WO2023272797A1 - Method for extracting alpinia oxyphylla miq essential oil for inhibiting listeria monocytogenes, and alpinia oxyphylla miq essential oil - Google Patents

Method for extracting alpinia oxyphylla miq essential oil for inhibiting listeria monocytogenes, and alpinia oxyphylla miq essential oil Download PDF

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WO2023272797A1
WO2023272797A1 PCT/CN2021/106837 CN2021106837W WO2023272797A1 WO 2023272797 A1 WO2023272797 A1 WO 2023272797A1 CN 2021106837 W CN2021106837 W CN 2021106837W WO 2023272797 A1 WO2023272797 A1 WO 2023272797A1
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essential oil
powder
water
listeria monocytogenes
alpinia oxyphylla
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PCT/CN2021/106837
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French (fr)
Chinese (zh)
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于福来
胡璇
王丹
陈悦
王凯
谢小丽
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中国热带农业科学院热带作物品种资源研究所
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Priority to JP2021560525A priority Critical patent/JP2023547742A/en
Publication of WO2023272797A1 publication Critical patent/WO2023272797A1/en

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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
    • C11B9/027—Recovery of volatiles by distillation or stripping
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A01N65/48—Zingiberaceae [Ginger family], e.g. ginger or galangal
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to a method for extracting a nootropic essential oil inhibiting Listeria monocytogenes and the nootropic essential oil.
  • Listeria monocytogenes (Listeria monocytogenes) is a zoonotic pathogen, it widely exists in nature, food-borne Listeria monocytogenes is harmful to human safety, the bacteria in the environment of 4 °C It can still grow and reproduce, and it is one of the main pathogenic bacteria that threaten human health in refrigerated food.
  • the bacteria has strong resistance to physical and chemical factors, can survive for a long time in different environments such as soil, feces, and silage feed, has strong resistance to alkali and salt, is not easy to be frozen and thawed, can withstand high osmotic pressure, and is not easy to suppressed or killed.
  • the applicant's research found that the nootropic essential oil extracted by a specific method showed obvious inhibitory effect on Listeria monocytogenes.
  • the present invention discloses a method for extracting intellectual essential oil that inhibits Listeria monocytogenes, which is of great significance for the popularization and application of intellectual essential oil.
  • the object of the present invention is to provide a method for extracting the nootropic essential oil that inhibits Listeria monocytogenes and the nootropic essential oil.
  • the present invention scheme comprises the following contents:
  • a kind of extracting method of the intelligence-inducing essential oil that suppresses Listeria monocytogenes comprises the following steps:
  • the powder obtained by crushing and sieving is first placed at 35 ⁇ 5° C. under the condition of relative humidity of 60-70%, and then soaked in water and steam distilled.
  • the standing time is 5-7d.
  • the sieve is a 40-60 mesh sieve.
  • the pH of the water is alkaline.
  • the pH of the water is 7.5-8.5.
  • the pH of the water is 8.5.
  • the temperature of steam distillation is 220-230°C.
  • the present invention provides a nootropic essential oil, which is prepared by the method provided by the present invention and exhibits inhibitory activity against Listeria monocytogenes.
  • the invention provides a method for extracting intelligence-inducing essential oil inhibiting Listeria monocytogenes.
  • the essential oil can be extracted efficiently through a steam distillation method, and the extraction rate can reach more than 1.5%.
  • the extraction method of the invention is easy to operate, does not need to use a large amount of chemical reagents, and is safe and efficient.
  • the nootropic essential oil obtained in the invention exhibits extremely strong inhibitory activity against Listeria monocytogenes.
  • the diameter of the inhibition zone was above 8.6 mm, and the MIC was below 15 ⁇ L/mL.
  • the said nootropic is the dried ripe fruit of the nootropic Alpinia oxyphylla Miq., and the samples were collected in Yongchu Village, Baisha County, Hainan province.
  • the results show that: when the soaking time is 0-2 hours, the soaking time and the extraction rate of the essential oil increase in a positive correlation. The greater the effect.
  • the immersion time is 2-5h, the immersion time and the extraction rate of the nootropic essential oil are negatively correlated, and the oil yield of the nootropic fruit essential oil is greatly lost.
  • the extraction rate of essential oil is reduced, so the soaking time is selected to be 2 to 3 hours.
  • the final conditions for the essential oil of the fruit of the fruit were determined as follows: the degree of crushing was 40 mesh, the ratio of solid to liquid was 1:10, soaked at 230°C for 2.4h and distilled for 5.5h; the extraction rate reached 1.8%.
  • essential oil 1 ⁇ 5 Take the dried and mature fruit of Noutropia chinensis, crush it through a 40-mesh sieve to get the powder; weigh 50g of the powder, and soak it in 10 times the amount of distilled water with pH 7.0, 7.5, 8.0, 8.5, and 9.0 respectively In, soak for 2.4h, steam distillation at 230°C for 5.5h.
