Journal of Ethnopharmacology 158 (2014) 358–363
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Research Paper
The protective activity of Conyza blinii saponin against acute gastric ulcer induced by ethanol Long Ma a,b,c,n, Jiangguang Liu d a Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, School of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China b Tianjin Key Laboratory of Industry Microbiology, School of Biotechnology, Tianjin University of Science & Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, China c Centre for Biomolecular Sciences, University of St Andrews, Room 4.11, North Haugh, St Andrews KY16 9ST, United Kingdom d School of Pharmaceutical Science and Technology, Tianjin University, No 92, Weijing Road, Nankai District, Tianjin 300072, China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 June 2014 Received in revised form 9 October 2014 Accepted 13 October 2014 Available online 4 November 2014
Ethnopharmacological relevance: Conyza blinii H.Lév., is a type of natural plant. Its dried overground section is used to treat infections and inflammations in traditional Chinese medicine. Triterpenoidal saponins have a wide range of bioactivities, for instance, anti-cancer, anti-virus and anti-anaphylaxis. Conyza blinii saponin (CBS), mainly composed of triterpenoidal saponins, is the total saponin of Conyza blinii H.Lév. It has been reported that CBS also has gastric mucous membrane protection activity. This study aims to test CBS's protective activity of gastric's mucous membrane against ethanol. This investigation may lead to the development of novel drug from natural products as anti-ulcer agent, or as gastric mucous protective against chemical damage. Materials and methods: CBS (Conyza blinii saponin) is the total saponin of Conyza blinii H.Lév., which was obtained as described previously. We tested the protective activity of CBS against ethanol-induced ulcer. Thirty six rats were grouped randomly as ‘NORMAL’, ‘CONTROL’, ‘MODEL’, ‘LOW DOSE’, ‘MEDIUM DOSE’ and ‘HIGH DOSE’. The ‘NORMAL’ group were rats with no pathological model established within it. The ‘CONTROL’ group was administrated with colloidal bismuth subcitrate, while ‘MODEL’ group was not given any active agents apart from absolute ethanol in order to obtain gastric ulcer model. The three ‘DOSE’ groups were treated with different concentrations of CBS (5, 10, 20 mg/mL) before administration followed by absolute ethanol. All rats were sacrificed after the experiment to acquire the gastric tissue. The ulcer index (UI), malondialdehyde (MDA) and superoxide dismutase (SOD) were measured to monitor the activity of CBS. Besides, the rat gastric tissue was made to paraffin section and stained using the Hematoxylin–Eosin (HE) method. The histopathology examination was carried out to examine CBS efficacy in terms of gastric mucous protection. Results: We found that CBS had a profound protection activity against acute gastric ulcer induced by ethanol and this activity displayed a concentration-dependent manner. The efficacy of 10 and 20 mg/mL CBS was comparable with colloidal bismuth subcitrate (Po0.05). All three level of CBS tested were able to significantly reduce UI, MDA and enhance SOD level (Po0.05). Conclusions: It was deduced that the mechanism for such activity would be anti-lipid peroxidation, facilitating free radicals clearance. In addition , histopathology examination of the gastric mucous membrane supported the same conclusion, that CBS can efficiently suppress the inflammatory reactions, bleeding and protect the gastric mucosa. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Conyza blinii H.Lév. Total saponin Gastric ulcer Superoxide dismutase (SOD) Malondialdehyde (MDA)
1. Introduction
n Corresponding author at: Tianjin Key Laboratory of Industry Microbiology, School of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, China. Tel.: þ 86 22 60602948; fax: þ 86 22 60602298. E-mail addresses: [email protected], [email protected] (L. Ma).
http://dx.doi.org/10.1016/j.jep.2014.10.052 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
Conyza blinii H.Lév., is a native herbaceous plant in Compositae. It is spread in Sichuan and Yunnan provinces of China and is commonly called Jin Long Dan Cao. Its dried overground section is valuable in folk medicine for the treatment of chronic bronchitis, gastroenteritis and some other inflammatory diseases (Materia Medica, 1998; Pharmacopoeia of the People's Republic of China, 1977, 2010). Chemical profiles of this species have revealed many
L. Ma, J. Liu / Journal of Ethnopharmacology 158 (2014) 358–363
natural products including diterpenoids, flavonoids, triterpenoids and saponins (Xu et al., 1999; Su et al., 2003). CBS has 17 known components, whose chemical profiles were all listed in Table SI-1 and Fig. SI-1 (Su et al., 2000, 2001a, 2001b, 2003). It has been reported that the CBS has significant protective activity against the gastric mucosal damage. The possible mechanisms account for such activity is to reduce the secretion of gastric collection and gastric acid, as well as to prevent free radicals formation (Su et al., 2001a, 2001b, 2007a, 2007b). Recently, it has shown that CBS induces the apoptosis of Hela and SPC-A1 cells, indicating an antineoplastic activity (Liu et al., 2011). Excessive consumption of ethanol-containing beverage exerts serious health problems in human. The direct damage of ethanol can lead to gastric lesion. Ethanol is destructive on gastric mucosa, which can be evidenced by gastric mucosa erosion and bleeding. This is caused by blood micro-circulation disorder in gastric wall, tissue necrosis, reduction in gastric mucosal blood flow and mucus secretion (Masuda et al., 1992; Morales et al., 1992; Ohta et al., 1997; Knoll et al., 1998; Kawano and Tsuji, 2000; Sibilia et al., 2003). This reinforces the need for developing new protective agents. Although CBS was originally shown to possess multiple anti-inflammatory and gastric mucosal protection activities, its effects on acute gastric ulcer has not been yet explored. The aim of the present study is to evaluate gastric mucosa protective properties and established the possible mechanisms.
