CN111450131A - Method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii - Google Patents

Method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii Download PDF

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CN111450131A
CN111450131A CN202010453803.8A CN202010453803A CN111450131A CN 111450131 A CN111450131 A CN 111450131A CN 202010453803 A CN202010453803 A CN 202010453803A CN 111450131 A CN111450131 A CN 111450131A
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ligusticum wallichii
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林晓琴
杨昕
青贤
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Sichuan Fufangji Biotechnology Co ltd
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    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

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Abstract

The invention discloses a method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of Ligusticum wallichii, comprising the following steps of homogenizing fresh stems and leaves of Ligusticum wallichii, mixing with distilled water and petroleum ether, extracting by ultrasonic method, filtering to obtain filtrate A and residue A; centrifuging the filtrate A to obtain a petroleum ether layer and a water layer B; recovering petroleum ether from petroleum ether layer, and purifying the residual oily liquid to obtain volatile oil of stem and leaf of rhizoma Ligustici Chuanxiong; mixing the water layer B with ethanol water solution and residue A, and performing ultrasonic extraction to obtain filtrate C; concentrating the filtrate C under reduced pressure, adsorbing with polyamide resin column, eluting with ethanol water solution to obtain a collected solution, concentrating, filtering the collected solution, and recrystallizing with ethyl acetate to obtain chlorogenic acid. The method has the advantages of reasonable design, simple process, high extraction efficiency, low production cost of chlorogenic acid, high content of effective components of volatile oil, energy conservation, environmental protection, good economy and suitability for popularization and application.

Description

Method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii
Technical Field
The invention relates to the technical field of plant extraction, in particular to a method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii.
Background
The common clinical rhizoma ligustici wallichii medicinal material is dry rhizome of an umbelliferae plant rhizoma ligustici wallichii L iguusticum chuanxiong Hort, is pungent in taste and warm in nature, and has the effects of activating blood and promoting qi circulation, dispelling wind and relieving pain.
Modern researches have shown that the stems and leaves of rhizoma Ligustici Chuanxiong are rich in volatile oil, organic acid, etc.
Volatile oil components in the stem and leaf of rhizoma Ligustici Chuanxiong include Z-ligustilide and E-ligustilide, and are easily soluble in petroleum ether; organic acid components in the stems and leaves of rhizoma Ligustici Chuanxiong, including chlorogenic acid and ferulic acid, are insoluble in petroleum ether and ethyl acetate and easily soluble in ethanol water solution, and chlorogenic acid is soluble in ethyl acetate.
The existing volatile oil extraction methods include steam distillation extraction, supercritical extraction, ultrasonic extraction and the like. Steam distillation extraction methods have the disadvantages of low yield, long time consumption and high energy consumption. Supercritical extraction has high requirements on equipment and cannot be generally used. The ultrasonic extraction method adopts ultrasonic wave to assist solvent for extraction, and the sound wave generates high-speed and strong cavitation effect and stirring effect to destroy the cells of the plant medicinal material, so that the solvent permeates into the medicinal material cells, the extraction time is shortened, the extraction rate is improved, and the extraction temperature is low.
Ligustilide is the main component in the volatile oil of the stem and leaf of Ligusticum wallichii, and has the functions of expanding blood vessel, relieving asthma, resisting platelet aggregation, relieving pain, protecting nerve, resisting thrombosis and resisting proliferation, but is unstable at high temperature.
The stem and leaf of the ligusticum wallichii is rich in chlorogenic acid, and the research on the determination of 3 active ingredients in the stem and leaf of the ligusticum wallichii by UP L C (Wu Yan et al, J. Huaxi pharmacy, 526 and 528 pages in 2018) reports that the content of the chlorogenic acid in the powder of the stem and leaf of the ligusticum wallichii is 15.96mg/g, and the content of the chlorogenic acid in the powder of the leaf is 16.41mg/g, but the process research on extracting the chlorogenic acid from the stem and leaf of the ligusticum wallichii is unavailable at present.
The prior art mainly extracts chlorogenic acid from honeysuckle and eucommia bark, has high production cost and can not meet market requirements, so the development of the process for extracting the chlorogenic acid from the stem and the leaf of the ligusticum wallichii has extremely important economic value; after the ligusticum wallichii is harvested, the rhizome is used as the medicine, the stem and the leaf are discarded frequently, the resources cannot be effectively utilized, and the comprehensive utilization of the ligusticum wallichii resources becomes an urgent problem to be solved by technical personnel in the field.