  • Essential oil 6 Take the dried and mature fruit of Yizhi, crush it through a 40-mesh sieve to get a powder; weigh 50g of the powder, and soak it in 10 times the amount of distilled water with pH 7.0, soak for 2.4h, and steam distill at 180°C for 5.5h .
  • Essential oils 7 ⁇ 10 Take the dried and mature fruits of Nootropics, crush them through a 40-mesh sieve to obtain powder; place the powder at 35 ⁇ 5°C and 65 ⁇ 5% relative humidity (thickness 2-3cm) 3, 5, 7, 9d; Weigh 50g of the powder, soak it in 10 times the amount of distilled water with pH 8.5, soak for 2.4h, and steam distill at 180°C for 5.5h.
  • strains and Listeria monocytogenes ATCC 19111 and Staphylococcus aureus ATCC 6538 were purchased from Beijing Beina Chuanglian Biotechnology Research Institute.
  • bacterial solution bacterial solution concentration 10 7 cfu/ml, coating amount 60 ⁇ L
  • make a mark and repeat each bacterium three times.
  • the negative control is sterile water
  • the positive control is sterile water.
  • the control was levofloxacin (concentration 15 ⁇ g/ml), and the culture condition was 24 hours in a constant temperature incubator at 37°C.
  • the result standard is: when the diameter of the antibacterial zone is greater than 20mm, it is extremely sensitive; when the diameter is between 15-20mm, it is high-sensitivity; Mei et al., 2018).
  • the MIC test was carried out on a 96-well plate using the micro-doubling dilution method.
  • A1 was used as the first well starting from left to right, and 1 to 12 wells on each plate were liquid medicines, and 3 wells were prepared on each plate.
  • the culture condition is to cultivate in a constant temperature incubator at 37°C for 24 hours, and then use a microplate reader to measure the light absorption value of the fungus.
  • the sample concentration at which the bacteriostatic rate reaches 80% is the minimum inhibitory concentration of the sample to the bacterium. (MIC).

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  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Dentistry (AREA)
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  • Agronomy & Crop Science (AREA)
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  • Microbiology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Organic Chemistry (AREA)
  • Fats And Perfumes (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Food Preservation Except Freezing, Refrigeration, And Drying (AREA)

Abstract

A method for extracting Alpinia oxyphylla Miq essential oil for inhibiting Listeria monocytogenes, and Alpinia oxyphylla Miq essential oil. The method comprises the following steps: taking Alpinia oxyphylla Miq, drying mature fruits, crushing same, and sieving to obtain a powder; and mixing the powder with water according to a material-liquid mass ratio of 1:10-12, standing still and soaking for 2-3 h, and then performing steam distillation at 220-300°C for 5-7 h. The high-efficiency extraction of the essential oil is achieved by a steam distillation method, and an extraction rate reaches 1.5% or more. The obtained Alpinia oxyphylla Miq essential oil shows extremely strong inhibitory activity on Listeria monocytogenes. The inhibition zone diameter reaches 8.6 mm or more, and the MIC reaches 15 μL/mL or less.

Description

一种抑制单增李斯特菌的益智精油的提取方法及益智精油A kind of extraction method of intellectual essential oil inhibiting listeria monocytogenes and intellectual essential oil 技术领域technical field
本发明属于生物医药技术领域，具体涉及一种抑制单增李斯特菌的益智精油的提取方法及益智精油。The invention belongs to the technical field of biomedicine, and in particular relates to a method for extracting a nootropic essential oil inhibiting Listeria monocytogenes and the nootropic essential oil.
背景技术Background technique
益智(拉丁学名：Alpinia oxyphylla Miq)，别名：益智仁、益智子，山姜属多年生草本植物。益智是我国四大南药之一，不仅可以作为中药材入药，还具有广泛的药理作用。云蕙莹等(益智仁精油抗氧化及抗菌能力分析研究[J].弘光学报,2013,(第71期))以超临界CO 2萃取法对益智精油进行提取并分析其抗氧化及抗菌能力，结果发现其提取所得益智精油对大肠杆菌及金黄色葡萄球菌具有显著抑制效果。罗琴等(益智仁挥发油的水蒸气蒸馏法提取工艺优化及其体外抑菌活性的研究[J].华西药学杂志,2011,26(02):147-149)采用水蒸气蒸馏法提取益智仁挥发油，结果发现其益智仁挥发油对大肠杆菌、金黄色葡萄球菌和绿脓杆菌均有明显的抑制作用。目前，未见关于益智精油对单增李斯特菌具有抑制作用的相关报道。 Puzzle (Latin scientific name: Alpinia oxyphylla Miq), alias: Yizhiren, Yizhizi, Alpine ginger is a perennial herb. Yizhi is one of the four major southern medicines in my country. It can not only be used as a traditional Chinese medicine, but also has a wide range of pharmacological effects. Yun Huiying et al. (Research on the antioxidant and antibacterial ability of the essential oil of Yizhiren[J]. Hongguang Journal, 2013, (No. 71)) extracted the essential oil of Yizhiren with supercritical CO 2 extraction and analyzed its antioxidant and antibacterial properties. Antibacterial ability, it was found that the extracted nootropic essential oil has a significant inhibitory effect on Escherichia coli and Staphylococcus aureus. Luo Qin et al. (Optimization of steam distillation extraction process of volatile oil from Yizhiren and research on its antibacterial activity in vitro[J]. West China Pharmaceutical Journal, 2011,26(02):147-149) Extracted Yizhiren volatile oil by steam distillation It was found that the volatile oil of Zhizhiren had obvious inhibitory effect on Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. At present, there are no related reports about the inhibitory effect of nootropic essential oil on Listeria monocytogenes.
