CN113197934B - Lindera aggregata leaf extract and preparation method and application thereof - Google Patents
Lindera aggregata leaf extract and preparation method and application thereof Download PDFInfo
- Publication number
- CN113197934B CN113197934B CN202110406430.3A CN202110406430A CN113197934B CN 113197934 B CN113197934 B CN 113197934B CN 202110406430 A CN202110406430 A CN 202110406430A CN 113197934 B CN113197934 B CN 113197934B
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- extraction
- lindera
- extract
- volatile oil
- leaf
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Abstract
The invention discloses a lindera aggregata leaf extract and a preparation method and application thereof, wherein lindera aggregata leaf is taken as a raw material, volatile oil is prepared by supercritical carbon dioxide extraction, flavone and alkaloid are extracted from extracted residues by ethanol reflux, and the flavone and the alkaloid are simultaneously refined by a macroporous resin composition consisting of D101 and NKA-9, so that lindera aggregata leaf volatile oil, a mixed extract of the flavone and the alkaloid and a mixture of the extracted extract and the mixed extract are obtained. Pharmacological research shows that the total alkaloids, the total flavonoids, the volatile oil and the mixture of the total flavonoids and the volatile oil have the activities of promoting blood circulation and relieving pain, and the activities are all higher than those of a direct water extract. The lindera aggregata leaf extract disclosed by the invention is simple in preparation process route, strong in quality controllability, high in extraction rate and suitable for large-scale production. The lindera aggregata leaf extract has excellent effects of promoting blood circulation and relieving pain, and can be applied to preparation of medicines or health-care foods with related effects.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a lindera aggregata leaf extract and a preparation method and application thereof.
Background
The combined spicebush root is one of new Zhejiang eight-ingredient medicinal materials, is a dry root tuber of combined spicebush root of Lauraceae plant, is originally recorded in Bencao Shiyi of Tang Dynasty, and has the effects of promoting qi circulation, relieving pain, warming kidney, dispelling cold and the like. It is mainly used for treating cold congealing and qi stagnation, chest and abdomen distending pain, adverse qi, dyspnea, bladder deficiency cold, enuresis, pollakisuria, hernia pain, cold channel, abdominal pain, etc. Radix Linderae tuberous root is rich in sesquiterpene, lactone, alkaloid, etc., and research shows that the main component of radix Linderae for promoting qi circulation and relieving pain is total alkaloids, but the components are complex and the content is low. The lindera aggregate leaf mainly contains flavonoids, alkaloids and volatile oil. Modern pharmacology mainly focuses on the research of the total flavonoids of the lindera aggregate leaves, has pharmacological effects of resisting oxidation, treating cardiovascular diseases and the like, relatively few researches on volatile oil and alkaloid components with low content, and needs to be further researched to improve the additional value of the full resource development and utilization of the lindera aggregate leaves.
Various components such as alkaloid, flavone and volatile oil in the lindera aggregate leaf are related to the activity of activating blood and relieving pain, if simple direct extraction or reflux extraction is adopted to produce simple alcohol extract or water extraction, the technical content is not high, the industrial upgrading of the added value is difficult to realize, and various functional components contained in the lindera aggregate leaf cannot be effectively extracted, so that the great waste of raw materials and functional component resources is caused. When a single component is extracted, the extraction conditions destroy other components or cause low extraction rate of other components, low purity and complicated subsequent treatment. Therefore, the method for continuously extracting multiple active ingredients of the combined spicebush root is developed by comprehensively considering the mutual influence, activity and extraction rate of various ingredients, and the utilization value of the active ingredients in the combined spicebush root leaves is developed to the maximum extent.
Disclosure of Invention
The invention aims to solve the technical defects and provide the lindera aggregata leaf extract which can continuously extract a plurality of active ingredients of the lindera aggregata and furthest develop the utilization value of the active ingredients with the functions of promoting blood circulation and relieving pain, as well as the preparation method and the application thereof.
