CN112245499B

CN112245499B - Chinese patent medicine for treating lumbar intervertebral disc protrusion, preparation method and application

Info

Publication number: CN112245499B
Application number: CN202011117148.5A
Authority: CN
Inventors: 石洪超; 覃仁安; 林建云; 何风雷; 杨能英; 雷胄熙; 刘海英; 蒋诗琪; 陈蓉蓉
Original assignee: Guangzhou Baiyunshan Chen Liji Pharmaceutical Factory Co ltd
Current assignee: Guangzhou Baiyunshan Chen Liji Pharmaceutical Factory Co ltd
Priority date: 2020-10-19
Filing date: 2020-10-19
Publication date: 2021-12-03
Anticipated expiration: 2040-10-19
Also published as: CN112245499A

Abstract

The invention provides a preparation method and application of a Chinese patent medicine for treating lumbar intervertebral disc protrusion, and relates to the technical field of Chinese medicine preparations. The preparation method comprises the following steps: extracting rhizoma Cibotii, and concentrating to obtain soft extract; decocting radix Millettiae Speciosae, fructus Rosae Laevigatae, caulis Spatholobi, radix Flemingiae Philippinensis, caulis Kadsurae Coccineae, herba Taxilli, and radix Zanthoxyli, and concentrating to obtain soft extract; pulverizing fructus Ligustri Lucidi, semen Cuscutae, and rhizoma corydalis into fine powder; heating and extracting cortex of Kadsura coccinea, collecting volatile oil, and concentrating the water extract to obtain soft extract; extracting volatile oil from Olibanum (preparata) and Myrrha (preparata); mixing and clathrating the volatile oil of Kadsura coccinea with the volatile oil of Olibanum and Myrrha; mixing all the soft extracts, fine powder and volatile oil clathrate. The method improves the preparation method based on the original Chinese medicinal composition, purifies and enriches the effective components, reduces gastrointestinal irritation, improves patient compliance, reduces dosage, is effective in treating prolapse of lumbar intervertebral disc, has better effect in improving dorsal root nerve pain, and increases diabetic symptoms of soreness of waist and knees patients.

Description

Chinese patent medicine for treating lumbar intervertebral disc protrusion, preparation method and application

Technical Field

The invention relates to the technical field of traditional Chinese medicine preparations, in particular to a Chinese patent medicine for treating lumbar disc herniation, a preparation method and application.

Background

Prolapse of lumbar intervertebral disc is a disease of a series of symptoms and signs caused by rupture of nucleus pulposus and further pressing and stimulation of nerve roots on the basis of degeneration of lumbar intervertebral disc. Lumbar intervertebral disc protrusion is one of the most common diseases in clinic, and is a common disease and a frequently encountered disease in the department of spine. The treatment methods for the lumbar intervertebral disc protrusion are various, and can be divided into non-operative treatment and operative treatment at present. The traditional Chinese medicine has obvious curative effect in the aspect of non-operative treatment, plays a unique important role and is a common method for non-operative treatment. Deficiency of liver and kidney and malnutrition of tendons and bones are the intrinsic factors of the disease, and wind, cold, heat, dampness, stasis and obstruction of meridians are the extrinsic factors of the disease, so the clinical treatment methods usually include tonifying liver and kidney, eliminating pathogenic factors and dredging collaterals.

The muscle-relaxing waist-strengthening pill is a unique wholesale variety and is prepared from 13 traditional Chinese medicines of rhizoma cibotii, fructus rosae laevigatae, caulis spatholobi, philippine flemingia root, black tiger, beautiful millettia root, glossy privet fruit, Chinese taxillus twig, semen cuscutae, rhizoma corydalis, radix zanthoxyli, frankincense and myrrh. The main functions are tonifying liver and kidney, strengthening bones and muscles, expelling wind and removing dampness, activating collaterals and relieving pain; can be used for treating soreness of waist and knees. The current execution criteria are: in the twelfth volume (Standard No. WS3-B-2441-97) of the Ministry of public health of the people's republic of China, the standard Chinese medicinal prescription preparation, the preparation method is as follows: decocting rhizoma Cibotii, fructus Rosae Laevigatae, caulis Spatholobi, radix Flemingiae Philippinensis and caulis Kadsurae Coccineae with water twice, filtering, mixing filtrates, and concentrating to obtain soft extract; pulverizing the rest materials and cortex of Kadsura coccinea into coarse powder, adding the above soft extract, mixing, drying, pulverizing into fine powder, sieving, mixing, adding appropriate amount of Mel and water, making into pill, coating with active carbon, drying, and polishing. The clinical dosage is large, 5g for one time and 3 times a day. Some patients feedback to take the pill for relaxing muscles and tendons and strengthening waist to cause gastrointestinal discomfort symptoms such as nausea, vomiting and the like.

Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia due to defective insulin secretion, insulin resistance, or both. Epidemiological survey data published by the international diabetes union in 2019 show that about 4.63 hundred million of worldwide adults aged 20-79 are diagnosed with diabetes, the incidence rate is as high as 9.3%, wherein Chinese diabetic patients are 1.16 hundred million, and the national diabetes is the most countries of worldwide diabetic patients. Diabetics need good blood sugar control for a long time and the risk of complications is reduced. The muscle-relaxing waist-strengthening pill is a concentrated water-honeyed pill containing about 30% of honey, so that the diabetic patients are not suitable for taking the pill for a long time.

Disclosure of Invention

Based on the above, there is a need to provide a Chinese patent medicine for treating lumbar disc herniation and a preparation method thereof, which is improved on the basis of the original traditional Chinese medicine composition, volatile oil is extracted from frankincense, myrrh and kadsura coccinea skin for inclusion, and irritation to gastrointestinal tract is reduced; the purification and enrichment of active ingredients are increased, the dosage is reduced, and the compliance of patients is improved; the sugar-free preparation can increase the suitable population of diabetic patients.

A preparation method of a Chinese patent medicine for treating lumbar intervertebral disc protrusion comprises the following raw materials in parts by weight:

preparing thick paste 1: extracting 800 parts of rhizoma cibotii 400 with water under reflux, filtering to obtain an extracting solution, adding the extracting solution into a macroporous adsorption resin column for adsorption, washing the resin with water after the extracting solution completely passes through the macroporous resin column until water eluent is colorless, continuously eluting with an ethanol solution with a certain concentration, collecting the ethanol eluent, and concentrating into thick paste;

preparing thick paste 2: soaking 350 parts of beautiful millettia root, 100 parts of cherokee rose fruit, 300 parts of suberect spatholobus stem, 100 parts of philippine flemingia root, 300 parts of black tiger (wood part thereof), 10-60 parts of shinyleaf pricklyash root and 100 parts of Chinese taxillus herb in water, decocting, filtering, collecting filtrate, and concentrating into thick paste;

preparing fine powder: pulverizing 10-60 parts of fructus Ligustri Lucidi, 10-60 parts of semen Cuscutae, and 10-60 parts of rhizoma corydalis into fine powder;

preparing a volatile oil clathrate compound: crushing bark of the black tiger (10-20 meshes), soaking in water, heating for extraction, collecting volatile oil of the black tiger, soaking 10-30 parts of frankincense (prepared) and 4-30 parts of myrrh (prepared) in water, heating for extraction, and collecting volatile oil of the frankincense and the myrrh; mixing the volatile oil of Kadsura coccinea with the volatile oil of Olibanum and Myrrha, and clathrating to obtain volatile oil clathrate.

Preparing a thick paste 3: filtering the water extract of the cortex kadsurae japonicae part after extracting volatile oil, collecting the filtrate, and concentrating to obtain soft extract;

mixing: and mixing the thick paste 1, the thick paste 2, the thick paste 3, the fine powder and the volatile oil inclusion compound to obtain the mixture of the Chinese patent medicines.

It is understood that the "wood" of Kadsura coccinea refers to the wood of Kadsura coccinea, and the "bark" of Kadsura coccinea is mainly the phloem of Kadsura coccinea. The cortex and wood of Kadsura coccinea can be separated by pulverizing machine.

