Inhibition of Saccharide Digestive Enzymes by Tea Polyphenols

Chapter 9

Inhibition of Saccharide Digestive Enzymes by Tea Polyphenols M . Honda, F. Nanjo, and Yukihiko Hara

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch009

Food Research Laboratories, Mitsui Norin Company Ltd., Fujieda 426, Japan

The inhibition of salivary α-amylase, intestinal sucrase and maltase by polyphenolic components of tea and their specificity were inves tigated. Theaflavins isolated from black tea and galloyl catechins from green tea showed potent inhibitory effects. Free catechins and gallic acid showed little or no effect. Based on these results, in vivo experiments were conducted. Crude catechin powder, obtained by purifying the catechin fraction from green tea, was administered to rats, followed by the administration of starch or sucrose. Rats were sacrificed at time intervals and their intestinal enzyme activities and glucose concentrations in the blood were determined. The results showed that after catechin administration, the increase in intestinal α-amylase activity caused by starch was markedly suppressed and that glucose levels in the blood were also suppressed dose -dependently by tea catechins. Similar in vivo results were observed with the administration of catechins prior to sucrose administration.

Tea polyphenols — or more broadly, tannins — are known to have a strong affinity to proteins. In the case of tea polyphenols this characteristic is indicated by the astringency felt on our palates when we drink tea. The inhibition of enzymes by the polyphenolic fraction of tea, therefore, has long been assumed. We have isolated and purified individual polyphenolic components from tea and subjected them to a series of enzyme inhibition tests (1,2). The inhibition of α-amylase by tea poly phenols was also examined and fairly potent inhibition was confirmed (3). In this paper, the in vitro inhibitory potency of tea polyphenols on α-amylase as well as on sucrase or maltase and their in vivo effects on blood glucose levels in rats are presented. In Vitro Inhibition of Saccharides by Tea Polyphenols Inhibition of α-Amylase by Tea Polyphenols, α-Amylase from human saliva was purchased from Sigma Chemical Co. Representing tea polyphenols, individual

0097-6156/94/0547-0083$06.00/0 © 1994 American Chemical Society

Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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catechins from green tea (4) and individual theaflavins from black tea (7) were prepared. For comparison, gallic acid also tested. The inhibitory effects of samples were measured according to a method described elsewhere (3). a-Amylase solution was incubated with the inhibitor solution for 10 min at 37 °C and the concentrations of samples that produced 50% inhibition of the enzyme were determined. The results are shown in Table I. Among the twelve samples, gallic acid, free catechins (EC and EGC) and their epimers did not have significant effects on the activity of α-amylase. A l l other samples were inhibitors and the inhibitory effects were in the descending order of TF3>TF2A>TF3B>TF 1 >Cg>GCg>EGCg.


Table I. Inhibition of a-Amylase by Tea Polyphenols and Gallic Acid Sample (-)-Epicatechin (EC) (-)-Catechin (C) (-)-Epigallocatechin (EGC) (-)-Gallocatechin (GC) (-)-Epicatechin gallate (ECg) (-)-Catechin gallate (Cg) (-)-Epigallocatechin gallate (EGCg) (-)-Gallocatechin gallate (GCg) (-)-Theaflavin (TF1) (-)-Theaflavin monogallate A (TF2A) Theaflavin monogallate B (TF2B) Theaflavin digallate (TF3) Gallic acid

IC50O1M)

>1000 >1000 >1000 >1000 130 20 260 55 18 1.0 1.7 0.6 >1000

Inhibition of Sucrase and Maltase. To determine inhibition of sucrase and maltase, the small intestinal brush border was removed from rats, carefully homogenized in a buffer and centrifuged. The supernatant was a crude enzyme solution containing sucrase and maltase, and was preincubated with the polyphenol solution. The sucrase activity was measured according to the standard assay of Bernfeld (5). Maltase activity was assayed by adding the substrate /?-nitrophenyl α-D-glucoside (NPG) to the enzyme-polyphenol solution. The inhibitory effects of tea polyphenols on the sucrase and maltase activity are shown in Table II. Among the samples, gallated polyphenols showed potent inhibition whereas non-gallated, free catechins and gallic acid showed little inhibitory activity. Their effects on sucrase were in the descending order of EGCg>TF3>ECg>TF2A>TF2B. The inhibition of maltase (determined as inhibition of NPG hydrolysis) by tea poly phenols was in the order of TF3>TF2B>TF2A>ECg>EGCg. The Effect of Tea Catechins on Starch Ingestion Crude catechin powder fractionated from Japanese green tea was administered orally to rats followed by administration of starch and the influence of this catechin powder on intestinal α-amylase and the blood glucose level was studied. The


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Saccharide Digestive Enzymes and Tea Polyphenols 85


composition of crude catechin powder (6) is shown in Table III. Crude catechin powder is abbreviated as catechin hereinafter. Male Wistar strain rats weighing 180-200 g (6 weeks of age) were fed a commercial diet for one week. Eighty rats were divided into 4 groups of 20 rats. One ml of a 40, 60 or 80 mg/ml catechin solution was administered orally to all the rats which were starved overnight. The control group was fed water instead of catechin. After 30 min, 4 ml of a 40% soluble starch solution was administered orally to the rats in each group. Following the administration of starch, five rats in each group were sacrificed at intervals of 0, 30, 60, and 120 min and the contents of the small intestine and the blood were collected.

