WO2018218717A1 - 一种具有提神功效精油或其产品的筛选方法 - Google Patents

一种具有提神功效精油或其产品的筛选方法 Download PDF

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WO2018218717A1
WO2018218717A1 PCT/CN2017/089697 CN2017089697W WO2018218717A1 WO 2018218717 A1 WO2018218717 A1 WO 2018218717A1 CN 2017089697 W CN2017089697 W CN 2017089697W WO 2018218717 A1 WO2018218717 A1 WO 2018218717A1
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refreshing
score
screening method
essential oil
brain
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PCT/CN2017/089697
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French (fr)
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肖蕾
陈宇
黄金莲
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无限极（中国）有限公司
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Priority to US16/467,478 priority Critical patent/US20190339256A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

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  • the invention relates to the field of daily chemical technology, in particular to a screening method for a refreshing essential oil or a product thereof.
  • Essential oils are volatile aromatic substances extracted from the flowers, leaves, stems, roots or fruits of plants by steam distillation, extrusion, cold soaking or solvent extraction. Aromatic essential oils can have an effect on the central nervous system of the human body. This effect is due to the effects of olfactory-sensing neuronal cells and their receptors on brain function. Such functions include behaviors such as refreshment and alertness. Essential oils are made up of small molecules that can be absorbed into the body by nasal mucosal tissue, send messages directly to the brain, and regulate the emotional and physical functions of the body through the limbic system of the brain.
  • Essential oils have the effects of stimulating sympathetic and parasympathetic nerves, sedation and hypnosis, refreshing, refreshing mental status, anti-depression, relieving psychological stress, and repairing the nervous system. Animal studies have shown that certain aromatic essential oils can increase the concentration of dopamine in body fluids to some extent.
  • the evaluation of the efficacy of aromatic essential oils is based on the raw material species, raw material origin, raw material composition, raw material content, raw material extraction and preparation process, physical properties of raw materials including freezing point, melting point, boiling point, density, refractive index, color, and raw material smell. , traditional experience in the application of raw materials, as well as a small amount of human body experience and certain specific body fluid measurements.
  • the above technical means for evaluating the refreshing efficacy of aromatic essential oils Based on the physical properties, preparation processes, traditional experience and sensory judgment and experience of aromatic essential oil raw materials.
  • the present invention provides a screening method having a refreshing effect essential oil or a product thereof.
  • the screening method has the characteristics of fast and accurate, data repeatability, method curable, standard objective and application safety.
  • the present invention provides the following technical solutions:
  • the invention provides a screening method with a refreshing essential oil or a product thereof, comprising the following steps:
  • the rate of change of the neurotransmitter concentration in the treated group compared with the neurotransmitter concentration in the blank control group was determined according to the following formula:
  • C 1 is the neurotransmitter concentration of the treatment group
  • C 0 is the neurotransmitter concentration of the blank control group
  • V c is the change rate of the neurotransmitter concentration of the treatment group compared with the neurotransmitter concentration of the blank control group
  • V c respectively serotonin, according to [gamma] -aminobutyric acid, melatonin, glycine, dopamine, glutamate, norepinephrine, acetylcholine and refreshing value X i; refreshing scores as follows:
  • X is the refreshing total score
  • X 1 is the refreshing score of serotonin
  • X 2 is the refreshing score of ⁇ -aminobutyric acid
  • X 3 is the refreshing score of melatonin
  • X 4 is the refreshing of glycine.
  • Score X 5 is the refreshing score of dopamine
  • X 6 is the refreshing score of glutamic acid
  • X 7 is the refreshing score of norepinephrine
  • X 8 is the refreshing score of acetylcholine;
  • the essential oil or its product is an essential oil or product thereof with refreshing effect
  • the essential oil or its product is an essential oil or a product thereof which does not have a refreshing effect.
  • the essential oil or a product thereof has a mass percentage concentration of 0.00005% to 0.05%.
  • the essential oil has a mass percent concentration of 0.0005%.
