic leaf extract of A. bilimbi (ABe) at 300 mg/kg or the water extract of the fruits of A. bilimbi (ABw) at 100 mg/kg or its cor- responding vehicle, daily for 7 days. Fasting blood glucose estimations and

F IGURE 2 The chemical structure of streptozotocin.

330 Pushparaj et al.

F IGURE 3 The mechanism of pancreatic h-cell destruction by streptozotocin.

food intake measurement were done on all the days of injection. The results of this preliminary study showed decreases in fasting blood glucose levels and mean daily food intake in the ABe- and ABw-treated STZ-diabetic rats (2).

B. Hypoglycemic Activity of A. bilimbi in STZ-diabetic Sprague-Dawley (SD) Rats

1. Effect on Oral Glucose Tolerance in Normal and Diabetic SD Rats

In the oral glucose tolerance test (OGTT) in both normal and STZ-diabetic SD rats, distilled water (control), a reference drug, metformin (500 mg/kg), or each of three different doses of ABe (125, 250, and 500 mg/kg) was orally administred to groups of four to five rats each after 16-hr fast. Thirty minutes later, glucose (3g/kg) was orally administered to each rat with a feeding syringe (10). The blood glucose levels of the normal and diabetic rats reached

a peak at 60 min after the oral administration of glucose and gradually decreased to preglucose load level. Of the three different doses, 125, 250, and 500 mg/kg, the lowest dose, i.e., 125 mg/kg, caused attenuation in the blood glucose level in both normal and diabetic rats (11). Similarly, its semipurified fractions, such as the aqueous fraction (AF) as well as the butanol fraction (BuF) at 125 mg/kg, caused attenuation in blood glucose level in STZ-diabetic

Averrhoa bilimbi 331

dose of 125 mg/kg twice a day for 14 days to STZ-diabetic SD rats caused a decrease in the blood glucose level (11,12).

2. Blood-Lipid and Cholesterol-Lowering Effect The daily administration of ABe per orally (125 mg/kg twice a day) for 14

days to STZ-diabetic SD rats caused a reduction in the serum triglycerides and an increase in HDL cholesterol. However, ABe did not decrease the serum cholesterol and LDL cholesterol. This leads to an increase in the antiathero- genic index and HDL cholesterol:total cholesterol ratio (11). Moreover, the daily administration of ABe (125 mg/kg) and metformin (500 mg/kg) to STZ- diabetic rats twice a day for 2 weeks caused a reduction in food and water intake and an increase in body weight (11). Since ABe increased HDL cholesterol, it significantly increased the antiatherogenic index and HDL cholesterol:total cholesterol ratio. ABe thus has the potential to prevent the formation of atherosclerosis and coronary heart disease, which are the sec- ondary diabetic complications of severe diabetes mellitus (13). In contrast, metformin failed to increase the HDL-cholesterol level and did not increase the antiatherogenic index and HDL cholesterol:total cholesterol ratio. How- ever, it has been reported that metformin can reduce the blood lipid param- eters in nondiabetic patients with coronary heart disease (14). Hence, ABe contains a hypolipidemic principle(s) that probably acts differently from metformin (11).

3. Effect of A. bilimbi on Liver and Kidney Thio-Barbituric-Acid-Reactive Substances (TBARS) in STZ-Diabetic SD Rats

ABe and AF treatment at a dose of 125 mg/kg in STZ-diabetic SD rats reduced the TBARS levels in the kidneys, but not in the liver. The lack of change in TBARS levels in the liver of ABe-, AF-, and BuF-treated diabetic rats could again reflect the resistance of the liver to the oxidative stress in the diabetic state. It is significant to note that neither AF nor BuF affects this capacity adversely. Since ABe and AF have the ability to reduce the formation of TBARS, they could potentially prevent platelet aggregation and throm- bosis (11,12).

4. Effect of A. bilimbi on Insulin in STZ-Diabetic Rats Similar to other hypoglycemic agents such as tungstate and vanadate (15–17),

AF caused a time-dependent hypoglycemic effect after twice-a-day oral administration of 125 mg/kg for 7 and 14 days in STZ-diabetic SD rats. On the other hand, when the STZ-diabetic rats were treated with 125 mg of BuF/ kg, the serum insulin level was higher on both day 7 and day 14. The elevation in serum insulin in the AF- and BuF-treated STZ-diabetic rats could be due

332 Pushparaj et al.

the residual functional h cells to produce insulin, or the protection of the functional h cells from further deterioration so that they remain active and produce insulin. However, except for the level in the AF-treated group on day

14, the insulin levels were well below the normal insulin level in control rats, suggesting that they may not be sufficient to lower the blood glucose to its normal level in STZ-diabetic rats (12). This indicates a possible insulin- releasing action of ABe in STZ-diabetic rats like the extracts of Medicago sativa (18), Eucalyptus globulus (19), and Sambucus nigra (20) which have been shown to possess insulin-releasing action both in vitro and in vivo.

5. Effect of A. bilimbi on Liver Glucose-6-Phosphatase Activity and Glycogen in STZ-Diabetic Rats

Glucose-6-phosphatase catalyzes the final step in glucose production by the liver and kidney. STZ has been reported to increase the expression of glucose- 6-phosphatase mRNA, which contributes to the increased glucose-6-phos- phatase activity in diabetes mellitus (21). Overproduction of glucose by the liver is the major cause of fasting hyperglycemia in both insulin-dependent and non-insulin-dependent diabetes mellitus. Ninety percent of partially pancreatectomized diabetic rats have a >5-fold increase in the messenger RNA and a 3–4 fold increase in the protein level of the catalytic subunit of hepatic glucose-6-phosphatase. Prolonged hyperglycemia may thus result in overproduction of glucose via increased expression of this protein (22). Normalization of the plasma glucose concentration in diabetic rats with either insulin or the glycosuric agent phlorizin normalized the hepatic glucose-6-phosphatase messenger RNA and protein within approximately