Table II. Effect of Tea Polyphenols on Rat Small Intestinal Sucrase and Maltase Relative activity (%) Sample

Concentration (mM)

Sucrase

Maltase

0.5 0.5 0.5 0.5 0.5 0.5 0.1 0.5 0.1 0.1 0.1 0.1 0.1

100 93 86 85 83 38 65 21 51 95 81 87 58

100 93 99 96 95 59 89 62 91 93 52 39 38

None Gallic acid (+)-Catechin (+C) (-)-Epicatechin (EC) (-)-Epigallocatechin (EGC) (-)-Epicatechin gallate (ECg) (-)-Epigallocatechin gallate (EGCg) Theaflavin (TF1) Theaflavin monogallate A (TF2A) Theaflavin monogallate B (TF2B) Theaflavin digallate (TF3)

Table III. Composition of Crude Catechins Catechin (+)-Gallocatechin (GC) (-)-Epigallocatechin (EGC) (-)-Epicatechin (EC) (-)-Epigallocatechin gallate (EGCg) (-)-Epicatechin gallate (ECg) Total

Absolute (%) 1.44 17.57 5.81 53.90 12.51 91.23

Relative (%) 1.6 19.3 6.4 59.1 13.7 100.0

α-Amylase Activity in the Intestine. The assay was carried out by the method of Willstatter and Schdel (7) with modification. The contents of the small intestine of


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starch-fed rats were filtered and the residue was removed to make an α-amylase solution. This sample solution was diluted with phosphate buffer and mixed with a fixed amount of soluble starch. The reaction mixture was incubated for 5 min at 37°C. One unit of α-amylase activity was defined as the amount of enzyme which liberated 1 μπιοί of maltose per min. The results are shown in Figure 1. As shown in the figure, α-amylase activity of the catechin-fed groups (40, 60 and 80 mg) scarcely increased in the 2 hrs after the starch-dosage, whereas the enzyme activity of the control group increased markedly peaking at a much higher point than the catechin groups. Glucose Concentration in the Plasma. The starch-fed rats were sacrificed and their blood was collected at intervals as described above. The blood was centrifuged at 3,000 rpm for 15 min and the plasma was stored at -20°C. Concen tration of glucose in the plasma was determined using the glucose oxidase kit. The results are shown in Figure 1. The figure shows an increase of concentration from the zero time. When 80 mg of catechin were given before the administration of starch, the increase of glucose concentrations in the plasma was significantly sup pressed as compared with that of the control group. The suppression depended on the quantity of catechin administered. When 40 mg of catechin was administered, there was only a slight suppressive effect on plasma glucose level.

α-Amylase Activity

Plasma Glucose Concentration

Time (hours) Figure 1. α-Amylase activity and plasma glucose levels in rats administered starch. * Statistically different from control (p TF2>TF1). In any case, it appears that a 3,4,5-trihydroxybenzoyl moiety at the 3-OH position is essential for the inhibition of α-amylase.




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Age (weeks)

Food intake

• 2% crude catechins

10 L"

1

5

6

»

7

1

8

1

9

1

1

1

1

'

1

'

'

10 11 12 13 14 15 16 17 18 Age (weeks)

Figure 3. The effect of catechins in the diet on body weight and food intake.

The above findings led us to further examine the possibility that tea poly phenols may suppress α-amylase activity in the intestine, resulting in the reduction of the glucose level in the blood. Since theaflavins are minor elements and hard to obtain, the catechin fraction from green tea was used for animal experiments. Oral administration of tea catechin prior to starch administration suppressed α-amylase activity otherwise elevated by starch. In addition, the increase in glucose concentration after starch administration was also arrested by the prior administra tion of catechin. Likewise, tea polyphenols suppressed sucrase and maltase activity in vitro as well as in vivo. Here again the galloyl moiety of tea polyphenols seems to determine the degree of inhibitory potency, and the effects are dose dependent. From these results we conclude that tea polyphenols are effective in sup pressing the activity of α-amylase and sucrase in the intestine when an excess amount of starch or sucrose is fed, thereby moderately suppressing the increase of the blood glucose level. Furthermore, it was confirmed that catechin supplemen tation in a normal diet over an extended period of time will not suppress the food


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Saccharide Digestive Enzymes and Tea Polyphenols 89

intake nor body weight gain. In another series of experiments, various prophylactic functions were noted with long term feeding of catechins (Hara, Y., "Prophylactic Functions of Tea Polyphenols," in this proceeding). These experimental facts seem to invite wider utilization of tea polyphenols for human health.


Literature Cited 1. Hara, Y.; Matsuzaki, T. Nippon Nogeikagaku Kaishi (in Japanese) 1987, 61, 803-808. 2. Hattori, M.; Kusumoto; I. T., Namba, T.; Ishigami, T.; Hara, Y. Chem. Pharm. Bull. 1990, 38, 717-720. 3. Hara, Y.; Honda, M. Agric. Biol. Chem. 1990, 54, 1939-1945. 4. Hara, Y.; Matsuzaki, S.; Nakamura, K. Nippon Eiyo Shokuryo Gakkaishi (in Japanese) 1989, 42, 39-45. 5. Bernfeld, P. In Methods of Enzymology; Colwick, S. B.; Kaplan, N. O., Eds.; Academic Press: New York, 1955; pp 149-150. 6. Hara, Y.; Tono-oka, F. Nippon Eiyo Syokuryo Gakkaishi (in Japanese) 1990, 43, 345-348. 7. Willstatter, R.; Schdel, G. Berichte 1918, 51, 780-781. R E C E I V E D May 4, 1 9 9 3


Inhibition of Saccharide Digestive Enzymes by Tea Polyphenols

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