Disclosure of Invention
Aiming at the problems in the prior technical scheme, the invention provides a method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii. The invention provides the following technical scheme:
a method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of Ligusticum wallichii comprises the following steps:
(1) pretreatment:
pulping fresh rhizoma Ligustici Chuanxiong stem and leaf, and mixing with petroleum ether and distilled water;
(2) extracting volatile oil from stems and leaves of Ligusticum wallichii by ultrasonic method:
extracting the mixture obtained in the step (1) by using an ultrasonic method, after extraction is finished, performing vacuum filtration to obtain filtrate A and residue A, centrifuging the filtrate A, and dividing the filtrate A into a petroleum ether layer B and a water layer B; adding petroleum ether layer B into a rotary evaporator, recovering petroleum ether to obtain light yellow oily liquid, placing in a fume hood, and volatilizing residual petroleum ether until the weight of the oily liquid is constant to obtain volatile oil of stem and leaf of rhizoma Ligustici Chuanxiong;
(3) purifying the volatile oil of the stem and leaf of the Ligusticum wallichii:
mixing the volatile oil of the stem and leaf of the ligusticum wallichii obtained in the step (2) with absolute ethyl alcohol, uniformly stirring, freezing, performing vacuum filtration, filtering precipitates to obtain a filtrate B, transferring the filtrate B into a rotary evaporator, performing rotary evaporation at 50-60 ℃, and recovering the ethyl alcohol in the filtrate B to obtain the purified light yellow transparent volatile oil of the stem and leaf of the ligusticum wallichii;
(4) extracting chlorogenic acid by an ultrasonic method:
mixing the water layer B in the step (2) with an ethanol water solution, then uniformly mixing the mixture with the residue A in the step (2), extracting by an ultrasonic method, and filtering to remove residues after ultrasonic treatment is finished to obtain a filtrate C;
(5) separation and purification of chlorogenic acid:
transferring the filtrate C into a rotary evaporator, and carrying out rotary evaporation at the temperature of 50-70 ℃ to obtain a concentrated solution; adjusting the pH value of the concentrated solution to 1-3, extracting twice with ethyl acetate with the volume of 4-6 times, and combining ethyl acetate extract liquor; placing the ethyl acetate extract in a pretreated polyamide resin column, washing with dilute hydrochloric acid with the pH value of 3-4, then isocratic eluting with 20-30 vol% ethanol water solution, collecting the eluent, concentrating, filtering, and recrystallizing with ethyl acetate to obtain chlorogenic acid crystals.
Further, the mixing ratio of the stem and leaf of the ligusticum wallichii to the distilled water and the petroleum ether in the step (1) is 1 kg: 1-2L: 2-4L.
Further, the ultrasonic power of the ultrasonic method in the step (2) is 100-250W, the ultrasonic time is 30-60 min, the ultrasonic temperature is 20-30 ℃, and stirring is carried out with a stirrer in the ultrasonic extraction process; the centrifugal rotating speed of the filtrate A is 3000-4000 r/min, and the centrifugal time is 10-15 min; the rotary evaporation temperature of the petroleum ether layer B is 30-60 ℃.
Preferably, the mixing ratio of the stems and leaves of the ligusticum wallichii to the distilled water to the petroleum ether in the step (1) is 1kg to 1L to 3L, the ultrasonic time in the step (2) is 60min, the ultrasonic power is 175W, and the ultrasonic temperature is 30 ℃.
Further, the using amount of the absolute ethyl alcohol in the step (3) is 2-4 times of the volume of the volatile oil; the freezing temperature is-20 ℃, and the freezing time is 8-24 h.
Further, the ethanol aqueous solution in the step (4) is 90-98 vol% ethanol aqueous solution, and the mixing volume ratio of the water layer B and the ethanol aqueous solution is 2: 1-6; the ultrasonic power of the ultrasonic method is 100-250W, the ultrasonic time is 30-60 min, and the ultrasonic temperature is 40-70 ℃.
Preferably, in the step (4), the mixing volume ratio of the water layer B to the 95 vol% ethanol aqueous solution is 1L: 3L, the ultrasonic time is 30min, the ultrasonic power is 250W, and the ultrasonic temperature is 70 ℃.
Further, the pretreatment method of the polyamide resin column in the step (5) comprises the steps of soaking the polyamide resin in 90-95 vol% ethanol water for 12-24 hours, washing with water to be neutral, soaking in 3-8 wt% sodium hydroxide water solution for 8-12 hours, washing with water to be neutral, soaking in 4-10 wt% acetic acid water solution for 8-12 hours, and washing with water to be neutral for later use.
Further, the dosage of the dilute hydrochloric acid in the step (5) is 1-2 BV, the dosage of the 20-30 vol% ethanol water solution is 3-5 BV, and the elution flow rate of the 20-30 vol% ethanol water solution is 1.5-2.5 BV/h, wherein BV represents the unit of column volume.
Further, the boiling range of the petroleum ether used in the step (1) and the step (2) is 30-60 ℃.