单增李斯特菌(Listeria monocytogenes)是一种人畜共患病的病原菌，它广泛存在于自然界中，食源性的单增李斯特氏菌对人类安全存在危害，该菌在4℃的环境中仍可生长繁殖，是冷藏食品威胁人类健康的主要病原菌之一。该菌对理化因素抵抗力较强，在土壤、粪便、青储饲料等不同环境下均能长期存活，对碱和盐抵抗力强，不易被冻融，能耐受较高的渗透压，不易被抑制或杀灭。申请人研究发现，经特定方法提取所得的益智精油对于单增李斯特菌表现出明显的抑制效果。Listeria monocytogenes (Listeria monocytogenes) is a zoonotic pathogen, it widely exists in nature, food-borne Listeria monocytogenes is harmful to human safety, the bacteria in the environment of 4 ℃ It can still grow and reproduce, and it is one of the main pathogenic bacteria that threaten human health in refrigerated food. The bacteria has strong resistance to physical and chemical factors, can survive for a long time in different environments such as soil, feces, and silage feed, has strong resistance to alkali and salt, is not easy to be frozen and thawed, can withstand high osmotic pressure, and is not easy to suppressed or killed. The applicant's research found that the nootropic essential oil extracted by a specific method showed obvious inhibitory effect on Listeria monocytogenes.
精油通过不同的提取方法进行提取时其化学成分等差别很大，需要根据精油的使用方向去选择适合的提取方法(王瑶等，2017；Bakkali et al，2008)。因此，本发明公开一种抑制单增李斯特菌的益智精油的提取方法对于益智精油的推广应用具有重要意义。When the essential oil is extracted by different extraction methods, its chemical composition varies greatly, and it is necessary to choose a suitable extraction method according to the direction of use of the essential oil (Wang Yao et al., 2017; Bakkali et al., 2008). Therefore, the present invention discloses a method for extracting intellectual essential oil that inhibits Listeria monocytogenes, which is of great significance for the popularization and application of intellectual essential oil.
发明内容Contents of the invention
鉴于现有技术的不足，为了解决上述问题，本发明的目的在于提供一种抑制单增李斯特菌的益智精油的提取方法及益智精油。In view of the deficiencies in the prior art, in order to solve the above problems, the object of the present invention is to provide a method for extracting the nootropic essential oil that inhibits Listeria monocytogenes and the nootropic essential oil.
本发明方案包括以下内容：The present invention scheme comprises the following contents:
一种抑制单增李斯特菌的益智精油的提取方法，包括以下步骤：A kind of extracting method of the intelligence-inducing essential oil that suppresses Listeria monocytogenes comprises the following steps:
取益智干燥成熟果实，粉碎过筛，得粉末；粉末与水按料液质量比1:10～12混合后静置浸泡2～3h，然后220～300℃下水蒸气蒸馏5～7h。Take dried and ripe fruits of Yizhi, crush and sieve to obtain powder; mix the powder and water according to the mass ratio of material to liquid 1:10-12, let stand and soak for 2-3 hours, and then steam distill at 220-300°C for 5-7 hours.
优选的，将粉碎过筛所得的粉末首先在35±5℃相对湿度60～70％条件下放置，再进行加水浸泡和水蒸气蒸馏。Preferably, the powder obtained by crushing and sieving is first placed at 35±5° C. under the condition of relative humidity of 60-70%, and then soaked in water and steam distilled.
优选的，所述放置时间为5～7d。Preferably, the standing time is 5-7d.
优选的，所述筛为40～60目筛。Preferably, the sieve is a 40-60 mesh sieve.
优选的，粉末与水混合时，水的pH为碱性。Preferably, when the powder is mixed with water, the pH of the water is alkaline.
优选的，粉末与水混合时，水的pH7.5～8.5。Preferably, when the powder is mixed with water, the pH of the water is 7.5-8.5.
优选的，粉末与水混合时，水的pH8.5。Preferably, when the powder is mixed with water, the pH of the water is 8.5.
优选的，水蒸气蒸馏的温度是220～230℃。Preferably, the temperature of steam distillation is 220-230°C.
