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Effects of Ya-hom on the gastric secretion in rats
Journal of Ethnopharmacology 94 (2004) 331–338
Effects of Ya-hom on the gastric secretion in rats W. Suvitayavat a,∗ , J. Kodchawongs a , S.S. Thirawarapan a , N. Bunyapraphatsara b a
b
Faculty of Pharmacy, Department of Physiology, Mahidol University, 447 Sri-ayudhaya Road, Bangkok 10400, Thailand Faculty of Pharmacy, Department of Pharmacognosy, Mahidol University, 447 Sri-ayudhaya Road, Bangkok 10400, Thailand Received 12 November 2003; received in revised form 30 March 2004; accepted 2 June 2004
Abstract The effects of Ya-hom, Thai traditional recipe, on gastric-secretory function was evaluated to verify its use for stomach discomfort treatment. The actions of Ya-hom on the gastric acid, pepsin and mucus secretions were studied in histamine- and carbachol-induced gastric fistula rats. After the first hour of collection, histamine (10 mg/kg, intramuscular injection) or carbachol (20 g/kg, intravenous injection) were injected to stimulate gastric secretion, and water or redissolved lyophilized Ya-hom extract (0.5, 1, 2 and 4 g/kg) were intraduodenally injected. The gastric samples were collected every hour for 5 h, at the end of which, the glandular part of the stomach was assayed for the visible mucus content. Ya-hom inhibited both histamine- and carbachol-induced gastric acid, pepsin and soluble mucus secretions in a dose-dependent manner. Ya-hom had a lower maximum inhibition on the acid-stimulating effects of the histamine than that of carbachol. Ya-hom had higher maximum inhibition on the pepsin and soluble mucus-stimulating effects of histamine than that of carbachol. Ya-hom also had a higher elevated effect on histamine-induced visible mucus than that of carbachol. This study reveals that Ya-hom inhibits the stimulatory effects of histamine and carbachol on the acid, pepsin and soluble mucus secretion but potentiates the visible mucus secretion. Attenuating gastric secretion by secretagogues and increasing gastric barrier effects are parts of Ya-hom’s action on stomach discomfort treatment. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Gastric acid secretion; Pepsin secretion; Mucous secretion; Gastric mucosal blood flow; Ya-hom
1. Introduction Among traditional medicines used in Thailand nowadays, Ya-hom is one of the most interesting folk formula used for the treatment of stomach discomfort. Ya-hom recipe selected for this study contains Agastache rugosa (Fisch. et Mey) O. Kuntze, Acorus gramineus Soland, Lysimachia foenum-graecum Hance, Citrus nobilis Lour., Magnolia officinalis Rehd. et Wils., Cinnamomum cassia Presl. (Chinese cinnamon), Mentha arvensis L. (Japanese mint), Asarum sieboldii Miq., Ligusticum wallichii Franch., Glycyrrhiza glabra L. (licorice), Eugenia caryophyllata Thunb. (clove), Saussuria lappa Clark, Aquilaria agallocha Roxb., Atractylis ovata Thunb., menthol, Borneo camphor, and Angelica anomala Lallem. The scientific reports of some Ya-hom’s ingredients have shown that licorice (Hakanson et al., 1973; Takagi and Harada, 1969; Yu, 1976; Shihata and Elghamry, 1963), Saussuria lappa Clark (Yoshikawa et al., 1993), Acorus gramineus Soland (Zhang et al., 1981) and ∗ Corresponding
author. E-mail address: [email protected] (W. Suvitayavat).
Chinese cinnamon (Tanaka et al., 1989) have antiulcer activity; licorice (Revers, 1956), Saussuria lappa Clark (Bose et al., 1961; Gupta and Ghatak, 1967), clove (Debelmas and Rochat, 1967) and Asarum sieboldii Miq. (Itokawa et al., 1983) have spasmolytic activity; and licorice possesses gastric secretion inhibitory effect (Hakanson et al., 1973; Takagi and Harada, 1969; Yu, 1976). However, some ingredients have no effect or no scientific data on their effects on the gastric function. Moreover, the effect and mechanism of action of the whole Ya-hom recipe on gastric function have not been reported. Since the stomach discomfort can be caused by interfering on the gastric function such as increasing gastric secretion, dyspepsia and stomach pain, the effect of Ya-hom on gastric secretion is investigated to clarify its action on decreasing stomach discomfort.
2. Materials and methods 2.1. Chemicals Urethane was obtained from Merck. Histamine, carbachol, hemoglobin, pepsin, bovine serum albumin,
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.06.003
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W. Suvitayavat et al. / Journal of Ethnopharmacology 94 (2004) 331–338
chondroitin sulfate and Alcian blue 8 GX were purchased from Sigma Chemical Co., St Louis, USA. Sodium hydroxide was purchased from Carlo Erba, Milano, Italy. Other reagents were of analytical grade. 2.2. Ya-hom Ya-hom was obtained from Five Pagodas Pharmacy Co. Ltd., Thailand. One hundred grams of Ya-hom contains Agastache rugosa (Fisch. et Mey) O. Kuntze (whole plant, Labiatae) 7.1 g, Acorus gramineus Soland (rhizomes, Araceae) 0.5 g, Lysimachia foenum-graecum Hance (whole plant, Primulaceae) 3.3 g, Citrus nobilis Lour. (outer yellow rind of the ripe fruit, Rutaceae) 7.1 g, Magnolia officinalis Rehd. et Wils. (bark of stem, Magnoliaceae) 11.8 g, Cinnamomum cassia Presl (Chinese cinnamon, bark, Lauraceae) 7.1 g, C L. (Japanese mint, whole plant, Lamiaceae) 0.5 g, Asarum sieboldii Miq. (whole plant, Aristolochiaceae) 2.3 g, Ligusticum wallichii Franch. (rhizomes, Umbelliferae) 9.3 g, Glycyrrhiza glabra L. (licorice, rhizomes, Leguminosae) 4.8 g, Eugenia caryophyllata Thunb. (clove, flower-bud, Myrtaceae) 9.3 g, Saussuria lappa Clark (rhizomes, Compositae) 7.1 g, Aquilaria agallocha Roxb. (wood, Thymelaeaceae) 7.1 g, Atractylis ovata Thunb. (rhizomes, Compositae) 9.3 g, menthol 4.7 g, Borneo camphor 1.4 g, Angelica anomala Lallem (rhizomes, Umbelliferae) 3.5 g. 2.3. Lyophilized Ya-hom water extract Ya-hom powder was mixed with boiled distilled water in the ratio of 1 kg:10 l and continuously boiled for 15 min. Ya-hom extract was filtered through cotton muslin cloth. The filtrate was lyophilized and kept at −20 ◦ C. One gram of Ya-hom powder yielded 0.136 g of lyophilized product. The test solution was freshly prepared by dissolving the lyophilized product in distilled water. Lyophilized Ya-hom water extract was dissolved in methanol, sonicated for 20 min and then adjusted to the concentration of 0.01 g/ml with methanol. Sample was filtered through Sep-pack® C18 cartridge before applying to HPLC using LiChrosphere® 100RP-18, Merck (5 m) column and eluted with deionized water for 5 min and followed by linear gradient of 10–100% acetonitrile for 15 min. HPLC analysis of lyophilized Ya-hom water extract was showed in Fig. 1. 2.4. Animal Male Wistar rats weighing between 200 and 220 g were obtained from the National Laboratory Animal Center at Salaya, Mahidol University. Rats were housed in hanging cage in the animal room at Faculty of Pharmacy, Mahidol University, for at least 1 week prior to the experiment. The animals were fed with commercial rat diet (C.P. Mice Feed; SWT. Co. Ltd., Thailand) and tab water ad libitum. The rats were fasted for 24 h before the experiment. Each experimental group was performed in 10 rats.
Fig. 1. HPLC analysis of lyophilized Ya-hom. Lyophilized Ya-hom in methanol was applied to HPLC using LiChrosphere® 100RP-18, Merck (5 m) column and eluted with deionized water and linear gradient of 10–100% acetonitrile.
2.5. Animal preparation Gastric fistula were performed as previously described by Limlomwongse et al. (1976). The fasted rat was anesthetized with urethane at the dose of 1.2 g/kg body weight intraperitoneally. The esophagus was ligated and the trachea was cannulated by using polyethylene tube No. 206. After opening the abdomen, the pyloduodenal junction was carefully ligated and the stomach was perforated and inserted the polyethylene tube No. 206 gastric fistula. The sample was collected through the gastric fistula. 2.6. Gastric sample collection The sample collection was taken in 1-h interval for 5 h and expressed as h1, h2, h3, h4 and h5. The first sample was collected and referred as the basal secretion. Then histamine (10 mg/kg, intramuscular injection) or carbachol (20 g/kg, intravenous injection) and Ya-hom extract (0.5–4 g/kg diluted to 1 ml/animal, intraduodenal injection) or water (1 ml/animal, intraduodenal injection) were administered. The gastric samples were collected at 1-h interval for 4 more hours (samples h2, h3, h4 and h5). At the end of the experiment, the stomach was removed and opened down the lesser curvature. The opened stomach was rinsed with normal saline pH 7.4 and dried with filter paper. Then the glandular portion was excised and determined the visible mucus. 2.7. Gastric sample analysis The gastric sample was weighed and centrifuged by Universal Centrifuge 16A, Hettich at 5000 rpm for 5 min to precipitate solid residue. The tube with residue was weighed again. The clear supernatant was separated and kept in ice bath. The supernatant was used for determining the gastric acid, pepsin activity, soluble mucus and protein.
