A volatile oil of Rhizoma Cynanchum and its application

Abstract

The present invention belongs to the technical field of research and development of Chinese herbal medicine products, discloses a volatile oil of the Herba Lycopodii and its application, and specifically relates to an extraction and purification method, a component analysis method, a chemical component composition and a study on the inhibition of acetylcholinesterase activity of the volatile oil of the Herba Lycopodii. The present invention purifies the volatile oil of the Herba Lycopodii for the first time, identifies its components, and verifies that it has good activity of inhibiting acetylcholinesterase through experiments. This lays the foundation for the development of new drugs for the treatment of Alzheimer's disease with acetylcholinesterase as the target. The extraction and purification method of the present invention has a simple process, low cost and is suitable for industrial production and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of research and development of Chinese herbal medicine products, and specifically relates to a herbal vine ( Jasminum elongatum (Bergius) Willd.) Volatile oils and their applications. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the premature accumulation of diffuse and neuritic plaques (composed of Aβ peptides) outside cells, followed by the appearance of neurofibrillary tangles (including hyperphosphorylated tau protein) inside the brain. Notably, oxidative stress caused by factors such as immune response and inflammation is the main culprit for the formation of Aβ plaques. Therefore, managing oxidative stress by alleviating the excessive production of ROS in AD patients is a promising therapeutic strategy, in which plant-derived antioxidants can serve as key ingredients. Another factor is the excessive activity of acetylcholinesterase (AChE), which rapidly hydrolyzes acetylcholine (ACh) to produce choline and acetate anions, thereby inhibiting the long-term excitation caused by the neurotransmitter at the postsynaptic membrane. This excessive activity leads to reduced ACh levels and cholinergic dysfunction, ultimately resulting in cognitive deficits in AD patients. Therefore, natural compounds with antioxidant activity and AChE inhibition have great promise in the treatment of AD.

[0004] Volatile oils (EO), also called essential oils, are complex mixtures of volatile, lipophilic compounds that are typically derived from plant secondary metabolism, are characterized by a unique aroma and flavor, and have been shown to possess a wide range of biological activities, including antimicrobial, antioxidant, anti-inflammatory, and enzyme inhibitory properties. Volatile oils have been used for therapeutic purposes since ancient times and have recently gained attention as a potential source of new drugs and therapeutics. Since EOs are mixtures of bio-organic small molecules, they exhibit a broad spectrum of activities in areas such as enzyme inhibition, disease treatment, and antimicrobial activity. The molecular diversity allows for extensive screening in related fields, providing opportunities for the discovery of potential therapeutic agents.

[0005] Twist-belly vine Jasminum elongatumBergius (Bergius) Willd.), also known as Xie Sanniang, white honeysuckle, Hainan jasmine, is a climbing shrub of the genus Jasmine in the family Oleaceae. The twigs are sparsely pubescent or densely yellow-brown; the leaves are simple, opposite, papery, ovate or ovate-lanceolate; the pedicels are long, hairy or hairless; the fruits are oblong or ovate, black when ripe; the flowering period is from April to December, and the fruiting period is from August to March of the following year. The twisted belly vine is distributed in the mixed forests and sandy lands of Guangdong, Hainan, Guangxi, and Yunnan. It is also distributed in Vietnam, Myanmar and the Himalayas. The "Lingnan Herbal Records" records that the twisted belly vine is slightly bitter and cool. Traditional Chinese medicine believes that it has the effects of clearing heat and promoting dampness, and is often used to treat damp-heat abdominal pain, enteritis, dysentery, paralysis and swelling of the limbs, deflation, scabies, etc.

[0006] At present, there is no report on the research of volatile oil of Thunb. The present invention studies the extraction, chemical component analysis and acetylcholinesterase inhibition activity of volatile oil of Thunb. branches and leaves, and provides ideas for the development and application of Thunb. Summary of the invention

[0007] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a volatile oil of the genus Phellodendron and its application. The present invention purifies the volatile oil of the genus Phellodendron for the first time and identifies its components. It is verified by experiments that it has good activity in inhibiting acetylcholinesterase, laying a foundation for the development of new drugs targeting acetylcholinesterase for the treatment of Alzheimer's disease.

[0008] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0009] The first aspect of the present invention provides a volatile oil of the strychnos nux vomica. Among the components of the volatile oil of the strychnos nux vomica, alcohols account for 14.2-16.5% of the total chromatographic elution peak area, aldehydes account for 2.9-3.5%, ketones account for 4.2-4.5%, esters account for 0.8-1.6%, acids account for 65.2-73.5%, hydrocarbons account for 1.3-1.8%, and amides account for 1.1-1.5%.