The preparation method improves the extraction method of the effective components of rhizoma Cibotii, radix Flemingiae Philippinensis, radix Zanthoxyli, herba Taxilli, Kadsura coccinea, Olibanum (processed) and Myrrha (processed) in the original pill for relaxing muscles and tendons and strengthening waist. Rhizoma Cibotii is dried rhizome of Cibotium barometz (L.) J.Sm of Unionidae, has effects of strengthening waist and knee, nourishing liver and kidney, and dispelling pathogenic wind and dampness, and contains abundant flavonoids such as kaempferide and chrysopodin. Millettia speciosa (Millettia speciosa Champ.) Merr is a dry root of Millettia speciosa (Millettia speciosa Champ.) Merr belonging to Millettia of Leguminosae, has the effects of tonifying deficiency, moistening lung, and strengthening and activating tendons, is widely used as a raw material for cooking soup in two broad areas, and has the main chemical components of Millettia speciosa polysaccharide, total flavonoids, alkaloids and the like. The Zanthoxylum nitidum (Zanthoxylum nitidum (Roxb.) DC.) dried root of Rutaceae plant has effects of promoting blood circulation for removing blood stasis, activating qi-flowing for relieving pain, dispelling pathogenic wind, dredging collaterals, removing toxic substance and relieving swelling, and has antibacterial, antiinflammatory, and antitumor effects, and alkaloid components have antiinflammatory, analgesic, ulcer inhibiting, and antitumor effects, and are main effective components of Zanthoxylum nitidum. Herba Taxilli is dry stem and branch with leaves of herba Taxilli (Taxillus chinensis (DC)) of Taxilliaceae, and has effects of nourishing liver and kidney, strengthening tendons and bones, dispelling pathogenic wind and dampness, dredging channels and collaterals, benefiting blood, and preventing miscarriage. The frankincense is resin exuded from the bark of Boswellia serrata (Boswellia carterii Birdw.) and plants of the same genus (Boswellia hhaw-dajiana Birdw.), and the myrrh is dry resin of the corydalis bungeana (Commiphora myrrha Engl.) and the corydalis bungeana (Commiphora molmol Engl.) of the olive family, which are commonly used in the formulas for promoting blood circulation to remove blood stasis, relaxing muscles and tendons and dredging collaterals, has very obvious modern pharmacological action, and has pharmacological activities of resisting inflammation, easing pain, resisting cancer and the like. The Kadsura coccinea (Lem.) A.C. Smith is dried root of Kadsura coccinea of Magnoliaceae, and volatile oil of Kadsura coccinea is important effective component of Kadsura coccinea, and has effects of activating qi-flowing, promoting blood circulation, dispelling pathogenic wind, and relieving pain, but is easily lost at high temperature during preparation process. Because the clinical patients feedback that the pill for relaxing muscles and tendons and strengthening waist has certain irritation to gastrointestinal tracts after taking the pill, in the process improvement, half of the water extract of frankincense and myrrh with smaller lethal dose and stronger toxicity is removed, the volatile oil of black tiger, frankincense and myrrh is coated with beta-cyclodextrin and then is taken into medicine, so that the irritation of the pill for relaxing muscles and tendons and strengthening waist is reduced, the stability of the volatile oil is improved, the solubility is increased, the bioavailability is improved, the odor of the volatile oil is covered up, and the use compliance of the patients is improved. The muscle-relaxing waist-strengthening pill (concentrated water pill) is a sugar-free dosage form and is suitable for people with diabetes.

In one embodiment, the raw material medicaments comprise the following components in part by weight: 360.0kg of rhizoma cibotii, 115.2kg of cherokee rose fruit, 216.0kg of suberect spatholobus stem, 86.4kg of philippine flemingia root, 216.0kg of kadsura coccinea, 144.0kg of beautiful millettia root, 18.0g of glossy privet fruit, 108.0kg of Chinese taxillus twig, 18.0kg of south dodder seed, 15.6kg of corydalis tuber, 15.6kg of shinyleaf pricklyash root, 7.2kg of frankincense and 12.0kg of myrrh.

In one embodiment, the step of preparing the thick paste 1 specifically comprises the following steps: reflux-extracting rhizoma Cibotii for 1-3 times for 1-3 hr, filtering, mixing filtrates to obtain extractive solution, adsorbing with macroporous adsorbent resin column, washing with water until the water eluate is colorless, eluting with 3-5 times column volume of 50-80% ethanol solution, collecting ethanol eluate, and concentrating to obtain soft extract. Preferably, the macroporous adsorption resin is D101.

In one embodiment, the step of preparing the thick paste 1 specifically comprises the following steps: reflux-extracting rhizoma Cibotii with water solution for 2 times, filtering, mixing filtrates to obtain extractive solution, adsorbing the extractive solution with D101 macroporous adsorbent resin column at flow rate of 3BV/h, washing with 5 times of water solution at flow rate of 4BV/h, eluting with 3 times of 70% ethanol solution at flow rate of 3BV/h, collecting eluate, and concentrating to obtain soft extract.

In one embodiment, the step of preparing the thick paste 2 specifically comprises the following steps: crushing radix Millettiae Speciosae, sieving with 5-12 mesh sieve, soaking with fructus Rosae Laevigatae, caulis Spatholobi, radix Flemingiae Philippinensis, caulis Kadsurae Coccineae, herba Taxilli, and radix Zanthoxyli in water for 0.5-3 hr, with the water amount being 8-12 times of solid mass, decocting for 1-3 times, each time for 2-4 hr, filtering, mixing filtrates, and concentrating into soft extract.

In a more preferred embodiment, the step of preparing the thick paste 2 specifically comprises the following steps: crushing radix Millettiae Speciosae, sieving with 8 mesh sieve, mixing with fructus Rosae Laevigatae, caulis Spatholobi, radix Flemingiae Philippinensis, caulis Kadsurae Coccineae, radix Zanthoxyli, and herba Taxilli, adding 8 times of water for the first time, soaking for 1 hr, extracting for 4 hr, adding 6 times of water for the second time, extracting for 2 hr, filtering, mixing filtrates, and concentrating to obtain soft extract.

In the embodiment, the step 3 of preparing the thick paste specifically comprises the following steps: filtering the water extractive solution of cortex kadsurae japonicae part after extracting volatile oil, collecting filtrate, and concentrating to obtain soft extract.

In one embodiment, in the step of preparing the volatile oil inclusion compound, the bark of the black tiger is crushed and sieved by a 10-20 mesh sieve, water is added for soaking for 0.5-1h, heating and extracting are carried out for 4-10h, and the volatile oil is collected. Soaking Olibanum and Myrrha in water for 0.5-1 hr, heating and extracting for 6-15 hr, and collecting volatile oil. Mixing volatile oils of Kadsura coccinea, Olibanum and Myrrha, and clathrating with beta-cyclodextrin saturated water solution or grinding.

In one embodiment, in the step of preparing the volatile oil inclusion compound, the proportion of the mixed volatile oil of the black tiger, the frankincense and the myrrh and the beta-cyclodextrin is 1:6-10, the inclusion temperature is 20-50 ℃, and the inclusion time is 10min-3 h.

In one embodiment, the step of preparing the volatile oil inclusion compound specifically comprises the following steps: crushing bark of Kadsura coccinea, sieving with 20 mesh sieve, soaking in 8 times of water for 0.5 hr, heating and extracting for 6 hr, and collecting Kadsura coccinea volatile oil; soaking Olibanum (preparata) and Myrrha (preparata) in 8 times of water for 0.5 hr, heating and extracting for 9 hr, and collecting volatile oil of Olibanum and Myrrha; mixing the Kadsura coccinea volatile oil with the frankincense and the myrrh volatile oil, adding equal amount of ethanol, uniformly mixing, clathrating the mixture with beta-cyclodextrin according to a ratio of 1:10 at 30 ℃ for 1h, cooling to room temperature, carrying out suction filtration on the clathrate solution, and drying the filter cake in a vacuum drying oven at 50 ℃ for 5h to obtain the Kadsura coccinea, frankincense and myrrh mixed volatile oil beta-cyclodextrin clathrate.

