Anti-Helicobacter pylori activity of acomplex mixture of Lactobacillus paracasei HP7 including the extract of Perilla frutescens var. acuta and Glycyrrhiza glabra

The effect of standard therapeutic strategies on Helicobacter pylori infection is diminished over time owing to the emergence of drug resistant strains. In this study, we would like to confirm the enhanced effect of L. paracasei HP7, which has been reported to exert antibacterial and gastric mucosal protective effects, in combination with Perilla frutescens var. acuta (P. frutescens)and Glycyrrhiza glabra (G. glabra) extracts. P. frutescens extract and G. glabra extract were found to inhibit the growth of H. pylori in a concentration-dependent manner, and the combination of L. paracasei HP7 and P. frutescens extract and G. glabra extract effectively inhibited H. pylori from attaching to AGS a gastric epithelial cells. Moreover, L. paracasei HP7 complex mixture containing P. frutescens and G. glabra extracts has been shown to inhibit H. pylori virulence genes such as AlpA, CagA, FlaA and UreA. When H. pylori-infected mice were administered a complex mixture of L. paracasei HP7 containing P. frutescens and G. glabra extract, the infection rate of H. pylori was significantly reduced. In addition, the L. paracasei HP7 complex mixture significantly reduced serum IL-8 levels and stomach inflammation in H. pylori infected mice. These results suggest that a complex mixture of L. paracasei HP7 containing P. frutescens and G. glabra extracts may be an alternative to treating diseases caused by H. pylori infection.


Introduction
Helicobacter pylori, a major causative pathogen of chronic gastritis [1] and gastric ulcers [2], is a spiral of gram-negative bacteria associated with an increased risk of gastric cancer [3,4]. Vaccination with antibiotics to remove gastric H. pylori can reduce H. pylori-associated gastrointestinal diseases [5,6] and reduce the risk of gastric cancer [7]. The standard recommended therapy for H. pylori uses two antibiotics, usually a triple combination therapy, including clarithromycin and a proton pump inhibitor with amoxicillin or metronidazole [8,9]. However, the efficacy of the triple therapy has currently reduced over time. Recent cure rates of less than 80% are mainly due to the increased prevalence of resistant H. pylori strains in metronidazole and clarithromycin [10][11][12]. In addition, some patients showed allergic side effects to antibiotics and can sometimes cause side effects if H. pylori is not treated [13]. Longterm vaccination with antibiotics is not recommended for the prevention of H. pylori infection. Therefore, it is important to develop new non-antibacterial agents for the treatment of H. pylori [14].
Lactobacillus spp. is recommended as an additive to the standard recommended treatment for H. pylori treatment, and it is possible to improve the patient's adaptability by reducing the side effects of antibacterial agents [15,16]. In our previous study, we reported that the lactic acid bacterium Lactobacillus paracasei HP7 (L. paracasei HP7) isolated from kimchi, a fermented vegetable dish widely consumed in Korea, had inhibitory effects against H. pylori in-vitro and in-vivo [17].
Recently, there has been a clear increase in demand for natural compounds from plant extracts that are effective antibacterial agents against a wide range of bacteria to control human infection and for the preservation of food [18]. Recently, the inhibitory effect of Glycyrrhiza glabra (G. glabra) on H. pylori and the therapeutic effect on infected patients have been reported [19][20][21]. In addition, antibacterial activities [22][23][24] and anti-inflammatory [25][26][27] effects of Perilla frutescens var. acuta (P. frutescens) have been reported.
In this study, we aimed to determine whether the combination of L. paracasei HP7 and P. frutescens and G. glabra extracts had a synergistic effect on the inhibition of H. pylori infection.

Herbal extract
Each of the herbal extracts of G. glabra and P. frutescens were obtained from Korea Yakult Co., Ltd.

Cell culture
Human gastric cell line AGS cells (human gastric adenocarcinoma) were obtained from the Korean Cell Line Bank (cellbank.snu.ac.kr) and used. For maintenance and proliferation of cells, passage was performed every 2 days at 37°C and 5% CO 2 using Ham's F-12 medium containing 10% FBS and 1% antibiotic. For analysis of H. pylori infection to gastric cells, antibiotics were not added to the culture medium.