[0010] Preferably, among the components of the volatile oil of the twisted belly vine, there are 10 compounds with a relative content greater than 1%, and the compound with the highest relative content is palmitic acid 40-46%, followed by linoleic acid 20-23%, linalool 4-5%, 1-hexanol 2-3%, phytol 2-3%, trans-nerolidol 1.5-2.5%, myristic acid 1-1.8%, palmitoleic acid 1-1.8%, 2-hydroxycyclopentadecanone 1-1.5%, and stearamide 0.8-1.2%.

[0011] Preferably, among the components of the volatile oil of the twisted belly vine, there are 8 compounds with a relative content greater than 0.5% and less than 1%, namely 0.5-1.0% pentadecanoic acid, 0.5-1.0% phyton, 0.5-1.0% wintergreen oil, 0.4-1.2% heptadecanoic acid, 0.4-1.2% damascus ketone, 0.2-0.8% n-pentadecanal, 0.2-0.8% stearic acid, and 0.2-0.8% 6-epi-calendulone.

[0012] Preferably, among the components of the volatile oil of the twisted belly vine, there are 42 compounds with a relative content of less than 0.5%, namely 1,2-epoxyhexadecane 0.2~0.8%, trans-2-decenal 0.2~0.8%, trans-2,4-decadienal 0.2~0.8%, citronellal 0.2~0.8%, octanol 0.2~0.8%, β-ionone 0.2~0.8%, bisabolol oxide B 0.2~0.5%, 2-undecenal 0.2~0.5%, furyl enol 0.2~0.5%, geranyl acetone 0.2~0.5%, trans-2-nonenal 0.2~0.5%, decanal 0.1~0.4%, 17-hydroxyheptadecanolactone 0.1~0.4%, farnesyl acetone 0.1~0.4%, citronellol 0.1~0.4%, (+)-calendula spirenone B 0.1~0.4%, 3-nonenol 0.1~0.4%, melaleuca alcohol 0.1~0.4%, terpineol 0.1~0.4%, lauric acid 0.1~0.4%, β-caryophyllene 0.1~0.4%, trans-squalene 0.1~0.4%, decahydrodimethylvinyl naphthol 0.1~0.4%, 5-dodecyldihydro-2(3H)-furanone 0.1~0.3%, (E,E)-2,4-nonadienal 0.1~0.3%, trans-farnesol acetate 0.1~0.3%, γ-cedrene 0.1~0.3%, tetradecaldehyde 0.1~0.3%, Tetradecane 0.1~0.3%, 2-camphene 0.1~0.3%, hexyl palmitate 0.1~0.3%, eucalyptus alcohol 0.1~0.3%, trans-9-octadecenoic acid methyl ester 0.1~0.3%, methyl palmitate 0.1~0.3%, β-eudesmol 0.1~0.2%, Dufoyol 0.1~0.2%, 4,8,12,16-tetramethylheptadecan-4-lactone 0.1~0.2%, zhianone 0.1~0.2%, n-pentacosane 0.1~0.2%, cis-7,10-hexadecadienal 0.1~0.2%, hexadecamide 0.1~0.2%.

[0013] Further preferably, among the components of the volatile oil of the Thunb. umbellata, alcohols account for 14.2% of the total chromatographic elution peak area, aldehydes account for 2.9%, ketones account for 4.2%, esters account for 0.8%, acids account for 73.1%, hydrocarbons account for 1.3%, and amides account for 1.1%.

[0014] Further preferably, among the components of the volatile oil of the twisted belly vine, there are 10 compounds with a relative content greater than 1%, and the compound with the highest relative content is palmitic acid 45.7%, followed by linoleic acid 22.7%, linalool 4.2%, 1-hexanol 2.4%, phytol 2.3%, trans-nerolidol 2.0%, myristic acid 1.4%, palmitoleic acid 1.3%, 2-hydroxycyclopentadecanone 1.1%, and stearamide 1.0%.

[0015] Further preferably, among the components of the volatile oil of the Wrightia ternata, there are 8 compounds with a relative content greater than 0.5% and less than 1%, namely 0.8% pentadecanoic acid, 0.8% phytonadione, 0.7% wintergreen oil, 0.6% heptadecanoic acid, 0.6% damascone, 0.5% pentadecanal, 0.5% stearic acid and 0.5% 6-epihydrocalamone.

