Alkaloids from Mahonia bealei posses anti-[H.sup.+]/[K.sup.+]-ATPase and anti-gastrin effects on pyloric ligation-induced gastric ulcer in rats.
The purpose of this study was to investigate the underlying mechanism(s) of the total alkaloids (TA) from Mahonia bealei in treating pyloric ligation-induced gastric ulcers in rats. Animals were sacrificed after 19 h of the ligation. Gastric acid, peptic activities, mucin levels, FT/IC-ATPase activities and the gastrin level were analyzed. To improve the accuracy of the observations, IPP 6.0 software was introduced to measure the area of ulcer. TA (18.56 mg/kg/day, i.g.) showed an antiulcer effect by significantly decreasing the gastric ulcer areas (11.28 [mm.sup.2]) compared with model group (26.36 [mm.sup.2]). The TA ulcer inhibition ratio was 57.2%, compared with the effect of the positive control, omeprazole (62.96%). The results also showed that TA had a significant effect in inhibiting the release of [H.sup.+]/[K.sup.+]-ATPase, reducing the content of gastrin and decreasing gastric acidity on experimental animals. However, the TA had no significant effects on gastric mucus secretion and pepsin activity. Data indicated that TA had gastric ulcer protective effects by modulating the [H.sup.+]/[K.sup.+]-ATPase activity and gastrin level. TA has a potential to be developed as a pharmacological agent for the treatment of gastric ulcers.
Mahonia is a flowering evergreen tree of the family Berberidaceae. It is recorded that there are about 70 species of the genera Mahonia growing around the world, and about 50 species growing in China. Mahonia, a traditional Chinese herb which was firstly recorded in Zhiwu Mingshi Tukao, derives from several species of the Mahonia genus. In Chinese folk medicine, the root, stem and leaves of Mahonia have been used as medicines to treat various ailments, such as dysentery and diarrhea for thousands of years in Chinese clinics for clearing heat, moistening aridity, purging fire and detoxifying toxins (Chinese Pharmacopoeia Commission 2010). Two species of Mahonia, M. bealei (Fort.) Carr, and M. fortunei (lindl.) Fedde. are included in the Chinese Pharmacopeia (2010). The alkaloids from Mahonia, M. bealei, include berberine, palmatine, magnoflorine, jatrorrhizine, oxyacanthine, berbamine, columbamine, isotertrandrine and epiberberine (Ji et al. 2000). These ingredients were also found to have significant antioxidant, anti-mutagenic, antibacterial, antifungal, antiproliferative and anticancer effects (Wiesenauer and Ludtke 1996; Wirth and Wagner 1997).
A gastric ulcer is a common form of peptic ulcer, mainly referring that its own digestive juices digests the stomach mucosa causing more than the muscularis mucosa tissue to be damaged. The inflammatory necrotic lesions occur between the cardia and pylorus, along with clinical manifestations of upper abdominal pain, loss of appetite, loss of weight, vomiting, heartburn and other symptoms (Lakshmi et al. 2010). The lesion becomes more complicated with bleeding, perforation, and obstruction, which occur due to the imbalance between attacking factors (pepsin, acid and Helicobacter pylori) and defensive (mucin, prostaglandin and bicarbonate) factors. The inhibition of gastric acid production as well as reinforcement of gastric mucosal protection is the major approaches in clinical treatment for the treatment of gastric ulcers. Gastric acid was identified as a key pathogenic factor in the gastric ulcer (Zhang et al. 2013).
Gastric [H.sup.+]/[K.sup.+]-ATPase is the proton pump in the stomach and the enzyme is primarily responsible for the acidification of the stomach contents. The proton pump is the common and final pathway of all stimulation of gastric acid production. Inhibiting gastric acid secretion by blocking the activity of [H.sup.+]/[K.sup.+]-ATPase is a commonly used clinical intervention for dyspepsia, peptic ulcer and gastroesophageal reflux disease (Zhang et al. 2013). In clinical practice, antiacids, anticholinergics, proton pump inhibitors and histamine [H.sub.2] receptor antagonists have been used to treat the gastric ulcer (Santin et al. 2010).