The invention has the beneficial effects that:
(1) the invention adopts an ultrasonic extraction process to extract the volatile oil and the chlorogenic acid of the stems and leaves of the ligusticum wallichii, utilizes the thermal effect, the cavitation effect, the mechanical effect and the like generated by ultrasonic radiation extraction solvent to effectively break the cell walls of the stems and leaves of the ligusticum wallichii, ensures that the extraction solvent permeates into cells, can accelerate the molecular motion of the extraction solvent, accelerates the dissolution of the effective components of the stems and leaves of the ligusticum wallichii in the extraction solvent, ensures that the extraction solvent and the components to be extracted are quickly contacted and dissolved, and has the advantages of high extraction efficiency and short extraction time;
(2) the method utilizes the solubility characteristic difference of each component in the stems and leaves of the ligusticum wallichii in different solvents, and simultaneously extracts and purifies the volatile oil and the chlorogenic acid from the discarded stems and leaves of the ligusticum wallichii after being harvested, comprehensively utilizes the ligusticum wallichii resource, has reasonable design and simple process, and reduces the production cost of the chlorogenic acid;
(3) the invention extracts the volatile oil by an ultrasonic method, avoids the problems of easy emulsification of oil and water, higher energy consumption and the like caused by traditional steam distillation, has low extraction temperature, and avoids the degradation of ligustilide by high temperature, thereby leading the active ingredient content of the volatile oil to be high;
(4) the extraction solvent used in the invention is easy to recycle, no solvent residue exists in the extract, the extraction solvent can be repeatedly used, and the method is energy-saving, environment-friendly, good in economical efficiency and suitable for popularization and application.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
A method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of Ligusticum wallichii comprises the following steps:
(1) pretreatment: pulping fresh rhizoma Ligustici Chuanxiong stem and leaf, and mixing with petroleum ether and distilled water.
(2) Extracting volatile oil from stems and leaves of Ligusticum wallichii by ultrasonic method:
extracting the mixture obtained in the step (1) by using an ultrasonic method, after extraction is finished, performing vacuum filtration to obtain filtrate A and residue A, centrifuging the filtrate A, and dividing the filtrate A into a petroleum ether layer B and a water layer B; and adding the petroleum ether layer B into a rotary evaporator to recover petroleum ether to obtain light yellow oily liquid, wherein the petroleum ether obtained by recovery can be recycled, placing the oily liquid in a fume hood, and volatilizing residual petroleum ether until the weight of the liquid is constant to obtain the volatile oil of the stem and leaf of the ligusticum wallichii.
After the ultrasonic extraction, the effective components of the volatile oil in the extracted material, including Z-ligustilide, E-ligustilide and the like, are completely extracted by petroleum ether, and the effective components of the volatile oil can also be dissolved in an ethanol water solution.
(3) Purifying the volatile oil of the stem and leaf of the Ligusticum wallichii:
and (3) mixing the volatile oil of the stem and leaf of the ligusticum wallichii obtained in the step (2) with absolute ethyl alcohol, uniformly stirring, freezing, performing vacuum filtration, filtering precipitates to obtain filtrate, transferring the filtrate into a rotary evaporator, performing rotary evaporation at the temperature of 50-60 ℃, and recovering the ethyl alcohol in the filtrate to obtain the purified light yellow transparent volatile oil of the stem and leaf of the ligusticum wallichii.
The volatile oil of the stem and leaf of the ligusticum wallichii, which is prepared by the method in the step (2), may contain some fat-soluble components such as resin, grease and the like, and the impurities can influence the purity of the volatile oil of the stem and leaf of the ligusticum wallichii, and if the high-purity volatile oil of the stem and leaf of the ligusticum wallichii is required to be obtained, the impurities are required to be removed.
(4) Extracting chlorogenic acid by an ultrasonic method:
and (3) mixing the water layer B in the step (2) with an ethanol water solution, then uniformly mixing with the residue A in the step (2), extracting by an ultrasonic method, and filtering to remove residues after ultrasonic treatment is finished to obtain a filtrate C.
(5) Separation and purification of chlorogenic acid:
transferring the filtrate C into a rotary evaporator, carrying out rotary evaporation at 50-70 ℃, recovering ethanol, wherein the recovered ethanol can be recycled to obtain a concentrated solution; adjusting the pH value of the concentrated solution to 1-3, extracting twice with ethyl acetate with the volume of 4-6 times, and combining ethyl acetate extract liquor; placing the ethyl acetate extract in a pretreated polyamide resin column, washing with dilute hydrochloric acid with the pH value of 3-4, isocratic eluting with 20-30 vol% ethanol water solution, collecting the eluent, concentrating, filtering, and recrystallizing with ethyl acetate to obtain chlorogenic acid, wherein the concentration can be reduced pressure concentration, the concentrated solution is cooled to-20 ℃, crystals are separated out and filtered, and the filter cake is recrystallized with ethyl acetate to remove impurities, such as ferulic acid, which are easily dissolved in ethyl acetate, so that the purity of the extracted and purified chlorogenic acid is improved. Before gradient elution, dilute hydrochloric acid with the pH value of 3-4 is used for washing the polyamide resin column, so that the loss of chlorogenic acid can be reduced, and the purity of chlorogenic acid crystals is further improved.
Further, in the step (1), the mixing ratio of the stem and leaf of the ligusticum wallichii to the distilled water and the petroleum ether is 1 kg: 1-2L: 2-4L.
Further, the ultrasonic power of the ultrasonic method in the step (2) is 100-250W, the ultrasonic time is 30-60 min, the ultrasonic temperature is 20-30 ℃, and the stirring is carried out by a stirrer in the ultrasonic extraction process, so that the organic solvent petroleum ether and water are in more sufficient contact with the materials, and the components are more sufficiently extracted; the centrifugal rotating speed of the filtrate A is 3000-4000 r/min, and the centrifugal time is 10-15 min; the rotary evaporation temperature of the petroleum ether layer B is 30-60 ℃.
