WO2023155315A1 - 一种具有抗炎活性的胡椒精油及其制备方法 - Google Patents

一种具有抗炎活性的胡椒精油及其制备方法 Download PDF

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WO2023155315A1
WO2023155315A1 PCT/CN2022/093758 CN2022093758W WO2023155315A1 WO 2023155315 A1 WO2023155315 A1 WO 2023155315A1 CN 2022093758 W CN2022093758 W CN 2022093758W WO 2023155315 A1 WO2023155315 A1 WO 2023155315A1
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essential oil
pepper
pepper essential
preparation
fresh
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French (fr)
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谷风林
吴桂苹
段梦雅
李鑫
朱红英
宗迎
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中国热带农业科学院香料饮料研究所
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Priority to GB2214556.9A priority Critical patent/GB2620210A/en
Publication of WO2023155315A1 publication Critical patent/WO2023155315A1/zh

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    • 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
    • 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]
    • 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
    • C11B9/025—Recovery by solvent extraction
    • 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
    • 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
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • 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
    • A61K2236/30—Extraction of the material
    • A61K2236/31—Extraction of the material involving untreated material, e.g. fruit juice or sap obtained from fresh plants
    • 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
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction

Definitions

  • the invention relates to the technical field of natural product extraction, in particular to a pepper essential oil with anti-inflammatory activity and a preparation method thereof.
  • Pepper (Piper nigrum L.) is a plant belonging to the family Piperaceae. It is the oldest and well-known spice crop. It contains volatile oil, piperine and other substances. It has a variety of functional activities and is widely used in the food and pharmaceutical industries.
  • Commonly used essential oil extraction methods include steam distillation, pressing, organic solvent extraction, and later ultrasonic-assisted extraction, supercritical CO2 extraction, and microwave-assisted steam distillation.
  • steam distillation equipment is simple, easy to operate, low in cost, and of good quality, and is the most commonly used method for extracting essential oils.
  • due to the operation at a higher temperature for a long time it is easy to cause thermal decomposition or hydrolysis of heat-sensitive compounds and easily hydrolyzed components in the essential oil, resulting in the loss of active ingredients of the essential oil, resulting in a low extraction rate.
  • the invention provides a pepper essential oil with anti-inflammatory activity and a preparation method thereof.
  • the method uses fresh pepper fruits as raw materials, undergoes ultra-low temperature freezing, rapid microwave thawing, grinding, ultrasonic-assisted enzymolysis, steam distillation, oil-water separation, and anhydrous sodium sulfate to remove water to obtain pepper essential oil with anti-inflammatory activity.
  • a preparation method of pepper essential oil comprises: ultra-low temperature freezing of fresh pepper fruit, microwave thawing, grinding, enzymatic hydrolysis, steam distillation, and oil-water separation to obtain pepper essential oil.
  • the maturity of the fresh pepper fruit is 50-70%.
  • the temperature of the ultra-low temperature freezing is -60 to -80°C, specifically, -80°C.
  • the freezing time is 24-60h, such as 24-48h.
  • the microwave power is 600-800W, such as 800W, when the microwave is thawing.
  • thaw to a core temperature of 4°C.
  • polar substances such as water in the cells absorb microwaves to generate heat, and the intracellular temperature rises rapidly. Water vaporization produces pressure that ruptures the thin walls, resulting in micropores and cracks, so that the intracellular substances are easier to freeze. Dissolution.
  • an appropriate amount of water may be added to the refining process, for example, the amount of water added may be 5-10 times the weight of the fresh pepper fruit.
  • Refining can be done by conventional methods.
  • refining is performed to a fineness of 5-20 mesh. The study found that through the refining treatment, the raw materials are more fully broken, which is conducive to the full extraction of fresh pepper fruit components.
  • the enzyme used in the enzymolysis is a compound enzyme composed of cellulase, hemicellulase, and pectinase, and the weight ratio of the three is preferably (1-2):(1-2) :(1-3), such as 1:1:2.
  • the use of the above compound enzyme enzymatic hydrolysis can cause changes such as local loosening and bulking of the intercellular substance, and reduce the mass transfer resistance of the intercellular substance and other mass transfer barriers to the diffusion of volatile components from the cell to the extraction medium.
  • cellulase hemicellulase and pectinase are all commercially available.
  • the cellulase enzyme activity is 50u/mg.
  • the activity of the hemicellulase is 20000 u/mg.
  • the activity of pectinase is 500u/mg.
  • the added amount of the compound enzyme is 0.05%-1% of the weight of the fresh pepper fruit.
  • the temperature during the enzymatic hydrolysis is 40-60°C, such as 45-50°C.
  • the enzymatic hydrolysis time is usually 1-2h.
  • the enzymatic hydrolysis is preferably performed under ultrasonic conditions, and the ultrasonic frequency is 400-500W.
  • conventional steam distillation and oil-water separation methods can be used.
  • anhydrous sodium sulfate can be used to remove water.
  • the steam distillation method includes: putting the enzymatically hydrolyzed material into a round bottom flask, adding glass beads, connecting the volatile oil receiver and the reflux condenser, heating in an electric heating mantle, and distilling until the essential oil Increase again, stop heating, read the volume of the essential oil after cooling to room temperature, collect the essential oil, and remove water with anhydrous sodium sulfate.