8 hr. However, phlorizin failed to decrease hepatic glucose-6-phosphatase gene expression in diabetic rats when the fall in the plasma glucose concen- tration was prevented by glucose infusion (22). This indicates that in vivo gene expression of glucose-6-phosphatase in the diabetic liver is regulated by glucose independently of insulin. The AF fraction, like the biguanide drug metformin, appears to control the increase in blood glucose in STZ-diabetic rats by decreasing the activity of glucose-6-phosphatase in the liver. This could be one of the mechanisms for the suppression of blood glucose concentration in the diabetic rats (12). Similarly, extracts of plants such as Zizyphus spina-christi reduced serum glucose level, liver phosphorylase, and glucose-6-phosphatase activities, and increased serum pyruvate level after 4 weeks of treatment. Likewise 60% ethanolic extract of Coccinia indica and 95% ethanolic extract of Momordica charantia extracts were found to lower blood glucose by depressing its synthesis, on the one hand, by depressing key gluconeogenic enzymes glucose-6-phosphatase and fructose-1, 6-bisphospha- tase and, on the other, by enhancing glucose oxidation by the shunt pathway through activation of its principal enzyme, glucose -6-phosphate dehydro-

Averrhoa bilimbi 333

affect hepatic glycogen content (12). Similarly, vanadate compounds have been shown to inhibit hepatic glucose-6-phosphatase activity thereby reduc- ing blood glucose levels in nonobese diabetic (NOD) mice (24).

III. FUTURE PROSPECTS The antidiabetic studies of A. bilimbi in STZ-diabetic rats demonstrate that

the leaves of A. bilimbi possess hypoglycemic, hypotriglyceridemic, anti-lipid peroxidative as well as antiatherogenic properties in STZ-diabetic rats. Hence it is believed that A. bilimbi leaves might have different types of active prin- ciples, each with a single or a diverse range of biological activities (11,12). However, the ethnopharmacological properties of A. bilimbi—such as anti- bacterial, antiscorbutic, astringent, postpartum protection, anti-inflammato- ry, as well as its use in traditional medicine for the treatment of itches, boils, rheumatism, cough, syphilis (paste of leaves); scurvy, bilious colic, whooping cough, hypertension, children’s cough (syrup of flowers); stomachache (fruits), mumps, and pimples (leaf decoction)—are yet to be justified by appropriate in vivo as well as in vitro studies.

REFERENCES 1. Goh SH, Chuah CH, Mok JSL, Soepadmo E. Malaysian Medicinal Plants for the

Treatment of Cardiovascular Diseases . Pelanduk Publications: Malaysia, 1995: 62–63. 2. Tan BKH, Fu P, Chow PW, Hsu A. Effects of A. bilimbi on blood sugar and food intake in streptozotocin-induced diabetic rats [abstr]. Phytomedicine 1996; 3(supp 1):271. 3. Junod A, Lambert AE, Atauffacher W, Renold AE. Diabetogenic action of streptozotocin: relationship of dose to metabolic response. J Clin Invest 1969; 48:2129–2139. 4. Johansson EB, Tjalve H. Studies on the tissue-disposition and fate of [14C] streptozotocin with special reference to the pancreatic islets. Acta Endocrinol 1978; 89:339–351. 5. Yamamoto H, Uchigata Y, Okamoto H. Streptozotocin and alloxan induce DNA strand breaks and poly (ADP-ribose) synthetase in pancreatic islets. Nature 1981; 294:284–286. 6. Pitkanen O, Martin J, Hallman M, Akerblom H. Free radical activity during development of insulin-dependent diabetes mellitus in the rat. Life Sci 1992; 50:335–339. 7. Nagy MV, Chan EK, Teruya M, Forrest LE, Likhite V, Charles MA. Macro- phage-mediated islet cell cytotoxicity in BB rats. Diabetes 1989; 38(10):1329– 1331. 8. Robbins MJ, Sharp RA, Slonim AE, Burr IM. Protectin against streptozotocin- induced diabetes by superoxide dismutase. Diabetologia 1980; 18:55–58.

334 Pushparaj et al. synthetase inhibitors administration to rats before and after injection of alloxan

and streptozotocin on islet proinsulin synthesis. Diabetes 1983; 32:316–318. 10. Al-awadi FM, Khattar MA, Gumaa A. On the mechanism of the hypoglycaemic effect of a plant extract. Diabetologia 1985; 28:432–434. 11. Pushparaj P, Tan CH, Tan BKH. Effects of Averrhoa bilimbi leaf extract on blood glucose and lipids in streptozotocin-diabetic rats. J Ethnopharmacol 2000; 72:69–76. 12. Pushparaj P, Tan BKH, Tan CH. The mechanism of hypoglycemic action of the semi-purified fractions of Averrhoa bilimbi in streptozotocin-diabetic rats. Life Sci 2001; 70:535–547. 13. Fontbonne A, Eschwege E, Cambien F, Richard JL, Ducimetiere P, Thibult N, Warnet JM, Claude JR, Rosselin GE. Hypertriglyceridaemia as a risk factor of coronary heart disease mortality in subjects with impaired glucose tolerance or diabetes. Diabetologia 1989; 32:300–304. 14. Carlsen SM, Rossvoll O, Bjerve KS, Folling I. Metformin improves blood lipid pattern in non-diabetic patients with coronary heart disease. J Intern Med 1996; 239:227–233. 15. Weirnsperger NF. Preclinical Pharmacology of Biguanides. Germany: Springer, 1996. 16. Barbera A, Rodriquez-Gil JE, Guinovart JJ. Insulin-like actions of tungstate in diabetic rats. J Biol Chem 1994; 269:20047–20053. 17. Gil J, Miralpeix M, Carreras J, Bartrons R. Insulin-like effects of vanadate on glucokinase activity and fructose-2,6-bisphosphate levels in the liver of diabetic rats. J Biol Chem 1988; 263:1868–1871. 18. Gray AM, Flatt PR. Pancreatic and extra-pancreatic effects of the traditional anti-diabetic plant, Medicago sativa (Lucerne). Br J Nutr 1997; 78:325–334. 19. Gray AM, Flatt PR. Antihyperglycemic actions of Eucalyptus globulus (Eucalyptus) are associated with pancreatic and extrapancreatic effects in mice. J Nutr 1998; 128:2319–2323. 20. Gray AM, Abdel-Wahab YHA, Flatt PR. The traditional plant treatment, Sambucus nigra (elder), exhibits insulin-like and insulin-releasing actions in vitro. J Nutr 2000; 130:15–20. 21. Liu Z, Barrett EJ, Dalkin AC, Zwart AD, Chou JY. Effect of acute diabetes on rat hepatic glucose-6-phosphatase activity and its messenger RNA level. Biochem Biophys Res Commun 1994; 205:680–686. 22. Massillon D, Barzilai N, Chen W, Hu M, Rossetti L. Glucose regulates in vivo glucose-6-phosphatase gene expression in the liver of diabetic rats. J Biol Chem 1996; 271(17):9871–9874. 23. Glombitza KW, Mahran GH, Mirhom YW, Michel KG, Motawi TK. Hypoglycemic and antihyperglycemic effects of Zizyphus spina-christi in rats. Planta Med 1994; 60:244–247. 24. Mosseri R, Waner T, Shefi M, Shafrir E, Meyerovitch J. Gluconeogenesis in non- obese diabetic (NOD) mice: in vivo effects of vanadate treatment on hepatic glucose-6-phosphatase and phosphoenopyruvate carboxykinase. Metab Clin Exp 2000; 49:321–325.