In one embodiment, the step of preparing the fine powder specifically comprises: pulverizing fructus Ligustri Lucidi, semen Cuscutae, and rhizoma corydalis into fine powder.

In one embodiment, the preparation method further comprises the preparation steps of: and preparing the mixture of the Chinese patent medicines into pills, capsules or tablets.

In one embodiment, the preparation of the pellets is obtained by: mixing the fine powder, the volatile oil clathrate, appropriate amount of starch and microcrystalline cellulose, adding soft extract 1, soft extract 2 and soft extract 3, mixing, making pill, coating with active carbon, drying, and polishing to obtain SHUJINJIANYAO pill (concentrated watered pill).

The invention provides a Chinese patent medicine obtained by the preparation method. Compared with the Chinese patent medicine prepared by the prior art, the Chinese patent medicine has the advantages of reducing the stimulation to the gastrointestinal tract, having less dosage, having better effect on treating the protrusion of the lumbar intervertebral disc and increasing the applicable population of the diabetic patients.

The invention also provides application of the Chinese patent medicine prepared by the preparation method in preparing a medicine for treating lumbar intervertebral disc protrusion.

Compared with the prior art, the invention has the following beneficial effects:

the preparation method of the invention improves the extraction method of the effective components of the raw materials of rhizoma cibotii, beautiful millettia root, shinyleaf pricklyash root, Chinese taxillus twig, kadsura root-bark, frankincense (prepared) and myrrh (prepared) in the original muscle-relaxing waist-strengthening pill. (1) Extracting rhizoma Cibotii with water, and efficiently enriching flavonoid effective substances in the extractive solution with macroporous resin to improve effective substance content. (2) Radix Millettiae Speciosae, radix Zanthoxyli and herba Taxilli are extracted with water instead of pulverizing into powder. The millettia speciosa champ is crushed before the millettia speciosa champ is extracted and passes through a screen with 5-12 meshes, the transfer rate of the millettia speciosa champ polysaccharide water extract reaches 80.0 percent, the transfer rate of the erythrina indica alkali is 68.1 percent, and the transfer rate of the total flavone is 67.8 percent. Extracting radix Zanthoxyli with water, and detecting nitidine in the final product SHU JING JIAN YAO WAN (concentrated watered pill). (3) The volatile oil of the bark of Kadsura coccinea is an important effective component of Kadsura coccinea. In the experiment of the median lethal dose of the zebra fish, the median lethal dose concentration of the frankincense volatile oil is greater than that of the emulsion perfume extract, the median lethal dose concentration of the myrrh volatile oil is greater than that of the non-aqueous extract, and the safety of the frankincense volatile oil and the myrrh volatile oil is higher than that of the aqueous extract of the medicinal materials, so that in the process improvement, the aqueous extract of the frankincense and the myrrh is removed. The volatile oil of the three medicinal materials of the kadsura coccinea bark, the frankincense and the myrrh is coated by beta-cyclodextrin to be used as a medicine, so that the irritation of the muscle-relaxing waist-strengthening pill on gastrointestinal tracts is reduced, the stability of the volatile oil is improved, the solubility is increased, the bioavailability is improved, the odor of the volatile oil is covered, and the use compliance of patients is improved. (4) The muscle-relaxing and waist-strengthening pill (concentrated water pill) prepared by the method is prepared by directly adding the medicinal powder and the auxiliary materials into thick paste for pill preparation, and does not need to be dried, crushed and prepared into pills, so that compared with the original muscle-relaxing and waist-strengthening pill, the energy consumption is lower, the production process is shorter, and the production efficiency is higher. (5) The muscle-relaxing waist-strengthening pill (concentrated water pill) obtained by the invention does not contain honey as an adhesive, and is suitable for long-term administration of patients with soreness of waist and knees with diabetes symptoms. (6) The muscle-relaxing waist-strengthening pill (concentrated water pill) disclosed by the invention is taken 1g each time (5 g each time of the original preparation), the dosage is less, and the compliance of patients is good. (7) The muscle-relaxing waist-strengthening pill (concentrated water pill) is effective in treating lumbar intervertebral disc protrusion and has a better effect in improving dorsal root neuropathic pain.

Drawings

FIG. 1 is a thin layer chromatogram of a volatile oil;

wherein, A, frankincense volatile oil, B, myrrh volatile oil, C, kadsura coccinea volatile oil and D, mixed volatile oil inclusion compound ultrasonic extracting solution.

FIG. 2 is a diagram showing the staining of zebra fish with calcein in vivo;

wherein the extract 11 is the beta-cyclodextrin inclusion compound of the mixed volatile oil of the black cutworm, the frankincense and the myrrh, and the extract 15 is the mixed volatile oil of the black cutworm, the frankincense and the myrrh.

FIG. 3 is a TIC stack diagram of erythrina alkali in the form of pill (concentrated water pill) for relaxing muscles and tendons and strengthening waist;

wherein, S1: erythrina base, S2: beautiful millettia root medicinal material, S3: muscle-relaxing waist-strengthening pills (concentrated water pills), S4: muscle-relaxing waist-strengthening pills (concentrated watered pills negative for beautiful millettia root).

FIG. 4 is a TICIN stack diagram of nitidine for treating lumbar and muscle diseases.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the preferred embodiments. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1

Example of Process Condition screening

1 screening of rhizoma Cibotii extraction method

1.1 method for detecting total flavone from rhizoma Cibotii water extractive solution and ethanol eluate

Preparing a standard solution: accurately weighing 20mg of rutin standard substance, dissolving with 60% ethanol, diluting to a constant volume in a 100mL volumetric flask, and shaking up.

Preparation of a standard curve: precisely measuring 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0mL of rutin standard solution respectively, placing into a 25mL volumetric flask, adding 2.0mL 30% ethanol and 1.0mL 5% NaNO₂Shaking the solution, and standing for 6 min; then adding 10% Al (NO)₃)₃1.0mL of the solution is shaken up and placed for 6min, then 10.0mL of 4% NaOH solution is added, 30% ethanol is used for fixing the volume to the scale, the solution is shaken up and placed for 15 min. And (3) taking a reagent blank as a reference, measuring the absorbance at the wavelength of 510nm, and drawing a standard curve by taking the absorbance as a vertical coordinate and the concentration as a horizontal coordinate.

And (3) measuring the content of the rhizoma cibotii total flavonoids: precisely measuring a proper amount of rhizoma Cibotii water extract or ethanol eluate, placing in a 25mL volumetric flask, measuring absorbance according to the method under the preparation item of the standard curve from the time of adding 2.0mL of 30% ethanol, reading out the amount of total flavonoids in the test solution from the standard curve, and calculating to obtain the product.

1.2 rhizoma Cibotii Process screening

1.2.1 selection of the number of reflux extractions of rhizoma Cibotii

Weighing 300g rhizoma Cibotii, extracting for 3 times, adding 8 times of water for the first time, 6 times of water for the second time, and 6 times of water for the third time, extracting for 2 hr each time, collecting extractive solution each time, and determining total flavone content in the extractive solution, as shown in Table 1.

TABLE 1 determination of extraction times

According to the test results, the extraction rate of the total flavone in the third extraction liquid is low, so the extraction times are selected to be 2 times.

1.2.2 reflux extraction Water addition multiple selection

Weighing 3 parts of 300g rhizoma Cibotii, adding (6 times, 4 times), (8 times, 6 times), (10 times, 8 times) water, extracting for 2 times, each time for 2h, mixing extractive solutions, and determining total flavone content in the extractive solution, as shown in Table 2.

TABLE 2 determination of Water addition factor

According to the experimental result, the difference of the extraction rate difference of (8 times, 6 times) and (10 times, 8 times) total flavone extraction is small, and the water adding times are selected to be 8 times and 6 times in consideration of production energy consumption.

