Zanthoxylum bungeanum maxim heavy oil component with antibacterial activity and application thereof

Abstract

The application discloses a heavy Zanthoxylum bungeanum essential oil component with antibacterial activity and a preparation method thereof. The Zanthoxylum bungeanum essential oil is deeply researched, and the Zanthoxylum bungeanum essential oil is separated into a light Zanthoxylum bungeanum essential oil component and the heavy Zanthoxylum bungeanum essential oil component through a molecular distillation instrument. The antibacterial and antifungal experimental results show that the heavy Zanthoxylum bungeanum essential oil component obtained through distillation refining has better bactericidal activity on Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus and corynebacterium striatum compared with the Zanthoxylum bungeanum essential oil and the light Zanthoxylum bungeanum essential oil component, and has better bactericidal activity on Candida tropicalis, Candida krusei, Candida glabrata and white Candida, and a good technical effect is achieved.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology. Specifically, this invention relates to the antibacterial activity of Sichuan pepper heavy oil components, their preparation methods, and applications. Background Technology

[0002] Sichuan pepper, also known as fried Sichuan pepper, is pungent and hot in nature. It enters the stomach, spleen, and kidney meridians. According to traditional Chinese medicine pharmacopoeias, Sichuan pepper warms the middle jiao (spleen and stomach), dispels cold, eliminates dampness, relieves pain, stops itching, kills parasites, and detoxifies. Sichuan pepper mainly contains volatile oil, which includes limonene, eucalyptol, myrcene, sapinene, linalool, etc. It also contains phytosterols and various unsaturated organic acids.

[0003] Sichuan pepper has many health benefits:

[0004] 1. Sichuan pepper has the effect of inhibiting platelet aggregation and anticoagulation. The extract of Sichuan pepper root has thrombotic activity and can be used as a hemostatic agent for hemophilia.

[0005] 2. Sichuan pepper has the effect of lowering blood pressure and protecting against stress-induced myocardial damage.

[0006] 3. Sichuan pepper oil can inhibit the contraction of tracheal smooth muscle and has a certain effect in relieving asthma.

[0007] 4. Sichuan pepper has anti-ulcer, liver-protective, anti-inflammatory, anti-functional diarrhea, and gastrointestinal dysfunction-correcting effects.

[0008] 5. Sichuan pepper has significant antibacterial and antifungal effects, can reduce inflammation and pain, enhance the body's immunity, and also has the effects of expelling roundworms and killing scabies mites.

[0009] 6. Sichuan pepper has anti-cancer activity and mutagenic effects.

[0010] 7. Sichuan pepper can reduce the excitability of isolated sciatic nerves in toads and reversibly block nerve conduction, producing a local anesthetic effect and having a sedative and calming effect on the central nervous system. Sichuan pepper also has anticonvulsant, salivary gland stimulant, papillary secretion inhibitory, lipid-lowering, and anti-arteriosclerosis effects.

[0011] Modern research shows that Sichuan pepper has anti-experimental gastric ulcer effects; it has a bidirectional regulatory effect on intestinal smooth muscle; the water extract of Sichuan pepper has a hepatoprotective effect; this product also has local anesthetic and analgesic effects; it has anti-platelet aggregation and anti-experimental cerebral thrombosis effects; and it has the effects of regulating gastrointestinal motility, anti-ulcer, anti-inflammatory, analgesic, and antibacterial effects.

[0012] Chinese patents 202210945055.4 and 202310047866.7 disclose a method for preparing antibacterial essential oils, respectively. Currently, research reports on Sichuan pepper volatile oil mainly focus on its extraction methods, chemical composition, and pharmacological activity. There are no in-depth reports on the separation, purification, and activity screening of the heavy and light oils in Sichuan pepper volatile oil. This invention conducts in-depth research on Sichuan pepper volatile oil, screens the optimal process for preparing heavy Sichuan pepper oil, and conducts antibacterial and antifungal experiments to screen the components of heavy Sichuan pepper oil with a broad antibacterial spectrum and good antibacterial effect. Summary of the Invention

[0013] Purpose of the invention: The purpose of this invention is to screen out the optimal process for preparing Sichuan pepper heavy oil through a large number of experiments, and to screen its antibacterial and antifungal activity, so as to develop Sichuan pepper heavy oil components with broad antibacterial spectrum and good antibacterial effect and their new applications.