[0016] Further preferably, among the components of the volatile oil of the twisted belly vine, there are 42 compounds with a relative content of less than 0.5%, namely 1,2-epoxyhexadecane 0.4%, trans-2-decenal 0.4%, trans-2,4-decadienal 0.4%, citronellal 0.4%, octanol 0.4%, β-ionone 0.4%, bisabolol oxide B 0.3%, 2-undecenal 0.3%, forkylenol 0.3%, geraniol 0.3%, geranylacetone 0.3%, trans-2-nonenal 0.3%, decanal 0.2%, 17-hydroxyheptadecanolactone 0.2%, farnesylacetone 0.2%, citronellol 0.2%, (+)-calamus spirenone B 0.2%, 3-nonenol 0.2%, melaleuca alcohol 0.2%, terpineol 2%, lauric acid 0.2%, β-caryophyllene 0.2%, trans-squalene 0.2%, decahydrodimethylvinyl naphthol 0.2%, 5-dodecyldihydro-2(3H)-furanone 0.1%, (E,E)-2,4-nonadienal 0.1%, trans-farnesol acetate 0.1%, γ-cedrene 0.1%, tetradecaldehyde 0.1%, Tetradecane 0.1%, 2-camphene 0.1%, hexyl palmitate 0.1%, eucalyptus alcohol 0.1%, trans-9-octadecenoic acid methyl ester 0.1%, methyl palmitate 0.1%, β-eudesmol 0.1%, Dufoyl alcohol 0.1%, 4,8,12,16-tetramethylheptadecan-4-lactone 0.1%, zhianone 0.1%, n-pentacosane 0.1%, cis-7,10-hexadecadienal 0.1%, hexadecamide 0.1%.

[0017] The second aspect of the present invention provides the use of the volatile oil of the first aspect of the present invention in the preparation of a product for inhibiting acetylcholinesterase.

[0018] Preferably, the product is a medicine.

[0019] Preferably, the volatile oil of the Rhizoma Cynanchum is combined with pharmaceutical excipients and various pharmaceutically or physiologically acceptable carriers and prepared into any pharmaceutical dosage form according to conventional Chinese medicine preparation technology.

[0020] The third aspect of the present invention provides an anti-Alzheimer's disease drug, the active ingredient of which includes the volatile oil of the Herba Lycopodii of the first aspect.

[0021] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0022] The present invention purifies the volatile oil of the Herba Lycopodii for the first time and identifies the components thereof. The volatile oil has good acetylcholinesterase inhibition activity and has broad application prospects as verified by experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 This is the total ion current diagram of the volatile oil of the Herba Lycopodii prepared in Example 1 of the present invention;

[0025] Figure 2 This is a graph showing the effect of an acetylcholinesterase inhibition experiment of the volatile oil of the Herba Lycopodii prepared in Example 1 of the present invention;

[0026] Figure 3 This is a graph showing the inhibition of acetylcholinesterase by the volatile oil of the Herba Lycopodii prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0028] As mentioned above, there is no report on the research of the volatile oil of the truncatum truncatum. Therefore, the present invention studies the extraction, chemical component analysis, and inhibitory activity of the volatile oil of the truncatum truncatum branches and leaves on acetylcholinesterase.

[0029] The first typical embodiment of the present invention provides a volatile oil of the strychnos nux vomica, wherein the components of the volatile oil of the strychnos nux vomica are 14.2-16.5% of the total chromatographic elution peak area of ​​alcohols, 2.9-3.5% of aldehydes, 4.2-4.5% of ketones, 0.8-1.6% of esters, 65.2-73.5% of acids, 1.3-1.8% of hydrocarbons, and 1.1-1.5% of amides.

[0030] In one or more embodiments of this implementation mode, among the components of the volatile oil of the twisted belly vine, there are 10 compounds with a relative content greater than 1%, and the compound with the highest relative content is palmitic acid 40~46%, followed by linoleic acid 20~23%, linalool 4~5%, 1-hexanol 2~3%, phytol 2~3%, trans-nerolidol 1.5~2.5%, myristic acid 1~1.8%, palmitoleic acid 1~1.8%, 2-hydroxycyclopentadecanone 1~1.5%, and stearamide 0.8~1.2%.

[0031] In one or more embodiments of this implementation mode, among the components of the volatile oil of the twisted belly vine, there are 8 compounds with a relative content greater than 0.5% and less than 1%, namely 0.5-1.0% pentadecanoic acid, 0.5-1.0% phyton, 0.5-1.0% wintergreen oil, 0.4-1.2% heptadecanoic acid, 0.4-1.2% damascus ketone, 0.2-0.8% pentadecanal, 0.2-0.8% stearic acid, and 0.2-0.8% 6-epihydrocalamine.

