In Vitro and In Vivo Antibacterial Activities of Patchouli Alcohol, a Natural-occurring Tricyclic

43 Pages·2017·7.8 MB·English

AAC Accepted Manuscript Posted Online 20 March 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.00122-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
In Vitro and In Vivo Antibacterial Activities of Patchouli
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Alcohol, a Natural-occurring Tricyclic Sesquiterpene against
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Helicobacter pylori Infection
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4 Xu YF a, Lian DW a, Chen YQ a, Cai YF a, Zheng YF b, Fan PL a, Ren WK a, Fu LJ a,
5 Li YC a, Xie JH c, Cao HYa, Tan Ba, Su ZR a,d,*, Huang P a,* D
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6 a School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, n
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7 Guangzhou, 510006, PR China d
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8 b Department of Mammary Disease, The Second Affiliated Hospital, Guangzhou fr
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9 University of Chinese Medicine, Guangzhou, 510120, PR China h
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10 c Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese ://
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11 Medicine Syndrome, The Second Affiliated Hospital, Guangzhou University of .
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12 Chinese Medicine, Guangzhou, 510120, PR China .
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13 d Dongguan Mathematical Engineering Academy of Chinese Medicine, Guangzhou /
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14 University of Chinese Medicine, Dongguan, 523808, PR China A
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15 * Corresponding author: Ziren Su, Address: School of Chinese Materia Medica, 2
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16 Guangzhou University of Chinese Medicine, 232# Wai Huan East Road, Guangzhou 1
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17 Higher Education Mega Center, Guangzhou 510006, China. Tel: [phone]. y

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18 Fax: [phone]. E-mail: [email protected]. e
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19 * Corresponding author: Ping Huang, Address: School of Chinese Materia Medica,
20 Guangzhou University of Chinese Medicine, 232# Wai Huan East Road, Guangzhou
21 Higher Education Mega Center, Guangzhou 510006, China. Tel: [phone].
22 E-mail: [email protected].
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23 Abstract
24 This study further evaluated the in vitro and in vivo anti-Helicobacter pylori activity
25 and potential underlying mechanism of patchouli alcohol (PA), a tricyclic
26 sesquiterpene. In in vitro assay, the capacities of PA to inhibit and kill H. pylori were
27 tested on three standard strains at different pH values and 12 clinical isolates. The D
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28 effects of PA on H. pylori adhesion (and its alpA, alpB and babA genes), motility (and n
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29 its flaA and flaB genes), ultrastructure and flagellation were investigated. Moreover, d
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30 the H. pylori resistance and post-antibiotic effect (PAE) of PA were determined. fr
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31 Furthermore, the in vivo effects of PA on H. pylori eradication and gastritis were h
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32 examined. Results showed that MICs of PA against three standard strains (pH 5.3–9) //
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33 and twelve clinical isolates were 25–75 and 12.5–50 μg/ml, respectively. The killing .
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34 kinetics of PA were time- and concentration- dependent, and its MBCs were 25–75 .
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35 μg/ml. Besides, H. pylori adhesion, motility, ultrastructure and flagellation were /
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36 significantly suppressed. PA also remarkably inhibited the expression of adhesion A
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37 genes (alpA and alpB), and motility genes (flaA and flaB). Furthermore, PA treatment 2
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38 caused a long PAE and less bacterial resistance as compared with clarithromycin and 1
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39 metronidazole. The in vivo study showed that PA can effectively eradicate H. pylori, y

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40 inhibit gastritis, and suppress the expression of inflammatory mediators (Cox-2, Il-1β, e
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41 Tnf-α and Inos). In conclusion, PA can efficiently kill H. pylori, interfere with its
42 infection process and attenuate gastritis with less bacterial resistance, making it a
43 potential candidate for new drug development.
44 Keywords: Patchouli alcohol; Helicobacter pylori; anti-adhesion; anti-motility;
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45 anti-gastritis.
46 INTRODUCTION
47 Helicobacter pylori is a Gram-negative and spiral bacterium proved to be a major
48 pathogen causing chronic gastritis, ulcer, and even gastric carcinoma (1, 2). Although
49 triple therapy (combined use of proton-pump inhibitor, clarithromycin (CLR), and D
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50 metronidazole (MET) or amoxicillin) showed a reliable therapeutic effect against H. n
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51 pylori, the prevalence of antibiotic-resistant H. pylori became a worldwide concern d
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52 (3). The eradication rate in patients who accepted triple treatment once has decreased fr
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53 (4). In addition, antibiotics can seriously disturb the microbiome, resulting in other h
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54 diseases (5). Therefore, alternative therapeutic methods should be explored in clinical //
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55 treatment. .
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56 .
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57 The motility and adhesion of H. pylori are essential factors influencing its /
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58 colonization. To avoid gastric acid and search for a suitable proliferation environment, A
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59 the flagella of H. pylori consisting of FlaA and FlaB proteins provide strong motility 2
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60 to penetrate gastric mucus (6, 7). During this process, some adhesive proteins from 1
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61 the H. pylori membrane are effected and form a strong attachment to gastric epithelial y