  • the time during which the essential oil or its product treats brain neuronal cells is 5 to 60 min.
  • the treatment time is 30 min.
  • the essential neuron or its product brain neuron cells are brain neuron cells cultured for 5 to 15 days.
  • the brain neuron cells are brain neuron cells cultured for 10 days.
  • the culture medium is a horse serum culture solution
  • the cell inoculation amount is 1 ⁇ 10 4 /mL
  • the culture temperature is 37 ° C
  • the culture condition is 5% CO 2 .
  • the brain neuron cells are human brain neuron cells or animal brain neuronal cells.
  • the animal brain neuronal cells are murine brain neuronal cells.
  • the animal brain neuronal cells are mouse brain neuronal cells.
  • the present invention provides a screening method for a refreshing essential oil or a product thereof.
  • the screening method comprises: treating brain neuron cells with essential oil or its products, detecting concentration of serotonin and the like, obtaining the neurotransmitter concentration of the treatment group; and comparing the neurotransmitter concentration of the treatment group with the neurotransmitter concentration of the blank control group.
  • Rate V c obtain the refreshing score X i of each neurotransmitter according to V c ; detect the total score X of the essential oil or its product according to the summation formula; when X ⁇ 44, the essential oil or its product is refreshing Efficient essential oil or its products.
  • the present invention has at least one of the following advantages:
  • the invention can quantitatively evaluate the efficacy and the refreshing effect of the aromatic essential oil raw materials and products from the biological cells and their physiological and biochemical aspects;
  • the invention can solve the biological objective data of the refreshing effect of the aromatic essential oil and its products, and quantify the evaluation of the refreshing efficacy of the aromatic essential oil and its products;
  • the invention can repeatedly verify the refreshing effect of the aromatic essential oil and its products
  • the method for evaluating the refreshing efficacy of the aromatic essential oil and the product thereof can effectively support the raw material selection, formula design and formulation and the function detection of the aromatic essential oil with refreshing effect;
  • the screening method of the invention is aimed at the biological evaluation of the aromatic essential oil and its products, and has the characteristics of fast and accurate, data repeatable, method curable, standard objective, and application safety.
  • the screening method of the invention can accelerate the screening and evaluation progress of the aromatic essential oil and its products, and quickly evaluate the establishment of the aromatic essential oil with refreshing effect and the formulation of the product thereof, and provide the product effect for the users of the aromatic essential oil and the product thereof with refreshing effect. Instructions, product safety instructions, product usage dose instructions, and instructions on how to use the product.
  • Figure 1 shows the growth status of brain neuron cell culture on day 10
  • Figures 2 to 4 show the levels of dopamine, gamma aminobutyric acid and melatonin released by neuronal cells in the culture medium from 0 to 60 minutes after the peppermint oil is placed in the brain neuron cells;
  • Figure 5 shows the effect of lemongrass oil on the neurotransmitter dopamine levels in brain neuronal cells
  • Figures 6 to 13 show the effects of different aromatic essential oils on the release of serotonin, ⁇ -aminobutyric acid, acetylcholine, norepinephrine, glycine, glutamic acid, melatonin and dopamine from nerve cells;
  • Figure 14 shows the process flow of the present invention.
  • the invention discloses a screening method with a refreshing effect essential oil or a product thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • each neurotransmitter level was carried out using a commercially available kit including Developer A and Developer B.
  • the essential oil or brain neuron cells used in the screening method of the essential oil or the product thereof provided by the present invention are commercially available.
  • Neonatal mice were selected for 1 day, and the neurons in the brain of the mice were isolated and dispersed in a medium containing Neurosal Medium (Thermo Fisher, Cat. #21103-049) containing horse serum, and then placed in a 37 ° C cell incubator. Culture, culture conditions are air: carbon dioxide is 95:5. Brain neuron cell culture fluid was replaced every other day. The growth status of brain neuron cell culture on day 10 is shown in Fig. 1.