[0032] In one or more embodiments of this embodiment, among the components of the volatile oil of the twisted belly vine, there are 42 compounds with a relative content of less than 0.5%, namely 1,2-epoxyhexadecane 0.2~0.8%, trans-2-decenal 0.2~0.8%, trans-2,4-decadienal 0.2~0.8%, citronellal 0.2~0.8%, octanol 0.2~0.8%, β-ionone 0.2~0.8%, bisabolol oxide B 0.2~0.5%, 2-undecenal 0.2~0.5%, furyl enol 0.2~0.5%, geranyl acetone 0.2~0.5%, trans-2-nonenal 0.2~0.5%, decanal 0.1~0.4%, 17-hydroxyheptadecanolactone 0.1~0.4%, farnesyl acetone 0.1~0.4%, citronellol 0.1~0.4%, (+)-calendula spirenone B 0.1~0.4%, 3-nonenol 0.1~0.4%, melaleuca alcohol 0.1~0.4%, terpineol 0.1~0.4%, lauric acid 0.1~0.4%, β-caryophyllene 0.1~0.4%, trans-squalene 0.1~0.4%, decahydrodimethylvinyl naphthol 0.1~0.4%, 5-dodecyldihydro-2(3H)-furanone 0.1~0.3%, (E,E)-2,4-nonadienal 0.1~0.3%, trans-farnesol acetate 0.1~0.3%, γ-cedrene 0.1~0.3%, tetradecaldehyde 0.1~0.3%, Tetradecane 0.1~0.3%, 2-camphene 0.1~0.3%, hexyl palmitate 0.1~0.3%, eucalyptus alcohol 0.1~0.3%, trans-9-octadecenoic acid methyl ester 0.1~0.3%, methyl palmitate 0.1~0.3%, β-eudesmol 0.1~0.2%, Dufoyol 0.1~0.2%, 4,8,12,16-tetramethylheptadecan-4-lactone 0.1~0.2%, zhianone 0.1~0.2%, n-pentacosane 0.1~0.2%, cis-7,10-hexadecadienal 0.1~0.2%, hexadecamide 0.1~0.2%.

[0033] In one or more embodiments of this implementation mode, among the components of the volatile oil of the twisted belly vine, alcohols account for 14.2% of the total chromatographic elution peak area, aldehydes account for 2.9%, ketones account for 4.2%, esters account for 0.8%, acids account for 73.1%, hydrocarbons account for 1.3%, and amides account for 1.1%.

[0034] In one or more embodiments of this implementation mode, among the components of the volatile oil of the twisted belly vine, there are 10 compounds with a relative content greater than 1%, and the compound with the highest relative content is palmitic acid 45.7%, followed by linoleic acid 22.7%, linalool 4.2%, 1-hexanol 2.4%, phytol 2.3%, trans-nerolidol 2.0%, myristic acid 1.4%, palmitoleic acid 1.3%, 2-hydroxycyclopentadecanone 1.1%, and stearamide 1.0%.

[0035] In one or more embodiments of this implementation mode, among the components of the volatile oil of the twisted belly vine, there are 8 compounds with a relative content greater than 0.5% and less than 1%, namely 0.8% pentadecanoic acid, 0.8% phytonadione, 0.7% wintergreen oil, 0.6% heptadecanoic acid, 0.6% damascus ketone, 0.5% pentadecanal, 0.5% stearic acid, and 0.5% 6-epihydrocalamone.

[0036] In one or more embodiments of this embodiment, among the components of the volatile oil of the twisted belly vine, there are 42 compounds with a relative content of less than 0.5%, namely 1,2-epoxyhexadecane 0.4%, trans-2-decenal 0.4%, trans-2,4-decadienal 0.4%, citronellal 0.4%, octanol 0.4%, β-ionone 0.4%, bisabolol oxide B 0.3%, 2-undecenal 0.3%, forkylenol 0.3%, geraniol 0.3%, geranylacetone 0.3%, trans-2-nonenal 0.3%, decanal 0.2%, 17-hydroxyheptadecanolactone 0.2%, farnesylacetone 0.2%, citronellol 0.2%, (+)-calamus spirenone B 0.2%, 3-nonenol 0.2%, melaleuca alcohol 0.2%, terpineol 0.2%, lauric acid 0.2%, β-caryophyllene 0.2%, trans-squalene 0.2%, decahydrodimethylvinyl naphthol 0.2%, 5-dodecyldihydro-2(3H)-furanone 0.1%, (E,E)-2,4-nonadienal 0.1%, trans-farnesol acetate 0.1%, γ-cedrene 0.1%, tetradecaldehyde 0.1%, Tetradecane 0.1%, 2-camphene 0.1%, hexyl palmitate 0.1%, eucalyptus alcohol 0.1%, trans-9-octadecenoic acid methyl ester 0.1%, methyl palmitate 0.1%, β-eudesmol 0.1%, Dufoyl alcohol 0.1%, 4,8,12,16-tetramethylheptadecan-4-lactone 0.1%, zhianone 0.1%, n-pentacosane 0.1%, cis-7,10-hexadecadienal 0.1%, hexadecamide 0.1%.

[0037] In one or more examples of this implementation, the method for preparing the volatile oil of the Thunb. ...

[0038] In one or more examples of this implementation mode, the cleaning is to soak the freshly collected vine branches in deionized water for cleaning; further preferably, the mass ratio of the vine branches to deionized water is 1:3-5.

[0039] In one or more examples of this implementation mode, the air-dried material is crushed into a powder with a particle size of 15 to 20 meshes.

