Activity of Amomum tasao-ko Fruits Essential Oil against Methicillin-Resistant Staphylococcus aureus in vivo

• OH: Hydroxyl radical

MRSA:  Methicillin-Resistant Staphylococcus Aureus

SPF:        Specific Pathogen-Free

MLD:     Minimum Lethal Dose

PH:         Preventative High dose

PM:        Preventative Medium dose

PL:          Preventative Low dose

TH:         Treatment High dose

TM:        Treatment Medium dose

TL:         Treatment Low dose

TNF-α:   Tumor Necrosis Factor alpha

IL:          Interleukin

GSH-Px:                Glutathione Peroxidase

MDA:     Malondialdehyde

PVL:       Panton-Valentine Leucocidin

ROS:      Reactive Oxygen Species

GSH:      Glutathione

In the early 1940s, before the introduction of penicillin for the treatment of the disease, blood infection caused by Staphylococcus aureus was usually fatal. The use of penicillin in controlling the infection resulted in the emergence of β-lactamase-producing S. aureus [1,2]. Thus, new drugs were developed against S. aureus infection, such as methicillin and oxacillin. Methicillin-Resistant S. Aureus (MRSA) has appeared as the abuse of methicillin [3]. At present, MRSA is one of the most important bacterial pathogens based on its incidence and the severity of the associated infections [4]. MRSA can transmit between humans and animals [5] and infection occurs mainly through surgical wounds, pneumonia, and trauma [6-9]. Because of the increased susceptibility and drug resistance of MRSA, it is necessary to develop new anti MRSA drugs. 

Botanical medicines were widely used in the prevention and treatment of various diseases for a long time [10,11]. Amomum tsao-ko, which is widely distributed in the south-west regions of China and Korea, is used for treatment of hemorrhoids, flatulence, vomiting, diarrhea, malaria, throat infections, stomach disorders, dyspepsia, nausea, and abdominal pain in China. It also is used in many culinary dishes, such as Chinese medicinal soup dishes, herbal beef dishes, and hotpots [12-15]. Several researchers have conducted pharmacological studies on A. tasao-ko oil and identified that it has anti-inflammatory, antioxidant, antitumor, antihepatitis B, antimicrobial, anti-apoptosis activities [16-21]. Our previous studies found that A. tasao-ko essential oil had strong anti-MRSA activity in vitro [22], so this study focused on the anti-MRSA activity of A. tasao-ko essential oil in vivo. We observed the protective effect of A. tasao-ko essential oil on mice by intraperitoneal injection of MLD MRSA. The in vivo anti-MRSA mechanism due to its bacterial inhibitory activity and decrease the inflammatory level and oxidative stress in mice. The ED₅₀ also calculated and these data make a foundation for further development of A. tasao ko essential oil.

Materials 

Vancomycin hydrochloride for injection was obtained from VIANEX S.A. (PLANT C) (Greece). Nutrient agar was purchased from Beijing Aobo Star Bio-technology Co., Ltd (China). Gastric mucin was obtained from Sigma-Aldrich (United States). Sodium chloride injection (0.9%) was purchased from Sichuan Kelun Pharmaceutical Co., Ltd (China). The inject able soybean oil was obtained from Zhejiang Tian Yu Shan Pharmaceutical Oil Co., Ltd (China) and MRSA was obtained from the American Type Culture Collection (ATCC 43300).

The extraction of Amomum tsao-ko essential oil

A. tsao-ko (1000 g) was purchased from Tong Ren Tang Co., Ltd (Beijing, China) and identified by Prof. Min Li at the Chengdu University of Traditional Chinese Medicine. The extract and GC-MS analysis method of A. tsao-ko essential oil were described in our previous study, and a total of 32 compounds, representing 81.89% of the essential oil, were identified, the major compounds of essential oil were 1,8-cineole (25.92%) and geraniol (13.69%) [23]. Air-dried A. tsao-ko (200 g) was subjected to hydro distillation for 4 h in a modified Clevenger-type apparatus with a water-coupled oil receiver to obtain the essential oil (approximately 4 mL). The oils were dried over sodium sulfate and stored in air-tight glass bottles in a refrigerator prior to experiments.

Animals 

All animal experiments were performed in accordance with the guidelines and approved by Chengdu Medical College Animal Care and Use Committee. A total of 160 Specific Pathogen-Free (SPF) strains of both male and female KM mice (weight: 18-22 g) were purchased from Chengdu Institute of Biological Products Co., Ltd (Chengdu, China) and acclimatized for 3 days Zprior to the start of the experiment. All animals were housed in cages (five animals of the same sex per cage) maintained at a constant temperature (25 ± 2 °C) and humidity (50 ± 10%) and given free to access food and water throughout the study.