2. Experimental and methods 2.1. Animals Male and female Sprague & Dawley rats, 180–200 g, supplied by the Tianjin Medical University, PRC, were used throughout this study. All animal experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People's Republic of China. The animals were fed with a certified diet with free access to water, and placed in cages under a 12 h light/dark cycles for 4 days before experiments. The temperature was maintained at 2471 1C and the humidity at 70–75% in a controlled room. 2.2. The preparation of CBS from dried Conyza blinii H.Lév. Conyza blinii H.Lév. the accepted name of a species in the genus Conyza, family Compositae, which has been checked against websites www.theplantlist.org (The Plant List), and www.ipni.org (The International Plant Names Index). A voucher specimen of Conyza blinii H.Lév. was identified by Dr. Yanfang Su, and deposited in the herbarium of the School of Pharmaceutical and Technology, Tianjin University. The method for CBS preparation was based on the protocols published before (Su et al., 2007a, 2007b). Simply, (1) the ground dried Conyza blinii H.Lév. was refluxed in 90% ethanol twice (each time for 2 h). The filter residue was then refluxed in 55–60% ethanol twice and filtered. The filtrates were combined and followed by evaporation under reduced pressure to completely remove the ethanol. (2) The distilled water was added to dilute the concentrated solution to make it 10–20 g per 100 mL. Ethyl acetate was added to fractionate the non-saponin components using separatory funnel. The ethyl acetate in the aqueous layer was totally evaporated under reduced pressure. Butanol was added to the resultant aqueous fraction using separatory funnel for three times until completion. Combined butanol fraction was
SOD ðU=mg proteinÞ ¼
359
concentrated until dried to obtain crude saponin. (3) The crude saponin was dissolved in water and columned using D101 macroporus resin chromatography. Water, 30%, 70% and 95% ethanol were subsequently used for column elution. The 70% ethanol elution was collected and concentrated to acquire CBS. 2.3. The grouping and administration plan Thirty six Sprague & Dawley rats were divided into six groups randomly as follows: ‘NORMAL’, ‘MODEL’, ‘CONTRL’, ‘LOW DOSE’, ‘MEDIUM DOSE’ and ‘HIGH DOSE’. Each group had six rats. All reagents were given using intragastric administration. For ‘NORMAL’ group, the rats were given 1 mL 0.5% carboxymethylcellulose sodium (CMC-Na) for consecutive two days. Before the first administration, all six rats were fasted for 24 h. Two hours after the last administration, all six rats were administered with 1 mL physiological saline, waited for another one hour before sacrification. ‘MODEL’ and ‘CONTROL’ groups were given 1 mL absolute ethanol and 14.4 mg/kg colloidal bismuth pectin respectively. For the other three ‘DOSE’ groups (LOW DOSE, MEDIUM DOSE and HIGH DOSE) designed to test CBS activity; 5, 10, 20 mg/mL CBS (dissolved in 0.5% CMC-Na) were given instead of physiological saline otherwise the same as ‘NORMAL’ group. 2.4. The preparation of rat gastric tissue homogenate The rat gastric mucosa was removed by scraping to make 10% tissue homogenate in physiological saline using homogeniser on ice, followed by centrifugation at 2000 rpm for 10 min. The supernatant was collected for subsequent use. 2.5. The protein quantification in rat gastric tissue homogenate Coomassie brilliant blue method was used to determine the protein concentration in gastric tissue homogenate as described previously (Sedmak and Grossberg, 1977). 2.6. The calculation of ulcer index The gastric ulcer index (UI) was conducted as follow: stomachs were taken from the sacrificed rats immediately and rinsed by cold physiological saline to remove the remnant. Stomachs were incised along greater gastric curvature. The spot gastric lesion was counted 1 point; linear gastric lesion less than 1 mm was counted 1 point, adding upon 1 point when the length of the ulcer lesion increasing 1 mm. If the width 41 mm, then the points were multiplied by factor 2. 2.7. Measurement of SOD Xanthine-xanthine oxidase system, as shown previously (Peskin and Winterbourn, 2000), was used and modified for SOD measurement. Simply, 2% rat gastric tissue homogenate was made in physiological saline and different amounts of (from 10 to 50 mL) which were tested to work out the optimal suppression rate. The sample gave a suppression rate ranged 48–50%, whose amount was chosen as the optimal amount for the assay. The ‘measured’ tube and ‘control’ tube were both used in the assay. The ‘measured’ tubes were defined as the one which contained the samples subjected to quantification, while the ‘control’ tubes only contained distilled water otherwise were exactly the same as the ‘measured’ ones. The SOD concentration was calculated using the following equation:
2 ODcontrol ODmeasured =ODcontrol total reaction volume=measured sampling volume protein concentration in tissue
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L. Ma, J. Liu / Journal of Ethnopharmacology 158 (2014) 358–363
Fig. 2. Effect of CBS on SOD activity and MDA concentration of gastric mucosa in rats. ★★★P o0.001 vs. NORMAL; ★★P o 0.01 vs. NORMAL; ▲▲▲Po 0.001 vs. MODEL; ▲ Po 0.05 vs. MODEL; ◆P 4 0.05 vs. CONTROL.