[0040] In one or more examples of this implementation mode, the soaking is to add ultrapure water to the powder, and the soaking time is 0.5-3 hours; the temperature of the water vapor distillation is 80-120° C., and the distillation time is 3-8 hours.

[0041] In one or more examples of this implementation, the mass ratio of the powder to ultrapure water is 1:1.5-2.5, preferably 1:2; the soaking time is 1 h, the temperature of the steam distillation is 100° C., and the distillation time is 6 h.

[0042] In one or more embodiments of this implementation, the method for analyzing the chemical components of the volatile oil of the Thunb. is specifically: analyzing and identifying the volatile oil of the Thunb. by gas chromatography-mass spectrometry (GC-MS), obtaining the relative content of each component in the component by peak area normalization method, and comparing the chemical components of different parts.

[0043] In one or more examples of this implementation, a dehydrating desiccant is added to the organic solvent containing the volatile oil of the Herba Lycopodii to dehydrate it, and then a gas chromatography-mass spectrometer is used to analyze the chemical components;

[0044] Wherein, the organic solvent is selected from n-pentane, n-hexane or dichloromethane, preferably n-pentane, and the volume concentration of the organic solvent containing the volatile oil of the scutellaria baicalensis is 1%; the dehydrating drying agent is selected from anhydrous sodium sulfate or anhydrous calcium sulfate.

[0045] In one or more examples of this embodiment, the dehydrating desiccant is anhydrous sodium sulfate.

[0046] In one or more embodiments of this implementation mode, the chromatographic conditions used in the gas chromatography-mass spectrometry analysis are as follows: the chromatographic column is a HP-5MS quartz capillary column; column temperature: program temperature rise 45~55°C and hold for 3~5 min, increase to 250~300°C at 5~10°C / min, hold for 2~5 min, injection port temperature 250~280°C, injection volume 0.15~0.25 μL, carrier gas He, flow rate 0.5~1.5 mL / min -1 , no split; mass spectrometry conditions: ionization mode is EI, electron energy is 50~80 eV, quadrupole temperature is 140~160℃, ion source temperature is 220~240℃, solvent delay time is 2~4 min, full scan acquisition mode, scanning mass number range is 25~550 m / z, and mass spectrum database is NIST / NIH / EPA EI 2023.

[0047] In one or more examples of this embodiment, the chromatographic conditions used in the gas chromatography-mass spectrometry analysis are as follows: the chromatographic column is a HP-5MS quartz capillary column; column temperature: program temperature rise of 50°C for 4 min, increase to 280°C at 7°C / min, maintain for 3 min, injection port temperature of 260°C, injection volume of 0.2 μL, carrier gas He, flow rate of 1 mL / min -1 , no split; mass spectrometry conditions: ionization mode is EI, electron energy is 70 eV, quadrupole temperature is 150℃, ion source temperature is 230℃, solvent delay time is 3.5 min, full scan acquisition mode, scanning mass number range is 25-550 m / z, and mass spectrum database is NIST / NIH / EPAEI 2023.

[0048] A second typical embodiment of the present invention provides the use of the above-mentioned Thunbergia truncatula volatile oil in the preparation of products for inhibiting acetylcholinesterase.

[0049] In one or more examples of this embodiment, the product is a drug.

[0050] In one or more examples of this implementation mode, the volatile oil of the Glehnia littoralis is combined with pharmaceutical excipients and various pharmaceutically or physiologically acceptable carriers and prepared into any pharmaceutical dosage form according to conventional Chinese medicine preparation technology.

[0051] A third typical embodiment of the present invention provides an anti-Alzheimer's disease drug, the active ingredient of which includes the above-mentioned Thunbergia volvacea volatile oil.

[0052] The volatile oil of the present invention has a good inhibitory effect on acetylcholinesterase. The volatile oil of the present invention can be directly or encapsulated into a variety of dosage forms: solutions (such as aromatic water, sprays, emulsions, etc.), injections, sustained-release preparations, nano-preparations, etc. The volatile oil of the present invention can be used as an active substance, mixed and combined with medically acceptable pharmaceutical excipients, excipients, etc., and prepared into the above-mentioned various dosage forms according to the methods in the prior art; it can also be used together with other drugs for treating Alzheimer's disease as active substances, mixed and combined with medically acceptable pharmaceutical excipients, excipients, etc., to prepare the above-mentioned various dosage forms.

[0053] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0054] Embodiment 1: This embodiment provides a volatile oil of Rhizoma Cynomorii and a preparation method thereof, comprising the following steps:

[0055] Take 1.5 kg of freshly collected leaves and branches of the Twist-belly Vine, soak and wash them with 5 L of deionized water, and then air-dry them in a cool place. Cut the air-dried Twist-belly Vine into 1 cm long segments with a clean knife, and then crush them into 15-20 mesh powder with a Chinese medicine grinder. Add it to a 5 L volatile oil extractor, then add 3 L of ultrapure water, soak for 1 hour, heat and distill for 6 hours until the volume of the volatile oil no longer increases, cool and stand for 1 hour, collect the upper layer of light yellow liquid in the extractor, and obtain the Twist-belly Vine volatile oil, with a volatile oil yield of 0.33% (volume (mL) / mass (g), v / w).

