GB2620210A - Pepper essential oil with anti-inflammatory activity and preparation method thereof - Google Patents
Pepper essential oil with anti-inflammatory activity and preparation method thereof Download PDFInfo
- Publication number
- GB2620210A GB2620210A GB2214556.9A GB202214556A GB2620210A GB 2620210 A GB2620210 A GB 2620210A GB 202214556 A GB202214556 A GB 202214556A GB 2620210 A GB2620210 A GB 2620210A
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- essential oil
- pepper
- pepper essential
- fruits
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- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 230000003110 anti-inflammatory effect Effects 0.000 title claims abstract description 17
- 235000002566 Capsicum Nutrition 0.000 title claims description 125
- 239000006002 Pepper Substances 0.000 title claims description 125
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- MXXWOMGUGJBKIW-YPCIICBESA-N piperine Chemical compound C=1C=C2OCOC2=CC=1/C=C/C=C/C(=O)N1CCCCC1 MXXWOMGUGJBKIW-YPCIICBESA-N 0.000 description 1
- 229940075559 piperine Drugs 0.000 description 1
- WVWHRXVVAYXKDE-UHFFFAOYSA-N piperine Natural products O=C(C=CC=Cc1ccc2OCOc2c1)C3CCCCN3 WVWHRXVVAYXKDE-UHFFFAOYSA-N 0.000 description 1
- 235000019100 piperine Nutrition 0.000 description 1
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- 235000013599 spices Nutrition 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
- C11B9/027—Recovery of volatiles by distillation or stripping
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/67—Piperaceae (Pepper family), e.g. Jamaican pepper or kava
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Botany (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Wood Science & Technology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
Provided is a preparation method of a Piper nigrum L. essential oil with anti-inflammatory activity, which comprises: performing ultralow-temperature freezing, microwave thawing, pulp grinding, enzymolysis, steam distillation, and oil-water separation on fresh Piper nigrum L. fruits to obtain a Piper nigrum L. essential oil. Experiments have shown that the Piper nigrum L. essential oil has an inhibitory effect on the release of nitric oxide (NO) by RAW 264.7 cells, the release of tumor necrosis factor (TNF‑ɑ), interleukin-6 (IL-6), interleukin-1β (IL‑1β) and prostaglandin E2 (PGE2), and the expression of cyclooxygenase-2 (COX-2), which indicates that the Piper nigrum L. essential oil has anti-inflammatory activity.
Description
PEPPER ESSENTIAL OIL WITH ANTI-INFLAMMATORY ACTIVITY AND PREPARATION METHOD THEREOF
Cross-reference to related application
The present application claims priority to Chinese Patent Application No. 2022101524579, entitled "pepper essential oil with anti-inflammatory activity and preparation method thereof', and filed on February 18, 2022, the entire disclosure of which is incorporated herein by reference in its entirety.
Technical Field
The present invention relates to the technical field of natural product extraction, specifically, to a pepper essential oil with anti-inflammatory activity and preparation method thereof.
Background Art
Pepper (Piper nigrum L.) is a plant of the Piperaccac family, which is one of the oldest and most famous spice crops. Pepper contains volatile oil, piperine and other substances.
Pepper has a variety of functional activities and is widely used in the food and pharmaceutical industries.
The extraction methods commonly used for the essential oil comprise steam distillation, pressing, organic solvent extraction, and later ultrasonic-assisted extraction, supercritical CO) extraction and microwave-assisted steam distillation. Among them, the steam distillation needs simple equipment, is easy to operate, costs lowly, and the quality of essential oils is good. The steam distillation is the most commonly used method for extracting essential oils. However, because the steam distillation needs to be operated at a high temperature for a long time, it is easy to cause thermal decomposition of heat-sensitive compounds and hydrolysis of easily hydrolyzed components in the essential oil, resulting in the loss of the effective components of the essential oil, and a low extraction rate.
At present, there have been reports on the processing of pepper stalk removal, peelirw, drying, oleoresin extraction, essential oil extraction, and preparation of condiment. Studies have also shown that black and white pepper essential oils have anti-tumor and anti-cough effects.
The extraction method and application of the pepper essential oil need further study.
Summary of the Invention
The present invention provides a pepper essential oil with anti-inflammatory activity and preparation method thereof. In the method, the fresh pepper fruits are used as raw material, and the pepper essential oil with anti-inflammatory activity is obtained by freezing at ultra-low temperature, rapidly thawing by microwave, pulp grinding, ultrasonic-assisted enzymolysis, steam distillation, oil-water separation, and removing water with anhydrous sodium sulfate.
