Hypoglycaemic effects of fermented mycelium of Paecilomyces farinosus (G30801) on high-fat fed rats with streptozotocin-induced diabetes.
Abstract: Background & objectives: Paceilomyces farinosus is an entomogenous fungus with a powerful insecticidal activity against the larvae of Lipidoptera, Coleoptera and Hymenoptera. However, the hypoglycaemic activity of P. farinosus extract has not been studied. This study was undertaken to investigate the hypoglycaemic and anti-diabetic effects of P. farinosus (G30801) in rats with streptozotocin (STZ)-induced diabetes given a high-fat and compared with normal rats.

Methods: Rats fed with high fat diet for 2 months and injected with (30 or 50 mg STZ/kg bw) showed raised level of plasma triglyceride (TG), cholesterol, D-glucose concentration and glycosylated haemoglobin (HbA1C) %. The STZ-induced type 1 (T1DM) and type 2 diabetes (T2DM) in rats was further confirmed using glucose tolerance test and insulin-glucose tolerance test. P. farinosus (G30801) was fermented in different media [soybean (S), black bean (B), and rice (R)] and their extracts were tested for hypoglycaemic effect using T1DM and T2DM rats.

Results: STZ (30 and 50 mg/kg bw) could successfully induce T2DM and T1DM in rats, respectively. No change in blood glucose levels were noted in P. farinosus (R medium) treated normal rats (P < 0.05). In addition, STZ-high fat fed diabetic (T1DM and T2DM) rats when treated with P. farinosus (R medium) showed decreased blood glucose level as compared with P. farinosus extracted from B and S medium.

Interpretation & conclusions: Our findings showed hypoglycaemic effect of fermented P. farinosus (G30801) in experimental diabetes rat model fed with high fat diet.

Key words Diabetes--high-fat--hypoglycaemia--insulin--Paecilomyces farinosus--streptozotocin
Article Type: Report
Subject: Mycelia (Research)
Streptozocin (Research)
Hypoglycemic agents (Research)
Authors: Lu, Huai-En
Jian, Chien-Huei
Chen, Shu-Fen
Chen, Tse-Min
Lee, Son-Tay
Chang, Chun-Sheng
Weng, Ching-Feng
Pub Date: 05/01/2010
Publication: Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2010 Indian Council of Medical Research ISSN: 0971-5916
Issue: Date: May, 2010 Source Volume: 131 Source Issue: 5
Topic: Event Code: 310 Science & research
Product: Product Code: 2834121 Antidiabetic Preparations NAICS Code: 325412 Pharmaceutical Preparation Manufacturing SIC Code: 2834 Pharmaceutical preparations
Geographic: Geographic Scope: Taiwan Geographic Code: 9TAIW Taiwan
Accession Number: 237135614
Full Text: It is estimated that 143 million people in the world live with diabetes and this number will probably double by the year 2030 (1). Type 2 diabetes mellitus (T2DM) currently affects more than 200 million individuals worldwide (2). The characteristic features of T2DM include hyperglycaemia, near normal insulin levels, varying degrees of insulin resistance, slightly raised levels of glucagons and almost no ketoacidosis (3). Coronary artery disease (CAD) is markedly increased in subjects with T2DM; dyslipidemia in T2DM is often characterized by increased triglycerides, small, dense low-density lipoprotein (LDL), and low concentrations of high-density lipoprotein (HDL) cholesterol (4). Increased LDL and decreased HDL cholesterol have been shown to associate with the development of CAD in T2DM (5). Management of these complications represents a huge financial burden (6). Investigations with oral anti-hyperglycaemic agents derived from plants used in traditional medicine have shown these plants with good antidiabetic activity (7,8). Conventional drugs used have rigid and multiple dosing regimen, high-cost, and untoward effects (9). There are more than 1200 plant species broadly used in the treatment of DM and many of these showed effective hypoglycaemic activity after laboratory testing (8).

