Optimal Extraction Conditions of Anti-obesity Lipase Inhibitor from Phellinus linteus and Nutritional Characteristics of the Extracts.

In an effort to develop novel mushroom-derived anti-obesity nutraceuticals, water and ethanol extracts containing the lipaseinhibitory compound from Phellinus linteus were prepared, and their nutritional components were determined. The optimal conditions for the extraction of P. linteus lipase inhibitor involved the treatment of the fruiting bodies with distilled water at 80℃ for 72 hr and 80% ethanol at 100℃ for 60 hr, respectively. The distilled water extract and ethanol extract contained 10.9% and 6.11% of crude protein, and 0.96% and 15.86% of crude fat, respectively. Additionally, the distilled water extract contained a large quantity of minerals, including 239.5 mg of K, 39.3 mg of Mg, and 39.3 mg of Na. The free amino acid content of the distilled water extracts was also higher than that of the ethanol extracts, and in particular, the distilled water extracts contained 5,139 mg of asparagine, 3,891 mg of tryptophan, 2,598 mg of alanine, and 2,066 mg of serine in 100 g of the distilled water extracts. 100 g of the distilled water and ethanol extracts were found to contain 12.31 g and 8.16 g of malic acid, respectively.

Phellinus linteus is classified as a member of the genus phellinus of the family Hymenochaetaceae. This mushroom is perennial and thrives in broad-leaved trees, particularly dead trees of Morus and Prunus sargentii Rehder. It has previously been evaluated for its immuno-stimulatory effects, as well as its polysaccharides and putative anti-cancer compounds [1-4]. Recently, its anti-obesity lipase inhibitor was also identified [5].

Obesity has been shown to induce serious cardiovascular diseases, cancers, and other conditions. Several anti-obesity agents have been developed thus far, including amylase and lipase inhibitors as well as commercial antiobesity products such as orlistat (Xenical). Among them, it is generally believed that a potent lipase inhibitor could prove extremely useful as an anti-obesity compound, because lipase (triacylglycerol hydrolase, EC 3.1.1.3) is a key enzyme for dietary fat adsorption, hydrolyzing triacylglycerols to 2-monoacylglycerols and fatty acids [6].

Many studies have been conducted involving lipase inhibitors derived from natural sources such as tannin [7], algae [8], pumpkin and Job's tear [9], saponins [10], Rhei Rhizoma and chunghyuldan [11] and Monascus pigment [12], etc. The orlistat from Streptomyces toxylricim, a potent lipase inhibitor, has proven useful in the treatment of obesity [13]. However, commercial lipase inhibitors have some troubling side effects, including fecal incontinence and low efficiency, among others. Therefore, it is clearly necessary to develop a potent anti-obesity lipase inhibitor with no side effects.

As part of a broader effort to develop new anti-obesity functional foods, we have already reported on the screening of a potent anti-obesity lipase inhibitor-containing mushroom, and assessed its characteristics to some degree [5]. In this paper, we attempted to determine the optimal water and ethanol extraction conditions of the lipase inhibitor from fruiting bodies of Phellinus linteus in order to develop new anti-obesity functional foods, and its nutritional characteristics were assessed.

Materials and Methods

Mushroom and chemicals

P. linteus was acquired from the Chungnam Agricultural Research and Extension Service of Yesan, in Chungnam province. Unless specified otherwise, all chemicals and solvents utilized herein were of analytical grade. Lipase (porcine pancreatic lipase, Type II), triolein as substrate, TES (N-Tris [hydroxymethyl] methyl-2-aminoethane-sulfonic acid), taurocholic acid, and gum arabic were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Extract preparation

The lyophilized powder of fruiting bodies of P. linteus was added to distilled water and 60% ethanol (1 : 20, w/v), then shaken for 18 hr at 40℃. These extracts were centrifuged for 20 min at 8,000 rpm, concentrated with a rotary vacuum evaporator, and lyophilized.