In order to achieve the purpose, the preparation method of the lindera aggregata leaf extract is characterized by comprising the following steps: the method comprises the following steps:
drying and crushing lindera aggregate leaves at 30-40 ℃, then placing the crushed lindera aggregate leaves in an extraction kettle in a supercritical extraction device, and performing carbon dioxide supercritical extraction on the dried and crushed lindera aggregate leaves in the supercritical extraction device by adopting carbon dioxide as a solvent: firstly, cooling introduced carbon dioxide through a condenser until the carbon dioxide is in a liquid state, pumping the carbon dioxide into an extraction kettle through a high-pressure pump, heating and pressurizing the extraction kettle in a supercritical extraction device to enable the carbon dioxide to be in a supercritical state for extraction, controlling the flow rate of the carbon dioxide to be 30L per hour during extraction, and performing circulating extraction; simultaneously keeping the extraction pressure at 22 +/-5 MPa, the extraction temperature at 40 +/-5 ℃ and the extraction time at 2-3 hours, carrying out two-stage separation in a separation kettle, wherein the first-stage separation pressure is 4 +/-0.5 MPa, the separation temperature is 40 +/-5 ℃, the second-stage separation pressure is 4 +/-0.5M MPa and the separation temperature is 35 +/-5 ℃, thus obtaining a yellow-green viscous extract, removing moisture in the extract to obtain the lindera aggregate leaf volatile oil, precisely weighing, and calculating the yield of the extract; drying and crushing lindera aggregate leaves at 30-40 ℃, wherein the crushing degree can be selected from 20-100 mesh sieve and then carrying out subsequent extraction;
step two, adding 80% ethanol containing 0.1% HCl into residue obtained after supercritical extraction of lindera aggregata leaf volatile oil according to the material-liquid ratio of 1: 10-15 g/mL, carrying out reflux extraction for 1 hour to 2 hours at the temperature of 60 ℃, carrying out extraction for 2 times to 3 times by the same method, combining filtrates, adjusting the pH to 7.0 by using NaOH solution, filtering, collecting the filtrate, concentrating volatile ethanol through rotary evaporation, and dissolving with distilled water to obtain a crude extract;
and step three, sampling the crude extraction liquid obtained in the step two at the flow rate of 2BV/h, wherein the concentration of the sampling liquid is 2mg/mL, the pH of the sampling is 6.0, adding the sampling liquid to a macroporous resin column, respectively washing the sampling liquid for 1 time by using 2BV water and 1BV 20% ethanol, respectively eluting the sampling liquid by using 3BV 60% ethanol and 2BV 80% ethanol containing 2% ammonia water at the flow rate of 2BV/h, collecting and combining the eluates, and obtaining the flavone and alkaloid mixed extract. Here, BV is the bed volume unit and h is the hour.
In order to effectively collect and extract the extracted flavone and alkaloid mixed extract, the resin adopted in the macroporous resin column in the third step is a macroporous resin composition consisting of D101 and NKA-9, and the mass ratio of D101 to NKA-9 is 7-8: 3-2.
The lindera aggregata leaf extract prepared by the method is a flavone and alkaloid mixed extract obtained by extracting lindera aggregata leaf volatile oil obtained by carbon dioxide supercritical extraction of lindera aggregata leaves or extracting residues obtained after the carbon dioxide supercritical extraction of the lindera aggregata leaf volatile oil with ethanol or a mixture of the flavone and alkaloid mixed extract.
The mixture of the lindera aggregata leaf volatile oil and the mixed extract of the flavone and the alkaloid can be obtained by emulsifying the lindera aggregata leaf volatile oil obtained in the step 1 and then mixing the extracted volatile oil with the mixed extract of the flavone and the alkaloid obtained in the step three.
The lindera aggregate leaf volatile oil obtained by the method mainly comprises curzerene, lindera aggregate leaf, beta-cineol and beta-cineole; the flavone and alkaloid mixed extract mainly comprises norisoboldine, norboldine boldine and reticuline, Karakoramine higenamine catechin, isoquercitrin and quercitrin.
The invention provides application of the lindera aggregata leaf extract obtained by the method, which is mainly used for promoting blood circulation and easing pain and corresponding health-care food, functional cosmetics and common food.
The lindera aggregata leaf extract and the preparation method and the application thereof have the advantages that: the extract with the activity of promoting blood circulation and relieving pain can be prepared from lindera aggregata leaves, and is a natural product which is natural, plant-derived, non-toxic and low in cost; the blood-activating analgesic activity of the flavone and alkaloid mixed extract, the lindera aggregate leaf volatile oil and the mixture of the two is obviously higher than that of the lindera aggregate leaf water extract prepared by the traditional method, and is similar to that of the prior chemical medicine aspirin; the chemical properties and extraction rates of various components related to the blood circulation promoting and analgesic activities of the lindera aggregate leaf are fully considered, supercritical extraction is adopted to prepare the lindera aggregate leaf volatile oil, the operation temperature is low, and the active ingredients are not easy to damage; the supercritical extraction residue is refined by adopting the macroporous resin composition, and the lindera strychnifolia leaf flavone and alkaloid with high purity and high yield are obtained at the same time, and can be directly used without subsequent purification treatment, so that the production cost can be effectively reduced.