P. farinosus an entomogenous fungus, is a powerful insecticidal against the larvae of Lipidoptera, Coleoptera and Hymenoptera. Bioactive metabolites of P. farinosus have been investigated for antioxidant and anti-tumour properties10-13. Studies on hypoglycaemic activity using P. farinosus polysaccharides are not yet reported. Thus the present study was undertaken to study the hypoglycaemic effect of P. farinosus extract from various fermented medium [soybean (S), black bean (B), and rice (R)] in rats with streptozotocin-induced diabetes and fed with high fat diet.

Material & Methods

The study was carried out at Department of Life Science, National Dong-Hwa University, Hualien, Taiwan.

Microorganism and media: P. farinosus G30801 was kindly provided by Prof. Lee Son-Tay (Department of Biotechnology, Southern Taiwan University of Technology). The voucher specimens were deposited at the culture collection laboratory of Department of Biotechnology, Southern Taiwan University of Technology, Tainan County, Taiwan. P. farinosus G30801 was initially grown on potato dextrose agar (PDA) medium in a petri dish at 17[degrees]C, and then transferred to 250 ml flasks containing 100 ml of seed culture potato dextrose agar broth (PDB) medium and incubated on a rotary shaker (100 rpm) at 17[degrees]C for 7 days. Soybean (S), black bean (B), and rice (R) were evaluated for their potential as the major solid substrate in solid-state fermentation (SSF), respectively. Beans were soaked in 2-3 volume of water at room temperature for 4 h. Rice was boiled in water at 100[degrees]C. SSF was carried out by taking 300 g of the treated solid substrate in 1000 ml wide-mouth plastic bottle, moistening with liquid media solution containing 2 per cent peptone, 0.1 per cent K[H.sub.2]P[O.sub.4] dissolved in distilled water. All plastic bottles were autoclaved at 121[degrees]C for 30 min and after cooling were inoculated with 10 ml of seed culture and incubated at 17[degrees]C for 20 days. The fermented product by SSF was freeze-dried (VirTis apparatus; Gardiner, NY, USA) and then ground into flour as the tested sample. The fermented products cultured from soybean, black bean and rice were named as S, B, and R powder, respectively.

Quantification of crude water-soluble polysaccharides: The fermented product of P. farinosus by solid-state fermentation was extracted with water (100[degrees]C, 4 h). The supernatant was obtained by centrifuging at 5000 xg to separate the solid from the crude extract. The supernatant were extracted by Sevag method to remove the dissociative protein (14). The supernatant was followed by three consecutive precipitations with 4 volumes of 95 per cent ethyl alcohol (v/v). The precipitated polysaccharides were dried under vacuum and the crude polysaccharides were obtained. The polysaccharide content was measured followed a phenolsulphuric acid method (15,16). The polysaccharide content was 0.0335 g/g P. farinosus. The polysaccharide content was taken as a token of index component for the quality control of batch fermentation. The polysaccharide contents of fermented products cultured from soybean (S), black bean (B), and rice (R) bore a resemblance to the original one (data not shown).

Experimental animals: Wistar rats (n=70 body weight 250 [+ or -] 20 g at 8 wk old) were obtained from National Laboratory Animal Center, Taipei, Taiwan. The rats were raised under a 12 h light/dark cycle and had free access to food and water and maintained on a standard laboratory diet (carbohydrates; 30%, proteins; 22%, lipids; 12%, vitamins; 3%) ad libitum (17,18). The protocol of animal care was recognized and approved from the Animal Ethics Committee of the institution.

Induction of diabetes in rats: Rats (n=40) were fed a high fat diet for 2 months and diabetes was induced by a single intraperitoneal injection of freshly prepared streptozotocin (STZ, 30 or 50 mg/kg bw) (Sigma, St. Louis, Mo, USA) in 0.1 M citrate buffer (pH 4.5) to overnight fasted rats (modified from Zhang et al (19) via various dosage for treating different types DM). After 2 wk of STZ administration, animals with fasting blood glucose levels >200 mg/dl were considered diabetic and included in this study.