  • the preparation method of the pepper essential oil comprises the following steps:
  • mixture A add a complex enzyme with 0.05% to 1% of the mass of fresh pepper fruit, and under the condition of 40-60°C, the ultrasonic frequency is 400-500W to assist enzymolysis for 1-2h to obtain mixture B; the complex enzyme is obtained from cellulose Enzyme, hemicellulase and pectinase are composed of 1:1:2 by weight;
  • the mixture B was extracted by steam distillation, the essential oil was collected, and the water was removed by anhydrous sodium sulfate.
  • step 3) includes: putting the mixture B into a round bottom flask, adding glass beads, connecting the volatile oil receiver and the reflux condenser, heating in an electric heating mantle, distilling until the essential oil does not increase, and stopping the heating After cooling to room temperature, read the volume of the essential oil, collect the essential oil, and remove water with anhydrous sodium sulfate.
  • the method of the present invention stores the fresh pepper fruit by ultra-low temperature freezing, and the substance metabolism and growth activities in the living cells are almost completely stopped, the physiological metabolic intensity is suppressed to the greatest extent, and the volatile components of the raw materials are better preserved;
  • the specific reason is that during the microwave rapid thawing process, polar substances such as water in the cells absorb microwaves to generate heat, the intracellular temperature rises rapidly, and the pressure generated by water vaporization causes the cell walls to rupture and produce micropores and cracks, so that the substances in the cells can be dissolved more easily; through grinding and homogenization, the raw materials are more fully broken, which is conducive to the full extraction of pepper fresh fruit components; then compound enzymes are added to it, causing changes such as local loosening and bulking of the intercellular substance , reduce the mass transfer resistance of mass transfer barriers such as the intercellular substance to the diffusion of volatile components from the cell to the extraction medium; ultrasonic-assisted enzymolysis speeds up the enzymatic hydrolysis time, and the above overall steps are
  • the invention is a method of decomposing the cell wall of fresh pepper fruit by using compound enzymes, reducing the mass transfer resistance, promoting the release of essential oil, and then extracting the essential oil by backflow of water vapor.
  • the fresh pepper essential oil prepared by the method is pale yellow, has obvious peppery aroma, has no peculiar smell, and has an essential oil yield as high as 3.14%, which is more than twice the essential oil yield of traditional black pepper and white pepper steam distillation methods.
  • the method of the present invention uses fresh pepper fruits as raw materials to directly extract natural products. Compared with traditional white pepper and black pepper extraction, the processing process is omitted, the utilization rate of pepper fresh fruits is effectively improved, and pepper promotes pepper resources to a certain extent. Development of value-added industries.
  • the present invention also includes 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 1-28 in Table 1 below.
  • the pepper essential oil contains ⁇ -thujene, with a relative content of 0.32%-0.34%, such as 0.34%.
  • the relative content in the present invention refers to the 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 studies and finds that the pepper essential oil has a better anti-inflammatory effect.
  • the pepper essential oil especially refers to the pepper essential oil prepared by the above method.
  • LSP lipopolysaccharide
  • pepper essential oil can inhibit the release of nitric oxide (NO), tumor necrosis factor (TNF- ⁇ ), interleukin-6 (IL-6), Interleukin-1 ⁇ (IL-1 ⁇ ), prostaglandin E2 (PGE2) and expression of cyclooxygenase-2 (COX-2) were inhibited, indicating that pepper essential oil has anti-inflammatory activity.
  • NO nitric oxide
  • TNF- ⁇ tumor necrosis factor
  • IL-6 interleukin-6
  • IL-1 ⁇ Interleukin-1 ⁇
  • PGE2 prostaglandin E2
  • COX-2 cyclooxygenase-2
  • the invention also includes the application of the pepper essential oil in the preparation of medicines with anti-inflammatory effects.
  • the anti-inflammatory effect includes inhibiting the release of nitric oxide (NO) from RAW 264.7 cells, inhibiting the release of tumor necrosis factor (TNF- ⁇ ), inhibiting the release of interleukin-6 (IL-6), inhibiting the release of interleukin- 1 ⁇ (IL-1 ⁇ ) release, inhibition of prostaglandin E2 (PGE2) release, inhibition of cyclooxygenase-2 (COX-2) expression in one or more.
  • NO nitric oxide
  • TNF- ⁇ tumor necrosis factor
  • IL-6 interleukin-6
  • IL-1 ⁇ interleukin- 1 ⁇
  • PGE2 prostaglandin E2
  • COX-2 cyclooxygenase-2
  • the invention also provides a pharmaceutical composition, which includes the pepper essential oil and a pharmaceutically acceptable carrier.
  • Fig. 1 Effect of pepper essential oil on cell viability in the experimental example of the present invention.
  • Figure 2 Effect of pepper essential oil on the release of nitric oxide (NO) from RAW 264.7 cells.
  • FIG 3 The effect of pepper essential oil on the release of tumor necrosis factor (TNF- ⁇ ), interleukin-6 (IL-6), interleukin-1 ⁇ (IL-1 ⁇ ), and prostaglandin E2 (PGE2) from RAW 264.7 cells.
  • TNF- ⁇ tumor necrosis factor
  • IL-6 interleukin-6
  • IL-1 ⁇ interleukin-1 ⁇
  • PGE2 prostaglandin E2
  • Figure 4 Effect of pepper essential oil on expression of cyclooxygenase-2 (COX-2) in RAW 264.7 cells.