[0014] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0015] A component of Sichuan pepper heavy oil with antibacterial activity is prepared by the following method:

[0016] (1) Extraction of Sichuan pepper essential oil:

[0017] Take a certain amount of Sichuan pepper powder, soak it in water, then extract it by steam distillation, centrifuge it, collect the essential oil, and store it in the dark at low temperature.

[0018] (2) Refining of Sichuan pepper essential oil: Put the Sichuan pepper essential oil from step (1) into the feed bottle of the molecular distillation apparatus, fill the cold trap with liquid nitrogen, turn on the refrigeration device, cool with reflux water, adjust the scraper speed, set a certain distillation temperature and distillation pressure, and collect the heavy oil components of Sichuan pepper and the heavy oil components.

[0019] As a preferred embodiment, the above-mentioned pepper oil component with antibacterial activity is prepared by the following method:

[0020] (1) Extraction of Sichuan pepper essential oil:

[0021] Take a certain amount of Sichuan pepper powder, add 8 to 12 times the amount of water, soak for 0.5 to 1 hour, then extract by steam distillation for 1 to 6 hours, centrifuge, collect the essential oil, and store at 4°C away from light.

[0022] (2) Refining of Sichuan pepper essential oil: Sichuan pepper essential oil is put into the feed bottle of a molecular distillation apparatus, liquid nitrogen is put into the cold trap, the refrigeration device is turned on, the temperature of the reflux water device is set to 5-10℃, the scraper speed is 200-240r / min, the material flow rate is 1-2mL / min, the distillation temperature is 50℃-70℃ and the distillation pressure is 60Pa-100Pa, and the light oil component and heavy oil component of Sichuan pepper are collected.

[0023] As a preferred embodiment, the above-mentioned pepper oil component with antibacterial activity is prepared by the following method:

[0024] (1) Extraction of Sichuan pepper essential oil: Take a certain amount of Sichuan pepper powder, add 12 times the amount of water, soak for 1 hour, extract by steam distillation for 6 hours, centrifuge, collect the essential oil, and store at 4℃ away from light.

[0025] (2) Refining of Sichuan pepper essential oil: Sichuan pepper essential oil is put into the feed bottle of a molecular distillation apparatus, liquid nitrogen is put into the cold trap, the refrigeration device is turned on, the temperature of the reflux water device is set to 5℃, the scraper speed is 240r / min, the material flow rate is 1mL / min, the distillation temperature is 80℃ and the distillation pressure is 80Pa, and the heavy oil component of Sichuan pepper is collected.

[0026] Through experimental screening, this invention has discovered the application of the prepared Sichuan pepper heavy oil component in the preparation of drugs with antibacterial activity, especially in the preparation of drugs with antibacterial and antifungal activity.

[0027] As a further preferred embodiment, the bacteria include Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Acinetobacter baumannii, and Corynebacterium banding. The fungi include Candida tropicalis, Candida krusei, Candida glabrata, and Candida albicans.

[0028] This invention can prepare the Sichuan pepper heavy oil component into capsules, tablets, granules, pills, powders, ointments, mixtures, oral liquids or injections using a pharmaceutically acceptable carrier.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0030] This invention conducts in-depth research on Sichuan pepper essential oil, separating the crude Sichuan pepper essential oil into light and heavy oil components using molecular distillation. The study found that the heavy oil in the Sichuan pepper essential oil not only exhibits better antibacterial and antifungal activity than the light and crude oil, but also has a broader antibacterial spectrum. By retaining the heavy oil component after molecular distillation, the required dosage is reduced while achieving better results, yielding unexpected and significant technological advancements. Attached Figure Description

[0031] Figure 1 This is a graph showing the results of the extraction and screening of heavy oil components from Sichuan pepper under different distillation temperatures and pressures.

[0032] Figure 2 This is a MIC diagram of the components of Sichuan pepper essential oil against Pseudomonas aeruginosa and Klebsiella pneumoniae.

