Isolation of a Wild Morchella spp. Strain and the Effects of its Extract on Ethanol-Induced Gastric Mucosal Lesions in Rats Wei Weia,b, Xia Luob, Linyong Zhengc, Mengyao Yub, Nan Jiangb, Xiao-yan Xub, and Zhi-rong Yanga,* a College of Life Sciences, Sichuan University, 610064, Chengdu, P. R. China. Fax: +86-28-8525-0783. E-mail: [email protected] b Sichuan Academy of Chinese Medicine Science, 610041, Chengdu, P. R. China c Sichuan Academy of Agricultural Sciences, 610066, Chengdu, P. R. China * Author for correspondence and reprint requests Z. Naturforsch. 66 c, 55 – 62 (2011); received April 1/August 24, 2010 A Morchella spp. strain was isolated from a wild morel mushroom, and the effects of its mycelia extract on the ethanol-induced gastric mucosal lesions of rats were investigated in vivo. Sequence analysis of internal transcribed spacer suggested that this Morchella spp. strain (strain No. M1) was clustered together with M. conica in the phylogenetic tree. The superoxide dismutase (SOD) activity increased signifi cantly compared to the control. How- ever, the malondialdehyde (MDA) level and myeloperoxidase (MPO) activity decreased signifi cantly compared to the control. These results indicated that M1 is one member of M. conica and the protective effects of M1 extract against the ethanol-induced gastric lesions may be related to the increased SOD activity and decreased MDA level and MPO activity in rats. Key words: Morchella sp., Ethanol-Induced Gastric Lesion, Malondialdehyde, Superoxide Dismutase Introduction evolving region of the nuclear rDNA nested in the rDNA repeat between the highly conserved The number of mushrooms on earth is estimat- sequences of 18 S, 5.8 S, and 28 S subunits. It is ed to be more than 140,000, although only 10% commonly believed that ITS regions are variable of them have been named. Recently, the extract between morphologically distinct species or even or bioactive constituents of mushrooms have isolates of the same species (Green et al., 2004). captured the attention of investigators because Members of the Morchella genus, commonly they exhibit hypolipidemic, hypoglycemic, im- known as morels, are edible mushrooms most munomodulatory, and antitumour activities. They highly priced for their high gastronomic quality. are widely used as nutritional foods and food-fl a- In China, morels have been used in traditional vouring materials in many regions since centuries Chinese medicines for their healthy properties (Negi, 2006). Despite the widespread apprecia- for thousands of years. It has been previously tion of these prized edibles, many aspects of their reported that some extracts from Morchella spp. molecular biology are poorly understood. Tra- have demonstrated many pharmaceutical effects, ditionally, identifi cation and characterization of such as fatigue-resisting, hepatoprotective, and macrofungi species have been based on morpho- antioxidant effects (Sun et al., 2001; Mau et al., logical characters, such as conidial and appresso- 2004; Zhou et al., 2006). However, prior to this rium shape and size, as well as pathogenicity tests study little was known on the molecular identi- (Gunnell and Gubler, 1992). Recently, molecular fi cation of Morchella strains and the effects of biological techniques have been applied success- their extracts on ethanol-induced gastric mucosal fully to identify fungi precisely and rapidly (Las lesions in vivo. Recent studies showed that acute Heras-Vazquez et al., 2003). The internal tran- ethanol challenge may induce oxidative stress, scribed spacer (ITS) region has been generally such as decreased superoxide dismutase (SOD) considered as extensively sequenced molecular activity and increased malondialdehyde (MDA) marker and an effective target for the molecular level in gastric mucosal cells (Nermina et al., identifi cation of fungi. The ITS region is a rapidly 2007). Ethanol could activate neutrophil infi ltra- 0939 – 5075/2011/0100 – 0055 $ 06.00 © 2011 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D 56 W. Wei et al. · Isolation of a Wild Morchella spp. Strain tion that produces oxygen radicals and injures Morphology characterization tissues, and myeloperoxidase (MPO) activity as Morphology studies were carried out by plating an enzyme marker of leukocytes may be induced. 