H. pylori growth inhibition
To confirm the anti-H.pylori activity of P. frutescens and G. glabra extracts, Alamar blue assay was performed by referring to the study of Tsukasa M et al. [28]. H. pylori was suspended in DMEM / F-12 containing 5 mM L-lactic acid with a turbidity of 0.005 (1 × 10 5 CFU/mL). One hundred microliter suspension was added to 96 well culture plate and then incubated for 4 h at 37°C with the test material (P. frutescens and G. glabra extracts) under micro-aerophilic conditions. After incubation, inhibition of H. pylori growth was measured by Alamar blue according to manufacturer's criteria (Alamar Bio-Sciences, Sacramento, CA, U.S.A.). H. pylori inhibitory activity of the tested material was calculated by the following formula: A: cultured without test sample. B: cultured with test sample. C: medium alone.
Inhibition of H. pylori adhesion to AGS cells AGS cells were cultured in 6-well plates for 16 h. When the cells reached 90% confluence, the medium was replaced with serum and antibiotics-free F-12 medium. An overnight cultured H. pylori SS1 was suspended in Ham's F-12 medium. For co-culture of bacteria and gastric epithelial cells, H. pylori SS1 (10 7 CFU) were added to wells containing 10 6 AGS cells and incubated for 4 h in the absence or presence of herbal extracts and L. paracasei HP7. The adhesion of H. pylori was measured using a Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) as in our previous paper [17]. Forward and reverse sequences of primers for amplifying the H. pylori 16S RNA gene were as follows: 5′-TCG GAA TCA CTG GGC GTA A-3′ and 5′-TTC TAT GGT TAA GCC ATA GGA TTT CAC-3′.
Detect of H. pylori virulence gene expression H. pylori SS1 cells were cultured in brain-heart infusion broth at~1.0 × 10 7 CFU/mL. Cultured H. pylori were treated with G. glabra extract (3μg / mL), P. frutescens extract (25μg/mL), and L paracasei HP7 (1.0 × 10 7 CFU/ mL) and incubate at 37°C for 2 h. cDNA was synthesized using murine leukemia virus reverse transcriptasptase with random hexamer. Primer sequence for H. pylori virulence genes are listed in Table 1. AlpA is genes that H. pylori attaches to the gastric mucosa, and CagA plays the role of H. pylori invading gastric cells. FlaA is related to the mobility of H. pylori, and UreA is genes that H. pylori uses to neutralize gastric acid [29].

Animals
Specific pathogen free (SPF) male C57BL/6 mice weighing 20-24 g were purchased from Samtako Co. (Osan, Korea) and were maintained at the inspection facility of Wonkwang University (Iksan, Korea) for 1 week before experiments. Thereafter, the mice were maintained in an SPF barrier room with regulated temperature (23°C ± 1°C) and humidity (50% ± 5%) and a 12:12-h light/dark cycle. The animals were fed a sterilized pellet diet (Purina, Seoul, Korea) and sterilized water ad libitum. All studies were performed in accordance with the Guide for Animal Experimentation of Wonkwang University and were approved by the Institutional Animal Care and Use Committee of Wonkwang University (approval no. WKU 2019-08-22).

H. pylori inoculation
Animals were intragastrically inoculated three times, with a 3-day interval between inoculations, with H. pylori at~1.0 × 10 9 CFU in 0.5 mL broth. The challenged animals were confirmed as H. pylori-positive by stool antigen analysis using the Bioline H. pylori Ag kit (Standard Diagnostics, Suwon City, Korea) as previously described [30].

Blood analysis
Blood samples were collected from the hearts of sacrificed animals,centrifuged at 1000×g for 15 min at 4°C, and the isolated plasma was stored at − 80°C. Serum titers of anti-H. pylori antibodies were measured using a mouse anti-H. pylori antibody (IgG-1) ELISA kit (Cusabio Biotech, Wuhan, China) in accordance with the manufacturer's instructions. IL-8 levels in mice were measured using the Mouse Interleukin 8 ELISA Kit (R&D System, Minneapolis, USA) in accordance with the manufacturer's instructions.

Statistical analysis
Experimental results were compared between groups using Minitab (State College, PA, USA) and one-way ANOVA, a parametric multiple comparison procedure. The results were expressed as mean ± standard error and statistically significant when P < 0.05.