2.9. Histopathology examination Fig. 1. Effect of CBS on ulcer index (UI) in rats. (A–D) show the gastric wall of rats from different groups, namely ‘HIGH DOSE’, ‘MODEL’, ‘CONTROL’ and ‘NORMAL’. Clearly, for ‘MODEL’ group, severe damage and lesion can be seen; however for ‘CONTROL’ group, this lesion is considerably alleviated. For ‘NORMAL’ group, a smooth and complete gastric wall can be seen as well as for ‘HIGH DOSE’ group. Fig. 1E shows the protective effect of CBS against gastric ulcer induced by ethanol in rats in terms of UI. There is a significant decrease in UI for all groups administrated with CBS, compared with ‘MODEL’ group. ★★★P o 0.001 vs. NORMAL; ▲▲P o 0.01 vs. MODEL; ▲▲▲Po 0.001 vs. MODEL; ◆P 4 40.05 vs. CONTROL.
2.8. Measurement of MDA MDA was measured using the thiobarbituric acid (TBA)-based method with modification, as previously published (Ohkawa et al., 1979). To be detailed, three types of samples were used, namely ‘standard’, ‘standard blank’ and ‘measured’. The ‘measured’ samples contained the rat gastric tissue homogenate for MDA quantification; ‘standard’ sample contained 10 nmol/mL standard MDA; while ‘standard blank’ contained none of the standard nor the rat gastric tissue homogenate. All samples were reacted with TBA in phosphate buffer (50 mM, pH 7.0) supplemented with absolute alcohol and hydrochloric acid where needed. All samples were incubated at 95 1C for 40 min followed by centrifugation at 4000 rpm for 10 min. The absorbance of supernatant was measured at 532 nm.
MDA ðnmol=mg protÞ ¼
ODmeasured ODcontrol blank =ODstandard ODstandard protein concentration in tissue
concentration of standard dilution f actor
blank
The rat gastric tissue was made to paraffin section and stained using the Hematoxylin–Eosin (HE) method. The histopathology examination was carried out using common optical microscope. 2.10. Statistical analysis GraphPad InStat software (version 3.05) was used for statistical analysis. One-way analysis of variance (one-way ANOVA) method was chosen. Bartlett statistic method was first used to test the homogeneity of variances, followed by comparison between each two individual groups using the Student–Newman–Keuls. α ¼0.05 was applied as level of significance test, which indicated that Pr 0.05 was considered to have significant statistical significance.
3. Results and discussion Tables SI-2–4 in supplementary information list all the experimental data discussed in this study. We established an animal model of acute gastric ulcer successfully, which was alike to ethanol-induced gastric ulcer in human. In the first place, results showed that all these three parameters UI, SOD and MDA for ‘MODEL’ group had significant statistical difference compared with ‘NORMAL’ group as shown in Fig. 1E and Fig. 2A and B. To be detailed, UI (112.72 for MODEL vs. 0 for NORMAL, P o0.001); SOD activity (46.41 for MODEL vs. 59.64 for NORMAL, Po 0.01), MDA concentration (2.57 for MODEL vs. 1.47 for NORMAL, Po 0.001). These imply that the gastric ulcer takes place and free radicals and lipid peroxidation reaction plays important roles in pathologic process for ethanol-induced ulcer as previously reported (Pandian et al., 2002). When comes to the comparison between three ‘DOSE’ groups and ‘MODEL’ group, there exist significant discrepancy.