Methods 

The experiments on antibacterial activity were conducted according to a previously described method, with a little modification [24]. Briefly, the mice were randomly divided into 16 groups (n = 10). The blank (control) group and the model group were infected with 6×10⁴ CFU/10gMRSA , two positive control groups (the mice were injected with vancomycin hydrochloride (0.30g/kg) before or after infection), gastric mucin control group (in which the mice were injected with the same amount of 5% gastric mucin), the oil solvent control group (in which the mice were injected with the same vehicle oil for injection), and the groups treated with A. tsao-ko essential oil. The A. tsao-ko essential oil was diluted with inject able soybean oil at different concentrations, with five pretreatment groups and five treatment groups (Figure 1). For the five experimental pretreatment groups, all the experimental doses design is based on our preliminary experiment and the ineffective and the full cure dose were 0.05, 0.07, 0.16, 0.24 and 0.37g/kg respectively. For the five experimental treatment groups, the dosage of reagents was 0.08, 0.13, 0.20, 0.30, 0.47g/kg. After the end of the experiment, blood samples were collected from the surviving mice, and labeled. 

Figure 1: Schematic diagram of administration scheme.

Determination of cytokines 

Blood obtained from the mice was centrifuged and the supernatant was separated to measure the levels of three cytokines Tumor Necrosis Factor alpha (TNF-α), Interleukin-6 (IL-6), and Interleukin-1 beta (IL-1β) by using ELISA kits (Elabscience Biotechnology Co., Ltd, China). The quantification of cytokines and the experiments were conducted in accordance with the manufacturer’s instructions. And the data were recorded by Thermo Scientific™ Varioskan™ LUX multimode microplate reader. In the pretreatment groups, the 0.37 g/kg, 0.24 g/kg and 0.16 g/kg dose groups were labeled as Preventative High dose (PH), medium dose (PM), low dose (PL), respectively. In the treatment groups, the 0.46 g/kg, 0.30 g/kg and 0.20 g/kg dose groups were labeled as high dose (CH), medium dose (CM) and low dose (CL), respectively.

Determination of oxidative factors 

The activity of Glutathione Peroxidase (GSH-Px) and the contents of Malondialdehyde (MDA), Hydroxyl radicals (·OH) in serum was determined according to the instructions of kits. Bloods were collected and its content was measured by GSH kits, MDA kits, and ·OH kits (Naijing Jiancheng Bioengineering Institute). And the data were recorded by Thermo Scientific™ Varioskan™ LUX multimode microplate reader.

Statistical analysis 

The ED₅₀ of A. tsao-ko essential oil against MRSA was calculated by Karber’s method. Other statistical analyses were analyzed by using SPSS. One-way ANOVA was used to evaluate the effects of each variable and to determine the statistical significance. Values of P < 0.05 and P < 0.01 were considered significant and very significant, respectively.

Prophylactic activity of Amomum tsao-ko oil 

In a pilot study, we discussed the effect of pretreatment time of A. tsao-ko essential oil on the survival rate of mice; we found that there was no significant difference in injection with A tsao-ko oil for 5 days or 3 days. Thus, we selected the pretreatment time was 3 days. A. tsao-ko essential oil showed potent in vivo activity against the injections of MRSA (Figure 2) as both a preventive and a treatment. The survival rates of mice raised along with the incensement of the injected doses, and the calculated ED₅₀ was 0.15 mg/kg. When the injected dose of A. tsao-ko essential oil was 0.37mg/kg, it can protect the entire mouse from MRSA infection. 

Figure 2: Survival rate of mice.

Notes: A: In vivo anti-MRSA activity of Amomum tsao-ko essential oil pretreatment administered by intramuscular injection; B: In vivo anti-MRSA activity of Amomum tsao-ko essential oil treatment administered by intramuscular injection. B, blank group; M, model group; P, positive group.

Treatment activity of tsao-ko oil 

For the treatment of MRSA infection, this oil demonstrated a good therapeutic effect and showed a significant dose-dependent relationship (Figure 2B). At different injected doses of 0.47 g/kg, 0.30 g/kg, 0.20 g/kg, 0.13 g/kg, and 0.08 g/kg for injection, the survival rates of mice infected with MRSA were 100, 70, 50, 40 and 10%. By using Karber’s method, the calculated ED₅₀ was 0.18 g/kg.

The effect of tsao-ko essential oil on the inflammatory factors in mouse serum 

The effect of A. tsao-ko essential oil on the levels of the inflammatory cytokines of TNF-α, IL-6, and IL-1β was measured in the serum of survived mice by using enzyme-linked immunosorbent assays. As shown in Figure 3, after MRSA infection, the level of TNF-α, IL-6 and IL-1β increased significantly. After the treatment of A. tsao-ko essential oil the contents of TNF-α, IL-6, and IL-1β was reduced significantly. We also found that the contents of TNF-α, IL-6 and IL-1β decreased along with the increasement of the injection dose of A. tsao-ko essential oil. There was no significant difference in the contents of TNF-α, IL-6 and IL-1β between the high dose group and the control group regardless of the methods of administration. These results indicated that A. tsao-ko essential oil showed obvious anti-inflammatory properties except for the antimicrobial activities. 

Figure 3: Effect of Amomum tsao-ko essential oil on inflammatory factors in MRSA-infected blood.

Notes: A: IL-1β factor blood concentration; B: IL-6 factor blood concentration; C: TNF-α factor blood concentration. Compared with the blank control group, * <0.05, ** <0.01.

