Extraction and Characteristics of Anti-obesity Lipase Inhibitor from Phellinus linteus.

To develop a potent anti-obesity lipase inhibitor from mushroom, the lipase inhibitory activities of various mushroom extracts were determined. Methanol extracts from Phellinus linteus fruiting body exhibited the highest lipase inhibitory activity (72.8%). The inhibitor was maximally extracted by treatment of a P. linteus fruiting body with 80% methanol at 40℃ for 24 hr. After partial purification by systematic solvent extraction, the inhibitor was stable in the range of 40~80℃ and pH 2.0~9.0. In addition to lipase inhibitory activity, the inhibitor showed 59.4% of superoxide dismutase-like activity and 56.3% of acetylcholinesterase inhibitory activity.

Various mushrooms have become attractive as functional foods and medicinal agents due to their content of nutraceuticals containing health-stimulating properties and medicinal effects [1]. Many polysaccharides and polysaccharideprotein complexes such as a-glucans and other peptides have been extracted and characterized as bioactive compounds from the fruiting bodies and mycelial of mushrooms [2-8].

Recently, obesity has received considerable attention due to its promotion of cardiovascular disease and cancer. There are several known anti-obesity agents such as amylase inhibitors and pancreatic lipase inhibitors; additionally, there are commercial anti-obesity products such as sibutramine and orlistat (Xenicol) [9]. Especially, lipase (triacylglycerol hydrolase, EC 3.1.1.3) is a key enzyme for dietary fat adsorption, hydrolyzing triacylglycerols to 2-monoacylglycerols and fatty acids. Therefore, it is generally thought that a potent lipase inhibitor could be very useful as an anti-obesity compound.

There have been many reports on lipase inhibitors derived from natural sources such as phytic acid [10], tannin [11], algae [12], pumpkin and Job's tear [13], saponins [14], Rhei Rhizoma and chunghyuldan [15], soybean and oil seeds [16] and Monascus pigment [17], etc. It is also well known that bovine serum albumin and β-lactoglobulin contain lipase inhibitors [18]. Recently, orlistat, a potent lipase inhibitor produced by Streptomyces toxytricini, has been proven useful for the treatment of obesity [19]. However, commercial lipase inhibitors have some side effects such as fecal incontinence and low efficiency, etc. Furthermore, there have been relatively few studies on the development of anti-obesity lipase inhibitors from natural sources without side effects or lipase inhibitors from edible or medicinal mushrooms.

In this paper, we describe the screening of a potent lipase inhibitor-containing mushroom as well as the optimal extraction conditions of lipase inhibitor from the fruiting body. Furthermore, we describe the properties of the partially purified inhibitor and determine the inhibitor may be an effective bioactive anti-obesity agent.

Materials and Methods

Mushrooms and chemicals

Mushrooms were obtained from Chungnam Agricultural Research and Extension Services (Yesan, Korea) and Baekma Agricultural Community of Buyea in Chungnam province. Unless otherwise specified, all chemicals and solvents were of analytical grade. Lipase (porcine pancreatic lipase, Type II), triolein as substrate, TES (N-Tris [hydroxymethyl] methyl-2-aminoethanesulfonic acid), taurocholic acid, gum arabic, HHL (Hip-His-Leu), pyrogallol, acetylcholinesterase, thrombin and fibrinogen were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of extracts

Fruiting bodies were lyophilized and powdered. Mycelial were inoculated in potato-dextrose broth and cultured at 28℃ for 5 days. The seed cultures were inoculated in whole grain and then cultured at 28℃ for 15 days. The cultured mycelial were lyophilized, powdered and filtered using a 20 mesh sieve.

The lyophilized powders of fruiting bodies and mycelial were added to methanol (1 : 20, w/v) and shaken for 20 hr at 40℃. These extracts were both centrifuged at 4,000 ×g for 20 min, concentrated using a rotary vacuum evaporator and then lyophilized.

Assay of lipase inhibitory activity

Lipase inhibitory activity was determined by measuring the rate of release of oleic acid from triolein according to the modified method of Bitou et al. [12]. A suspension of 120 mg of triolein, 90 mg of gum arabic, 10.16 mg of taurocholic acid in 9 mL of 0.1 N TES buffer (pH 7.0) containing 0.1M NaCl was sonicated for 5 min. A mixture of 50 µL of pancreatic lipase (500 U/mL), 50 µL of mushroom extracts (4 mg/mL) and 300 µL of substrate mixture was incubated for 30 min at 37℃, and the amount of oleic acid produced was determined by the method of Zapf et al. [20] with slight modification. The 400 µL incubation mixture was added to 3 mL of chloroform/hexane (1 : 1) containing 2% (v/v) ethanol and extracted by shaking for 10 min. The mixture was centrifuged (2,000 ×g) for 10 min followed by the addition of copper reagent to the lower organic layer and shaking for 10 min. The mixture was centrifuged (2,000 ×g, 10 min), and 1 mL of the upper organic layer containing the copper salts of the extracted oleic acid was reacted for 10min with 0.5mL of 0.1% (w/v) bathocuproine-chloroform solution containing 0.05% (w/v) 3-tert-butyl-4-hydroxyanisol. The absorbance was determined at 480 nm. The inhibition (%) was calculated using the equation.

Inhibitory activity (%) = (A - B)/A × 100,

where A is lipase activity in the reaction solution without sample and B is lipase activity in the reaction solution containing sample.

Determination of physiological function

The partially purified lipase inhibitor (inhibitory activity; 60%/mg solid) was dissolved in 50 µL of D.W and physiological functions were determined as follows. We used the Blois method [21] and DPPH to assay antioxidant activity. Superoxide dismutase (SOD) - like activity was assayed by the method of Marklund et al. [22].