Preferably, the mixing ratio of the stems and leaves of the ligusticum wallichii to the distilled water to the petroleum ether in the step (1) is 1L: 1L: 3kg, the ultrasonic time in the step (2) is 60min, the ultrasonic power is 175W, and the ultrasonic temperature is 30 ℃.
Further, the using amount of the absolute ethyl alcohol in the step (3) is 2-4 times of the volume of the volatile oil; the freezing temperature is-20 ℃, and the freezing time is 8-24 h.
Further, the ethanol aqueous solution in the step (4) is 90-98 vol% ethanol aqueous solution, and the mixing volume ratio of the water layer B and the ethanol aqueous solution is 2: 1-6; the ultrasonic power of the ultrasonic method is 100-250W, the ultrasonic time is 30-60 min, and the ultrasonic temperature is 40-70 ℃.
Preferably, in the step (4), the mixing volume ratio of the water layer B and the 95 vol% ethanol aqueous solution is 1L: 3L, the ultrasonic time is 30min, the ultrasonic power is 250W, and the ultrasonic temperature is 70 ℃.
Further, the pretreatment method of the polyamide resin column in the step (5) comprises the steps of soaking the polyamide resin in 90-95 vol% ethanol water for 12-24 hours, then washing with water to neutrality, soaking with 3-8 wt% sodium hydroxide water solution for 8-12 h, washing with water to neutrality, soaking with 4-10 wt% acetic acid water solution for 8-12 h, then washing the polyamide resin with water to be neutral, soaking the polyamide resin in 90-95 vol% ethanol water solution, can remove alcohol-soluble impurities in the polyamide resin column, soaking the polyamide resin column in 3-8 wt% sodium hydroxide aqueous solution for 8-12 h, impurities such as tannin adsorbed on the polyamide resin can be removed, the polyamide resin is soaked in 4-10 wt% acetic acid aqueous solution for 8-12 h, impurities adsorbed on the polyamide resin and eluted by the acetic acid aqueous solution can be removed, and the regeneration and the repeated use of the polyamide resin column are realized through the operation.
Further, the dosage of the dilute hydrochloric acid in the step (5) is 1-2 BV, the dosage of the ethanol aqueous solution with each concentration is 3-5 BV, the flow rate of the ethanol aqueous solution with each concentration is 1.5-2.5 BV/h, wherein BV represents the unit of column volume.
Furthermore, the boiling range of the petroleum ether used in the step (1) and the step (2) is 30-60 ℃, the petroleum ether with a low boiling range is selected, the extraction temperature of the volatile oil of the stem and leaf of the ligusticum wallichii can be reduced, the active ingredients of the volatile oil are protected from being damaged or volatilized, meanwhile, the saturated vapor pressure of the petroleum ether is very low, the rotary evaporation is very easy, and after the extraction operation is finished, the residual petroleum ether in the volatile oil can be simply and quickly removed through natural volatilization, so that no solvent residue is left in the volatile oil, and the purity of the volatile oil is improved.
The following are specific examples of the present invention:
example 1: optimization of method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii
1. Orthogonal test optimized volatile oil extraction method
1.1 selection of factor level
Using quadrature test L9(34) Orthogonal table, examining the influence of petroleum ether dosage, ultrasonic power, ultrasonic temperature and ultrasonic time on the extraction rate of volatile oil, and optimizing the ultrasonic process for extracting volatile oil by ultrasonic method, wherein the orthogonal factor level table is shown in table 1, the parameters of the subsequent steps are fixed, 10kg of fresh ligusticum wallichii stems and leaves are taken to be pulped, distilled water 10L is added, 20-40L of petroleum ether with the boiling range of 30-60 ℃ is added for mixing, the ultrasonic method is used for extraction, the ultrasonic power is 100-250W, the ultrasonic time is 30-60 min, the ultrasonic temperature is 20-30 ℃, a stirrer is used for stirring in the ultrasonic extraction process, after the extraction is finished, the reduced pressure suction filtration is carried out, filtrate A and residue A are obtained, the filtrate A is centrifuged for 10min at the rotating speed of 3000r/min, the filtrate A is divided into a petroleum ether layer B and a water layer B after the centrifugation, the petroleum ether layer B is added into a rotary evaporator for rotary evaporation, the petroleum ether is recovered, the rotary evaporation temperature is 50 ℃, the petroleum ether is evaporated until no obvious oily ether escapes, the yellowish liquid is placed in a liquid cabinet, and the volatile oil is.
TABLE 1 orthogonal test factor horizon
Figure BDA0002508486540000081
1.2 orthogonal test Schedule
According to L9(34) The orthogonal table arrangement, 9 test arrangements are shown in table 2, each time 10kg is fed.
TABLE 2 orthogonal test Schedule
Figure BDA0002508486540000082
1.3 determination of evaluation index
The weight of volatile oil extracted from 10kg of stems and leaves of Ligusticum wallichii is selected as evaluation index.
1.4 results of orthogonal experiments
The results of the orthogonal tests are shown in tables 3 and 4.