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Fig. 3. Results and analysis of histological tests on gastric wall of rats. (A) Normal gastric wall of rats magnified by (10 4). It shows a complete gastric wall structure of normal rat, which resembles human's. A: epithelial layer and lamina propria mucosae; B: lamina muscularis mucosae; C: submucosa muscular layer; D: muscular layer. (B) Normal gastric mucosa magnified by (10 10). It shows submucous layer and muscular layer of rat. (C) Gastric mucosa of rats in ‘MODEL’ group magnified by (10 10). It can be seen that incomplete glandular organs, inflammatory reaction and the exudation of red cells are present in the surface of mucosa. A: exudation of red cells; B: the remains of mucosa after the disintegration of glandular organ; C: the disintegration of glandular organ cell. (D, E) Gastric mucosa of rats in ‘MODEL’ group magnified by (10 10) and (10 40) respectively. The red spots are substantial red blood cells, indicative of severe bleeding caused by the damage of ethanol. (F) Gastric mucosa of rats in ‘HIGH DOSE’ group magnified by (10 10). It can be seen that CBS has a profound protection of mucosa against ethanol-induced gastric lesion, as there is no obvious damage and erosion, neither observable bleeding. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
To be specific, it can be noticed that for UI (LOW DOSE 74.32 vs. MODEL 112.72, P o0.01; MEDIUM DOSE 43.85 vs. MODEL 112.72, P o0.001; HIGH DOSE 19.13 vs. MODEL 112.72, P o0.001). SOD activity (HIGH DOSE 55.61 vs. MODEL 46.41, Po 0.05). MDA concentration (LOW DOSE 1.79 vs. 2.57, P o0.001; MEDIUM DOSE 1.51 vs. MODEL 2.57, P o0.001; HIGH DOSE 1.14 vs. MODEL 2.57, P o0.001). These results suggest that order of severity of gastric ulcer for ‘DOSE’ groups is fairly different from ‘MODEL’ group. Even for the ‘LOW DOSE’ group, the UI and MDA had a remarkable decline in comparison with ‘MODEL’ group. In addition, the SOD’s level in ‘HIGH DOSE’ group evidently mounted up. All means CBS
has a striking preventive activity against ethanol-induced gastric ulcer. Last, we intended to compare the three parameters of the ‘DOSE’ groups with CONTROL group. UI (MEDIUM 43.85 vs. CONTROL 51.37, P4 0.05; HIGH DOSE 19.13 vs. CONTROL 51.37, P4 0.05), SOD activity (MEDIUM DOSE 50.86 vs. CONTROL 56.40, P4 0.05; HIGH DOSE 55.61 vs. CONTROL 56.40, P 40.05), MDA concentration (LOW DOSE 1.79 vs. CONTROL 1.50, P 40.05; MEDIUM 1.51 vs. CONTROL 1.50, P4 0.05; HIGH DOSE 1.14 vs. CONTROL 1.50, P 40.05). It thus can be demonstrated that for ‘MEDIUM DOSE’ and ‘HIGH DOSE’ group, UI and SOD activity have no significant difference with ‘CONTROL’ group; besides, MDA
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concentration in all three ‘DOSE’ groups has no significant difference with ‘CONTROL’ group. All above imply that the protection of CBS is comparable with colloidal bismuth pectin. Amongst all three ‘DOSE’ groups, the UI, SOD and MDA observables varied noticeably, suggesting a concentration-dependent manner of CBS. There are increasing evidences that the gastric mucosa damage is highly related to free radicals (Granger et al., 1981). It has been pointed out that superoxide radicals play pivotal role in gastric lesion induced by both ethanol and aspirin (Pihan et al., 1987). SOD is one type of crucial enzymes in animals, whose function is clearing excessive superoxide radicals as to protect the damage of cells (McCord et al. 1971). The level of damage of gastric mucosa can be reflected by the decrease of SOD in activity (Granger et al., 1981; Itoh and Guth, 1985; Nartey et al., 2012). Pre-treated with SOD can efficiently protects gastric mucosa. All these results support the fact that free radicals act on cell membrance, triggers the lipid peroxidation reaction (La Casa et al., 2000) and results in MDA level elevation, the damage of mitochondria and lysosome (Kwiecień et al. 2002; Kanter et al. 2005; Abbas and Sakr, 2013). All these changes leads to denature in epithelial cell, increase in cell permeability and acceleration of ulcer formation. Therefore, SOD activity determination, together with MDA measurement is deemed as ‘gold standard’ to reveal the severity of gastric lesion caused by lipid peroxidation and superoxide radicals. In the gastric lesion, the activity of SOD declines and MDA concentration rises accordingly (Rahim et al., 2014). Normal gastric mucosa of rat is composed of mucosa layer, submucosa, muscular layer and placenta percreta from inside to outside as shown in Fig. 3A and B. Mucosa consists of epithelial layer, lamina propria mucosae and lamina muscularis mucosae. In lamina propria mucosae, there were large numbers of bodies of gland (including leukomonocyte, plasma cells, eosinophile granulocyte, etc). The ethanol-induced ulcer always takes place at mucosa layer. The mucosa of ‘MODEL’ group had clear inflammatory reaction, with break-down of glandular organ as shown in Fig. 3C. Gastric mucosa surface had obvious necrosis and exfoliation; additionally, there were a mass of blood cells, indicative of severe bleeding as shown in Fig. 3D and E. In contrast, for ‘HIGH DOSE’ group (20 mg/kg CBS), the structure of gastric wall remained complete, there was no distinct erosive damage and bleeding as shown in Fig. 3F.
4. Conclusions To our knowledge, our study is the first report up to now, which evaluates the effect of CBS in protection of ethanol induced ulcer. We established the pathological model successfully in rats, UI, SOD and MDA were used as three parameters to indicate the severity of gastric ulcer. SOD and MDA are considered as ‘gold standard’ for free radicals and lipid peroxidation generation, which seems to be one possible mechanism for ethanol to damage gastric mucosa. Indeed CBS showed a striking gastroprotective activity against ethanol-induced gastric ulcer, which is in line with the fundamental activity when using Conyza blinii H.Lév. as folk medicine. No doubt that further pharmacological studies could be undertaken in order to unveil more action mechanisms involved in this activity. These preliminary results render us to believe that CBS could be further explored and has the potential to be used for drug candidate.
