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��ࡱ�>��	|~����{�������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������)`	�R���bjbj��;��{�{[%�������.......BJJJ8�$�,B/'6��"%%%%%%%$e(h�*�<%�.���<%..�&�$�$�$��..%�$�%�$�$..�$����4���J�"�$%�&0/'�$�+�#��+�$�+.�$XRR��$�TN�<%<%D$X/'����BBB�&$BBB&BBB......����Potential Effect of Spirulina Extract on Enzyme Activities Participated in
Lipid and Carbohydrate Digestion Processes


Reina Kishibuchi 1, Rie Matsumoto 1, Naoyoshi Nishibori 2, Takefumi Sagara 3
and
Kyoji Morita 1,*

1 Laboratory of Neuropharmacology, Department of Nursing, Shikoku University
School of Health Sciences, Tokushima 771-1192, Japan.
2 Laboratory of Cell Biology and Toxicology, Department of Food Science and Nutrition, 
Shikoku Junior College, Tokushima 771-1192, Japan.
3 Department of Food and Nutrition, Shokei Junior College, Kumamoto 862-8678, Japan.



	* Correspondence to :	K. Morita, Ph.D.
				Laboratory of Neuropharmacology, Department of Nursing,
				Shikoku University School of Health Sciences,
				Furukawa, Ohjin, Tokushima 771-1192, Japan.
	   			Phone: +81-88-665-9257,   Fax: +81-88- 665-8037
	   			e-mail: kmorita@shikoku-u.ac.jp
Abstract
	Cyanobacterium Spirulina platensis is highly nutritious material, and known to contain plenty of proteins, vitamins, minerals and dietary fiber, thereby being useful as an ingredient of diet foods in dietetic treatment for metabolic syndrome. On the other hand, the excessive calorie intake is generally considered to be possible as one of the primary cause of metabolic syndrome, and therefore the well-balanced diet and the moderate exercise is expected as the most effective measures to avoid the disorder of energy utilization and storage, which is commonly accepted as a primary cause of metabolic syndrome. Then, as one of the measures to improve the disorder of energy balance, it can be effective to delay and lower the digestion and/or absorption of energy sources, carbohydrates and lipids. Then, to search for novel effective substance to delay and/or lower the digestion process among natural materials, the aqueous extract was prepared from the algae, and the effect of this extract on the activities of a�-glucosidase and lipase was examined in vitro. Consequently, Spirulina extract was shown to inhibit lipase activity, but not a�-glucosidase activity, suggesting that the extract may be partially effective to improve the disorder of energy balance in metabolic syndrome.
	The present study provided evidence for suggesting that Spirulina may contain putative effective substances to prevent a postprandial increase in the blood triglyceride concentration by suppressing the absorption of lipids from the intestinal tract, thus resulting in the improvement of hyperglycemic conditions, thereby being beneficial to patients with lifestyle-related diseases.


	Key words:	Blu-green algae, Lipase inhibition, a�-Glucosidase activity, Obesity
			Calorie balance, Metabolic syndrome
Introduction