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2.7.1. Gastric volume determination The difference between the weights of tube containing gastric sample and tube with residue represented the collected gastric volume by assuming that the specific gravity of gastric sample was 1.00. 2.7.2. Gastric acid-secretory rate determination The collected gastric acid was examined by titration of gastric sample with 0.01N sodium hydroxide to the end point at pH 5.0. The gastric acid-secretory rate was calculated from the collected gastric acid dividing by the weight of stomach glandular part and expressed as equiv. HCl/g stomach/h. 2.7.3. Gastric pepsin-secretory rate determination Pepsin secretion was determined by measuring the proteolytic activity of pepsin in gastric sample by using hemoglobin as a substrate and pepsin (480 units/mg solid) as a standard. The activity was determined according to the method described by Miller et al. (1975). The pepsin activity was expressed as units/g stomach/h. 2.7.4. Gastric protein-secretory rate determination The amount of protein in gastric sample was measured by colorimetric method (Lowry et al., 1951) using bovine serum albumin as a standard. The protein-secretory rate was determined from the amount of protein in collected gastric sample and expressed as mg/g stomach/h. 2.7.5. Gastric soluble mucus-secretory rate determination Gastric soluble mucus was determined according to the method described by Whiteman (1973) and by using chondroitin sulfate as a standard. The gastric soluble mucus-secretory rate was calculated from the amount of soluble mucus and express as g chondroitin sulfate/g stomach/h. 2.7.6. Gastric visible mucus determination Visible mucus on the gastric mucosal surface was determined according to the modification of the method previously described by Corne et al. (1974). The Alcian blue 8 GX (Sigma chemical Co., St Louis, USA) dissolved in MgCl2 was used as a standard. The amount of gastric visible mucus was expressed as mg Alcian blue (AB)/g stomach. 2.8. Gastric mucosal blood flow Animal preparation was similar to that of gastric sample collection. Gastric mucosal blood flow was measured by Laser Doppler Flowmeter. The probe was inserted through the fistula until the distance between the probe and the mucosa was about 2 mm. The mucosal blood flow was expressed as perfusion unit (PU). After basal blood flow measurement, histamine (10 mg/kg, intramuscular injection) and Ya-hom extract (4 g/kg, intraduodenal injection) or water (1 ml, intraduodenal injection) were administered. The gastric blood flow was detected in 15 min interval for 2 h and then in 30 min interval for 2 more hours.
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2.9. Statistical analysis One-way analysis of variance (ANOVA) was used to compare the values for each of experimental groups. Tukey’s honestly significant difference (HSD) test was used to differentiate the difference between two experimental groups. Paired sample t-test was to compare the secretory rates at h2, h3 h4 and h5 with the basal secretion (h1) in each experimental group. The P-value of less than 0.05 (P < 0.05) was considered to statistical significant difference.
3. Results 3.1. Effects of Ya-hom on histamine-induced gastric secretion The gastric acid-secretory rate profile pattern of histamine control group was increased after histamine injection and highest at h2 and then gradually decreased to the basal level at h5 (Table 1). The gastric acid-secretory rates at h2, h3 and h4 were significantly higher than its basal level. All the histamine + Ya-hom treated groups had similar gastric acid-secretory rate profile patterns as control group. However, they were lower than the control group at the same time point. Ya-hom inhibited histamine-stimulated gastric acid secretion in a dose-dependent manner with a maximum inhibition at h2 (Table 1). In histamine control group, the gastric pepsin, protein and soluble mucus-secretory rate profile patterns were similar to the histamine-induced gastric acid secretion except that the pepsin- and soluble mucus-secretory rates at h5 were declined to the level which significantly lower than its basal level as shown in Tables 2–4, respectively. All the histamine + Ya-hom-treated groups showed gastric pepsin- (Table 2), protein- (Table 3) and soluble mucus (Table 4)-secretory rate patterns similar to the histamine control group but the secretory rates were lower than the control in a dose-dependent manner. Ya-hom also inhibited the gastric pepsin, protein and soluble mucus secretions. All the Ya-hom-treated rats had significantly lower pepsin- and protein-secretory rates than histamine-induced rats at the same time point from h2 to h5 (Tables 2 and 3). However, Ya-hom significantly decreased soluble mucus-secretory rates only from h2 to h4 (Table 4). In contrast to other gastric secretions, the visible mucus content on the gastric mucosal surface of all histamine + Ya-hom-treated groups were significantly higher than that of the histamine control group (Fig. 2). 3.2. Effects of Ya-hom on carbachol-induced gastric secretion In carbachol-induced rats, the gastric acid-secretory rate was significantly increased to the peak at h2 and then declined to the value which was still significantly higher than
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Table 1 Dose-dependent effects of Ya-hom (YH) on histamine- and carbachol-induced gastric acid-secretory rate (equiv./g stomach/h) at h1, h2, h3, h4 and h5 Treatment
Gastric acid-secretory rate at time (h) 1
2
3
4
5
Control
Histamine Carbachol
1.4 ± 0.1 1.6 ± 0.1
193.1 ± 4.9∗∗ 76.4 ± 5.9∗∗
95.6 ± 4.5∗∗ 43.3 ± 2.5∗∗
21.9 ± 2.0∗∗ 22.2 ± 2.2∗∗
2.8 ± 1.0 11.3 ± 1.4∗∗
0.5 g/kg YH
Histamine Carbachol
1.3 ± 0.1 1.5 ± 0.1
182.4 ± 2.3∗∗ 74.2 ± 1.5∗∗
71.3 ± 1.8∗∗∗,∗∗ 41.2 ± 2.1∗∗
21.3 ± 0.4∗∗ 22.2 ± 1.8∗∗
1.5 ± 0.1 9.4 ± 1.1∗∗
1 g/kg YH
Histamine Carbachol
1.3 ± 0.1 1.4 ± 0.0
138.5 ± 4.4a,∗∗∗,∗∗ 61.4 ± 2.4a,∗∗∗,∗∗
82.1 ± 6.6∗∗ 33.9 ± 1.3a,∗∗∗,∗∗
21.0 ± 3.0∗∗ 23.6 ± 0.9∗∗
2.2 ± 0.3∗ 8.9 ± 0.5∗∗
2 g/kg YH
Histamine Carbachol
1.2 ± 0.1 1.4 ± 0.0
116.8 ± 2.9a,b,∗∗∗,∗∗ 42.0 ± 1.0a,b,∗∗∗,∗∗
64.2 ± 4.4b,∗∗∗,∗∗ 29.6 ± 0.3a,∗∗∗,∗∗
11.5 ± 1.3a,b,∗∗∗,∗∗ 19.9 ± 0.5∗∗
1.6 ± 0.2∗ 7.0 ± 0.2∗∗
4 g/kg YH
Histamine Carbachol
1.2 ± 0.1 1.4 ± 0.0
105.3 ± 1.8a,b,∗∗∗,∗∗ 35.3 ± 1.4a,b,∗∗∗,∗∗
58.3 ± 2.6b∗∗∗,∗∗ 17.0 ± 1.0a,b,c,∗∗∗,∗∗
9.1 ± 1.0a,b,∗∗∗,∗∗ 8.8 ± 0.8a,b,c,∗∗∗,∗∗
1.7 ± 0.2∗∗ 3.5 ± 0.2a,b,c,∗∗∗,∗∗
All values were expressed as mean ± S.E.M. ∗ P < 0.05: significant difference from their basal secretion within group. ∗∗ P < 0.01: significant difference from their basal secretion within group. Comparison at the same time point: ∗∗∗ P < 0.05: significant difference from their control groups. a P < 0.05: significantly lower than 0.5 g/kg Ya-hom-treated group (within group). b P < 0.05: significantly lower than 1 g/kg Ya-hom-treated group (within group). c P < 0.05: significantly lower than 2 g/kg Ya-hom-treated group (within group).
the basal level (Table 1). The gastric acid-secretory rates of all carbachol + Ya-hom-treated groups also showed significantly higher gastric acid-secretory rates than their basal rates at h2 to h5 but lower than that of control at the same time point. Ya-hom decreased gastric acid secretion in a dose-dependent manner with a maximum inhibition at h2 (Table 1). The profile pattern of gastric pepsin-secretory rate in the carbachol control group was similar to that of histamine-induced pepsin secretion, the gastric-secretory rate was highest at h2 and then gradually decreased to the
significantly lower rate than its basal rate at h5 (Table 2). The secretory patterns of all carbachol + Ya-hom-treated groups were similar to that of carbachol control group (Table 2). Comparing the gastric pepsin secretion of the carbachol + Ya-hom-treated groups with the carbachol control group at the same time point showed that Ya-hom decreased gastric pepsin secretion in a dose-dependent manner (Table 2). The gastric protein- and mucus-secretory rates of carbachol-induced rats were similar to carbachol-induced gastric acid secretion. The protein- and mucus-secretory rates increased to the peak at h2 and declined but still
Table 2 Dose-dependent effects of Ya-hom (YH) on histamine- and carbachol-induced gastric pepsin-secretory rate (units/g stomach/h) at h1, h2, h3, h4 and h5 Treatment
Gastric pepsin-secretory rate at time (h) 1
2
3 44.6∗∗
4 36.9∗∗
5
Control
Histamine Carbachol
264.5 ± 10.8 230.5 ± 6.6
1402.7 ± 1260.3 ± 50.0∗∗
679.9 ± 639.7 ± 18.7∗∗
326.4 ± 329.4 ± 6.0∗∗
173.1 ± 7.5∗∗ 197.7 ± 4.1∗∗
0.5 g/kg YH
Histamine Carbachol
249.3 ± 8.0 239.4 ± 5.4
1143.7 ± 20.1∗∗∗,∗∗ 1214.4 ± 42.8∗∗
486.2 ± 35.6∗∗∗,∗∗ 618.8 ± 17.8∗∗
230.2 ± 13.1∗∗∗ 296.8 ± 11.9∗∗∗,∗∗
139.1 ± 7.4∗∗∗,∗∗ 192.4 ± 6.2∗∗
1 g/kg YH
Histamine Carbachol
250.3 ± 10.9 233.6 ± 5.7
824.7 ± 33.3a,∗∗∗,∗∗ 956.0 ± 38.7a,∗∗∗,∗∗
411.1 ± 30.0∗∗∗,∗∗ 575.6 ± 11.3∗∗
229.2 ± 16.3∗∗∗ 256.6 ± 7.7a,∗∗∗,∗∗
135.0 ± 8.2∗∗∗,∗∗ 179.1 ± 4.9∗∗
2 g/kg YH
Histamine Carbachol
253.2 ± 8.6 225.0 ± 5.8
725.9 ± 30.5a,∗∗∗,∗∗ 764.5 ± 20.4a,b,∗∗∗,∗∗
342.9 ± 20.9a,∗∗∗,∗∗ 494.1 ± 26.8a,b,∗∗∗,∗∗
158.1 ± 5.7a,b,∗∗∗,∗∗ 208.27 ± 4.9a,b,∗∗∗,∗∗
104.4 ± 7.3a,b,∗∗∗,∗∗ 159.9 ± 4.9a,b,∗∗∗,∗∗
4 g/kg YH
Histamine Carbachol
261.3 ± 11.5 240.5 ± 7.2
580.2 ± 19.3a,b,c,∗∗∗,∗∗ 655.9 ± 30.1a,b,∗∗∗,∗∗
243.3 ± 10.8a,b,∗∗∗ 433.9 ± 15.9a,b,∗∗∗,∗∗
131.8 ± 6.3a,b,∗∗∗,∗∗ 185.9 ± 4.1a,b,∗∗∗,∗∗
92.1 ± 3.0a,b,∗∗∗,∗∗ 152.5 ± 3.2a,b,∗∗∗,∗∗
All values were expressed as mean ± S.E.M. ∗∗ P < 0.01: significant difference from their basal secretion within group. Comparison at the same time point: ∗∗∗ P < 0.05: Significant difference from their control group. a P < 0.05: significantly lower than 0.5 g/kg Ya-hom-treated group (within group). b P < 0.05: significantly lower than 1 g/kg Ya-hom-treated group (within group). c P < 0.05: significantly lower than 2 g/kg Ya-hom-treated group (within group).
