CN110664962B - Pure traditional Chinese medicine oil for relaxing tendons and activating collaterals and preparation method thereof - Google Patents

Pure traditional Chinese medicine oil for relaxing tendons and activating collaterals and preparation method thereof Download PDF

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CN110664962B
CN110664962B CN201911003599.3A CN201911003599A CN110664962B CN 110664962 B CN110664962 B CN 110664962B CN 201911003599 A CN201911003599 A CN 201911003599A CN 110664962 B CN110664962 B CN 110664962B
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parts
oil
extract
chinese medicine
traditional chinese
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CN110664962A (en
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吴哲
宋学才
张继迁
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Anhui Xintai Pharmaceutical Co ltd
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Anhui Xintai Pharmaceutical Co ltd
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/888—Araceae (Arum family), e.g. caladium, calla lily or skunk cabbage
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/19—Acanthaceae (Acanthus family)
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
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    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/282—Artemisia, e.g. wormwood or sagebrush
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    • A61K36/18—Magnoliophyta (angiosperms)
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    • A61K36/57—Magnoliaceae (Magnolia family)
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
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    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

Abstract

The invention discloses pure traditional Chinese medicine oil for relaxing tendons and activating collaterals, which is prepared from the following raw materials in parts by weight: 20-40 parts of grassleaf sweelflag rhizome, 15-20 parts of pseudo-ginseng, 35-50 parts of ligusticum wallichii, 25-35 parts of clove, 35-50 parts of cortex moutan, 30-60 parts of Chinese sage herb, 10-18 parts of curcuma zedoary, 50-80 parts of polygonum cuspidatum, 20-35 parts of wormwood, 60-90 parts of red paeony root, 20-40 parts of Chinese mosla herb, 10-20 parts of Chinese anise, 10-15 parts of cablin potchouli herb, 15-45 parts of common andrographis herb, 10-30 parts of musk and 50-100 parts of essential oil; the preparation method combines the supercritical carbon dioxide extraction and distillation with the purification of silica gel column, extracts the effective active ingredients in the crude drug raw materials to the maximum extent, improves the content level of monoterpene, sesquiterpene, paeoniflorin, eugenol, resveratrol and other substances, can effectively resist inflammation and ease pain, and has better application prospect.

Description

Pure traditional Chinese medicine oil for relaxing tendons and activating collaterals and preparation method thereof
Technical Field
The invention relates to the field of traditional Chinese medicine compound preparations, in particular to pure traditional Chinese medicine oil for relaxing tendons and activating collaterals and a preparation method thereof.
Background
The collateral-flow-activating oil has effects of relaxing muscles and tendons, activating collateral flow, dispelling pathogenic wind, and removing blood stasis, and is suitable for treating various traumatic injury, arthralgia, soreness of waist, backache, and tense tendons and bones. The collateral activating oil is mainly prepared from traditional Chinese medicines, can play a role in integrally regulating each joint, and has good efficacy. Has certain help to rheumatism bone pain. The main components of the collateral activating oil comprise wintergreen oil (methyl salicylate), menthol, oleum Terebinthinae, Camphora, oleum Eucalypti, oleum Cinnamomi, oleum Caryophylli, thymol, etc.
Traumatic injuries are mainly caused by soft tissue injuries and are accompanied by various swelling pain and distending pain. This is because traumatic injury causes capillary exudation or bleeding, which results in the formation of blood precipitates in the tissue, causing aseptic inflammation, and thus causing pain and swelling of the tissue. Rheumatic bone pain is caused by cold wind and damp invading human body, which can cause tendon obstruction, thereby causing pain, redness and distending pain of joints, muscles and other parts. The channels and collaterals are the ways in the human body to contact the human body, and if the channels and collaterals can be dredged, the normal operation of the blood and qi of the human body can be ensured, so that the pain can be reduced, and the effect of quickly treating diseases can be achieved.
At present, the extraction methods of traditional Chinese medicines mainly comprise a water decoction extraction method, an ethanol reflux extraction method, a supercritical fluid extraction method and the like. The supercritical fluid extraction method is a new traditional Chinese medicine extraction process and is increasingly accepted in recent yearsPeople pay attention to the method. Supercritical CO2As an extraction technology widely applied to the extraction of plant natural compounds, the method has the following remarkable advantages: the operation is close to room temperature (34-39 ℃), and oxidation and degradation of heat-sensitive substances are effectively prevented. The biological activity can be completely retained, and substances with high boiling point, low volatility and easy pyrolysis can be extracted at the temperature far lower than the boiling point of the substances; extraction and separation are combined into one, and CO saturated with dissolved substances2The fluid is subjected to pressure or temperature regulation to make CO2The extract and the extract quickly become gas-liquid two phases and are separated immediately, so that the extraction efficiency is high and the energy consumption is low; CO 22As an inert gas, the gas is odorless, nontoxic and very safe; the extraction capacity is strong, and the extraction rate is high; the extraction time is short, and the production period is short. The pressure and temperature can be parameters for adjusting the extraction process, the extraction purpose can be achieved only by changing the extraction temperature and pressure, and the separation purpose can be achieved by changing the separation pressure or temperature, so the process is simple and easy to master, and the extraction speed is high.
But supercritical CO2The application of extraction techniques in plant extracts also presents certain limitations: for example, when there is a large difference in relative volatility or polarity between components in a mixture, it is necessary to fractionate the mixture at different extraction pressures; compounds containing strong polar groups (carbonyl or multiple hydroxyl groups), such as flavone and polyphenol, are difficult to extract; strongly polar substances, such as sugars, are difficult to extract at pressures below 40MPa, and compounds with too high molecular weights, such as polysaccharides, are also difficult to extract.
At present, most of the traditional Chinese medicine collateral activating oil is prepared by mixing traditional Chinese medicine plant extracts or directly purifying traditional Chinese medicine raw materials after mixing, although the traditional Chinese medicine collateral activating oil has a certain effect of promoting blood circulation to remove blood stasis, the components in the collateral activating oil are complex, various main components are volatile mixtures, the content of effective components is not clear, the main treatment components cannot be improved in a targeted manner, and the further improvement of the drug effect is difficult. The traditional Chinese medicine components are mostly medicines for promoting blood circulation and removing blood stasis, but few anti-inflammatory and antioxidant repair are mentioned, and the traditional Chinese medicine composition is still deficient in relieving chronic pain and repairing after pain.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at various problems existing in the preparation process of the traditional Chinese medicine collateral-activating oil at present, the invention provides the pure traditional Chinese medicine tendon-relaxing collateral-activating oil and the preparation method thereof, wherein a target extract is concentrated on compounds such as phenols and terpenoids, the content of active substances is greatly improved, the activity of the active substances is greatly kept, and the effects of analgesia, anti-inflammation, blood circulation promotion and repair of the compounds are utilized to treat traumatic injury and other chronic pains.