1.2.3 selection of reflux extraction time

Weighing 3 parts of 300g rhizoma Cibotii each, extracting for 2 times, adding 8 times of water for the first time, adding 6 times of water for the 2 nd time, with the extraction time of (0.5h ), (1h, 1h), (2h, 2h), and determining the total flavone content in the extractive solution, as shown in Table 3.

TABLE 3 reflux extraction time determination

According to the experimental results, the total flavone is extracted more fully by 2 times, 8 times of water is added for the first time for 2 hours, and 6 times of water is added for the second time for 2 hours.

1.2.4 concentration selection of eluting ethanol

Taking 3 parts of each 100mL of the liquid medicine extracted according to the optimal extraction process, respectively loading the liquid medicine on 3D 101 resin columns with processed wet volumes of 130mL, loading the liquid medicine for adsorption, then washing the liquid medicine by using aqueous solution with 5 times of column volumes until effluent liquid is colorless, respectively eluting the liquid medicine by using 50% ethanol, 70% ethanol and 85% ethanol with 3 times of column volumes, starting collecting eluent when the color of the effluent liquid is obviously darkened, and measuring the total flavone content of the collected ethanol eluent, wherein the results are shown in Table 4.

TABLE 4 determination of eluting ethanol concentration

According to the test result, the ethanol concentration is eluted at 70% and 85%, the total flavone content is high, the difference is not large, and 70% ethanol is optimal in consideration of the cost.

1.2.5 selection of type of macroporous adsorbent resin

Taking 100mL of each 4 parts of the liquid medicine extracted by the optimal process method, respectively loading the liquid medicine on 4 different types of resin columns with processed wet volumes of 130mL, loading the liquid medicine for adsorption, then washing the liquid medicine by using aqueous solution with 5 times of column volume at the flow rate of 4BV/h, respectively eluting the liquid medicine by using 70 percent ethanol with 3 times of column volume, and measuring the total flavone content of the collected ethanol eluent, wherein the results are shown in Table 5.

TABLE 5 determination of the type of macroporous adsorbent resin

According to the test results, the adsorption rates of the 4 macroporous resins are D101, FL-3, AB-8 and HPD100 from high to low, and the type of the macroporous resin D101 is preferred.

1.2.6 choice of ethanol amount to elute

Taking 3 parts of each 100mL of the liquid medicine extracted according to the optimal extraction process, respectively loading the liquid medicine on 3D 101 resin columns with processed wet volumes of 130mL, loading the liquid medicine for adsorption, then washing the liquid medicine by using aqueous solution with 5 times of column volumes until effluent liquid is colorless, respectively eluting the liquid medicine by using 70% ethanol with 2 times, 3 times and 5 times of column volumes, starting collecting eluent when the color of the effluent liquid is obviously darker, and measuring the total flavone content of the collected ethanol eluent, wherein the results are shown in Table 6.

TABLE 6 determination of eluting ethanol concentration

According to the test results, the total flavone is basically completely eluted by eluting with 70% ethanol in 3 times of the column volume.

1.2.7 Dry paste yield before and after passing the water extract of rhizoma Cibotii through D101 macroporous resin column

Weighing 300g of rhizoma cibotii, extracting for 2 times, adding 8 times of water for reflux extraction for 2 hours for the first time, adding 6 times of water for reflux extraction for 2 hours for the second time, combining filtrates, taking 100mL2 parts of filtrate, respectively adding into an evaporation dish, evaporating to dryness, and measuring the average dry extract 2.19g, wherein the dry extract yield is 25.9%; and taking 2 parts of each 100mL filtrate, respectively adding into a treated D101 macroporous resin column with the wet volume of 130mL for adsorption, adding 5 times of column volume of aqueous solution for washing until effluent is colorless, eluting with 3 times of column volume of 70% ethanol solution, collecting eluate, recovering ethanol under reduced pressure, and evaporating to dryness to obtain 0.63g of average dry extract, wherein the yield of the dry extract is 7.5%.

The experiment can determine that the optimal enrichment process conditions of the cibotium total flavonoids are as follows: reflux-extracting rhizoma Cibotii for 2 times, adding 8 times of water for the first time and 6 times of water for the second time, extracting for 2 hr each time, passing the extractive solution through D101 macroporous resin column for adsorption, washing with 5 times column volume of water solution until the effluent is colorless, eluting with 3 times column volume of 70% ethanol solution, collecting eluate, recovering ethanol under reduced pressure, and concentrating to obtain extract.

2 Water extraction of beautiful millettia root

2.1 method of measurement (results are each converted to the amount of each component per 1g of beautiful Millettia root)

2.1.1 beautiful Millettia root polysaccharide assay

2.1.1.1 measurement of polysaccharide content in beautiful millettia root medicinal material

Preparation of a test solution: taking 5g of millettia speciosa champ powder (sieved by a sieve of No. four), precisely weighing, adding 320mL of distilled water into a 500mL round-bottom flask, precisely weighing, heating and refluxing for 106min, cooling, weighing again, complementing the lost weight with water, shaking up, filtering, precisely absorbing 5mL of filtrate, precisely adding 20mL of absolute ethyl alcohol, continuously stirring to obtain a solution with the alcohol content of 80%, obtaining uniform milky precipitate, placing the precipitate in a refrigerator for standing overnight, centrifuging, dissolving the precipitate in a 50mL volumetric flask with boiling water, and fixing the volume.

Preparing a reagent: 10g of phenol was precisely weighed, dissolved in 200mL of water, and mixed to obtain a 5% phenol solution, which was stored in a brown bottle.

Preparation of a reference solution: accurately weighing 10mg of anhydrous glucose, adding water to a constant volume of 100mL, and shaking up to obtain the glucose-lowering oral liquid.

Preparation of a standard curve: respectively precisely measuring 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9mL of glucose standard solution of 0.1mg/mL, placing the glucose standard solution into a 10mL test tube with a plug, sequentially adding water to make the volume be 1.0mL, adding 1.0mL of water as a blank control and 1.0mL of 5% phenol solution respectively, fully shaking and uniformly mixing, rapidly adding 6.0mL of concentrated sulfuric acid, shaking, standing at room temperature for 30min, measuring the absorption value (A) at 490nm wavelength, and drawing a standard curve by taking the concentration (C) of the glucose reference substance as an abscissa and the A as an ordinate.

And (3) measuring the content of the test solution: taking 1mL of test solution, determining absorbance according to the method from the point that 1.0mL of 5% phenol solution is added respectively according to the method under the preparation item of the standard curve, reading the amount of polysaccharide in the test solution from the standard curve, and calculating to obtain the polysaccharide-containing test solution.

2.1.1.2 content detection of polysaccharide in extractive solution

Taking appropriate amount of extractive solution, and detecting by the above method.

2.1.2 determination of Total Flavonoids in beautiful Millettia root

2.1.2.1 method for determining total flavone content in radix Millettiae Speciosae

Preparation of a test solution: precisely weighing 10g of beautiful millettia root medicinal material powder (sieved by a sieve IV), ultrasonically extracting for 2 times, adding 200mL of 80% ethanol each time, extracting for 60 minutes, standing and filtering, combining filtrates, concentrating under reduced pressure, and fixing the volume to 25mL by using 80% ethanol.

The content determination of the beautiful millettia root medicinal material total flavonoids: precisely measuring 0.2mL of test solution, placing the test solution in a 25mL volumetric flask, measuring absorbance according to the method under the preparation item of the standard curve from the time of adding 2.0mL of 30% ethanol, reading out the amount of total flavonoids in the test solution from the standard curve, and calculating to obtain the total flavonoids.

2.1.2.2 detection of total flavone content in extractive solution

Concentrating the extractive solution to obtain extract, and detecting with the above method.