Mahonia was used to treat gastrointestinal disorders as a traditional Chinese medicine. However, the underlying mechanisms for antiulcer remedies are far from fully clarified. In this study, we identified the main active chemical constituents from Mahonia bealei using LC-MS/MS and investigated its anti-gastric ulcer activities and underlying mechanisms.
Materials and methods
Preparation of the alkaloids
The stems of M. bealei (Fort), collected in Qujing City, Yunnan Province, China, were identified by professor Ping Li, Department of Traditional Chinese Medicine, China Pharmaceutical University.
Voucher specimens were deposited in the State Key laboratory of Natural Products and Functions, China Pharmaceutical University. The smashed stems were extracted by 50% ethanol two times. These two extractants were put together and concentrated to a certain concentration. The total alkaloids (TA, TA >50%, Lot. No. 20120807) were obtained by weak acid cation exchange resin D152 (HCl/50% ethanol = 1:25, v/v).
Omprazole (OL, Jiangsu Yangtze River Pharmaceutical Co., Zhunzi H20067433, Lot. No. 20120304) and TA (Lot. No. 20120807) was suspended in carboxymethylcellulose (CMC, Sigma), and the compounds were stored at 4[degrees]C. Alcian blue (8GX) was obtained from Shanghai YuanYe Biotechnology Co., Ltd. The Rat Pepsin ELISA kits, Rat Gastrin ELISA kits, [H.sup.+]/[K.sup.+] ATPase ELISA kits and Protein assay kits were obtained from Nanjing jiancheng Bioengineering Institute. All chemicals used were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Male Sprague Dawley rats weighing 180-220 g were purchased from the Laboratory Animal Services Centre, Jiangsu University, Jiangsu, China. The animals were housed in raised bottom mesh cages to prevent coprophagy and kept in environmentally controlled rooms (25[+ or -]2[degrees]C, 12 h light and dark cycle), fed with standard diet and water ad libitum. When necessary, animals were deprived of food allowing access to water freely for 24 h before the experiments. All the experiments were performed in accordance with Guides for the Care and Use of Laboratory Animals of China, and were approved by the ethics committee.
Determination of TA
The contents of alkaloids in Mahonia were determined by HPLC. The optimum separation for alkaloids was achieved using acetonitrile-0.2% formic acid solution (25:75, v/v) as the mobile phase and a Hedera ODS-2 C18 column.
The anti-ulcer study
Ulcer model induced by pyloric ligation (PL)
The animals were divided into six groups: control (CTR), model (MOD), TA-L(TA, 6.19 mg/kg/day, i.g.), TA-M (TA, 18.56mg/kg/day, i.g.), TA-H (TA, 55.68 mg/kg/day, i.g.) and omprazole (OL, 4.2 mg/kg/day, i.g.), eight rats in each group. These drugs were administered intragastrically to rats once a day, and continuously for seven days. 1 h after the last drug administration, the animals were deprived of food allowing free access to water for 24 h. Pyloric ligation was done under anesthesia with 10% chloral hydrate (0.33 ml/100g, i.p.). The abdomen was opened below the xiphoid process, and the pyloric end of the stomach was ligated avoiding any injuries to the adjacent blood vessels. The stomach was replaced carefully and the abdomen was sutured. After being deprived of food and water for 19h, the animals were sacrificed by cervical dislocation after collecting blood from the orbit, and then the stomachs were dissected out. Lesions were scored and gastric fluid was collected and centrifuged at 3500 rpm for 15 min. The supernatant was used for the estimation of gastric secretion, mucin estimation and peptic activity.
Measuring ulcer areas (UA)
IPP 6.0 software was chosen as image analysis for its allowing a high degree of control and replication of image-processing steps. The stomachs were incised along the greater curvature and observed ulcers. Digital pictures of the mucosa surface of each stomach were taken by a digital camera (SAMSUNG PL170, SAMSUNG ELECTRONICS CO., LTD, made in China). The count of ulcer areas consisted of five steps in IPP: (1) changing to intensity style, (2) building a new calibration, (3) drawing object regions using 'Irregular AOI', (4) convert AOI(s) to Object(s), (5) count the areas.