In order to solve the technical problems, the invention provides the following technical scheme:
a pure traditional Chinese medicine oil for relaxing tendons and activating collaterals is prepared from the following raw materials in parts by weight: 20-40 parts of grassleaf sweelflag rhizome, 15-20 parts of pseudo-ginseng, 35-50 parts of ligusticum wallichii, 25-35 parts of clove, 35-50 parts of cortex moutan, 30-60 parts of Chinese sage herb, 10-18 parts of curcuma zedoary, 50-80 parts of polygonum cuspidatum, 20-35 parts of wormwood, 60-90 parts of red paeony root, 20-40 parts of Chinese mosla herb, 10-20 parts of Chinese anise, 10-15 parts of cablin potchouli herb, 15-45 parts of common andrographis herb, 10-30 parts of musk and 50-100 parts of essential oil;
the pure traditional Chinese medicine oil for relaxing tendons and activating collaterals consists of a crude drug extract and essential oil, wherein the crude drug extract consists of recovered oil and traditional Chinese medicine oil, and the recovered oil comprises the following components in parts by weight: traditional Chinese medicine oil: the weight ratio of the essential oil is 1-3: 5-10: 5; the crude drug extract contains 15.33-24.57 wt% of terpenoids, 3.21-5.34 wt% of eugenol, 7.56-11.23 wt% of flavonoids and 22.54-31.25 wt% of polyphenols.
Preferably, the terpenoids comprise 33.56 to 45.27wt% monoterpenes, 30.29 to 43.11wt% sesquiterpenes; the polyphenol compounds comprise 56.88-74.53 wt% of resveratrol; the flavonoid compound comprises 33.25-50.34 wt% of paeoniflorin.
Preferably, the essential oil comprises one or more of wormwood essential oil, rose essential oil, lavender essential oil, safflower essential oil, peppermint oil, camphor essential oil, wintergreen essential oil, capsicum essential oil; the rhizoma Acori Graminei, Notoginseng radix, rhizoma Ligustici Chuanxiong, flos Caryophylli, cortex moutan, herba Salviae chinensis, Curcumae rhizoma, rhizoma Polygoni Cuspidati, folium Artemisiae Argyi, radix Paeoniae Rubra, herba Moslae, fructus Anisi Stellati, herba Agastaches, herba Andrographitis, and Moschus are dried products or Chinese medicinal decoction pieces.
A preparation method of the pure traditional Chinese medicine oil for relaxing tendons and activating collaterals comprises the following specific steps:
(1) crushing raw materials of crude drugs except essential oil, sieving the raw materials with a sieve of 80-100 meshes, soaking the raw materials in 60-80% ethanol aqueous solution with the volume of 1-3 times of that of the raw materials for 24-48 h, carrying out suction filtration, filtering the filtrate with filter paper, placing the filtrate on a rotary evaporator for reduced pressure distillation at 35 ℃, removing ethanol in the solution to obtain extract aqueous solution, repeatedly extracting the extract aqueous solution with petroleum ether for 3 times (100 mL each time), collecting the extract aqueous solution in a conical flask, taking out the lower-layer aqueous solution, repeatedly extracting with ethyl acetate for 3 times (100 mL each time), collecting the extract aqueous solution in the conical flask, taking out the lower-layer aqueous solution, repeatedly extracting with n-butyl alcohol for 3 times, collecting the extract aqueous solution in the conical flask, and collecting the rest part in the conical flask;
(2) after suction filtration, the raw material powder is put into CO2In a supercritical extraction kettle, according to the extraction temperature of 40-60 ℃, the extraction pressure of 2-30 MPa, 5-10 wt% of entrainer and CO2Extracting at the flow rate of 10-30 kg/h for 0.5-1.5 h, filtering the extract with a 0.22-micron filter membrane, extracting the filtrate with n-hexane in a separating funnel, mixing the n-hexane extract with the petroleum ether extract, the ethyl acetate extract and the n-butanol extract in the step (1), and concentrating at 60 ℃ in a rotary evaporator to remove the organic extractant to obtain traditional Chinese medicine oil;
(3) carrying out co-water distillation on the dregs of the extraction of the crude drug raw materials according to the weight ratio of the raw materials to water of 1: 10-60, continuously adding distilled water in the distillation process to supplement distillate, distilling for 1-3 h, collecting the distillate by taking 50-500 mL as a unit, extracting for 1h by using equal volume of anhydrous ether after collecting, then separating the liquid and collecting ether, adding anhydrous sodium sulfate for drying, recovering the solvent by using ether for extraction at 50 ℃ in water bath, and collecting a crude extract;
(4) preparing a chromatographic column by using a 150-plus-250-mesh silica gel packed column, packing the column according to a diameter-height ratio of 1: 8-16, weighing a crude extract, dissolving the crude extract in an eluent according to a weight ratio of 1:3, loading the sample at room temperature, eluting by using the eluent as a mobile phase, collecting the eluent by using 10-30 mL as a unit at an eluent flow rate of 1.0-3.8 mL/min, carrying out sample application tracking analysis by using thin-layer chromatography while eluting, collecting fractions according to sample application conditions, and volatilizing a solvent at room temperature under reduced pressure to obtain recovered oil after collecting effective fractions; the eluent is prepared from ethyl acetate and petroleum ether according to the volume ratio of 1: 10-20;
(5) and (3) recovering oil: traditional Chinese medicine oil: mixing the essential oils according to the weight ratio of 1-3: 5-10: 5, adding 15-25 wt% of activated carbon for decoloring for 1-7 d, centrifuging at 10000rpm for 30min, and taking supernatant to obtain the pure traditional Chinese medicine oil for relaxing tendons and activating collaterals.
Preferably, the CO is2The extraction time in the supercritical extraction is composed of dynamic extraction time and static extraction time, wherein the dynamic extraction time is as follows: and (3) the static extraction time is 2-4: 1, and the mass ratio of the crude extract to the silica gel in the step (4) is 1: 45-200.