Specifically, a preparation method of a pepper essential oil comprises the following steps: freezing fresh pepper fruits at ultra-low temperature, thawing by microwave, pulp grinding, enzymolysis, steam distillation, oil-water separation, and obtaining the pepper essential oil. According to an embodiment of the present invention, the ripeness of the fresh pepper fruits is 50% to 70%. Studies found that the fresh pepper fruits with ripeness of 50% to 70% have high content of essential oil, which is more conducive to the extraction of the pepper essential oil.
According to an embodiment of the present invention, the freezing at ultra-low temperature is performed at a temperature of -60°C to -80°C, for example, -80°C. Usually, the freezing is performed for 24 to 60 h, for example, 24 to 48 h. Studies found that by freezing fresh pepper fruits at ultra-low temperature, the material metabolism and growth activities in living cells are almost completely stopped, which suppresses physiological metabolism intensity to the greatest extent, and the volatile components of the raw materials are better preserved.
According to an embodiment of the present invention, the frozen fruits are thawed by microwave with a microwave power of 600 to 800 W, for example. 800W. Usually the frozen fruits are thawed until a core temperature of the fruits is 4°C. Studies found that in the process of rapid thawing by microwave, polar substances such as water in the cells absorb microwaves and then generate heat, the temperature in the cells rises rapidly, and the pressure of water vaporization causes the cell walls to rupture, resulting in micropores and cracks, such that the intracellular substances can be more easily dissolved.
According to an embodiment of the present invention, an appropriate amount of water can be added during pulp grinding, for example, the amount of added water is 5 to 10 limes of the weight of the fresh pepper fruits. The pulp grinding can be performed by conventional methods. In some specific embodiments, the pulp grinding is performed to a fineness of 5 to 20 mesh. Studies found that the raw materials were more fully broken through the treatment of pulp grinding, which is conducive to the full extraction of components of fresh pepper fruits.
According to an embodiment of the present invention, an enzyme used in the enzymolysis is a compound enzyme consisting of cellulase, hemicellulase and pectinase, with a weight ratio of (1 to 2):(1 to 2):(1 to 3), for example, 1:1:2. Studies found that enzymolysis with the above-mentioned compound enzyme can cause changes such as local loosening and bulking of the intercellular substance, and reduce the mass transfer resistance of the mass transfer barrier such as the intercellular substance to the diffusion of volatile components from the cell to the extraction medium.
In the present invention cellulase, hemicellulase, and pectinase are all commercially available.
In some embodiments, the enzymatic activity of cellulase is 50 u/mg.
In some embodiments, the enzymatic activity of hemicellulase is 20000 u/mg.
In some embodiments, the enzymatic activity of pectinase is 500 u/mg.
According to an embodiment of the present invention, an added amount of the compound enzyme is 0.05% to 1% of the weight of the fresh pepper fruits.
According to an embodiment of the present invention, the enzymolysis is performed for 1 to 2 h at 40 to 60°C, for example, 45 to 50°C.
According to an embodiment of the present invention, the enzymolysis is preferably performed with an ultrasonic frequency of 400 to 500W. Studies found that ultrasonic-assisted enzymolysis accelerated the enzymolysis, which was beneficial to improve the yield of the pepper essential oil.
In the present invention, the conventional methods of steam distillation and oil-water separation can be used. For example, anhydrous sodium sulfate can be used to remove water. According to an embodiment of the present invention, the method of steam distillation comprises the following steps: putting the enzymatically hydrolyzed material into a round-bottomed flask and adding glass beads into the flask, connecting a volatile oil receiver and a reflux condenser, putting the flask in an electric heating mantle for heating, and distilling until the essential oil is no longer increased more, stopping heating, cooling to room temperature, and then reading the volume of the essential oil, collecting the essential oil, and removing water with anhydrous sodium sulfate.