另一方面，本发明提供了一种益智精油，该精油由本发明所提供的方法制得，表现出对单增李斯特菌的抑制活性。On the other hand, the present invention provides a nootropic essential oil, which is prepared by the method provided by the present invention and exhibits inhibitory activity against Listeria monocytogenes.
本发明所取得的有益效果：The beneficial effect that the present invention obtains:
本发明提供了一种抑制单增李斯特菌的益智精油的提取方法，通过水蒸气蒸馏法实现该精油的高效提取，提取率达到1.5％以上。The invention provides a method for extracting intelligence-inducing essential oil inhibiting Listeria monocytogenes. The essential oil can be extracted efficiently through a steam distillation method, and the extraction rate can reach more than 1.5%.
本发明提取方法，操作简便，不需要使用大量的化学试剂，安全高效。The extraction method of the invention is easy to operate, does not need to use a large amount of chemical reagents, and is safe and efficient.
本发明所得益智精油对单增李斯特菌表现出极强的抑制活性。抑菌圈直径达到8.6mm以上，MIC达到15μL/mL以下。The nootropic essential oil obtained in the invention exhibits extremely strong inhibitory activity against Listeria monocytogenes. The diameter of the inhibition zone was above 8.6 mm, and the MIC was below 15 μL/mL.
具体实施方式detailed description
为了更好理解本发明技术内容，下面提供具体实施例，对本发明做进一步的说明。In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.
所述益智为益智Alpinia oxyphylla Miq.的干燥成熟果实，样品收集于海南省白沙县拥处村。The said nootropic is the dried ripe fruit of the nootropic Alpinia oxyphylla Miq., and the samples were collected in Yongchu Village, Baisha County, Hainan Province.
试验例1 粉碎度考察Test Example 1 Investigation on the Degree of Grinding
取益智干燥成熟果实，分别粉碎过10、20、30、40、50、60目筛，得不同颗粒大小的粉末；分别称取不同颗粒大小的粉末50g，加入10倍质量的pH6.8蒸馏水，浸泡3h，220℃下水蒸气蒸馏5h；三次重复试验。按照2020年版《中国药典》精油测定法“甲法”，测定精油提取率。精油提取率(％)＝精油体积(mL)/植物粉末质量(g)×100％。Take the dry and mature fruits of Yizhi, crush them through 10, 20, 30, 40, 50, and 60 mesh sieves to obtain powders of different particle sizes; weigh 50g of powders with different particle sizes, and add 10 times the mass of distilled water with pH6.8 , soaked for 3h, and steam distilled at 220°C for 5h; the test was repeated three times. According to the 2020 edition of "Chinese Pharmacopoeia" essential oil determination method "Method A", the extraction rate of essential oil was determined. Essential oil extraction rate (%)=essential oil volume (mL)/plant powder mass (g)×100%.
结果显示：当样品颗粒从10目减小到40目时，益智精油的提取率快速上升，根据扩散定律可知，益智果实的粉碎度越高，与蒸馏水的接触越多，浸出效果越好，精油提取率越高。当样品颗粒从40目减小到60目时，益智精油的提取率慢慢下降直至趋于平缓，这可能是因为益智果实的粉碎度过高，在水蒸气蒸馏的过程中，益智果实中浸出的成分随粉碎度的增高而增加，浸于水中的成分过多会增加精油随水蒸气蒸出的阻力，从而导致精油提取率的下降。所以选择粉碎度为40目(提取率达到1.7％)。The results show that when the sample particles are reduced from 10 mesh to 40 mesh, the extraction rate of the essential oil of the fruit increases rapidly. According to the law of diffusion, the higher the degree of crushing of the fruit of the fruit, the more contact with distilled water, the better the extraction effect , the higher the extraction rate of essential oil. When the sample particles were reduced from 40 mesh to 60 mesh, the extraction rate of the nootropic essential oil slowly decreased until it became flat, which may be because the pulverization of the nootropic fruit was too high. The components extracted from the fruit increase with the increase of the degree of crushing, and too many components immersed in water will increase the resistance of the essential oil to evaporate with water vapor, resulting in a decline in the extraction rate of the essential oil. Therefore, the degree of crushing is selected to be 40 mesh (the extraction rate reaches 1.7%).
试验例2 料液比考察Test Example 2 Investigation of the ratio of solid to liquid
取益智干燥成熟果实，粉碎过40目筛，得粉末；称取粉末50g，分别加入7、8、9、10、11、12倍质量的pH6.8蒸馏水，浸泡3h，220℃下水蒸气蒸馏5h；三次重复试验。按照2020年版《中国药典》精油测定法“甲法”，测定精油提取率。精油提取率(％)＝精油体积(mL)/植物粉末质量(g)×100％。Take the dried and mature fruit of Yizhi, crush it through a 40-mesh sieve, and obtain the powder; weigh 50g of the powder, add 7, 8, 9, 10, 11, and 12 times the mass of distilled water at pH 6.8, soak for 3 hours, and steam distill at 220°C 5h; repeat the test three times. According to the 2020 edition of "Chinese Pharmacopoeia" essential oil determination method "Method A", the extraction rate of essential oil was determined. Essential oil extraction rate (%)=essential oil volume (mL)/plant powder mass (g)×100%.