Since lindera aggregate leaf is a new resource food with high safety, the extract prepared from the lindera aggregate leaf is used as a raw material to prepare health food, functional cosmetics, common food and the like for activating blood circulation and relieving pain, and meets the national requirements of restriction and regulation on the raw materials of the health food and the common food, so that the extract prepared by the invention has wide application and development prospects.
The medicinal part of the combined spicebush root is underground root tuber growing for 8 to 10 years, and the harvesting of the medicinal materials is at the cost of the death of the whole plant, so that the market demand can not be met. And a large amount of stems and leaves are generated in a long growth period, most of the stems and leaves are discarded, the resource utilization rate is low, and the method conforms to the idea of realizing resource-saving and environment-friendly circular economy development.
In the theory of traditional Chinese medicine, "postpartum general pain" and "dysmenorrhea" are caused by stagnation of blood stasis in meridians or skin or other viscera, which is likely to cause pain due to stagnation of qi and blood. The patient has no abnormity in laboratory examination, the western medicine has no corresponding disease name, the medicine is mainly used for symptomatic treatment, the symptoms can be temporarily improved by using hormone and antipyretic analgesic, but the defects of easy relapse after stopping the medicine, large side effect and the like exist, the breast-feeding of the patient is influenced, and the compliance is poor. The components which can warm yang and activate blood and warm meridians and free of arthralgia are found from natural medicines or medicinal and edible traditional Chinese medicines, and have a wide application prospect.
The invention takes lindera aggregate leaf as raw material, prepares lindera aggregate leaf volatile oil extraction residue on the basis of supercritical extraction of volatile oil, and then adopts macroporous resin to refine flavone and alkaloid in combination, thereby obtaining the continuous extraction process of lindera aggregate leaf volatile oil, flavone and alkaloid. The invention not only enriches the sources of pure natural blood-activating and pain-relieving functional components, but also develops new efficacy and application value of non-medicinal parts of the combined spicebush root, improves the additional value of development and utilization of combined spicebush root resources, and has very positive practical significance for further development of products in the fields of blood-activating and pain-relieving medicines, health-care foods, functional foods and the like.
Drawings
FIG. 1 is a statistical chart comparing the average total moving distance of zebra fish after being treated by each experimental group with that of a model control group;
FIG. 2 is a statistical chart comparing the average number of the zebra fish tail blood stasis occurrences of each experimental group with the model control group.
Detailed Description
In order to more clearly understand the technical scheme of the invention, the invention is further illustrated by the following embodiments in combination with the accompanying drawings.
Example 1:
this example provides a method for preparing lindera aggregata leaf extract, which comprises the following steps:
step one, drying and crushing lindera aggregate leaves at 30-40 ℃, sieving with a 40-mesh sieve, weighing 1000 g of the crushed lindera aggregate leaves, placing the crushed lindera aggregate leaves in an extraction kettle of a supercritical extraction device, and performing carbon dioxide supercritical extraction on the dried and crushed lindera aggregate leaves in the supercritical extraction device by adopting carbon dioxide as a solvent: firstly, cooling introduced carbon dioxide through a condenser until the carbon dioxide is in a liquid state, pumping the carbon dioxide into an extraction kettle through a high-pressure pump, heating and pressurizing the extraction kettle in a supercritical extraction device to enable the carbon dioxide to be in a supercritical state for extraction, controlling the flow rate of the carbon dioxide to be 30L per hour during extraction, and performing circulating extraction; meanwhile, the extraction pressure is kept to be 17MPa, the extraction temperature is kept to be 35 ℃, the extraction time is kept to be 3 hours, two-stage separation is carried out in a separation kettle, the first-stage separation pressure is 4 +/-0.5 MPa, the separation temperature is 40 +/-5 ℃, the second-stage separation pressure is 4 +/-0.5M MPa, and the separation temperature is 35 +/-5 ℃, so that a yellow-green viscous extract is obtained, water in the extract is removed to obtain lindera aggregate leaf volatile oil, 27 g of lindera aggregate leaf volatile oil (number: WH1) is obtained through precise weighing, the extract yield is calculated to be 2.7%, and the yellow-green viscous extract mainly contains 12.6% of curzerene, 5.06% of lindera aggregate leaf, 4.82% of beta-eucalyptol and 2.18% of beta-eucalyptol through detection;
step two, adding 80% ethanol containing 0.1% HCl into residue obtained after supercritical extraction of lindera aggregata leaf volatile oil according to the material-liquid ratio of 1: 10g/mL, carrying out reflux extraction for 1 hour at the temperature of 60 ℃, extracting for 2 times by the same method, combining filtrates, adjusting the pH to 7.0 by using NaOH solution, filtering, collecting the filtrate, concentrating and volatilizing the ethanol by rotary evaporation, and dissolving by using distilled water to obtain crude extract;
step three, loading the crude extraction liquid obtained in the step two at the flow rate of 2BV/h, wherein the concentration of the loading liquid is 2mg/mL, the pH of the loading liquid is 6.0, adding the crude extraction liquid to a macroporous resin column, respectively washing the column for 1 time by using 2BV water and 1BV 20% ethanol, wherein the macroporous resin column is filled with a macroporous resin composition consisting of D101 and NKA-9, and the mass ratio of the D101 to the NKA-9 is 7: 3; eluting with 3BV of 60% ethanol and 2BV of 80% ethanol containing 2% ammonia water at a flow rate of 2BV/h, collecting the combined eluates, concentrating the eluates by rotary evaporation or adding anhydrous ethanol to obtain 1000 ml of mixed extract of flavone and alkaloid (No. WY 1). Here, BV is the bed volume unit and h is the hour.