Acknowledgement The authors would like to thank Dr. Yanfang Su (School of Pharmaceutical Science and Technology, Tianjin University, PRC) for providing the CBS sample in this study. We would also like to
thank the anonymous referees for their comments. This work is supported by a start-up funding from Tianjin University of Science & Technology.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.10.052.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Research Paper
The protective activity of Conyza blinii saponin against acute gastric ulcer induced by ethanol Long Ma a,b,c,n, Jiangguang Liu d a Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, School of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China b Tianjin Key Laboratory of Industry Microbiology, School of Biotechnology, Tianjin University of Science & Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, China c Centre for Biomolecular Sciences, University of St Andrews, Room 4.11, North Haugh, St Andrews KY16 9ST, United Kingdom d School of Pharmaceutical Science and Technology, Tianjin University, No 92, Weijing Road, Nankai District, Tianjin 300072, China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 June 2014 Received in revised form 9 October 2014 Accepted 13 October 2014 Available online 4 November 2014
Ethnopharmacological relevance: Conyza blinii H.Lév., is a type of natural plant. Its dried overground section is used to treat infections and inflammations in traditional Chinese medicine. Triterpenoidal saponins have a wide range of bioactivities, for instance, anti-cancer, anti-virus and anti-anaphylaxis. Conyza blinii saponin (CBS), mainly composed of triterpenoidal saponins, is the total saponin of Conyza blinii H.Lév. It has been reported that CBS also has gastric mucous membrane protection activity. This study aims to test CBS's protective activity of gastric's mucous membrane against ethanol. This investigation may lead to the development of novel drug from natural products as anti-ulcer agent, or as gastric mucous protective against chemical damage. Materials and methods: CBS (Conyza blinii saponin) is the total saponin of Conyza blinii H.Lév., which was obtained as described previously. We tested the protective activity of CBS against ethanol-induced ulcer. Thirty six rats were grouped randomly as ‘NORMAL’, ‘CONTROL’, ‘MODEL’, ‘LOW DOSE’, ‘MEDIUM DOSE’ and ‘HIGH DOSE’. The ‘NORMAL’ group were rats with no pathological model established within it. The ‘CONTROL’ group was administrated with colloidal bismuth subcitrate, while ‘MODEL’ group was not given any active agents apart from absolute ethanol in order to obtain gastric ulcer model. The three ‘DOSE’ groups were treated with different concentrations of CBS (5, 10, 20 mg/mL) before administration followed by absolute ethanol. All rats were sacrificed after the experiment to acquire the gastric tissue. The ulcer index (UI), malondialdehyde (MDA) and superoxide dismutase (SOD) were measured to monitor the activity of CBS. Besides, the rat gastric tissue was made to paraffin section and stained using the Hematoxylin–Eosin (HE) method. The histopathology examination was carried out to examine CBS efficacy in terms of gastric mucous protection. Results: We found that CBS had a profound protection activity against acute gastric ulcer induced by ethanol and this activity displayed a concentration-dependent manner. The efficacy of 10 and 20 mg/mL CBS was comparable with colloidal bismuth subcitrate (Po0.05). All three level of CBS tested were able to significantly reduce UI, MDA and enhance SOD level (Po0.05). Conclusions: It was deduced that the mechanism for such activity would be anti-lipid peroxidation, facilitating free radicals clearance. In addition , histopathology examination of the gastric mucous membrane supported the same conclusion, that CBS can efficiently suppress the inflammatory reactions, bleeding and protect the gastric mucosa. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Conyza blinii H.Lév. Total saponin Gastric ulcer Superoxide dismutase (SOD) Malondialdehyde (MDA)
1. Introduction
n Corresponding author at: Tianjin Key Laboratory of Industry Microbiology, School of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, China. Tel.: þ 86 22 60602948; fax: þ 86 22 60602298. E-mail addresses: [email protected], [email protected] (L. Ma).
http://dx.doi.org/10.1016/j.jep.2014.10.052 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
Conyza blinii H.Lév., is a native herbaceous plant in Compositae. It is spread in Sichuan and Yunnan provinces of China and is commonly called Jin Long Dan Cao. Its dried overground section is valuable in folk medicine for the treatment of chronic bronchitis, gastroenteritis and some other inflammatory diseases (Materia Medica, 1998; Pharmacopoeia of the People's Republic of China, 1977, 2010). Chemical profiles of this species have revealed many
L. Ma, J. Liu / Journal of Ethnopharmacology 158 (2014) 358–363
natural products including diterpenoids, flavonoids, triterpenoids and saponins (Xu et al., 1999; Su et al., 2003). CBS has 17 known components, whose chemical profiles were all listed in Table SI-1 and Fig. SI-1 (Su et al., 2000, 2001a, 2001b, 2003). It has been reported that the CBS has significant protective activity against the gastric mucosal damage. The possible mechanisms account for such activity is to reduce the secretion of gastric collection and gastric acid, as well as to prevent free radicals formation (Su et al., 2001a, 2001b, 2007a, 2007b). Recently, it has shown that CBS induces the apoptosis of Hela and SPC-A1 cells, indicating an antineoplastic activity (Liu et al., 2011). Excessive consumption of ethanol-containing beverage exerts serious health problems in human. The direct damage of ethanol can lead to gastric lesion. Ethanol is destructive on gastric mucosa, which can be evidenced by gastric mucosa erosion and bleeding. This is caused by blood micro-circulation disorder in gastric wall, tissue necrosis, reduction in gastric mucosal blood flow and mucus secretion (Masuda et al., 1992; Morales et al., 1992; Ohta et al., 1997; Knoll et al., 1998; Kawano and Tsuji, 2000; Sibilia et al., 2003). This reinforces the need for developing new protective agents. Although CBS was originally shown to possess multiple anti-inflammatory and gastric mucosal protection activities, its effects on acute gastric ulcer has not been yet explored. The aim of the present study is to evaluate gastric mucosa protective properties and established the possible mechanisms.