  • Human brain neuron cells were purchased commercially, and the cells were placed in a Neurobasal Medium (Thermo Fisher, Cat. #21103-049) culture medium containing horse serum, and then cultured in a 37 ° C cell culture incubator. For air: carbon dioxide is 95:5. Brain neuron cell culture fluid was replaced every other day. The growth of brain neuron cell culture on day 10 was similar to that of Figure 1.
  • Neurobasal Medium Thermo Fisher, Cat. #21103-049
  • Mint Peppermint Yakima Oil affects the release of neurotransmitter levels in brain neurons. Adding peppermint essential oil to the brain neuron cell culture system on the 10th day of culture, and The final concentrations of peppermint essential oil in cerebral neuron cell culture systems were 0.0005%, 0.0050%, and 0.050%.
  • GABA gamma aminobutyric acid
  • MELATONIN melatonin
  • Example 3 Effect of peppermint essential oil on the release levels of various neurotransmitters in brain neuronal cells
  • Mint Peppermint Yakima Oil The effects of Mint Peppermint Yakima Oil on the release levels of different neurotransmitters in neuronal cell culture systems at 30 minutes from brain mineral cells at different concentrations are shown in Table 1.
  • the neurotransmitters involved in this embodiment include dopamine (DOPAMINE), serotonin (5HYDROXY-TRYPTAMINE), gamma-aminobutyric acid (GAMMA AMINOBUTYRIC ACID), acetylcholine (ACETYLCHOLINE), norepinephrine (NORADRENALINE), glycine (GLYCINE). , glutamate (GLUTAMINE) and melatonin (MELATONIN).
  • peppermint essential oil can promote the release of melanin, dopamine and norepinephrine, and inhibit the release of serotonin, ⁇ -aminobutyric acid, glycine, acetylcholine and glutamic acid.
  • results of this experiment show that different doses of peppermint essential oil have a dose-effect relationship for promoting or inhibiting the release of different neurotransmitters.
  • This example relates to the effect of lemongrass oil on the neurotransmitter dopamine levels in brain neuronal cells and the analysis of the efficacy.
  • Lemongrass oil was added to the brain neuron cell culture system on the 10th day of culture, and the final concentration of lemongrass oil in the brain neuron cell culture system was 0%, 0.00005%, 0.0005%, and the essential oil was measured.
  • This example investigates the effects of different aromatic essential oils on neurotransmitter levels in a variety of brain neuronal cells.
  • concentration of the aromatic essential oils involved in the examples was 0.0005%.
  • Test method The essential oil was added to the brain neuron cell culture system on the 10th day of culture, and the final concentration of the essential oil in the brain neuron cell culture system was 0.0005%, and the culture oil was placed in the culture medium 30 minutes after the brain cells were placed. The level of neurotransmitters released by neuronal cells.
  • the neurotransmitters involved in this embodiment include dopamine (DOPAMINE), serotonin (5HYDROXY-TRYPTAMINE), gamma-aminobutyric acid (GAMMA AMINOBUTYRIC ACID), acetylcholine (ACETYLCHOLINE), norepinephrine (NORADRENALINE), glycine (GLYCINE) , glutamate (GLUTAMINE) and melatonin (MELATONIN).
  • DOPAMINE dopamine
  • serotonin 5HYDROXY-TRYPTAMINE
  • GMMA AMINOBUTYRIC ACID gamma-aminobutyric acid
  • ACETYLCHOLINE acetylcholine
  • NORADRENALINE norepinephrine
  • GLYCINE glutamate
  • MELATONIN melatonin
  • aromatic essential oils involved in this embodiment include eucalyptus oil (HOHE) and rosemary oil (ROSEMARY) OIL), BASIL OIL, LEMONGRASS OIL, YLANG III MADAGASCAR EO, MINT PEPPERMINT YAKIMA OIL, Lemon Oil (LEMON OIL ITALY), Eucalyptus Oil (EUCALYPTUS) GLOBULUS OIL).