The eluent of the mixed extract of flavone and alkaloid obtained by constant volume in the embodiment is tested to mainly comprise 0.0067% of norisoboldine, boldine and reticuline, 3.4518% of Karakoramine higenamine catechin, 0.1113% of isoquercitrin and 0.9705% of quercitrin.
The percentage content of the total flavone of the purified lindera aggregata leaves is 3.8 times of that of the crude extract, and the percentage content of the total alkaloid is 6.5 times of that of the crude extract.
Example 2:
the invention provides a preparation method of lindera aggregata leaf extract with blood circulation promoting and analgesic activities, which comprises the following steps:
step one, drying and crushing lindera aggregate leaves at 30-40 ℃, sieving with a 40-mesh sieve, weighing 1000 g of the crushed lindera aggregate leaves, placing the crushed lindera aggregate leaves in an extraction kettle of a supercritical extraction device, and performing carbon dioxide supercritical extraction on the dried and crushed lindera aggregate leaves in the supercritical extraction device by adopting carbon dioxide as a solvent: firstly, cooling introduced carbon dioxide through a condenser until the carbon dioxide is in a liquid state, pumping the carbon dioxide into an extraction kettle through a high-pressure pump, heating and pressurizing the extraction kettle in a supercritical extraction device to enable the carbon dioxide to be in a supercritical state for extraction, controlling the flow rate of the carbon dioxide to be 30L per hour during extraction, and performing circulating extraction; keeping the extraction pressure at 27MPa, the extraction temperature at 45 ℃ and the extraction time at 2h, performing two-stage separation in a separation kettle, wherein the first-stage separation pressure is 4 +/-0.5 MPa, the separation temperature is 40 +/-5 ℃, the second-stage separation pressure is 4 +/-0.5M MPa, and the separation temperature is 35 +/-5 ℃, obtaining yellow-green viscous lindera aggregate leaf volatile oil, removing moisture in an extract, precisely weighing to obtain 28.8 g of lindera leaf volatile oil (numbered as WH2), and the extraction yield is 2.88%;
the extraction yield of the lindera aggregate leaf volatile oil obtained in the embodiment is 2.85%, and tests show that the lindera aggregate leaf volatile oil mainly comprises 12.8% of curzerene, 5.12% of lindera aggregate leaf, 4.9% of beta-cineol and 2.20% of beta-cineol;
step two, adding 80% ethanol containing 0.1% HCl into residue obtained after supercritical extraction of lindera aggregata leaf volatile oil according to the material-liquid ratio of 1: 15g/mL, extracting for 2 hours at 60 ℃ under reflux, extracting for 3 times by the same method, combining filtrates, adjusting the pH to 7.0 by NaOH solution, filtering, collecting the filtrate, concentrating the volatile ethanol by rotary evaporation, and dissolving by distilled water to obtain crude extract.
Step three, loading the crude extraction liquid obtained in the step two at the flow rate of 2BV/h, wherein the concentration of the loading liquid is 2mg/mL, the pH of the loading liquid is 6.0, adding the crude extraction liquid to a macroporous resin column, respectively washing the column for 1 time by using 2BV water and 1BV 20% ethanol, wherein the macroporous resin column is filled with a macroporous resin composition consisting of D101 and NKA-9, and the mass ratio of the D101 to the NKA-9 is 8: 2; eluting with 3BV of 60% ethanol and 2BV of 80% ethanol containing 2% ammonia water at a flow rate of 2BV/h, collecting the combined eluates, concentrating the eluates by rotary evaporation or adding anhydrous ethanol to obtain 1000 ml of mixed extract of flavone and alkaloid (No. WY 2).
