In vitro and in vivo bactericidal activity of Tinospora sagittata (Oliv.) Gagnep. var. craveniana (S.Y.Hu) Lo and its main effective component, palmatine, against porcine Helicobacter pylori

TSG plants were collected from Mount Emei (Sichuan, China) between the months of September and October, 2014. The plants were identified and authenticated by Professor Qiao-jia Fan of the College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China. A voucher specimen (TSG2014) was deposited in the Veterinary Medicine Department of Sichuan Agricultural University. We prepared an extract of TSG from the plants according to reported methods [21]. Air-dried TSG (500 g) was refluxed with 95 % ethanol (v/v) three times each for 2 h. The solvent was completely removed in vacuo to generate a semisolid residue, which yielded 14.72 % (w/w) dry starting material. Stock solutions of the freeze-dried extracts were prepared for the initial screening by reconstitution in 1 % dimethyl sulfoxide (Amresco, America) to a final concentration of 1 g/mL.

Palmatine, amoxicillin, omeprazole, clarithromycin, and vancomycin were purchased from Shanghai Yuanye Bio-technology, Co., Ltd (Shanghai, China). Brucella broth, Columbia blood agar, and Mueller-Hinton agar were purchased from OXOID (Hampshire, UK).

One hundred seventy gastric samples were randomly selected from pigs sent to the Xingrui slaughterhouse (A29180101) in Ya’an, Sichuan Province, China, during August 28, 2014. Gastric tissue samples of 0.5 to 1 cm were obtained from the pyloric stomach immediately after slaughtering. The samples were placed in 3 mL of sterile Brucella broth with 30 % glycerol and transported rapidly to the laboratory.

The samples were homogenized in 1 mL Brucella broth with 30 % glycerol by the use of sterile mortar grinders. A 500 μL homogenate of each biopsy specimen was inoculated on selective agar plates (Columbia blood agar supplemented with 7 % fresh defibrinated sheep blood and 3 % vancomycin). Plates were incubated at 37 °C in a microaerobic atmosphere (5 % O₂, 10 % CO₂, 85 % N₂) for 3 to 7 days. Isolates were presumptively identified as H. pylori on the basis of colony morphology (gray, small, and translucent), Gram staining, rapid urease test and biochemical reactions (oxidase, catalase, and urease positive). The bacterial colonies were collected and prepared in Brucella broth with 10 % fetal calf serum for tests.

Susceptibility of H. pylori strains to TSG and palmatine was determined by use of the agar cup diffusion technique as described [22]. Inoculated plates (Mueller-Hinton agar plates containing 5 % sheep blood) were incubated at 37 °C in an incubator under microaerophilic conditions for 3 to 7 days, after which the diameters of the zones of inhibition (mm) were measured. One percent of dimethyl sulfoxide was included in each plate as a solvent (negative) control; amoxicillin was included as positive controls.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the use of agar dilution methods according to the Clinical and Laboratory Standards Institute as described [23, 24]. Briefly, the H. pylori strains were inoculated on Mueller-Hinton agar plates containing 5 % sheep blood for 3 to 7 days. The bacterial colonies were collected and prepared in Brucella broth with 10 % fetal calf serum. Inoculates were prepared at a density adjusted to a 1.0 McFarland turbidity (3 × 10⁸ CFU/mL) standard. Serial dilutions (1:2) of TSG were added to the Mueller-Hinton agar for final concentrations from 200 mg/mL down to 0.39 mg/mL, and palmatine and clarithromycin (as a positive control) were added to the Mueller-Hinton agar for final concentrations from 200 μg/mL down to 0.39 μg/mL. The no-drug containing agar and 1 % dimethyl sulfoxide agar served as negative controls. Agar plates were inoculated with H. pylori strains and cultured for 3 days. The MIC was regarded as the lowest concentration that prevented visible growth from a duplicate experiment, and the MBC was the lowest concentration that completely inhibited bacterial growth.

Bactericidal activities were evaluated by the use of time-kill curves with 0.5, 1, and 2 × MIC of drugs, and with blank, clarithromycin, and 1 % dimethyl sulfoxide controls. H. pylori SCYA201401 and H. pylori SS1 (0.1 mL at 1 × 10⁸ CFU/mL) were added to 90-mm plates with the calculated concentrations of drugs or dimethyl sulfoxide and Brucella broth with 10 % fetal calf serum (final volume 10 mL), and cultured with gentle shaking at 37 °C in a microaerobic atmosphere. At 0, 4, 8, 12, 16, 20, 24, 32, 40 and 48 h, 0.1 mL of liquid was removed, serially diluted, and plated on Columbia blood agar plates (n = 2 per group). Colonies were counted and averaged after 3 days of incubation [25].