TABLE 3 results of orthogonal experiments
Figure BDA0002508486540000091
TABLE 4 analysis of variance
Figure BDA0002508486540000092
F0.01(1,2)＝99；F0.05(1,2)＝19.00
As can be seen from Table 3, the range R value C > B > A > D, namely the influence factor ultrasonic power > ultrasonic time > petroleum ether dosage > ultrasonic temperature; the best extraction method is A judged by test indexes and K2B3C2D3Namely, the preferred technological parameters for extracting the volatile oil of the invention are 10kg of fresh stem and leaf of the ligusticum wallichii, the dosage of the petroleum ether is 30L, the ultrasonic time is 60min, the ultrasonic power is 175W, and the ultrasonic temperature is 30 ℃.
2. Method for optimizing chlorogenic acid extraction by orthogonal test
2.1 selection of factor level
Using quadrature test L9(34) And (3) an orthogonal table, wherein the influence of the dosage of the ethanol aqueous solution, the ultrasonic power, the ultrasonic temperature and the ultrasonic time on the extraction rate of the chlorogenic acid is examined, the ultrasonic process for extracting the chlorogenic acid is optimized, and the levels of orthogonal factors are shown in a table 5.
Taking 100kg of fresh ligusticum wallichii stem leaves, pulping, adding 100L of distilled water, mixing with petroleum ether 300L with a boiling range of 30-60 ℃, uniformly mixing, extracting by an ultrasonic method, wherein the ultrasonic power is 175W, the ultrasonic time is 60min, the ultrasonic temperature is 30 ℃, stirring with a stirrer in the ultrasonic extraction process, after extraction is finished, carrying out vacuum filtration to obtain a filtrate A and a residue A, centrifuging the filtrate A at the rotating speed of 3000r/min for 10min, dividing the filtrate into a petroleum ether layer B and a water layer B after centrifugation, and enabling the volume of the water layer B to be 152L.
Adding the petroleum ether layer B into a rotary evaporator, carrying out rotary evaporation at 50 ℃, carrying out rotary evaporation until no obvious petroleum ether escapes to obtain light yellow oily liquid, placing the light yellow oily liquid in a fume hood, volatilizing residual petroleum ether until the weight of the liquid is constant to obtain the volatile oil of the stem and leaf of the ligusticum wallichii, placing the volatile oil of the stem and leaf of the ligusticum wallichii into a reaction vessel, adding absolute ethyl alcohol with the volume of 4 times of that of the volatile oil, uniformly stirring, freezing at-20 ℃ for 12 hours, carrying out reduced pressure suction filtration after freezing, filtering out a precipitate to obtain a filtrate C, transferring the filtrate C into the rotary evaporator, carrying out rotary evaporation at 50 ℃, and recovering the ethyl alcohol in the filtrate C to obtain light yellow transparent refined volatile oil of the stem and leaf of the.
Soaking polyamide resin in 95 vol% ethanol water solution for 24 hr, washing with water to neutrality, soaking in 8 wt% sodium hydroxide water solution for 12 hr, washing with water to neutrality, soaking in 10 wt% acetic acid water solution for 12 hr, and washing with water to neutrality.
Mixing 15L water layer B and 95 vol% ethanol water solution according to the volume ratio of 2: 1-6, then uniformly mixing with 4kg of residue A, extracting by an ultrasonic method, wherein the ultrasonic time is 30-60 min, the ultrasonic power is 100-250W, the ultrasonic temperature is 40-70 ℃, filtering to remove residues after ultrasonic processing is finished, obtaining filtrate C, transferring the filtrate C into a rotary evaporator, and carrying out rotary evaporation at the temperature of 60 ℃ to obtain concentrated solution.
Adjusting the pH value of the concentrated solution to 3, extracting twice with ethyl acetate with the volume of 4 times of the concentrated solution, and combining ethyl acetate extract liquor; placing the ethyl acetate extract in a pretreated polyamide resin column for adsorption for 3h, washing with 2BV of dilute hydrochloric acid with the pH value of 3, eluting with 5BV of 20 vol% ethanol water solution at the flow rate of 2BV/h, collecting the eluent, concentrating, filtering the eluent, and recrystallizing with ethyl acetate for 1 time to obtain the chlorogenic acid.
TABLE 5 orthogonal test factor horizon
Figure BDA0002508486540000111
2.2 orthogonal test Schedule
According to L9(34) The orthogonal tables are arranged, and 9 test arrangements are shown in Table 6.
TABLE 6 orthogonal test Schedule
Figure BDA0002508486540000112
2.3 determination of evaluation index
Selecting the weight of chlorogenic acid crystal extracted by different process parameters as evaluation index.
2.4 results of orthogonal experiments
The results of the orthogonal tests are shown in tables 7 and 8.
TABLE 7 results of orthogonal experiments
Figure BDA0002508486540000113
Figure BDA0002508486540000121
TABLE 8 analysis of variance
Figure BDA0002508486540000122
F0.01(1,2)＝99；F0.05(1,2)＝19.00
As can be seen from Table 9, the range R value C > A > B > D, namely the influence factor ultrasonic power > 95 vol% ethanol aqueous solution dosage > ultrasonic time > ultrasonic temperature; the best extraction method is A judged by test indexes and K3B1C3D3Namely, the preferable technological parameters for extracting the chlorogenic acid are that 10kg of fresh stems and leaves of the ligusticum wallichii, 95 vol% of ethanol water solution are used in an amount of 45L, the ultrasonic time is 30min, the ultrasonic power is 250W, and the ultrasonic temperature is 70 ℃.