Lentinus edodes Shiitake Mushrooms

Ann-Teck Yap and Mah-Lee Ng National University of Singapore

Singapore, Republic of Singapore

I. INTRODUCTION For many years, mushrooms have been used as both a food and medicine in

many cultures, especially in the East. The use of mushrooms to maintain health was formally recorded as early as A . D . 100 in China. Mushrooms have a longhistory of use in folk medicine and have been incorporated into health tonics, tinctures, teas, soups, and healthty food dishes, as well as herbal for- mulae (1). Within the framework of traditional medicine, mushrooms have been applied to lubricate the lungs (Tremella fuciformis), tone the kidneys (Cordyceps sinesis), reduce excessive dampness (Grifola umbellate), and in- vigorate the spleen (Poria cocos).

Higher Basidiomycetes mushrooms with medicinal values, such as Ganoderma lucidum (Curt:Fr) P. Karst. (Reishi) Dendropolyporyus umbellatus (Pers.:Fr) Ju¨l., and Tremella fuciformis Berk. (1–6), were recorded in Shen Norg Ben Cao Jin (Compendium of Materia Medica of the godly farmer, of

East Han dynasty in China, A . D . 100–200).

Large numbers of mushroom-derived compounds, both cellular com- ponents and secondary metabolites, have also stirred up much interest in the

336 Yap and Ng

medical field. Many have been shown to affect the immune system and could

be used to treat a variety of disease states (7–14). There is vast amount of lit- erature on a large diversity of mushrooms and their medicinal properties. This chapter will concentrate on the Lentinus edodes and its medicinal properties.

II. LENTINUS EDODES In recent years, there has been growing awareness and interest in the

medicinal value of the biologically active components extracted from higher Basidiomycoto mushrooms (15–18). One such mushroom is the Lentinus edodes , better known as shiitake mushroom, which belongs to the family Tricholomataceae (order Agaricales and class Basidiomycetes).

The edible part of raw L. edodes contains about 90% water. Carbohy- drates make up the major component of dried shiitake (59.2%), followed by 22.7% protein (digestibility of 80–87%), 10% fiber, 3.2% lipids (primarily linoleic acid), and 4.7% ash. L. edodes is currently second in terms of global production, next to Agaricus bisporus. For centuries, this mushroom has been consumed not only as a delicacy but also for its beneficial effects.

Shiitake mushrooms (L. edodes, Berk Sing, Tricholomataceae fungus) is

a cultured edible fungus popular in Japan for the past three centuries. The curative powers of shiitake mushrooms are legendary. It was stated in Ri Yong Ben Cao , Volume 3 (1620), written by Wu-Rui of the Mingdynasty (1368– 1644), that ‘‘shiitake accelerates vital energy, wards off hunger, cures colds, and defeats body fluid energy.’’ Thus, shiitake is treated as an elixir of life, but without scientific verification (12,13). Recently, it has attracted considerable medical interest because of a multitude of medical effects, in particular for its anticancer activity and control of cholesterol level and blood pressure. In addition, it was reported to have antiviral, antibacterial, and antiparasitic activities (19).

The active substances include polysaccharides (e.g., h-glucans), nucleic acid derivatives, the hypocholesterolemic eritadenine, lipids, peptides, pro- teins, and glycoproteins. From the carbohydrate portion, the most important component of shiitake, several polysaccharides have been identified. They consist of water-soluble polysaccharides (1–5% of dried mushrooms), glyco-

gen-like polysaccharides, (1!4)-, (1!6)-a- D -glucans (antitumor polysac- charides), lentinan, (1!3)-, (1!6)-h- D -glucans, (1!6)-h- D -glucan with (1!3)- and (1!4)-h-bonded heteroglucans, heterogalactan, heteromannan, and xyloglucan (8). L. edodes also contains vitamins (e.g., provitamin D 2 , niacin, vitamin B 2 and B 1 ), free sugars such as trehalose, glycerol, mannitol, arabitol, glucose, mannose, and arabinose including minerals such as Cd, Zn, Cu, Fe, Mn, and Ni.

Lentinus edodes: Shiitake Mushrooms 337

III. LENTINAN-h h h - D -GLUCAN The variety of polysaccharides that have the ability to enhance the immune

system are pharmacologically classified as biological response modifiers (BRM). The most active BRM has (1!3)-h- D -glucans, sometimes referred

to as (1!3), (1!6)-h- D -glucans. Chihara and co-workers (7,20,21) first isolated a water-soluble antitumor polysaccharide from fruitingbodies of

L. edodes , which was named ‘‘lentinan’’ after the generic name of this mushrooms. The amount of pure lentinan extracted was low. In 2001, Yap and Ng(22) designed a more efficient and simple extraction procedure that resulted in yield of more that 100-fold of pure lentinan from the same amount of mushrooms.