H. pylori growth inhibition
We measured the H. pylori growth inhibitory activity of 140 plant extracts including G. glabra and P. frutescens. Excluding non-edible plants, G. glabra and P. frutescens extracts showed the best inhibitory effect on the growth of H. pylori. In particular, G. glabra 90% ethanol extract and P. frutescens 50% ethanol extract showed high activity (data not shown).
There have been several reports of antibacterial and Helicobacter pylori inhibitory activity of P. frutescens and G. glabra [19][20][21][22][23][24]. However, there have been few reports of synergistic effects of H. pylori inhibitory activity of P. frutescens and G. glabra. Therefore, the H. pylori growth inhibitory activity of each of the P. frutescens extract (PFE) and G. glabra extracts (GGE) was investigated, and whether the two extracts had a synergistic effect on H. pylori inhibition was examined. PFE and GGE inhibited the growth of H. pylori in a concentration-dependent manner. PFE and GGE almost completely inhibited the growth of H. pylori at concentrations of 12.5 μg/mL and 50 μg/mL, respectively, and the IC 50 of each extract was 23.84 μg/mL and 2.88 μg/mL. (Fig. 1a). When the extract corresponding to IC 50 was co-treated, the growth of H. pylori was inhibited by about 90% (Fig. 1b). This suggests that P. frutescens and G. glabra are synergistic in inhibiting the growth of H. pylori.

Suppression of H. pylori adhesion to gastric epithelial cells
In a previous study, we confirmed that hp7 inhibits Helicobacter pylori adhesion to gastric epithelial cells [17]. The complex mixture of L. paracasei HP7 containing PFE and GGE significantly inhibited H. pylori adhesion to gastric cells than L. paracasei HP7 or PFE or GGE alone (Fig. 2a). These results demonstrate that L. paracasei HP7 and P. frutescens and G. glabra extracts are synergistic in inhibiting bacterial adhesion to gastric epithelial cells.
Inhibition of H. pylori virulence factor H. pylori produces urease to decompose the urea in the stomach, reduce the acidity around it, move using flagella, and attach to epithelial cells through adhesion factors such as AlpA. In addition, the CagA protein secreted by H. pylori inflames gastric epithelial cells and causes gastric cell changes known as the "hummingbird phenomenon" [1,2,29]. Therefore, we investigated the effect of a complex mixture of L. paracasei HP7 containing PFE and GGE on the mRNA expression of genes encoding AlpA, Cag, FlaA, and UreA of H. pylori. PFE significantly reduced ureA and GGE decreased alpA and cagA. HP7 significantly reduced flaA associated with H. pylori motility. Meanwhile, the HP7 complex mixture significantly reduced H. pylori virulence genes compared to PPE or GGE or HP7 alone (Fig. 2b).

Anti-H. pylori antibody titer in serum
To confirm the colonization of H. pylori in mice, the absorbance of IgG serum against H. pylori was also related to H. pylori colonization, so anti-Helicobacter IgG-1 serum levels were measured [31]. The serum antibody titers were elevated 4 weeks after H. pylori inoculation, to values of 1.48 ± 0.06, 0.94 ± 0.07, and 0.95 ± 0.04 in the H. pylori infection (Group C), positive control DGL (Group D), and H. pylori infection/L. paracasei.
These results indicated that H. pylori infection was significantly reduced by treatment with a complex mixture of L. paracasei HP7 containing extracts of PPE and GGE.