  • the following pepper varieties are Indonesian large-leaved species, provided by the Institute of Spices and Beverages, Chinese Academy of Tropical Agricultural Sciences.
  • Cellulase, hemicellulase, and pectinase were all purchased from source leaf organisms, and the enzyme activities were 50u/mg, 20000u/mg, and 500u/mg, respectively.
  • the water content is measured with reference to the azeotropic distillation method (third method) in GB 5009.3-2016 "Determination of Moisture in Food Safety National Standard”.
  • Example 1 The results show that the dry base extraction yield of pepper fresh fruit essential oil in Example 1 is 3.511mL/100g, which is about twice that of comparative example 1 black pepper (1.710mL/100g) and comparative example 2 white pepper (1.853mL/100g), indicating that The extraction yield of the fresh pepper fresh fruit essential oil on a dry basis is significantly higher than that of black pepper and white pepper.
  • GC-MS method Dilute the essential oil 50 times with n-hexane, dehydrate it with anhydrous sodium sulfate, pass through a 0.45 ⁇ m microporous membrane, and wait for the test.
  • Chromatographic column J&W DB-5 quartz capillary column (30m ⁇ 0.25mm, 0.25 ⁇ m). Heating program: column temperature is 50°C, raised to 75°C at 3°C/min, then raised to 140°C at 1.5°C/min, then raised to 230°C at 10°C/min, kept for 2 minutes, and finally raised to 20°C/min To 280°C, keep for 3min.
  • the flow rate of carrier gas (He) was 1 mL/min, and the injection volume was 1 ⁇ L, splitless.
  • Example 1 pepper fresh fruit, comparative example 1 black pepper, and comparative example 2 white pepper essential oils are shown in Table 1. From Table 1, it can be seen that the essential oil components of pepper fresh fruit and black and white pepper are mainly terpene compounds, but the relative content There were some differences, and ⁇ -thujene was detected in the essential oil of pepper fresh fruit, but not in black and white pepper.
  • Dexamethasone was purchased from Yuanye Biotechnology
  • lipopolysaccharide LPS
  • macrophage RAW264.7 was purchased from Shanghai Cybercon Biotechnology
  • fetal bovine serum was purchased from BOVOGEN (South America)
  • trypsin -EDTA was purchased from GIBCO
  • PBS was purchased from Boster
  • CCK-8 was purchased from MCE
  • NO colorimetric assay kit TNF- ⁇ ELISA Kit, IL-6 ELISA Kit, IL-1 ⁇ ELISA Kit, PGE2 ELISA Kit were purchased from Elabscience .
  • the pepper essential oil sample prepared in Example 1 was sterilized by filtering through a 0.22 ⁇ m filter. Discard the old medium in the culture flask before seeding the plate, wash with PBS and then add 0.25% trypsin to digest the cells. Pour off the trypsin, add DMEM medium containing 10% fetal bovine serum and blow the cells repeatedly, and transfer to the sample tank to blow evenly. Count with a cell counting plate, add complete medium to dilute, suck 100 ⁇ L with a row gun, inoculate the cells on a 96-well plate (5 ⁇ 10 3 cells/well), and culture overnight at 37°C in a 5% CO 2 constant temperature incubator.
  • control represents the control group, that is, adding 0 ⁇ g/mL pepper essential oil.
  • the results showed that 200 ⁇ g/mL dexamethasone (DXMS) had no significant effect on the cell viability.
  • DXMS dexamethasone
  • the concentration of pepper essential oil was chosen to be 7.5 ⁇ g/mL, 15 ⁇ g/mL, and 30 ⁇ g/mL in subsequent experiments.
  • the essential oil samples were filter sterilized through a 0.22 ⁇ m filter. Discard the old medium in the culture flask before seeding the plate, wash with PBS and then add 0.25% trypsin to digest the cells. Pour off the trypsin, add DMEM medium containing 10% fetal bovine serum and blow the cells repeatedly, and transfer to the sample tank to blow evenly. Count with a cell counting plate, add complete medium to dilute, draw 1 mL with a row gun, inoculate the cells on a 12-well plate (2 ⁇ 10 5 cells/well), and culture overnight at 37°C in a 5% CO 2 incubator. Aspirate the medium, add the sample to the working concentration, and pretreat the cells for 1 h.
  • Blank indicates the blank group, which was not induced by 1 ⁇ g/mL LSP (the same below);
  • Model indicates the model group, which was induced by 1 ⁇ g/mL LSP (the same below).
  • TNF- ⁇ tumor necrosis factor
  • IL-6 interleukin-6
  • IL-1 ⁇ interleukin-1 ⁇
  • PGE2 prostaglandin E2
  • the essential oil samples were filter sterilized through a 0.22 ⁇ m filter. Discard the old medium in the culture flask before seeding the plate, wash with PBS and then add 0.25% trypsin to digest the cells. Pour off the trypsin, add DMEM medium containing 10% fetal bovine serum and blow the cells repeatedly, and transfer to the sample tank to blow evenly. Count with a cell counting plate, add complete medium for dilution, suck 500 ⁇ L with a row gun, inoculate the cells on a 24-well plate (1 ⁇ 10 5 cells/well), and culture overnight at 37°C in a 5% CO 2 incubator. Aspirate the medium, add the sample and DXM to the working concentration, and pretreat the cells for 1 h.