20 ml PDA medium in a 10-cm (i.d.) Petri dish The objectives of the present study are to identify and inoculating with 0.5 cm2 mycelial disc. The Morchella strains based on ITS sequence analysis mycelia were observed after culturing on a mi- and to investigate the effects of the water extract croscope slide with PDA medium at 25 °C for 3 d, of Morchella strains on the MDA level, SOD and and then colouring by fungus staining solution. MPO activity in rats. The solution consisted of 20.0 g phenol, 20.0 ml lactic acid, 40.0 ml glycerin, 20.0 ml distilled wa- ter, and 0.5 g fuchsin acid. Material and Methods Animals DNA extraction Male Wistar rats (180 – 200 g) were obtained Total genomic DNA was extracted from about from the Animal Facility of the Institute of Chi- 50 mg mycelia following the modifi ed cetyltri- nese Traditional Medicine, Sichuan, China. The methylammonium bromide (CTAB) method protocols of feeding were formed in accordance (Zheng el al., 2007). The DNA was estimated with the Guidelines of Institute of Chinese Tra- spectrophotometrically (Bio-Rad Laboratories, ditional Medicine Animals Research Committee. Hercules, CA, USA), and quality was checked by The rats, 5 per cage, were housed in a SPF level agarose gel electrophoresis. laboratory at (20 2) °C with a 12-h light/dark cycle. They were fed with a standard rat chaw, and PCR amplifi cation, sequencing, and analysis drinking water was available ad libitum. PCR primers were universal sense primer ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and anti- Microorganism and culture sense primer ITS4 (5’-TCCTCCGCTTATTGA- The morel strain (designated M1) was isolated TATGC-3’) for fungi. Primers were synthesized from the fruit body of a wild Morchella sp. from by Shanghai Generay Biotech Co., Ltd, China. a forest in the north region, Sichuan, China. The PCR reactions were performed in 50 μl total vol- strain was maintained on synthetic potato dextrose ume, containing about 100 ng of DNA, 5 μl of agar (PDA) plates at 4 °C. Before the experiment, PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, the slant was transferred from the active slant and 0.2 μM of each primer, and 1.5 U of Taq DNA maintained on a newly prepared synthetic PDA polymerase (Promega, Madison, WI, USA). The medium in a Petri dish at 25 °C for 7 d. Then it amplifi cation was incubated in a Mycycler ther- was transferred into the seed culture by punching mal cycler (Bio-Rad Laboratories). The PCR re- out mycelia mat (ca. 1 cm2) from the Petri dish action mixture was denatured at 94 °C for 5 min, and incubated on a rotary shaker incubator un- followed by 30 cycles of 1 min at 94 °C, 1 min at der agitation at 150 rev/min for 7 d at 25 °C. The 56 °C, 1 min at 72 °C, and a fi nal extension step seeds were grown in 250-ml Erlenmeyer flasks of 10 min at 72 °C. The amplifi ed products were containing 100 ml liquid culture medium. The visualized in 1% (w/v) agarose gel in Tris-Bo- submerged fermentation experiment was carried rate-EDTA (TBE), and then were purifi ed and out in 500-ml flasks containing 200 ml of liquid sequenced by Invitrogen Biotechnology Co., Ltd culture medium after inoculating with 10% (v/v) (Shanghai, China). of the seed culture on a rotary shaker incubator DNA sequence similarity searching was per- under agitation at 150 rev/min for 6 d at 27 °C. formed using the BLAST standard nucleotide- The liquid culture medium was composed of nucleotide basicloc alignment search tool. A 200 g/l potato, 20 g/l glucose, 2 g/l peptone, 1.5 g/l total of 20 Morchella spp. sequences and 2 out- KH2PO4, 5 g/l MgSO4. Mycelia were separated by group sequences (Verpa bohemica, acession No. centrifugation at 3,000 × g for 15 min. The precip- AM269502, and Disciotis venosa, acession No. itate was washed three times with a large amount DQ491503) were obtained from GenBank for of distilled water, freeze-dried, and stored at 4 °C phylogenetic analysis. Sequences were aligned us- for further use. ing the multiple alignment program CLUSTAL X W. Wei et al. · Isolation of a Wild Morchella spp. Strain 57 1.83. Their phylogenetic analysis was performed lesion. The total area of lesions was calculated using maximum parsimony as implemented by the and expressed in mm2 (Ancha et al., 2003). PAUP* (Phylogenetic Analysis Using Parsimony, Version 4.0b) computer program. A heuristic Histological studies search analysis was run with tree bisection-recon- nection branch swapping to infer branch lengths For histological study, the stomach tissues of with MULTREES option on, with ADDSEQ set animals were examined by light microscopy. The at random and 1,000 randomized replicates. All stomach tissues were fi xed in 10% neutral forma- characters were weighted equally. Bootstrap val- line, dehydrated in ethanol, and then em bedded ues from 1,000 replicates were calculated using in paraffi n. Sections from tissue blocks taken the same settings as for heuristic searches (Yu et from ulcerated areas were stained with hematox- al., 2008). yline/eosin for routine histological examination (Ahmet et al., 2003). Preparation of M1 extract Lipid peroxidation assay After fermentation, the fermentation broth was MDA is an important toxic byproduct of li- collected and centrifuged for 10 min at 1,157 × g. pid peroxidation in animal and plant tissues. The resulting precipitate was washed repeatedly The measurement of the MDA content has been with distilled water, and the mycelial pellets were widely used as an index of lipid peroxidation.