Lentinan is a neutral polysaccharide isolated from L. edodes. It is a purified h-1,3- D -glucan with h-1,6 branches in a triple helical structure. Its physical and chemical properties have been widely characterized. The molec- ular formula for lentinan is (C 6 H 10 O 5 ) n , and the mean molecular mass is 500 kDa (23–25). The structure of lentinan, as shown by electrophoresis, ultra- centrifugation, and other instrumental analyses, was confirmed as h-(1,3)- D - glucopyranan with a branched chain of h-(1,6)-monoglycosyl, showing a right-handed helix. The properties of lentinan are listed by Chihara and colleagues (26).

Only those that consist of a (1!3)-linked h-glucan backbone with (1!6)-linked h- D -glucopyranosyl units as branches produce strong inhibi- tion of tumor growth (27,28). Degree of branching plays a role in producing the antitumor activity. The most active polymers [(1!3)-h- D -glucans] have degrees of branching between 0.20 and 0.33. Immunomodulating activity also depends on the distribution of the branch units alongthe backbone chain (29).

A triple helical tertiary conformation of h-(1!3)-glucans appears to play an important role in the immunostimulatingactivity of medicinal mushrooms.

Various degrees of antitumor activities were observed according to the structure of glucans (26,30–33).

Lentinan is insoluble in cold water, acid, and almost all of organic solvents such as alcohol, ether, chloroform, and pyridine; slightly soluble in hot water, and soluble in aqueous alkali at a raised temperature and formic acid. Infrared spectra and nuclear magnetic resonance spectra show absorp- tions at 890 cm -1 and H 5.4, respectively, and the presence of h-glycoside linkage (21,25).

IV. OPTIMAL DOSAGE DETERMINATION To achieve the desired highest antitumor effect, the amount of lentinan given

to the experimental model should be optimized. Initially, lentinan showed

338 Yap and Ng

little effectiveness, primarily because the dosages used were above or below what was later found to be optimal. Chihara and co-workers (7) and Aoki (34) showed that lentinan caused complete regression of sarcoma 180 transplanted into the mice at a dose of 1 mg/kg for 10 days (intraperitoneal route), while

a larger dose of 80 mg/kg for 5 days yielded no antitumor activity in com- parison with untreated control mice (28). Yap and Ng(22) demonstrated that 150 mg/kg lentinan is the most effective dose when administered by the oral route and achieved a tumor inhibition rate of 94.44% in murine lymphoma. Doses higher or lower than that resulted in a lower tumor inhibition rate (76– 85%). These observations suggested that there might be a relationship between lentinan, types of cancer under study, route of administration, and the host’s immune system, whereby a threshold of saturation for activation of lymphocytes exists. So the optimal dosage determined is a crucial consider- ation in treatment with lentinan.

V. THE MEDICINAL AND THERAPEUTIC VALUE OF LENTINAN The beneficial effects of lentinan have been known for a longtime and their

activities have been identified. Many experiments have been conducted to demonstrate the usefulness of lentinan. Results from various experiments are promising. In Japan, lentinan is currently classified as a drug for its known effects. Lentinan has been marketed for the past 10 years in Japan for the treatment of gastric cancer, in combination with tegafur. It was safely administrated to 50,000 patients in Japan, involvinga total of more than 2,000,000 doses. The usual dose in Japan is 2 mg/week (35).

A. Anticancer/Antitumor Effects Lentinan isolated by Chihara and co-workers (7,20,26) resulted in complete

regression of tumor induced from sarcoma 180 ascites cells implanted in Swiss albino mice with no cytotoxicity when given intraperitoneally. Lentinan was effective in limitingthe tumor size, regardless of whether it was in an allogenic tumor-host system, syngenic tumor-host system, or autochthonous host system (31,32,36,37).

Other studies showed tumor regression in C3H/He mice when MM46 mammary carcinoma cells were subcutaneously inoculated (38). Complete regression of large Madison 109 lung carcinoma in syngeneic BALB/c mice was achieved. Evaluation of the effects of lentinan against Lewis Lung (LL) and Madison 109 (M109) lungcarcinomas that were implanted in the foot- pads of syngeneic mice was carried out by Rose and co-workers (39). Intraperitoneal administration of lentinan was curative to mice bearing M109 lungcarcinomas (50–70%) though it had no substantial effect on LL

Lentinus edodes: Shiitake Mushrooms 339

carcinomas. This contradicted the findings of Suzuki et al. (40). Their study showed inhibition of pulmonary metastasis of LL carcinoma, also in mice.

Lentinan would also be effective for patients with advanced or recurrent breast cancer (41,42). Side effects have been transitional and not serious. Use of lentinan in a combined treatment of patients with advanced or recurrent gastric or colorectal cancer has also resulted in an increased life span (26,43). In addition, lentinan was able to prevent chemical oncogenesis as shown by its suppressive effect on 3-methylcholanthrene-induced carcinogenesis (36).

For all the above studies cited so far, lentinan was administered via in- jections. Studies (44–48) were carried out usingoral administration of powdered, dried mushroom fruitingbodies. Oral administration may be

important for eliminatingthe side effects of (1!3)-h- D -glucans, including the pain that accompanies parenteral administration. The tumor-growth-

inhibitory activity increased with the concentration of the shiitake mushroom powder. When 10% L-feed (feed containingpowdered shiitake fruit bodies) was used, the rate of tumor inhibition was 39.6%. Inhibition rates of 53.2% and 58.9% were achieved when 20% L-feed and 30% L-feed, respectively, were used. The degree of inhibition was proportional to the experimental diet. Administration schedule of the lentinan also influenced the rate of tumor inhibition.