[0033] Figure 3 This is a MIC diagram of the light oil component of Sichuan pepper against Pseudomonas aeruginosa and Klebsiella pneumoniae.

[0034] Figure 4 This is a MIC diagram of the heavy oil component of Sichuan pepper against Pseudomonas aeruginosa and Klebsiella pneumoniae.

[0035] Figure 5 This is the MIC diagram of the components of Sichuan pepper essential oil against methicillin-resistant Staphylococcus aureus and Staphylococcus aureus.

[0036] Figure 6 This is the MIC diagram of the light oil component of Sichuan pepper against methicillin-resistant Staphylococcus aureus and Staphylococcus aureus.

[0037] Figure 7 This is the MIC diagram of the heavy oil component of Sichuan pepper against methicillin-resistant Staphylococcus aureus and Staphylococcus aureus.

[0038] Figure 8 This is the MIC diagram of the components of Sichuan pepper essential oil against Acinetobacter baumannii.

[0039] Figure 9 This is the MIC diagram of the light oil component of Sichuan pepper against Acinetobacter baumannii.

[0040] Figure 10 This is the MIC diagram of the heavy oil component of Sichuan pepper against Acinetobacter baumannii.

[0041] Figure 11 This is the MIC diagram of the components of Sichuan pepper essential oil against Corynebacterium styloides.

[0042] Figure 12 This is the MIC diagram of the heavy oil component of Sichuan pepper against Corynebacterium styloides.

[0043] Figure 13 This is the MIC diagram of the components of Sichuan pepper essential oil against Candida glabrata and Candida albicans.

[0044] Figure 14 This is the MIC diagram of the light oil component of Sichuan pepper against Candida glabrata and Candida albicans.

[0045] Figure 15 This is the MIC diagram of the heavy oil component of Sichuan pepper against Candida glabrata and Candida albicans.

[0046] Figure 16 This is a MIC diagram of the components of Sichuan pepper essential oil against Candida tropicalis and Candida krusei.

[0047] Figure 17 This is the MIC diagram of the light oil component of Sichuan pepper against Candida tropicalis and Candida krusei.

[0048] Figure 18 This is the MIC diagram of the heavy oil component of Sichuan pepper against Candida tropicalis and Candida krusei.

[0049] Figure 19 This is an MBC diagram showing the effects of Sichuan pepper essential oil, Sichuan pepper light oil component, and Sichuan pepper heavy oil component on Pseudomonas aeruginosa, Klebsiella pneumoniae, and methicillin-resistant Staphylococcus aureus.

[0050] Figure 20 This is an MBC diagram showing the effects of Sichuan pepper essential oil, Sichuan pepper light oil component, and Sichuan pepper heavy oil component on Staphylococcus aureus, Acinetobacter baumannii, and Corynebacterium banding.

[0051] Figure 21 This is an MBC diagram showing the effects of Sichuan pepper essential oil, Sichuan pepper light oil component, and Sichuan pepper heavy oil component on Candida albicans, Candida tropicalis, and Candida glabrata.

[0052] Figure 22 This is an MBC diagram of the effects of Sichuan pepper essential oil, Sichuan pepper light oil component, and Sichuan pepper heavy oil component on Candida krusei. Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments, but these embodiments should not be construed as limiting the present invention.

[0054] Example 1: Screening of Refining Process Conditions for Sichuan Pepper Oil

[0055] 1. Instruments and Equipment: Short-path thin-film distillation apparatus (UIC GmbH, Germany, KDL2)

