Sources and authentication of herbs
All the herbs of HZJW formula were obtained from Yifang Chinese Material Medica Business Department, Yulin City, all of which are authenticated by Prof. Lai Xiaoping at Guangzhou University of Chinese Medicine. The authenticated voucher specimens (Voucher #20090924) were kept in School of Chinese Materia Medica, Guangzhou University of Chinese Medicine (GUCM). Assurance of quality control for all the materials was validated according to Pharmacopoeia of the People's Republic of China.
Preparation of HZJW
HZJW was composed of 12 medicinal herbs as shown in Table 1. Coptidis Rhizoma and Corydalis Rhizoma were ground to obtain fine powder while others ground to be coarse powder. Cynanchi Paniculati Radix et Rhizoma was distilled with water (1:8, w/v) for 4 h and 30% NaCl was added into the obtained distillate to yield salt-outings. After filtration, the residues of Cynanchi Paniculati Radix et Rhizoma and the other nine medicinal herbs (except Coptidis Rhizoma and Corydalis Rhizoma) were extracted with water (1:10, w/v) twice, 1 h each, of which Scutellariae Barabtae Herba was added post boiling. The resulting supernatant was concentrated to a relative density (RD) of 1.20 (60°C), mixed together with the abovementioned fine powder thoroughly, and then dried out under vacuum. Finally, the above dry extract was blended thoroughly with the salt-outings and an appropriate amount of microcrystalline cellulose to produce HZJW. The yield of HZJW extract was 31.75% (w/w) compared with the original herbs. Chemical profile of HZJW was analyzed by HPLC (see Additional file 1: Figure S1). A voucher specimen was deposited at GUCM, with the registration number 20091102. HZJW was stored at 4°C and diluted to the desired concentrations in distilled water at the time of administration.
Bacterial strains and cultivation
Helicobacter pylori strain ATCC 43504 (Vac A and Cag A positives) employed for the assay was obtained from American Type Culture Collection (Rockville, MD), and stored at −80°C in Muller–Hinton broth (OXOID) containing 15% (w/v) glycerol until experimentation. Frozen H. pylori isolate was thawed and grown on Brucella agar supplemented with bovine serum albumin (BSA) for 72 h at 37°C under a microaerophilic atmosphere (85% N2, 10% CO2, 5% O2) and 98% humidity. Each plate was swabbed with a sterile cotton-tipped applicator, and the tested organism inocula used for the dilution tests were prepared by suspending 72 h colonies in 2 mL of sterile distilled water (DW) to obtain turbidity equivalent to a 2.0 McFarland standard (~108 CFU/mL).
Experimental animals and maintenance
Sprague–Dawley (SD) rats (180 ± 20 g), Balb/c mice (18 ± 2 g), Kun-ming (KM) mice (18 ± 2 g) of both sexes at the initiation of treatment were obtained from the Medical Experimental Animal Center of Guangzhou University of Chinese Medicine and Evaluation and Research Center for Toxicology Institute of Disease Control and Prevention PLA. Licences for rats and mice were SCXK (YUE) 2008–0020, 2009–0210 and SCXK(JUN)2007-004, respectively. For the experiment, the animals were transferred for the laboratory and submitted to adaptation by period of 7 days. They were acclimatized under controlled temperature (25 ± 2°C) and humidity (50-70%) on a 12-h light/12-h dark cycle (artificial lighting from 08:00 to 20:00) and had free access to standard chow and drinking water. In all experiments, the animals were kept in cages with raised, wide-mesh floors to prevent coprophagy. The experimental protocols involved were in accordance to the rules and guidelines of the Experimental Animal Center of Guangzhou University of Chinese Medicine and, approved by the Animal Care and Use Ethics Committee of our institution, with reference to European Community guidelines for the use of experimental animals.