15.8∗∗
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Table 3 Dose-dependent effects of Ya-hom (YH) on histamine- and carbachol-induced gastric protein-secretory rate (mg/g stomach/h) at h1, h2, h3, h4 and h5 Treatment
Gastric protein-secretory rate at time (h) 1
2
3
4
5
Control
Histamine Carbachol
1.03 ± 0.05 1.01 ± 0.02
5.44 ± 0.17∗∗ 5.14 ± 0.20∗∗
3.06 ± 0.08∗∗ 3.47 ± 0.16∗∗
1.54 ± 0.06∗∗ 2.28 ± 0.13∗∗
0.98 ± 0.03 1.41 ± 0.10∗∗
0.5 g/kg YH
Histamine Carbachol
1.00 ± 0.03 1.00 ± 0.01
3.67 ± 0.17∗∗∗,∗∗ 5.10 ± 0.08∗∗
1.53 ± 0.06∗∗∗,∗∗ 3.36 ± 0.16∗∗
0.89 ± 0.04∗∗∗,∗∗ 2.18 ± 0.10∗∗
0.75 ± 0.02∗∗∗,∗∗ 1.39 ± 0.06∗∗
1 g/kg YH
Histamine Carbachol
1.04 ± 0.03 1.03 ± 0.01
2.88 ± 0.11a,∗∗∗,∗∗ 4.40 ± 0.13a,∗∗∗,∗∗
1.31 ± 0.04∗∗∗,∗∗ 2.75 ± 0.05a,∗∗∗,∗∗
0.68 ± 0.01a,∗∗∗,∗∗ 1.94 ± 0.06∗∗∗,∗∗
0.55 ± 0.02a,∗∗∗,∗∗ 1.28 ± 0.04∗∗
2 g/kg YH
Histamine Carbachol
1.04 ± 0.02 1.02 ± 0.01
2.13 ± 0.06a,b,∗∗∗,∗∗ 3.65 ± 0.09a,b,∗∗∗,∗∗
1.19 ± 0.05a,∗∗∗,∗ 2.19 ± 0.04a,b,∗∗∗,∗∗
0.52 ± 0.01a,b,∗∗∗,∗∗ 1.87 ± 0.03∗∗∗,∗∗
0.34 ± 0.02a,b,∗∗∗,∗∗ 1.15 ± 0.02a,∗∗∗,∗∗
4 g/kg YH
Histamine Carbachol
1.05 ± 0.02 1.04 ± 0.01
2.12 ± 0.06a,b,∗∗∗,∗∗ 3.11 ± 0.04a,b,c,∗∗∗,∗∗
1.08 ± 0.06a,∗∗∗ 2.11 ± 0.03a,b,∗∗∗,∗∗
0.49 ± 0.01a,b,∗∗∗,∗∗ 1.70 ± 0.02a,∗∗∗,∗∗
0.36 ± 0.02a,b,∗∗∗,∗∗ 1.10 ± 0.02a,∗∗∗,∗∗
All values were expressed as mean ± S.E.M. ∗ P < 0.05: significant difference from their basal secretion within group. ∗∗ P < 0.01: significant difference from their basal secretion within group. Comparison at the same time point: ∗∗∗ P < 0.05: significant difference from their control groups. a P < 0.05: significantly lower than 0.5 g/kg Ya-hom-treated group (within group). b P < 0.05: significantly lower than 1 g/kg Ya-hom-treated group (within group). c P < 0.05: significantly lower than 2 g/kg Ya-hom-treated group (within group).
significantly higher than its basal level until h5 (Tables 3 and 4). The protein-secretory rates of all carbachol + Ya-hom-treated rats showed the similar pattern as carbachol control group but lower than carbachol control group at the same time point. Ya-hom at the dose of 1, 2 and 4 g/kg significantly inhibited gastric protein- and soluble mucus-secretory rates (Tables 3 and 4). Similar to the effect of Ya-hom on histamine-induced visible mucus secretion, all of carbachol + Ya-hom-treated rats had more gastric visible mucus than the carbachol con-
trol group (Fig. 2). However, only Ya-hom at doses of 1, 2 and 4 g/kg significantly increased gastric visible mucus in carbachol-treated rats. 3.3. Effects of Ya-hom on gastric mucosal blood flow Ya-hom at a dose of 4 g/kg that had the highest gastric-secretory inhibition in this present experiment was used for investigation the effect of Ya-hom on histamine-induced gastric mucosal blood flow. As shown
Table 4 Dose-dependent effects of Ya-hom (YH) on histamine- and carbachol-induced gastric soluble mucus-secretory rate (g chondroitin sulfate/g stomach/h) at h1, h2, h3, h4 and h5 Treatment
Gastric soluble mucus-secretory rate at time (h) 1
2
3 3.22∗∗
4 2.40∗∗
5
Control
Histamine Carbachol
4.53 ± 0.15 11.63 ± 0.51
97.19 ± 105.79 ± 2.30∗∗
56.71 ± 59.11 ± 2.39∗∗
27.59 ± 31.88 ± 1.32∗∗
3.99 ± 0.21∗∗ 16.91 ± 0.77∗∗
0.5 g/kg YH
Histamine Carbachol
4.47 ± 0.09 12.02 ± 0.46
79.32 ± 1.74∗∗∗,∗∗ 104.68 ± 2.70∗∗
47.49 ± 1.05∗∗∗,∗∗ 58.68 ± 2.03∗∗
22.42 ± 1.46∗∗∗,∗∗ 31.75 ± 1.61∗∗
3.95 ± 0.29 16.99 ± 0.77∗∗
1 g/kg YH
Histamine Carbachol
4.47 ± 0.08 11.54 ± 0.53
64.94 ± 2.61a,∗∗∗,∗∗ 95.26 ± 2.85∗∗∗,∗∗
42.23 ± 0.92∗∗∗,∗∗ 46.79 ± 1.34a,∗∗∗,∗∗
18.07 ± 0.30a,∗∗∗,∗∗ 26.62 ± 1.08∗∗∗,∗∗
3.76 ± 0.25∗ 13.94 ± 0.73a,∗∗∗,∗∗
2 g/kg YH
Histamine Carbachol
4.33 ± 0.13 11.45 ± 0.37
54.48 ± 1.40a,b,∗∗∗,∗∗ 77.66 ± 2.95a,b,∗∗∗,∗∗
31.02 ± 1.66a,b,∗∗∗,∗∗ 41.32 ± 1.86a,∗∗∗,∗∗
14.08 ± 0.20a,b,∗∗∗,∗∗ 25.11 ± 1.43a,∗∗∗,∗∗
3.73 ± 0.22∗ 11.71 ± 0.60a,∗∗∗
4 g/kg YH
Histamine Carbachol
4.47 ± 0.11 11.57 ± 0.44
46.00 ± 0.74a,b,∗∗∗,∗∗ 71.61 ± 1.71a,b,∗∗∗,∗∗
26.54 ± 0.95a,b,∗∗∗,∗∗ 39.70 ± 1.46a,∗∗∗,∗∗
13.52 ± 0.40a,b,∗∗∗,∗∗ 20.56 ± 0.80a,b,∗∗∗,∗∗
3.70 ± 0.05∗∗ 10.73 ± 0.47a,b,∗∗∗
All values were expressed as mean ± S.E.M. ∗ P < 0.05: significant difference from their basal secretion within group. ∗∗ P < 0.01: significant difference from their basal secretion within group. Comparison at the same time point: ∗∗∗ P < 0.05: significant difference from their control groups. a P < 0.05: significantly lower than 0.5 g/kg Ya-hom-treated group (within group). b P < 0.05: significantly lower than 1 g/kg Ya-hom-treated group (within group).