In the most recent study on oral administration of lentinan conducted by Yap and Ng(22,49), male ARK mice (5–6 weeks old) were used. K36 cells (a murine lymphoma cell line) were used to induce the tumors. The mice were divided into three cohorts, namely, prefeedingfor 7 days prior to inoculation of K36 cells, simultaneous feedingof the lentinan with inoculation of K36 cells, and postfeedingafter 7 days of tumor induction with K36 cells. Three milligrams of lentinan were resuspended in buffer and force-fed to the mice daily.

Table 1 summarizes the data collected. It was clearly shown that lentinan feedingwas very effective in preventingtumor development (between

83 and 94%). Prefeedingwas the most effective regime when compared with simultaneous and postfeeding. The percentage of 83% from the postfeeding cohort indicated the regression rate of the developed tumors.

Crude mushroom homogenates also resulted in some degree of anti- tumor efficacy but at a much lower percentage (43–55%) The buffer-fed mice were controls (placebo). The mice with tumors are shown in Figure 1. Comparison of the sizes of the excised tumors is clearly shown.

In addition to the strongantitumor properties, the lentinan appeared to interfere with the morphogenesis of the murine lymphoma retrovirus (Fig. 2). The virus from the tumor cells of the control groups was electron-dense. However, in the lentinan-fed cohort, the virus particles from the tumor cells were empty. This indicated that the virus particles lacked the genomes and,

340 Yap and Ng

T ABLE 1 Antitumor Properties of Lentinan

Tumor inhibition Feeding with

Average weight

rate (TIR) % Pure lentinan

of tumors (g)

0.124 (prefeed)

94.44 ( p < 0.001)

0.265 (S/F)

88.59 ( p < 0.001)

83.14 ( p < 0.001) Crude mushroom

0.398 (postfeed)

55.20 ( p < 0.001) homogenate

0.997 (prefeed)

1.272 (S/F)

45.32 ( p < 0.001)

43.06 ( p < 0.001) Buffer solution

1.345 (postfeed)

2.230 (prefeed) 2.386 (S/F) 2.362 (postfeed)

thus, were defective. This could be the reason that the induced tumors (if they occurred) were very small (Table 1) and well localized, unlike the control mice.

Past studies have shown that the efficacy of lentinan in the treatment of cancer is increased when used in conjunction with other therapies. The highest antitumor effect was achieved when bacterial lipopolysaccharide was used with lentinan (50,51). Lentinan could be used in combination with IL-2 for treatingcancer (52–56). The combined administration of IL-2 and lentinan was effective against IL-2-resistant established murine tumors. Hamuro and co-workers (57) also investigated the antimetastatic effects of combined treatment with lentinan and IL-2 in spontaneous metastatic systems using murine fibrosarcoma and also showed the fruitful effects.

Marked antitumor effects were achieved when lentinan was used in conjunction with chemotherapy. The activity of pyrimidine nucleoside phos- phorylase (an enzyme that converts 5V-deoxy-5-fluorouridine (5V-DFUR) to 5-fluorouracil) was induced by lentinan in tumors, but not in the spleen, thereby increasingthe susceptibility of tumor cells to 5V-DFUR (58). Con- siderable improvement was seen in gastric and breast cancers when lentinan was used in combination with chemotherapy (59,60). Lentinan also enhanced the sensitivity of mouse colon 26 tumor to cis-diamminedichloro platinum (61). An in vivo study in rats with peritonitis involvingthe combination of lentinan with gentamicin showed a significantly better survival rate than controls (13). The tumor-regressive effect of lentinan was markedly reduced by X-irradiation of mice. 6-Benzyl thioguanosine reduced the antitumor activity of lentinan when it was injected after sarcoma 180 implantation (62). Such immunosuppressive agents should be avoided when lentinan is used to treat cancer patients.

Lentinus edodes: Shiitake Mushrooms 341

F IGURE 1 Comparison of tumor sizes between lentinan-, crude-mushroom- homogenate-, and buffer-fed mice. K36 cells (a murine lymphoma cell line) were used to induce the tumors. It is clearly demonstrated that the tumor excised from the buffer-fed mouse is several times larger than that from mouse fed with lentinan. Crude mushroom homogenate also offers some antitumor properties as the tumor size is smaller than that of the buffer-fed cohort. This observation is statistically significant as presented in Table 1.

The immunopotentiatingability of lentinan was postulated to play a key role in the antitumor process. Administration of lentinan to gastric cancer patients undergoing chemotherapy inhibited suppressor T-cell activity and increased the ratio of activated T cells and cytotoxic T cells in the spleen (63,64). Lentinan also increases peritoneal macrophage cytotoxicity against metastatic tumor cells in mice (65). Antimacrophage agents such as carra- geenan also inhibited the effect of lentinan (26,66–68). The antitumor effect of lentinan is abolished by neonatal thymectomy and decreased by the admin- istration of antilymphocyte serum, supportingthe concept that lentinan requires immunocompetent T-cell compartments.

342 Yap and Ng

F IGURE 2 Electron micrograph of murine retrovirus particles budding from tumor cells. (a) In the buffer-fed regime, electron-dense infectious virus particles (Vi) are seen budding out from the plasma membrane. (b) In contrast, the virus par- ticles budding out from the lentinan-fed cohort exhibit empty core structures. This indicates that the virus particles are defective (dVi) and lack the virus genomes.

Lentinus edodes: Shiitake Mushrooms 343

B. Immune-Modulating Effects of Lentinan The antitumor activity of lentinan resulted from activation of the host’s im-

mune functions rather than direct cytotoxicity to target cells (22,26,66,67, 69,70). The mechanism is postulated to involve bindingof h-glucan to the surface layer of lymphocyte or specific serum protein. This activated macro- phages, T cells, NK cells, and other effector cells, as well as increasing production of antibodies, interleukins, and interferons (45,71,72). Lentinan is considered to be phagocytosed by cells of macrophage lineage present in organs such as liver, spleen, and lung, and activates these cells (54,55,70,73– 75). Macrophages are the first to recognize foreign bodies as nonself and give this information to lymphocytes to activate the immune system. They prob- ably respond acutely to BRMs such as lentinan. Lentinan has been shown to facilitate the infiltration of lymphocytes and macrophages into tumor tissues (64).