[0056] 2. Experimental Methods

[0057] By adjusting the evaporation temperature and distillation pressure of the molecular distillation apparatus, the molecular free path of each component can be altered, achieving separation of different components. Light oil and heavy oil components are collected separately, and the process conditions are determined by calculating the recovery rate of the light oil. Sichuan pepper essential oil is loaded into a molecular distillation feed flask, liquid nitrogen is placed in the cold trap, the refrigeration device is turned on, the reflux water temperature is set to 5℃, the scraper rotation speed is 240 r / min, and the material flow rate is 1 mL / min. The evaporation intensity is adjusted by changing the distillation temperature (50℃, 60℃, 70℃, 80℃, 90℃) and distillation pressure (20Pa, 40Pa, 60Pa, 80Pa, 100Pa) to determine the suitable conditions for molecular distillation. Based on the optimal molecular distillation conditions, the light oil and heavy oil components of Sichuan pepper are collected. Figure 1As shown, adjusting the evaporation temperature and distillation pressure can alter the molecular free path of each component, achieving component separation. Light oil and heavy oil components were collected separately, and the process conditions were determined by calculating the recovery rate. When the feed flow rate was 1 mL / min, the scraper rotation speed was 240 rpm, the condensation temperature was 5℃, and the distillation pressure was 80 Pa, increasing the temperature decreased the recovery rate of the heavy oil component; the highest recovery rate was achieved at 50℃, therefore, the distillation temperature was chosen to be 50℃. When the feed flow rate was 1 mL / min, the scraper rotation speed was 240 rpm, the condensation temperature was 5℃, and the distillation temperature was 50℃, higher pressure was beneficial for improving the recovery rate of Sichuan pepper heavy oil; the recovery rate of the heavy oil component was relatively high at a distillation pressure of 80 Pa, therefore, the distillation pressure was chosen to be 80 Pa. Based on these results, the final refining process conditions for Sichuan pepper essential oil were determined to be: feed flow rate 1 mL / min, scraper rotation speed 240 rpm, condensation temperature 5℃, distillation temperature 50℃, and distillation pressure 80 Pa.

[0058] Example 2

[0059] A component of Sichuan pepper heavy oil with antibacterial activity is prepared by the following method:

[0060] (1) Extraction of Sichuan pepper essential oil: Take a certain amount of Sichuan pepper powder, add 12 times the amount of water, soak for 1 hour, then extract by steam distillation for 6 hours, centrifuge, collect the essential oil, and store at 4℃ away from light.

[0061] (2) Refining of Sichuan pepper essential oil: Sichuan pepper essential oil is put into the feed bottle of a molecular distillation apparatus, liquid nitrogen is put into the cold trap, the refrigeration device is turned on, the temperature of the reflux water device is set to 5℃, the scraper speed is 240r / min, the material flow rate is 1mL / min, the distillation temperature is 80℃ and the distillation pressure is 80Pa, and the heavy oil component and light oil component of Sichuan pepper are collected respectively.

[0062] Example 3: Determination of the minimum inhibitory concentration (MIC) of Sichuan pepper essential oil

[0063] 1. Activation of bacterial strains: The bacterial strains were inoculated onto sterilized culture medium using the streak method. Bacteria were incubated at 35°C for 24 hours, Candida albicans at 28°C for 48 hours, and Aspergillus at 28°C for 3-4 days to obtain activated test strains for later use.

[0064] 2. Pretreatment of test solutions: The Sichuan pepper essential oil, Sichuan pepper light oil component and Sichuan pepper heavy oil component prepared according to the method in Example 2 were filtered through a 0.22 μM filter membrane for sterilization. The Sichuan pepper essential oil, Sichuan pepper light oil component and Sichuan pepper heavy oil component were diluted with 1% Tween-80 distilled aqueous solution to 128 μL / mL (bacteria) and 64 μL / mL (Candida) respectively, and then set aside.

[0065] 3. Preparation of test bacterial suspension: (1) Bacteria: Take a single colony and dilute it with sterile 0.45% physiological saline to a bacterial suspension of 0.5 McFarland units, that is, the bacterial suspension concentration is about 10. 8 CFU / mL. Dilute the above bacterial suspension 1000 times with sterile physiological saline to obtain a ready-to-use bacterial suspension with a concentration of approximately 1 × 10⁻⁶. 5 (2) Candida: Take a single colony and dilute it with sterile 0.85% physiological saline to a bacterial suspension of 1.8-2.2 McFarland units; dilute the above bacterial suspension 1000 times with RPMI 1640 medium to obtain a ready-to-use bacterial suspension with a concentration of 1×10⁻⁶. 3 ~5×10 3 cfu / mL.