2. Experimental and methods 2.1. Animals Male and female Sprague & Dawley rats, 180–200 g, supplied by the Tianjin Medical University, PRC, were used throughout this study. All animal experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People's Republic of China. The animals were fed with a certified diet with free access to water, and placed in cages under a 12 h light/dark cycles for 4 days before experiments. The temperature was maintained at 2471 1C and the humidity at 70–75% in a controlled room. 2.2. The preparation of CBS from dried Conyza blinii H.Lév. Conyza blinii H.Lév. the accepted name of a species in the genus Conyza, family Compositae, which has been checked against websites www.theplantlist.org (The Plant List), and www.ipni.org (The International Plant Names Index). A voucher specimen of Conyza blinii H.Lév. was identified by Dr. Yanfang Su, and deposited in the herbarium of the School of Pharmaceutical and Technology, Tianjin University. The method for CBS preparation was based on the protocols published before (Su et al., 2007a, 2007b). Simply, (1) the ground dried Conyza blinii H.Lév. was refluxed in 90% ethanol twice (each time for 2 h). The filter residue was then refluxed in 55–60% ethanol twice and filtered. The filtrates were combined and followed by evaporation under reduced pressure to completely remove the ethanol. (2) The distilled water was added to dilute the concentrated solution to make it 10–20 g per 100 mL. Ethyl acetate was added to fractionate the non-saponin components using separatory funnel. The ethyl acetate in the aqueous layer was totally evaporated under reduced pressure. Butanol was added to the resultant aqueous fraction using separatory funnel for three times until completion. Combined butanol fraction was
SOD ðU=mg proteinÞ ¼
359
concentrated until dried to obtain crude saponin. (3) The crude saponin was dissolved in water and columned using D101 macroporus resin chromatography. Water, 30%, 70% and 95% ethanol were subsequently used for column elution. The 70% ethanol elution was collected and concentrated to acquire CBS. 2.3. The grouping and administration plan Thirty six Sprague & Dawley rats were divided into six groups randomly as follows: ‘NORMAL’, ‘MODEL’, ‘CONTRL’, ‘LOW DOSE’, ‘MEDIUM DOSE’ and ‘HIGH DOSE’. Each group had six rats. All reagents were given using intragastric administration. For ‘NORMAL’ group, the rats were given 1 mL 0.5% carboxymethylcellulose sodium (CMC-Na) for consecutive two days. Before the first administration, all six rats were fasted for 24 h. Two hours after the last administration, all six rats were administered with 1 mL physiological saline, waited for another one hour before sacrification. ‘MODEL’ and ‘CONTROL’ groups were given 1 mL absolute ethanol and 14.4 mg/kg colloidal bismuth pectin respectively. For the other three ‘DOSE’ groups (LOW DOSE, MEDIUM DOSE and HIGH DOSE) designed to test CBS activity; 5, 10, 20 mg/mL CBS (dissolved in 0.5% CMC-Na) were given instead of physiological saline otherwise the same as ‘NORMAL’ group. 2.4. The preparation of rat gastric tissue homogenate The rat gastric mucosa was removed by scraping to make 10% tissue homogenate in physiological saline using homogeniser on ice, followed by centrifugation at 2000 rpm for 10 min. The supernatant was collected for subsequent use. 2.5. The protein quantification in rat gastric tissue homogenate Coomassie brilliant blue method was used to determine the protein concentration in gastric tissue homogenate as described previously (Sedmak and Grossberg, 1977). 2.6. The calculation of ulcer index The gastric ulcer index (UI) was conducted as follow: stomachs were taken from the sacrificed rats immediately and rinsed by cold physiological saline to remove the remnant. Stomachs were incised along greater gastric curvature. The spot gastric lesion was counted 1 point; linear gastric lesion less than 1 mm was counted 1 point, adding upon 1 point when the length of the ulcer lesion increasing 1 mm. If the width 41 mm, then the points were multiplied by factor 2. 2.7. Measurement of SOD Xanthine-xanthine oxidase system, as shown previously (Peskin and Winterbourn, 2000), was used and modified for SOD measurement. Simply, 2% rat gastric tissue homogenate was made in physiological saline and different amounts of (from 10 to 50 mL) which were tested to work out the optimal suppression rate. The sample gave a suppression rate ranged 48–50%, whose amount was chosen as the optimal amount for the assay. The ‘measured’ tube and ‘control’ tube were both used in the assay. The ‘measured’ tubes were defined as the one which contained the samples subjected to quantification, while the ‘control’ tubes only contained distilled water otherwise were exactly the same as the ‘measured’ ones. The SOD concentration was calculated using the following equation:
2 ODcontrol ODmeasured =ODcontrol total reaction volume=measured sampling volume protein concentration in tissue
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Fig. 2. Effect of CBS on SOD activity and MDA concentration of gastric mucosa in rats. ★★★P o0.001 vs. NORMAL; ★★P o 0.01 vs. NORMAL; ▲▲▲Po 0.001 vs. MODEL; ▲ Po 0.05 vs. MODEL; ◆P 4 0.05 vs. CONTROL.