Suzuki et al. (55–57) also showed that the antitumor effect was dependent on the CD-positive T lymphocytes. Lentinan stimulates the macrophages to augment their antitumor activity (76). More macrophages and T cells were found in lentinan-fed mice compared to the control group. Suzuki and co-workers (55) and Hamuro et al. (57) suggested that in addition to the augmentation of immune effector cell activity against tumors, infiltra- tion of these cells into the tumor sites might also be involved in eradication of tumors by lentinan. Tumor-induced immunosuppression could also be overcome by lentinan treatment through enhancement of the macrophage migration inhibitory factor production (32).

The effector cells might act either selectively or nonselectively on target cells (8). Various kinds of bioactive serum factors, associated with immunity and inflammation, (such as IL-2, IL-3, vascular dilation inducer, and acute- phase protein inducer), appeared immediately after the administration of lentinan. Increments in the production of antibodies as well as interleukins (IL-1, IL-2) and interferon (IFN-g) have also been observed (12,13,22,26,57, 63,67,70,77–86). The mechanism of lentinan-enhanced antibody-dependent, cell-mediated cytotoxicity through helper T cells (77,84) remains uncertain.

Yap and Ngfurther conducted a time-sequenced study on the induction of various cytokines in AKR mouse after oral feedingwith lentinan. Four cytokines levels, namely IL-1a (interleukin-1 produced mainly by monocytes, promote proliferation of Th2 CD4+ T cells), IL-2 (interleukin-2 produced mainly by Th0 and Th1 CD4+ cells, as T-cell growth factor), TNF-a (produced mainly by monocytes, activates endothelial cells and other cells of immune and nonimmune systems), and IFN-g (interferon-gamma, pro- duced by Th1 CD4+ cells, activates NK cells, macrophages, and killer cells), rose significantly after feeding with lentinan. They peaked at different hours

344 Yap and Ng

after feedingwith lentinan but returned to baseline after 24 hr (Fig. 3a–d). The IL-1a (Fig. 3a) and IL-2 (Fig. 3b) peaked at 2 hr postfeeding with lentinan. Both the TNF-a and IFN-g peaked at 4 hr postfeeding(Fig. 3c and 3d).

These results suggested that oral administration of lentinan may serve as

a means of activatingthe immune system, provokingthe immune responses required for disease prevention. Lentinan, once ingested, may encounter the gut-associated lymphoid tissue (GALT), which is a well-developed immune network, evolved to protect the host from infectingpathogens. Lentinan may also be absorbed into the systemic circulation, thereafter, involved in inducing immune systems against future pathogenic attack. Quantitative analysis of orally administered lentinan in murine blood carried out usinglimulus colorimetric test demonstrated that pure lentinan was detected in the murine blood and peaked at 0.2 mg(equivalent to the usual intravenous or intraperi- toneal inoculation dosage) 30 min after feeding (Yap and Ng, unpublished data).

When fed with crude mushroom homogenates, IL-1a, IL-2, and TNF-a levels were also induced but to much lower levels when compared to lentinan- fed cohort. The IFN-g level was not significantly induced. The lipid and protein fractions from the mushrooms did not result in noticeable induction of any of the four cytokines tested and was at the baseline like the buffer-fed cohort (control).

Many interestingbiological activities of lentinan have been reported. This included an increase in the activation of nonspecific inflammatory responses such as APP (acute-phase protein) production (87), vascular dilation, and hemorrhagic necrosis of the tumors (88). Bradykinin-induced skin reaction could be used as an index of vascular reactions against lentinan and this skin reaction could be used to moniter the host sensitivity to lentinan in antitumor responses (89).

Activation and generation of helper and cytotoxic T cells (10,67,79,90–

93) is an essential aspect of lentinan treatment. The augmentation of immune mediators such as IL-1 and IL-3, colony-stimulatingfactor(s) (94), migration inhibitory factor (32,94,95), and increasingthe capacity of PBM (peripheral blood mononuclear) cells (52,53) contributed additively to the antitumor efficacy. Wangand Lin (96) postulated that the immunomodulatingeffect of lentinan might be relevant to change of T-cell subpopulation and increase of TNF production.

The recent study of Yap and Ngconfirmed that T-lymphocytes were increased significantly (fourfold, p < 0.001, Student’s t-test) after feeding with lentinan (Table 2). The CD3, CD4 (T-helper), and CD8 (T-cytotoxic) lymphocytes were isolated usingT-cell-enrichment columns. All three types of CD lymphocytes were activated in comparison with the buffer-fed controls. It was noted that the placebo (controls) effects did result in some degree of

Lentinus edodes: Shiitake Mushrooms 345

F IGURE 3 Cytokine profiles from mice fed with lentinan, crude mushroom homogenate, lipid fraction, protein fraction, and buffer (placebo). (a) IL1-a profile. There is a sharp rise 2 hr after feeding with lentinan. A small increase in level of IL1-a is seen in the mice fed with the crude mushroom homogenate. For

the other three cohorts of mice, the level is at the baseline. (b) IL-2. Similar to (a), a significant rise in IL-2 level is seen in the lentinan-fed mice. This is followed in modest amount in the crude-mushroom-homogenate-fed popula- tion. Again the lipid-, protein-, and buffer-fed mice do not show any change in the level over the experimental period. (c) TNF-a. The rise in TNF-a level is seen at 4 hr after lentinan feeding. The level is also quite significant for the crude- mushroom-homogenate-fed mice. The usual low level of stimulation is seen with the other three regimes. (d) IFN-g. The profile obtained is similar to (c) with rise at 4 hr in the lentinan-fed mice. Other than a low level of induction seen in the crude-mushroom-homogenate-fed population, the other three cohorts show minimal changes in the level of production over the 24-hr period.

346 Yap and Ng

F IGURE 3 Continued.

activations of the lymphocytes. This could be due to the response of stress from force feedingof the fluid. The most activated lymphocytes were the CD4 helper lymphocytes (100%) followed by almost similar degree of activation in CD3 and CD8 lymphocytes.

