ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2013, том 49, № 2, с. 181-189
UDC 577.154.3
INHIBITORY EFFECT OF COMPONENTS FROM Streptomyces SPECIES ON a-GLUCOSIDASE and a-AMILASE OF DIFFERENT ORIGIN
© 2013 P. Meng, C. Xie, P. Geng, X. Qi, F. Zheng, F. Bai
Nankai University, Tianjin 300071, China e-mail: baifang1122@nankai.edu.cn Received April 26, 2012
The search for the effective and safe a-glucosidase and a-amylase inhibitors from Actinomycetaceae being antidiabetic agents is actual problem. Twenty one Streptomyces spp. of soil samples collected from different places of China were screened for the ability to produce this kind of inhibitory activities. Fermentation broth of isolated strains had absorbance between 350—190 nm. The Streptomyces strains PW003, ZG636, and ZG731 were characterized by special absorption at 280, 275, and 400 nm, respectively. Ten of the collected actinomycete strains had the ability to inhibit a-glucosidase or/and a-amylase and the fermentation broth of the same strain had inhibitory activity varied greatly depending on the enzyme source. In the process to screen the leading compounds used as antidiabetic agents, human a-glucosidase and a-amylase were revealed as the best used in trail compared with the same enzymes from other sources. Active a-glucosidase inhibitor was isolated from Streptomyces strain PW638 fermentation broth and identified as acarviostatin I03 by MS and NMR spectrometry. Its IC50 value was 1.25 and 12.23 p.g/mL against human intestinal N-terminal maltase-glu-coamylase and human pancreatic a-amylase, respectively.
DOI: 10.7868/S0555109913020104
a-Glucosidase inhibitors were used to treat type II diabetes and obesity by suppressing the absorption of glucose and reducing postprandial hyperglycemia [1]. Current interest in these compounds has been extended to a diverse range of diseases including lysosomal storage disorders, cancer and AIDS [2]. It has been well acknowledged that microbial extracts and phy-tochemicals are potential alternatives to synthetic inhibitors against a-glucosidase. The use of kinetic data combined with variations in potential inhibitor structural information can elucidate the enzyme activity [3]. Such knowledge is fundamental to the discovery of lead compounds, because of their promising therapeutic potential.
The genus Streptomyces was described by Waksman and Henrici [4] for aerobic and spore forming Actino-mycetes and emended by Kim et al. [5]. These microorganisms remain an important source of drug research for the future. Streptomyces were able to degrade relatively complex and recalcitrant plant and animal residues. Currently, about 10,000 antibiotics had been discovered from microorganisms. It had been estimated that approximately two thirds of these naturally occurring antibiotics were isolated from Ac-tinomycetes [6]. All Streptomycetes strains were acid fast negative and Gram stain positive. They differed greatly in their morphology, physiology, and biochemical activities, producing the majority ofknown antibiotics.
The main digestible carbohydrates in the human diet are starch and sucrose [7]. Human intestinal N-terminal maltase—glucoamylase (MGAMnt, EC 3.2.1.20) is a membrane anchored glycoprotein of the intestinal epithelial cells and can be solubilized by papain digestion [8]. The enzyme is more heat-stable than the other disaccharidases [9] and is the key enzyme which catalyses the final step in the digestive process of carbohydrates. Hence, a-glucosidase inhibitors can reduce postprandial plasma glucose levels and suppress postprandial hyperglycaemia, because of retarding the liberation of ^-glucose of oligosaccharides and disaccharides from dietary complex carbohydrates and delay glucose absorption [10]. Glucosidase inhibitors delay the breakdown of carbohydrates in the small intestine and thus diminish the postprandial increase of blood glucose in diabetic subjects [11, 12].
In the course of screening for new bioactive compounds, studies are currently oriented towards the isolation of new Streptomyces species from uncommon habitats. It has been reported that Streptomyces species such as S. hygroscopicus [13], S. griseus [14], S. fradiae [15], and S. lavendulae [16] produce many bioactive compounds, while there are also many Streptomyces species that have not yet been shown to produce them. To select new Streptomyces species, several methods have been developed to identify this genus of bacteria. These include selective plating technique [17], construction of genetic marker systems [18], a combination of chemical markers, the presence of L,L-diami-
nopimelic acid and the absence of characteristic sugars in the cell wall [19]. In addition, 16S rRNA sequence data have proved invaluable in Streptomycet-es systematic, in which they have been used to identify several newly isolated Streptomyces species [20, 21].
In order to develop physiological functional compounds for use as antidiabetic agents, much effort has been expended in the search for effective a-glucosi-dase inhibitors from natural materials. In a series of our studies on extracting inhibitor from Streptomyces species, we previously reported that acarviostatins from Streptomyces coelicoflavus ZG0656 were a new class of a-glucosidase inhibitors [22].