  • HOHE eucalyptus oil
  • ROSEMARY rosemary oil
  • BASIL OIL LEMONGRASS OIL
  • YLANG III MADAGASCAR EO MINT PEPPERMINT YAKIMA OIL
  • Lemon Oil LEMON OIL ITALY
  • Eucalyptus Oil EUCALYPTUS
  • Es32 bitter orange essential oil 6% + super lavender essential oil 8% + ylang ylang oil 4% + geranium essential oil 4% + cypress essential oil 3% + rose grass essential oil 4% + Roman chamomile essential oil 6% + orange flower essential oil 7% + benzoin Essential oil 8% + sweet almond oil 50%.
  • the invention divides the boundary line by 44, and the higher the total score, the better the refreshing effect of the sample.
  • Table 3 shows that some concentrations of peppermint oil and lemongrass oil have a total score of 44 for neurotransmitter release from brain neurons, suggesting that the sample may have a better refreshing effect at the corresponding concentration;
  • the total scores of neurotransmitter release levels of brain neuronal cells in other essential oil samples did not exceed 44 points at the corresponding concentrations, suggesting that the samples may not have a good refreshing effect at the corresponding concentrations.
  • samples Es32 and Es10 were negative and positive controls, respectively.
  • the total score of Es32 on neurotransmitter release from brain neurons was 28, and the total score of Es10 on neurotransmitter release from brain neurons was 51. It is suggested that this evaluation method can be used to obtain a judgment of the refreshing effect consistent with the control sample.

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Abstract

本申请提供了一种具有提神功效的精油或其产品的筛选方法，包括使用精油或其产品处理大脑神经元细胞；检测其中5-羟色胺等神经递质的浓度；通过处理组和对照组浓度的变化获得各个神经递质的提神分值；计算总提神分值并以此筛选提神功效的精油或其产品。

Description

一种具有提神功效精油或其产品的筛选方法