1.29∗∗
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4. Discussion
Fig. 2. Effects of Ya-hom on histamine- (open circle) and carbachol-induced (closed circle) gastric visible mucus secretion (mg AB/g stomach). All values were expressed as mean ± S.E.M. ∗ P < 0.05: significant difference from their control group. a P < 0.05: significantly higher than 0.5 g/kg Ya-hom-treated group (within group). b P < 0.05: significantly higher than 1 g/kg Ya-hom-treated group (within group). c P < 0.05: significantly higher than 2 g/kg Ya-hom-treated group (within group).
Fig. 3. Effects of Ya-hom 4 g/kg (YH4) on histamine-induced gastric mucosal blood flow (perfusion unit). ∗ P < 0.05, ∗∗ P < 0.01: significant difference from the base line within group. a P < 0.05: significant difference from their control group at the same time point.
in Fig. 3, the gastric mucosal blood flow of the control gastric mucosa was significantly increased after histamine injection to the peak at 45 min and then declined to the basal level at 150 min. Ya-hom-treated rats had no increase in gastric mucosal blood flow after histamine administration except at 150 min and had significantly lower gastric mucosal blood flow than its base line at 210 and 240 min. The gastric mucosal blood flow of Ya-hom-treated rat was significantly lower than those of control rat at 30– 90 min.
The present study attempted to investigate the effect of Ya-hom on secretagogue-induced gastric secretion in rats to verify its use for stomach discomfort. The gastric-secretory profiles in histamine- and carbachol-induced rats were the same. The dose of carbachol was limited by its stimulatory effect on salivary and mucus secretion in respiratory tract. Most of the effect of histamine was higher but shorter duration of action than carbachol. The efficiency of histamine on stimulation of gastric acid and visible mucus was higher than carbachol. However, the stimulatory efficiency on pepsin and soluble mucus secretions of histamine and carbachol were similar. This indicated that histamine and carbachol had different effects on each secretory cell types. In addition, carbachol is known to act both directly on the secretory cells and indirectly through stimulation on histamine-released ECL cells (Hirschowitz et al., 1995). Ya-hom inhibited histamine- and carbachol-induced gastric acid, pepsin, protein and soluble mucus secretion in a dose-dependent manner. Our preliminary experiment found that Ya-hom at a dose of higher than 4 g/kg did not show any significant higher inhibition, so the dose of 4 g/kg was the highest dose using in the present experiment. The maximum inhibition of Ya-hom was at h2, which was the period of the highest response of histamine or carbachol. The maximum inhibition of Ya-hom on histamine-induced gastric acid, pepsin, protein and soluble mucus secretions were 45.5, 58.6, 61.0 and 52.7% whereas its maximum inhibition on carbachol-induced secretions were 53.8, 48.0, 39.5 and 32.3%, respectively. In contrast to the effects on acid, pepsin and soluble mucus secretion, Ya-hom potentiated both histamine- and carbachol-induced gastric visible mucus secretion with the maximum stimulation of 77.4 and 58.2%. Except for gastric acid secretion, Ya-hom had higher attenuation and potentiation on histamine-induced gastric secretion than carbachol. This indicated that Ya-hom’s action on gastric secretion mainly involved histamine pathway. Even though the effect of Ya-hom on histamine and acetylcholine have not been reported, there were some studies on its ingredients: licorice (Fumio, 1973; Paris and Guillot, 1955), Saussuria lappa Clarke (Bose et al., 1961; Gupta and Ghatak, 1967), clove (Apisariyakul, 1984) and Acorus gramineus Soland (Zhang et al., 1981) showed that each plant extract antagonized the effects on histamine and acetylcholine-stimulated smooth muscle contraction. In addition, clove has shown to stimulate gastric mucus secretion (Enrique, 1951). So, the effect of Ya-hom may be due to these ingredients. In addition to Ya-hom’s inhibitory effect on gastric secretion, Ya-hom at a dose of 4 g/kg completely inhibited histamine-induced gastric mucosal blood flow from 30 to 90 min. The blood flow in each area of the gastrointestinal tract as well as in each layer of the gut is directly related
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to the level of local activity. In this study, Ya-hom inhibited histamine-induced gastric mucosal blood flow from 30 to 90 min which related to its maximum inhibitory effect on gastric acid, pepsin and soluble mucus secretion at h2 (1 h after secretagogue administration). Since histamine itself can cause a vasodilation via H1 receptor whereas it stimulates gastric acid secretion via H2 receptor, it is possible that effect of Ya-hom on gastric mucosal blood flow may or may not directly related to the gastric secretion inhibitory effect. However, the decrease in blood flow caused by Ya-hom potentiates its inhibitory effect on gastric secretion. Inhibition of gastric acid secretion causes by direct action on secretory cell such as M3 or H2 receptor antagonists or indirect action through the releasing of the inhibitory hormone, somatostatin or the inhibition of histamine-released ECL cells (Hirschowitz et al., 1995). Ya-hom composes of several ingredients, so it is possible that one or several compounds in Ya-hom cause direct and/or indirect effects on gastric-secretory functions. Furthermore, the stomach discomfort may cause by oversecretion, motility or gas production. It is shown here that Ya-hom inhibits gastric acid secretion about 50% which is enough to attenuate the discomfort feeling if it is caused by overgastric acid secretion. As known that, the integrity of the gastric mucosa and gastric ulcer are regulated by the balance between factors that promote and protect the ulcer development (Lloyd and Soll, 1994). The ulcer can also be the cause of stomach pain. Ya-hom has been shown here to decrease in aggressive factors (acid and pepsin) and increase in protective factor secretion (visible mucus). This effect of Ya-hom also causes a pain relief in case of peptic ulcer. It is possible that Ya-hom has more than one effect for this treatment. This gastric-secretory inhibition is a part of Ya-hom’s action on this treatment. The exact mechanism of Ya-hom’s action on the gastric cells and other effects of Ya-hom on the stomach should be further investigated to clarify its stomach discomfort treatment efficiency. In human, Ya-hom is taken by mixing 3 g of Ya-hom powder with warm water and drinking the whole preparation as needed. The dose of Ya-hom used in this experiment (4 g/kg) is about 80 times of human dose if it is assumed that a man weighs 60 kg. However, it is about 1 g per rat (weighing 250 g) when compares with 3 g per person in human. Since Ya-hom in the experiment uses the lyophilized product of boiling Ya-hom solution for 15 min, the extract may not contain total amount of each active compound form the powder. Boiling Ya-hom longer than 15 min is avoided to prevent the loss the volatile oil. This dose of Ya-hom extract should be able to represent the action of Ya-hom in human. The dose of Ya-hom used in this experiment is in the safety amount, since oral administration of the water extract of this Ya-hom recipe has been shown to possess LD50 more than 5 g/kg. It did not show any adverse effect when given to the rats at a dose of 2 g/kg per day for 5 weeks (Thongpraditchote et al., 1999).
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5. Conclusion Ya-hom inhibited the effect of histamine- and carbacholstimulated gastric acid, pepsin and soluble mucus secretion but potentiated gastric visible mucus secretion in a dose-dependent manner. Ya-hom also inhibited histamineinduced gastric mucosal blood flow that related to its inhibitory effect on gastric secretion. This study clarifies the claimed efficacy of Ya-hom ingestion for stomach discomfort because it not only decreases in aggressive factors (acid and pepsin) but also increases in protective factor secretion (visible mucus).
Acknowledgements This work was partly supported by the M.S. research grant, Faculty of Pharmacy, Mahidol University.
References Apisariyakul, A.C., 1984. A pharmacological screening of the plants on isolated rat ileum. Chiang Mai Phesatchasat 3, 8–16. Bose, B.C., Saifi, A.Q., Vijayvargiya, R., Shama, S.K., 1961. Phytochemical and pharmacological studies of Saussurea lappa. Journal of Pharmaceutical Sciences (Washington, DC) 50, 679–680. Corne, S.J., Morrisey, S.M., Wood, R.J., 1974. The method for the quantitative estimation of gastric barrier mucus. Journal of Physiology (Cambridge) 242, 116P–117P. Debelmas, A.M., Rochat, J., 1967. Pharmacologic study of the essential oils antispasmodic activity of fifty samples. Plantes Medicinales et Phytotherapie (Frances) 1, 23–27. Enrique, S.P., 1951. Mucous barrier and its significance. II. Changes in the gastric mucous produced by the local actions of spices and other irritative agents. Gastroenterology 18, 269–286. Fumio, M., 1973. Glycyrrhizin and flavonoid from licorice. Japan Kokai 1.30A.31. Gupta, O.P., Ghatak, B.J.R., 1967. Pharmacological investigations on Saussurea lappa. Indian Journal of Medical Research 55, 1078–1083. Hakanson, R., Liedberg, G., Oscarson, J., Rehfeld, J.F., Stadil, F., 1973. Effect of deglycyrrhizinized licorice on gastric acid secretion, histidine decarboxylase activity and serum gastrin level in the rat. Experientia (Basel) 29, 570–571. Hirschowitz, B.I., Keeling, D., Lewin, M., Okabe, S., Parsons, M., Sewing, K., Wallmark, B., Sacha, G., 1995. Pharmacological aspects of acid secretion. Digestive Disease Sciences 40, 3S–23S. Itokawa, H., Mihashi, S., Watanabe, S., Natsumoto, H., Hamanaka, T., 1983. Studies on the constituents of crude drugs having inhibitory activity against contraction of the ileum caused by histamine or barium chloride. 1. Screening test for the activity of commercially available crude drugs and the related plant materials. Shoyakugaku Zasshi 37, 223–238. Limlomwongse, L., Nophakoon, J., Thithapanda, A., 1976. Mechanism of inhibition of gastric acid secretion by hypertonic solutions and vasopressin Pfluegers Archiv. European Journal of Physiology (Berlin) 366, 159–165. Lloyd, K.C., Soll, A.H., 1994. Multiple pathways controlling acid secretion. Journal of Gastroenterology (Tokyo) 29, 77–79. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 266–275.