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62 cells (8). Outer membrane proteins including blood group antigen-binding adhesion e
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63 (BabA), adherence-associated lipoprotein A (AlpA) and adherence-associated
64 lipoprotein B (AlpB) reportedly contribute to this process (9). Therefore, H. pylori
65 could be eradicated by suppressing its motility or adhesion.
66
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67 Pogostemonis Herba, the dried aerial part of Pogostemon cablin (Blanco) Benth.
68 (Labiatae), has been used to treat gastrointestinal diseases for thousands of years in
69 many Asian countries (10). The major active ingredient in Pogostemonis Herba is
70 patchouli alcohol (PA, the structure is shown in Fig. 1). Modern researches revealed
71 that Pogostemonis Herba and its extract exhibited anti-bacterial, anti-inflammatory, D
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72 gastroprotective, and anti-emetic effects (11–15). In our previous study, PA exerts n
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73 gastroprotective and H. pylori urease inhibitory effects (16, 17). Besides, PA protects d
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74 against urease-induced cytotoxicity in gastric epithelial cells (18). fr
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75 h
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76 In this follow-up study, we endeavored to further illustrate the anti-H. pylori property //
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77 and the potential mechanism of PA from other aspects. First, the minimum inhibitory .
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78 concentration (MIC), minimum bactericidal concentration (MBC) and killing kinetics .
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79 experiments were undertaken to evaluate the anti-H. pylori effect of PA. Second, the /
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80 possible effects of PA on the motility, adhesion and ultrastructure of H. pylori were A
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81 investigated. Third, the bacterial resistance and post-antibiotic effect (PAE) of PA on 2
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82 H. pylori were examined. Finally, the bacterial eradication rate and effect of PA on H. 1
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83 pylori-induced gastritis were investigated in vivo. y

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84 RESULTS e
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85 MIC and MBC of PA against H. pylori
86 The MICs of PA against the three standard H. pylori strains of NCTC11637,
87 NCTC26695 and SS1 were 25–75 μg/ml at pH 5.3–9. Twelve clinical strains were
88 obtained from three sites in China, and the MICs were 12.5–50 μg/ml. Furthermore,
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89 the MBCs of PA against three standard strains were 50 μg/ml, and 25–75 μg/ml
90 against eleven clinical strains. (Tables 2 and 3). The effective bacterial inhibitory
91 effect of PA was also observed in the CLR resistance (Hp 1870 and Hp 1876) and
92 MET resistance (Hp 1869, Hp 1870, Hp 1871 Hp 1872 and Hp 1876) isolates.
93 Killing kinetics analysis of PA against H. pylori D
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94 As shown in Fig. 2, the time- and dose-dependent killing kinetics of PA were n
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95 observed in two H. pylori standard strains (NCTC11637 and SS1). Under both weak d
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96 acidic and neutral conditions, PA at 2–5 times MIC showed a reliable bactericidal fr
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97 ability. For NCTC11637, PA at 5 times MIC cleared all H. pylori in only 15 min at h
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98 pH 5.3 and 7. For SS1, PA at 5 times MIC killed H. pylori in 15 min at pH 7. The time //
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99 required to kill H. pylori shortened with increasing PA concentration. These data .
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100 suggest that PA was efficaciously bactericidal against H. pylori in a dose- and .
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101 time-dependent manner under different pHs. /
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102 Effect of PA on H. pylori adhesive capacity A
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103 Anti-adhesive ability experiment was carried out in three different ways, including H. 2
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104 pylori pretreatment with PA, GES-1 cell pretreatment with PA and the three above 1
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105 cocultures simultaneously (Supplementary Material, point 5 Fig. S2). The adhesive y