Body weights were determined gravimetrically, food and water intakes were recorded at 2 days and 2 wk following STZ or vehicle injection (saline), respectively. Blood glucose levels were determined using an automatic analyzer-Glucometer Elite XL (Bayer Incorporation, Toronto, Ont., Canada) with glucose oxidase/potassium ferricyanide reagent strips. Following plasma separation, an aliquot was taken for measuring triglyceride, cholesterol (CHOL), HDL, LDL, HbA1c by blood auto analyzer (Sysmex SF3000, Sysmex Corp; Kobe, Japan).

Experimental design

Glucose tolerance test--Two weeks after STZ injection, rats were administrated (ip) 0.5 g glucose / kg bw after 15 h fasting. At approximately 0, 30, 60, 90 and 120 min following glucose injection, blood was sampled by venipuncture from the caudal vein for determining blood glucose.

Insulin-glucose tolerance test--To determine the response of the diabetic rats to insulin action, they were injected with 0.5 g glucose /kg bw ip, immediately followed by insulin (25.2 USP unit/mg, Sigma, MO, USA) at a dose of 0.2 U/kg bw. At approximately 0, 30, 60, 90 and 120 min following insulin injection, blood was sampled by venipuncture from the caudal vein and the percentage changes in blood glucose were calculated for each group. The STZ-induced diabetic rats were followed glucose tolerance test and insulin-glucose tolerance test (20) and further defined as T1DM and T2DM based on amount of changed plasma glucose.

Assessment anti-diabetic activity of P. farinosus extract from various fermented medium in T1DM and T2DM rats--Two weeks after glucose tolerance and glucose-insulin tolerance tests, normal (n=30), T1DM (n=15) and T2DM (n=15) rats were fasted for 15 h and tested for blood glucose. Rat was orally given tested sample (R powder, S powder or B powder, 0.06 g/kg bw in water) and tested for blood glucose. After 30 min, rats were administrated (ip) 0.5 g glucose/kg bw. At approximately 0, 30, 60, 90 and 120 min following glucose injection, blood was sampled by venipuncture from the caudal vein for determining glucose. The percentage changes of glucose were calculated for each group.

Statistical analysis: The control and treatment groups were compared by one-way ANOVA after performing the Duncan multiple range tests.

Results

Rats fed with high fat diet showed increased body weight, CHOL, plasma TG levels (Table I) as compared to the normal rats (P<0.05). From the lipid profile it was evident that high fat fed rats showed decreased LDL-C, and increased HDL-C levels as compared with the control rats.

High-fat fed rat injected with STZ (30 and 50 mg/ kg bw) were found to have (P<0.05) high TG levels in blood plasma. Contrary, high-fat fed rat with STZ induction (50 mg/kg bw) showed the significantly (P<0.05) higher percentage of HbA1c than that of the normal group (Table II). As compared with normal rat, high-fat fed rats showed significant difference (P<0.05) in the plasma total cholesterol.

To confirm the success of STZ-induced diabetes, rats were checked for glucose tolerance and glucose-insulin tolerance tests, respectively, at 2 wk following STZ administration. STZ-injected animals had blood glucose exceeding 200 mg/dl, compared to a normal range of between 50 and 135 mg/dl following glucose injection at approximately 60 min in relation to 0 min (Fig. 1). Further, insulin-glucose tolerance test demonstrated that high-fat and STZ-induced rats had higher glucose level following insulin administration (Fig. 2).

To further investigate the hypoglycaemic effects of P. farinosus (G30801) fermented from various media (R, S and B) on diabetic rats, 30 min before glucose administration rats were treated with R, S or B extracts. Single oral administration of R, S or B extracts of P. farinosus (G30801) failed to alter the blood sugar in the normal rats (P<0.05; Fig. 3). Compared to the normal rats, P. farinosus extract from rice powder showed most significant hypoglycaemic effect at 120 min in T2DM (P<0.01) and in T1DM (P<0.05) rats, respectively (Fig. 4 and 5).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Discussion

The aim of the current work was to assess hypoglycaemic effect of P farinosus in experimental diabetes model. Due to the high prevalence of diabetes worldwide, extensive research is still being performed to develop new antidiabetic agents and determine their mechanisms of action, consequently, a number of diabetic animal models have been developed and improved over the years (21).