Chemicals and drugs
Aspirin was obtained from Bayer HealthCare AG (Lot. BTA8RR3), dexamethasone was obtained from Zhejiang Xianju pharmaceutical Co., LTD (Lot. 091032);ranitidine was purchased from O tevez (foshan) pharmaceutical Co., LTD (Lot. 0908521); amoxicillin was from Zhuhai federal pharmaceutical Co., LTD (Lot. 00800208). Azithromycin was purchased from Suzhou Changzheng Hinkay pharmaceutical Co., LTD (Lot. 100604); gentamycin was obtained from Henan topfond pharmaceutical Co., LTD (Lot. 100630950); ampicillin was obtained from Shandong Lukang pharmaceutical Co., LTD (Lot. L100708). Formaldehyde and phenol red were from Guangzhou Chemical Reagent Factory (Lot. 20100302 & 20091021). Hydrochloric acid, sodium hydroxide and alcohol were purchased from Guangzhou chemical reagent factory (Lot. 20091014, 20090401 & 20090304). H. pylori urease Immunogold Testing kit was from Beijing Tian Hong Sig biotechnology Co., Ltd (Lot. 010652); H. pylori infection test paper (Rapid Urease Test, RUT) was from Zhuhai Kedi science and technology development Co., Ltd (Lot. 110401). Scutellarin (Lot. 110842–200605), berberine hydrochloride (Lot. 110713–200910) and paeonol (Lot. 110708–200505) were purchased from National Institute of Food and Drug Control (Beijing, China) with purity over 98% based on HPLC analysis. HPLC-grade ethanol and acetonitril were obtained from Honeywell (Honeywell, USA). All reagents were at least of analytic grade and applied according to the specific instruction manual.
The experiment was carried out according to the method of Morimoto et al. [18
], with the following modifications. Rats of each sex were randomly divided into five groups of ten animals each. The first group was given 1 mL of vehicle (normal saline), and the second group was treated with ranitidine (250 mg/kg, p
.). The remaining groups received 260, 520 and 1040 mg/kg (p
.) of HZJW respectively. All the treatments were administered daily for 3 consecutive days. All rats were subjected to abrosia 24 h prior to the ulcerogenic challenge. On the 4th
day, one hour after the last administration, all rats received an oral dose of 1 mL of 0.15 M HCl in 60% ethanol to induce gastric ulcer. One hour post ulcerogenic challenge, all animals were sacrificed by cervical dislocation, and stomach of each rat was removed and inflated with 10 mL of 1% buffered formalin solution to fix for 10 min. Subsequently, each stomach was incised along the greater curvature and rinsed with normal saline to remove stomach contents, then the lengths of the necrotizing lesions were measured under a dissecting microscope, to access the formation of ulcers (hemorrhagic lesions).The maximum length of each lesion was determined and the sum of lengths of all lesions (mm) for each stomach was expressed as the ulcer index (UI), and the inhibition percentage was calculated by the following formula:
Nonsteroidal anti-inflammatory drug (NSAID)-induced ulcer
The experiment was carried out according to the method by Nwafor et al.  with a few modifications. Rats of either sex were randomly divided into five groups of ten animals each. The first group was given 1 mL of vehicle (normal saline), and the second group was treated with ranitidine (250 mg/kg, p.o.). The remaining groups received 260, 520 and 1040 mg/kg (p.o.) of HZJW respectively. All the treatments were administered daily for 3 consecutive days. All rats were subjected to fast 24 h before the ulcerogenic challenge. On the 4th day, one hour after the last administration, all the rats received an oral dose of aspirin (100 mg/kg) to induce gastric ulcer. Seven hours after the aspirin challenge, all animals were sacrificed by cervical dislocation, and stomach of each rat was excised and inflated with 10 mL of 1% buffered formalin solution to fix for 10 min. Thereafter, the greater curvature of each stomach was incised, and the extent of gastric damage in the glandular region was evaluated according to the ulcerative lesion index and inhibition percentage as described above.
In vitro anti-H. pylori assay
Agar dilution test was employed to analyze the susceptibility of reference strain H. pylori to HZJW in two-fold serial dilution in the range of 500–0.5 mg/mL. H. pylori were cultivated for 72 hours on blood agar, harvested and suspended in Brucella broth at a final concentration of 2.48 × 109 CFU/mL. Thereafter, a volume of 100 μL bacterial suspension was inoculated in the Brucella medium containing HZJW of serial decreasing concentrations (500–0.5 mg/mL). In addition to the tested agent, amoxicillin was employed as the positive control. Under the same condition, surfaces that were inoculated with a suspension of H. pylori without any tested materials and non-inoculated media were used as controls. Inocula were incubated at 37 °C under a microaerophilic atmosphere for 72 hours. Minimum inhibitory concentration (MIC), the lowest concentration that inhibits the visible growth of bacterium was determined. This experiment was performed in triplicate.