结果显示：料液比1:5～12时，提取率逐渐升高，料液比1:10时，提取率达到最大1.7％。The results showed that when the ratio of solid to liquid was 1:5-12, the extraction rate increased gradually, and when the ratio of solid to liquid was 1:10, the extraction rate reached a maximum of 1.7%.
当料液比在7～10倍时，益智精油提取率快速上升。当料液比在10～12倍时，益智精油提取率先下降随后不变(提取率1.5～1.7％)由于随着水量的增加，样品与蒸馏水的接触面积越大，有利于精油的浸出和往水中的扩散。当液料比继续增大、精油量不再增加时，过多的水溶液增加了精油在蒸馏水中的溶解，使得精油不易随水蒸气挥发出，从而使得精油提取率减小，所以选择料液比为10倍。When the ratio of solid to liquid is 7-10 times, the extraction rate of Nootropic essential oil increases rapidly. When the ratio of solid to liquid is 10 to 12 times, the extraction of the nootropic essential oil first declines and then remains unchanged (extraction rate 1.5 to 1.7%). As the amount of water increases, the contact area between the sample and distilled water is larger, which is conducive to the leaching and extraction of essential oils. Diffusion into water. When the liquid-to-material ratio continues to increase and the amount of essential oil does not increase, too much aqueous solution increases the dissolution of the essential oil in distilled water, making it difficult for the essential oil to volatilize with water vapor, thereby reducing the extraction rate of the essential oil, so choose the material-liquid ratio 10 times.
试验例3 浸泡时间考察Test example 3 Soaking time investigation
取益智干燥成熟果实，粉碎过40目筛，得粉末；称取粉末50g，并将其浸泡于10倍量的pH6.8蒸馏水，浸泡0、1、2、3、4、5h，220℃下水蒸气蒸馏5h；三次重复试验。按照2020年版《中国药典》精油测定法“甲法”，测定精油提取率。精油提取率(％)＝精油体积(mL)/植物粉末质量(g)×100％。Take the dried and mature fruit of Yizhi, crush it through a 40-mesh sieve to get the powder; weigh 50g of the powder, and soak it in 10 times the amount of distilled water with pH6.8, soak for 0, 1, 2, 3, 4, 5h, 220°C Under steam distillation for 5h; repeat the test three times. According to the 2020 edition of "Chinese Pharmacopoeia" essential oil determination method "Method A", the extraction rate of essential oil was determined. Essential oil extraction rate (%)=essential oil volume (mL)/plant powder mass (g)×100%.
结果显示：浸泡时间0～2h时，浸泡时间和益智精油提取率正相关增长，这可能是因为在一定时间范围内浸泡时间可以决定精油在水中的水散作用，浸泡时间越长，水散作用越大。当浸泡时间为2～5h时，浸泡时间和益智精油提取率呈负相关，益智果实精油的出油量损失很多，这可能是因为浸泡时间过长会使得部分精油组分挥发，从而大大降低了精油的提取率，所以选择浸泡时间为2～3h。The results show that: when the soaking time is 0-2 hours, the soaking time and the extraction rate of the essential oil increase in a positive correlation. The greater the effect. When the immersion time is 2-5h, the immersion time and the extraction rate of the nootropic essential oil are negatively correlated, and the oil yield of the nootropic fruit essential oil is greatly lost. The extraction rate of essential oil is reduced, so the soaking time is selected to be 2 to 3 hours.
试验例4 蒸馏时间考察Test example 4 Distillation time investigation
取益智干燥成熟果实，粉碎过40目筛，得粉末；称取粉末50g，并将其浸泡于10倍量的pH6.8蒸馏水，浸泡2h，220℃下分别水蒸气蒸馏2、3、4、5、6、7、8h；三次重复试验。按照2015年版《中国药典》精油测定法“甲法”，测定精油提取率。精油提取率(％)＝精油体积(mL)/植物粉末质量(g)×100％。Take the dry and mature fruit of Yizhi, crush it through a 40-mesh sieve, and obtain the powder; weigh 50g of the powder, soak it in 10 times the amount of distilled water with pH6. , 5, 6, 7, 8h; repeat the test three times. According to the 2015 edition of "Chinese Pharmacopoeia" essential oil determination method "Method A", the extraction rate of essential oil was determined. Essential oil extraction rate (%)=essential oil volume (mL)/plant powder mass (g)×100%.