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Miller, L.L., Segal, H.L., Plumb, E.J., 1975. Proteolytic enzyme activity. Gastroenterology 33, 566–574. Paris, R.A., Guillot, M., 1955. Liquiritoside, flavonoside of the root of Glycyrrhiza glabra. Annales Pharmaceutiques Francaises (Paris) 13, 592–595. Revers, F.E., 1956. Pharmacological investigations on extract of licorice. Acta Medica Scandinavica 154, 749–751. Shihata, I.M., Elghamry, M.I., 1963. Experimental studies on the effect of Glycyrrhiza glabra. Planta Medica (Stuttgart) 11, 37–43. Takagi, K., Harada, M., 1969. Pharmacological studies on the herb peony root. II. Antiinflammatory effects, inhibitory effect on gastric juice secretion, preventive effect on stress ulcer, antidiuretic effect of peoniflorin and combined effects with licorice component Fm 100. Yakugaku Zasshi 89, 887–892. Tanaka, S., Yoon, Y.H., Fukui, H., Tabata, M., Akira, T., Okano, K., Iwai, M., Iga, Y., Yakoyama, K., 1989. Antiulcerogenic compounds
isolate from Chinese cinnamon. Planta Medica (Stuttgart) 55, 245– 248. Thongpraditchote, S., Wongkrajang, Y., Suvitayavat, W., Kitpati, C., Atisuk, K., 1999. A toxicity of Ya-hom. Thai Journal of Phytopharmacy 6, 1–10. Whiteman, P., 1973. The quantitative measurement of Alcian blue glycosaminoglycan. Biochemical Journal 131, 343–350. Yoshikawa, M., Hatakeyama, S., Inoue, Y., Yasuhiro, I., Yamahara, J., 1993. Saussureamines A, B, C, D and E, new antiulcer principles from Chinese Saussurea Radix. Chemical and Pharmaceutical Bulletin 41, 214–216. Yu, T.H., 1976. Pharmaceutical for peptic ulcer. Japan Kokai Tokyo Koho 7976, 815. Zhang, H., Wang, J., Doo-Yau, Y., Li, S., An, D., 1981. Survey on the content and composition of volatile oil in Acorus gramineus Soland from different growing areas. Yao Hsueh T’ung Pao 16, 15–17.
Effects of Ya-hom on the gastric secretion in rats W. Suvitayavat a,∗ , J. Kodchawongs a , S.S. Thirawarapan a , N. Bunyapraphatsara b a
b
Faculty of Pharmacy, Department of Physiology, Mahidol University, 447 Sri-ayudhaya Road, Bangkok 10400, Thailand Faculty of Pharmacy, Department of Pharmacognosy, Mahidol University, 447 Sri-ayudhaya Road, Bangkok 10400, Thailand Received 12 November 2003; received in revised form 30 March 2004; accepted 2 June 2004
Abstract The effects of Ya-hom, Thai traditional recipe, on gastric-secretory function was evaluated to verify its use for stomach discomfort treatment. The actions of Ya-hom on the gastric acid, pepsin and mucus secretions were studied in histamine- and carbachol-induced gastric fistula rats. After the first hour of collection, histamine (10 mg/kg, intramuscular injection) or carbachol (20 g/kg, intravenous injection) were injected to stimulate gastric secretion, and water or redissolved lyophilized Ya-hom extract (0.5, 1, 2 and 4 g/kg) were intraduodenally injected. The gastric samples were collected every hour for 5 h, at the end of which, the glandular part of the stomach was assayed for the visible mucus content. Ya-hom inhibited both histamine- and carbachol-induced gastric acid, pepsin and soluble mucus secretions in a dose-dependent manner. Ya-hom had a lower maximum inhibition on the acid-stimulating effects of the histamine than that of carbachol. Ya-hom had higher maximum inhibition on the pepsin and soluble mucus-stimulating effects of histamine than that of carbachol. Ya-hom also had a higher elevated effect on histamine-induced visible mucus than that of carbachol. This study reveals that Ya-hom inhibits the stimulatory effects of histamine and carbachol on the acid, pepsin and soluble mucus secretion but potentiates the visible mucus secretion. Attenuating gastric secretion by secretagogues and increasing gastric barrier effects are parts of Ya-hom’s action on stomach discomfort treatment. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Gastric acid secretion; Pepsin secretion; Mucous secretion; Gastric mucosal blood flow; Ya-hom
1. Introduction Among traditional medicines used in Thailand nowadays, Ya-hom is one of the most interesting folk formula used for the treatment of stomach discomfort. Ya-hom recipe selected for this study contains Agastache rugosa (Fisch. et Mey) O. Kuntze, Acorus gramineus Soland, Lysimachia foenum-graecum Hance, Citrus nobilis Lour., Magnolia officinalis Rehd. et Wils., Cinnamomum cassia Presl. (Chinese cinnamon), Mentha arvensis L. (Japanese mint), Asarum sieboldii Miq., Ligusticum wallichii Franch., Glycyrrhiza glabra L. (licorice), Eugenia caryophyllata Thunb. (clove), Saussuria lappa Clark, Aquilaria agallocha Roxb., Atractylis ovata Thunb., menthol, Borneo camphor, and Angelica anomala Lallem. The scientific reports of some Ya-hom’s ingredients have shown that licorice (Hakanson et al., 1973; Takagi and Harada, 1969; Yu, 1976; Shihata and Elghamry, 1963), Saussuria lappa Clark (Yoshikawa et al., 1993), Acorus gramineus Soland (Zhang et al., 1981) and ∗ Corresponding
author. E-mail address: [email protected] (W. Suvitayavat).
Chinese cinnamon (Tanaka et al., 1989) have antiulcer activity; licorice (Revers, 1956), Saussuria lappa Clark (Bose et al., 1961; Gupta and Ghatak, 1967), clove (Debelmas and Rochat, 1967) and Asarum sieboldii Miq. (Itokawa et al., 1983) have spasmolytic activity; and licorice possesses gastric secretion inhibitory effect (Hakanson et al., 1973; Takagi and Harada, 1969; Yu, 1976). However, some ingredients have no effect or no scientific data on their effects on the gastric function. Moreover, the effect and mechanism of action of the whole Ya-hom recipe on gastric function have not been reported. Since the stomach discomfort can be caused by interfering on the gastric function such as increasing gastric secretion, dyspepsia and stomach pain, the effect of Ya-hom on gastric secretion is investigated to clarify its action on decreasing stomach discomfort.
2. Materials and methods 2.1. Chemicals Urethane was obtained from Merck. Histamine, carbachol, hemoglobin, pepsin, bovine serum albumin,
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.06.003
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chondroitin sulfate and Alcian blue 8 GX were purchased from Sigma Chemical Co., St Louis, USA. Sodium hydroxide was purchased from Carlo Erba, Milano, Italy. Other reagents were of analytical grade. 2.2. Ya-hom Ya-hom was obtained from Five Pagodas Pharmacy Co. Ltd., Thailand. One hundred grams of Ya-hom contains Agastache rugosa (Fisch. et Mey) O. Kuntze (whole plant, Labiatae) 7.1 g, Acorus gramineus Soland (rhizomes, Araceae) 0.5 g, Lysimachia foenum-graecum Hance (whole plant, Primulaceae) 3.3 g, Citrus nobilis Lour. (outer yellow rind of the ripe fruit, Rutaceae) 7.1 g, Magnolia officinalis Rehd. et Wils. (bark of stem, Magnoliaceae) 11.8 g, Cinnamomum cassia Presl (Chinese cinnamon, bark, Lauraceae) 7.1 g, C L. (Japanese mint, whole plant, Lamiaceae) 0.5 g, Asarum sieboldii Miq. (whole plant, Aristolochiaceae) 2.3 g, Ligusticum wallichii Franch. (rhizomes, Umbelliferae) 9.3 g, Glycyrrhiza glabra L. (licorice, rhizomes, Leguminosae) 4.8 g, Eugenia caryophyllata Thunb. (clove, flower-bud, Myrtaceae) 9.3 g, Saussuria lappa Clark (rhizomes, Compositae) 7.1 g, Aquilaria agallocha Roxb. (wood, Thymelaeaceae) 7.1 g, Atractylis ovata Thunb. (rhizomes, Compositae) 9.3 g, menthol 4.7 g, Borneo camphor 1.4 g, Angelica anomala Lallem (rhizomes, Umbelliferae) 3.5 g. 2.3. Lyophilized Ya-hom water extract Ya-hom powder was mixed with boiled distilled water in the ratio of 1 kg:10 l and continuously boiled for 15 min. Ya-hom extract was filtered through cotton muslin cloth. The filtrate was lyophilized and kept at −20 ◦ C. One gram of Ya-hom powder yielded 0.136 g of lyophilized product. The test solution was freshly prepared by dissolving the lyophilized product in distilled water. Lyophilized Ya-hom water extract was dissolved in methanol, sonicated for 20 min and then adjusted to the concentration of 0.01 g/ml with methanol. Sample was filtered through Sep-pack® C18 cartridge before applying to HPLC using LiChrosphere® 100RP-18, Merck (5 m) column and eluted with deionized water for 5 min and followed by linear gradient of 10–100% acetonitrile for 15 min. HPLC analysis of lyophilized Ya-hom water extract was showed in Fig. 1. 2.4. Animal Male Wistar rats weighing between 200 and 220 g were obtained from the National Laboratory Animal Center at Salaya, Mahidol University. Rats were housed in hanging cage in the animal room at Faculty of Pharmacy, Mahidol University, for at least 1 week prior to the experiment. The animals were fed with commercial rat diet (C.P. Mice Feed; SWT. Co. Ltd., Thailand) and tab water ad libitum. The rats were fasted for 24 h before the experiment. Each experimental group was performed in 10 rats.