本申请要求于2017年05月27日提交中国专利局、申请号为201710391002.1、发明名称为“一种具有提神功效精油或其产品的筛选方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及日化技术领域，特别涉及一种具有提神功效精油或其产品的筛选方法。
背景技术
随着社会的发展，现代生活节奏不断加快，生存竞争日趋激烈，工作学习压力越来越大，尤其是商务人士、白领及学生，经常会感到疲惫、头脑昏沉。有些人在春天、炎热夏季、秋季容易犯困，特别是驾驶人员，更需要在疲乏困倦时提神醒脑，避免危险。因此，消费者对具有提神醒脑功效的产品需求量增加。
精油是从植物的花、叶、茎、根或果实中，通过水蒸气蒸馏法、挤压法、冷浸法或溶剂提取法提炼萃取的挥发性芳香物质。芳香精油对人体中枢神经系统可以产生影响，这种影响源于嗅觉感应神经元细胞及其受体对大脑功能的影响，这类功能包括了提神和警觉等行为。精油是由一些很小的分子所组成，这些高挥发物质，可由鼻腔粘膜组织吸收进入身体，将讯息直接送到脑部，通过大脑的边缘系统，调节情绪和身体的生理功能。精油具有刺激交感神经及副交感神经、镇静及催眠、兴奋提神、调整精神状况、抗忧郁、缓解心理压力、修复神经系统的作用。动物研究显示，某些芳香精油可以在一定程度上增加体液中多巴胺浓度。
目前，芳香精油功效的评估多基于原料种属，原料产地，原料成分，原料含量，原料提取和制备工艺，包括凝固点、熔点、沸点、密度、折光度、颜色在内的原料物理属性，原料气味，原料应用的传统经验，以及少量人体使用感受和某些特定体液测量等。上述对芳香精油提神功效进行评估的技术手段，多 基于芳香精油原料的物理属性、制备工艺、传统经验以及感官判断和体验。但针对芳香精油生物学功效评估的技术手段，缺乏在生物学水平的客观标准支持，无法固化和量化芳香精油提神功效的相关生物学功效，导致在工业应用中在挑选具有提神功效的芳香精油原料、配方应用和功效宣传等方面，受到很大局限性和不确定性。
发明内容
有鉴于此，本发明提供了一种具有提神功效精油或其产品的筛选方法。该筛选方法具有快速准确、数据可重复、方法可固化、标准客观以及应用安全的特点。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种具有提神功效精油或其产品的筛选方法，包括如下步骤：
采用精油或其产品处理大脑神经元细胞，检测5-羟色胺、γ-氨基丁酸、褪黑激素、甘氨酸、多巴胺、谷氨酸、去甲肾上腺素和乙酰胆碱的浓度，获得处理组神经递质浓度；
根据如下公式检测处理组神经递质浓度与空白对照组神经递质浓度相比较的变化率：
Figure PCTCN2017089697-appb-000001
其中，C1为处理组神经递质浓度，C0为空白对照组神经递质浓度，Vc为处理组神经递质浓度与空白对照组神经递质浓度相比较的变化率；
根据Vc分别获得5-羟色胺、γ-氨基丁酸、褪黑激素、甘氨酸、多巴胺、谷氨酸、去甲肾上腺素和乙酰胆碱的提神分值Xi；提神分值如下：
Figure PCTCN2017089697-appb-000002
Figure PCTCN2017089697-appb-000003
根据如下公式检测精油或其产品的提神总分值X：
X＝X1+X2+X3+X4+X5+X6+X7+X8
其中，X为提神总分值，X1为5-羟色胺的提神分值，X2为γ-氨基丁酸的提神分值，X3为褪黑激素的提神分值，X4为甘氨酸的提神分值，X5为多巴胺的提神分值，X6为谷氨酸的提神分值，X7为去甲肾上腺素的提神分值，X8为乙酰胆碱的提神分值；
当X≥44时，则精油或其产品为具有提神功效的精油或其产品；
当X＜44时，则精油或其产品为不具有提神功效的精油或其产品。
作为优选，精油或其产品的质量百分比浓度为0.00005％～0.05％。
优选地，精油的质量百分比浓度为0.0005％。
作为优选，精油或其产品处理大脑神经元细胞的时间为5～60min。
优选地，处理的时间为30min。
作为优选，精油或其产品大脑神经元细胞为培养5～15天的大脑神经元细胞。
优选地，大脑神经元细胞为培养10天的大脑神经元细胞。
作为优选，培养的培养基为马血清培养液，细胞接种量为1×104个/mL，培养温度为37℃，培养条件为5％CO2。
作为优选，大脑神经元细胞为人体大脑神经元细胞或动物大脑神经元细胞。