Streptozotocin-induced diabetic rats are one of the animal models of human insulin-dependent diabetes mellitus (22-24). As in human type 2 DM, diet has a great influence on the development of overt diabetes as well as hypertension, hyperlipidaemia, and eventually nephropathy in experimental models (25). In the present study, rats were fed with high-fat diet and then induced with both low/high dose of STZ; high-fat diet fed rats showed higher plasma TG, HDL-C, and lower LDL-C concentration compared to normal rats. Similar phenomenon was previously reported (26). The clinical symptoms of T2DM rats are closer to those of diet and obesity related diabetes. STZ treatment induces weight loss related to diabetes severity. The reduction of body weight might be due to the low utilization of uptake blood sugar in cell.

Hyperlipidaemia has been reported to accompany hyperglycaemia states (27), high levels of TC; importantly LDL cholesterol is one of the major coronary risk factors (28) which is the major cause of morbidity and deaths in diabetic subjects (29). A significance difference (P<0.05) in the plasma total cholesterol was observed in STZ induced diabetes rats as compared with the untreated group. The present experimental data demonstrate that a high-fat diet rat can successfully induce T2DM and T1DM via low/high dose STZ administration.

Some substances express anti-diabetic property by influencing cells to stimulate insulin secretion and restore insulin sensitivity (30). In our study, treatment with P. farinosus significantly reduced fasting blood glucose levels in high-fat fed/ (50 mg/kg bw) STZ-induced T1DM rats. Kiho et al31 demonstrated that the cultured mycelium of Cordyceps sinensis had hypoglycaemic activity and lowered plasma glucose concentration in normal, STZ-induced diabetic mice and epinephrine-induced hyperglycaemic mice. Total flavonoids of Polygonatum odoratum significantly reduced fasting blood glucose levels in STZ-induced T1DM mice and alloxan-induced T2DM rats (32). The hypoglycaemic mechanism of P. farinosus aqueous extract remains unclear and further studies are required to elucidate site(s), cellular and molecular mechanisms of P. farinosus extract. Our results demonstrate that fermented mycelia of P. farinosus (G30801) possess the hypoglycaemic effect in an experimental diabetes model and can be further evaluated for its use in alternative system of medicine.

Acknowledgment

This study was partially funded (92Agr-5.1.3-Food-Z1(10) & 93Agr -5.1.3-Food-Z1(10)) by Council of Agriculture, Taiwan, R.O.C. The authors thank Dr V. Bharath Kumar for critical comments.

Received January 28, 2009

References

(1.) Boyle JP, Honeycutt AA, Narayan KM, Hoerger TJ, Geiss LS, Chen H, et al. Projection of diabetes burden through 2050: impact of changing demography and disease prevalence in the U.S. Diabetes Care 2001; 24: 1936-40.

(2.) Ruchat SM, Elks CE, Loos RJ, Vohl MC, Weisnagel SJ,

Rankinen T, et al. Association between insulin secretion, insulin sensitivity and type 2 diabetes susceptibility variants identified in genome-wide association studies. Acta Diabetol 2009; 46: 217-26.

(3.) Emerson P, Van Haeften TW, Pimenta W, Plummer E, Woerle HJ, Mitrakou A, et al. Different pathophysiology of impaired glucose tolerance in first-degree relatives of individuals with type 2 diabetes mellitus. Metabolism 2009; 58: 602-7.

(4.) Gong W, Lu B, Yang Z, Ye W, Du Y, Wang M, et al. Earlystage atherosclerosis in newly diagnosed, untreated type 2 diabetes mellitus and impaired glucose tolerance. Diabetes Metab 2009; 35: 458-62.

(5.) Haffner SM. Lipoprotein disorders associated with type 2 diabetes mellitus and insulin resistance. Am J Cardiol 2002; 90: 55i-61i.

(6.) Gloyn AL. The search for type 2 diabetes genes. Ageing Res Rev 2003; 2: 111-27.

(7.) Kesari AN, Kesari S, Singh SK, Gupta RK, Watal G. Studies

on the glycemic and lipidemic effect of Murraya koenigii in experimental animals. JEthnopharmacol 2007; 112: 305-11.