2.1.3 determination of erythrina alkali in Millettia speciosa Roxb

2.1.3.1 determination of the content of the beautiful millettia root medicinal material erythrina alkali (QDA determination)

The preparation method of the test sample comprises the following steps: precisely weighing 1g of radix Millettiae Speciosae powder (sieved by a sieve IV), placing in a 100mL conical flask, precisely adding 50mL of 50% ethanol, weighing, ultrasonically extracting for 40min, cooling to room temperature, adding 50% ethanol to make up the weight, shaking up, and filtering to obtain the final product.

Preparation of control solutions: precisely weighing 1.50mg of erythrina alkali in a 25mL volumetric flask, dissolving with methanol, and fixing the volume to obtain the final product.

Chromatographic conditions are as follows: the column xBridge Amide (250 mm. times.4.6 mm, 3.5 μm) was eluted with a gradient according to the following table using acetonitrile as mobile phase A and 0.1% aqueous formic acid as mobile phase B: the instrument comprises the following steps: waters ARC ultra high performance liquid chromatograph; a detector: an ACQUITY QDa mass spectrometer detector; the detection wavelength is 280 nm; the flow rate is 0.5 mL/min; the column temperature was 30 ℃. The sample volume was 2. mu.l.

TABLE 7 elution conditions

2.1.3.2 determination of the content of erythrine in the extract

2.2 test methods

2.2.1 Effect of Millettia speciosa champ medicinal material granularity size on extraction

Weighing 300g of 2 parts of beautiful millettia root medicinal materials which are respectively conventional decoction pieces and crushed materials (8-mesh sieve), respectively adding water for extraction for 2 times, wherein the water addition amount is 8 times and 6 times, the extraction time is 4 hours and 2 hours, filtering, and combining the filtrates. The contents of polysaccharides, total flavonoids and erythrina alkali in the extract were determined, and the results are shown in Table 8.

TABLE 8 determination of particle size of herbs

According to the experimental result, the components of the crushed material (8-mesh sieve), the beautiful millettia root polysaccharide, the total flavone, the erythrina alkali and the like are extracted more fully.

2.2.2 selection of the number of times of extraction of beautiful Millettia root medicinal materials

Weighing 300g of 1 part of millettia speciosa champ crushed material (8-mesh sieve), adding water for extraction for 3 times, adding 8 times of water for the first time, soaking for 1 hour, extracting for 4 hours, adding 6 times of water for the second time, extracting for 2 hours, adding 6 times of water for the 3 rd time, extracting for 2 hours, respectively filtering, collecting filtrate, and measuring the contents of polysaccharide, total flavone and erythrina alkali, wherein the results are shown in a table 9.

TABLE 9 number of extractions investigation

According to the test results, the extraction rate of each component in the third extract was low, and therefore, the number of times of extraction was selected to be 2.

2.2.3 selection of extraction time for Millettia speciosa Roxb

Weighing 300g of 3 parts of beautiful millettia root crushed material (8-mesh sieve), respectively adding water for extraction for 2 times, wherein the water addition amount is 8 times and 6 times, the extraction time is (1 hour ), (2 hours, 2 hours), (4 hours and 2 hours), respectively filtering, and combining 2 times of filtrate. The contents of polysaccharide, total flavone and erythrina alkali were measured, and the results are shown in Table 10.

TABLE 10 examination of extraction times

According to the test results, the extraction time (2 hours ) and the extraction rate (4 hours, 2 hours) of each component of the beautiful millettia root are not obviously different.

2.2.4 transfer rates of beautiful millettia root water-extracted polysaccharide, total flavone and erythrina alkali

The contents of polysaccharide, total flavone and erythrina alkali in the medicinal materials are measured according to the content measuring method of each component of the beautiful millettia root medicinal material, and the transfer rate is calculated. The results are shown in Table 11.

TABLE 11 Millettia speciosa champ water extraction transfer rate conditions

From the above table, the transfer rate of the beautiful millettia root water-extracted polysaccharide is 80.0%, the transfer rate of the total flavone is 67.8%, and the transfer rate of the erythrina alkali is 68.1%.

3 screening of process for extracting volatile oil from black tiger

3.1 Effect of degree of pulverization on the extraction of Kadsura coccinea bark volatile oil

Respectively weighing 300g of non-broken material, broken material (10 mesh screen) and broken material (20 mesh screen) of kadsura coccinea, respectively placing in 5000mL round-bottomed flask, adding 10 times of water, connecting with volatile oil detector, soaking for 0.5 hr, heating and extracting for 5 hr, wherein the extraction amount of volatile oil is 0.62mL, 2.15mL and 4.25mL respectively. The extraction amount of volatile oil of the crushed material (20 meshes) of the black tiger skin is obviously higher than that of the uncrushed material and the crushed material (10 meshes of sieve) of the black tiger skin, so the crushed material (20 meshes of sieve) of the black tiger skin is preferably selected for extraction.

3.2 Effect of Water addition on the extraction of Kadsura coccinea bark volatile oil

Weighing 300g of black tiger skin broken materials (20-mesh screen), 3 parts in total, putting the black tiger skin broken materials into a 5000mL round-bottom flask, adding 8 times, 10 times and 12 times of water respectively, connecting the black tiger skin broken materials with a volatile oil tester, soaking for 0.5 hour, heating and extracting for 5 hours, wherein the extraction amounts of the volatile oil are 4.15mL, 4.08mL and 4.25mL respectively. The added water has little influence on the extraction of the kadsura coccinea volatile oil, so 8 times of water is selected for extraction.

3.3 determination of extraction time of Kadsura coccinea bark volatile oil

Weighing 300g of crushed black tiger skin (20-mesh screen), placing the crushed black tiger skin in a 5000mL round-bottom flask, adding 8 times of water, connecting the flask with a volatile oil tester, soaking for 0.5 hour, heating and extracting, recording the extraction amount of the volatile oil every 1 hour, and recording the data as shown in Table 12.

TABLE 12 extraction of kadsura coccinea volatile oil at different extraction times

As is clear from Table 12, the volatile oil amount extracted from Kadsura coccinea bark was increased little after 6 hours of extraction, indicating that the extraction was carried out for 6 hours.

The experiment can confirm that the extraction process of the kadsura coccinea volatile oil is as follows: crushing the black tiger skin (20-mesh screen), adding 8 times of water, soaking for 0.5 hour, and heating and extracting for 6 hours.

4 screening process for extracting volatile oil from frankincense and myrrh

4.1 Effect of Water addition on the extraction of Olibanum and Myrrha volatile oil

Weighing 48g of frankincense and 80g of myrrh according to a formula proportion, 3 parts in total, putting the frankincense and the myrrh into a 2000mL round-bottom flask, adding 8 times, 10 times and 12 times of water respectively, connecting the round-bottom flask with a volatile oil tester, soaking for 0.5 hour, and heating and extracting for 6 hours, wherein the extraction amount of the volatile oil is 3.62mL, 3.64mL and 3.63mL respectively. The added water has little influence on the extraction of the volatile oil of the frankincense and the myrrh, so 8 times of water is selected for extraction.

4.2 determination of extraction time of Olibanum and Myrrha volatile oil

Weighing Olibanum 48g and Myrrha 80g according to formula ratio, placing in 2000mL round bottom flask, adding 8 times of water, connecting with volatile oil tester, soaking for 0.5 hr, heating for extraction, recording volatile oil extraction amount every 1 hr, and recording data as shown in Table 13.

TABLE 13 extraction of volatile oil of Olibanum and Myrrha at different extraction times

As can be seen from Table 13, after 9 hours of extraction, the volatile oil amount from the extracts of Olibanum and Myrrha increased very little, indicating that 9 hours of extraction was sufficient.

The above experiment can confirm that the extraction process of the frankincense and myrrh volatile oil is as follows: soaking Olibanum and Myrrha in 8 times of water for 0.5 hr, and extracting under heating for 9 hr.

5 study on inclusion process of mixed volatile oil of Kadsura coccinea, frankincense and myrrh

5.1 preparation of volatile oils

Black tiger volatile oil: weighing 2880g of black tiger, separating skin part from wood part, crushing the skin part (20-mesh screen), adding 8 times of water, soaking for 0.5 hour, heating and extracting for 6 hours, and collecting volatile oil.