Lentinan-activated lymphocytes were extracted from lentinan-fed AKR mice and reinoculated into nude (athymic) and SCID (lack immuno- globulin and lymphocytes) mice before the inoculation of human colon carcinoma cells. Six different cell lines were used representingdifferent stages of differentiation of the carcinoma cells. Tabulated in Figure 4, the data showed that the ‘‘lentinan-activated’’ lymphocytes did indeed protect the immunocompromised mice effectively against human colon carcinoma development (49). These experiments showed beyond doubt that T-lym- phocytes were truly activated by lentinan administration. The antitumor mechanism was a T-cell-mediated process. Figure 5 shows the proposed immunomodulatingpathway after lentinan administration. Activation of

Lentinus edodes: Shiitake Mushrooms 347

T ABLE 2 Cluster of Differentiation of T-Lymphocytes

Total cell

count

Cell count

(CD25— Percentage of Mice fed with

(CD90—

activated) activation (%) CD3

normal)

Lentinan 6 6 69.20 T-lymphocytes

Crude 6 6 64.24 isolated using

mushroom CD3 T-cell-

homogenate enrichment

Buffer solution 6 6 50.69 column

CD4 T helper Lentinan 6 6 100.00 lymphocytes

Crude 6 6 49.85 isolated using

mushroom CD4 T-cell-

homogenate enrichment

Buffer solution 6 6 40.33 column

CD8 T Lentinan 6 6 66.80 cytotoxic

Crude 6 6 39.29 lymphocytes

mushroom isolated using

homogenate CD8 T-cell-

Buffer solution 6 6 34.54 enrichment

column

the immune responses against the tumor cells resulted in the process of apoptosis in these cells leadingto tumor regression (49).

C. Antiviral Effects Lentinan showed marked antiviral activity and increased host resistance

against various kinds of viral infections, such as adenovirus, vesicular stoma- titis virus (VSV)-encephalitis virus, Abelson virus, human immunodeficiency virus (HIV), and influenza virus.

The effect of lentinan on influenza virus infection has also been deter- mined and reported by Irinoda et al. (97) and Maeda et al. (93). A significant level of protection in NMRI mice was noticed, even at low dosage (50 Ag ) of lentinan when administrated intranasally or intravenously. In 2001, Yap and Ng(22) showed that the administration of lentinan to AKR mice inter- fered with the maturation process of the murine leukemia retrovirus (Fig. 2).

348 Yap and Ng

F IGURE 4 The efficacy of ‘‘lentinan-activated’’ lymphocytes in nude and SCID mice. Lymphocytes from AKR mice that had been fed with lentinan for 7 days were extracted and inoculated into nude or SCID mice. After inoculation of the

‘‘lentinan-activated’’ lymphocytes, human colon carcinoma cells were also inoculated into these mice to induce tumors. Six different human colon carcinoma cell lines were used. LoVo and SW48 represent a well-differentiated stage with signet ring formation, SW620 and SW480 are moderately differ- entiated, and SW403 and SW1116 are poorly differentiated with little or no signet ring formation. The tumor from the buffer-fed mouse is rather large when compared to that of the mouse that is fed with lentinan. The summary table shows that ‘‘lentinan-activated’’ lymphocytes do have good efficacy against tumor formation in these immunodeficient mice.

The progeny virus particles found in the induced murine lymphoma cells were defective as observed usingelectron microscopy.

Many AIDS patients die of opportunistic infections due to immuno- dysfunction, and prevention of the development of symptoms by enhancing the immune system would be useful. Effects against HIV have been found in

some of the carcinostatic h- D -glucans (lentinan and others) isolated from shiitake mushrooms (98–101). A tolerance study of lentinan in HIV patients has been carried out and showed that lentinan was well tolerated and produced increases in neutrophils and natural killer (NK) cells activity (35). Lentinan also produced a trend toward improvement in CD4 cells generation and a decline in p24 antigen levels.

Tochikura et al. (101) and Iizuka (102) reported that when used in combination with azidothymidine (AZT), lentinan suppressed the surface expression of HIV on T cells more efficiently than AZT alone. It enhanced the effects against viral replication in vitro. Lentinan therefore qualified as a participant in future multidrugstudies in HIV (35).

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F IGURE 5 A proposed immunomodulation pathway of lentinan. Lentinan first comes in contact with macrophages, which in turn stimulate and activate the T cells, resulting in cytokine production. The immune response acts on the tumor cells causing apoptosis and regression of the tumor.

350 Yap and Ng

Lentinan has been used as an immune modulator in combination with didanosine (DDI) in a controlled study with HIV-positive patients. In vitro studies have indicated that anti-HIV effects of lentinan and DDI were additive. With promisingresults, it is reasonable to test the combined use of lentinan and DDI in HIV-infected individuals as well (35).

D. Antibacterial Effects Lentinan’s effectiveness against bacterial infections has been demonstrated

using Mycobacterium tuberculosis as the test organism in IRC mice. A marked anti-infective activity was observed, and all mice completely survived when lentinan was injected intravenously once from 4 to 10 days before the infection (103). In mouse experiments, it has also been proven to be effective against relapse of tuberculosis infections (60). Peritoneal macrophages secre- tory activity of active oxygen was activated by lentinan and the cytokines produced enhanced the ability of polymophonuclear leukocytes to produce active oxygen with bactericidal effect (104).

E. Antiparasitic Activity of Lentinan Lentinan was shown to be effective against parasitic infections caused by

Schistosoma mansoni , S. japonicum, and Mesocestoides corti (93,105,106). The mechanism was concluded to be through cell-mediated immunity and was T-cell-dependent as the antiparasitic activities were not illustrated in nude mice.

VI. OTHER COMPONENTS OF LESSER INTEREST Another active component, a-mannan peptide (KS-2), extracted from cul-

tured mycelium of L. edodes was shown to be effective against sarcoma 180 and Ehrlich’s carcinoma. Antitumor activity of KS-2 in mice has demon- strated a macrophage tumoricidal effect, though the actual mechanism of action of KS-2 is not clear (100).