Ulcer inhibition ratio (U1R) was calculated as follows: UIR = ([U.sub.m] - [U.sub.t])/[U.sub.m] x 100%. [U.sub.m] was the average ulcer areas of the model group; [U.sub.t] was the average ulcer areas of the tested group.
Gastric secretion study
Free and total acids in the gastric juice were titrated with 0.01 mol/L NaOH, using Topfer's reagent and phenolphthalein as indicators, respectively (Anoop and Jegadeesan 2003), and expressed as mmol/L. Peptic activity was determined through the colorimetric method by measuring the amount of liberated tyrosine and expressed as U/ml.
Estimation of mucin
The free mucin level was quantified by adding Alcian blue solution to the gastric juice. The amount of Alcian blue was determined at a wavelength of 615 nm. The difference of Alcian blue was the amount of gastric cavity free mucin, which was expressed as mg Alcian blue per ml of gastric juice.
Gastric wall mucus was determined as follows: gastric mucosa was weighted and immediately transferred to 0.1 % Alcian blue solution and stained for 2 h at 25[degrees]C. After segments were rinsed with PBS, 5 ml incubation fluid was taken and centrifuged at 3500 rpm for 10 min, and the amount of Alcian blue was determined from supernatant colorimetric assay at a wavelength of 615 nm. The difference of Alcian blue was the amount of gastric wall mucus and was expressed as mg Alcian blue per g of gastric mucosa.
The stomach tissue of rat was weighed and a 10% homogenate was made in accordance with the proportion of gastric mucosal tissue weight with saline in a 1:9 ratio. The homogenate was centrifuged at 3500 rpm for 10 min, and the supernatant was processed for protein estimation using Protein assay kit, following the manufacturer's instructions. Results were expressed as g protein/1 solution.
In vivo assay of [H.sup.+]/[K.sup.+]-ATPase activity
[H.sup.+]/[K.sup.+]-ATPase activity was analyzed by measuring the inorganic phosphate release after hydrolysis of ATP. The homogenate of gastric mucosal tissue was made as seen in Section "Protein determination", the homogenate was centrifuged at 3500 rpm for 10 min. The inorganic phosphate release was determined from the resulting supernatant spectrophotometrically at 450 nm. Results were expressed as [micro]mol Pi/mg prot/hour.
Blood was centrifuged at 3000 rpm for 10 min and the supernatant was used for determination of gastrin levels using a Rat Gastrin ELISA kit, following the manufacturer's instructions. The results were expressed as pg/ml.
All values were shown as means [+ or -]SE in the figures. Statistical analysis were performed with the SPSS 16.0 for Windows software package using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. A value of p < 0.05 was considered statistically significant.
Analysis of TA
As shown in Figs. 1-3, peak 1 was identified as columbamine by comparison with the reference compound. Peak 1 mainly gives the [[M.sup.+]] (m/z: 338.3), ion [[M-15].sup.+] (m/z: 323.2) and [[M-44].sup.+] (m/z: 294.1). In Fig. 2, peak 2 gives the same ions as columbamine, and was identified as jatrorrhizine. And peak 3 gives the [[M.sup.+]] (m/z: 352.3), ion [[M-15].sup.+] (m/z: 337.2) and [[M-44].sup.+] (m/z: 308.2), which belong to palmatine. Similarly, peak 4, including the [[M.sup.+]] (m/z: 336.3), ion [[M-15].sup.+] (m/z: 321.2) and [[M-44].sup.+] (m/z: 292.2), was identified as berberine.
Compounds in TA were found to be mainly columbamine, jatrorrhizine, palmatine and berberine by the means of HPLC chromatogram. Their contents were 2.7%, 31.6%, 29.9% and 29.3% (Fig. 3C), respectively, and total content of these four alkaloids was about 93.5%.
Ulcer areas obtained with 1PP were clear and repeatable (Fig. 4C and D). It showed that graded doses of TA (TA-L, TA-M, TA-H) had different effects on PL induced gastric ulcers. Compared with the model group, omprazole (OL) and TA-M showed a significant decrease of ulcer areas (Figs. 4A and 5, p < 0.01), and the UIR were 62.96%, 57.20% (Fig. 4B), respectively. There is no significant difference between TA-M (18.56mg/kg) and OL (p>0.05). The UIR of TA-L and TA-H were 18.72%, 25.27%, respectively. The color of the ulcers as well as the areas of the ulcers were visibly ameliorated in the TA-M treated group.