  • the content of IL-6 in the cell supernatant of each mass concentration of pepper essential oil was significantly lower than that of the model group in a dose-dependent manner, and there was no significant difference in IL-6 content between 30 ⁇ g/mL pepper essential oil and 200 ⁇ g/mL dexamethasone in the control group.
  • the content of IL-1 ⁇ in the cell supernatant of 7.5 ⁇ g/mL pepper essential oil was not significantly different from that of the model group, and the content of IL-1 ⁇ in the cell supernatant of 15 ⁇ g/mL and 30 ⁇ g/mL pepper essential oil was significantly different from that of the model group.
  • the essential oil samples were filter sterilized through a 0.22 ⁇ m filter. Discard the old medium in the culture flask before seeding the plate, wash with PBS and then add 0.25% trypsin to digest the cells. Pour off the trypsin, add DMEM medium containing 10% fetal bovine serum and blow the cells repeatedly, and transfer to the sample tank to blow evenly.
  • Count with a cell counting plate add complete medium to dilute, suck 2 mL with a row gun, inoculate the cells on a 6-well plate (5 ⁇ 10 5 cells/well), and culture overnight in a 37°C, 5% CO 2 constant temperature incubator; Aspirate the medium, add the adjusted sample and DXM to the working concentration, and pretreat the cells for 1 h; add 1 ⁇ g/mL LPS to continue culturing for 24 h; wash the cells with PBS, lyse the cells with ripa, and collect the cell lysate; use BCA The quantitative method was used to adjust the protein concentration of each group to be consistent, and the COX-2 protein content was detected by WB.
  • the invention provides a pepper essential oil with anti-inflammatory activity and a preparation method thereof.
  • the preparation method of the pepper essential oil of the present application comprises: ultra-low temperature freezing of the fresh pepper fruit, microwave thawing, grinding, enzymatic hydrolysis, steam distillation, and oil-water separation to obtain the pepper essential oil.
  • pepper essential oil can inhibit RAW 264.7 cells from releasing nitric oxide (NO), tumor necrosis factor (TNF- ⁇ ), interleukin-6 (IL-6), interleukin-1 ⁇ (IL-1 ⁇ ), prostate Pepper essential oil has anti-inflammatory activity and has good economic value and application prospect.
  • NO nitric oxide
  • TNF- ⁇ tumor necrosis factor