Preferably, the entrainer is absolute ethyl alcohol; the step (3) also comprises ultrasonic treatment during co-water distillation, and the specific steps are as follows: carrying out ultrasonic treatment with the frequency of 25-45 kHz 15min after the distillation is started, taking the operation of 5min and the stop of 10min as a treatment period, and continuously treating for 3-10 periods.
The invention has the following beneficial effects:
(1) the traditional Chinese medicine composition is prepared from multiple Chinese herbal medicines with analgesic, blood circulation promoting and anti-inflammatory effects, wherein clove, wormwood, herba elsholtziae, Chinese anise, patchouli, common andrographis herb and musk are rich in monoterpene, sesquiterpene and other volatile terpenoids, can promote blood circulation and relieve pain, and achieve the effects of promoting blood circulation and removing blood stasis, the ligusticum wallichii and the polygonum cuspidatum are rich in ligustrazine, resveratrol and other compounds with anti-inflammatory and analgesic effects, the red peony root and the cortex moutan are rich in paeoniflorin and other anti-inflammatory and analgesic compounds, and the pseudo-ginseng, the acorus gramineus, the salvia chinensis and the curcuma zedoary are used as guiding medicines, so that the absorption of the medicines can be obviously improved, the toxicity of the medicines can be reduced, and the analgesic, blood circulation promoting and post-pain repairing effects of the traditional Chinese medicine composition can be obviously improved.
(2) By means of carbon dioxide supercritical extraction, extraction of different substances is effectively improved, technological parameters such as extraction pressure, extraction temperature and the like of supercritical extraction are optimized, the extraction rate and activity of most of traditional Chinese medicine active substances are remarkably improved, and the defects that the extraction rate of complex traditional Chinese medicine components is low, the active substances are easy to thermally inactivate and oxidize in conventional extraction methods such as thermal reflux and the like are overcome.
(3) The terpenoids, flavonoids such as paeoniflorin and the like, and polyphenol compounds such as resveratrol and the like in the active ingredients are compounds containing a plurality of carbonyl groups and hydroxyl groups, the substances are difficult to extract by carbon dioxide supercritical extraction, the extraction rate is low, so that the active substances in the dregs after supercritical extraction can be extracted, separated and extracted in a targeted manner by assisting in extraction of a plurality of organic solvents and purification of silica gel columns, the defect of low extraction rate of individual effective active substances by supercritical extraction is overcome, the extraction rate and activity of the total terpenoids, the total flavonoids and the total polyphenols are obviously higher than those of the extraction by a single method due to mutual matching of conventional distillation extraction and supercritical extraction, the main active ingredients of the medicine are extracted to the maximum, and the utilization rate of crude drug raw materials is improved.
Detailed Description
The following examples are included to provide further detailed description of the present invention and to provide those skilled in the art with a more complete, concise, and exact understanding of the principles and spirit of the invention.
Example 1: the pure traditional Chinese medicine oil for relaxing tendons and activating collaterals is prepared by the following method:
firstly, preparation of raw materials
The entrainer is absolute ethyl alcohol;
the essential oil is Capsici fructus essential oil.
Rhizoma Acori Graminei, Notoginseng radix, rhizoma Ligustici Chuanxiong, flos Caryophylli, cortex moutan, herba Salviae chinensis, Curcumae rhizoma, rhizoma Polygoni Cuspidati, folium Artemisiae Argyi, radix Paeoniae Rubra, herba Moslae, fructus Anisi Stellati, herba Agastaches, herba Andrographitis, and Moschus are dried or Chinese medicinal decoction pieces.
Preparation of pure Chinese medicine oil for relaxing muscles and tendons and activating the flow of energy
(1) Weighing the raw materials in parts by weight as follows: 20 parts of grassleaf sweelflag rhizome, 15 parts of pseudo-ginseng, 35 parts of szechuan lovage rhizome, 25 parts of clove, 35 parts of tree peony bark, 30 parts of Chinese sage herb, 10 parts of zedoary, 50 parts of giant knotweed rhizome, 20 parts of wormwood, 60 parts of red paeony root, 20 parts of Chinese mosla herb, 10 parts of Chinese anise, 10 parts of patchouli, 15 parts of common andrographis herb and 10 parts of musk are crushed and sieved by a 80-mesh sieve, 60 percent ethanol water solution with the volume of 1 time is soaked for 24 hours, the filtration is carried out, the filtrate is filtered by filter paper, the filtrate is placed on a rotary evaporator for reduced pressure distillation at 35 ℃ to remove the ethanol in the solution to obtain extract water solution, the extract water solution is repeatedly extracted by petroleum ether for 3 times, each time is 100mL, the extract water solution is collected into a conical flask, taking out the lower layer water solution, repeatedly extracting with ethyl acetate for 3 times, each time 100mL, collecting in a conical flask, taking out the lower layer water solution, repeatedly extracting with n-butanol for 3 times, collecting into a conical flask, and collecting the rest part into the conical flask;
(2) after suction filtration, the raw material powder is put into CO2In a supercritical extraction kettle, extracting at 40 deg.C under 2MPa, 5wt% entrainer and CO2The flow rate is 10kg/h, dynamic extraction is carried out firstly, the extraction time is 20min, then static extraction is carried out, the extraction time is 10min, and the dynamic extraction time is as follows: static extraction time is 2:1, the extract passes through a 0.22 mu m filter membrane, filtrate is extracted by equal volume of normal hexane in a separating funnel, the normal hexane extract is mixed with the petroleum ether extract, the ethyl acetate extract and the normal butanol extract in the previous step, and a rotary evaporator is used for removing the organic extractant at 60 ℃ to obtain traditional Chinese medicine oil;
(3) carrying out co-hydro distillation on the dregs of the extraction of the crude drug raw materials according to the weight ratio of the raw materials to the water of 1:10, continuously adding distilled water in the distillation process to supplement distillate, distilling for 1h, and carrying out ultrasonic treatment simultaneously by the co-hydro distillation, wherein the specific steps are as follows: ultrasonic treatment with 25kHz frequency is carried out 15min after the distillation is started, the operation is carried out for 5min, the operation is stopped for 10min to serve as a treatment period, and 3 periods are continuously treated. Collecting 50mL of distillate, extracting with equal volume of anhydrous ether for 1h, separating, collecting ether, adding anhydrous sodium sulfate, drying, recovering solvent from the ether at 50 deg.C in water bath, and collecting crude extract;
(4) preparing a chromatographic column by using 150-mesh silica gel packed column, packing the column according to the diameter-height ratio of 1:8, weighing a crude extract, dissolving the crude extract in an eluent according to the weight ratio of 1:3, loading the sample at room temperature, wherein the loading amount is 1:45 of the mass ratio of the crude extract to the silica gel, eluting by using the eluent as a mobile phase, collecting the eluent by using 10mL as a unit at the eluent flow rate of 1.0mL/min, carrying out tracking analysis by using thin layer chromatography sample application while eluting, collecting the fraction according to the sample application condition, collecting an effective fraction, and then volatilizing a solvent at room temperature under reduced pressure to obtain recovered oil; the eluent is prepared from ethyl acetate and petroleum ether according to the volume ratio of 1: 10;
(5) and (3) recovering oil: traditional Chinese medicine oil: mixing the essential oils at a weight ratio of 1:5:5, adding 15 wt% of activated carbon for decolorizing for 1d, centrifuging at 10000rpm for 30min, and collecting supernatant to obtain the final product.