(8.) Marles RJ, Farnsworth NR. Antidiabetic plants and their active constituents. Phytomed 1995; 2: 137-89.

(9.) de Melo Junior EJ, Raposo MJ, Lisboa Neto JA, Diniz MF, Marcelino Junior CA, Sant'Ana AE. Medicinal plants in the healing of dry socket in rats: microbiological and microscopical analysis. Phytomedicine 2002; 9: 109-16.

(10.) Cheng Y, Schneider B, Riese U, Schubert B, Li Z, Hamburger M. Neurotrophic alkaloidal metabolites from the entomogenous deuteromycete Paecilomyces farinosus. J Nat Prod 2005; 67: 1854-8.

(11.) Lang G, Blunt JW, Cummings NJ, Cole AL, Munro MH. Paecilosetin, a new bioactive fungal metabolite from a New Zealand isolate of Paecilomyces farinosus. J Nat Prod 2005; 68: 810-1.

(12.) Jiang YH, Jiang XL, Wang P, Hu XK. In vitro antioxidant activities of water-soluble polysaccharides extracted from Isaria farinosa B05. J Food Biochem 2005; 29: 323-35.

(13.) Jiang YH, Jiang XL, Wang P, Mou HJ, Hu XK, Liu SQ. The antitumor and antioxidative activities of polysaccharides isolated from Isaria farinosa B05. Microbiol Res 2008; 163: 424-30.

(14.) Staub AM. Removal of protein--Sevag method. Methods Carbohyd Chem 1965; 5: 5-6.

(15.) Xiao JH, Chen DX, Xiao Y, Liu JW, Liu ZL, Wan WH, et al. Optimization of submerged culture conditions for mycelia polysaccharide production in Cordyceps pruinosa. Process Biochem 2004; 39: 2241-7.

(16.) Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956; 28: 350-6.

(17.) Eddouks M, Maghrani M, Michel JB. Hypoglycaemic effect of Triticum repens P. Beauv. in normal and diabetic rats. J Ethnopharmacol 2005; 102: 228-32.

(18.) Kamgang R, Mboumi RY, N'dille GP, Yonkeu JN. Cameroon local diet-induced glucose intolerance and dyslipidemia in adult Wistar rat. Diabetes Res Clin Pract 2005; 69: 224-30.

(19.) Zhang F, Ye C, Li G, Ding W, Zhou W, Zhu H, et al. The rat model of type 2 diabetic mellitus and its glycometabolism characters. J Exp Sci Anim 2003; 52: 401-7.

(20.) Zhang M, Lv XY, Li J, Xu ZG, Chen L. The characterization of high-fat diet and multiple low-dose streptozotocin induced type 2 diabetes rat model. Exp Diabetes Res 2008; 2008: 7040-5.

(21.) Islam MS, Loots DT. Experimental rodent models of type 2 diabetes: A review. Methods Find Exp Clin Pharmacol 2009; 31: 249-61.

(22.) Bach JF. Insulin-dependent diabetes mellitus as an autoimmune disease. Endocrine Revs 1994; 15: 516-42.

(23.) JunodA, LambertAE, Stauffacher W, Renold AE. Diabetogenic action of streptozotocin: relationship of dose to metabolic response. J Clin Invest 1969; 48: 2129-39.

(24.) Fisher J. Drugs and chemicals that produce diabetes. Trends Pharmacol Sci 1985; 6: 72-5.

(25.) Sugano M, Yamato H, Hayashi T, Ochiai H, Kakuchi J, Goto S, et al. High-fat diet in low-dose-streptozotocin-treated heminephrectomized rats induces all features of human type 2 diabetic nephropathy: A new rat model of diabetic nephropathy. NutrMetab Cardiovasc Dis 2006; 16: 477-84.

(26.) Danda RS, Habiba NM, Rincon-Choles H, Bhandari BK, Barnes JL, Abboud HE, et al. Kidney involvement in a nongenetic rat model of type 2 diabetes. Kidney Int 2005; 68: 2562-71.