结果显示：蒸馏时间在2～5h时，益智精油提取率快速上升，精油的浸出量与蒸馏时间成正比，在5h时达到最大值并保持不变，这主要是因为随着蒸馏时间的增加，精油随水蒸气不断挥发出来，在达到一定时间后益智果实中的精油成分已全部浸出，浸出量达到最高值。当浸泡了5h时提取率达到最大值(1.65％)并保持不变。蒸馏时间5～8h时，提取率变化不明显。所以通过单因素试验并从节约能源和时间的基础上选择蒸馏时间为5h。The results show that: when the distillation time is 2 to 5 hours, the extraction rate of the essential oil increases rapidly, and the leaching amount of the essential oil is proportional to the distillation time, and reaches the maximum value at 5 hours and remains unchanged. , the essential oil is continuously volatilized with the water vapor, and after a certain period of time, all the essential oil components in the fruit of the puzzle have been leached out, and the leached amount reaches the highest value. When soaked for 5h, the extraction rate reached the maximum value (1.65%) and remained unchanged. When the distillation time is 5-8 hours, the extraction rate does not change significantly. Therefore, the distillation time was selected as 5h through a single factor test and on the basis of saving energy and time.
试验例5 蒸馏温度考察Test Example 5 Distillation Temperature Investigation
取益智干燥成熟果实，粉碎过40目筛，得粉末；称取粉末50g，并将其浸泡于10倍量的pH6.8蒸馏水中，浸泡2h，分别100、140、180、220、260、300℃下水蒸气蒸馏5h；三次重复试验。按照2015年版《中国药典》精油测定法“甲法”，测定精油提取率。精油提取率(％)＝精油体积(mL)/植物粉末质量(g)×100％。Take the dry and mature fruit of Yizhi, crush it through a 40-mesh sieve, and obtain the powder; weigh 50g of the powder, and soak it in 10 times the amount of distilled water with pH6. Steam distillation at 300°C for 5h; repeat the test three times. According to the 2015 edition of "Chinese Pharmacopoeia" essential oil determination method "Method A", the extraction rate of essential oil was determined. Essential oil extraction rate (%)=essential oil volume (mL)/plant powder mass (g)×100%.
结果显示：蒸馏温度100～220℃时，提取率呈现上升趋势，蒸馏温度220～300℃时出现缓慢下降，由于随着温度的增加，样品中具有挥发性的成分不断蒸出，但当蒸馏的温度过高时样品中的少部分挥发性成分来不及冷却收集，挥发于空气中，所以当温度过高时，精油提取率会缓慢下降，蒸馏温度为220℃时益智果实精油的提取率最高，高达1.75％。The results show that: when the distillation temperature is 100-220°C, the extraction rate shows an upward trend, and when the distillation temperature is 220-300°C, it slowly decreases. As the temperature increases, the volatile components in the sample continue to evaporate, but when the distillation When the temperature is too high, a small part of the volatile components in the sample have no time to cool and collect, and volatilize in the air. Therefore, when the temperature is too high, the extraction rate of essential oil will slowly decrease. When the distillation temperature is 220 ° C, the extraction rate of the essential oil of the fruit of the fruit is the highest. Up to 1.75%.
经单因素实验及星点实验，最终确定益智果实精油的条件为：粉碎度40目，料液比1:10，230℃下浸泡2.4h蒸馏5.5h；提取率达到1.8％。After the single factor experiment and the star point experiment, the final conditions for the essential oil of the fruit of the fruit were determined as follows: the degree of crushing was 40 mesh, the ratio of solid to liquid was 1:10, soaked at 230°C for 2.4h and distilled for 5.5h; the extraction rate reached 1.8%.
另，经研究发现部分样品对单增李斯特菌表现出更高的抑菌活性。对比试验结果如下：In addition, the research found that some samples showed higher antibacterial activity against Listeria monocytogenes. The comparative test results are as follows:
试验例6 抑菌活性研究Test Example 6 Bacteriostatic Activity Research
6.1材料、菌种、试剂6.1 Materials, strains, reagents
材料：精油①～⑤：取益智干燥成熟果实，粉碎过40目筛，得粉末；称取粉末50g，并将其分别浸泡于10倍量的pH7.0、7.5、8.0、8.5、9.0蒸馏水中，浸泡2.4h，230℃下水蒸气蒸馏5.5h。精油⑥：取益智干燥成熟果实，粉碎过40目筛，得粉末；称取粉末50g，并将其浸泡于10倍量的pH7.0蒸馏水中，浸泡2.4h，180℃下水蒸气蒸馏5.5h。精油⑦～⑩：取益智干燥成熟果实，粉碎过40目筛，得粉末；将粉末在35±5℃相对湿度65±5％条件下分别 放置(厚度2～3cm)3、5、7、9d；称取粉末50g，并将其浸泡于10倍量的pH 8.5蒸馏水中，浸泡2.4h，180℃下水蒸气蒸馏5.5h。Materials: essential oil ①～⑤: Take the dried and mature fruit of Noutropia chinensis, crush it through a 40-mesh sieve to get the powder; weigh 50g of the powder, and soak it in 10 times the amount of distilled water with pH 7.0, 7.5, 8.0, 8.5, and 9.0 respectively In, soak for 2.4h, steam distillation at 230°C for 5.5h. Essential oil ⑥: Take the dried and mature fruit of Yizhi, crush it through a 40-mesh sieve to get a powder; weigh 50g of the powder, and soak it in 10 times the amount of distilled water with pH 7.0, soak for 2.4h, and steam distill at 180°C for 5.5h . Essential oils ⑦～⑩: Take the dried and mature fruits of Nootropics, crush them through a 40-mesh sieve to obtain powder; place the powder at 35±5°C and 65±5% relative humidity (thickness 2-3cm) 3, 5, 7, 9d; Weigh 50g of the powder, soak it in 10 times the amount of distilled water with pH 8.5, soak for 2.4h, and steam distill at 180°C for 5.5h.