The eluent of the mixed extract of flavone and alkaloid obtained in the embodiment is detected to mainly comprise 0.0071% of norisoboldine, boldine and reticuline, 3.4620% of Karakoramine higenamine catechin, 0.1120% of isoquercitrin and 0.9711% of quercitrin.
Example 3:
the invention provides a preparation method of lindera aggregata leaf extract with blood circulation promoting and analgesic activities, which comprises the following steps:
step one, drying and crushing lindera aggregate leaves at 30-40 ℃, sieving with a 40-mesh sieve, weighing 1000 g of the crushed lindera aggregate leaves, placing the crushed lindera aggregate leaves in an extraction kettle of a supercritical extraction device, and performing carbon dioxide supercritical extraction on the dried and crushed lindera aggregate leaves in the supercritical extraction device by adopting carbon dioxide as a solvent: firstly, cooling introduced carbon dioxide through a condenser until the carbon dioxide is in a liquid state, pumping the carbon dioxide into an extraction kettle through a high-pressure pump, heating and pressurizing the extraction kettle in a supercritical extraction device to enable the carbon dioxide to be in a supercritical state for extraction, controlling the flow rate of the carbon dioxide to be 30L per hour during extraction, and performing circulating extraction; keeping the extraction pressure at 22MPa, the extraction temperature at 40 ℃ and the extraction time at 2.5h, performing two-stage separation in a separation kettle, wherein the first-stage separation pressure is 4 +/-0.5 MPa, the separation temperature is 40 +/-5 ℃, the second-stage separation pressure is 4 +/-0.5M MPa and the separation temperature is 35 +/-5 ℃, obtaining a yellow-green viscous extract, removing water in the extract, precisely weighing to obtain 28.2 g of lindera aggregate leaf volatile oil (numbered as WH3), and obtaining the extraction yield of the lindera aggregate leaf volatile oil of 2.82%;
step two, adding 80% ethanol containing 0.1% HCl into residue obtained after supercritical extraction of lindera aggregata leaf volatile oil according to the material-liquid ratio of 1: 12g/mL, performing reflux extraction at 60 ℃ for 1.5h, extracting for 2 times by the same method, combining filtrates, adjusting the pH to 7.0 by using NaOH solution, filtering, collecting the filtrate, concentrating volatile ethanol by rotary evaporation, and dissolving with distilled water to obtain crude extract;
step three, loading the crude extraction liquid obtained in the step two at the flow rate of 2BV/h, wherein the concentration of the loading liquid is 2mg/mL, the pH of the loading liquid is 6.0, adding the crude extraction liquid to a macroporous resin column, respectively washing the column for 1 time by using 2BV water and 1BV 20% ethanol, wherein the macroporous resin column is filled with a macroporous resin composition consisting of D101 and NKA-9, and the mass ratio of the D101 to the NKA-9 is 7: 2; eluting with 3BV of 60% ethanol and 2BV of 80% ethanol containing 2% ammonia water at a flow rate of 2BV/h, collecting the combined eluates, concentrating the eluates by rotary evaporation or adding anhydrous ethanol to obtain 1000 ml of mixed extract of flavone and alkaloid (No. WY 3).
The mixed extract of flavone and alkaloid obtained in the embodiment is detected to be 0.0068% of norisoboldine, 0.0068% of norboldine boldine and reticuline, 3.4519% of Karakoramine higenamine catechin, 0.1116% of isoquercitrin and 0.9709% of quercitrin.
Example 4:
this example is a method of repeating examples 1, 2 and 3, and mixing the volatile oil of lindera strychnifolia leaves obtained in examples 1, 2 and 3 with the mixed extract of flavones and alkaloids obtained in step three, respectively, to obtain liquid mixtures of the volatile oil of lindera strychnifolia leaves and the mixed extract of flavones and alkaloids, which are respectively numbered as WHY1, WHY2 and WHY3, and the average number thereof is WHY.
The liquid mixture of the lindera aggregata leaf volatile oil and the mixed extract of the flavone and the alkaloid is obtained by adopting polyoxyethylene 40 hydrogenated castor oil as an emulsifier, mixing the lindera aggregata leaf volatile oil obtained in each embodiment with the polyoxyethylene 40 hydrogenated castor oil according to the weight ratio of 1:2 to 2.5, then respectively incorporating the flavone and alkaloid mixed extract with the volume of 1000 ml in each embodiment, and stirring at high speed until the mixture is in a transparent state.