A water-soluble, pale-brownish powder designated LEM was prepared from the culture medium of shiitake mushroom mycelia before the formation of fruit bodies. LEM is a glycoprotein containing glucose, galactose, xylose, arabinose, mannose, fructose, and various nucleic acid derivatives, vitamin B

compounds, especially B 1 (thiamine), B 2 (riboflavin), and ergosterol (107). Two alcohol-insoluble fractions, LAP1 and LAP2, can be separated from LEM (108,109). LAP is a glycoprotein containing glucose, galactose, xylose, arabinose, mannose, and fructose (102). In turn, a heteroglycan fraction

Lentinus edodes: Shiitake Mushrooms 351

(LAF1) can be prepared from LAP1 by DEAE-sepharose CL-6B column chromatography. Xylose is the major sugar in LAP or LAP1. The fraction LAF1 is composed mainly of xylose, arabinose, mannose, glucose, and galactose (111).

Both LEM and LAP components displayed strongantitumor activities (111,112). The growth of cancerous liver tumors was slowed in rats when LEM was given by injection (60). Administration of LEM (intraperitoneally) suppressed the cell proliferation of ascites hepatoma. The LEM fraction was also observed to activate the murine macrophage functions and promoted the proliferation of bone marrow cells in vitro. Cytotoxicity of NK cells was noted, and macrophages and T cells were also activated (112). LEM was found to inhibit the expression of cytopathic effects of herpes simplex virus, western equine enchepalitis virus, poliovirus, measles virus, mumps virus, and HIV (100,101,113).

The same effect was achieved when LAP was administered. The fraction LAP2 also suppressed cell proliferation of the ascites hepatoma, but the survival rate of hepatoma-bearingrats was not improved (108,109). Thus, the action of LAP1 was not cytocidal. Both LAP1 and LAF1 fractions might act as mitogens for mouse splenic macrophages and/or monocytes that are involved in cytokine induction (109,110). A protein fraction of L. edodes fruitingbodies known as fruitingbody protein (FBP) is noted to prevent infection of plants with tobacco mosaic virus (TMV) (14).

The antiviral and immunopotentiatingactivities of the water-soluble, lignin-rich fraction of LEM, JLS-18, was proven. JLS-18 showed about 70 times higher antiviral activity than LEM in vitro. Release of herpes simple virus type 1 in animals is blocked by JLS-18 (9).

Studies have shown that L. edodes is also able to lower blood serum cholesterol (BSC) via a factor known as eritadenine (also called ‘‘lentinacin’’

or ‘‘lentysine’’). Eritadenine was isolated from 80% ethanol extraction of L. edodes mushroom fruitingbodies by absorption on an Amberlite IR-120

(H + ) column, followed by elution with 4% NH 4 OH (21). Addition of eritadenine (0.005%) to the diet of rats caused a 25% decrease in total cholesterol in 1 week. Eritadenine apparently reduces serum cholesterol in mice by acceleratingthe excretion of ingested cholesterol and its metabolic decomposition (13,114).

VII. MYCOVIRUS Mycovirus or virus-like particles have been found in various species of fungi

and most possess double-stranded RNA (ds-RNA) (115–122). These myco- viruses are heritable viruses (25) and are unusual in that they do not lyse or cause any detrimental effects in their hosts (119). The spores are an effective

352 Yap and Ng

means for serial transmission. Three morphologically groups (25, 30, and 39 nm in diameter) have been found in partially purified preparations from both fruit bodies and mycelia of L. edodes (122–126).

In 1979 and 1981, Takehara and co-workers (127,128) demonstrated that viruses isolated from L. edodes mushrooms (includingtheir RNA) exhibited antiviral activities in cell culture studies. It was proposed that the efficacy was from its interferon-inductive effect. Certain ds-RNA of natural and synthetic origin has been found to inhibit the growth of various trans- planted tumors or leukemia in animals (58,129,130).

Suzuki and co-workers (131) reported that ds-RNA fractions extracted from L. edodes are highly active as an interferon inducer. They also showed the inhibitory activity was against solid tumor but not against the ascites form of the tumor, which suggested the involvement of a host-mediated immunological response to the tumor. However, Takehara and co-workers (127) demonstrated antitumor activity against Ehrlich ascites carcinoma in mice.

Owingto the lack of detailed study on the medical significance of these mycovirus, Sudhir and Ng(132) and Subha and Ng(unpublished data) carried out several experiments on this component of the L. edodes mush- rooms. Prefeedingof AKR mice with mycovirus-extract conferred the best antitumor activity with a tumor inhibition rate (TIR) of 80.7%. Simultaneous feedingand administration of extract on induced tumors were also effective with TIR of 73.8% and 67.6%, respectively. The tumor cells used to induce the tumor were K36 murine lymphoma cells. Tumor cells in the prefeeding regime also exhibited extensive apoptotic cell death, which could be the mechanism of destroyingthe tumor cells (132). In addition, elevated produc- tion of T-helper lymphocytes, IFN-g, and TNF-a was observed after oral administration of mycovirus extract.

The antiviral effect of the produced interferons appeared to interfere with the infectious retrovirus morphogenesis. The virus particles found in the mycovirus-fed tumor cells were mostly empty, i.e., without the virus genome. The defective progeny virus particles could explain the strong inhibition of tumor development in the prefed cohort.

Antiviral activity of the mycovirus was demonstrated with avian influenza virus in primary chick embryo cells. Cytopathic effects caused by the influenza virus were inhibited when the chick cells were pretreated with a suspension of the mycovirus before exposure to influenza virus (Subha and Ng, unplublished data). The mycovirus suspension was also tested against a range of bacteria (Bacillus subtilis, Corynebacterium diptheriae, Micrococcus luteus , Streptococcus pyogenes, Staphylococcus aureus, Enterobacter aero- genes , Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Vibrio para- chaemolyticus , Bacteroides fragilis, Clostridium perfringes, and Clostridium

Lentinus edodes: Shiitake Mushrooms 353

sporogenes ). The mycovirus suspension was not effective in the inhibition of any bacterial growth.