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106 bacteria markedly decreased using the first and third incubation methods, indicating e
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107 that the effect of PA against H. pylori adhesion might be closely associated with H.
108 pylori itself. The results of H. pylori pretreatment with PA were shown in Figs. 3-A–
109 3-D, and green fluorescence indicated FITC-labeled H. pylori. Compared with the
110 control, the values of fluorescent area/cell area were significantly decreased with the
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111 treatment of PA at 5, 10 and 20 μg/ml for 1 h (P < 0.01 or P < 0.05), indicating that
112 PA could remarkably inhibit the adhesion of H. pylori to GES-1 cells (Fig. 3 E).
113 While CLR and MET exerted no significant effects on the adhesive capacity of H.
114 pylori.
115 Effect of PA on H. pylori motility D
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116 In 3 days’ cultivation, H. pylori could use its flagella to move in the swarm agar plate. n
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117 In the control group, obvious diffusion traces of H. pylori were observed (13.42 ± d
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118 1.44 mm2). By contrast, PA treatment (10 or 20 μg/ml) significantly decreased the fr
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119 diffusion in a concentration-dependent manner, with diffusion areas of 6.12 ± 0.80 h
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120 mm2 and 5.65 ± 0.32 mm2 (P < 0.05), respectively (Fig. 4). CLR and MET bot ://
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121 presented with remarkable anti-motility capacity on H. pylori (P < 0.01), which was .
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122 in accord with the previous report (19). .
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123 Effect of PA on H. pylori flagella formation /
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124 As shown in Fig. 5, most H. pylori cells displayed normal morphology of long A
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125 flagella and spiral shape. However, treatment with 10 μg/ml PA decreased the number 2
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126 of H. pylori flagella. Meanwhile, 20 μg/ml PA significantly decreased the formation 1
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127 of H. pylori flagella, and the formed flagella were obviously shorter than the normal y

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128 ones. The result suggested that PA can suppress the flagellation of H. pylori. e
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129 Effect of PA on H. pylori ultrastructure
130 As shown in Fig. 6, H. pylori displayed normal spiral and coccoid morphology after
131 exposure to DMSO (control) for 1 or 2 h (A-1–A-4). However, treatment with 25
132 μg/ml PA for 1 h evidently reduced the numbers of normal spiral rod isoforms, and
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133 led to bacterial cell lysis and bleb formation (B-1 and B-2). Furthermore, after 2 h,
134 bacterial cell lysis was aggravated and most H. pylori cells became coccoid (B-3 and
135 B-4). Treatment with 50 μg/ml PA for 1 and 2 h induced considerable conversion
136 from spiral to coccoid morphology. Thus, spiral H. pylori cells were rarely observed.
137 Most H. pylori cells became swollen, and the separation between the cell wall and the D
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138 inner membrane was also observed (C-1 and C-3). Meanwhile, some bacterial cells n
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139 were shown with cell wall damage and lysis of the cytoplasmic membrane (C-2 and d
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140 C-4). fr
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141 Effect of PA on H. pylori adhesion and motility-related gene expression h
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142 As shown in Figs. 7A–7C, treatment with PA at 5, 10, or 20 μg/ml significantly //
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143 downregulated the expression levels of alpA and alpB, two important H. pylori .
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144 adhesion-related genes (all P < 0.01). Nevertheless, PA exerted no significant effect .
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145 on babA gene expression (P > 0.05). Besides, as shown in Figs. 7 D and 7 E, /
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146 treatment with PA at 10 and 20 μg/ml significantly decreased the expression levels of A
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147 H. pylori motility-related genes flaA and flaB (P < 0.01). However, CLR and MET 2
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148 exerted no significant effects on the expression levels of adhesion- and 1
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149 motility-related genes of H. pylori. y

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150 H. pylori resistance toward PA e
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151 As shown in Table 4, the MICs of MET, CLR and PA against the three standard
152 original strains were shown in the first line at the table. The MICs of MET, CLR and
153 PA against three control groups remained constant (data not shown). The MICs of
154 MET against NCTC11637 and SS1 both increased from 0.5 to 512 μg/ml after 30
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155 passages. The MICs of CLR against NCTC11637 and SS1 increased from 0.0156 to
156 0.2496 μg/ml after 12 passages. And the MICs of MET and CLR against NCTC26695
157 increased from 0.5 to 256 μg/ml and 0.0156 to 7.9872 μg/ml after 27 passages,
158 respectively. However, the MICs of PA against all tested strains remained the same
159 after 9 passages at 12.5 μg/ml (1/2MIC), indicating that PA carries a small risk of D
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160 inducing bacterial resistance. n
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161 Post-antibiotics effect of PA on H. pylori d
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162 As shown in Fig. 8, pre-treatment with the three drugs for 2 h obviously delayed the fr
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163 logarithmic phases of H. pylori. In addition, the PAE times for twofold MIC MET and h
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164 CLR were 6.3 and 12.4 h, respectively. For PA, at 1.5 MIC, the PAE time was 7.5 h. //
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165 However, when the concentration was raised to twofold MIC, H. pylori lost its .
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166 proliferation ability after 2 h PA treatment, indicating the prolonged PAE induced by .
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167 PA. /
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168 Effect of PA on H. pylori eradication A
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169 Establishment of H. pylori infection was also confirmed by PCR assay using DNA 2
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170 isolated from H. pylori-infected stomach tissues and in vitro culture in blood agar 1
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171 plates. (Supplementary Material, Fig. S3). As shown in Table 5, the results of RUT y