在本发明提供的实施例中，动物大脑神经元细胞为鼠大脑神经元细胞。
在本发明提供的实施例中，动物大脑神经元细胞为小鼠大脑神经元细胞。
本发明提供了一种具有提神功效精油或其产品的筛选方法。该筛选方法包括：采用精油或其产品处理大脑神经元细胞，检测5-羟色胺等浓度，获得处理组神经递质浓度；检测处理组神经递质浓度与空白对照组神经递质浓度相比较 的变化率Vc；根据Vc分别获得各个神经递质的提神分值Xi；根据求和公式检测精油或其产品的提神总分值X；当X≥44时，则精油或其产品为具有提神功效的精油或其产品。本发明至少具有如下优势之一：
本发明从生物体外细胞及其生理和生化层面，对芳香精油原料和产品进行与提神提神功效相关的可量化评估；
本发明可以解决芳香精油及其产品提神功效的生物学客观数据，对芳香精油及其产品的提神功效评估进行量化；
本发明可以对芳香精油及其产品提神功效进行重复验证；
本发明对芳香精油及其产品提神功效的量化评估和验证均在体外进行，便于操作，同时，这一体外评估方法具有可重复的固化特点；
本发明对芳香精油及其产品提神功效的评估方法，可以有效支持具有提神功效芳香精油的原料挑选、配方设计和调配及其功效检测；
可见，本发明筛选方法针对芳香精油及其产品，进行提神功效的生物学体外评估，具有快速准确、数据可重复、方法可固化、标准客观、以及应用安全的特点。
本发明筛选方法可以加速对芳香精油及其产品的筛选评估进度，对建立具有提神功效的芳香精油及其产品配方调配进行快速评估，为具有提神功效的芳香精油及其产品的使用者提供产品功效说明、产品安全说明、产品使用剂量说明以及产品使用方法说明。
附图说明
图1示脑神经元细胞培养第10天生长状况；
图2～4分别示薄荷精油放置大脑神经元细胞后0～60分钟培养液中神经元细胞所释放的多巴胺、γ氨基丁酸和褪黑激素水平；
图5示柠檬草油对大脑神经元细胞神经递质多巴胺水平的影响；
图6～13分别示不同芳香精油对神经细胞释放5-羟色胺、γ-氨基丁酸、乙酰胆碱、去甲肾上腺素、甘氨酸、谷氨酸、褪黑激素、多巴胺水平的影响；
图14示本发明的工艺流程。
具体实施方式
本发明公开了一种具有提神功效精油或其产品的筛选方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
以下实施案例中的％，如无特别说明，均为质量百分比。
在以下实施例中，在检测各神经递质水平时采用市售试剂盒进行检测，试剂盒中包括显色剂A和显色剂B。
本发明提供的具有提神功效精油或其产品的筛选方法中所用精油或脑神经元细胞均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1脑神经元细胞的培养
1、培养原代小鼠脑神经元细胞
选择新生1天小鼠，分离提取小鼠大脑中神经元细胞，分散于含有马血清的Neurobasal Medium (Thermo Fisher,Cat.#21103-049)培养液中，然后置于37℃细胞培养箱中进行培养，培养条件为空气:二氧化碳为95:5。每隔一天更换脑神经元细胞培养液。脑神经元细胞培养第10天生长状况如图1所示。
2、培养原代人脑神经元细胞
通过商业途径购买人脑神经元细胞，并将细胞置于含有马血清的Neurobasal Medium(Thermo Fisher,Cat.#21103-049)培养液中，然后置于37℃细胞培养箱中进行培养，培养条件为空气:二氧化碳为95:5。每隔一天更换脑神经元细胞培养液。脑神经元细胞培养第10天生长状况与图1相近。
实施例2薄荷精油对大脑神经元细胞3种神经递质释放水平影响
薄荷精油(Mint Peppermint Yakima Oil)对大脑神经元细胞神经递质释放水平影响时间。将薄荷精油加入培养第10天的大脑神经元细胞培养体系中，并 使薄荷精油在大脑神经元细胞培养体系中的最终浓度为0.0005％、0.0050％和0.0500％，测量薄荷精油放置大脑神经元细胞后0～60分钟培养液中神经元细胞所释放的多巴胺(DOPAMINE)、γ氨基丁酸(GABA)和褪黑激素(MELATONIN)水平。本实施例中的神经递质样本收集时间为第5分钟、第10分钟、第15分钟、第20分钟、第30分钟、第40分钟、第50分钟和第60分钟。结果如图2-4所示。