Example 2: research on ethology, anti-inflammation and antioxidation effects of ligusticum wallichii stem and leaf volatile oil on stress depression rats
1 Material
1.1 Experimental instruments
Emax Plus microplate reader (Biochrom L td cambride); L500 desk low speed centrifuge (Hunan instrument laboratory Instrument development Co., Ltd.).
1.2 materials of the experiment
Fluoxetine hydrochloride capsules (lilac pharmaceutical co., ltd., national drug standard J20130010), serum tumor necrosis factor α (TNF- α) kit (lot number: a38290222), interleukin 6(I L-6) kit (lot number: a30690153) were all purchased from hangzhou co-ordinated biotechnology ltd., glutathione peroxidase (GSH-PX) colorimetric test kit (lot number: WPTB4MYG33), and catalase colorimetric test kit (lot number: T6VRJP72VA) were all purchased from wuhan yi reiry biotechnology ltd.
1.3 Experimental drugs
1.3.1 preparation of Ligusticum wallichii Stem and leaf volatile oil
The volatile oil is the volatile oil of the stem and leaf of Ligusticum wallichii prepared in example 1.
1.3.2 preparation of Fluoxetine hydrochloride solution
And removing the capsule shell of the fluoxetine hydrochloride capsule, weighing 100mg, putting into a 50m L volumetric flask, diluting to a scale with normal saline, and shaking up to obtain a fluoxetine hydrochloride solution with the concentration of 2mg/m L for later use.
1.3.3 preparation of L PS solution
An appropriate amount of L PS (lipopolysaccharide) was weighed, phosphate buffered saline PBS (1 ×) was added to prepare a L PS solution of 10mg/m L, and the solution was stored in a refrigerator at 4 ℃ for further use.
1.4 Experimental animals
60 male SPF SD rats with weight of 160-180 g are purchased from Soudouzhuoshu laboratory animals Co., Ltd, and the animal production license number is as follows: SCXK (Chuan) 2015-030. The experimental animals are raised in a constant-temperature and constant-humidity animal experiment raising room, are fed with water freely, and are raised adaptively for one week.
2 method of experiment
2.1 grouping of Experimental animals, modeling of stress Depression rat model, administration and sample Collection
All rats were randomly divided into a blank group (K), a model group (M), a volatile oil high dose group (DH), a volatile oil medium dose group (DM), a volatile oil low dose group (D L), and a yang drug group (Y, fluoxetine hydrochloride).
2.2 model modeling of stress Depression rats
All rats began restraint stress at 9: 00 a day for 6h on a continuous basis for 14d on day 15, and were administered with 6h restraint stress on a day, except for the blank group, and after half an hour of the last administration on day 19, all rats were given an intraperitoneal injection of L PS solution at a dose of 10 μ g/kg.
2.3 administration to laboratory animals
The administration is started on day 15, the administration dose of the yang medicine group (Y) is 0.1m L/10 g, the administration dose of the volatile oil high dose group (DH) is 3m L, the administration dose of the volatile oil medium dose group (DM) is 2m L, the administration dose of the volatile oil low dose group (D L) is 1m L, and the administration doses of the blank group and the model group are 1 time per day and 4 days continuously, wherein the administration dose of the blank group and the model group is 0.1m L/10 g of physiological saline.
2.4 open field experiments
The experimental method comprises the steps of injecting L PS solution 2h on the 19 th day, carrying out an open field experiment, keeping a laboratory dark light, avoiding direct light, keeping the temperature constant at about 23 +/-2 ℃ and keeping the environment quiet, dividing an open field experimental box into 16 grids, slightly and slowly placing the head of an experimental rat back to an experimenter into a central area of the open field experimental box at the beginning, adapting the rat to 30s in the open field, recording the total crossing number, the vertical standing times and the modification times of the rat within 5min, taking the crossing of double hind limbs as a standard in crossing the grids, taking the time as counted by the hind limbs, simultaneously taking the double forelimbs off in the vertical standing mode as a standard, and taking the modification to comprise uplifting of the forelimbs, scratching, face washing and foot licking, and cleaning of the olfactory stimulation of the animal odor, and cleaning of a maze platform by 10% of alcohol before each measurement so as to eliminate information, such as odor, excrement and the like, urine and the like, left by the rat at the last time.
2.5 forced swimming test
Forced swimming experiments were performed immediately after the end of the open field experiment on day 19. The test was carried out using a clear glass jar 60cm in height and 50cm in diameter, the water temperature being controlled at 23. + -. 2 ℃. Swimming pre-training is carried out 24 hours before testing, and the time is 15 min. The test is carried out for 5min in a formal way, and video records the desperate time of the rat floating on the water surface. A state of floating is defined as the limbs no longer rowing water, but only with minor movements of the limbs and the head and tail to maintain balance.
2.6 serum sample Collection and measurement of serum inflammatory factor levels and antioxidant indicators
On the 19 th day, L PS solution is injected for 5h, all rats are anesthetized with urethane, blood is taken from a blood collecting tube, after the blood coagulates, serum is taken out, and the blood is subpackaged and transferred to a refrigerator at the temperature of 80 ℃ below zero for freezing storage for later use.