According to an embodiment of the present invention, the preparation method of a pepper essential oil comprises the following steps: 1) pretreatment of raw material freezing and storing the fresh pepper fruits at ultra-low temperature at -80°C; rapidly thawing the frozen fruits with a microwave of 600 to 800W, and adding distilled water for pulp grinding to obtain mixture A; 2) ultrasonic-assisted enzymolysis IC adding the compound enzyme at an amount of 0.05% to 1% of the mass of fresh pepper fruits into the mixture A. and subjecting the resultant to enzymolysis for 1 to 2 h at 40 to 60°C assisted by an ultrasonic at a frequency of 400 to 500 W to obtain mixture B; the compound enzyme consists of cellulase, hemicellulase and pectinase with a weight ratio of 1:1:2; 3) extraction by steam distillation extracting the mixture B by steam distillation to collect the essential oil, and removing water with anhydrous sodium sulfate.
In some specific embodiments, step 3) includes the following steps: putting mixture B into a round-bottomed flask and adding glass heads into the flask, connecting a volatile oil receiver and a reflux condenser, putting the flask in an electric heating mantle for heating, and distilling until the essential oil is no longer increased more, slopping heatin2, cooling to room temperature, and then reading the volume of the essential oil, collecting the essential oil, and removing water with anhydrous sodium sulfate.
In die method of the present invention, the material metabolism and growth activities in living cells are almost completely stopped by freezing and storing fresh pepper fruits at ultra-low temperature, which suppresses physiological metabolism intensity to the greatest extent, and the volatile components of the raw materials are better preserved. By the method combined with rapidly thawing by microwave, pulp winding, and ultrasonic-assisted enzymolysis, specifically the polar substances such as water in the cells absorb microwaves in the process of rapid thawing by microwave and then generate heat, the temperature in the cells rises rapidly, and the pressure of water vaporization causes the cell walls to rupture, resulting in micropores and cracks, such that the intracellular substances can he more easily dissolved. Through treatment of pulp grinding, the raw materials are more fully broken, which is conducive to the full extraction of the components of fresh pepper fruits. Then the compound enzyme is added to it, which causes changes such as local loosening and bulking of the intercellular substance, and reduces the mass transfer resistance of the mass transfer barrier such as the intercellular substance to the diffusion of volatile components from the cell to the extraction medium. The ultrasonic-assisted enzymolysis accelerates enzymolysis, and the above overall steps are all conducive to the extraction of the essential oil of fresh pepper fruits In the method of the present invention, the compound enzyme is used to decompose the cell wall of fresh pepper fruits, reduce the mass transfer resistance, and promote the release of the essential oil, and then the water vapor is refluxed to extract the essential oil. The essential oil of fresh pepper fruits prepared by this method is light yellow, has obvious pepper aroma and no peculiar smell, with a yield of up to 3.14%, which is more than twice yield of the essential oil of Hack pepper and white pepper by traditional steam distillation.
In the method of the present invention, the fresh pepper fruits are used as the raw material to directly extract the natural products. Compared with the traditional extraction of white pepper and black pepper, the processing process is omitted, which effectively improves the utilization rate of the fresh pepper fruits. Moreover, the pepper promotes the development of value-added industries of pepper resources in a certain extent.
The present invention also comprises the pepper essential oil prepared by the above method. The pepper essential oil contains at least one or more of the components in the serial numbers 1 to 28 in Table 1 below. In particular, the pepper essential oil contains a-thujene with a relative content of 0.32% to 0.34%, for example, 0.34%.
The relative content in the present invention refers to a peak area of a certain compound divided by the sum of all integrated peak areas, that is, the percentage content of the compound.
The present invention also finds that the pepper essential oil has better anti-inflammatory effect. The pepper essential oil especially refers to the pepper essential oil prepared by the above method. Experiments show that, the pepper essential oil has inhibitory effects on the release of nitric oxide (NO), tumor necrosis factor (TNF-a), interlcukin-6 (IL-6), i nterlcuki n113 (IL-113), prostaglandin E2 (PGE2) from RAW 264.7 cells and expression of cyclooxygenase-2 (COX-2) in RAW 264.7 cells in a cell model induced by lipopolysaccharide (LSP), which indicates that the pepper essential oil has anti-inflammatory activity The present invention also comprises use of a pepper essential oil in preparing pharmaceuticals with anti-inflammatory effect. Specifically, the anti-inflammatory effect comprises inhibiting one or more of release of nitric oxide (NO), release of tumor necrosis factor (TNF-a), release of interleukin-6(IL-6), release of interleukin-113(IL-10), release of prostaglandin E2(PGE2) from RAW 264.7 cells, and expression of cyclooxygenase-2(COX2) in RAW 264.7 cells.