2.9. Histopathology examination Fig. 1. Effect of CBS on ulcer index (UI) in rats. (A–D) show the gastric wall of rats from different groups, namely ‘HIGH DOSE’, ‘MODEL’, ‘CONTROL’ and ‘NORMAL’. Clearly, for ‘MODEL’ group, severe damage and lesion can be seen; however for ‘CONTROL’ group, this lesion is considerably alleviated. For ‘NORMAL’ group, a smooth and complete gastric wall can be seen as well as for ‘HIGH DOSE’ group. Fig. 1E shows the protective effect of CBS against gastric ulcer induced by ethanol in rats in terms of UI. There is a significant decrease in UI for all groups administrated with CBS, compared with ‘MODEL’ group. ★★★P o 0.001 vs. NORMAL; ▲▲P o 0.01 vs. MODEL; ▲▲▲Po 0.001 vs. MODEL; ◆P 4 40.05 vs. CONTROL.
2.8. Measurement of MDA MDA was measured using the thiobarbituric acid (TBA)-based method with modification, as previously published (Ohkawa et al., 1979). To be detailed, three types of samples were used, namely ‘standard’, ‘standard blank’ and ‘measured’. The ‘measured’ samples contained the rat gastric tissue homogenate for MDA quantification; ‘standard’ sample contained 10 nmol/mL standard MDA; while ‘standard blank’ contained none of the standard nor the rat gastric tissue homogenate. All samples were reacted with TBA in phosphate buffer (50 mM, pH 7.0) supplemented with absolute alcohol and hydrochloric acid where needed. All samples were incubated at 95 1C for 40 min followed by centrifugation at 4000 rpm for 10 min. The absorbance of supernatant was measured at 532 nm.
MDA ðnmol=mg protÞ ¼
ODmeasured ODcontrol blank =ODstandard ODstandard protein concentration in tissue
concentration of standard dilution f actor
blank
The rat gastric tissue was made to paraffin section and stained using the Hematoxylin–Eosin (HE) method. The histopathology examination was carried out using common optical microscope. 2.10. Statistical analysis GraphPad InStat software (version 3.05) was used for statistical analysis. One-way analysis of variance (one-way ANOVA) method was chosen. Bartlett statistic method was first used to test the homogeneity of variances, followed by comparison between each two individual groups using the Student–Newman–Keuls. α ¼0.05 was applied as level of significance test, which indicated that Pr 0.05 was considered to have significant statistical significance.
3. Results and discussion Tables SI-2–4 in supplementary information list all the experimental data discussed in this study. We established an animal model of acute gastric ulcer successfully, which was alike to ethanol-induced gastric ulcer in human. In the first place, results showed that all these three parameters UI, SOD and MDA for ‘MODEL’ group had significant statistical difference compared with ‘NORMAL’ group as shown in Fig. 1E and Fig. 2A and B. To be detailed, UI (112.72 for MODEL vs. 0 for NORMAL, P o0.001); SOD activity (46.41 for MODEL vs. 59.64 for NORMAL, Po 0.01), MDA concentration (2.57 for MODEL vs. 1.47 for NORMAL, Po 0.001). These imply that the gastric ulcer takes place and free radicals and lipid peroxidation reaction plays important roles in pathologic process for ethanol-induced ulcer as previously reported (Pandian et al., 2002). When comes to the comparison between three ‘DOSE’ groups and ‘MODEL’ group, there exist significant discrepancy.