In vivo anti-H. pylori assay
Balb/c mice of either sex (except the normal group that was given normal saline) were pretreated with an oral dose of 0.5 mL antibiotic mixture (ampicillin 10 mg/mL, gentamicin 1.2 mg/mL, azithromycin 10 mg/mL) for 3 consective days, to make sure that they were free from any H. pylori-like organisms that could have been acquired through natural infection. On the 4th day, mice were intragastricly administrated with 0.5 mL freshly prepared suspension of H. pylori (108 CFU/mL) daily for 7 days. During this interval, mice were deprived of chew supplement but free access to drinking water was maintained for 12 h each day. On the second day following the last inoculation, the infected animals were randomly grouped according to the following doses: 520, 1040 and 2080 mg/kg HZJW, and were treated for 8 successive weeks (one administration per day), while the control group was given 20 mg/kg normal saline. Amoxicillin, suspended in 0.5% w/v CMC, was used as reference drug, and was administered orally to the mice for 8 successive weeks at a dose of 670 mg/kg body weight, which was approximately 10 times of the maximum recommended human dose. After the last administration, all mice were fasted for 24 h, blood samples were collected from retro-orbital and sacrificed by cervical dislocation. Serum was prepared and subjected to H. pylori urease Immunogold Testing kit to assess H. pylori clearance. All procedures were performed as described according to the manufacturers’ recommendation in the kit manuals.
At the same time, the stomach was excised, and the gastric mucosa tissue was submitted to rapid urease test (RUT). In brief, with the aid of tweezers, a fragment of gastric tissue was inserted in the centre of a minitube containing urease gel. Inoculation times were recorded. The minitubes were kept at room temperature and the colour change was evaluated within 3 min. Test result was considered strong positive if an alkaline reaction had developed (from yellow to dark pink within the tissue margin) in 1 min, and weak positive if in 3min, while negative if the colour failed to exhibit any colour variation within 3 min in the medium (yellow or light orange). The rest of the stomach tissues were fixed with a 10% formalin solution, dehydrated, embedded in paraffin, sectioned, deparaffinized, and stained with carbolic acid and basic fuchsin for H. pylori detection.
Acute toxicity test of HZJW
Acute oral toxicity study was performed according to fixed dose procedure. Two experimental groups of mice (10 mice of each sex in each group) were treated orally with a single dose of 0 and 18.0 g/kg body weight. Animals receiving the vehicle (saline) served as control. Animals were observed individually at least once during the first 30 min after administration, periodically during the first 24 h (with special attention during the first 4 h) and daily thereafter for a period of 14 days. The observation principally included changes in skin and fur, eyes and mucous membrane (nasal), autonomic changes (salivation, lacrimation, perspiration, piloerection, urinary volume, and defecation) and alterations of the central nervous system (ptosis, drowsiness, gait, tremors and convulsion). Food and water were provided throughout the experiment. For 14 days the animals were weighed and the number of deaths noted.
Experimental schedule for general toxicity of HZJW
To generally detect potential long-term, repeat-dose toxicity of HZJW and thereby define the characteristic, extent, dose and time-dependent relationship, as well as target organs and tissues of potentially toxic effects, 80 male and 80 female SD rats were assigned randomly and evenly to 4 experimental groups. The animals were separated by gender and housed five in each cage. Group I animals (control) were orally administered with distilled water throughout the course of the study. Animals in Groups II (1000 mg/kg body weight/day), III (2500 mg/ kg body weight/day), IV (5000 mg/kg body weight/day) received orally administered HZJW dissolved in distilled water once-daily for a period of 13 or 26 weeks. The dosages selected in the present study were based on existing data on the effective dose, results of acute toxicity study of HZJW in mice, and suggested human dose (equivalent to 10, 25, and 50 times of the normal human dose in clinical prescription, respectively). Clinical signs (general behavior, fur condition, breathing and nose conditions, eyes and oral secretions, urine and faeces), toxic reactions, and mortality were monitored daily after the initiation of HZJW treatment. Body weight, food and water consumption were recorded once a week. After 13 weeks, 5 rats per each sex in each group were sacrificed. Another 10 rats per each sex in each group were sacrificed at the end of the 26th week. The remaining rats were sacrificed after 4 weeks of recovery. The animals of the recovery groups were observed for reversibility, persistence and delayed occurrence of toxic effects. All examinations were conducted as described above.
At the end of the tested period, all animals were sacrificed and subjected to hematology and clinical chemistry assays, ophthalmic testing (cornea, conjunctiva, iris, pupil, atria, len, fundus, eyelid), necropsy examination, organ weighing, and histopathologic examination. This toxicity study was carried out in compliance with the Testing Guidelines for Safety Evaluation of Drugs (Notification [Z] GPT3-1 issued by China Food and Drug Administration (SFDA) on March 2005) and Good Laboratory Practice Regulations for Nonclinical Laboratory Studies.