Assay of lipase inhibitory activity

Lipase inhibitory activity was assessed by determining the rate of release of oleic acid from triolein, via a modified version of the method developed by Bitou et al. [8]. A suspension of 120 mg of triolein, 90 mg of gum arabic, 10.16 mg of taurocholic acid in 9 mL 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 quantity of oleic acid generated was determined via the method developed by Zapf et al. [14] with some slight modifications. The 400 µL incubation mixtures were added to 3 mL of chloroform/hexane (1 : 1) containing 2% (v/v) ethanol, then extracted by 10 min of shaking in a shaker. The mixture was then centrifuged (2,000 × g) for 10 min and copper reagent was added to the lower organic layer, and then shaken for 10 min. The mixture was centrifuged (2,000 × g, 10 min), and 1 mL of the upper organic layer, which contained the copper salts of the extracted oleic acid, was allowed to react for 10 min with 0.5 mL of 0.1% (w/v) bathocuproine-chloroform solution containing 0.05% (w/v) 3-tert-butyl-4-hydroxyanisol, and the absorbance was determined at 480 nm. The inhibition (%) was calculated via the following equation:

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

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

Determination of nutritional components

The general components of distilled water extract and ethanol extract from the fruiting bodies of P. linteus were determined via the methods of Helrich [15]. The mineral contents were analyzed via ICP (Varian, Nederland) after wet degradation. The free sugar and organic acid contents of the distilled water extract and ethanol extract were determined by HPLC (2695; Waters Corp., Milford, MA, USA), and the amino acid contents were also determined via HPLC (2965; Waters Corp., FLD) after pretreatment with the Accq-tag method (Table 1).

Table 1

Analysis conditions of HPLC for determination of free sugars, organic acids, and amino acids

Results and Discussion

Optimal conditions for extraction of lipase inhibitor

To collect data regarding the application of P. linteus lipase inhibitor for use in the functional food industry, the effects of temperature and time on the extraction of the lipase inhibitor from P. linteus by distilled water and ethanol were determined. As is shown in Figs 1 and 2, the lipase inhibitory activities of the fruiting bodies of P. linteus were higher in the ethanol extracts than in the distilled water extracts, and the inhibitor extraction yield increased with increasing extraction temperature and time. Furthermore, the optimal ethanol concentration for lipase inhibitor extraction was 80% (data not shown).

Fig. 1

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

Fig. 2

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

The lipase inhibitor was optimally extracted when P. linteus was treated with distilled water at 80℃ for 72 hr (52.7%) and 80% ethanol at 100℃ for 60 hr (73.2%), respectively. It has been determined that even when the same inhibitor is used, the optimal extraction temperature between plants and microorganisms differs. For example, the acetylcholinesterase (AChE) inhibitor from Umbilicaria esculenta [16] and Job's tears [7] was optimally extracted at 40℃; however, the AChE inhibitors from some plants were most efficiently extracted with hot water [17, 18].

Nutritional characteristics of lipase inhibitor-containing extracts

The lipase inhibitor-containing distilled water extract and ethanol extract from P. linteus contained crude protein contents of 10.9% and 6.11%, and crude fat contents of 0.96% and 15.86%, respectively (Table 2). The mineral contents of the distilled water extract were higher than those of the ethanol extract--specifically, the distilled water extract contained 239.5 mg of K, 36.3 mg of Mg and 39.3 mg of Na (Table 3).

Table 2

General component contents of lipase inhibitor-containing extracts from Phellinus linteus (unit: %)

Table 3

Mineral contents of lipase inhibitor-containing extracts from Phellinus linteus (unit: mg/100 g, dry basis)

The free amino acid contents of the distilled water extract and ethanol extract were determined (Table 4) and included tryptophan (3,891 mg/100 g solid), alanine (2,598 mg/100 g solid), serine (2,066 mg/100 g solid) and interestingly, asparagines (5,139 mg/100 g solid). These amino acid contents were higher than those of the ethanol extract, with the notable exception of the asparagine contents (6,137 mg/100 g solid). However, these amino acid contents were 2.0~6.0 times higher than those of the corresponding tryptophan (60 mg/100 g solid) and serine (1,400 mg/100 g solid) contents in the methanol extracts from Umbilicaria esculenta [16], and were also similar to the alanine contents (3,400 mg/100 g solid) and lower than the glutamic acid contents (44,400 mg/100 g solid). The glutamic acid (17.19 mg/g solid) and histidine contents (6.42 mg/g solid) of the distilled water extract from P. linteus were similar to those of Lentinus edodes (15.84 mg/g solid and 7.95 mg/g solid, respectively).

Table 4

Free amino acid contents of lipase inhibition-containing extracts from Phellinus linteus (unit: mg/100 g, dry basis)

an.d, not detected.

The principal organic acid detected in the distilled water extract and ethanol extract of P. linteus was malic acid (12,312 mg/100 g solid, 8,160 mg/100 g solid, respectively). The distilled water extract also contained 590 mg/100 g solid of oxalic acid, and the ethanol extracts contained tartaric acid (620 mg/100 g solid); no other organic acids were detected (data not shown).