菌种和单增李斯特菌(Listeria monocytogenes ATCC 19111)、金黄色葡萄球菌(Staphylococcus aureus ATCC 6538)购自于北京北纳创联生物技术研究院。The strains and Listeria monocytogenes ATCC 19111 and Staphylococcus aureus ATCC 6538 were purchased from Beijing Beina Chuanglian Biotechnology Research Institute.
试剂：培养基、左氧氟沙星等均购自于北京索莱宝科技有限公司。Reagents: culture medium, levofloxacin, etc. were purchased from Beijing Suolaibao Technology Co., Ltd.
6.2试验方法6.2 Test method
6.2.1抑菌圈的测定6.2.1 Determination of inhibition zone
采用滤纸片琼脂平板扩散法，在无菌的环境条件下用移液枪取精油或对照20μL滴于无菌滤纸片(d＝6mm)上，待纸片充分吸收后贴在已均匀涂布各菌液(菌液浓度10 7cfu/ml、涂布量60μL)的培养基平板(TSA，d＝6cm)上，做好标记，每个菌进行3次重复，阴性对照为无菌水，阳性对照为左氧氟沙星(浓度15μg/ml)，培养条件为恒温培养箱37℃中培养24h。培养结束后，观察并记录抑菌圈的有无以及直径大小作为判断敏感度高低的标准，采用十字交叉法量取抑菌圈直径大小，取其平均值作为测定结果。结果标准为：抑菌圈直径大于20mm时为极敏、直径在15～20mm之间时为高敏，在10～15mm之间的为中敏，抑菌圈直径小于10mm的为低敏(兰仕梅等，2018)。 Using the filter paper agar plate diffusion method, use a pipette gun to take 20 μL of the essential oil or the control and drop it on the sterile filter paper (d=6mm) under sterile environmental conditions. On the medium plate (TSA, d=6cm) of bacterial solution (bacterial solution concentration 10 7 cfu/ml, coating amount 60 μL), make a mark, and repeat each bacterium three times. The negative control is sterile water, and the positive control is sterile water. The control was levofloxacin (concentration 15 μg/ml), and the culture condition was 24 hours in a constant temperature incubator at 37°C. After the cultivation, observe and record the presence or absence of the bacteriostatic zone and its diameter as the standard for judging the sensitivity, use the cross method to measure the diameter of the bacteriostatic zone, and take the average value as the measurement result. The result standard is: when the diameter of the antibacterial zone is greater than 20mm, it is extremely sensitive; when the diameter is between 15-20mm, it is high-sensitivity; Mei et al., 2018).
6.2.2最低抑菌浓度(MIC)6.2.2 Minimum Inhibitory Concentration (MIC)
在96孔板上采用微量二倍稀释法进行MIC试验，将A1作为起始的第1孔，从左到右依次进行，每个板上1～12孔为药液，每个板均做3个空白、阳性对照和阴性对照，阴性对照为无菌水，阳性对照为左氧氟沙星。首先用移液枪向每排第1孔加入180μL各菌悬液(菌液浓度10 7cfu/ml、涂布量60μL)，第2～12个孔各加入100μL各菌悬液，再分别吸取20μL精油至第1孔，依次对第1～12孔做梯度稀释，使其精油浓度分别为100、50、25、12.5、6.25、3.125、1.563、0.781、0.391、0.195、0.097μL/mL，充分混匀，重复三次，培养条件为恒温培养箱37℃中培养24h，而后使用酶标仪测定真菌的吸光值，抑菌率达到80％的样品浓度就为该样品对该菌的最低抑菌浓度(MIC)。 The MIC test was carried out on a 96-well plate using the micro-doubling dilution method. A1 was used as the first well starting from left to right, and 1 to 12 wells on each plate were liquid medicines, and 3 wells were prepared on each plate. A blank, positive control and negative control, the negative control is sterile water, and the positive control is levofloxacin. First, add 180 μL of each bacterial suspension to the first well of each row with a pipette gun (bacteria concentration: 107 cfu /ml, coating volume: 60 μL), add 100 μL of each bacterial suspension to each of the second to 12th wells, and then pipette Add 20 μL of essential oil to the first well, and sequentially make serial dilutions for the first to 12th wells, so that the concentrations of the essential oils are 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, and 0.097 μL/mL. Mix well and repeat three times. The culture condition is to cultivate in a constant temperature incubator at 37°C for 24 hours, and then use a microplate reader to measure the light absorption value of the fungus. The sample concentration at which the bacteriostatic rate reaches 80% is the minimum inhibitory concentration of the sample to the bacterium. (MIC).