Serum frozen at-80 ℃ is taken, and serum inflammatory factors including serum tumor necrosis factor α (TNF- α) and interleukin 6(I L6) and serum antioxidant indexes including glutathione peroxidase (GSH-PX) and Catalase (CAT) are detected according to an Elisa kit specification.
2.7 data processing
Statistical software SPSS20.0 is adopted for data analysis, and the mean value plus or minus standard deviation is adopted for each group of data
Figure BDA0002508486540000153
The expression that the homogeneity of variance is satisfied is analyzed by L SD or Dunnett method, the homogeneity of variance is not satisfied is analyzed by Dunnett's T3 or Tamhane' sT2 method, and the analysis results show that the difference is statistically significant when P < 0.05.
3 results of the experiment
3.1 Effect of Ligusticum wallichii Stem and leaf volatile oil on Depression induced rat behaviourology
As can be seen from Table 9, compared with the normal group, the number of horizontal cross lattices, the number of vertical stands and the number of modifications of the rat in the model group are all significantly reduced (P < 0.05), and the swimming resting time is significantly increased (P < 0.05). The swimming resting time of the yang medicine group of the volatile oil high dose group (DH) is significantly reduced (P < 0.05), the number of horizontal cross lattices is significantly increased (P < 0.05), the number of vertical cross lattices of the volatile oil high dose group (DH), the volatile oil medium dose group (DM), the volatile oil low dose group (D L) and the yang medicine group is significantly increased (P < 0.05), and the number of vertical stands and the number of modifications of the volatile oil high dose group (DH), the volatile oil medium dose group (DM) and the yang medicine group are significantly increased (P < 0.05). The above results show that the volatile oil of the rhizome and the rhizome of chuanxiong has an improving effect on the behavior of.
TABLE 9 comparison of results of open field and forced swimming experiments in respective rats
Figure BDA0002508486540000151
Figure BDA0002508486540000152
Note: in comparison with the normal group,a)p is less than 0.05; in comparison with the set of models,b)P＜0.05；
3.2 influence of Ligusticum wallichii stem and leaf volatile oil on stress depression rat serum inflammation and antioxidant factor
As can be seen from Table 10, after the model group (M) is modeled by combining restraint stress with L PS, the inflammatory factors TNF- α and I L-6 of the stress depressed rat are remarkably increased (P is less than 0.05), the oxidation resistance related factors GSH-PX and CAT are remarkably reduced (P is less than 0.05), compared with the model group (M), after the ligusticum wallichii stem and leaf volatile oil is administered for treatment, the volatile oil high dose group (DH), the volatile oil medium dose group (DM) and the TNF- α of the yang medicine group are remarkably reduced (P is less than 0.05), the volatile oil high dose group (DH), the volatile oil medium dose group (DM), the volatile oil low dose group (D L) and the I L-6 of the yang medicine group are remarkably reduced (P is less than 0.05), and the GSH-PX and CAT are remarkably increased (P is less than 0.05), the results show that the ligusticum wallichii stem and leaf volatile oil has a regulating effect on the inflammation and oxidation resistance system imbalance of.
TABLE 10 stress inhibition by volatile oil of stem and leaf of Ligusticum wallichiiSerum inflammation and antioxidant factor effects in depressed rats
Figure BDA0002508486540000161
Figure BDA0002508486540000162
Figure BDA0002508486540000163
Note: in comparison with the normal group,a)p is less than 0.05; in comparison with the set of models,b)P＜0.05；
the research proves that the Ligusticum wallichii stem and leaf volatile oil has the effect of improving the behavioural science of stress-depressed rats through forced swimming experiments and open field experiments, and the effects are related to the regulation of organism inflammation and the unbalance of an oxidation-antioxidation system, the component research indicates that the Ligusticum wallichii stem and leaf volatile oil contains Z-ligustilide, E-ligustilide, ligustrazine, α -pinene, β -pinene, cis-ocimene and other components, the document reports that the ligustilide has better pharmacological effects on cardiovascular and cerebrovascular systems, a circulatory system, an immunologic function, a nervous system and the like, the anti-depression effect of the aerial part of Ligusticum wallichii volatile oil can be directly related to the ligustilide, the ligustrazine has a remarkable anti-depression effect by regulating a neuroendocrine system and the like, so the anti-depression effect of the aerial part of Ligusticum wallichii volatile oil can be directly related to the ligustrazine, in addition, α -pinene has an anxiolytic drug activity, and the anti-depression effect of the aerial part of the Ligusticum wallichii can be related to α -pinene.