Frankincense and myrrh volatile oil: weighing 96g of frankincense (prepared) and 160g of myrrh (prepared), adding 8 times of water, soaking for 0.5 hour, heating and extracting for 9 hours, and collecting volatile oil.

Mixing the volatile oil of Kadsura coccinea with the volatile oil of Olibanum and Myrrha, adding anhydrous sodium sulfate to remove water, and refrigerating the volatile oil in a refrigerator at 4 deg.C to obtain mixed volatile oil.

5.2 preparation of the essential oils

Precisely measuring the mixed volatile oil of the kadsura coccinea, the frankincense and the myrrh, precisely measuring the absolute ethyl alcohol with the same amount of the mixed volatile oil, adding the absolute ethyl alcohol into the mixed volatile oil, and uniformly mixing.

5.3 preparation method of beta-cyclodextrin inclusion compound of mixed volatile oil of kadsura coccinea, frankincense and myrrh

Weighing beta-cyclodextrin required by an experiment, placing the beta-cyclodextrin into a conical flask, weighing 10 times of distilled water, adding the distilled water into the beta-cyclodextrin, heating the beta-cyclodextrin to 60 ℃ by using a magnetic stirrer, stirring until the beta-cyclodextrin is dissolved, and cooling to the temperature required by the experiment to prepare a saturated aqueous solution of the beta-cyclodextrin. Precisely measuring 2mL of a mixed volatile oil preparation solution of the kadsura coccinea, the frankincense and the myrrh, slowly adding the mixed volatile oil preparation solution into a beta-cyclodextrin saturated aqueous solution under the stirring state, and adding a rubber stopper. Mixing Kadsura coccinea, Olibanum and Myrrha, and cooling to room temperature, placing in a refrigerator at 4 deg.C, and refrigerating overnight. And (3) carrying out suction filtration on the inclusion compound solution, placing the filter cake in a vacuum drying oven at 50 ℃, drying for 3.5h, taking out the filter cake, slightly pressing the filter cake into powder, carrying out suction filtration and washing by using 20mL of petroleum ether I, repeating the process for 3 times, placing the filter cake in an oven at 50 ℃, and drying for 0.5h to obtain the mixed volatile oil beta-cyclodextrin inclusion compound of the kadsura coccinea, the frankincense and the myrrh.

5.4 preparation of the beta-cyclodextrin inclusion compound of the mixed volatile oil of black tiger, frankincense and myrrh

Selecting three factors including inclusion time (h), cyclodextrin-volatile oil ratio (g/mL) and inclusion temperature (deg.C), selecting 3 levels of each factor, and determining the inclusion concentration according to L₉(3⁴) Orthogonality table orthogonality experiments were performed as shown in table 14.

TABLE 14 orthogonal factor horizon

Evaluation indexes are as follows: inclusion rate (%) - (recovery amount of volatile oil)/(addition amount of volatile oil + blank recovery rate) × 100%

Recovery amount of volatile oil: adding the mixed volatile oil beta-cyclodextrin inclusion compound sample into a 1000mL round-bottom flask, adding 500mL distilled water and zeolite, extracting for 2h by using a steam distillation method, and standing for 1h to accurately read.

Blank recovery rate: accurately measuring 2mL of mixed volatile oil by using a pipette, adding the mixed volatile oil into a 1000mL round-bottom flask, adding 500mL of distilled water, adding zeolite, extracting for 2h by using a steam distillation method, standing for 1h, accurately reading, respectively taking 3 samples, measuring for 3 times, and measuring the average value of blank recovery. The blank recovery (%) ═ actual recovery of the mixed volatile oil)/(added amount of mixed volatile oil) × 100%.

The results of the orthogonal experiments are shown in Table 15, and the results of the analysis of variance are shown in Table 16.

TABLE 15 results of orthogonal experiments

TABLE 16 ANOVA TABLE

Note: f_0.01(2，2)＝99，F_0.05(2，2)＝19

Derived from the value of K, A₃Factor level of (A) is better than that of (A)₂And A₁，B₃Factor level of (B) is better than that of (B)₁And B₂，C₁Factor level of (2) is better than that of (C)₂And C₃The optimum horizontal combination is A₃B₃C₁. From the range results, the range Ra is 11, Rb is 50.5, Rc is 10.5, and Rd is 8.5, which affects the inclusion rate level by Rb>Ra>Rc, namely the proportion of the mixed volatile oil of the black tiger, the frankincense and the myrrh to the beta-cyclodextrin has the greatest influence, the inclusion time is less, and the inclusion temperature is the smallest. Further analysis of variance of the experimental results showed that there was no significant difference (p) between inclusion rates at each level of factor A>0.05), the factor A has no significant influence on the inclusion rate; there was no significant difference between inclusion rates at various levels of factor C (p)>0.05), the factor C has no significant influence on the inclusion rate; there was a significant difference (p) between inclusion rates at each level of factor B<0.05), factor B has a significant effect on inclusion rate. In summary, the highest inclusion rate combination is A₃B₃C₁Namely, the inclusion condition is 1.5h, preferably 1:10, 30 ℃.

For the analysis of the result of the inclusion rate variance of the experimental result, because the inclusion time has no significant influence (p is less than 0.05), the inclusion conditions are further optimized by combining the consideration of production energy saving, and the inclusion rates under the inclusion conditions of 1.5h, 1:10 and 30 ℃ and 1h, 1:10 and 30 ℃ are compared. The inclusion rate under the conditions of 1.5h, 1:10 and 30 ℃ is 80 percent, the inclusion rate under the conditions of 1h, 1:10 and 30 ℃ is 79.5 percent, and the inclusion rates under the conditions are very close, so the inclusion conditions under the conditions of 1h, 1:10 and 30 ℃ are selected as the optimal conditions in consideration of the production cost and the production benefit.

Rechecking and verifying the inclusion conditions of 1h, 1:10 and 30 ℃ respectively to obtain the inclusion rates of 79.5 percent, 80 percent and 79 percent and the RSD of 0.62 percent. Shows that under the inclusion condition, the inclusion rate is stable; the inclusion rate is high, which indicates that the inclusion method is stable and feasible.

5.5 characteristic thin-layer spots of volatile oils of Kadsura coccinea, Olibanum and Myrrha in the clathrate

Taking 1g of mixed volatile oil clathrate, adding 10mL of methanol, performing ultrasonic treatment for 30min, and taking supernatant as clathrate sample. Respectively taking 100 mu L of kadsura coccinea volatile oil, myrrh volatile oil and frankincense volatile oil, and diluting to 10mL with petroleum ether to obtain a kadsura coccinea volatile oil control solution, a myrrh volatile oil control solution and a frankincense volatile oil control solution. Testing by thin layer chromatography (0502 of the four ministerial rules of the design reside in the Chinese pharmacopoeia 2015), sucking 5 μ l of the above solutions, respectively dropping on the same silica gel G thin layer plate, developing with cyclohexane-diethyl ether (4: 1) as developing agent, taking out, air drying, spraying 5% vanillin, concentrated sulfuric acid and ethanol solution, heating at 105 deg.C for color development, and inspecting under fluorescent lamp. The results are shown in FIG. 1 as a thin layer chromatogram.

A, B, C, D refers to Olibanum volatile oil, Myrrha volatile oil, Kadsura coccinea volatile oil, and thin-layer speckle of mixed volatile oil clathrate, as shown in figure 1. The spots in the frame a are the characteristic spots of myrrh volatile oil, and the spots in the frame b are the characteristic spots of black tiger volatile oil, which can be obtained from the thin-layer chromatogram.

6 toxicity study of water extract and volatile oil of Olibanum and Myrrha

6.1 Experimental materials and instruments

Methylcellulose and tricaine were purchased from Nanjing chemical reagent one, phenylmethylsulfonyl fluoride (Biyuntian Biotechnology research institute), Penaeus chinensis (Tianjin Fengyuan Aquaculture Co., Ltd.), AB wild type zebra fish (national zebra fish resource center), and triple distilled water (self-made in laboratory).