Clinical observation and feed consumption
The animals were observed daily prior to and following administration for signs of toxicity and mortality throughout the experimental period. Detailed clinical signs were assessed and recorded, including changes in skin and fur, eyes and mucous membranes, manure, psyche states and behavior patterns, etc. The body weight was measured at the initiation of treatment and once a week during the treatment period thereafter. The amounts of feed were weighed before they were supplied to each cage and their remnants were measured the next day. The differences were calculated and regarded as daily feed consumption (g/animal/day).
Haematological and biochemical parameters
Samples of blood were obtained from all animals on Week 13 and Week 26 of the investigation, and after recovery. All rats were fasting but allowed access to water ad libitum for more than 12 h prior to blood sample collecting. Blood samples were collected into two tubes: (1) heparinized centrifuge tubes and (2) dry non-heparinized centrifuge tubes. The heparinized blood was used for a hematological determination, which include red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular HGB (MCH), mean corpuscular HGB concentration (MCHC), red cell distribution width (RDW), platelets (PLT), mean platelet volume (MPV), platelet distribution width (PDW), white blood cell (WBC) counts, reticulocytes (RET), neutrophils (NE), lymphocytes (LYM), monocytes (MON), eosinophils (EOS), and basophils (BAS) with a MEK-7222K Hematology analyzer (Nihon Kohden, Japan) according to the manufacturer’s operator manual. Plasma was isolated and used to determine the prothrombin time (PT) using a coagulometer (CA-50; Sysmex, Japan).
The non-heparinized blood was allowed to coagulate before being centrifuged and the serum separated. Serum was analyzed for changes in biochemistry using an automatic biochemical analyzer (RA-1000, Technico, USA), which measured aspartate aminotransferase activity (AST), alanine aminotransferase activity (ALT), alkaline phosphatase activity (ALP), albumin (ALB), total protein (TP), total cholesterol (CHOL), creatine phosphokinase activity (CPK), total bilirubin (T-BIL), direct bilirubin (D-BIL), creatinine (Cre), triglycerides (TG), urea (Ure) and glucose (GLU). The concentrations of sodium ions (Na+), potassium ions (K+) and chloride ions (Cl-) were measured with an electrolyte autoanalyzer (EasyLyte Na+/K+/Cl- Analyzer, MEDICA, USA). All parameters of blood chemistry and hematology were measured following standard procedures.
Organ weights, gross necropsy and histopathological examination
All animals were fasted overnight before scheduled necropsies. A complete necropsy was conducted on all animals and consisted of an external examination, including the identification of all clinically recorded lesions, and a detailed internal examination. The rats were anesthetized with diethyl ether and sacrificed by decapitation after blood collection from the abdominal aorta. After dissection to remove fat and connective tissue, the following organs were carefully dissected out and weighed: brain (cerebrum, cerecellum and brainstem), thymus glands, heart, lungs, liver, spleen, kidneys, adrenal glands, testis/ovary and epididymis/uterus. The relative organ weight was calculated based on the terminal body weight before fixation.
The following organs of all animals were fixed in 10% neutral formalin: brain, thoracic spinal cord, thyroid glands, Gley's glands, pituitary gland, thymus, adrenal glands, esophagus, salivary glands, stomach, small intestine (duodenum, jejunum and ileum), large intestine (cecum, colon and rectum), pancreas, spleen, lungs, trachea, kidneys, liver, heart, aorta, bladder, testes, epididymides, ovaries, uterus, prostate gland, vagina, mammary gland, sciatic nerve, urinary mesenteric lymph node, sternum, sub-mandibular lymph nodes, bone marrow.
The fixed organs of the vehicle control and high dose group and any organs from the other groups that displayed gross abnormalities were subjected to histopathological examination. These samples were Paraffin-embedded, microsectioned at a nominal thickness of 4 μm and then stained with hematoxylin and eosin for histopathological examination.
The data obtained by the various parameters was statistically evaluated by one way analysis of variance (ANOVA), and presented as means ± standard deviation (S.D.) for the indicated number of independently performed experiments. The body and organ weight, food and water consumption, hematological parameters, and blood biochemical parameters were analyzed for homogeneity of dispersion by ANOVA. The parameters found to be significant in ANOVA were assessed by the Dunnett test. Significant differences (as shown in the plots) were classified as: * for P < 0.05; more significant ** for P < 0.01.