The foregoing is a detailed description of the invention, which is described in more detail and specific, but is not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the spirit of the invention, which falls within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of Ligusticum wallichii is characterized in that: the method comprises the following steps:
(1) pretreatment:
pulping fresh rhizoma Ligustici Chuanxiong stem and leaf, and mixing with petroleum ether and distilled water;
(2) extracting volatile oil from stems and leaves of Ligusticum wallichii by ultrasonic method:
extracting the mixture obtained in the step (1) by using an ultrasonic method, after extraction is finished, performing vacuum filtration to obtain filtrate A and residue A, centrifuging the filtrate A, and dividing the filtrate A into a petroleum ether layer B and a water layer B; adding petroleum ether layer B into a rotary evaporator, recovering petroleum ether to obtain light yellow oily liquid, placing in a fume hood, and volatilizing residual petroleum ether until the weight of the oily liquid is constant to obtain volatile oil of stem and leaf of rhizoma Ligustici Chuanxiong;
(3) purifying the volatile oil of the stem and leaf of the Ligusticum wallichii:
mixing the volatile oil of the stem and leaf of the ligusticum wallichii obtained in the step (2) with absolute ethyl alcohol, uniformly stirring, freezing, performing vacuum filtration, filtering precipitates to obtain a filtrate B, transferring the filtrate B into a rotary evaporator, performing rotary evaporation at 50-60 ℃, and recovering the ethyl alcohol in the filtrate B to obtain the purified light yellow transparent volatile oil of the stem and leaf of the ligusticum wallichii;
(4) extracting chlorogenic acid by an ultrasonic method:
mixing the water layer B in the step (2) with an ethanol water solution, then uniformly mixing the mixture with the residue A in the step (2), extracting by an ultrasonic method, and filtering to remove residues after ultrasonic treatment is finished to obtain a filtrate C;
(5) separation and purification of chlorogenic acid:
transferring the filtrate C into a rotary evaporator, and carrying out rotary evaporation at the temperature of 50-70 ℃ to obtain a concentrated solution; adjusting the pH value of the concentrated solution to 1-3, extracting twice with ethyl acetate with the volume of 4-6 times, and combining ethyl acetate extract liquor; placing the ethyl acetate extract in a pretreated polyamide resin column, washing with dilute hydrochloric acid with the pH value of 3-4, then isocratic eluting with 20-30 vol% ethanol water solution, collecting the eluent, concentrating, filtering, and recrystallizing with ethyl acetate to obtain chlorogenic acid crystals.
2. The method for simultaneously extracting volatile oil and chlorogenic acid from ligusticum wallichii stems and leaves according to claim 1, characterized in that the mixing ratio of the ligusticum wallichii stems and leaves, distilled water and petroleum ether in step (1) is 1 kg: 1-2L: 2-4L.
3. The method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii according to claim 1, which is characterized in that: the ultrasonic power of the ultrasonic method in the step (2) is 100-250W, the ultrasonic time is 30-60 min, the ultrasonic temperature is 20-30 ℃, and stirring is carried out with a stirrer in the ultrasonic extraction process; the centrifugal rotating speed of the filtrate A is 3000-4000 r/min, and the centrifugal time is 10-15 min; the rotary evaporation temperature of the petroleum ether layer B is 30-60 ℃.
4. The method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of Ligusticum wallichii according to claim 1, wherein the mixing ratio in step (1) is Ligusticum wallichii stems and leaves, distilled water and petroleum ether are 1 kg: 1L: 3L, the ultrasonic time in step (2) is 60min, the ultrasonic power is 175W, and the ultrasonic temperature is 30 ℃.
5. The method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii according to claim 1, which is characterized in that: the amount of the absolute ethyl alcohol in the step (3) is 2-4 times of the volume of the volatile oil; the freezing temperature is-20 ℃, and the freezing time is 8-24 h.
6. The method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii according to claim 1, which is characterized in that: the ethanol aqueous solution in the step (4) is 90-98 vol% ethanol aqueous solution, and the mixing volume ratio of the water layer B to the ethanol aqueous solution is 2: 1-6; the ultrasonic power of the ultrasonic method is 100-250W, the ultrasonic time is 30-60 min, and the ultrasonic temperature is 40-70 ℃.
7. The method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of Ligusticum wallichii according to claim 1, wherein in step (4), the mixing volume ratio of water layer B and 95 vol% ethanol aqueous solution is 1L: 3L, the ultrasonic time is 30min, the ultrasonic power is 250W, and the ultrasonic temperature is 70 ℃.
8. The method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii according to claim 1, which is characterized in that: the pretreatment method of the polyamide resin column in the step (5) comprises the steps of soaking the polyamide resin in 90-95 vol% ethanol water solution for 12-24 hours, then washing the polyamide resin with water to be neutral, then soaking the polyamide resin in 3-8 wt% sodium hydroxide water solution for 8-12 hours, washing the polyamide resin with water to be neutral, then soaking the polyamide resin in 4-10 wt% acetic acid water solution for 8-12 hours, and then washing the polyamide resin with water to be neutral for later use.
9. The method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii according to claim 1, which is characterized in that: the dosage of the dilute hydrochloric acid in the step (5) is 1-2 BV, the dosage of the 20-30 vol% ethanol water solution is 3-5 BV, and the elution flow rate of the 20-30 vol% ethanol water solution is 1.5-2.5 BV/h, wherein BV represents the unit of column volume.
10. The method for simultaneously extracting volatile oil and chlorogenic acid from stems and leaves of ligusticum wallichii according to claim 1, which is characterized in that: the boiling range of the petroleum ether used in the step (1) and the step (2) is 30-60 ℃.
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