Fig. 1. HPLC analysis of lyophilized Ya-hom. Lyophilized Ya-hom in methanol was applied to HPLC using LiChrosphere® 100RP-18, Merck (5 m) column and eluted with deionized water and linear gradient of 10–100% acetonitrile.
2.5. Animal preparation Gastric fistula were performed as previously described by Limlomwongse et al. (1976). The fasted rat was anesthetized with urethane at the dose of 1.2 g/kg body weight intraperitoneally. The esophagus was ligated and the trachea was cannulated by using polyethylene tube No. 206. After opening the abdomen, the pyloduodenal junction was carefully ligated and the stomach was perforated and inserted the polyethylene tube No. 206 gastric fistula. The sample was collected through the gastric fistula. 2.6. Gastric sample collection The sample collection was taken in 1-h interval for 5 h and expressed as h1, h2, h3, h4 and h5. The first sample was collected and referred as the basal secretion. Then histamine (10 mg/kg, intramuscular injection) or carbachol (20 g/kg, intravenous injection) and Ya-hom extract (0.5–4 g/kg diluted to 1 ml/animal, intraduodenal injection) or water (1 ml/animal, intraduodenal injection) were administered. The gastric samples were collected at 1-h interval for 4 more hours (samples h2, h3, h4 and h5). At the end of the experiment, the stomach was removed and opened down the lesser curvature. The opened stomach was rinsed with normal saline pH 7.4 and dried with filter paper. Then the glandular portion was excised and determined the visible mucus. 2.7. Gastric sample analysis The gastric sample was weighed and centrifuged by Universal Centrifuge 16A, Hettich at 5000 rpm for 5 min to precipitate solid residue. The tube with residue was weighed again. The clear supernatant was separated and kept in ice bath. The supernatant was used for determining the gastric acid, pepsin activity, soluble mucus and protein.
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2.7.1. Gastric volume determination The difference between the weights of tube containing gastric sample and tube with residue represented the collected gastric volume by assuming that the specific gravity of gastric sample was 1.00. 2.7.2. Gastric acid-secretory rate determination The collected gastric acid was examined by titration of gastric sample with 0.01N sodium hydroxide to the end point at pH 5.0. The gastric acid-secretory rate was calculated from the collected gastric acid dividing by the weight of stomach glandular part and expressed as equiv. HCl/g stomach/h. 2.7.3. Gastric pepsin-secretory rate determination Pepsin secretion was determined by measuring the proteolytic activity of pepsin in gastric sample by using hemoglobin as a substrate and pepsin (480 units/mg solid) as a standard. The activity was determined according to the method described by Miller et al. (1975). The pepsin activity was expressed as units/g stomach/h. 2.7.4. Gastric protein-secretory rate determination The amount of protein in gastric sample was measured by colorimetric method (Lowry et al., 1951) using bovine serum albumin as a standard. The protein-secretory rate was determined from the amount of protein in collected gastric sample and expressed as mg/g stomach/h. 2.7.5. Gastric soluble mucus-secretory rate determination Gastric soluble mucus was determined according to the method described by Whiteman (1973) and by using chondroitin sulfate as a standard. The gastric soluble mucus-secretory rate was calculated from the amount of soluble mucus and express as g chondroitin sulfate/g stomach/h. 2.7.6. Gastric visible mucus determination Visible mucus on the gastric mucosal surface was determined according to the modification of the method previously described by Corne et al. (1974). The Alcian blue 8 GX (Sigma chemical Co., St Louis, USA) dissolved in MgCl2 was used as a standard. The amount of gastric visible mucus was expressed as mg Alcian blue (AB)/g stomach. 2.8. Gastric mucosal blood flow Animal preparation was similar to that of gastric sample collection. Gastric mucosal blood flow was measured by Laser Doppler Flowmeter. The probe was inserted through the fistula until the distance between the probe and the mucosa was about 2 mm. The mucosal blood flow was expressed as perfusion unit (PU). After basal blood flow measurement, histamine (10 mg/kg, intramuscular injection) and Ya-hom extract (4 g/kg, intraduodenal injection) or water (1 ml, intraduodenal injection) were administered. The gastric blood flow was detected in 15 min interval for 2 h and then in 30 min interval for 2 more hours.
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2.9. Statistical analysis One-way analysis of variance (ANOVA) was used to compare the values for each of experimental groups. Tukey’s honestly significant difference (HSD) test was used to differentiate the difference between two experimental groups. Paired sample t-test was to compare the secretory rates at h2, h3 h4 and h5 with the basal secretion (h1) in each experimental group. The P-value of less than 0.05 (P < 0.05) was considered to statistical significant difference.
3. Results 3.1. Effects of Ya-hom on histamine-induced gastric secretion The gastric acid-secretory rate profile pattern of histamine control group was increased after histamine injection and highest at h2 and then gradually decreased to the basal level at h5 (Table 1). The gastric acid-secretory rates at h2, h3 and h4 were significantly higher than its basal level. All the histamine + Ya-hom treated groups had similar gastric acid-secretory rate profile patterns as control group. However, they were lower than the control group at the same time point. Ya-hom inhibited histamine-stimulated gastric acid secretion in a dose-dependent manner with a maximum inhibition at h2 (Table 1). In histamine control group, the gastric pepsin, protein and soluble mucus-secretory rate profile patterns were similar to the histamine-induced gastric acid secretion except that the pepsin- and soluble mucus-secretory rates at h5 were declined to the level which significantly lower than its basal level as shown in Tables 2–4, respectively. All the histamine + Ya-hom-treated groups showed gastric pepsin- (Table 2), protein- (Table 3) and soluble mucus (Table 4)-secretory rate patterns similar to the histamine control group but the secretory rates were lower than the control in a dose-dependent manner. Ya-hom also inhibited the gastric pepsin, protein and soluble mucus secretions. All the Ya-hom-treated rats had significantly lower pepsin- and protein-secretory rates than histamine-induced rats at the same time point from h2 to h5 (Tables 2 and 3). However, Ya-hom significantly decreased soluble mucus-secretory rates only from h2 to h4 (Table 4). In contrast to other gastric secretions, the visible mucus content on the gastric mucosal surface of all histamine + Ya-hom-treated groups were significantly higher than that of the histamine control group (Fig. 2). 3.2. Effects of Ya-hom on carbachol-induced gastric secretion In carbachol-induced rats, the gastric acid-secretory rate was significantly increased to the peak at h2 and then declined to the value which was still significantly higher than
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Table 1 Dose-dependent effects of Ya-hom (YH) on histamine- and carbachol-induced gastric acid-secretory rate (equiv./g stomach/h) at h1, h2, h3, h4 and h5 Treatment
Gastric acid-secretory rate at time (h) 1
2
3
4
5
Control
Histamine Carbachol
1.4 ± 0.1 1.6 ± 0.1
193.1 ± 4.9∗∗ 76.4 ± 5.9∗∗
95.6 ± 4.5∗∗ 43.3 ± 2.5∗∗
21.9 ± 2.0∗∗ 22.2 ± 2.2∗∗
2.8 ± 1.0 11.3 ± 1.4∗∗
0.5 g/kg YH
Histamine Carbachol
1.3 ± 0.1 1.5 ± 0.1
182.4 ± 2.3∗∗ 74.2 ± 1.5∗∗
71.3 ± 1.8∗∗∗,∗∗ 41.2 ± 2.1∗∗
21.3 ± 0.4∗∗ 22.2 ± 1.8∗∗
1.5 ± 0.1 9.4 ± 1.1∗∗
1 g/kg YH
Histamine Carbachol
1.3 ± 0.1 1.4 ± 0.0
138.5 ± 4.4a,∗∗∗,∗∗ 61.4 ± 2.4a,∗∗∗,∗∗
82.1 ± 6.6∗∗ 33.9 ± 1.3a,∗∗∗,∗∗
21.0 ± 3.0∗∗ 23.6 ± 0.9∗∗
2.2 ± 0.3∗ 8.9 ± 0.5∗∗
2 g/kg YH
Histamine Carbachol
1.2 ± 0.1 1.4 ± 0.0
116.8 ± 2.9a,b,∗∗∗,∗∗ 42.0 ± 1.0a,b,∗∗∗,∗∗
64.2 ± 4.4b,∗∗∗,∗∗ 29.6 ± 0.3a,∗∗∗,∗∗
11.5 ± 1.3a,b,∗∗∗,∗∗ 19.9 ± 0.5∗∗
1.6 ± 0.2∗ 7.0 ± 0.2∗∗
4 g/kg YH
Histamine Carbachol
1.2 ± 0.1 1.4 ± 0.0
105.3 ± 1.8a,b,∗∗∗,∗∗ 35.3 ± 1.4a,b,∗∗∗,∗∗
58.3 ± 2.6b∗∗∗,∗∗ 17.0 ± 1.0a,b,c,∗∗∗,∗∗
9.1 ± 1.0a,b,∗∗∗,∗∗ 8.8 ± 0.8a,b,c,∗∗∗,∗∗
1.7 ± 0.2∗∗ 3.5 ± 0.2a,b,c,∗∗∗,∗∗
All values were expressed as mean ± S.E.M. ∗ P < 0.05: significant difference from their basal secretion within group. ∗∗ P < 0.01: significant difference from their basal secretion within group. Comparison at the same time point: ∗∗∗ P < 0.05: significant difference from their control groups. a P < 0.05: significantly lower than 0.5 g/kg Ya-hom-treated group (within group). b P < 0.05: significantly lower than 1 g/kg Ya-hom-treated group (within group). c P < 0.05: significantly lower than 2 g/kg Ya-hom-treated group (within group).