[0056] Example 2: This example is an analysis of the chemical composition of the volatile oil of the Herba Lycopodii prepared in Example 1. The details are as follows:

[0057] Anhydrous sodium sulfate was added to the volatile oil of the Herba Lycopodii for dehydration, and then the volatile oil was dissolved in mass spectrometry grade n-pentane or n-hexane to prepare a volatile oil n-pentane solution with a concentration of 1% (v / v).

[0058] The gas chromatography-mass spectrometry was used for detection, wherein the chromatographic conditions were as follows: the quartz capillary column was HP-5MS (30m*0.25* mm*0.25 μm), the column temperature was programmed to rise to 50°C for 4 min; then to 280°C at 7°C / min and held for 3 min; the injection port temperature was 260°C, the injection volume was 0.2 μL; the carrier gas was He, the flow rate was 1.0 mL / min -1, without splitting the flow; Mass spectrometry conditions: Ionization mode is EI; Electron energy is 70 eV; Quadrupole temperature is 150 °C; Ion source temperature is 230 °C; Volume delay is 3.5 min; Full scan acquisition mode; Scanning mass number range is 25 - 550 m / z. The total ion current chromatogram of the volatile oil of Jasminum elongatum is as Figure 1 shown.

[0059] The analysis conditions for the chemical components of the volatile oil of Jasminum elongatum are as follows: The GC-MS detection data is analyzed using Agilent MassHunter Workstation 10.0 in combination with the NIST / NIH / EPA EI 2023 mass spectrometry database.

[0060] First, set the peak area threshold to 50000 on the total ion current chromatogram and integrate the entire chromatographic peak; then extract the mass spectrometry diagram at the vertex of each substance peak and subtract the background data to form the mass spectrometry diagram corresponding to the chromatographic peak; then compare the mass spectrometry diagram with the standard spectrum diagram in the NIST spectral library. Peaks with a similarity matching threshold greater than 600 (the highest is 1000), that is, peaks with both the forward matching value and the reverse matching value greater than 600 are recognized; in addition, calculate the relative retention index of the chromatographic peak as an auxiliary means for qualitative analysis, with an error range of ±5%. Relative retention index (RI = 100n + 100(tx - tn) / (tn+1 - tn), where n is the number of carbon atoms of the n-alkane, tx and tn are the retention times of the eluted peaks, and tn < tx < tn+1). The relative content is analyzed by relative quantitative analysis through the FID detection integral value of the peak area, and the area normalization method is used to calculate the relative content. Through analysis, a total of 60 compounds were identified, with a total content of 97.6%. The analysis results of the components of the volatile oil of Jasminum elongatum are shown in Table 1.

[0061] Table 1 Analysis results of the components of the volatile oil of Jasminum elongatum by GC-MS

[0062]

[0063] Experimental Example 1: This experimental example is a test for the acetylcholinesterase inhibitory activity of the volatile oil of Jasminum elongatum prepared in Example 1

[0064] I. Experimental materials

[0065] 1. Medicinal materials and reagents

[0066] Tris base (77-86-1), sodium dihydrogen phosphate dihydrate (13472-35-0), disodium hydrogen phosphate dodecahydrate (10039-32-4), acetylcholinesterase (AChE) (9000-81-1, C3389), bovine serum albumin (BSA) (9048-46-8), 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) (69-78-3), iodothioacetylcholine (ATCI) (1866-15-5), galantamine hydrobromide (1953-04-4). All the above reagents are of AR or BR grade from Sigma.

[0067] 2. Instruments and equipment

[0068] Microplate reader (Epoch, Biotek, USA), water bath (OSB-2200, Tokyo Rika, Suzhou), pipette (eppendorf, USA), 96-well microplate (Corning, USA).

[0069] 2. Experimental Methods

[0070] 1. In a 96-well plate:

[0071] Enzyme inhibition assay: Add 145 μL of PBS buffer (0.1 M, pH = 8.0), 20 μL of the sample solution to be tested and 15 μL of the enzyme solution to the wells of the ELISA plate, mix well and store at 4°C for 20 min. Take out and add 10 μL of DTNB (2mM) and 10 μL of ATCI (15mM), mix well for 60 seconds, and measure the absorbance at 412 nm for 6 minutes at intervals of 60 seconds. After the enzymatic reaction is completed, Figure 2 As shown. With absorbance as the ordinate and time as the abscissa, the initial reaction rate K of the enzyme under inhibition conditions is obtained after linear regression.