试验结果表明：薄荷精油对大脑神经元细胞胞释放多巴胺(DOPAMINE)、γ氨基丁酸(GABA)和褪黑激素(MELATONIN)等神经递质的时间可以在加入样品后第5分钟开始变得明显，这一过程可以持续60分钟。数据提示，芳香精油样品对大脑神经元细胞神经递质释放的样本收集时间在第5分钟到第30分钟之间更具有一定的线性关系。
实施例3薄荷精油对大脑神经元细胞多种神经递质释放水平影响
在不同浓度下，薄荷精油(Mint Peppermint Yakima Oil)对大脑神经元细胞在第30分钟时，对神经元细胞培养体系中不同神经递质释放水平的影响，见下表1。本实施例所涉及的神经递质包括多巴胺(DOPAMINE)、五羟色胺(5HYDROXY-TRYPTAMINE)、γ-氨基丁酸(GAMMA AMINOBUTYRIC ACID)、乙酰胆碱(ACETYLCHOLINE)、去甲肾上腺素(NORADRENALINE)、甘氨酸(GLYCINE)、谷氨酸盐(GLUTAMINE)和褪黑激素(MELATONIN)。
表1薄荷精油对大脑神经元细胞的不同神经递质释放水平的影响
Figure PCTCN2017089697-appb-000004
上述试验结果显示，在一定浓度下，薄荷精油可促进退黑色素、多巴胺、去甲肾上腺素的释放，同时抑制5-羟色胺、γ-氨基丁酸、甘氨酸、乙酰胆碱、谷氨酸的释放。此外，这一实验结果显示，不同剂量薄荷精油对促进或者抑制不同神经递质的释放具有一定的量效关系。
实施例4柠檬草油对大脑神经元细胞神经递质多巴胺水平的影响
本实施例涉及了柠檬草油对大脑神经元细胞神经递质多巴胺水平的影响以及提神功效的分析。
试验方法：将柠檬草油加入培养第10天的大脑神经元细胞培养体系中，并使柠檬草油在大脑神经元细胞培养体系中的最终浓度为0％、0.00005％、0.0005％，测量精油放置大脑神经元细胞后30分钟培养液中神经元细胞所释放的神经递质多巴胺水平。
检测结果如图5所示。实验数据显示，不同浓度的柠檬草油，可以提升大脑神经元细胞多巴胺水平。多巴胺对人的感觉、兴奋、记忆等信息传递密切相关，因此，本申请通过这项测量可以从数据上得知，柠檬草油在500ppm浓度时，对脑神经元细胞的兴奋性刺激作用提升了32.22％，在这一浓度下调配相关产品，可能获得对人体较好的提神作用。
实施例5提神功效精油的筛选方法
本实施例研究了不同芳香精油对多种大脑神经元细胞神经递质水平的影响。本实施例所涉及的芳香精油浓度均为0.0005％。
试验方法：将精油加入培养第10天的大脑神经元细胞培养体系中，并使精油在大脑神经元细胞培养体系中的最终浓度为0.0005％，测量精油放置大脑神经元细胞后30分钟培养液中神经元细胞所释放的神经递质水平。
本实施案例所涉及的神经递质包括多巴胺(DOPAMINE)、五羟色胺(5HYDROXY-TRYPTAMINE)、γ-氨基丁酸(GAMMA AMINOBUTYRIC ACID)、乙酰胆碱(ACETYLCHOLINE)、去甲肾上腺素(NORADRENALINE)、甘氨酸(GLYCINE)、谷氨酸盐(GLUTAMINE)和褪黑激素(MELATONIN)。
本实施例所涉及的芳香精油包括芳樟油(HOHE)，迷迭香油(ROSEMARY  OIL)，罗勒油(BASIL OIL)，柠檬草油(LEMONGRASS OIL)，依兰油(YLANG III MADAGASCAR EO)，薄荷油(MINT PEPPERMINT YAKIMA OIL)，柠檬油(LEMON OIL ITALY)，桉叶油(EUCALYPTUS GLOBULUS OIL)。
不同芳香精油对神经细胞处理后的多种神经递质水平检测结果如图6-13所示。
根据上述检测结果，采用如下的评估体系计算各种芳香精油在提神方面的分值。工艺流程图见图14。提神作用评估体系的标准如表2所示，计算结果如表3所示。
表2芳香精油对大脑神经元细胞在提神作用评估体系的标准
Figure PCTCN2017089697-appb-000005
注：评分数值越高，说明兴奋型神经递质浓度越高或抑制型神经递质浓度越低。
表3芳香精油对大脑神经元细胞的提神分值
Figure PCTCN2017089697-appb-000006