  • IL-6 interleukin-6
  • IL-1 ⁇ interleukin-1 ⁇
  • prostate Pepper essential oil has anti-inflammatory activity and has good economic value and application prospect.

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Abstract

一种具有抗炎活性的胡椒精油的制备方法，包括：将胡椒鲜果超低温冷冻、微波解冻、磨浆、酶解、水蒸气蒸馏、油水分离，得到胡椒精油。实验证明，胡椒精油对抑制RAW 264.7细胞释放一氧化氮(NO)、肿瘤坏死因子(TNF‑ɑ)、白细胞介素‑6(IL‑6)、白细胞介素‑1β(IL‑1β)、前列腺素E2(PGE2)和表达环氧合酶‑2(COX‑2)具有抑制作用，说明胡椒精油具有抗炎活性。

Description

一种具有抗炎活性的胡椒精油及其制备方法
交叉引用
本申请要求2022年2月18日提交的专利名称为“一种具有抗炎活性的胡椒精油及其制备方法”的第2022101524579号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及天然产物提取技术领域，特别涉及一种具有抗炎活性的胡椒精油及其制备方法。
背景技术
胡椒(Piper nigrum L.)为胡椒科胡椒属植物，是一种最古老而闻名的香辛料作物，含有挥发油、胡椒碱等物质，具有多种功能活性，广用于食品和医药工业。
常用的精油提取方法有水蒸气蒸馏法、压榨法、有机溶剂萃取法以及后来出现的超声波辅助萃取、超临界CO 2萃取和微波辅助水蒸气蒸馏等方法。其中，水蒸气蒸馏设备简单、易操作、成本低、精油品质好，是提取精油最常用的方法。但由于要在较高温度下操作很长时间，容易引起精油中热敏性化合物和易水解成分的热分解或水解，造成精油有效成分的损失，导致提取率不高。
目前已有胡椒脱梗、脱皮、干燥、油树脂提取、精油提取、调味品制作等加工方面的报道。也有研究表明，黑、白胡椒精油在抗肿瘤、防止咳嗽作用。
胡椒精油的提取方法及应用有待进一步研究。
发明内容
本发明提供一种具有抗炎活性的胡椒精油及其制备方法。本方法以胡椒鲜果为原料，经超低温冷冻、微波快速解冻、磨浆、超声波辅助酶解、水蒸气蒸馏，油水分离，无水硫酸钠除水，得到具有抗炎活性的胡椒精油。
具体而言，一种胡椒精油的制备方法，包括：将胡椒鲜果超低温冷冻、微波解冻、磨浆、酶解、水蒸气蒸馏、油水分离，得到胡椒精油。
根据本发明实施例，所述胡椒鲜果的成熟度为5-7成。研究发现，5-7成熟的胡椒鲜果精油含量更高，且更有利于胡椒精油的提取。
根据本发明实施例，所述超低温冷冻的温度为-60至-80℃，具体例如-80℃。通常冷冻的时间为24-60h，例如24-48h。研究发现，通过超低温冷冻胡椒鲜果，活细胞内的物质代谢和生长活动几乎完全停止，最大限度地抑制了生理代谢强度，较好地保存了原料的挥发性成分。
根据本发明实施例，所述微波解冻时微波功率为600-800W，例如800W。通常解冻至中心温度为4℃。研究发现，微波快速解冻过程中细胞内的水等极性物质吸收微波后产生热量，胞内温度迅速上升，水气化产生压力使细壁破裂，产生微孔和裂痕，从而使细胞内物质更易溶出。
根据本发明实施例，所述磨浆过程可加入适量的水，例如加水量可为胡椒鲜果重量的5-10倍。可采用常规方法磨浆。在一些具体实例中，磨浆至细度为5-20目。研究发现，通过磨浆处理，原料破碎更充分，有利于胡椒鲜果成分充分提取。
根据本发明实施例，所述酶解所用的酶为由纤维素酶、半纤维素酶、果胶酶所组成的复合酶，三者重量比例优选为(1-2):(1-2):(1-3)，例如1:1:2。研究发现，采用上述复合酶酶解，引起细胞间质局部疏松、膨松等变化，减小细胞间质等传质屏障对挥发性成分从细胞内向提取介质扩散的传质阻力。
本发明中，纤维素酶、半纤维素酶、果胶酶均可市售购得。
在一些实例中，纤维素酶的酶活为50u/mg。
在一些实例中，半纤维素酶的酶活为20000u/mg。
在一些实例中，果胶酶的酶活为500u/mg。
根据本发明实施例，所述复合酶的添加量为胡椒鲜果重量的0.05％-1％。
根据本发明实施例，所述酶解时的温度为40-60℃，例如45-50℃。酶解时间通常为1-2h。
根据本发明实施例，所述酶解优选在超声条件下进行，超声频率为400-500W。研究发现，超声波辅助酶解加速了酶解时间，有利于提高胡椒精油得率。
本发明中，可采用常规水蒸气蒸馏及油水分离方法。例如可采用无水硫酸钠除水。
根据本发明实施例，水蒸气蒸馏的方法包括：将酶解后的物料装入圆底烧瓶中，加玻璃珠，连接挥发油接收器与回流冷凝管，置于电热套中加热，蒸馏至精油不再增加，停止加热，冷却至室温后读取精油体积，收集精油，无水硫酸钠除水。
根据本发明实施例，所述胡椒精油的制备方法，包括以下步骤：
1)原料预处理
将胡椒鲜果于-80℃超低温冷冻储存；600-800W微波快速解冻，加入蒸馏水磨浆处理得到混合物A；
2)超声波辅助酶解
在混合物A中，添加胡椒鲜果质量0.05％～1％的复合酶，在40-60℃条件下，超声波超声频率为400-500W辅助酶解1-2h得到混合物B；所述复合酶由纤维素酶、半纤维素酶、果胶酶按重量比1:1:2组成；
3)水蒸气蒸馏提取
将混合物B水蒸气蒸馏提取，收集精油，无水硫酸钠除水。