The present invention also provides a pharmaceutical composition comprising a pepper essential oil and a pharmaceutically acceptable carrier.
Brief Description of the Drawings
Figure 1: Effect of the pepper essential oil on cell viability in the experimental example of the present invention.
Figure 2: Effect of the pepper essential oil on the release of nitric oxide (NO) from RAW 264.7 cells.
Figure 3: Effect of the pepper essential oil on the release of tumor necrosis factor (TNF-a), interleukin-6 (IL-6), interleukin-113 (11E-1f3), and prostaglandin E2 (PGE2) from RAW 264.7 cells.
Figure 4: Effect of the pepper essential oil on the expression of cyclooxygenase-2 (COX-2) in RAW 264.7 cells.
Specific Modes for Carrying Out the Embodiments In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the examples. It should be understood that the specific examples described herein are only used to explain the present invention, but not to limit the present invention.
Unless otherwise specified, the following pepper varieties are Indonesian large-leaf varieties, provided by Flavor Beverage Institute, Chinese Academy of Tropical Agriculture Science.
Cellulase, hemicellulase, and pectinase were purchased from Yuanye Bio-Technology Co., Ltd, and their enzyme activities were 50 u/mg, 20000 u/mg, and 500 u/mg, respectively.
Example 1: The preparation method of a pepper essential oil (fresh pepper fruits) The fresh pepper fruits (ripeness of 50% to 70%) were freezed and stored for 24 h at ultra-low temperature freezing at -80°C, rapidly thawed for 10 min with microwave (power, 800 W). Distilled water with a weight of 6 times the weight of the fresh pepper fruits was added for pulp grinding to obtain mixture A. In mixture A, the compound enzyme (consisting of cellulase, hemicellulase, pectinase in a weight ratio of 1:1:2) with a weight of 1% the weight of the fresh pepper fruits was added, and ultrasonic (power, 500W) assisted enzymolysis was performed for 1 h at 50°C to obtain mixture B. Mixture B was put into a round-bottomed flask, glass beads were added into this flask, the volatile oil receiver and the reflux condenser were connected, and this flask was put in an electric heating mantle for heating and distilled until the essential oil was no longer increased more, heating was stopped, this flask was cooled to room temperature, and then the volume of the essential oil was read, the essential oil was collected and dried with anhydrous sodium sulfate to obtain the pepper essential oil.
The formula for calculating the extraction yield of the essential oil on a dry weight basis of pepper fresh fruits is as follows: 41,;(1;,fi of eNgenti,i1 oil1.) 10Q Extraction yield of essentiA oil Or:11100g dry v.,&ght - x 100 1,4,2 Cce freQh poppte fruits tztent The water content was determined with reference to the azeotropic distillation method (the third method) in GB 5009.3-2016 "National Food Safety Standard -Determination of Moisture in Foods".
Comparative Example I Preparation and content determination of a black pepper essential GB/T 17527-2009 "Determination of content of pepper essential oil" was used.
Comparative Example 2 Preparation and content determination of a white pepper essential GB/T 17527-2009 "Determination of content of pepper essential oil" was used. Experiment I The results showed that the extraction yield of the essential oil on a dry weight basis of fresh pepper fruits in Example 1 was 3.511 mL/1008, which was about twice of black pepper in Comparative Example 1 (1.710 mL/100g) and white pepper in Comparative Example 2 (1.853 mL/100g). This indicates that the extraction yield of the essential oil on a dry weight basis of the fresh pepper fruits of the present invention is significantly higher than those of black pepper and white pepper.
Determination by GC-MS method: the essential oil was diluted 50 times with n-hexane, dehydrated by anhydrous sodium sulfate, and passed through a 0.45 pm microporous membrane to be tested. Column was: J&W DB-5 quartz capillary (30 m x 0.25 mm, 0.25 pm). Heating program was as follows: column temperature was 50°C, and increased to 75°C at 3°C/min, then increased to 140°C at 1.5°C/min, then increased to 230°C at 10°C/min, held for 2 min, and finally increased to 280°C at 20°C/min and held for 3 min. The flow rate of carrier gas (He) was 1 inUmin, and the injection volume was 1 p L, splitless. Electron bombardment ion source was used with electron energy of 70 eV; transmission line temperature of 280°C; ion source temperature of 230°C; and mass scanning range of m/z 35-450 mu.