The transparent liquid mixture of the lindera strychnifolia leaf volatile oil and the mixed extract of the flavone and the alkaloid is formed because the oil-phase lindera strychnifolia leaf volatile oil containing various components is mixed and dissolved with the mixed extract of the flavone and the alkaloid which takes ethanol as eluent under the action of polyoxyethylene 40 hydrogenated castor oil, the ethanol plays a role in eluting the mixed extract of the flavone and the alkaloid from a macroporous resin composition and also plays a role of an auxiliary emulsifier, so that various substances contained in the mixed extract of the flavone and the alkaloid and various oil-soluble substances contained in the lindera strychnifolia leaf volatile oil form micro-emulsion distribution in an ethanol water solution, the average particle size of the micro-emulsion distribution is detected to be about 15.25 +/-1.22 nm, the formed liquid mixture of the mixed extract of the lindera strychnifolia leaf volatile oil and the flavone and the alkaloid can realize good dispersibility when being dropped into a water phase substance, and the following test data show that the activity of the compound can be improved, and the compound has better effects in promoting blood circulation and relieving pain.
In order to fully embody the use of the lindera aggregata leaf extract obtained by the invention in activating blood and relieving pain, the following will further perform the test for pain relieving experiments and the test for activating blood respectively on the lindera aggregata leaf volatile oil with the serial numbers of WH1, WH2 and WH3, the flavone and alkaloid mixed extract with the serial numbers of WY1, WY2 and WY3 obtained in the above examples, and the liquid mixture of the lindera aggregata leaf volatile oil with the serial numbers of WHY1, WHY2 and WHY3, and the flavone and alkaloid mixed extract, respectively, and obtain the average value of WH1, WH2 and WH3 as WH; the average values of WY1, WY2 and WY3 are WY and the average values of WHY1, WHY2 and WHY3 are WHY, and the technical effects achieved by the invention are further embodied by the aid of the graphs shown in fig. 1 and fig. 2.
The lindera aggregata leaf extract obtained by the invention has the functions of promoting blood circulation and relieving pain, so the lindera aggregata leaf extract provided by the invention can also be used for being added into corresponding fields of medicines for promoting blood circulation and relieving pain, health-care food, functional food and the like.
Firstly, the method is used for analgesic experiment detection:
the experimental detection is to use the combined extract of the lindera aggregate leaf volatile oil, the flavone and the alkaloid obtained in the above embodiment and the mixture thereof in the analgesic experimental detection, wherein the conventional water extract (WS) and aspirin are adopted for comparing the effects of activating blood and relieving pain.
The aqueous extract (WS) of lindera aggregate leaf is prior art and is used here for comparison purposes only.
Comparative analgesic effect experiments were as follows:
3dpf wild type AB strain zebrafish was randomly selected in six-well plates with 30 tails per well (per experimental group) and a volume of 3mL per well. WH1, WH2, WH3, WY1, WY2, WY3, WHY1, WHY2 and WHY3 were respectively dissolved in water, the concentration of the mixture was 31.25. mu.g/mL, the concentration of positive control aspirin was 100. mu.g/mL, and a normal control group (water treatment group for fish farming) and a model control group were set. After each experimental group is treated for 1 hour, the zebra fish is immediately transferred to a 96-well plate, the number of the zebra fish is 1, the zebra fish is 100 mu L/well, the other experimental groups except a normal control group are dissolved in water and are given glacial acetic acid, a behavior analyzer is used for detecting the total movement distance of the zebra fish within 0.5 hour, and the analgesic effect of the three extracts on the zebra fish is evaluated according to the statistical analysis result of the total movement distance. Statistical treatment results are expressed as mean ± SE. The analgesic effect calculation formula is as follows:
statistical analysis using the TTEST test indicated significant differences with p < 0.05.
The results are shown in table 1 and fig. 1, wherein fig. 1 shows the total distance of zebrafish movement after treatment of each experimental group compared with the model control group, p <0.01, p < 0.001.
Table 1 total distance traveled by zebra fish after treatment in each experimental group (n ═ 10)
P <0.01, p <0.001, compared to model control group
The total movement distance (6866mm) of the zebra fish in the model control group is compared with that in the normal control group (1611mm) by p <0.001, so that the successful construction of the model is prompted. The total movement distance of the zebra fish in the positive control group with aspirin of 100 mu g/mL concentration is 4393mm, compared with the model control group, p is less than 0.001, the analgesic effect is 47.1 percent, and the aspirin is prompted to have obvious analgesic effect.