[0066] 4. MIC Determination: The MIC was determined using the micro-broth dilution method (96-well plate method). 50 μL of sterilized MH broth (bacteria) or 50 μL of sterilized RPMI 1640 medium (Candida) was added to each well of the 96-well plate. The first column served as a positive control, containing 50 μL each of MH broth and the test bacterial culture (bacteria), or RPMI 1640 medium. 50 μL each of RPMI 1640 medium and test bacterial suspension (Candida); the second column is the negative control, with 50 μL each of MH broth medium and Tween 80 (bacteria), or 50 μL each of RPMI 1640 medium and Tween 80 (Candida); in columns 3-12, add 50 μL of the test bacterial suspension to each well, followed by 50 μL each of sequentially diluted Sichuan pepper essential oil, Sichuan pepper light oil component, and Sichuan pepper heavy oil component. The concentrations of the Sichuan pepper essential oil, Sichuan pepper light oil component, and Sichuan pepper heavy oil component for bacterial testing are 32, 16, 8, 4, 2, and 1, respectively. The concentrations of the fungal test components of Sichuan pepper essential oil, light Sichuan pepper oil, and heavy Sichuan pepper oil were 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03 μL / mL, respectively. After loading the drug and bacterial solution, the plate was covered, and the bacteria were incubated at 35℃ for 24 hours, while Candida was incubated at 28℃ for 48 hours. The lowest concentration at which the culture medium was clear and no bacterial growth was observed was determined as the minimum inhibitory concentration of the drug.

[0067] 5. Minimum bactericidal concentration (MBC) determination

[0068] Use sterile cotton to absorb the mixed solution in the wells of a 96-well plate that is higher than the MIC (including the MIC concentration), spread it on the corresponding culture medium sections, incubate the bacteria at 35°C for 24 hours, and incubate the Candida at 28°C for 48 hours. Observe the results with the naked eye. The lowest sample concentration with less than 5 colonies or no colony growth is determined as the MBC value.

[0069] 6. Experimental Results

[0070] 6.1 The minimum inhibitory concentrations (MICs) of Sichuan pepper essential oil, Sichuan pepper light oil component, and Sichuan pepper heavy oil component tested in this invention are shown in Table 1 below. Figures 2 to 18 As shown.

[0071] Table 1. MIC (μL / mL) of Sichuan pepper essential oil, light oil component, and heavy oil component.

[0072]

[0073]

[0074] 6.2 The minimum bactericidal and antimicrobial concentrations (MBCs) of Sichuan pepper essential oil, light Sichuan pepper oil component, and heavy Sichuan pepper oil component are shown in Table 2 below. Figures 19-22 As shown.

[0075] Table 2. MBC (μL / mL) of Sichuan pepper essential oil, light oil component, and heavy oil component

[0076]

[0077] The above experimental results demonstrate that, according to this invention, the heavy oil component of Sichuan pepper obtained through redistillation and refining exhibits better bactericidal activity against Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, and Corynebacterium banding compared to raw Sichuan pepper oil and light Sichuan pepper oil. Furthermore, it demonstrates better bactericidal activity against fungi such as Candida tropicalis, Candida krusei, Candida glabrata, and Candida albicans.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Sichuan pepper heavy oil component as the sole active ingredient in the preparation of drugs with antibacterial and antifungal activities; wherein the bacteria is Corynebacterium striatum and the fungus is Candida krusei; The Sichuan pepper heavy oil component is prepared by the following method: (1) Extraction of Sichuan pepper essential oil: Take a certain amount of Sichuan pepper powder, add 12 times the amount of water, soak for 1 hour, extract by steam distillation for 6 hours, centrifuge, collect the essential oil, and store at 4℃ away from light. (2) Refining of Sichuan pepper essential oil: Sichuan pepper essential oil is put into the feed bottle of a molecular distillation apparatus, liquid nitrogen is put into the cold trap, the refrigeration device is turned on, the temperature of the reflux water device is set to 5℃, the scraper speed is 240r / min, the material flow rate is 1mL / min, the distillation temperature is 80℃ and the distillation pressure is 80Pa, and the heavy oil component of Sichuan pepper is collected.

2. The application according to claim 1, characterized in that, The heavy oil component of Sichuan pepper is prepared into capsules, tablets, granules, pills, powders, ointments, mixtures, oral liquids or injections using pharmaceutically acceptable carriers.

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