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172 showed that the clearing rates of triple therapy and PA (5 mg/kg) were 100% and 80%, e
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173 respectively (P < 0.01 or P < 0.05, Chi-Square test). While the results of BAMB
174 staining indicated the clearing rate of the triple therapy and PA (5 mg/kg) were both
175 40%, suggesting that the H. pylori detection sensitivity of histologic section was
176 higher than that of RUT (Supplementary Material, Fig. S4). Therefore, PA could
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177 effectively attenuate H. pylori colonization in C57BL/6 mice.
178 Quantitative analysis of cytokine gene expression
179 As shown in Fig. 9, the expression of the inflammatory genes Cox-2, Inos, Il-1β and
180 Tnf-α were significantly up-regulated in model group as compared with control (P <
181 0.01). Administration with triple drugs and PA for two weeks significantly decreased D
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182 the gene expression of Inos, Il-1β and Tnf-α (P < 0.01) in the stomach mucosa. In n
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183 addition, PA remarkably down-regulated the gene expression of Cox-2 (P < 0.01). d
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184 The effect of PA on the gene expression of the tested cytokines was stronger than that fr
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185 of the positive control. Hence, PA can exert anti-inflammatory effects on H. h
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186 pylori-induced gastritis in mice by suppressing inflammatory mediators. //
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187 DISCUSSION .
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188 Owing to the antibiotic-resistance and declining eradication rate of H. pylori in clinic, .
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189 more alternative therapeutic protocols are urgently necessary (20). In the present /
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190 research, we aimed to investigate the anti-H. pylori activity and mechanism of PA. A
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191 The results of MIC, MBC and killing kinetic assays showed the effective anti-H. 2
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192 pylori abilities of PA, and indicated the therapeutic effect of PA against clinical 1
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193 antibiotic-resistant strains. For acid MIC experiment, pH 5.3 was employed since H. y

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194 pylori usually lives under mildly acidic condition between gastric epithelial cell and e
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195 the gastric mucosa with pH ranging from 5.3 to 6.9 (21, 22). Potent antibacterial
196 activity of PA was also found even in low-pH environments.
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198 In our previous investigation, PA was shown to arrest the growth of H. pylori by
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199 inhibition of its urease (17). In continuation of our work, the potential effect of PA on
200 the two vital infections processes–motility and adherence of H. pylori were analyzed.
201 We determined the non-toxic concentration of PA on H. pylori and GES-1 cell and
202 found that PA (5, 10, 20 μg/ml) exerted no significant effects on H. pylori and GES-1
203 proliferation (Supplementary Material, Fig. S5). Adhesive proteins BabA and AlpAB D
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204 are the key factors in H. pylori adhesion, that specifically bind to the cell surface n
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205 proteins LewisB and laminin, respectively (23, 24). Result showed that PA d
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206 significantly suppressed the adherence of H. pylori to the GES-1 cell, and inhibited fr
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207 alpA and alpB gene expression. In addition, the results of swarm agar plate, Q-PCR h
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208 and SEM assay showed that PA significantly decreased H. pylori motility and //
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209 downregulated the expression of flaA and flaB. Flagella movement is driven by the .
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210 energy supplied by a [H+] gradient, which is triggered by urea hydrolysis (25). In this .
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211 context, the significant inhibition of PA on H. pylori motility might be associated with /
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212 the urease inhibitory effect of PA observed in our previous investigation. The A
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213 inhibitory effect of PA on the docking process of H. pylori to the stomach tissue and 2
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214 bacterial motility could be important mechanisms against H. pylori infection and 1
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215 colonization, leading to a diminished incidence of infection. y

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216 e
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217 The effect of PA on H. pylori ultrastructure was further investigated via TEM. The
218 physiologically unfavorable conditions for H. pylori such as avoidance of its
219 metabolic pathways and/or a lack of energy source result in transformation from the
220 spiral to the coccoid form (26). PA treatment reduced the spiral rods and promoted the
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