6.2 Experimental methods

6.2.1 Zebra fish feeding and reproduction

The zebra fish is fed according to The method guided in The Zebraphis Book, The feeding environment water temperature is kept at 28.5 ℃, The daily illumination time is kept at 14h, and The zebra fish is fed with The brine shrimp once in The morning and at night. And (3) determining the night before the embryo collection time, arranging partition plates in the spawning tank, and placing equal amounts of male and female zebra fishes on two sides of the partition plates in the spawning tank in the night before the day. Under normal lighting, feeding requirements, the light source was turned off at 22:30 and turned back on the next morning at 8: 30. At this time, the partition plate in the spawning tank can be pulled away, and male and female zebra fishes can be contacted. Zebrafish embryos can be collected after 30min in spawning tanks, washed with Egg water and then placed in a light incubator at 28.5 ℃ for rearing.

6.2.2 median lethal dose test

Observing the collected zebra fish embryos, and selecting the zebra fish embryos with normal development of 1dpf (day post fertilization) by microscopic observation, wherein each group of the fed emulsion perfume extract, the frankincense volatile oil, the myrrh volatile oil extract and the myrrh volatile oil are all 30 embryos, placing the embryos in a 24-pore plate, each pore is 10 embryos, each pore is 2.0mL solution, using a culture medium as a blank control, and replacing the liquid medicine every 24 hours. And counting the death rate of the zebra fish embryo/juvenile fish after 24 hours, 48 hours, 72 hours, 96 hours and 120 hours of administration of the milk perfume extract, the frankincense volatile oil, the myrrh volatile oil extract and the myrrh volatile oil with different administration amounts, and calculating the median lethal dose.

6.3 results of the experiment

The median lethal dose of Olibanum, Myrrha extract and volatile oil is shown in Table 17.

TABLE 17 median lethal dose (mg/mL)

When the administration time is 48h, 72h and 96h, the median lethal dose of the frankincense volatile oil is more than that of the frankincense perfume extract; the administration time is 48h, 72h, 96h and 120h, and half of lethal dose of myrrh volatile oil is larger than that of the immersion liquid extract. At the time of 48h administration, the median lethal dose of the volatile oil of the frankincense is 16 times that of the aqueous extract of the frankincense, and at the time of 48h administration, the median lethal dose of the volatile oil of the myrrh is 4.7 times that of the aqueous extract of the myrrh. Therefore, the safety of the frankincense volatile oil and the myrrh volatile oil is obviously higher than that of the medicinal material water extract.

In the invention, the water extract of frankincense and myrrh is removed, and the volatile oil of frankincense and myrrh is used as the medicine.

Research on irritation of mixed volatile oil and inclusion compound of kadsura coccinea, frankincense and myrrh on gastrointestinal tracts of zebra fish

7.1 materials and instruments

Calcein and alcian blue were purchased from Shanghai Michelin Biochemical technology Ltd.

XW-80A vortex mixer (Shanghai medical university instrumentation), BSA224S electronic balance (Sartourus, USA), Research plus pipettor (Eppendorf, Germany), Pax-250B Intelligent light incubator (Ningbo Saifu laboratory instruments Co., Ltd.), SZX16 Zebra fish stereoscope (Olympus Olympus).

7.2 Experimental methods

7.2.1 Zebra fish feeding and reproduction

7.2.2 measurement of gastrointestinal motility

Observing the collected zebra fish juvenile fish, and selecting the zebra fish juvenile fish with normal development of 4dpf (day post fertilization) by microscopic observation, placing the zebra fish juvenile fish in a 24-pore plate, adding 10 juvenile fishes in each pore, adding mixed volatile oil and mixed volatile oil beta-cyclodextrin inclusion compounds with different concentrations in each pore, wherein 2.0mL of solution in each pore is used as a blank control by using a culture medium. The liquid medicine is replaced every 24 h. Experiments were performed in parallel for 3 times. The preparation method comprises the steps of administering the medicine to 5dpf, removing the medicine liquid, adding 0.2% of calcein solution, culturing for 10min at a constant temperature of 25 ℃, cleaning with artificial seawater, and continuously dripping tricaine solution (mass fraction is 0.02%) for anesthesia after 3 times of cleaning. Taking a double-concave glass slide, firstly dripping 3% methyl cellulose into the groove position, transferring the anesthetized zebra fish juvenile fish to the surface of the double-concave glass slide by using a suction pipe, and properly adjusting the position by using a hair ring, so that the zebra fish keeps a lateral and horizontal state, the eyes and the body joints of the zebra fish coincide, and the gastrointestinal peristalsis times within 1min can be conveniently observed and recorded by using a fluorescence microscope.

7.3 results of the experiment

The zebra fish is subjected to living body staining by calcein, and is shown in figure 2.

Times of gastrointestinal peristalsis of zebra fish in table 181 min

Note: p <0.05, significant differences from the control group.

As can be seen from Table 18, the number of peristalsis times of the mixed volatile oil beta-cyclodextrin inclusion compound in the gastrointestinal tract of the juvenile fish within 1min is (11.1 + -1.57), (11.5 + -1.62) and (13.4 + -1.95) times in sequence along with the increase of the concentration. The gastrointestinal tract peristalsis times of juvenile fish within 1min are not obviously abnormal under the administration doses of 0.0001mg/mL and 0.001mg/mL, and the enterokinesia times of juvenile fish in stomach and intestine within 1min are obviously abnormal under the administration doses of 0.01mg/mL and 0.01 mg/mL. The peristalsis times of the gastrointestinal tract of the juvenile fish within 1min are (13.2 +/-1.71), (14.1 +/-1.88) and (15.8 +/-2.01) times in sequence along with the increase of the concentration of the mixed volatile oil, the peristalsis times of the gastrointestinal tract of the juvenile fish can be obviously abnormal under the administration doses of 0.001mg/mL, 0.01mg/mL and 0.1mg/mL of the mixed volatile oil, and the mixed volatile oil can stimulate the peristalsis of the gastrointestinal tract under the three concentrations.

Under the administration dosage of 0.001mg/mL, the mixed volatile oil can cause remarkable abnormal gastrointestinal motility of juvenile fish, but the beta-cyclodextrin inclusion compound of the mixed volatile oil can not be generated; under the administration dosage of 0.01mg/mL, the gastrointestinal peristalsis of the juvenile fish is abnormal due to the mixed volatile oil beta-cyclodextrin inclusion compound and the mixed volatile oil, but the peristalsis frequency of the mixed volatile oil is larger, and the abnormal condition is more serious. Therefore, the irritation of the mixed volatile oil to the gastrointestinal tract of the juvenile fish is greater than that of the mixed volatile oil beta-cyclodextrin inclusion compound.

The above is a test for screening part of process conditions of the pharmaceutical composition of the invention, the labor of the invention is not limited to the above test, and the above test shows that the pharmaceutical composition of the invention can obtain a product with better effect than the original tendon-relaxing and waist-strengthening pill through the processes of continuous thinking and verification of the inventor.

Example 2

A Chinese patent medicine for treating lumbar intervertebral disc protrusion comprises the following raw material medicines: 360.0kg of rhizoma cibotii, 115.2kg of cherokee rose fruit, 216.0kg of suberect spatholobus stem, 86.4kg of philippine flemingia root, 216.0kg of kadsura coccinea, 144.0kg of beautiful millettia root, 18.0g of glossy privet fruit, 108.0kg of Chinese taxillus twig, 18.0kg of south dodder seed, 15.6kg of corydalis tuber, 15.6kg of shinyleaf pricklyash root, 7.2kg of frankincense and 12.0kg of myrrh.