[0072] Enzyme activity determination: Replace 20 μL of the sample solution with 20 μL of solvent, and keep other conditions unchanged. Measure the absorbance at 412 nm for 6 minutes at intervals of 60 seconds. Use absorbance as the ordinate and time as the abscissa to calculate the initial reaction rate K0 of the enzyme under inhibition conditions after linear regression.

[0073] 2. Calculate the inhibition rate of the sample to be tested according to the following formula:

[0074] Inhibition rate I = (K0-K) / K0*100%

[0075] 3. Sample IC 50The values ​​are obtained by measuring the inhibition rate of samples on acetylcholinesterase at at least 6 different concentrations. Therefore, the acetylcholinesterase inhibition rate corresponding to the final concentrations of 50, 100, 250, 500, 750, and 1000 μg / mL was determined. Galantamine hydrobromide was used as a positive control drug. All experiments were repeated three times and the mean and standard deviation were calculated.

[0076] 4. According to the corresponding inhibition rate at different concentrations, use Graphpad prism 10.1 and nonlinear regression to calculate the corresponding IC 50 value.

[0077] 3. Experimental Results

[0078] 1. The experimental data of the inhibition of acetylcholinesterase by the volatile oil of Thunbergia truncatula at different concentrations are as follows:

[0079]

[0080] 2. Conclusion Analysis

[0081] Based on the relationship between concentration and inhibition rate, the curves of the two were obtained by nonlinear regression, such as Figure 3 As shown. The IC is obtained by calculation 50 The value is 123.7 μg / mL.

[0082] Thus, it was found that the volatile oil of Rhizoma Angustifolia has a significant AChE inhibitory effect. Compared with previous related studies, the results of the present invention are stronger than those of Rhizoma Angustifolia (IC 50 =570 μg / mL), basil (IC 50 =220 μg / mL) and Uncaria rhynchophylla (IC 50 =175 μg / mL). Preliminary structure-activity relationship analysis showed that its inhibitory effect on AChE may be attributed to monoterpenes and ketone compounds. These secondary metabolites generally have potent but reversible acetylcholinesterase inhibitory ability.

[0083] The above effect evaluation proves that the volatile oil extracted from the present invention has significant inhibitory activity on acetylcholinesterase.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A volatile oil of Rhizoma Cynanchum, characterized in that: Among the components of the volatile oil of the twisted belly vine, alcohols account for 14.2-16.5% of the total chromatographic elution peak area, aldehydes account for 2.9-3.5%, ketones account for 4.2-4.5%, esters account for 0.8-1.6%, acids account for 65.2-73.5%, hydrocarbons account for 1.3-1.8%, and amides account for 1.1-1.5%.

2. The volatile oil of Rhizoma Cynanchum as claimed in claim 1, characterized in that Among the components of the volatile oil of the twisted belly vine, there are 10 compounds with a relative content greater than 1%, and the compound with the highest relative content is palmitic acid 40-46%, followed by linoleic acid 20-23%, linalool 4-5%, 1-hexanol 2-3%, phytol 2-3%, trans-nerolidol 1.5-2.5%, myristic acid 1-1.8%, palmitoleic acid 1-1.8%, 2-hydroxycyclopentadecanone 1-1.5%, and stearamide 0.8-1.2%.

3. The volatile oil of Rhizoma Cynanchum as claimed in claim 1, characterized in that Among the components of the volatile oil of the twisted belly vine, there are 8 compounds with a relative content greater than 0.5% and less than 1%, namely 0.5-1.0% pentadecanoic acid, 0.5-1.0% phyton, 0.5-1.0% wintergreen oil, 0.4-1.2% heptadecanoic acid, 0.4-1.2% damascus ketone, 0.2-0.8% n-pentadecanal, 0.2-0.8% stearic acid and 0.2-0.8% 6-epi-calendulone.

4. The volatile oil of Rhizoma Cynanchum as claimed in claim 1, characterized in that Among the components of the volatile oil of the twisted belly vine, there are 42 compounds with a relative content of less than 0.5%, namely 1,2-epoxyhexadecane 0.2-0.8%, trans-2-decenal 0.2-0.8%, trans-2,4-decadienal 0.2-0.8%, citronellal 0.2-0.8%, octanol 0.2-0.8%, β-ionone 0.2-0.8%, bisabolol oxide B 0.2~0.5%, 2-undecenal 0.2~0.5%, furyl enol 0.2~0.5%, geranyl acetone 0.2~0.5%, trans-2-nonenal 0.2~0.5%, decanal 0.1~0.4%, 17-hydroxyheptadecanolactone 0.1~0.4%, farnesyl acetone 0.1~0.4%, citronellol 0.1~0.4%, (+)-calendula spirenone B 0.1~0.4%, 3-nonenol 0.1~0.4%, melaleuca alcohol 0.1~0.4%, terpineol 0.1~0.4%, lauric acid 0.1~0.4%, β-caryophyllene 0.1~0.4%, trans-squalene 0.1~0.4%, decahydrodimethylvinyl naphthol 0.1~0.4%, 5-dodecyldihydro-2(3H)-furanone 0.1~0.3%, (E,E)-2,4-nonadienal 0.1~0.3%, trans-farnesol acetate 0.1~0.3%, γ-cedrene 0.1~0.3%, tetradecaldehyde 0.1~0.3%, Tetradecane 0.1~0.3%, 2-camphene 0.1~0.3%, hexyl palmitate 0.1~0.3%, eucalyptus alcohol 0.1~0.3%, trans-9-octadecenoic acid methyl ester 0.1~0.3%, methyl palmitate 0.1~0.3%, β-eudesmol 0.1~0.2%, Dufoyol 0.1~0.2%, 4,8,12,16-tetramethylheptadecan-4-lactone 0.1~0.2%, zhianone 0.1~0.2%, n-pentacosane 0.1~0.2%, cis-7,10-hexadecadienal 0.1~0.2%, hexadecamide 0.1~0.2%.