在一些具体实例中，步骤3)包括：将混合物B装入圆底烧瓶中，加玻璃珠，连接挥发油接收器与回流冷凝管，置于电热套中加热，蒸馏至精油不再增加，停止加热，冷却至室温后读取精油体积，收集精油，无水硫酸钠除水。
本发明方法通过超低温冷冻储存胡椒鲜果，活细胞内的物质代谢和生长活动几乎完全停止，最大限度地抑制了生理代谢强度，较好地保存了原 料的挥发性成分；通过微波快速解冻、研磨匀浆、超声波辅助酶解相结合的方法，具体原因是微波快速解冻过程中细胞内的水等极性物质吸收微波后产生热量，胞内温度迅速上升，水气化产生压力使细壁破裂，产生微孔和裂痕，从而使细胞内物质更易溶出；通过研磨匀浆处理，原料破碎更充分，有利于胡椒鲜果成分充分提取；接着向其中加入复合酶，引起细胞间质局部疏松、膨松等变化，减小细胞间质等传质屏障对挥发性成分从细胞内向提取介质扩散的传质阻力；超声波辅助酶解加速了酶解时间，以上整体步骤均有利于胡椒鲜果精油提取。
本发明是利用复合酶分解胡椒鲜果细胞壁，减小传质阻力，促进精油释放，再利用水蒸气回流提取精油的一种方法。本方法制得的胡椒鲜果精油呈淡黄色，具有明显的胡椒香，无异味，精油得率高达3.14％，是传统黑胡椒和白胡椒的水蒸气蒸馏法精油得率的2倍多。
本发明方法采用胡椒鲜果为原料直接进行天然产物的提取，相比传统的白胡椒、黑胡椒提取省去了加工过程，有效提高了胡椒鲜果的利用率，且胡椒在一定程度上促进了胡椒资源附加值产业发展。
本发明还包括上述方法制备得到的胡椒精油。该胡椒精油至少含有下文表1中的序号1-28中的成分中的一种或几种。特别地，该胡椒精油中含有α-侧柏烯，相对含量为0.32％-0.34％，例如0.34％。
本发明所述相对含量是指某个化合物的峰面积除以所有积分到的峰面积的和，也就是该化合物的百分比含量。
本发明还研究发现，胡椒精油具有较好的抗炎效果。所述胡椒精油尤其是指上述方法制备得到的胡椒精油。实验证明，在脂多糖(LSP)诱导的细胞模型中，胡椒精油对抑制RAW 264.7细胞释放一氧化氮(NO)、肿瘤坏死因子(TNF-ɑ)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、前列腺素E2(PGE2)和表达环氧合酶-2(COX-2)具有抑制作用，说明胡椒精油具有抗炎活性。
本发明还包括胡椒精油在制备具有抗炎作用的药物方面的应用。具体 地，所述抗炎作用包括抑制RAW 264.7细胞释放一氧化氮(NO)，抑制肿瘤坏死因子(TNF-ɑ)释放，抑制白细胞介素-6(IL-6)释放，抑制白细胞介素-1β(IL-1β)释放，抑制前列腺素E2(PGE2)释放，抑制环氧合酶-2(COX-2)表达中的一种或几种。
本发明还提供一种药物组合物，包括胡椒精油和药学上可用的载体。
附图说明
图1：本发明实验例中胡椒精油对细胞活力的影响。
图2：胡椒精油对RAW 264.7细胞释放一氧化氮(NO)的影响。
图3：胡椒精油对RAW 264.7细胞释放肿瘤坏死因子(TNF-ɑ)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、前列腺素E2(PGE2)的影响。
图4：胡椒精油对RAW 264.7细胞表达环氧合酶-2(COX-2)的影响。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
若无特殊指明，以下胡椒品种为印尼大叶种，由中国热带农业科学院香料饮料研究所提供。
纤维素酶、半纤维素酶、果胶酶均购自源叶生物，酶活分别为50u/mg、20000u/mg、500u/mg。
实施例1 胡椒精油制备方法(胡椒鲜果)
将胡椒鲜果(5-7成熟)放入-80℃超低温冷冻储存24h，微波(功率800W)快速解冻10min，加入胡椒鲜果6倍重量蒸馏水，磨浆处理得到混合物A；在混合物A中，添加胡椒鲜果重量1％复合酶(由纤维素酶、半纤维素酶、果胶酶按重量比1:1:2组成)，在50℃条件下，超声波(功率500W)辅助酶解1h得到混合物B；将混合物B装入圆底烧瓶中，加玻璃珠，连接挥发油接收器与回流冷凝管，置于电热套中加热，蒸馏至精 油不再增加，停止加热，冷却至室温后读取精油体积，收集精油，无水硫酸钠除水，得胡椒精油。
胡椒鲜果精油干基提取得率的计算公式为：
Figure PCTCN2022093758-appb-000001
含水量参照GB 5009.3-2016《食品安全国家标准食品中水分的测定》中的共沸蒸馏法(第三法)进行测量。
对比例1
黑胡椒精油的制备及含量测定：采用GB/T 17527-2009《胡椒精油含量的测定》。
对比例2
白胡椒精油的制备及含量测定：采用GB/T 17527-2009《胡椒精油含量的测定》。
实验1
结果显示，实施例1胡椒鲜果精油干基提取得率为3.511mL/100g，为对比例1黑胡椒(1.710mL/100g)、对比例2白胡椒(1.853mL/100g)的两倍左右，说明本发明的胡椒鲜果精油干基提取得率显著高于黑胡椒、白胡椒。
GC-MS方法测定：正己烷将精油稀释50倍经无水硫酸钠脱水后过0.45μm微孔膜，待测。色谱柱：J&W DB-5石英毛细柱(30m×0.25mm，0.25μm)。升温程序：柱温50℃，以3℃/min升至75℃，再以1.5℃/min升至140℃，然后以10℃/min升至230℃，保持2min，最后以20℃/min升至280℃，保持3min。载气(He)流速1mL/min，进样量1μL，不分流。电子轰击离子源；电子能量70eV；传输线温度280℃；离子源温度230℃；质量扫描范围m/z 35～450amu。