The components of the pepper essential oils in pepper fresh fruits of Example 1, black IC pepper of Comparative Example I and white pepper of Comparative Example 2, are shown in Table 1. As can be seen from Table 1, the components of the essential oil of fresh pepper fruits, black pepper and white pepper are all based on terpenes, but the relative contents are different, and a-thujene was detected in the essential oil of fresh pepper fruits, hut not in black pepper and white pepper.
Table 1: Composition of the pepper essential oil No. CAS Compound Molecular Relative content 167 Formula Essential oil of Essential oil of Essential oil of black pepper white pepper fresh pepper (Comparative (Comparative fruits (Example Example 1) Example 2) 1) 1 2867-05-2 a-thujene C101116 0.34 1 7785-70-8 (+)-u-pinene CulH 16 4.22 4.64 5.63 127-91-3 13-pin ne Ciolito 8.65 10.16 9.61 4 123-35-3 myrcene C101-116 5.14 6.32 7.45 13466-78-9 3-carene CloH16 13.88 13.19 13.81 6 527-84-4 o-cymene C10li14 0.16 1.18 0.64 7 5989-27-5 D-limonene Ciollio 9.53 13.90 10.18 8 99-85-4 y-terpinene C101-116 0.73 1.03 1.47 9 586-62-9 terpinolene Ciollio 3.22 4.56 3.96 78-70-6 Linalool Cloth KO 1.04 1.18 1.43 11 562-74-3 4-terpene alcohol C12E150 0.12 0.26 0.15 12 98-55-5 a-terpineol CroRAO 0.34 0.54 0.29 13 20307-84-0 5-elemene C15H24 7.68 9.09 5.53 14 17699-14-8 (-)-a-cubebene CI; H 24 0.47 0.31 0.46 3856-25-5 (-)-a-copaene C15H24 5.23 4.26 3.60 16 13744-15-5 D-cubebene CI5H24 0.42 0.16 0.54 17 515-13-9 li-elemene CA-124 1.74 1.86 1.85 18 87-44-5 13-caryophyllene C15Hs4 24.31 6.31 18.18 19 6753-98-6 a -humulene C 1 4-i24 3.02 5.12 3.10 18794-84-8 trans-I3-famesene CI5H 24 0.25 0.32 0.38 21 23986-74-5 germacrene C151424 0.42 0.45 0.51 22 17066-67-0 (±)-13-selinene C1;H24 0.96 0.41 1.31 23 473-13-2 (-)-a-selinene Ci5H24 0.95 0.35 1.34 24 495-61-4 fl-bisabolene C15H24 0.34 0.23 0.57 483-76-1 A-cadinene C151124 2.57 2.30 2.19 26 1139-30-6 (-)-caryophyllene oxide C141240 1.05 3.20 1.02 27 77171-55-2 0-apocarotenal C151-1240 1.70 4.09 1.04 28 515-69-5 u-bisabolol C151-1260 0.13 0.23 0.15 Note: "-" means not detected.
Experiment 2: Determination of anti-inflammatory activity 1. Instruments and materials 1.1 Instruments
Table 2: Instruments
Instrument Manufacturer Constant temperature incubator SHEL LAB Clean workbench Suzhou Purification Equipment Co., Ltd. Enzyme label detector B iotek 1.2 Experimental materials Dexamethasone (DXMS) was purchased from Yuanye Bio-Technology Co., Ltd. Lipopolysaccharide (LPS) was purchased from Sigma Company in the United States. RAW264.7 macrophages were purchased from Shanghai iCell Bioscience Inc. Fetal bovine serum was purchased from BOVOGEN (South America). Trypsin -EDTA was purchased from GIBCO. PBS was purchased from Boster Biological Technology., Ltd. CCK-8 was purchased from MCE, NO color' metric test kit, and TNF-a ELISA Kit, IL-6 ELISA Kit, IL-1[3 ELISA Kit, and PGE2 ELISA Kit were purchased from Elabscience.
2 Experimental content Cell recovery: 0 The RAW264.7 cells frozen at -80°C were placed in a water bath at 37°C and shaken quickly until thawed. The thawed cells and 7 mL of medium were added to a 15 mL stmile centrifuge tube, which was centrifuged at 1000 rpm for 5 min The supernatant was discarded, and the remaining was transferred to a new 25 T cell culture flask containing 5 mL of 10% FBS medium and cultured for 8 h at 37°C with 7.5% CO2. Then the old medium was discarded and a new medium replaced the old one for continuing to culture until the cells were full of the culture flask to proceed with the experiment.