The recovered oil and the Chinese medicinal oil are mixed in proportion, and then the components are measured according to a gas chromatography (appendix VE of the second part of the Chinese pharmacopoeia 2015 edition).
The chromatographic column is HP-5MS type quartz capillary chromatographic column (30m × 0.25mm × 0.25, um), the column flow is 1.0mL/min, the carrier gas is high-purity helium (99.999%), and the column front pressure is 7.65 psi. Temperature programming, namely, the initial temperature is 130 ℃, the temperature is raised to 150 ℃ at the rate of 1.5 ℃ per minute, the temperature is raised to 165 ℃ at the rate of 1 ℃ per minute, the temperature of an injection port is 280 ℃, and the split ratio is 20: 1. The ion source is an EI source, the temperature of the ion source is 230 ℃, the temperature of the quadrupole rod is 150 ℃, the electron energy is 70eV, the emission current is 34.6uA, and the mass range is 20-800 amu.
And (3) retrieving a Nist standard mass spectrum gallery through an Agilent 5975C MSD chemical workstation, and simultaneously analyzing by combining related mass spectrum literature to identify chemical components in the mixed oil. And calculating the peak area relative percentage content of each chemical component according to a peak area normalization method by using an Agilent 5975C MSD chemical workstation data processing system.
The mixture of Chinese medicinal oil and recovered oil contains 15.33 wt% terpenoids, wherein 33.56 wt% monoterpene and 30.29 wt% sesquiterpene; contains eugenol 3.21 wt%, and flavonoids 7.56 wt%, wherein penoniflorin accounts for 33.25 wt%. Contains 22.54 wt% of polyphenol compounds, wherein resveratrol accounts for 56.88 wt%;
example 2: the pure traditional Chinese medicine oil for relaxing tendons and activating collaterals is prepared by the following method:
firstly, preparation of raw materials
The entrainer is absolute ethyl alcohol;
the essential oil is wormwood essential oil: safflower essential oil: the wintergreen essential oil is prepared according to the weight ratio of 1:3: 1;
rhizoma Acori Graminei, Notoginseng radix, rhizoma Ligustici Chuanxiong, flos Caryophylli, cortex moutan, herba Salviae chinensis, Curcumae rhizoma, rhizoma Polygoni Cuspidati, folium Artemisiae Argyi, radix Paeoniae Rubra, herba Moslae, fructus Anisi Stellati, herba Agastaches, herba Andrographitis, and Moschus are dried or Chinese medicinal decoction pieces.
Preparation of pure Chinese medicine oil for relaxing muscles and tendons and activating the flow of energy
(1) Weighing the raw materials in parts by weight as follows: 40 parts of grassleaf sweelflag rhizome, 20 parts of pseudo-ginseng, 50 parts of szechuan lovage rhizome, 35 parts of clove, 50 parts of tree peony bark, 60 parts of Chinese sage herb, 18 parts of zedoary, 80 parts of giant knotweed rhizome, 35 parts of wormwood, 90 parts of red paeony root, 40 parts of Chinese mosla herb, 20 parts of Chinese anise, 15 parts of patchouli, 45 parts of common andrographis herb and 30 parts of musk are crushed and then sieved by a 100-mesh sieve, 80 percent ethanol water solution with the volume being 3 times of that of the crushed raw materials is soaked for 48 hours, the filtered solution is filtered by filter paper, the filtered solution is placed on a rotary evaporator for reduced pressure distillation at 35 ℃ to remove ethanol in the solution to obtain extract water solution, the extract water solution is repeatedly extracted by petroleum ether for 3 times, each time is 100mL, the extract water solution is collected into a conical flask, taking out the lower layer water solution, repeatedly extracting with ethyl acetate for 3 times, each time 100mL, collecting in a conical flask, taking out the lower layer water solution, repeatedly extracting with n-butanol for 3 times, collecting into a conical flask, and collecting the rest part into the conical flask;
(2) after suction filtration, the raw material powder is put into CO2In a supercritical extraction kettle, extracting at 60 deg.C under 30MPa with 10wt% entrainer and CO2The flow rate is 30kg/h, dynamic extraction is carried out firstly, the extraction time is 1.2h, then static extraction is carried out, the extraction time is 0.3h, and the dynamic extraction time is as follows: static extraction time is 4:1, the extract passes through a 0.22 mu m filter membrane, filtrate is extracted by equal volume of normal hexane in a separating funnel, the normal hexane extract is mixed with the petroleum ether extract, the ethyl acetate extract and the normal butanol extract in the step (1), and a rotary evaporator is used for removing the organic extractant at 60 ℃ to obtain traditional Chinese medicine oil;
(3) carrying out co-hydro distillation on the dregs of the extraction of the crude drug raw materials according to the weight ratio of the raw materials to the water of 1:60, continuously adding distilled water in the distillation process to supplement distillate, distilling for 3 hours, and carrying out ultrasonic treatment simultaneously by the co-hydro distillation, wherein the specific steps are as follows: ultrasonic treatment with 45kHz frequency is carried out 15min after the distillation is started, the operation is carried out for 5min, the operation is stopped for 10min to serve as a treatment period, and 10 periods are continuously treated. Collecting 500mL of distillate, extracting with equal volume of anhydrous ether for 1h, separating, collecting ether, adding anhydrous sodium sulfate, drying, recovering solvent from the ether at 50 deg.C in water bath, and collecting crude extract;
(4) preparing a chromatographic column by using a 250-mesh silica gel packed column, packing the column according to a diameter-height ratio of 1:16, weighing a crude extract, dissolving the crude extract in an eluent according to a weight ratio of 1:3, loading the sample at room temperature, wherein the loading amount is 1:200 of the mass ratio of the crude extract to the silica gel, eluting by using the eluent as a mobile phase, collecting the eluent by using 30mL as a unit at the eluent flow rate of 3.8mL/min, carrying out sample application tracking analysis by using thin layer chromatography while eluting, collecting fractions according to the sample application condition, collecting effective fractions, and then volatilizing a solvent at room temperature under reduced pressure to obtain recovered oil; the eluent is prepared from ethyl acetate and petroleum ether according to the volume ratio of 1: 20;
(5) and (3) recovering oil: traditional Chinese medicine oil: mixing the essential oils at a weight ratio of 3:10:5, adding 25wt% of activated carbon for decolorization for 7d, centrifuging at 10000rpm for 30min, and collecting supernatant to obtain the pure traditional Chinese medicine oil for relaxing muscles and tendons and activating collaterals.