the basal level (Table 1). The gastric acid-secretory rates of all carbachol + Ya-hom-treated groups also showed significantly higher gastric acid-secretory rates than their basal rates at h2 to h5 but lower than that of control at the same time point. Ya-hom decreased gastric acid secretion in a dose-dependent manner with a maximum inhibition at h2 (Table 1). The profile pattern of gastric pepsin-secretory rate in the carbachol control group was similar to that of histamine-induced pepsin secretion, the gastric-secretory rate was highest at h2 and then gradually decreased to the
significantly lower rate than its basal rate at h5 (Table 2). The secretory patterns of all carbachol + Ya-hom-treated groups were similar to that of carbachol control group (Table 2). Comparing the gastric pepsin secretion of the carbachol + Ya-hom-treated groups with the carbachol control group at the same time point showed that Ya-hom decreased gastric pepsin secretion in a dose-dependent manner (Table 2). The gastric protein- and mucus-secretory rates of carbachol-induced rats were similar to carbachol-induced gastric acid secretion. The protein- and mucus-secretory rates increased to the peak at h2 and declined but still
Table 2 Dose-dependent effects of Ya-hom (YH) on histamine- and carbachol-induced gastric pepsin-secretory rate (units/g stomach/h) at h1, h2, h3, h4 and h5 Treatment
Gastric pepsin-secretory rate at time (h) 1
2
3 44.6∗∗
4 36.9∗∗
5
Control
Histamine Carbachol
264.5 ± 10.8 230.5 ± 6.6
1402.7 ± 1260.3 ± 50.0∗∗
679.9 ± 639.7 ± 18.7∗∗
326.4 ± 329.4 ± 6.0∗∗
173.1 ± 7.5∗∗ 197.7 ± 4.1∗∗
0.5 g/kg YH
Histamine Carbachol
249.3 ± 8.0 239.4 ± 5.4
1143.7 ± 20.1∗∗∗,∗∗ 1214.4 ± 42.8∗∗
486.2 ± 35.6∗∗∗,∗∗ 618.8 ± 17.8∗∗
230.2 ± 13.1∗∗∗ 296.8 ± 11.9∗∗∗,∗∗
139.1 ± 7.4∗∗∗,∗∗ 192.4 ± 6.2∗∗
1 g/kg YH
Histamine Carbachol
250.3 ± 10.9 233.6 ± 5.7
824.7 ± 33.3a,∗∗∗,∗∗ 956.0 ± 38.7a,∗∗∗,∗∗
411.1 ± 30.0∗∗∗,∗∗ 575.6 ± 11.3∗∗
229.2 ± 16.3∗∗∗ 256.6 ± 7.7a,∗∗∗,∗∗
135.0 ± 8.2∗∗∗,∗∗ 179.1 ± 4.9∗∗
2 g/kg YH
Histamine Carbachol
253.2 ± 8.6 225.0 ± 5.8
725.9 ± 30.5a,∗∗∗,∗∗ 764.5 ± 20.4a,b,∗∗∗,∗∗
342.9 ± 20.9a,∗∗∗,∗∗ 494.1 ± 26.8a,b,∗∗∗,∗∗
158.1 ± 5.7a,b,∗∗∗,∗∗ 208.27 ± 4.9a,b,∗∗∗,∗∗
104.4 ± 7.3a,b,∗∗∗,∗∗ 159.9 ± 4.9a,b,∗∗∗,∗∗
4 g/kg YH
Histamine Carbachol
261.3 ± 11.5 240.5 ± 7.2
580.2 ± 19.3a,b,c,∗∗∗,∗∗ 655.9 ± 30.1a,b,∗∗∗,∗∗
243.3 ± 10.8a,b,∗∗∗ 433.9 ± 15.9a,b,∗∗∗,∗∗
131.8 ± 6.3a,b,∗∗∗,∗∗ 185.9 ± 4.1a,b,∗∗∗,∗∗
92.1 ± 3.0a,b,∗∗∗,∗∗ 152.5 ± 3.2a,b,∗∗∗,∗∗
All values were expressed as mean ± S.E.M. ∗∗ P < 0.01: significant difference from their basal secretion within group. Comparison at the same time point: ∗∗∗ P < 0.05: Significant difference from their control group. a P < 0.05: significantly lower than 0.5 g/kg Ya-hom-treated group (within group). b P < 0.05: significantly lower than 1 g/kg Ya-hom-treated group (within group). c P < 0.05: significantly lower than 2 g/kg Ya-hom-treated group (within group).
15.8∗∗
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Table 3 Dose-dependent effects of Ya-hom (YH) on histamine- and carbachol-induced gastric protein-secretory rate (mg/g stomach/h) at h1, h2, h3, h4 and h5 Treatment
Gastric protein-secretory rate at time (h) 1
2
3
4
5
Control
Histamine Carbachol
1.03 ± 0.05 1.01 ± 0.02
5.44 ± 0.17∗∗ 5.14 ± 0.20∗∗
3.06 ± 0.08∗∗ 3.47 ± 0.16∗∗
1.54 ± 0.06∗∗ 2.28 ± 0.13∗∗
0.98 ± 0.03 1.41 ± 0.10∗∗
0.5 g/kg YH
Histamine Carbachol
1.00 ± 0.03 1.00 ± 0.01
3.67 ± 0.17∗∗∗,∗∗ 5.10 ± 0.08∗∗
1.53 ± 0.06∗∗∗,∗∗ 3.36 ± 0.16∗∗
0.89 ± 0.04∗∗∗,∗∗ 2.18 ± 0.10∗∗
0.75 ± 0.02∗∗∗,∗∗ 1.39 ± 0.06∗∗
1 g/kg YH
Histamine Carbachol
1.04 ± 0.03 1.03 ± 0.01
2.88 ± 0.11a,∗∗∗,∗∗ 4.40 ± 0.13a,∗∗∗,∗∗
1.31 ± 0.04∗∗∗,∗∗ 2.75 ± 0.05a,∗∗∗,∗∗
0.68 ± 0.01a,∗∗∗,∗∗ 1.94 ± 0.06∗∗∗,∗∗
0.55 ± 0.02a,∗∗∗,∗∗ 1.28 ± 0.04∗∗
2 g/kg YH
Histamine Carbachol
1.04 ± 0.02 1.02 ± 0.01
2.13 ± 0.06a,b,∗∗∗,∗∗ 3.65 ± 0.09a,b,∗∗∗,∗∗
1.19 ± 0.05a,∗∗∗,∗ 2.19 ± 0.04a,b,∗∗∗,∗∗
0.52 ± 0.01a,b,∗∗∗,∗∗ 1.87 ± 0.03∗∗∗,∗∗
0.34 ± 0.02a,b,∗∗∗,∗∗ 1.15 ± 0.02a,∗∗∗,∗∗
4 g/kg YH
Histamine Carbachol
1.05 ± 0.02 1.04 ± 0.01
2.12 ± 0.06a,b,∗∗∗,∗∗ 3.11 ± 0.04a,b,c,∗∗∗,∗∗
1.08 ± 0.06a,∗∗∗ 2.11 ± 0.03a,b,∗∗∗,∗∗
0.49 ± 0.01a,b,∗∗∗,∗∗ 1.70 ± 0.02a,∗∗∗,∗∗
0.36 ± 0.02a,b,∗∗∗,∗∗ 1.10 ± 0.02a,∗∗∗,∗∗
All values were expressed as mean ± S.E.M. ∗ P < 0.05: significant difference from their basal secretion within group. ∗∗ P < 0.01: significant difference from their basal secretion within group. Comparison at the same time point: ∗∗∗ P < 0.05: significant difference from their control groups. a P < 0.05: significantly lower than 0.5 g/kg Ya-hom-treated group (within group). b P < 0.05: significantly lower than 1 g/kg Ya-hom-treated group (within group). c P < 0.05: significantly lower than 2 g/kg Ya-hom-treated group (within group).