	Metabolic syndrome is the term indicating the particular medical conditions observed in human of middle age, and commonly characterized by revealing central obesity, elevated blood glucose, high blood pressure, elevated serum triglycerides and elevated plasma cholesterol levels, thereby increasing the risk of developing cardiovascular disease [1-3]. On the other hand, the term "metabolic syndrome" is originally proposed to the pathological process of insulin resistance or hyperinsulinemia, and type 2 diabetes mellitus is known as another major outcome of metabolic syndrome [4]. Therefore, metabolic syndrome is regarded as one of the most serious problems in the point of maintaining human health, and the prevention and the improvement of energy imbalance is practically recognized to be important and necessary for leading healthy human life. Since the critical cause of metabolic syndrome, particularly the obesity, is considered to be simply due to the over-intake and/or the under-consumption of calorie, it can be predicted to be theoretically easy, but practically difficult to work out this energy imbalance, because the imbalance is usually considered to have roots in the dietary habits of patients, therefore quite difficult to make the fundamental alteration in their daily life style. Then, to make it much easier to help the improvement of calorie balance, it would be necessary to introduce supplementary materials modulating the dietary function and restoring the normal calorie balance for the prevention and improvement of metabolic syndrome. Currently, the untiring effort to find out such kind of effective substances from natural materials is extensively and enthusiastically carried out.
	In general, both carbohydrates and lipids are highly nutritious, and known to be major sources of food energy, thereby largely contributing to the fluctuations in energy balance. Therefore, the digestion and absorption of carbohydrates and lipids may be connected with the prevention and improvement of the energy balance disorder, and biologically active substances to suppress and delay their digestion and absorption can be considered to be effective and practically useful for preventing and improving the pathological conditions of metabolic syndrome [5]. For example, the dietary fiber, which can delay the absorption of carbohydrates and lipids from the digestive tract, is commercially available and commonly used as a food supplement to suppress the postprandial elevation of blood glucose and triglyceride levels. On the other hand, a�-glycosidase and lipase are generally known to play key roles in the digestion and absorption of carbohydrates and lipids, respectively, and substances to inhibit these enzymes are naturally expected to reduce and delay the digestion and absorption of these nourishments, thereby being effective for the prevention and improvement of metabolic syndrome [6]. In previous studies, the aqueous extract prepared from the non-edible joint part of lotus root has been reported to inhibit a�-glucosidase activity in vitro, and suggested to have a potential activity to reduce the carbohydrate digestion and absorption in the intestinal tract [7]. Furthermore, the extract has been found to inhibit lipase activity in vitro, thereby being speculated to have a potential ability to suppress the lipid digestion (unpublished data). In addition, the extract has also been reported to have the antioxidant and free radical-scavenging activities, which may be derived from large amounts of polyphenolic compounds in the extract, and proposed to protect the cells and tissues against oxidative toxic insults [8]. In contrast, we have recently found that the extract of cyanobacterium Spirulina platensis, particularly the fraction of non-protein components may have a variety of biological activities, such as the antioxidant and free radical-scavenging activities and the cytoprotective action (unpublished data), and then speculated that the algae extract has similar properties to the lotus root extract, thereby proposing the possibility that both of them may contain the common biologically active substances. Based on this speculation, the effect of Spirulina extract on the carbohydrate- and lipid-digesting enzymes was examined as the first screening for its potential effectiveness to reduce the over-intake of calorie in patients with metabolic syndrome.
	Basically, metabolic syndrome is considered to be primarily associated with the calorie imbalance brought by the excessive intake of the energy sources, and therefore speculated to be efficiently improved by suppressing the digestion and absorption of lipids and carbohydrates from intestinal tract. Then, the effect of Spirulina extract on lipase and a�-glucosidase activities was examined to confirm its inhibitory activity, which could be speculated to effectively contribute to the delay and reduction of lipid and carbohydrate digestion. Thus, the active substances inhibiting these enzymes may be able to effectively prevent the calorie over-intake, thereby improving the energy imbalance in the metabolic syndrome.
MATERIALS AND METHODS

Chemicals
	p-Nitrophenyl-a�-D-glucopyranoside, DTNB [5,5'-Dithio-bis(2-nitrobenzoic Acid)], DMPTB [2,3-Dimercapto-1-propanol tributyrate] and Candida rugosa lipase were obtained from Sigma-Aldrich (St. Louis, MO, USA). Yeast a�-glucosidase was purchased from Oriental Yeast Co. Ltd. (Tokyo, Japan). Other chemicals used were commercially available reagent grade.

Preparation of Spirulina protein-deprived extract
	The dry powder of Spirulina platensis was kindly gifted from DIC Life-Tec Co., Ltd. (Tokyo, Japan). The non-protein components of the algae were extracted with water as follows: The dry powder (10 g) was suspended in 100 ml of distilled water, and the suspension was autoclaved at 121oC for 30 min, and then kept in an autoclave chamber for overnight to allow it cool down. The suspension was filtered through a Whatman No. 1 filter paper, and the obtained filtrate was centrifuged at 5,000 x g for 20 min to remove the dregs. The supernatant fraction was acidified with acetic acid to pH 4.0, and kept at 5oC for overnight to precipitate soluble proteins. Then, the solution was centrifuged at 12,000 x g for 20 min, and the obtained supernatant fraction was neutralized with sodium hydroxide, and the extract was divided into small portions (5 ml), and kept in a freezer until use. The obtained extract containing mostly non-protein components of the algae was designated "protein-deprived extract".
	The amounts of proteins in the extract were directly determined using the dye binding method [9]. On the other hand, proteins contained in the extract were precipitated with 0.4M perchloric acid, and the precipitate was dissolved in the same volume of 1M NaOH to the original extract to avoid the interference of colored non-protein components, and the dye-binding assay was carried out to estimate the protein amounts contained in the extract. In some cases, the extract was dialyzed against distilled water (50-volume, 3-times exchanged) at 4oC for overnight to remove the small non-protein components as much as possible.