The total moving distances of the zebra fishes corresponding to WY1, WY2, WY3, WH1, WH2, WH3, WHY1, WHY2, WHY3 and WS groups are 4854, 4939, 4790, 5031, 5010, 5201, 4482, 4790, 4602 and 5569mm respectively, so that the average value of the total moving distances of the zebra fishes corresponding to WY1, WY2 and WY3 is 4861, and the group is set as WY; the average value of the total moving distances of the zebra fishes corresponding to WH1, WH2 and WH3 is 5081, and the group is set as WH; the average value of the total movement distance of zebrafish corresponding to WHY1, WHY2 and WHY3 is 4625, the group is set as WHY, and p <0.001& p <0.001& p <0.01 and the analgesic effect is 38.3%, 36.7%, 39.5%, 34.9%, 35.3%, 31.7%, 45.4%, 39.5%, 43.1% and 24.7% respectively compared with the model control group, so that under the condition of the experimental concentration, the groups of WY1, WY2, WY3, WH1, WH2, WH3, WHY1, WHY2 and WHY3 have obvious analgesic effect, and the average analgesic effect is higher than that of the group of WY1, WHY2 and WHY3, especially the group of WHY1, WHY2 and WHY3, and is close to aspirin 47.1%, and the synergistic effect of the mixed extract of the lindera leaf volatile oil and the alkaloid is shown in the graph 1.
Secondly, the method is used for the blood circulation activating experiment detection:
in this example, the combined extracts of essential oil, flavone and alkaloid and their mixture obtained in the above examples were tested in blood-activating experiments, wherein conventional aqueous extract (WS) and aspirin were used for blood-activating (described below as qi stagnation and blood stasis) comparison.
The efficacy comparison experiment for treating qi stagnation and blood stasis is as follows:
randomly selecting 3dpf melanin allele mutant type translucent Albino strain zebra fish in a six-well plate, wherein 30 fishes per well (per experimental group) have the volume of 3 mL. WH1, WH2, WH3, WY1, WY2, WY3, WHY1, WHY2 and WHY3 were respectively added in water at a concentration of 3.9. mu.g/mL, and a normal control group (a fish-farming water treatment group) and a model control group were set. After each experimental group is treated for 48 hours, the generation condition of the 30 tail zebra fish blood stasis of each experimental group is observed under an anatomical microscope, typical pictures are photographed, the tail blood stasis generation mantissa and the tail blood stasis generation rate (%) of each experimental group are statistically analyzed, and the treatment effect of the two crude extracts on the zebra fish qi stagnation and blood stasis is evaluated according to the statistical significance of the tail blood stasis mantissa.
The experimental results are shown in table 2 and fig. 2, wherein fig. 2 shows the number of the blood stasis at the tail of the zebra fish after the treatment of each experimental group compared with the model control group, p is less than 0.01, and p is less than 0.001.
Table 2 statistics of the occurrence of blood stasis in the tail of zebra fish after treatment in each experimental group (n is 30)
P <0.01, p <0.001, compared to model control group
Compared with the normal control group (0 tail), the tail blood stasis occurrence mantissa (25 tail) of the zebra fish in the model control group is less than 0.001, and the tail blood stasis occurrence rate is 83 percent (25/30), so that the successful construction of the model is prompted. Compared with a model control group, the tail blood stasis occurrence mantissa (6 tails) of a positive control drug aspirin group with the concentration of 30 mu g/mL is less than 0.001, and the tail blood stasis occurrence rate is 20% (6/30), which indicates that aspirin has obvious effect of treating qi stagnation and blood stasis.