The recovered oil and the Chinese medicinal oil are mixed in proportion, and then the components are measured according to a gas chromatography (appendix VE of the second part of the Chinese pharmacopoeia 2015 edition).
The chromatographic column was an HP-5MS type quartz capillary chromatographic column (30 m. times.0.25 mm. times.0.25 μm), the column flow was 1.0mL/min, the carrier gas was high purity helium (99.999%), and the column front pressure was 7.65 psi. Temperature programming, namely, the initial temperature is 130 ℃, the temperature is raised to 150 ℃ at the rate of 1.5 ℃ per minute, the temperature is raised to 165 ℃ at the rate of 1 ℃ per minute, the temperature of an injection port is 280 ℃, and the split ratio is 20: 1. The ion source is an EI source, the temperature of the ion source is 230 ℃, the temperature of the quadrupole rod is 150 ℃, the electron energy is 70eV, the emission current is 34.6uA, and the mass range is 20-800 amu.
And (3) retrieving a Nist standard mass spectrum gallery through an Agilent 5975C MSD chemical workstation, and simultaneously analyzing by combining related mass spectrum literature to identify chemical components in the mixed oil. And calculating the peak area relative percentage content of each chemical component according to a peak area normalization method by using an Agilent 5975C MSD chemical workstation data processing system.
The mixture of Chinese medicinal oil and recovered oil contains 24.57wt% terpenoids, wherein 45.27wt% is monoterpene and 43.11wt% is sesquiterpene; contains eugenol 5.34 wt%; contains flavonoids 11.23wt%, wherein paeoniflorin accounts for 50.34 wt%; contains 31.25wt% of polyphenol compounds, wherein the resveratrol accounts for 74.53 wt%;
example 3: the pure traditional Chinese medicine oil for relaxing tendons and activating collaterals is prepared by the following method:
firstly, preparation of raw materials
The entrainer is absolute ethyl alcohol;
the essential oil is rose essential oil: lavender essential oil: peppermint oil: the camphor essential oil is prepared according to the weight ratio of 1:1:3: 2;
rhizoma Acori Graminei, Notoginseng radix, rhizoma Ligustici Chuanxiong, flos Caryophylli, cortex moutan, herba Salviae chinensis, Curcumae rhizoma, rhizoma Polygoni Cuspidati, folium Artemisiae Argyi, radix Paeoniae Rubra, herba Moslae, fructus Anisi Stellati, herba Agastaches, herba Andrographitis, and Moschus are dried or Chinese medicinal decoction pieces.
Preparation of pure Chinese medicine oil for relaxing muscles and tendons and activating the flow of energy
(1) Weighing the raw materials in parts by weight as follows: 30 parts of grassleaf sweelflag rhizome, 18 parts of pseudo-ginseng, 43 parts of szechuan lovage rhizome, 30 parts of clove, 42 parts of tree peony bark, 45 parts of Chinese sage herb, 14 parts of zedoary, 65 parts of giant knotweed rhizome, 27 parts of wormwood, 75 parts of red paeony root, 30 parts of Chinese mosla herb, 15 parts of Chinese anise, 13 parts of patchouli, 30 parts of common andrographis herb and 20 parts of musk are crushed and sieved by a 90-mesh sieve, 2 times of volume of 70 percent ethanol water solution is soaked for 36 hours, the filtrate is filtered by filter paper, the filtrate is placed on a rotary evaporator for reduced pressure distillation at 35 ℃ to remove ethanol in the solution to obtain extract water solution, the extract water solution is repeatedly extracted by petroleum ether for 3 times, each time is 100mL, the extract water solution is collected into a conical flask, taking out the lower layer water solution, repeatedly extracting with ethyl acetate for 3 times, each time 100mL, collecting in a conical flask, taking out the lower layer water solution, repeatedly extracting with n-butanol for 3 times, collecting into a conical flask, and collecting the rest part into the conical flask;
(2) after suction filtration, the raw material powder is put into CO2In a supercritical extraction kettle, extracting at 50 ℃ under 16MPa and 8 wt% of entrainer and CO2The flow rate is 20kg/h, dynamic extraction is carried out firstly, the extraction time is 45min, then static extraction is carried out, the extraction time is 15min, and the dynamic extraction time is as follows: static extraction time is 3:1, the extract passes through a 0.22 mu m filter membrane, the filtrate is extracted by n-hexane with 0.5 volume times in a separating funnel, the n-hexane extract is mixed with the petroleum ether extract, the ethyl acetate extract and the n-butanol extract in the step (1), and the mixture is subjected to rotary evaporation at 60 DEG CConcentrating to remove organic extractant to obtain Chinese medicinal oil;
(3) carrying out co-hydro distillation on the dregs of the extraction of the crude drug raw materials according to the weight ratio of the raw materials to the water of 1:35, continuously adding distilled water in the distillation process to supplement distillate, distilling for 2 hours, and carrying out ultrasonic treatment simultaneously by the co-hydro distillation, wherein the specific steps are as follows: ultrasonic treatment with 35kHz frequency is carried out 15min after the distillation is started, the operation is carried out for 5min, the operation is stopped for 10min to serve as a treatment period, and 6 periods are continuously treated. Collecting 275mL of distillate, extracting with equal volume of anhydrous ether for 1h, separating liquid, collecting ether, adding anhydrous sodium sulfate, drying, recovering solvent from the extraction ether at 50 ℃ in water bath, and collecting crude extract;
(4) preparing a chromatographic column by using 200-mesh silica gel packed column, packing the column according to the diameter-height ratio of 1:12, weighing a crude extract, dissolving the crude extract in an eluent according to the weight ratio of 1:3, loading the sample at room temperature, wherein the loading amount is 1:125 of the mass ratio of the crude extract to the silica gel, eluting by using the eluent as a mobile phase, collecting the eluent by using 20mL as a unit at the eluent flow rate of 2.4mL/min, carrying out sample application tracking analysis by using thin layer chromatography while eluting, collecting the fractions according to the sample application condition, collecting the effective fractions, and then decompressing and volatilizing the solvent at room temperature to obtain recovered oil; the eluent is prepared from ethyl acetate and petroleum ether according to the volume ratio of 1: 15;
(5) and (3) recovering oil: traditional Chinese medicine oil: mixing the essential oils at a weight ratio of 2:7.5:5, adding 20 wt% of activated carbon for decolorization for 4d, centrifuging at 10000rpm for 30min, and collecting supernatant to obtain the pure traditional Chinese medicine oil with effects of relaxing muscles and tendons and activating collaterals.