实施例1胡椒鲜果、对比例1黑胡椒、对比例2白胡椒精油成分见表1，从表1中可知，胡椒鲜果精油成分与黑、白胡椒均以萜烯类化合物为主，但相对含量有所差异，且在胡椒鲜果精油中检测出了α-侧柏烯，在 黑、白胡椒中未检测到。
表1 胡椒精油成分表
Figure PCTCN2022093758-appb-000002
Figure PCTCN2022093758-appb-000003
注：“-”表示未检测出。
实验2 抗炎活性检测
1.仪器与材料
1.1仪器
表2 仪器
Figure PCTCN2022093758-appb-000004
1.2实验材料
地塞米松(DXMS)购自源叶生物，脂多糖(LPS)，购自美国Sigma公司，巨噬细胞RAW264.7购买自上海赛柏慷生物，胎牛血清购买自BOVOGEN(南美)，胰酶-EDTA购买自GIBCO，PBS购买自博士德生物，CCK-8购买自MCE，NO比色法测试盒、TNF-αELISA Kit、IL-6 ELISA Kit、IL-1βELISA Kit、PGE2 ELISA Kit均购买自Elabscience。
2实验内容
细胞复苏：
将-80℃冻存的RAW264.7细胞置于37℃的水浴锅中，快速晃动至融化。将融化后的细胞和7mL培养基加入到15mL无菌离心管，1000rpm离心5min，弃去上清液，转移至含有5mL 10％FBS培养基的全新25T细胞培养瓶中；7.5％CO 2，37℃条件下，培养8h后弃去旧培养基更换新培养基，继续培养。等到细胞长满培养瓶即可进行实验。
2.1胡椒精油对细胞活力的影响
将实施例1制备的胡椒精油样品(下同)经0.22μm过滤器过滤除菌。 种板前弃去培养瓶中的旧培养基，用PBS洗后加入0.25％胰蛋白酶消化细胞。倒掉胰蛋白酶，加入含10％胎牛血清的DMEM培养基反复吹打细胞，并转入加样槽中吹匀。用细胞计数板计数，加入完全培养基稀释，用排枪吸取100μL，把细胞接种到96孔板上(5×10 3个/孔)，在37℃，5％CO 2恒温培养箱内培养过夜。弃去96孔板中的旧培养基，加入样品至工作浓度，继续培养120h。吸走原有培养基，PBS冲洗2次，把96孔板中的培养基换成100μL新鲜的含10％胎牛血清的DMEM，在每孔中分别加入10μL CCK-8溶液。在培养箱中培养3h后，在450nm测吸光度。细胞存活率的计算公式如下：
细胞存活率(％)＝(A sample)/(Blank)×100％
结果见图1。control表示对照组，即添加0μg/mL胡椒精油。结果表明，200μg/mL地塞米松(DXMS)对细胞活性无明显影响，当胡椒精油浓度在0～30μg/mL时，对RAW 264.7细胞的生长抑制无显著影响，当胡椒精油浓度大于30μg/mL时，对RAW 264.7细胞的生长抑制有显著影响。因此，后续实验选择胡椒精油浓度为7.5μg/mL、15μg/mL、30μg/mL。
2.2胡椒精油对RAW 264.7细胞释放一氧化氮(NO)的影响
将精油样品经0.22μm过滤器过滤除菌。种板前弃去培养瓶中的旧培养基，用PBS洗后加入0.25％胰蛋白酶消化细胞。倒掉胰蛋白酶，加入含10％胎牛血清的DMEM培养基反复吹打细胞，并转入加样槽中吹匀。用细胞计数板计数，加入完全培养基稀释，用排枪吸取1mL，把细胞接种到12孔板上(2×10 5个/孔)，在37℃，5％CO 2恒温培养箱内培养过夜。吸去培养基，加入样品至工作浓度，预处理细胞1h。加入1μg/mL的LPS继续培养24h。用胰酶将细胞消化，并用PBS清洗细胞，最后用ripa裂解细胞，收集细胞裂解液。利用NO比色法测试盒检测胞内NO含量。
结果见图2。Blank表示空白组，未采用1μg/mL LSP诱导刺激(下同)；Model表示模型组，采用1μg/mL LSP诱导刺激(下同)。
结果表明，模型组NO分泌量明显增加，与空白组相比，具有显著性差异，说明LSP炎症模型建立成功。胡椒精油各质量浓度细胞上清液中的NO含量明显低于模型组，呈现剂量依赖性，且30μg/mL胡椒精油比对照组200μg/mL地塞米松抑制NO抑制效果更佳，说明胡椒精油有良好抑制NO释放作用。
2.3胡椒精油对RAW 264.7细胞释放肿瘤坏死因子(TNF-ɑ)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、前列腺素E2(PGE2)的影响
将精油样品经0.22μm过滤器过滤除菌。种板前弃去培养瓶中的旧培养基，用PBS洗后加入0.25％胰蛋白酶消化细胞。倒掉胰蛋白酶，加入含10％胎牛血清的DMEM培养基反复吹打细胞，并转入加样槽中吹匀。用细胞计数板计数，加入完全培养基稀释，用排枪吸取500μL，把细胞接种到24孔板上(1×10 5个/孔)，在37℃，5％CO 2恒温培养箱内培养过夜。吸去培养基，加入样品和DXM至工作浓度，预处理细胞1h。加入1μg/mL的LPS继续培养24h。收集细胞上清。利用ELISA检测试剂盒测上清中TNF-α、IL-6、IL-1β、PGE2的含量。