Figure PCTCN2017089697-appb-000007
*注：
Es10：薄荷精油12％+柠檬草油7％+柠檬桉精油4％+葡萄柚精油5％+佛手柑精油8％+罗勒精油6％+没药精油8％+甜杏仁油50％；
Es32：苦橙精油6％+超级薰衣草精油8％+依兰精油4％+天竺葵精油4％+丝柏精油3％+玫瑰草精油4％+罗马洋甘菊精油6％+橙花精油7％+安息香精油8％+甜杏仁油50％。
本发明以44分为分界线，总分越高，样品的提神效果越好。表3数据显示，某些浓度的薄荷油和柠檬草油对脑神经元细胞神经递质释放水平的总分值超过44分，提示在相应浓度下，该样品可能具有较好的提神作用；而其他精油样品在相应浓度下对脑神经元细胞神经递质释放水平的总分值未超过44分，则提示在相应浓度下，该样品可能不具有较好的提神作用。实验3中样品Es32和Es10分别为阴性和阳性对照，Es32对脑神经元细胞神经递质释放水平的总分值为28分，Es10对脑神经元细胞神经递质释放水平的总分值为51分，提示采用这一评估方法，可以获得与对照样品相一致的提神作用的判断。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰， 这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种具有提神功效精油或其产品的筛选方法，其特征在于，包括如下步骤：
    采用精油或其产品处理大脑神经元细胞，检测5-羟色胺、γ-氨基丁酸、褪黑激素、甘氨酸、多巴胺、谷氨酸、去甲肾上腺素和乙酰胆碱的浓度，获得处理组神经递质浓度；
    根据如下公式检测处理组神经递质浓度与空白对照组神经递质浓度相比较的变化率：
    Figure PCTCN2017089697-appb-100001
    其中，C1为处理组神经递质浓度，C0为空白对照组神经递质浓度，Vc为处理组神经递质浓度与空白对照组神经递质浓度相比较的变化率；
    根据所述Vc分别获得5-羟色胺、γ-氨基丁酸、褪黑激素、甘氨酸、多巴胺、谷氨酸、去甲肾上腺素和乙酰胆碱的提神分值Xi；所述提神分值如下：
    Figure PCTCN2017089697-appb-100002
    根据如下公式检测所述精油或其产品的提神总分值X：
    X＝X1+X2+X3+X4+X5+X6+X7+X8
    其中，X为提神总分值，X1为5-羟色胺的提神分值，X2为γ-氨基丁酸的提神分值，X3为褪黑激素的提神分值，X4为甘氨酸的提神分值，X5为多巴胺的提神分值，X6为谷氨酸的提神分值，X7为去甲肾上腺素的提神分值，X8为乙酰胆碱的提神分值。
  2. 根据权利要求1所述的筛选方法，其特征在于，所述精油或其产品的质量百分比浓度为0.00005％～0.05％。
  3. 根据权利要求2所述的筛选方法，其特征在于，所述精油的质量百分比浓度为0.0005％。
  4. 根据权利要求1所述的筛选方法，其特征在于，所述处理的时间为5～60min。
  5. 根据权利要求4所述的筛选方法，其特征在于，所述处理的时间为30min。
  6. 根据权利要求1所述的筛选方法，其特征在于，所述大脑神经元细胞为培养5～15天的大脑神经元细胞。
  7. 根据权利要求6所述的筛选方法，其特征在于，所述大脑神经元细胞为培养10天的大脑神经元细胞。
  8. 根据权利要求6或7所述的筛选方法，其特征在于，所述培养的培养基为马血清培养液，细胞接种量为1×104个/mL，培养温度为37℃，培养条件为5％CO2。
  9. 根据权利要求1所述的筛选方法，其特征在于，所述大脑神经元细胞为人体大脑神经元细胞或动物大脑神经元细胞。
  10. 根据权利要求9所述的筛选方法，其特征在于，所述动物大脑神经元细胞为鼠大脑神经元细胞。
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CN105441391A (zh) * 2015-12-10 2016-03-30 湖南中医药大学 一种糖尿病并发抑郁症的细胞模型及建立方法和应用

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