Gastric secretion study
The effects of TA on different factors such as free acidity, total acidity, gastric juice and peptic activity, that play a crucial role in the occurrence of gastric ulcers, were studied with PL-induced rats. OL, TA-M significantly reduced the volume of gastric juice (Fig. 6D) by 43.33% (p<0.01) and 36.65% (p<0.01), respectively, compared with the model group. Simultaneously, TA-L and TA-H reduced the volume by 29.56% (p<0.05) and 27.12% (p<0.05), respectively.
As shown in Fig. 6A and B, OL has reduced free acidity and total acidity by 31.04% (p < 0.05) and 30.10% (p < 0.01), respectively, and TA-M has the same function in the reduction of free acidity and total acidity of 29.05% (p <0.05) and 26.79% (p <0.05), respectively. The TA-L and TA-H groups could also reduce the content of free acidity and total acidity, but there was no significant difference (p > 0.05).
It was no difference between tested groups and model group (53.71 U/ml, p > 0.05) for peptic activity. The results are represented in Fig. 6C.
Estimation of mucus
The gastric mucus is one of the most important protective features for the gastric mucosa. As shown in Fig. 7A and B, there was no significant difference in mucus between the tested groups and model group (p > 0.05), which indicated that the anti-ulcer function of TA was not mainly through the reinforcement of gastric mucosal protection.
Effect of TA on [H.sup.+]/[K.sup.+]-ATPase activity
The anti-secretion mechanism of TA-M had been confirmed through the inhibition of [H.sup.+]/[K.sup.+]-ATPase activity of 14.71 [micro]mol Pi/mg prot/h (p < 0.05), which was much lower than the model group (19.88 [micro]mol Pi/mg prot/h, p<0.05). OL reduced the enzyme activity as 14.94 p,mol Pi/mg prot/h (p < 0.05) (Fig. 7C). Furthermore, intragastric administration treatment with the standard drug, OL and TA-M (18.56mg/kg, i.g.) significantly reduced the [H.sup.+]/[K.sup.+]-ATPase activity by 24.85% and 26.01% respectively, in comparison with the model group. And the [H.sup.+]/[K.sup.+]-ATPase activity in the TA-L and TA-H groups was decreased by 15.39% and 23.54%, but there was no significant difference when comparing with the model group (p > 0.05).
Effect of TA on gastrin hormone concentration
Gastrin hormone is a known modulator of gastric acid secretion, resulting in the development of gastric ulcer when overexpressed. Compared with model group (50.52 pg/ml), TA (18.56 and 55.68 mg/kg, i.g.) significantly reduced the plasma gastrin level to 44.15pg/ml (p<0.05) and 41.87pg/ml (p<0.01), respectively (Fig. 7D). OL was used as reference drug by reducing the gastrin level to 43.72 pg/ml (p < 0.01) significantly.
The methods of conventional treatment for an ulcer, such as endoscopic methods, proton pump inhibitors and [H.sub.2] receptor antagonists have more side effects than plant extracts. Gastric bleeding and perforation are serious side effects, which are observed in long terms NSAIDs therapy (Izzettin et al. 2012; Ji et al. 2012). Chinese medicines are being used more and more widely throughout the world, but the separation and determination of the active chemical constituents is generally recommended for the standardization and quality control of herbal products and herb related investigations (Tseng et al. 2007), and the authentication and preparation of the major compounds of an herb may be helpful in elucidating its pharmacological activity and underlying mechanisms (Chao et al. 2009). Components extracted from gastro-protective plants have the balance within, and can moderately treat the disease from several possible directions. Thus, new potential agents identified from natural sources are essential for safer and more effective anti-ulcer drugs. In the study, we identified the main alkaloids of Mahonia by HPLC and LC-MS/MS, found that columbamine, jatrorrhizine, palmatine and berberine were suitable TA biomarkers, and they were responsible for the anti-ulcer activity of TA.