Alleviation of gastric mucosal lesions caused by H. pylori
Pathological changes in the gastric mucosa were minimal in animals not infected with H. pylori (Group NC). In contrast, Group C (H. pylori inoculated) mice exhibited gastric atrophy and severely shortened villi. However, mice in Group COM3 (H. pylori infected/L. paracasei HP7 + PPE + GGE) showed a significant improvement in gastric mucosa. These results were confirmed by an increase in villus length in Group COM3 compared with Group C (Fig. 5).
A new Lactobacillus spp. isolated from kimchi by Korea Yakult Co. Ltd. was identified as L. paracasei and was named strain HP7. Kimchi is considered a healthy food as it is enriched in vitamins A, B, and C, and is high in fiber, but also contains a number of lactic acid bacteria [37]. The two herbs selected in this study were P. frutescens var. acuta and G. glabra, which showed the strong antibacterial activity of H. pylori by measuring the Helicobacter antibacterial activity (growth suppression) in the extraction of natural product candidates through the inhibitory clear zone test of H. pylori (data not shown).
G. glabra (licorice) was reported to exhibit antimicrobial activity against several gram-negative and gram- Fig. 3 Results of Helicobacter pylori antibody (IgG) test with mice serum. *Significantly different from the infection control Group C (P < 0.05). **Significantly different from the infection control Group C (P < 0.01). ***Significantly different from the infection control Group C (P < 0.001) positive bacterial strains including H. pylori [38]. In addition, licorice also exerted beneficial effects against H. pylori through its antiadhesive properties [39]. Activity against ulcer and cancer, and clinical outcomes of H. pylori infection were also exhibited by licorice. The curative effect of deglycyrrhizinated licorice (DGL) on ulcers has been reported in vivo and in clinical studies [40][41][42], and the anticancer effect of licorice extract was shown in an in vitro study [43]. G. glabra was shown to possess anti-ulcerogenic properties that may be conferred by the cytoprotective mechanism of its antioxidant properties. These results supported the ethnomedical uses of licorice in the treatment of gastric ulcer [44].
In this study, we confirmed in vitro and in vivo experiments of H. pylori inhibitory activity of a L. paracasei HP7 complex mixture containing P. frutescens var. acuta and G. glabra extracts. P. frutescens extract and G. glabra extract inhibited the growth of H. pylori in a dosedependent manner, and the H. pylori growth inhibitory effect was increased when the two extracts were mixed at IC 50 concentration. In addition, the inhibitory effect of adhesion of gastric epithelial AGS cells of H. pylori by the L. paracasei HP7 or P. frutescens extract and G. glabra extract, when applied in a complex mixture, rather than each individually, was confirmed to be larger. Also, we confirmed the inhibitory activity of a complex mixture of L. paracasei HP7 including the extracts of P. frutescens and G. glabra against H. pylori in a mouse model; a rapid urease test of mouse stomachs showed decreased H. pylori colonization. Thus, the eradication of H. pylori reduced inflammation and epithelial damage in the stomach, although it is also possible that a complex mixture of L. paracasei HP7 including the extract of P. frutescens and G. glabra had direct antiinflammatory effects on the gastric mucosa.  Although triple therapy consisting of two antibiotics and a proton pump inhibitor is effective over a short term and helps to maintain patient compliance, many patients experience undesirable side effects such as diarrhea, epigastric pain, nausea, and bloating [45].
In comparison, a complex mixture of L. paracasei HP7, including the extracts of P. frutescens and G. glabra, is safe and therefore appropriate for the prevention and treatment of H. pylori infection. In this study, the therapeutic effect of a complex mixture of L. paracasei HP7 including the extract of P. frutescens and G. glabra, was partial, at 90%. However, H. pylori adhesion and a reduced inflammatory response was shown. Other researchers reported also that probiotics alone could not completely eliminate H. pylori, but could reduce the load of H. pylori in the stomach, and alleviate gastric mucosal inflammation [46,47]. Accumulating evidence suggests an important role of IL-8 in H. pylori infection-associated chronic atrophic gastritis, peptic ulcer and gastric cancer [48]. The suppression of IL-8 by a complex mixture of L. paracasei HP7, including the extract of P. frutescens and G. glabra, can potentially prevent H. pylori-induced gastritis and carcinogenesis in the stomach.
Previously, the results of our study reported that L. paracasei HP7 alone was able, to some extent, suppress H. pylori infection [17]. This study was performed to confirm the elevation effect of compounds mixed with P. frutescens and G. glabra extract, which are known to have antibacterial and gastric mucosal protective effects other than L. paracasei HP7.

Conclusions
The administration of a complex mixture of L. paracasei HP7 containing an extract of P. frutescens and G. glabra was more effective than that of L. paracasei HP7 alone or P. frutescens extract or G. glabra extract, and the administration of a higher antibacterial effect of H. pylori and inflammation induced by H. pylori or it was confirmed to reduce the damage to the mucous membrane. The mechanism of this action resulted from the inhibitory effect of L. paracasei HP7 on the adhesion of H. pylori to the gastric mucosa, the antibacterial effect and antioxidative effect of G. glabra and P. frutescens extract, and the increased secretion of gastric mucosal mucin. It can be assumed that the anti-H.pylori effect and the protective effect on the gastric mucosa were induced. Thus, a complex mixture of L. paracasei HP7, including the extract of Perilla frutescens and Glycyrrhiza glabra can be used to treat patients with gastric symptoms, including ulcers caused by H. pylori.
These results demonstrated that treatment with a complex mixture of L. paracasei HP7, including the extract of P. frutescens and G. glabra could inhibit the growth of H. pylori and is thus a promising treatment for patients with gastric symptoms, such as gastritis, that are caused by H. pylori infection. Fig. 6 Serum IL-8 levels of mice infected with H. pylori followed by treatment with complex mixture of L. paracasei HP7 and herbal extracts. **Significantly different from the infection control Group C (P < 0.01). ***Significantly different from the infection control Group C (P < 0.001)