The aim of the study was to isolate of 21 Streptomyces spp. of soil samples collected from different places of China and to test them for ability to inhibit the activity of MGAMnt and human pancreatic a-amylase (HPA, EC 3.2.1.1). Active a-glucosidase inhibitor, ac-arviostatin I03, was prepared from the Streptomyces strain PW638 broth. In the process to screen the lead compounds for use as antidiabetic agents, mammalian a-glucosidase and a-amylase were revealed as the best to be used in trail.
MATERIALS AND METHODS
Biological material. Streptomycetes were collected from the following places of China: (i) 3 strains were isolated from a wet black mud sample collected at An-shan, Liaoning province; (ii) 7 strains were collected directly from soil samples at campus of the University of Nankai (CN); (iii) 3 strains were collected in the cave of Tai mountain; (iv) 4 strains were isolated from a paddy field, Gutian, Fujian province; (v) 4 strains were isolated from a soil sample, Shenzhou, Fujian province.
Sample collection, isolation and storage of Streptomyces spp. For each collected sample, 3.0 g of soil were suspended in 100 mL of 0.85% NaCl and allowed to stand for 15 min. Three different dilutions (1 : 10, 1 : 100 and 1 : 1000) were prepared using sterile saline solutions in a total volume of 10 mL. An aliquot of 0.1 mL of each dilution was plated on Gause's No. 1 synthetic medium [23]. Plates were incubated at 28°C, and monitored after 48, 72, and 96 h. Representative colonies were selected and streaked on new plates of Gause's No. 1 synthetic medium. The isolated Streptomyces species were preserved on Gause's No. 1 synthetic medium plates at 4°C until use. This procedure led to pure colonies of Streptomyces. The isolated Streptomyces strains were maintained as suspensions of spores and mycelial fragments in 10% glycerol (v/v) at 4°C in the Nankai University Collection of Pharmaceutical Sciences (China).
Genus identification and morphological characteristics. Visual observation of both morphological and microscopic characteristics using light microscopy and Gram staining were performed. All morphological
properties were observed on Gause's No. 1 synthetic medium and used for classification and differentiation.
Preparation of the fermentation complex. The culture of Streptomyces strain was filtered by hollow cellulose membrane (MOTIMO, China) with 100000 MWCO (Molecular Weight Cutoff) and the mycelium was discarded. The impurities were removed by ultrafiltration using hollow cellulose membrane with 360 and 10000 MWCO (MOTIMO, China). The effluent liquid passed through a D301R macroporous resin column (300 x 40 mm) (The Chemical Plant of Nan-kai University, China) to partly remove pigments, followed by a column of 001*7 cation-exchange resin (200 x 20 mm), washed with water, eluted with 0.1 M ammonia (The Chemical Plant of Nankai University, China). Then about a ninefold volume of EtOH was added to the concentrated eluate, and the supernatant was discarded after centrifugation at 3000 g, 10 min. The pellet was lyophilized to give the fermentation complex.
Purication and structure analysis of the Streptomyces strain PW638 fermentation complex. The Streptomyces strain PW638 fermentation complex was dissolved in water and filtered through a 0.45 ^m membrane (Sangon Biotech, China), then separated by semi-preparative reversed phase HPLC using a stainless steel column filled with Kromasil C18 (250 x 10 mm, i.d., 10 |m) at 25°C. The mobile phase was MeCN: water (10 : 90) at a fow rate of 5 mL/min with UV detection at 205 nm. The active fraction containing inhibitors was collected at 10.1 min. This fraction was further puried on the Waters (USA) Spherisorb S5 SCX semi-preparative column at 25°C with water: ammonia: acetic acid (1000 : 8 : 8) as the mobile phase. One active fraction containing inhibitors was collected with peak at 9.7 min.
Mass spectrometric detection was performed on a ThermoFinnigan LCQ Advantage mass spectrometer (USA) equipped with an ESI source and a mass range up to m/z 2000. Positive ion mode was employed, and the spray voltage was set at 4.5 kV. The capillary voltage was fixed at 5.0 V, and its temperature was maintained at 220°C. The solvent was nebulized using N2 as both the sheath gas and the auxiliary gas at a flow rate of 0.8 and 0.08 L/min, respectively. NMR spectrome-try was also used in the identification. The MS and NMR data then compared with the known a-glucosi-dase inhibitors.
The a-glucosidase inhibitory activity assay. Fifty |L of dissolved fermentation complex and 50 |L of 0.1 M phosphate buffer, pH 6.9, containing a-glucosidase solution (1.0 U/mL) were incubated in 96 well plates
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