结果见图3。结果表明，LSP作用于RAW264.7细胞，细胞上清液中的TNF-α、IL-6、IL-1β、PGE2含量明显增加，与空白组相比，具有显著性差异，说明LSP炎症模型建立成功。7.5μg/mL胡椒精油细胞上清液中TNF-ɑ含量明显与模型组无显著差异，15μg/mL和30μg/mL胡椒精油细胞上清液中TNF-ɑ含量与模型组具有显著差异，呈现剂量依赖性，且15μg/mL胡椒精油与对照组200μg/mL地塞米松TNF-ɑ含量无显著性差异。胡椒精油各质量浓度细胞上清液中的IL-6含量显著低于模型组，呈现剂量依赖性，且30μg/mL胡椒精油与对照组200μg/mL地塞米松IL-6含量无显著性差异。7.5μg/mL胡椒精油细胞上清液中IL-1β含量明显与模型组无显著差异，15μg/mL和30μg/mL胡椒精油细胞上清液中IL-1β含量与模型组具有显著差异，呈现剂量依赖性，且30μg/mL胡椒精油与对照组200μg/mL地塞米松TNF-ɑ含量无显著性差异。7.5μg/mL胡椒精油细 胞上清液中PGE2含量明显与模型组无显著差异，15μg/mL和30μg/mL胡椒精油细胞上清液中PGE2含量与模型组具有显著差异，呈现剂量依赖性，且30μg/mL胡椒精油与对照组200μg/mL地塞米松TNF-ɑ含量无显著性差异。以上整体，说明胡椒精油有良好抑制TNF-α、IL-6、IL-1β、PGE2释放作用。
2.4胡椒精油对RAW 264.7细胞表达环氧合酶-2(COX-2)的影响
将精油样品经0.22μm过滤器过滤除菌。种板前弃去培养瓶中的旧培养基，用PBS洗后加入0.25％胰蛋白酶消化细胞。倒掉胰蛋白酶，加入含10％胎牛血清的DMEM培养基反复吹打细胞，并转入加样槽中吹匀。用细胞计数板计数，加入完全培养基稀释，用排枪吸取2mL，把细胞接种到6孔板上(5×10 5个/孔)，在37℃，5％CO 2恒温培养箱内培养过夜；吸去培养基，加入调整好浓度的样品和DXM至工作浓度，预处理细胞1h；加入1μg/mL的LPS继续培养24h；用PBS清洗细胞后，用ripa裂解细胞，收集细胞裂解液；用BCA定量法调整各组样品蛋白浓度一致，用WB检测COX-2蛋白含量。
结果见图4。结果表明，模型组与空白组COX-2蛋白含量差异显著，说明LSP炎症模型建立成功。胡椒精油各质量浓度细胞上清液中的COX-2蛋白含量明显低于模型组，呈现剂量依赖性，且30μg/mL胡椒精油与对照组200μg/mL地塞米松抑制COX-2蛋白表达效果无显著差异，说明胡椒精油有良好抑制COX-2蛋白表达作用。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种一种具有抗炎活性的胡椒精油及其制备方法。本申请的胡椒精油的制备方法包括：将胡椒鲜果超低温冷冻、微波解冻、磨浆、 酶解、水蒸气蒸馏、油水分离，得到胡椒精油。实验证明，胡椒精油对抑制RAW 264.7细胞释放一氧化氮(NO)、肿瘤坏死因子(TNF-ɑ)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、前列腺素E2(PGE2)和表达环氧合酶-2(COX-2)具有抑制作用，说明胡椒精油具有抗炎活性，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种胡椒精油的制备方法，其特征在于，包括：将胡椒鲜果超低温冷冻、微波解冻、磨浆、酶解、水蒸气蒸馏、油水分离，得到胡椒精油。
  2. 根据权利要求1所述胡椒精油的制备方法，其特征在于，所述胡椒鲜果的成熟度为5-7成。
  3. 根据权利要求1或2所述胡椒精油的制备方法，其特征在于，所述超低温冷冻的温度为-60至-80℃，可选为-80℃；进一步优选冷冻的时间为24-60h。
  4. 根据权利要求1-3任一项所述胡椒精油的制备方法，其特征在于，所述微波解冻时微波功率为600-800W，可选为800W。
  5. 根据权利要求1-4任一项所述胡椒精油的制备方法，其特征在于，所述磨浆时加水量可为胡椒鲜果重量的5-10倍；和/或，磨浆至细度为5-20目。
  6. 根据权利要求1-5任一项所述胡椒精油的制备方法，其特征在于，所述酶解所用的酶为由纤维素酶、半纤维素酶、果胶酶所组成的复合酶，三者重量比例为(1-2):(1-2):(1-3)，进一步优选为1:1:2；和/或，
    所述复合酶的添加量为胡椒鲜果重量的0.05％-1％；
    纤维素酶的酶活为50u/mg；和/或，
    半纤维素酶的酶活为20000u/mg；和/或，
    果胶酶的酶活为500u/mg。
  7. 根据权利要求1-6任一项所述胡椒精油的制备方法，其特征在于，包括以下步骤：
    1)原料预处理
    将胡椒鲜果于-80℃超低温冷冻储存；600-800W微波快速解冻，加入蒸馏水磨浆处理得到混合物A；
    2)超声波辅助酶解
    在混合物A中，添加胡椒鲜果质量0.05％～1％的复合酶，在40-60℃ 条件下，超声波超声频率为400-500W辅助酶解1-2h得到混合物B；所述复合酶由纤维素酶、半纤维素酶、果胶酶按重量比1:1:2组成；
    3)水蒸气蒸馏提取
    将混合物B水蒸气蒸馏提取，收集精油，无水硫酸钠除水。
  8. 胡椒精油，其特征在于，由权利要求1-7任一项所述方法制备得到；可选地，所述胡椒精油中含有α-侧柏烯，相对含量为0.32％-0.34％，可选0.34％。
  9. 胡椒精油在制备具有抗炎作用的药物方面的应用；可选地，所述抗炎作用包括抑制RAW 264.7细胞释放一氧化氮(NO)，抑制肿瘤坏死因子(TNF-ɑ)释放，抑制白细胞介素-6(IL-6)释放，抑制白细胞介素-1β(IL-1β)释放，抑制前列腺素E2(PGE2)释放，抑制环氧合酶-2(COX-2)表达中的一种或几种；和/或，所述胡椒精油为权利要求8所述的胡椒精油。
  10. 一种药物组合物，其特征在于，包括胡椒精油和药学上可用的载体；可选地，所述胡椒精油为权利要求8所述的胡椒精油。
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