Determination of lipase activity
	Lipase activity was determined by a colorimetric microplate assay as described previously [10]. Briefly, the reaction mixture containing 80 m�l of the master mixture [0.2 mM DMPTB, 0.8 mM DTNB, 1 mM EDTA, 0.05% Triton X-100, 50 mM Tris-HCl, pH 7.5], 20 m�l of lipase solution [700 units of Candida rugosa lipase, 10 mM KCl, 50 mM Tris-HCl, pH7.5] and 0 - 100 m�l of sample solution in total volume of 200 m�l was incubated at 37oC for 60 min, and the absorbance at 405 nm was determined using a ChroMate microplate reader (Awareness Technology, Inc., Palm City, FL, USA). The mixture containing the same components except the enzyme was processed through the whole assay procedure, and the absorbance was measured as a blank. 

Determination of a�-glucosidase activity
	The enzyme activity was determined according to the method described previously [7, 11]. The sample solution (20 m�l) was mixed with 100 m�l of the enzyme solution (10 mU of the enzyme in 1 ml of 100 mM phosphate buffer, pH7.0), and preincubated at 37oC for 5 min. This reaction mixture was chilled on ice, and mixed with 100 m�l of the substrate solution (0.5 mM p-nitrophenyl-a�-D-glucopyranoside in 100 mM phosphate buffer, pH7.0). The complete reaction mixture was further incubated at 37oC for 20 min, and the reaction was stopped by adding 100 m�l of 1 M Na2CO3. The mixture was diluted with 400 m�l of distilled water, and the absorbance at 405 nm was then determined.

Statistical analysis
The results were presented as the mean � SEM, and the statistical analysis was carried out using a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The differences between two groups at p < 0.05 were regarded as statistically significant.
RESULTS AND DISCUSSION

	Many pieces of researches have previously been done to understand the nutritional effectiveness of blue-green algae [12-20]. However, the benefits of these algae for human health are not completely elucidated yet, and still necessary to be further investigated. Particularly, the biological activity of the extract containing mostly non-protein components of the algae (protein-deprived extract) is now being watched with great interest, because this extract can be expected to contain a wide variety of active substances. In the present study, as a series of our studies searching for effective components in the prevention and/or improvement of metabolic syndrome, the protein-deprived extract was prepared from blue-green algae Spirulina platensis, and the effect of this extract on the activities of lipase and a�-glucosidase, which are known as the key enzymes in the digestion and absorption of lipids and carbohydrates, respectively. As shown in Fig. 1, lipase activity was significantly inhibited by incubating the enzyme with the protein-deprived extract for at least 30 min, and an approximately 50% inhibition was observed by 200 m�l/well of the extract under the assay conditions used. Furthermore, the inhibitory effect of the extract was observed in a manner depending upon its concentration, and the maximal inhibition was obtained at the concentration of 200 m�l/well in these assays (Fig. 2). However, it would be possible to obtain more remarkable inhibitory effect by using highly concentrated extract, and therefore the extract can be expected to express its effectiveness in the digestive tract.
	Possible effect of the protein-deprived extract on the carbohydrate digestion was furthermore examined by determining the direct effect of the extract on a�-glucosidase activity in vitro, and the extract failed to cause any significant effect on the enzyme activity under the conditions used here (Fig. 3). Therefore, it seems possible to speculate that the non-protein fraction of blue-green algae may contain potentially effective components to modulate the digestion and absorption of lipids, but not carbohydrates, thereby being considered to possibly reduce only the over-intake of lipid-derived energy in patients with metabolic syndrome. Not surprisingly, because most of the potentially active components contained in the protein-deprived extract could be predicted to be relatively small molecules, which might be easily removal by dialysis. Practically, the inhibitory effect of the protein-deprived extract was completely abolished by dialyzing it against distilled water for overnight (Fig. 4). Thus, potentially active lipase-inhibitory components contained in the non-protein fraction of the algae was considered to be probably non-protein small molecules, but the molecular size and other properties were entirely uninvestigated, and still remains to be one of the critical issues in this research project.
	Kinetic analysis of the inhibitory effect on lipase activity was carried out to characterize the effective components contained in the protein-deprived extract, and the double-reciprocal plot analysis clearly indicated that the extract could reduce the Vmax value without any substantial change in the Km value (Fig. 5), thereby proposing the possibility that the extract might be able to cause the reversible, non-competitive inhibition of lipase under the experimental conditions used here. Thus, it can be possible to speculate that the inhibitory substances may be able to bind to the site different from the substrate-binding site on the enzyme molecule, and therefore seems to give a suggestive notion that the inhibitory substances may not necessarily to have the same lipid-soluble properties to triglycerides and other enzyme substrates. Moreover, it seems possible and may be necessary to consider the possibility that the inhibitory potency of the extract on the enzyme activity can be altered by using the different kinds of the substrates in the in vitro assay system. Therefore, it seems desirable, or rather may be required to further confirm the inhibitory effect of the protein-deprived extract on lipase activity in the presence of different kinds of the enzyme substrate.
	In the present study, the aqueous extract containing the non-protein components of blue-green algae Spirulina platensis was shown to cause the inhibition of lipase activity in vitro, thereby providing evidence for suggesting that the protein-deprived extract of the algae may be able to reduce the digestion and absorption of lipids in the digestive tract, thus proposing the possibility that the extract may be able to somewhat prevent and improve the over-intake of lipid energy in patients with metabolic syndrome. Therefore, it seems reasonable to search for potentially effective components from a variety of natural food materials by evaluating their inhibitory effects on the activities of lipase and a�-glucosidase, known as the key enzymes participating in the digestion and absorption of lipids and carbohydrates in the digestive tract. Thus, we believe that this method or way may be effective and useful to search for potentially active substances which can be probably beneficial to the prevention and improvement of metabolic syndrome.
Acknowledgments
	This work was supported in part by the funds provided from Kohken Co. Inc. (Sapporo, Japan) and Shikoku Kakoki Co. Ltd. (Tokushima, Japan).