2.1 The effect of the pepper essential oil on cell viability The pepper essential oil sample prepared in Example 1 (the same below) was sterilized by filtration through a 0.22 gm filter. Before seeding, the old medium in the culture flask was discarded, the sample was washed with PBS, and 0.25% trypsin was added to digest the cells.
The trypsin was poured out, DMEM medium containing 10% fetal bovine serum was added, and the cells were pipetted repeatedly, transferred to the sample tank and blew evenly. Counting with a cell counting plate, a complete medium was added to dilute, and 100 hiL of cells were pipetted into a 96-well plate (5 x 103 cells/well) for inoculation and cultured overnight in a constant temperature incubator with 5% CO2 at 37°C. The old medium in the 96-well plate was discarded, the sample was added to the working concentration, and the culture was continued for 120 h. The original medium was sucked out, the sample was rinsed twice with PBS, and the medium in the 96-well plate was replaced with 100 pL of fresh DMEM containing 10% fetal bovine serum, and 10 htL of CCK-8 solution was added to each well, respectively. After culturing for 3 h in the incubator, the absorbance was measured at 450 nm. The formula for calculating cell viability is as follows: Cell viability (%) = (A sample) / (Blank) x 100% The results are shown in Figure 1. Control represents the control group, that is, 0 p.g/mL pepper essential oil was added. The results showed that 200 gghnL Dexamethasone (DXMS) had no significant effect on cell viability. When the concentration of the pepper essential oil was between 0 and 30 pg/mL, it had no significant effect on the growth inhibition of RAW 264.7 cells. When the concentration of the pepper essential oil was greater than 30 pg/mL, it had a significant effect on the growth inhibition of RAW 264.7 cells. Therefore, the concentration of the pepper essential oils was selected as 7.5 pg/mL, 15 Rg/mL and 30 pg/mL in subsequent experiments.
2.2 The effect of the pepper essential oil on the release of nitric oxide (NO) from RAW 264.7 cells The essential oil samples were sterilized by filtration through a 0.22 pm filter. Before seeding, the old medium in the culture flask was discarded, the sample was washed with PBS, and 0.25% trypsin was added to digest the cells. The trypsin was poured out. DMEM medium containing 10% fetal bovine serum was added, and the cells were pipetted repeatedly, transferred to the sample tank and blew evenly. Counting with a cell counting plate, a complete medium was added to dilute, and 1 mL of cells were pipetted into a 12-well plate (2 x 105 cells/well) for inoculation and cultured overnight in a constant temperature incubator with 5% C01 at 37°C. The medium was aspirated, the sample was added to the working concentration, and cells were pretreated for 1 h. 1 p.g/mL LPS was added to continue the culture for 24 h. The cells were trypsinized, washed with PBS, and finally lyscd with ripa lytic cells, and the cell lysate was collected. The intracellular NO content was detected by the NO colorimetric assay kit.
The results are shown in Figure 2. Blank represents the blank group, which was not induced and stimulated by 1 ttg/mL LSP (the same below). Model represents the model group, which was induced and stimulated by 1 ttg/mL LSP (the same below).
The results showed that the NO secretion in the model group was significantly increased, and compared with the blank group, there was a significant difference, which indicates that the LSP inflammation model was successfully established. The NO content in the cell supernatant of each concentration of pepper essential oil was significantly lower than that of the model group, which showed a dose-dependent manner, and 30 pg/mL pepper essential oil was more effective than 200 pg/mL dexamethasone in the control group in inhibiting NO secretion, which indicates that the pepper essential oil has a good inhibitory effect on NO release.
2.3 The effect of the pepper essential oil on the release of tumor necrosis factor (TNF-a), interleukin-6 (IL-6), interleukin-113 (IL-113) and prostaglandin E2 (PGE2) from RAW 264.7 cells The essential oil samples were sterilized by filtration through a 0.22 pm filter. Before seeding, the old medium in the culture flask was discarded, the sample was washed with PBS, and 0.25% trypsin was added to digest the cells. The trypsin was poured out. DMEM medium containing 10% fetal bovine serum was added, and the cells were pipetted repeatedly, transferred to the sample tank and blew evenly. Counting with a cell counting plate, a complete medium was added to dilute, and 500 ut of cells were pipetted into a 24-well plate ( lx 105 cells/well) for inoculation and cultured overnight in a constant temperature incubator with 5% CO2 at 37°C. The medium was aspirated, the sample and DXM were added to the working concentration, and cells were pretreated for 1 h. 1 utg/mL LPS was added to continue the culture for 24 h. The cell supernatant was collected. The contents of TNF-a, 1L-6, TL-113 and PGE2 in the supernatant were measured by ELISA detection kit.