significantly higher than its basal level until h5 (Tables 3 and 4). The protein-secretory rates of all carbachol + Ya-hom-treated rats showed the similar pattern as carbachol control group but lower than carbachol control group at the same time point. Ya-hom at the dose of 1, 2 and 4 g/kg significantly inhibited gastric protein- and soluble mucus-secretory rates (Tables 3 and 4). Similar to the effect of Ya-hom on histamine-induced visible mucus secretion, all of carbachol + Ya-hom-treated rats had more gastric visible mucus than the carbachol con-
trol group (Fig. 2). However, only Ya-hom at doses of 1, 2 and 4 g/kg significantly increased gastric visible mucus in carbachol-treated rats. 3.3. Effects of Ya-hom on gastric mucosal blood flow Ya-hom at a dose of 4 g/kg that had the highest gastric-secretory inhibition in this present experiment was used for investigation the effect of Ya-hom on histamine-induced gastric mucosal blood flow. As shown
Table 4 Dose-dependent effects of Ya-hom (YH) on histamine- and carbachol-induced gastric soluble mucus-secretory rate (g chondroitin sulfate/g stomach/h) at h1, h2, h3, h4 and h5 Treatment
Gastric soluble mucus-secretory rate at time (h) 1
2
3 3.22∗∗
4 2.40∗∗
5
Control
Histamine Carbachol
4.53 ± 0.15 11.63 ± 0.51
97.19 ± 105.79 ± 2.30∗∗
56.71 ± 59.11 ± 2.39∗∗
27.59 ± 31.88 ± 1.32∗∗
3.99 ± 0.21∗∗ 16.91 ± 0.77∗∗
0.5 g/kg YH
Histamine Carbachol
4.47 ± 0.09 12.02 ± 0.46
79.32 ± 1.74∗∗∗,∗∗ 104.68 ± 2.70∗∗
47.49 ± 1.05∗∗∗,∗∗ 58.68 ± 2.03∗∗
22.42 ± 1.46∗∗∗,∗∗ 31.75 ± 1.61∗∗
3.95 ± 0.29 16.99 ± 0.77∗∗
1 g/kg YH
Histamine Carbachol
4.47 ± 0.08 11.54 ± 0.53
64.94 ± 2.61a,∗∗∗,∗∗ 95.26 ± 2.85∗∗∗,∗∗
42.23 ± 0.92∗∗∗,∗∗ 46.79 ± 1.34a,∗∗∗,∗∗
18.07 ± 0.30a,∗∗∗,∗∗ 26.62 ± 1.08∗∗∗,∗∗
3.76 ± 0.25∗ 13.94 ± 0.73a,∗∗∗,∗∗
2 g/kg YH
Histamine Carbachol
4.33 ± 0.13 11.45 ± 0.37
54.48 ± 1.40a,b,∗∗∗,∗∗ 77.66 ± 2.95a,b,∗∗∗,∗∗
31.02 ± 1.66a,b,∗∗∗,∗∗ 41.32 ± 1.86a,∗∗∗,∗∗
14.08 ± 0.20a,b,∗∗∗,∗∗ 25.11 ± 1.43a,∗∗∗,∗∗
3.73 ± 0.22∗ 11.71 ± 0.60a,∗∗∗
4 g/kg YH
Histamine Carbachol
4.47 ± 0.11 11.57 ± 0.44
46.00 ± 0.74a,b,∗∗∗,∗∗ 71.61 ± 1.71a,b,∗∗∗,∗∗
26.54 ± 0.95a,b,∗∗∗,∗∗ 39.70 ± 1.46a,∗∗∗,∗∗
13.52 ± 0.40a,b,∗∗∗,∗∗ 20.56 ± 0.80a,b,∗∗∗,∗∗
3.70 ± 0.05∗∗ 10.73 ± 0.47a,b,∗∗∗
All values were expressed as mean ± S.E.M. ∗ P < 0.05: significant difference from their basal secretion within group. ∗∗ P < 0.01: significant difference from their basal secretion within group. Comparison at the same time point: ∗∗∗ P < 0.05: significant difference from their control groups. a P < 0.05: significantly lower than 0.5 g/kg Ya-hom-treated group (within group). b P < 0.05: significantly lower than 1 g/kg Ya-hom-treated group (within group).
1.29∗∗
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4. Discussion
Fig. 2. Effects of Ya-hom on histamine- (open circle) and carbachol-induced (closed circle) gastric visible mucus secretion (mg AB/g stomach). All values were expressed as mean ± S.E.M. ∗ P < 0.05: significant difference from their control group. a P < 0.05: significantly higher than 0.5 g/kg Ya-hom-treated group (within group). b P < 0.05: significantly higher than 1 g/kg Ya-hom-treated group (within group). c P < 0.05: significantly higher than 2 g/kg Ya-hom-treated group (within group).
Fig. 3. Effects of Ya-hom 4 g/kg (YH4) on histamine-induced gastric mucosal blood flow (perfusion unit). ∗ P < 0.05, ∗∗ P < 0.01: significant difference from the base line within group. a P < 0.05: significant difference from their control group at the same time point.
in Fig. 3, the gastric mucosal blood flow of the control gastric mucosa was significantly increased after histamine injection to the peak at 45 min and then declined to the basal level at 150 min. Ya-hom-treated rats had no increase in gastric mucosal blood flow after histamine administration except at 150 min and had significantly lower gastric mucosal blood flow than its base line at 210 and 240 min. The gastric mucosal blood flow of Ya-hom-treated rat was significantly lower than those of control rat at 30– 90 min.
The present study attempted to investigate the effect of Ya-hom on secretagogue-induced gastric secretion in rats to verify its use for stomach discomfort. The gastric-secretory profiles in histamine- and carbachol-induced rats were the same. The dose of carbachol was limited by its stimulatory effect on salivary and mucus secretion in respiratory tract. Most of the effect of histamine was higher but shorter duration of action than carbachol. The efficiency of histamine on stimulation of gastric acid and visible mucus was higher than carbachol. However, the stimulatory efficiency on pepsin and soluble mucus secretions of histamine and carbachol were similar. This indicated that histamine and carbachol had different effects on each secretory cell types. In addition, carbachol is known to act both directly on the secretory cells and indirectly through stimulation on histamine-released ECL cells (Hirschowitz et al., 1995). Ya-hom inhibited histamine- and carbachol-induced gastric acid, pepsin, protein and soluble mucus secretion in a dose-dependent manner. Our preliminary experiment found that Ya-hom at a dose of higher than 4 g/kg did not show any significant higher inhibition, so the dose of 4 g/kg was the highest dose using in the present experiment. The maximum inhibition of Ya-hom was at h2, which was the period of the highest response of histamine or carbachol. The maximum inhibition of Ya-hom on histamine-induced gastric acid, pepsin, protein and soluble mucus secretions were 45.5, 58.6, 61.0 and 52.7% whereas its maximum inhibition on carbachol-induced secretions were 53.8, 48.0, 39.5 and 32.3%, respectively. In contrast to the effects on acid, pepsin and soluble mucus secretion, Ya-hom potentiated both histamine- and carbachol-induced gastric visible mucus secretion with the maximum stimulation of 77.4 and 58.2%. Except for gastric acid secretion, Ya-hom had higher attenuation and potentiation on histamine-induced gastric secretion than carbachol. This indicated that Ya-hom’s action on gastric secretion mainly involved histamine pathway. Even though the effect of Ya-hom on histamine and acetylcholine have not been reported, there were some studies on its ingredients: licorice (Fumio, 1973; Paris and Guillot, 1955), Saussuria lappa Clarke (Bose et al., 1961; Gupta and Ghatak, 1967), clove (Apisariyakul, 1984) and Acorus gramineus Soland (Zhang et al., 1981) showed that each plant extract antagonized the effects on histamine and acetylcholine-stimulated smooth muscle contraction. In addition, clove has shown to stimulate gastric mucus secretion (Enrique, 1951). So, the effect of Ya-hom may be due to these ingredients. In addition to Ya-hom’s inhibitory effect on gastric secretion, Ya-hom at a dose of 4 g/kg completely inhibited histamine-induced gastric mucosal blood flow from 30 to 90 min. The blood flow in each area of the gastrointestinal tract as well as in each layer of the gut is directly related
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to the level of local activity. In this study, Ya-hom inhibited histamine-induced gastric mucosal blood flow from 30 to 90 min which related to its maximum inhibitory effect on gastric acid, pepsin and soluble mucus secretion at h2 (1 h after secretagogue administration). Since histamine itself can cause a vasodilation via H1 receptor whereas it stimulates gastric acid secretion via H2 receptor, it is possible that effect of Ya-hom on gastric mucosal blood flow may or may not directly related to the gastric secretion inhibitory effect. However, the decrease in blood flow caused by Ya-hom potentiates its inhibitory effect on gastric secretion. Inhibition of gastric acid secretion causes by direct action on secretory cell such as M3 or H2 receptor antagonists or indirect action through the releasing of the inhibitory hormone, somatostatin or the inhibition of histamine-released ECL cells (Hirschowitz et al., 1995). Ya-hom composes of several ingredients, so it is possible that one or several compounds in Ya-hom cause direct and/or indirect effects on gastric-secretory functions. Furthermore, the stomach discomfort may cause by oversecretion, motility or gas production. It is shown here that Ya-hom inhibits gastric acid secretion about 50% which is enough to attenuate the discomfort feeling if it is caused by overgastric acid secretion. As known that, the integrity of the gastric mucosa and gastric ulcer are regulated by the balance between factors that promote and protect the ulcer development (Lloyd and Soll, 1994). The ulcer can also be the cause of stomach pain. Ya-hom has been shown here to decrease in aggressive factors (acid and pepsin) and increase in protective factor secretion (visible mucus). This effect of Ya-hom also causes a pain relief in case of peptic ulcer. It is possible that Ya-hom has more than one effect for this treatment. This gastric-secretory inhibition is a part of Ya-hom’s action on this treatment. The exact mechanism of Ya-hom’s action on the gastric cells and other effects of Ya-hom on the stomach should be further investigated to clarify its stomach discomfort treatment efficiency. In human, Ya-hom is taken by mixing 3 g of Ya-hom powder with warm water and drinking the whole preparation as needed. The dose of Ya-hom used in this experiment (4 g/kg) is about 80 times of human dose if it is assumed that a man weighs 60 kg. However, it is about 1 g per rat (weighing 250 g) when compares with 3 g per person in human. Since Ya-hom in the experiment uses the lyophilized product of boiling Ya-hom solution for 15 min, the extract may not contain total amount of each active compound form the powder. Boiling Ya-hom longer than 15 min is avoided to prevent the loss the volatile oil. This dose of Ya-hom extract should be able to represent the action of Ya-hom in human. The dose of Ya-hom used in this experiment is in the safety amount, since oral administration of the water extract of this Ya-hom recipe has been shown to possess LD50 more than 5 g/kg. It did not show any adverse effect when given to the rats at a dose of 2 g/kg per day for 5 weeks (Thongpraditchote et al., 1999).
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5. Conclusion Ya-hom inhibited the effect of histamine- and carbacholstimulated gastric acid, pepsin and soluble mucus secretion but potentiated gastric visible mucus secretion in a dose-dependent manner. Ya-hom also inhibited histamineinduced gastric mucosal blood flow that related to its inhibitory effect on gastric secretion. This study clarifies the claimed efficacy of Ya-hom ingestion for stomach discomfort because it not only decreases in aggressive factors (acid and pepsin) but also increases in protective factor secretion (visible mucus).
Acknowledgements This work was partly supported by the M.S. research grant, Faculty of Pharmacy, Mahidol University.
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