Effects of Amomum tsao-ko essential oil on oxidative stress 

Oxidative stress refers to the exposure of cells to high concentrations of reactive oxygen species caused by cell damage. As shown in figure 4, A. tsao-ko essential oil increased the activity of GSH-Px, reduced the levels of MDA and hydroxyl radicals, especially the activity of GSH-Px after high dose of A. tsao-ko essential oil treatment, was increased significantly when compared to the control group, which indicates that the A. tsao-ko essential oil showed antioxidant activity except for the antimicrobial activity. The activity of GSH-Px and the contents of MDA and hydroxyl radicals showed significant dose-dependent effect. High doses restored oxidative stress markers to normal levels.

Figure 4: Effect of Amomum tsao-ko essential oil on oxidative stress factors.

Notes: A: concentration of GSH-Px in the blood; B: concentration of MDA in the blood; C: concentration of ·OH in the blood. Compared with the blank control group, * <0.05, ** <0.01.

S. aureus causes a wide range of infections, such as skin and soft tissue infections, implant infections, pneumonia, septicemia, and toxic shock syndrome, but can also be the source of more serious bacteremia [25,26]. In recent years, several researchers are looking for natural products as potential antibacterial agents in the treatment of MRSA infection. Several essential oils which was extracted from several plants has got researchers attention due to its excellent antimicrobial activity, such as patchouli, tea tree, geranium, lavender essential oils, citricidal and wilhelmsii essential oil [10,27]. These essential oils have good anti-MRSA activity in vitro but have no antibacterial activity in vivo. Compared with in vitro bioactivity study, in vivo bioactivity study is more instructive for clinical use, which is also the significance of this study. In this study, we explored the exact efficacy of A. tsao-ko essential oil against MRSA infection by establishing a model of systemic infection in mice. The result showed that both the prevention and treatment modes of administration resulted in a 100% survival rate. Studies have shown that the antibacterial activities of various plant extracts are related to their monomer components, for example, quinolone derivatives [28]. The main components in the essential oil of A. tsao-ko were 1,8-cineole (25.92%) and geraniol (13.69%) [23] and some research showed that geraniol was the main active ingredient [29-31]. In the follow-up studies, we will continue to focus on the anti-MRSA activity of the monomers in the essential oil of straw fruit and hopefully find more effective anti-MRSA drugs. 

In previous studies, MRSA infection was reported on the surface of the body and can cause inflammation [32,33]. MRSA can express Panton-Valentine Leukocidin (PVL), which is an additional cause for concern [25], the infection often causes an inflammatory response. During the process of infection in diseases, the concentration of cytokines and disease severity and prognosis are related [34]. Among them, IL-6 is considered a sign of inflammatory response [35]. Compared with the blank group, the inflammatory factors of the model group were significantly increased (P < 0.01). MRSA infection was found to cause this inflammatory reaction in mice and increased the inflammatory factors. In the group administered the higher dose, the factors were restored to the level of the control group (P > 0.05); the high dose resulted in significantly more reduction than the lower dose, which showed a significant dose-effect relationship. Recent studies have shown that two novel components isolated from the dried fruits of A. tsao-ko displayed significant in vitro anti-inflammatory activity in a dose-dependent manner [36]. This finding was also observed in this in vivo study. 

Under normal physiological conditions, cells have antioxidant systems that protect against the harmful effects of Reactive Oxygen Species (ROS) [37]. Studies have shown that oxidative stress is associated with a variety of diseases [38]. When the body is subjected to oxidative stress, the main changes in the endogenous oxidation system are an increase in MDA and decreases in SOD, GSH-Px, CAT activity of T-AOC and the endogenous antioxidant system; they were also important markers of oxidative stress [39-41]. In this study, when the model group was compared with the control group, GSH decreased but MDA and ·OH increased, which indicated that the oxidation reactions of the mice in the model group were outside of the normal range (i.e., oxidative stress). In the absence of any drug in the model group, the oxidative stress response was not improved, which lead to the death of the mice. In the experimental group, the oxidative stress factor improved and the oxidative response improved compared with the model group. The high dose oil restored oxidative stress to a normal level (P > 0.05). Therefore, A. tsao-ko essential oil has an ameliorative role on antioxidant stress in vivo. This may be related to geraniol, it has the effect of regulating oxidative stress [42].

The present study found that A. tsao-ko essential oil has strong anti-MRSA activity in vivo and exerts anti-inflammatory activity through the reduction of inflammatory factors. Based on these results, A. tsao-ko essential oil appears to show strong potential as an anti-MRSA drug for the treatment of various human and animal diseases.

The study was supported by a grant from the Fund of the National Natural Science Foundation of China (no. 31970137), the Benefit People Project of Science and Technology of Chengdu Science and Technology Bureau (no.2016-HM01-00362-SF), the Province Training Program of Innovation and Entrepreneurship for Undergraduate, China (no.201813705049), and Supported by Sichuan Science and Technology Program (no.2020JDRC0071), the Open-Study Funds of State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine, Chengdu University of Traditional Chinese Medicine.