The results are shown in Figure 3. The results showed that LS P acted on RAW 264.7 cells, and the contents of TNF-a, IL-6, IL-1[3 and PGE2 in the cell supernatant were significantly increased, and compared with the blank group, there were significant differences, which indicates that the LS P inflammation model was successfully established. The content of TNFa in the cell supernatant of 7.5 p.g/mL pepper essential oil was not significantly different from that of the model group, and the contents of TNF-a in the cell supernatants of 15 pg/mL and 30 itg/mL pepper essential oils were significantly different from that of the model group, and showed a dose-dependent manner. Moreover, there was no significant difference in TNF-a content between 15 pg/mL pepper essential oil and 200 pg/mL dexamethasone in the control group. The IL-6 content in the cell supernatant of each concentration of pepper essential oil was significantly lower than that of the model group, and showed a dose-dependent manner, and there was no significant difference in the IL-6 content of 30 pg/mL pepper essential oil and 200 Rg/mL dexamethasone in the control group. The IL-113 content in the cell supernatant of 7.5 pg/mL pepper essential oil was not significantly different from that of the model group, and the IL-1 contents in the cell supernatants of 15 Rg/mL and 30 It.g/mL pepper essential oil were significantly different from that in the model group, and showed a dose-dependent manner. Moreover, there was no significant difference in TNF-a content between 30 itg/mL pepper essential oil and 200 p,g/mL dexamethasone in the control group. The content of PGE2 in the cell supernatant of 7.5 p.g/mL pepper essential oil was not significantly different from that of the model group, while the contents of PGE2 in the cell supernatant of 15 pg/mL and 30 pg/mL pepper essential oils were significantly different from that of the model group, and showed a dose-dependent manner. Moreover, there was no significant difference in TNF-a content between 30 jtg/mL pepper essential oil and 200 jtg/mL dexamethasone in the control 16 group. The above overall shows that pepper essential oil has a good inhibitory effect on the release of TNF-a, IL-6, IL-113 and PGE2.
2.4 The effect of the pepper essential oil on the expression of cyclooxygenase-2 (COX-2) in RAW 264.7 cells The essential oil samples were sterilized by filtration through a 0.22 pm filter. Before seeding, the old medium in the culture flask was discard, the sample was washed with PBS, and 0.25% trypsin was added to digest the cells. The trypsin was poured out, DMEM medium containing 10% fetal bovine serum was added, and the cells were pipetted repeatedly, transferred to the sample tank and blew evenly. Counting with a cell counting plate, a complete medium was added to dilute, and 2 mL of cells were pipetted into a 6-well plate (5x 105 cells/well) for inoculation and cultured overnight in a constant temperature incubator with 5% CO, at 37°C. The medium was aspirated, the adjusted sample and DXM were added to the working concentration, and cells were pretreated for 1 h. 1 j.tg/mL LPS was added to continue the culture for 24 h. The cells were washed with PBS, and then lysed with ripa lytic cells to collect the cell lysate. the protein concentration of samples of each group was adjusted to be consistent by BCA quantitative method, and the content of COX-2 protein was detected by WB.
The results are shown in Figure 4. The results showed that there were significant differences in COX-2 protein content between the model group and the blank group, which indicates that the LSP inflammation model was successfully established. The COX-2 protein content in the cell supernatant of various concentrations of pepper essential oil was significantly lower than that in the model group, and showed a dose-dependent manner.
Moreover, there was no significant difference in inhibiting the expression of COX-2 protein between 30 i.tg/mL pepper essential oil and 200 p.g/naL dexamethasone in the control group, which indicates that the pepper essential oil has a good effect on inhibiting the expression of COX-2 protein.
Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to a person skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.