The recovered oil and the Chinese medicinal oil are mixed in proportion, and then the components are measured according to a gas chromatography (appendix VE of the second part of the Chinese pharmacopoeia 2015 edition).
The chromatographic column was an HP-5MS type quartz capillary chromatographic column (30 m. times.0.25 mm. times.0.25 μm), the column flow was 1.0mL/min, the carrier gas was high purity helium (99.999%), and the column front pressure was 7.65 psi. Temperature programming, namely, the initial temperature is 130 ℃, the temperature is raised to 150 ℃ at the rate of 1.5 ℃ per minute, the temperature is raised to 165 ℃ at the rate of 1 ℃ per minute, the temperature of an injection port is 280 ℃, and the split ratio is 20: 1. The ion source is an EI source, the temperature of the ion source is 230 ℃, the temperature of the quadrupole rod is 150 ℃, the electron energy is 70eV, the emission current is 34.6uA, and the mass range is 20-800 amu.
And (3) retrieving a Nist standard mass spectrum gallery through an Agilent 5975C MSD chemical workstation, and simultaneously analyzing by combining related mass spectrum literature to identify chemical components in the mixed oil. And calculating the peak area relative percentage content of each chemical component according to a peak area normalization method by using an Agilent 5975C MSD chemical workstation data processing system.
The mixture of Chinese medicinal oil and recovered oil contains 20.22 wt% terpenoids, 39.11 wt% monoterpene and 36.54 wt% sesquiterpene; 4.28 wt% eugenol; contains 9.39 wt% of flavonoids, wherein paeoniflorin accounts for 42.08 wt%; contains 25.89 wt% of polyphenol compounds, wherein resveratrol accounts for 66.34 wt%;
example 4: the rest was the same as example 3 except that the co-water distillation was not carried out with ultrasonic treatment. The entrainer was analytically pure methanol.
The mixture of Chinese medicinal oil and recovered oil contains terpenoids 14.43 wt%, wherein monoterpene 22.17 wt% and sesquiterpene 25.14 wt%; 3.28 wt% eugenol; contains 8.26 wt% of flavonoids, wherein paeoniflorin accounts for 43.25 wt%; contains 12.66 wt% of polyphenol compounds, wherein resveratrol accounts for 47.28 wt%;
comparative example 1: the rest is the same as example 3, except that only the traditional Chinese medicine oil and the essential oil are mixed according to the weight ratio of 9.5: 5, compounding.
The Chinese medicinal oil contains terpenoids 10.16 wt%, wherein monoterpene 18.45 wt% and sesquiterpene 16.29 wt%; 1.11 wt% eugenol; contains flavonoids 3.98 wt%, wherein paeoniflorin accounts for 22.19 wt%; contains 13.26 wt% of polyphenol compounds, wherein resveratrol accounts for 33.98 wt%;
comparative example 2: plant essential oil was prepared according to the method disclosed in chinese patent CN103393854B as a control in subsequent performance experiments.
Gas chromatography component detection results show that, compared with example 3, the content of active ingredients of main drugs such as polyphenols, flavonoids and terpenoids is significantly reduced, the ratio of paeoniflorin, eugenol, terpenoids and resveratrol is also significantly reduced, and the reduction range is larger than that of polyphenols, flavonoids and terpenoids, as compared with example 1, the carbon dioxide supercritical extraction has a low extraction rate of paeoniflorin, eugenol, terpenoids and resveratrol, when subsequent preparation of recovered oil is not performed, a large amount of paeoniflorin and other substances exist in the herb residue and are not recovered, the ratio of other substances in the supercritical extraction is high, and the content of the herb residue is low, so that the ratio of paeoniflorin, eugenol, terpenoids and resveratrol is significantly reduced. Example 4 does not adopt ultrasonic auxiliary treatment, the extraction rate of each substance is also obviously lower than that of examples 1-3, which shows that ultrasonic treatment obviously helps the dissolution and extraction of target substances and can obviously improve the extraction amount of active substances such as polyphenols, flavonoids, terpenoids and the like.
The following experiments were carried out on the collateral-activating oils or essential oils prepared in examples 1 to 3 and comparative examples 1 to 2:
1. experiment for mouse auricle swelling caused by xylene
The test animals were: KM mice, clean grade, 18-20g, male.
The experimental method comprises the following steps: the mice were randomly grouped into groups of 10 mice each, namely a negative control group, a positive control group, examples 1-3 groups, and control examples 1-2 groups. The left ear of the negative control group mouse is evenly smeared with 0 mu L of soybean oil on both sides; the positive control group (dexamethasone) is evenly coated with 10 mu L of Hongli Huoluo oil on both sides of the left ear, and the examples 1-3 and the control examples 1-2 are respectively evenly coated with 0uL of the tested drug on both sides of the left ear of the mouse.
The experimental steps are as follows:
(1) uniformly smearing corresponding medicines on both sides of the left ear of each experimental group of mice according to the required dosage;
(2) after administration for 90min, wiping off the reagent applied to the left ear with cotton ball, and uniformly applying xylene (50u L/ear) to both sides of the left ear to cause inflammation;
(3) after 30min of inflammation, the mice were sacrificed, both ears were cut off, the ears were punched out at the same positions of the mouse ears with a 7mm punch, weighed, and recorded.