5. The volatile oil of Rhizoma Cynanchum as claimed in claim 1, characterized in that Among the components of the volatile oil of the Thunb. Thunb., alcohols account for 14.2% of the total chromatographic elution peak area, aldehydes account for 2.9%, ketones account for 4.2%, esters account for 0.8%, acids account for 73.1%, hydrocarbons account for 1.3%, and amides account for 1.1%.

6. The volatile oil of Rhizoma Cynanchum as claimed in claim 2, characterized in that: Among the components of the volatile oil of the twisted belly vine, there are 10 compounds with a relative content greater than 1%, and the compound with the highest relative content is palmitic acid 45.7%, followed by linoleic acid 22.7%, linalool 4.2%, 1-hexanol 2.4%, phytol 2.3%, trans-nerolidol 2.0%, myristic acid 1.4%, palmitoleic acid 1.3%, 2-hydroxycyclopentadecanone 1.1%, and stearamide 1.0%.

7. The volatile oil of Rhizoma Cynanchum as claimed in claim 3, characterized in that Among the components of the volatile oil of the twisted belly vine, there are 8 compounds with a relative content greater than 0.5% and less than 1%, namely 0.8% pentadecanoic acid, 0.8% phyton, 0.7% wintergreen oil, 0.6% heptadecanoic acid, 0.6% damascone, 0.5% pentadecanal, 0.5% stearic acid and 0.5% 6-epihydrocalamone.

8. The volatile oil of Rhizoma Cynanchum as claimed in claim 4, characterized in that Among the components of the volatile oil of the twisted belly vine, there are 42 compounds with a relative content of less than 0.5%, which are 0.4% of 1,2-epoxyhexadecane, 0.4% of trans-2-decenal, 0.4% of trans-2,4-decadienal, 0.4% of citronellal, 0.4% of octanol, 0.4% of β-ionone, 0.3% of bisabolol oxide B, 0.3% of 2-undecenal, 0.3% of fukienol, 0.3% of geraniol, 0.3% of geranylacetone, 0.3% of trans-2-nonenal, 0.2% of decanal, 0.2% of 17-hydroxyheptadecanolactone, 0.2% of farnesylacetone, 0.2% of citronellol, and (+)-calamus spirenone B 0.2%, 3-nonenol 0.2%, melaleuca alcohol 0.2%, terpineol 0.2%, lauric acid 0.2%, β-caryophyllene 0.2%, trans-squalene 0.2%, decahydrodimethylvinyl naphthol 0.2%, 5-dodecyldihydro-2(3H)-furanone 0.1%, (E,E)-2,4-nonadienal 0.1%, trans-farnesol acetate 0.1%, γ-cedrene 0.1%, tetradecaldehyde 0.1%, Tetradecane 0.1%, 2-camphene 0.1%, hexyl palmitate 0.1%, eucalyptus alcohol 0.1%, trans-9-octadecenoic acid methyl ester 0.1%, methyl palmitate 0.1%, β-eudesmol 0.1%, Dufoyl alcohol 0.1%, 4,8,12,16-tetramethylheptadecan-4-lactone 0.1%, zhianone 0.1%, n-pentacosane 0.1%, cis-7,10-hexadecadienal 0.1%, hexadecamide 0.1%.

9. Use of the volatile oil of the Herba Lycopodii of any one of claims 1 to 8 in the preparation of a product for inhibiting acetylcholinesterase; in, The product is a medicine; the volatile oil of the genus Thunb is mixed with pharmaceutical excipients and various pharmaceutically or physiologically acceptable carriers and prepared into any pharmaceutical dosage form according to conventional Chinese medicine preparation technology.

10. An anti-Alzheimer's disease drug, characterized in that: The active ingredient comprises the volatile oil of the Herba Lycopodii of any one of claims 1 to 8.

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