(27.) Taskinen MR. Lipoprotein and apoproteins in diabetes. In: Belfiore F, Bergnan RN, Molinatt GM, editors. Current topics in diabetes research, vol 12. Informa Health Care. 1996. p. 122-34.

(28.) Temme EH, Van HPG, Schouten EG, Kesteloot H. Effect of a plant sterol-enriched spread on serum lipids and lipoprotein in mildly hypercholesterolaemic subjects. Acta Cardiol 2002; 57: 111-5.

(29.) Baynes JW. Role of oxidative stress in the development of complications in diabetes. Diabetes 1991; 40: 405-12.

(30.) Yolanda BL, Adriana GC. Effects of dietary polyunsaturated n-3 fatty acids on dyslipidemia and insulin resistance in rodents and humans. J Nutr Biochem 2006; 17: 1-13.

(31.) Kiho T, Ookubo K, Usui S, Ukai S, Hirano K. Structural features and hypoglycemic activity of a polysaccharide (CSF10) from the cultured mycelium of Cordyceps sinensis. Biol Pharm Bull 1999; 22: 966-70.

Reprint requests: Dr Ching-Feng Weng, Institute of Biotechnology, National Dong-Hwa University, Hualien 974, Taiwan e-mail: cfweng@mail.ndhu.edu.tw

Huai-En Lu, Chien-Huei Jian, Shu-Fen Chen, Tse-Min Chen *, Son-Tay Lee **, Chun-Sheng Chang ** & Ching-Feng Weng

Institute of Biotechnology, National Dong-Hwa University, Hualien, * Hualien Armed Forces General Hospital Hualien & ** Department of Biotechnology, Southern Taiwan University of Technology, Yung-Kang City Tainan County, Taiwan
Table I. Body weight and blood biochemistry in normal (n=30)
and high-fat fed rats (n=40) before STZ induction

                          Body weight (g)            CHOL (mg/dl)

High-fat fed          463.92 [+ or -] 32.01 **   69.81 [+ or -] 9.80 *
  rats (n= 40)
Normal rats (n= 30)   357.50 [+ or -] 20.07      55.00 [+ or -] 6.49

                             TG (mg/dl)              HDL-C (mg/dl)

High-fat fed           78.13 [+ or -] 26.07 (+)  51.18 [+ or -] 9.92
  rats (n= 40)
Normal rats (n= 30)    37.25 [+ or -] 2.57        45.00 [+ or -] 4.69

                           LDL-C (mg/dl)

High-fat fed           5.16 [+ or -] 1.72 **
  rats (n= 40)
Normal rats (n= 30)    8.30 [+ or -] 3.87

Values are mean [+ or -] SE. P< values * <0.05; ** <0.01;
(+) <0.005 as compared to the normal rats

Table II. Blood biochemical parameters in normal, type I diabetic-high
dosage and type II diabetic-low dosage STZ induced rats

                          CHOL (mg/dl)               TG (mg/dl)

Type I diabetic        67.30 [+ or -] 6.42     143.00 [+ or -] 32.41 *
  rats (n=15)
Type II diabetic       75.27 [+ or -] 19.08    124.68 [+ or -] 38.55
  rats (n=15)
Normal Rats (n=30)     56.33 [+ or -] 7.23      32.00 [+ or -] 5.56

                          HDL-C (mg/dl)             LDL-C (mg/dl)

Type I diabetic       51.33 [+ or -] 4.50 *      6.00 [+ or -] 3.00
  rats (n=15)
Type II diabetic      56.66 [+ or -] 16.50       6.00 [+ or -] 3.46
  rats (n=15)
Normal Rats (n=30)    46.00 [+ or -] 5.29        6.33 [+ or -] 2.88

                            HbA1c (%)

Type I diabetic        8.10 [+ or -] 0.65
  rats (n=15)
Type II diabetic       8.33 [+ or -] 1.06
  rats (n=15)
Normal Rats (n=30)     4.16 [+ or -] 0.15

High and low dosages of streptozotocin (STZ, 50 and 30 mg/kg bw)
were used in this experiment

Values are mean [+ or -] SE. * P<0.05 presents the significantly
different levels between type I and type II diabetic rats
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