6.2.3最低杀菌浓度(MBC)的测定6.2.3 Determination of minimum bactericidal concentration (MBC)
采用琼脂培养基平板法，取“6.2.2”下的各样品MIC值所对应的溶液均匀涂布于相应的培养基中，培养条件为恒温培养箱28℃培养，而后观察培养基中有无菌株生长，该药物的MBC为菌落数低于5个的平板所对应孔中的最低浓度。Using the agar medium plate method, take the solution corresponding to the MIC value of each sample under "6.2.2" and evenly spread it on the corresponding medium. The strain grows, and the MBC of the drug is the lowest concentration in the well corresponding to the plate with less than 5 colonies.
6.3结果及分析6.3 Results and Analysis
结果见表1～2。结果显示，所得益智精油对单增李斯特菌表现出良好的抑制作用，无杀菌活性。其中精油⑧和精油⑨表现出更高的抑制活性。The results are shown in Tables 1-2. The results showed that the obtained nootropic essential oil showed good inhibitory effect on Listeria monocytogenes, but had no bactericidal activity. Among them, essential oil ⑧ and essential oil ⑨ showed higher inhibitory activity.
表1各样品对细菌的抑菌圈直径The diameter of the inhibition zone of each sample in table 1 to bacteria
Figure PCTCN2021106837-appb-000001
Figure PCTCN2021106837-appb-000001
同列不同字母表示清除率差异显著(p<0.05)；“—”为无抑菌圈Different letters in the same column indicate significant difference in clearance rate (p<0.05); "—" means no inhibition zone
表3各样品对细菌的MIC值Table 3 The MIC value of each sample to bacteria
Figure PCTCN2021106837-appb-000002
Figure PCTCN2021106837-appb-000002
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (9)

  1. 一种抑制单增李斯特菌的益智精油的提取方法，其特征在于，包括以下步骤：A kind of extracting method of the intelligence-inducing essential oil of suppressing listeria monocytogenes, is characterized in that, comprises the following steps:
    取益智干燥成熟果实，粉碎过筛，得粉末；粉末与水按料液质量比1:10～12混合后静置浸泡2～3h，然后220～300℃下水蒸气蒸馏5～7h。Take dried and ripe fruits of Yizhi, crush and sieve to obtain powder; mix the powder and water according to the mass ratio of material to liquid 1:10-12, let stand and soak for 2-3 hours, and then steam distill at 220-300°C for 5-7 hours.
  2. 根据权利要求1所述的提取方法，其特征在于，将粉碎过筛所得的粉末首先在35±5℃相对湿度60～70％条件下放置，再进行加水浸泡和水蒸气蒸馏。The extraction method according to claim 1, characterized in that the powder obtained by crushing and sieving is first placed at 35±5° C. under the condition of relative humidity of 60-70%, and then soaked in water and steam distilled.
  3. 根据权利要求2所述的提取方法，其特征在于，所述放置时间为5～7d。The extraction method according to claim 2, characterized in that, the storage time is 5-7d.
  4. 根据权利要求1所述的提取方法，其特征在于，所述筛为40～60目筛。The extraction method according to claim 1, characterized in that, the sieve is a 40-60 mesh sieve.
  5. 根据权利要求1所述的提取方法，其特征在于，粉末与水混合时，水的pH为碱性。The extraction method according to claim 1, characterized in that, when the powder is mixed with water, the pH of the water is alkaline.
  6. 根据权利要求1所述的提取方法，其特征在于，粉末与水混合时，水的pH为7.5～8.5。The extraction method according to claim 1, characterized in that when the powder is mixed with water, the pH of the water is 7.5-8.5.
  7. 根据权利要求1所述的提取方法，其特征在于，粉末与水混合时，水的pH为8.5。The extraction method according to claim 1, characterized in that, when the powder is mixed with water, the pH of the water is 8.5.
  8. 根据权利要求1所述的提取方法，其特征在于，水蒸气蒸馏的温度是220～230℃。The extraction method according to claim 1, characterized in that the temperature of steam distillation is 220-230°C.
  9. 一种抑制单增李斯特菌的益智精油，其特征在于，由权利要求1～8任一项所述的提取方法制得。A nootropic essential oil inhibiting Listeria monocytogenes, characterized in that it is prepared by the extraction method described in any one of claims 1-8.
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