Figure BDA0002242062980000091
Figure BDA0002242062980000092
The results are shown in Table 1:
TABLE 1 anti-inflammatory effect of collateral-activating oil
Figure BDA0002242062980000093
Figure BDA0002242062980000101
2. Analgesic effect experiment of collateral activating oil
The test animals were: ICR mice, clean grade, 18-20g, female
The experimental method comprises the following steps: the initial pain threshold of the mouse is tested at the temperature of 55 +/-0.5 ℃ (the after-licking time of the mouse is used as the pain threshold), the mouse with the pain threshold of 5-30s is selected as qualified, and the mice with the pain threshold of more than 30s and less than 5s jump within 5-30s are discarded without use.
The mice with qualified initial pain threshold are randomly grouped, and each group comprises 10 mice, namely a negative control group, a positive control group (Honglihuoluo oil), examples 1-3 groups and control examples 1-2 groups. The experimental steps are as follows:
(1) according to grouping, respectively and uniformly smearing medicines on four paws of a mouse in an experimental group;
(2) respectively measuring the pain threshold of each group of mice (the time of the mice licking the expected afterpaw is the pain threshold) at the time of administration for 60min and 120min, and recording the pain threshold for 60s when the pain threshold exceeds 60 s;
(3) the inhibition rate was calculated as follows:
Figure BDA0002242062980000102
the results are shown in Table 2:
TABLE 2 hot plate analgesic test results for mice with collateral flow activating oil
Group of Number only Time to threshold(s) Inhibition ratio (%)
Blank control group 10 26.3±5.4 ------
Positive control group 10 32.1±6.1 22.05
Example 1 10 34.3±5.1 30.41
Example 2 10 33.2±4.9 26.23
Example 3 10 33.9±3.7 28.89
Comparative example 1 10 29.9±7.2 13.68
Comparative example 2 10 31.8±4.7 20.91
3. Acetic acid writhing analgesia experiment
Experimental animals and experimental groups are experiments on the analgesic effect of the collateral-activating oil, and the experimental steps are as follows:
(1) shaving the abdomen of the mouse with a razor to leave an exposed area of skin of about 2cm X2 cm (without damaging the skin of the mouse);
(2) uniformly smearing corresponding medicines to the abdominal skin bare area of the experimental mouse according to groups, and continuously administering for three days;
(3) after the last administration for 1 hour, 0.2 mL/mouse is injected with 0.6% glacial acetic acid water solution into the abdominal cavity, and the times of body twisting of the mouse within 20min are recorded;
(4) the inhibition rate was calculated as follows:
Figure BDA0002242062980000111
the results are shown in Table 3:
TABLE 3 Experimental results of pain relief of collateral flow oil to acetic acid writhing of mice
Group of Number only Number of twisting bodies Inhibition ratio (%)
Blank control group 10 21.3±3.3 ------
Positive control group 10 11.1±2.3 47.88
Example 1 10 12.3±1.8 42.25
Example 2 10 10.5±2.8 50.70
Example 3 10 13.2±2.2 38.03
Comparative example 1 10 16.4±3.0 23.00
Comparative example 2 10 15.9±5.3 25.35
The results in tables 1-3 show that the traditional Chinese medicine composition has excellent analgesic and anti-inflammatory effects, the drug effect is remarkably better than that of the control example, and the traditional Chinese medicine composition is equivalent to positive anti-inflammatory and analgesic drugs. By combining the content of effective active substances, the drug effect is in positive correlation with the content of compounds such as paeoniflorin, monoterpene, sesquiterpene, resveratrol and the like, and conforms to the theory of anti-inflammatory and analgesic of traditional Chinese medicines.
In conclusion, a plurality of Chinese herbal medicines with analgesic, blood-activating and anti-inflammatory effects are adopted as raw materials, wherein clove, wormwood, elsholtzia, star anise, patchouli, common andrographis herb and musk are rich in monoterpene, sesquiterpene and other volatile terpenoids, blood circulation can be promoted, pain can be relieved, blood circulation can be promoted, blood stasis can be promoted, ligusticum wallichii and polygonum cuspidatum are rich in ligustrazine, resveratrol and other compounds with anti-inflammatory and analgesic effects, red peony root and cortex moutan are rich in paeoniflorin and other anti-inflammatory and analgesic compounds, and pseudo-ginseng, rhizoma acori graminei, salvia chinensis and curcuma zedoary are used as guiding medicines, so that medicine absorption can be remarkably improved, medicine toxicity can be reduced, and analgesic, blood-activating and post-pain repairing effects of the traditional Chinese medicine formula can be remarkably improved. By means of carbon dioxide supercritical extraction, extraction of different substances is effectively improved, technological parameters such as extraction pressure, extraction temperature and the like of supercritical extraction are optimized, the extraction rate and activity of most of traditional Chinese medicine active substances are remarkably improved, and the defects that the extraction rate of complex traditional Chinese medicine components is low, the active substances are easy to thermally inactivate and oxidize in conventional extraction methods such as thermal reflux and the like are overcome. The terpenoids, flavonoids such as paeoniflorin and the like, and polyphenol compounds such as resveratrol and the like in the active ingredients are compounds containing a plurality of carbonyl groups and hydroxyl groups, the substances are difficult to extract by carbon dioxide supercritical extraction, the extraction rate is low, so that the active substances in the dregs after supercritical extraction can be extracted, separated and extracted in a targeted manner by assisting in extraction of a plurality of organic solvents and purification of silica gel columns, the defect of low extraction rate of individual effective active substances by supercritical extraction is overcome, the extraction rate and activity of the total terpenoids, the total flavonoids and the total polyphenols are obviously higher than those of the extraction by a single method due to mutual matching of conventional thermal reflux extraction and supercritical extraction, the main active ingredients of the medicine are extracted to the maximum, and the utilization rate of crude drug raw materials is improved.
The above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby, and any modification made on the basis of the technical scheme according to the technical idea proposed by the present invention falls within the protection scope of the present invention; the technology not related to the invention can be realized by the prior art.