Declaration of interest
	There is no conflict of interest associated with the authors of this paper, and the fund sponsor did not cause any inappropriate influence on this work.


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Figure legends

Figure 1: Inhibitory effect of Spirulina protein-deprived extract on lipase �activity. The enzyme was incubated with the extract (200 m�l) at 37oC for different period, and the enzyme activity was then determined by measuring the absorbance at 405 nm as described in the text. Values are the mean � SEM (*p < 0.01, n = 9).


Figure 2: Concentration-dependent effect of Spirulina protein-deprived extract on lipase activity. The enzyme was incubated with different concentrations of the extract at 37oC for 60 min, and the enzyme activity was then determined by measuring the absorbance at 405 nm as described in the text. Results were presented as a percent of the control. Values are the mean � SEM (*p < 0.01, n = 9).


Figure 3: Effect of Spirulina protein-deprived extract on a�-glucosidase activity. The enzyme was incubated with the extract (20 m�l) at 37oC for 20 min, and the enzyme activity was determined by measuring the absorbance at 405 nm as described in the text. Results were presented as a percent of the control. Values �y'z�zv{=|}~�~q9�����	�
����������N�P�R�������r�t����������������������������
$d�a$gdqed�gd�lare the mean � SEM (*p < 0.01, n = 9).


Figure 4: Inhibitory effect of dialyzed Spirulina protein-deprived extract on lipase �activity. The extract was dialyzed against distilled water (50-volume x 3-times) at 4oC for overnight, and the inhibitory effect of the dialyzed extract on the enzyme activity was determined as described in the text. Results were expressed as a percent of the control. Values are the mean � SEM (*p < 0.01, n = 9).


Figure 5: Lineweaver-Burk analysis of inhibitory effect of Spirulina protein-deprived extract on lipase�����������t�v�����r�t�v�z�|����������������������������������������������������������������������hj=�0JmHnHuhj=�
hj=�0Jjhj=�0JUhTB�jhTB�UhCbh�o(
hCbEHhCbOJQJ^JUhCb6�]�hCbhqe# �activity. The enzyme was incubated with the extract (200 m�l) at 37oC for 60 min in the assay mixture containing different concentrations of the substrate, and the enzyme activity was then determined as described in the text. The enzyme kinetics was graphically represented as the double-reciprocal plot (n = 9).









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