Helicobacter pylori were first discovered in the stomachs of patients with gastritis and stomach ulcers in 1982 by Dr. Barry Marshall and Dr. Robin Warren of Perth, Western Australia. Individuals infected with H. pylori have a 10-20% lifetime risk of developing peptic ulcers and 1 to 2% risk of acquiring stomach cancer (Kusters et al. 2006). So H. pylori eradication has been established in the treatment of peptic ulcers and reduction of the relapse of the disease. Eradication of H. pylori significantly reduced the incidence of ulcer recurrence at 8wk and 1 y post-operation, and eradication therapy should be provided to patients with H. pylori infection after simple closure of perforated gastroduodenal ulcers (Wonget al. 2013). In 1994, the National Institutes of Health (USA) published an opinion stating most recurrent duodenal and gastric ulcers were caused by H. pylori, and recommended antibiotics to be included in the treatment regimen (NIH Consensus Statement Online 1994). Appropriate antibiotic treatment combined with proton pump inhibitors or histamine (H2) blockers eradicates H. pylori infection in more than 90% of cases (Graham 1993).
The anti-ulcer effects of TA observed in this study can be attributed to the presence of alkaloids in Mahonia given the antibacterial and anti-inflammatory effects of this species. Columbamine, jatrorrhizine hydrochloride, berberine hydrochloride and palmatine chloride are natural isoquinoline alkaloids and the main ingredients of Mahonia. There is a similar main molecular structure in the four alkaloids, the difference between them being the functional groups (Fig. 2B). As a result, they have some similar functions. For example, colubamine, jatrorrhizine, berberine and plamatine possess significant anti-inflammatory (Fan et al. 2012), hepatoprotective (Kupeli et al. 2002) and analgesic activities (Chao et al. 2009).
Besides berberine, jatrorrhizine and palmatine inhibiting the multiplication of bacteria, fungi and viruses, they can also affect several molecular targets of bacteria simultaneously, such as DNA intercalation, inhibition of DNA synthesis, protein biosynthesis and uncoupling of oxidative phosphorylation (Li et al. 2013; Lee et al. 2010). Enzymes such as [H.sup.+]/[K.sup.+]-ATPase, COX-2 could be modulated to a normal level by berberine and palmatine (Schmeller et al. 1997). Therefore the possible antibacterial activity showed by TA can be the responsible for its important gastroprotective effect.
A PL induced ulcer is widely used to study the effect of drugs on gastric secretion and mucus secretion. It was believed that the excessive secretion of hydrochloric acid was stress-induced. The ligation of the pyloric end of the stomach caused the accumulation of gastric acid and lead to the stomach ulcer. In PL induced model, gastric acid is an important factor for the genesis of ulceration. Gastric acid is the secretory product of the parietal cells, and [H.sup.+]/[K.sup.+]-ATPase is a key player in acid secretion. Before the discovery of H. pylori, it was widely believed that peptic ulcer disease was caused by excess acid secretion (Ghosh et al. 2011). Results showed PL-induced rats had gastric mucosal damage, lesion ulcer, over-expression of acidity, gastrin and [H.sup.+]/[K.sup.+]-ATPase, but the oral administration of TA effectively protected the stomach tissues against attack factors. It is well established that gastric acid secretion plays a role in gastric ulcers, and we found that the anti-secretion effect of TA-M (24.86%) was almost similar to that of the reference antiulcer drug omeprazole (26.01%), which is a selective and irreversible proton pump inhibitor. However, the prolonged gastric acid-free state of the stomach of omeprazole is conducive to the propagation of bacteria and increases the chance of relapse. The alkaloids of mahonia have anti-acid effect as well as inhibiting bacteria, thus having a good effect on an ulcer and regulating the whole body.
In the study, UA quantified with IPP are more accurate than the previous method, which was quantification by measuring the width, length or diameter of the ulcer, and converting into the score. IPP allows a high degree of control and replication of image-processing steps (Blatt et al. 2004). The lesions of a gastric ulcer usually were received as Ulcer Index, which was converted from ulcer areas, and gastric ulcer area ([mm.sup.2]) = length (mm) x width (mm) of injury. As gastric lesions' borders are irregular, areas quantified by length or width are inaccurate. The UA of TA-M reached 11.28 [mm.sup.2], which significantly reduced UA compared with model group (26.36 [mm.sup.2]).