A total 108 mice C57BL/6 mice (6–8 week old, male/female = 1:1, weight 25 ± 5 g) were purchased from the specific pathogen-free facility at Chengdu Dossy Experimental Animals Co., Ltd. (license No. SCXK, Sichuan, 2013–24). All experimental procedures involving animals were approved by the Sichuan Agricultural University Animal Care and Use Committee (registration No. SCAU2015101801). In accordance with the Guidelines of the International Committee on Laboratory Animals, the animals were maintained in environmentally controlled rooms at 20–25 °C with a relative humidity of 55 ± 5 % and 12 h light/dark cycle. Food and water were available ad libitum. Mice were provided with sawdust bedding material and housed under these conditions for one week, then fasted for 12 h prior to the experiments.

A group of 12 mice, selected randomly as blank control, were inoculated with sterile liquid medium (Brucella broth with 10 % fetal calf serum). A group of 48 mice (the SCYA201401experimental group) was inoculated intragastrically with 0.3 mL of H. pylori SCYA201401 (1 × 10⁸ CFU/mL) on three alternate days. Another 48 mice (the SS1experimental group) were inoculated intragastrically with 0.3 mL of H. pylori SS1 (1 × 10⁸ CFU/mL) on three alternate days. Animals were fasted 24 h before and 2 h after each inoculation. After 24 days, 8 mice from the SS1 experimental group, 8 mice from the SCYA201401experimental group, and 2 mice from the control group were sacrificed by cervical dislocation without anesthesia prior to the end of the experiment, and used for assessing the by use of a rapid urea test (RUT) and bacterial culture. Only if there was a 100 % infection rate (all 18 mice positive in the RUT and bacterial culture) was the experiment continued.

Mice of two experimental groups were randomly divided into four groups: TSG, palmatine, triple therapy (omeprazole, clarithromycin, and amoxicillin), and model groups. Drugs dosages used in in vivo experiments were equivalent to those that are used clinically. The TSG and palmatine groups were treated intragastrically with 4 g/kg and 50 mg/kg daily for 1 week, respectively. The triple therapy group was treated intragastrically with a suspension of omeprazole (0.8 μg/kg), clarithromycin (20 μg/kg), and amoxicillin (40 μg/kg) daily for 1 week [26]. The SS1 model group, the SCYA201401 model group, and blank control group were given saline solution.

Forty-two H. pylori strains were isolated from the 170 samples, a culture-positive rate of 24.7 %. A total of 128 biopsy samples were negative for H. pylori by cultivation. A clinical H. pylori strain, containing the gene of cagA and vacA, was named H. pylori SCYA201401and was used to following studies.

As illustrated in Table 2, the results of antimicrobial susceptibility tests (zone of inhibition, MIC, and MBC) showed that TSG and palmatine had good bactericidal activity in vitro. There was no significant different in susceptibility of H. pylori strains SCYA201401 and SS1. The two strains had no visible bacterial colonies after 32-h incubation on the plate containing 6250 μg/mL TSG. The H. pylori SCYA201401 had no visible bacterial colonies after 40-h incubation with 6.25 μg/mL palmatine, and H. pylori SS1 had no growth with 3.12 μg/mL palmatine. Those concentrations defined the MBCs of these agents. The results of the antimicrobial susceptibility tests revealed also that the bactericidal activity of palmatine against the H. pylori in vitro was significantly more than that of clarithromycin.

Figures 1 and 2 show the time-kill curves of H. pylori SCYA201401 exposed to TSG or palmatine. The average colony count of the blank control and dimethyl sulfoxide control groups increased progressively at every time point. In contrast, the colony counts of the TSG and palmatine groups decreased steadily and did so in a dose-dependent manner. Clarithromyin similarly killed H. pylori SCYA201401.

The establishment of the H. pylori-infected mice model was evaluated in two ways: identification of H. pylori isolated from gastric tissues and the RUT. The bacteria were identified via Gram staining, RUT, and oxidase test and catalase tests. These experiments documented that a H. pylori-infected model was successfully established.

H. pylori eradication rates in the mouse model, determined with RUT and bacterial culture, are presented in Table 3. TSG was as effective as triple therapy, but palmatine was modestly less effective than those two treatments.