The inhibition of angiotensin I-converting enzyme (ACE) by the partially purified lipase inhibitor was assayed according to the modified method of Cushman and Cheung [4, 23]. Acetylcholinesterase (AChE) inhibitory activity was measured spectrophotometrically using the technique of Ellman et al. [24]. Fibrinolytic activity was also assayed by the method of Fayek and El-Sayed [25].

Results and Discussion

Screening of a potent lipase inhibitor-containing mushroom

To select the most potent lipase inhibitor-containing mushroom, methanol extracts from 14 species of mushroom fruiting bodies and 108 species of mushroom mycelial were tested for their lipase inhibitory activities. Among the fruiting bodies tested, P. linteus (Goryo No. 1) showed the highest lipase inhibitory activity (72.5%). Methanol extracts of Fomitopsis pinicola (68.0%) and Innotus obliquus (62.9%) also showed high inhibitory activities (Table 1). Generally, methanol extracts from mycelial showed higher lipase inhibitory activities when compared to fruiting bodies. Methanol extract of P. linteus (ASI 26082) mycelial showed the highest lipase inhibitory activity (70.5%), although mycelial extracts of Ganoderma applanatum ACTC 44053 (67.1%), Agrocybe aegerita ASI 19003 (65.3%), Pleurotus ostreatus Choogwang (64.7%) and Ganoderma lucidum MR15008 (64.7%) also showed high inhibitory activities (Table 2). Since methanol extracts of P. linteus (Goryo No. 1) showed the highest lipase inhibitory activity, the P. linteus fruiting body was selected for the production of lipase inhibitor. This is the first report on the development of an anti-obesity nutraceutical based on lipase inhibitor from P. linteus, even though P. linteus has been studied for its immune-stimulating action by polysaccharides and anti-cancer activity [26-29].

Table 1

Lipase inhibitory activities of methanol extracts from mushroom fruiting bodies

Table 2

Lipase inhibitory activities of methanol extracts from mushroom mycelial

n.d, not detected.

Therefore, P. linteus (Goryo No. 1) may play a functional role in the food industry. Meanwhile, the lipase inhibitory activity of P. linteus was similar or lower than those of several other lipase inhibitors such as Monascus pigment derivatives from Monascus fermentation [17], soybean and other oil seeds [16], Rhei Rhizoma and Chunghyuldan [15], Acanthopanax sessiliflorus leaves [14] and marine algae [12].

Optimal extraction conditions

Optimal extraction conditions of lipase inhibitor from P. linteus fruiting body were found to be in the range of 40~100℃ and from 6 hr to 48 hr using methanol. As the methanol concentration and extraction time were increased, lipase inhibitory activity was increased. Lipase inhibitor was maximally extracted by 80% methanol (1 : 20) for 24 hr at 40℃ and then remained constant without any change (Figs 1 and 2). Seo et al. [1] reported antidementia β-secretase inhibitor was maximally extracted at 40℃ for 24 hr by 50% methanol. Further studies on extraction by water or ethanol are necessary for application of lipase inhibitor from P. linteus to the food industry.

Fig. 1

Effects of methanol concentration on the extraction of lipase inhibitor from Phellinus linteus.

Fig. 2

Effects of extraction time on the extraction of lipase inhibitor from Phellinus linteus.

Characteristics of the lipase inhibitor

The lipase inhibitor from P. linteus, partially purified by systematic solvent extraction, was soluble in water and methanol, with maximal absorption spectra of 225.1 nm (data not shown). Stability was investigated in the temperature range of 40~100℃ and pH 2.0~9.0.

Treatment for 60 min at 40℃ and 60℃ did not affect inhibitory activity, and only 16.2% of the relative inhibitory activity was decreased by treatment at 100℃ for 60 min (Fig. 3).

Fig. 3

Thermal stability of the partially purified lipase inhibitor from Phellinus linteus. The partially purified lipase inhibitor was treated for 60 min at various temperatures and its relative inhibitory activity was determined.

Furthermore, the partially purified lipase inhibitor showed over 80% relative inhibitory activity in the range of pH 2.0~9.0 (Fig. 4). From these results, we conclude that lipase inhibitor from P. linteus is very stable under heat (40~80℃) and a wide pH range (2.0~9.0), suggesting it may be very useful in the food or medicinal industries.

Fig. 4

pH stability of the partially purified lipase inhibitor from Phellinus linteus. The partially purified lipase inhibitor was treated for 60 min at various pH and its relative inhibitory activity was determined.

We compared the activities of lipase inhibitor from P. linteus and the commercial lipase inhibitor Olistat at various concentrations (Fig. 5). Olistat showed over 83% inhibitory activity at 1.0 µg per mL and 10 µg per mL, whereas the partially purified lipase inhibitor from P. linteus showed only 71.5% inhibitory activity at 10.0 µg per mL, lower than that of commercial Olistat.

Some physiological functions of the partially purified lipase inhibitor were determined (Table 3). In addition to lipase inhibitory activity, the inhibitor showed 59.4% SODlike activity and 56.3% anti-dementia acetylcholinesterase inhibitory activity. However, antioxidant activity was 11.2%, and antihypertensive ACE inhibitory activity and fibrinolytic activity of P. lenteus were not detected, even though some ACE inhibitors are present in several mushrooms including Pholiota adiposa [4].

Table 3

Physiological functionalities of methanol extract from Phellinus linteus

ACE, angiotensin I-converting enzyme; AChE, acetylcholinesterase.; n.d, not detected.