Gastrin is a linear peptide hormone produced by gastrin cells, having regulatory functions of acute gastric acid secretion as well as the gastric epithelial cell population (Kovac et al. 2011). The circulating gastrin, as well as vagal excitation, can stimulate acid secretion (Ericsson et al. 2010). Alkaloids (18.56 and 55.68 mg/kg, i.g.) of mahonia reduced the plasma gastrin level to 44.15 pg/ml (p < 0.05) and 41.87 pg/ml (p < 0.01) respectively, indicating that its antiulcer effect might be by blocking the stimulating factors.
Pepsin is a monomeric L-protein with a high percentage of acidic residues (43 out of 327) leading to a very low pH of 1.5-2.0, and is expressed as a prototype of zymogen and pepsinogen and is released by the chief cells in the stomach to degrade food proteins into peptides (Wang et al. 2013). Compared with the model group (53.71 U/ml), graded concentration of TA had no significantly effects (p>0.05) on peptic activity. Mucus is a complex hydrogel composed of proteins, carbohydrates, lipids, antibodies, bacteria and so on. Gastric mucus is well known to play an important role in mucosal protection, as it is continuously secreted to remove pathogens and lubricate the epithelium as material passes through (Ensign et al. 2012). The unchanged parameters of mucus in this study strongly indicate the absence of mucus involvement in TA gastroprotection, eliminating another possible gastroprotective mechanism in PL-induced rats.
TA had an effect on the [H.sup.+]/[K.sup.+]-ATPase, the secretion of acid and gastrin level, but had little effect on the mucosal repair and the activity of pepsin in a PL induced ulcer.
In conclusion, our data indicated that TA from Mahonia has antiulcer activities, mainly by inhibiting [H.sup.+]/[K.sup.+]-ATPase activity and the secretion of gastrin and gastric acid in PL-induced rats. The protective action against various ulcerogenic factors is related to the functions of columbamine, jatrorrhizine, palmatine and berberine in TA. These four main active compounds are suitable for use as biomarkers of TA. TA has a potential to be developed as a pharmacological agent for the treatment of gastric ulcers.
Conflict of interest
The authors declare that there are no conflicts of interest.
Received 8 April 2014
Received in revised form 26 May 2014
Accepted 2 July 2014
This work was supported in part by a grant (BK 20131309) from The Natural Science Foundation of Jiangsu Province (CHN) and Tang Center for Herbal Medicine Research (U.S.A.).
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Su-Li Zhang (a), Hui Li (a), Xin He (b), Run-Qi Zhang (a) Yu-He Sun (a), Chun-Feng Zhang (a,c),* Chong-Zhi Wang (c), Chun-Su Yuan (c)
(a) State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, JS 210009, China
(b) School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, JS 210000, China
(c) Tang Center of Herbal Medicine and Department of Anesthesia & Critical Care, University of Chicago, Chicago, IL 60637, USA
Abbreviations: TA, total alkaloids from Mahonia bealei; IPP, Image-Pro Plus; OL, omprazole; PL, pyloric ligation; CTR, control group; MOD, model group; TA-L, low level of total alkaloids; TA-M, middle level of alkaloids; TA-H, high level of total alkaloids; UA, ulcer areas; UIR, ulcer inhibition ratio; ANONA, analyzed by oneway analysis of variance; NSAIDs, nonsteroidal antiinflammatory drugs; COX-2, cyclooxygenase-2; SMMC, spontaneous monocyte-mediated cytotoxicity.
* Corresponding author at: State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24Tongjia Lane, Nanjing, Jiangsu 210009, China.
Tel.: +86 25 86185129.
E-mail addresses: email@example.com, firstname.lastname@example.org, email@example.com (C.-F. Zhang).
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|Author:||Zhang, Su-Li; Li, Hui; He, Xin; Zhang, Run-Qi; Sun, Yu-He; Zhang, Chun-Feng; Wang, Chong-Zhi; Yuan,|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Sep 25, 2014|
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