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Fig. 3. Results and analysis of histological tests on gastric wall of rats. (A) Normal gastric wall of rats magnified by (10 4). It shows a complete gastric wall structure of normal rat, which resembles human's. A: epithelial layer and lamina propria mucosae; B: lamina muscularis mucosae; C: submucosa muscular layer; D: muscular layer. (B) Normal gastric mucosa magnified by (10 10). It shows submucous layer and muscular layer of rat. (C) Gastric mucosa of rats in ‘MODEL’ group magnified by (10 10). It can be seen that incomplete glandular organs, inflammatory reaction and the exudation of red cells are present in the surface of mucosa. A: exudation of red cells; B: the remains of mucosa after the disintegration of glandular organ; C: the disintegration of glandular organ cell. (D, E) Gastric mucosa of rats in ‘MODEL’ group magnified by (10 10) and (10 40) respectively. The red spots are substantial red blood cells, indicative of severe bleeding caused by the damage of ethanol. (F) Gastric mucosa of rats in ‘HIGH DOSE’ group magnified by (10 10). It can be seen that CBS has a profound protection of mucosa against ethanol-induced gastric lesion, as there is no obvious damage and erosion, neither observable bleeding. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
To be specific, it can be noticed that for UI (LOW DOSE 74.32 vs. MODEL 112.72, P o0.01; MEDIUM DOSE 43.85 vs. MODEL 112.72, P o0.001; HIGH DOSE 19.13 vs. MODEL 112.72, P o0.001). SOD activity (HIGH DOSE 55.61 vs. MODEL 46.41, Po 0.05). MDA concentration (LOW DOSE 1.79 vs. 2.57, P o0.001; MEDIUM DOSE 1.51 vs. MODEL 2.57, P o0.001; HIGH DOSE 1.14 vs. MODEL 2.57, P o0.001). These results suggest that order of severity of gastric ulcer for ‘DOSE’ groups is fairly different from ‘MODEL’ group. Even for the ‘LOW DOSE’ group, the UI and MDA had a remarkable decline in comparison with ‘MODEL’ group. In addition, the SOD’s level in ‘HIGH DOSE’ group evidently mounted up. All means CBS
has a striking preventive activity against ethanol-induced gastric ulcer. Last, we intended to compare the three parameters of the ‘DOSE’ groups with CONTROL group. UI (MEDIUM 43.85 vs. CONTROL 51.37, P4 0.05; HIGH DOSE 19.13 vs. CONTROL 51.37, P4 0.05), SOD activity (MEDIUM DOSE 50.86 vs. CONTROL 56.40, P4 0.05; HIGH DOSE 55.61 vs. CONTROL 56.40, P 40.05), MDA concentration (LOW DOSE 1.79 vs. CONTROL 1.50, P 40.05; MEDIUM 1.51 vs. CONTROL 1.50, P4 0.05; HIGH DOSE 1.14 vs. CONTROL 1.50, P 40.05). It thus can be demonstrated that for ‘MEDIUM DOSE’ and ‘HIGH DOSE’ group, UI and SOD activity have no significant difference with ‘CONTROL’ group; besides, MDA
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concentration in all three ‘DOSE’ groups has no significant difference with ‘CONTROL’ group. All above imply that the protection of CBS is comparable with colloidal bismuth pectin. Amongst all three ‘DOSE’ groups, the UI, SOD and MDA observables varied noticeably, suggesting a concentration-dependent manner of CBS. There are increasing evidences that the gastric mucosa damage is highly related to free radicals (Granger et al., 1981). It has been pointed out that superoxide radicals play pivotal role in gastric lesion induced by both ethanol and aspirin (Pihan et al., 1987). SOD is one type of crucial enzymes in animals, whose function is clearing excessive superoxide radicals as to protect the damage of cells (McCord et al. 1971). The level of damage of gastric mucosa can be reflected by the decrease of SOD in activity (Granger et al., 1981; Itoh and Guth, 1985; Nartey et al., 2012). Pre-treated with SOD can efficiently protects gastric mucosa. All these results support the fact that free radicals act on cell membrance, triggers the lipid peroxidation reaction (La Casa et al., 2000) and results in MDA level elevation, the damage of mitochondria and lysosome (Kwiecień et al. 2002; Kanter et al. 2005; Abbas and Sakr, 2013). All these changes leads to denature in epithelial cell, increase in cell permeability and acceleration of ulcer formation. Therefore, SOD activity determination, together with MDA measurement is deemed as ‘gold standard’ to reveal the severity of gastric lesion caused by lipid peroxidation and superoxide radicals. In the gastric lesion, the activity of SOD declines and MDA concentration rises accordingly (Rahim et al., 2014). Normal gastric mucosa of rat is composed of mucosa layer, submucosa, muscular layer and placenta percreta from inside to outside as shown in Fig. 3A and B. Mucosa consists of epithelial layer, lamina propria mucosae and lamina muscularis mucosae. In lamina propria mucosae, there were large numbers of bodies of gland (including leukomonocyte, plasma cells, eosinophile granulocyte, etc). The ethanol-induced ulcer always takes place at mucosa layer. The mucosa of ‘MODEL’ group had clear inflammatory reaction, with break-down of glandular organ as shown in Fig. 3C. Gastric mucosa surface had obvious necrosis and exfoliation; additionally, there were a mass of blood cells, indicative of severe bleeding as shown in Fig. 3D and E. In contrast, for ‘HIGH DOSE’ group (20 mg/kg CBS), the structure of gastric wall remained complete, there was no distinct erosive damage and bleeding as shown in Fig. 3F.
4. Conclusions To our knowledge, our study is the first report up to now, which evaluates the effect of CBS in protection of ethanol induced ulcer. We established the pathological model successfully in rats, UI, SOD and MDA were used as three parameters to indicate the severity of gastric ulcer. SOD and MDA are considered as ‘gold standard’ for free radicals and lipid peroxidation generation, which seems to be one possible mechanism for ethanol to damage gastric mucosa. Indeed CBS showed a striking gastroprotective activity against ethanol-induced gastric ulcer, which is in line with the fundamental activity when using Conyza blinii H.Lév. as folk medicine. No doubt that further pharmacological studies could be undertaken in order to unveil more action mechanisms involved in this activity. These preliminary results render us to believe that CBS could be further explored and has the potential to be used for drug candidate.
Acknowledgement The authors would like to thank Dr. Yanfang Su (School of Pharmaceutical Science and Technology, Tianjin University, PRC) for providing the CBS sample in this study. We would also like to
thank the anonymous referees for their comments. This work is supported by a start-up funding from Tianjin University of Science & Technology.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.10.052.
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