Industrial applicability
The present invention provides a pepper essential oil with anti-inflammatory activity and preparation method thereof. The preparation method of a pepper essential oil of the present invention comprises the following steps: freezing fresh pepper fruits at ultra-low temperature, thawing by microwave, pulp grinding, enzymolysis, steam distillation, oil-water separation, and obtaining a pepper essential oil. Experiments show that the pepper essential oil has inhibitory effects on the release of nitric oxide (NO), tumor necrosis factor (TNF-a), interleukin-6 (IL-6), interleukin-113 (IL-113), and prostaglandin E2 (PGE2) from RAW 264.7 cells, and expression of cyclooxy2enase-2 (COX-2) in RAW 264.7 cells, which indicates that the pepper essential oil has anti-inflammatory activity, and has good economic value and application prospects.
Claims (10)
- What is claimed is: 1. A preparation method of a pepper essential oil, comprising the following steps: freezing fresh pepper fruits at ultra-low temperature, thawing by microwave, pulp grinding, enzymolysis, steam distillation, oil-water separation, and obtaining the pepper essential oil.
- 2. The preparation method of a pepper essential oil according to claim 1, wherein a ripeness of the fresh pepper fruits is 50% to 70%.
- 3. The preparation method of a pepper essential oil according to claim 1 or 2, wherein the freezing at ultra-low temperature is performed at a temperature of -60°C to -80°C, optionally -80°C; further preferably, the freezing is performed for 24 to 60 h.
- 4. The preparation method of a pepper essential oil according to any one of claims 1 to 3, wherein frozen fruits are thawed by microwave with a microwave power of 600 to 800 W, optionally 800 W.
- 5. The preparation method of a pepper essential oil according to any one of claims 1 to 4, wherein water is added at an amount of 5 to 10 times of the weight of die fresh pepper fruits during pulp grinding; and/or, the pulp grinding is performed to a fineness of 5 to 20 mesh.
- 6. The preparation method of a pepper essential oil according to any one of claims 1 to 5, wherein an enzyme used in the enzymolysis is a compound enzyme consisting of cellulase, hemicellulase and pectinase, with a weight ratio of (1 to 2):(1 to 2):(1 to 3), further preferably 1:1:2; and/or, an added amount of the compound enzyme is 0.05% to 1% of the weight of the fresh pepper fruits; the cellulase has an enzymatic activity of 50 u/mg; and/or, the hemicellulase has an enzymatic activity of 20000 u/mg; and/or, the pectinase has an enzymatic activity of 500 u/mg.
- 7. The preparation method of a pepper essential oil according to any one of claims Ito 6, wherein the preparation method comprises the following steps: 1) pretreatment of raw material freezing and storing the fresh pepper fruits at ultra-low temperature at -80°C; rapidly thawing the frozen fruits with a microwave of 600 to 800 W, and adding distilled water for pulp grinding to obtain mixture A; 2) ultrasonic-assisted enzymolysis adding the compound enzyme at an amount of 0.05% to 1% of the mass of fresh pepper fruits into the mixture A, and subjecting the resultant to enzymolysis for 1 to 2 h at 40°C to 60°C assisted by an ultrasonic at a frequency of 400 to 500 W to obtain mixture B; the compound enzyme consists of cellulase, hemicellulase and pectinase with a weight ratio of 1:1:2; 3) extraction by steam distillation extracting the mixture B by steam distillation to collect the essential oil, and removing water with anhydrous sodium sulfate.
- 8. A pepper essential oil, prepared by the method according to any one of claims 1 to 7; optionally, the pepper essential oil contains a-thujene with a relative content of 0.32% to 0.34%, optionally 0.34%.
- 9. Use of a pepper essential oil in preparing pharmaceuticals with anti-inflammatory effect; optionally, the anti-inflammatory effect comprises inhibiting one or more of release of nitric oxide (NO), release of tumor necrosis factor (TNF-a), release of interleukin-6(IL-6), release of interleukin-113(IL-10), release of prostaglandin E2(PGE2) from RAW 264.7 cells, and expression of cyclooxygenase-2(COX-2) in RAW 264.7 cells; and/or, the pepper essential oil is the pepper essential oil according to claim 8.
- 10. A pharmaceutical composition, comprising a pepper essential oil and a pharmaceutically available carrier; optionally, the pepper essential oil is the pepper essential oil according to claim 8.
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| PCT/CN2022/093758 WO2023155315A1 (en) | 2022-02-18 | 2022-05-19 | Piper nigrum l. essential oil with anti-inflammatory activity and preparation method therefor |
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| GB202214556D0 (en) | 2022-11-16 |
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