Claims (4)

1. A pure traditional Chinese medicine oil for relaxing tendons and activating collaterals is characterized in that: the feed is prepared from the following raw materials in parts by weight: 20-40 parts of grassleaf sweelflag rhizome, 15-20 parts of pseudo-ginseng, 35-50 parts of ligusticum wallichii, 25-35 parts of clove, 35-50 parts of cortex moutan, 30-60 parts of Chinese sage herb, 10-18 parts of curcuma zedoary, 50-80 parts of polygonum cuspidatum, 20-35 parts of wormwood, 60-90 parts of red paeony root, 20-40 parts of Chinese mosla herb, 10-20 parts of Chinese anise, 10-15 parts of cablin potchouli herb, 15-45 parts of common andrographis herb, 10-30 parts of musk and 50-100 parts of essential oil;
the pure traditional Chinese medicine oil for relaxing tendons and activating collaterals consists of a crude drug extract and essential oil, wherein the crude drug extract consists of recovered oil and traditional Chinese medicine oil, and the recovered oil comprises the following components in parts by weight: traditional Chinese medicine oil: the weight ratio of the essential oil is 1-3: 5-10: 5; the crude drug extract contains 15.33-24.57 wt% of terpenoids, 3.21-5.34 wt% of eugenol, 7.56-11.23 wt% of flavonoids and 22.54-31.25 wt% of polyphenols;
the pure traditional Chinese medicine oil for relaxing tendons and activating collaterals is prepared by the following specific steps:
(1) crushing raw materials of crude drugs except essential oil, sieving the raw materials with a sieve of 80-100 meshes, soaking the raw materials in 60-80% ethanol aqueous solution with the volume of 1-3 times of that of the raw materials for 24-48 h, carrying out suction filtration, filtering the filtrate with filter paper, placing the filtrate on a rotary evaporator for reduced pressure distillation at 35 ℃, removing ethanol in the solution to obtain extract aqueous solution, repeatedly extracting the extract aqueous solution with petroleum ether for 3 times (100 mL each time), collecting the extract aqueous solution in a conical flask, taking out the lower-layer aqueous solution, repeatedly extracting with ethyl acetate for 3 times (100 mL each time), collecting the extract aqueous solution in the conical flask, taking out the lower-layer aqueous solution, repeatedly extracting with n-butyl alcohol for 3 times, collecting the extract aqueous solution in the conical flask, and collecting the rest part in the conical flask;
(2) filtering the solution to obtain a filtrateCharging CO into the powder2In a supercritical extraction kettle, according to the extraction temperature of 40-60 ℃, the extraction pressure of 2-30 MPa, 5-10 wt% of entrainer and CO2Extracting at the flow rate of 10-30 kg/h for 0.5-1.5 h, filtering the extract with a 0.22-micron filter membrane, extracting the filtrate with n-hexane in a separating funnel, mixing the n-hexane extract with the petroleum ether extract, the ethyl acetate extract and the n-butanol extract in the step (1), and concentrating at 60 ℃ in a rotary evaporator to remove the organic extractant to obtain traditional Chinese medicine oil;
(3) carrying out co-water distillation on the dregs of the extraction of the crude drug raw materials according to the weight ratio of the raw materials to water of 1: 10-60, continuously adding distilled water in the distillation process to supplement distillate, distilling for 1-3 h, collecting the distillate by taking 50-500 mL as a unit, extracting for 1h by using equal volume of anhydrous ether after collecting, then separating the liquid and collecting ether, adding anhydrous sodium sulfate for drying, recovering the solvent by using ether for extraction at 50 ℃ in water bath, and collecting a crude extract;
(4) preparing a chromatographic column by using a 150-plus-250-mesh silica gel packed column, packing the column according to a diameter-height ratio of 1: 8-16, weighing a crude extract, dissolving the crude extract in an eluent according to a weight ratio of 1:3, loading the sample at room temperature, eluting by using the eluent as a mobile phase, collecting the eluent by using 10-30 mL as a unit at an eluent flow rate of 1.0-3.8 mL/min, carrying out sample application tracking analysis by using thin-layer chromatography while eluting, collecting fractions according to sample application conditions, and volatilizing a solvent at room temperature under reduced pressure to obtain recovered oil after collecting effective fractions; the eluent is prepared from ethyl acetate and petroleum ether according to the volume ratio of 1: 10-20;
(5) and (3) recovering oil: traditional Chinese medicine oil: mixing the essential oils according to the weight ratio of 1-3: 5-10: 5, adding 15-25 wt% of activated carbon for decoloring for 1-7 d, centrifuging at 10000rpm for 30min, and taking supernatant to obtain pure traditional Chinese medicine oil for relaxing tendons and activating collaterals;
the entrainer is absolute ethyl alcohol; the step (3) also comprises ultrasonic treatment during co-water distillation, and the specific steps are as follows: carrying out ultrasonic treatment with the frequency of 25-45 kHz 15min after the distillation is started, taking the operation of 5min and the stop of 10min as a treatment period, and continuously treating for 3-10 periods.
2. The pure traditional Chinese medicine oil for relaxing tendons and activating collaterals as claimed in claim 1, is characterized in that: the terpenoids comprise 33.56-45.27 wt% of monoterpenes and 30.29-43.11 wt% of sesquiterpenes; the polyphenol compounds comprise 56.88-74.53 wt% of resveratrol; the flavonoid compound comprises 33.25-50.34 wt% of paeoniflorin.
3. The pure traditional Chinese medicine oil for relaxing tendons and activating collaterals as claimed in claim 1, is characterized in that: the essential oil comprises one or more of wormwood essential oil, rose essential oil, lavender essential oil, safflower essential oil, peppermint oil, camphor essential oil, wintergreen essential oil and capsicum essential oil; the rhizoma Acori Graminei, Notoginseng radix, rhizoma Ligustici Chuanxiong, flos Caryophylli, cortex moutan, herba Salviae chinensis, Curcumae rhizoma, rhizoma Polygoni Cuspidati, folium Artemisiae Argyi, radix Paeoniae Rubra, herba Moslae, fructus Anisi Stellati, herba Agastaches, herba Andrographitis, and Moschus are dried products or Chinese medicinal decoction pieces.
4. The pure traditional Chinese medicine oil for relaxing tendons and activating collaterals as claimed in claim 3, is characterized in that: the CO is2The extraction time in the supercritical extraction is composed of dynamic extraction time and static extraction time, wherein the dynamic extraction time is as follows: the static extraction time = 2-4: 1, and the mass ratio of the crude extract to the silica gel in the step (4) is 1: 45-200.
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CN101838253A (en) * 2009-03-20 2010-09-22 马良伟 Preparation method of ligustilide and butylidene phthalide
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