The tail blood stasis occurrence mantissas of the zebra fish corresponding to WY1, WY2, WY3, WH1, WH2, WH3, WHY1, WHY2, WHY3 and WS concentration groups are respectively 10, 12, 9, 12, 11, 12, 7, 8 and 14 tails, so that the average value of the tail blood stasis occurrence mantissas of the zebra fish corresponding to WY1, WY2 and WY3 is 10.33, and the group is WY; the average value of the tail blood stasis occurrence mantissas of zebra fish corresponding to WH1, WH2 and WH3 is 11.67, and the group is set as WH; the average value of the tail blood stasis occurrence mantissas of zebra fish corresponding to WHY1, WHY2 and WHY3 was 7.67, and the group was WHY, and compared with the model control group, p was <0.001& p <0.001& p <0.01, and the tail blood stasis occurrence rates were 33%, 40%, 30%, 40%, 37%, 40%, 23%, 27% and 47%, respectively. The data show that under the condition of the experimental concentration, the WH1, WH2, WH3, WY1, WY2, WY3, WHY1, WHY2 and WHY3 groups all have obvious effects of treating qi stagnation and blood stasis, and the WH1, WH2, WH3, WY1, WY2, WY3, WHY1, WHY2 and WHY3 are all higher than those of the 'WS' group, particularly the average incidence rate of the tail blood stasis of the WHY1, WHY2 and WHY3 groups is 25.67% and is close to aspirin 20%, which indicates that the effects of treating qi stagnation and blood stasis are close to aspirin, and the mixture of the lindera aggregate volatile oil, flavone and alkaloid mixed extract plays a synergistic effect on treating qi stagnation and blood stasis, and is shown in fig. 2.
Claims (4)
1. A preparation method of lindera aggregata leaf extract is characterized by comprising the following steps: the method comprises the following steps:
drying and crushing lindera aggregate leaves at 30-40 ℃, then placing the crushed lindera aggregate leaves in an extraction kettle in a supercritical extraction device, and performing carbon dioxide supercritical extraction on the dried and crushed lindera aggregate leaves in the supercritical extraction device by using carbon dioxide as a solvent: firstly, cooling introduced carbon dioxide through a condenser until the carbon dioxide is in a liquid state, pumping the carbon dioxide into an extraction kettle through a high-pressure pump, heating and pressurizing the extraction kettle in a supercritical extraction device to enable the carbon dioxide to be in a supercritical state for extraction, controlling the flow rate of the carbon dioxide to be 30L per hour during extraction, and performing circulating extraction; simultaneously keeping the extraction pressure at 22 +/-5 MPa, the extraction temperature at 40 +/-5 ℃ and the extraction time at 2-3 hours, and performing two-stage separation in a separation kettle, wherein the first-stage separation pressure is 4 +/-0.5 MPa, the separation temperature is 40 +/-5 ℃, the second-stage separation pressure is 4 +/-0.5 MMPa and the separation temperature is 35 +/-5 ℃, so as to obtain a yellow-green viscous extract, and removing water in the extract to obtain the lindera strychnifolia leaf volatile oil;
step two, adding 80% ethanol containing 0.1% HCl into residue obtained after supercritical extraction of lindera aggregata leaf volatile oil according to the material-liquid ratio of 1: 10-15 g/mL, carrying out reflux extraction for 1 hour to 2 hours at the temperature of 60 ℃, carrying out extraction for 2 times to 3 times by the same method, combining filtrates, adjusting the pH to 7.0 by using NaOH solution, filtering, collecting the filtrate, concentrating volatile ethanol through rotary evaporation, and dissolving with distilled water to obtain a crude extract;
step three, sampling the crude extraction liquid obtained in the step two at the flow rate of 2BV/h, wherein the concentration of the sampling liquid is 2mg/mL, the pH value of the sampling is 6.0, adding the sampling liquid to a macroporous resin column, respectively washing the sampling liquid for 1 time by using 2BV water and 1BV 20% ethanol, respectively eluting the sampling liquid by using 3BV 60% ethanol and 2BV 80% ethanol containing 2% ammonia water at the flow rate of 2BV/h, collecting and combining the eluates, and obtaining a flavone and alkaloid mixed extract; the resin adopted in the macroporous resin column is a macroporous resin composition consisting of D101 and NKA-9, and the mass ratio of D101 to NKA-9 is 7-8: 3-2.
2. An extract of lindera strychnifolia leaves prepared by the method of claim 1, which is a mixed extract of flavones and alkaloids obtained by the carbon dioxide supercritical extraction of lindera strychnifolia leaves volatile oil of claim 1 or the ethanol reflux extraction of the residue after the carbon dioxide supercritical extraction of lindera strychnifolia leaves volatile oil, or a mixture of lindera strychnifolia leaves volatile oil obtained by the method and flavones and alkaloids obtained by the method.
3. The lindera aggregata leaf extract as claimed in claim 2, wherein the mixture of the lindera aggregata leaf volatile oil and the mixed extract of flavone and alkaloid is obtained by emulsifying the lindera aggregata leaf volatile oil obtained in the first step and then mixing the extracted mixture of flavone and alkaloid obtained in the third step.
4. Use of the lindera strychnifolia leaf extract of claim 2 in preparing medicines for promoting blood circulation and relieving pain.
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