1. The preparation process comprises the following steps:

(1) extracting rhizoma Cibotii with water under reflux for 2 times, adding 8 times of water for the first time and 6 times of water for the second time, extracting for 2 hours each time, filtering, mixing filtrates to obtain extractive solution, adding the extractive solution into macroporous adsorbent resin column at flow rate of 3BV/h for adsorption, washing with 5 times of column volume of aqueous solution at flow rate of 4BV/h until effluent is colorless, eluting with 3 times of column volume of 70% ethanol at flow rate of 3BV/h, collecting ethanol eluate, recovering ethanol under reduced pressure, and concentrating to obtain thick paste 34.56kg (relative density 1.30, 82 deg.C, solid content 68%);

(2) crushing radix Millettiae Speciosae, sieving with 8 mesh sieve, mixing with fructus Rosae Laevigatae, caulis Spatholobi, radix Flemingiae Philippinensis, herba Taxilli, radix Zanthoxyli, and caulis Kadsurae Coccineae, adding 8 times of water for the first time, soaking for 1 hr, extracting for 4 hr, adding 6 times of water for the second time, extracting for 2 hr, filtering, mixing filtrates, and concentrating to obtain 75.63kg of soft extract (relative density 1.32, 79 deg.C, solid content 70%);

(3) crushing the skin of the black tiger, sieving the crushed skin with a 20-mesh sieve, adding 8 times of water to soak the crushed skin for 0.5 hour, heating and extracting the crushed skin for 6 hours, and collecting the volatile oil of the black tiger; soaking Olibanum (preparata) and Myrrha (preparata) in 8 times of water for 0.5 hr, heating and extracting for 9 hr, and collecting volatile oil of Olibanum and Myrrha; mixing the kadsura coccinea volatile oil with the frankincense and the myrrh volatile oil, adding ethanol with the same amount, uniformly mixing, clathrating the volatile oil and the beta-cyclodextrin according to a ratio of 1:10 of the volatile oil to the beta-cyclodextrin by a water saturated solution method at the temperature of 30 ℃ for 1h, carrying out suction filtration on the clathrate solution, and drying a filter cake in a vacuum drying oven at the temperature of 50 ℃ for 5h to obtain 16.31kg of the beta-cyclodextrin clathrate compound of the mixed volatile oil of the kadsura coccinea, the frankincense and the myrrh;

(4) filtering the water extract of the kadsura coccinea part from which the volatile oil is extracted, collecting the filtrate, and concentrating to obtain 10.89kg of thick paste (the relative density is 1.29, the temperature is 75 ℃, and the solid content is 63%);

(5) pulverizing the rest fructus Ligustri Lucidi, semen Cuscutae, and rhizoma corydalis to obtain fine powder 49.02kg, adding volatile oil clathrate, starch 2.50kg and microcrystalline cellulose 2.50kg, mixing, adding soft extract, mixing, making pill, coating with active carbon, drying, and polishing to obtain pill (concentrated watered pill) 150 kg. The dosage of the original muscle-relaxing waist-strengthening pill can be about 750kg, the dosage is 5 g/time, and the dosage of the muscle-relaxing waist-strengthening pill (concentrated water-paste pill) can be reduced to about 1g per time.

2. Detection of erythrine in sinkiang arnebia root pill (concentrated water pill) for relaxing muscles and tendons and strengthening waist

Precisely weighing a proper amount of erythrina alkali reference substance, adding methanol for dissolving, and preparing a reference substance solution with a certain concentration (S1); precisely weighing 1g of beautiful millettia root medicinal material (S2), 2.5g of sinews relaxing and waist strengthening pills (concentrated water pills) (S3) and 2.5g of sinews relaxing and waist strengthening pills (concentrated water pills are negative to beautiful millettia root), respectively adding 50mL of 50% ethanol, and determining according to the content determination (2.1.3.1) of the beautiful millettia root medicinal material erythrina alkali to obtain a plasma (TIC) diagram, wherein the result is shown in a TIC superposed diagram of the sinews relaxing and waist strengthening pills (concentrated water pills) (Table 19).

TABLE 19 transfer rate of erythrina base in samples

According to the experimental result, after the millettia speciosa champ is subjected to water extraction, the sineurine is detected by the sinewing and waist-strengthening pill (concentrated water pill), and the sineurine is not detected by the sinewing and waist-strengthening pill (concentrated water pill negative), so that the sinewing and waist-strengthening pill (concentrated water pill) can be detected after the millettia speciosa champ is subjected to water extraction, and the transfer rate of the sineurine in a finished product is 67.5%.

3. Radix Zanthoxyli detection of pill (concentrated watered pill) for relaxing muscles and tendons and strengthening waist

Preparing a test sample: taking about 1g of muscle-relaxing waist-strengthening pills (concentrated water pills), muscle-relaxing waist-strengthening pills (the two sides of the concentrated water pills are negative), and double-side needle medicinal material powder (passing through a third sieve), precisely weighing, placing in a 50mL conical flask with a plug, adding 20mL of 70% methanol by using a pipette, carrying out ultrasonic treatment for 30min, cooling, filtering, placing filtrate in a 50mL volumetric flask, adding 20mL of 70% methanol into filter residue and filter paper, carrying out ultrasonic treatment for 30min, cooling, filtering, placing the filtrate in the same volumetric flask, washing for 2 times by using a proper amount of 70% methanol, merging the washing liquor into the same volumetric flask, fixing the volume to the scale by using 70% methanol, and shaking uniformly to obtain the traditional Chinese medicine.

Preparation of a reference substance: taking a proper amount of nitidine chloride reference substance, precisely weighing, and preparing 1mL of solution containing 5 μ g with 70% methanol to obtain the final product.

Chromatographic conditions and chromatographic methods: using octadecylsilane chemically bonded silica filler as chromatographic column, at 35 deg.C and flow rate of 0.5mL/min, mobile phase A is acetonitrile, B is 0.1% formic acid water solution, and the mobile phase conditions are shown in Table 20:

TABLE 20 conditions of mobile phase

Mass spectrum conditions: a positive ion mode; probe; temperature: 600, preparing a mixture; capillary: 0.8; mode (2): SIR; mass: 348.12, respectively; cone Voltage: 15V.

The determination method comprises the following steps: respectively and precisely sucking 2 mul of the solution to be injected into a Waters Arc-PUV-QDa ultra performance liquid chromatography for determination.

The results are shown in figure 4, a stack of nitidine and TIC of Shujin Jianyao Wan (concentrated watered pill).

The experiments show that: the nitidine is detected in the muscle-relaxing and waist-strengthening pill (concentrated water pill) by water extraction, and the nitidine is not detected in the muscle-relaxing and waist-strengthening pill (negative on the two sides of the concentrated water pill), which indicates that the negative is not interfered.

4. Examination of properties, appearance, water content, dissolution time limit and weight difference of muscle relaxing and waist strengthening pill (concentrated watered pill)

4.1 Properties

The muscle relaxing and waist strengthening pill (concentrated water pill) is black coated concentrated water pill, and appears black brown after removing the coating; sweet, astringent and slightly bitter.

4.2 appearance

The muscle-relaxing waist-strengthening pill (concentrated water pill) is round, uniform in size and color, and has no adhesion phenomenon.

4.3 moisture content

The water content of the muscle-relaxing waist-strengthening pill (concentrated water pill) is 6.1 percent, the water content requirement of 0108 pill rules in the fourth part of China pharmacopoeia of 2020 edition is met, and the water content of the concentrated water pill is not more than 9.0 percent.

4.4 time limit of dissolution

The time limit of dissolving and dispersing of the muscle-relaxing waist-strengthening pill (concentrated water-paste pill) is 45 minutes, which meets the requirement of 0108 pill rules of the fourth part of the world in the 2020 edition of Chinese pharmacopoeia, and the concentrated water-paste pill should be completely dissolved and dispersed within 2 hours.

4.5 weight Difference

The weight of each pill of the muscle-relaxing waist-strengthening pills (concentrated water pills) is 0.05g, and the weight difference meets the general regulation requirement (+/-12%) of 0108 pills in the fourth part of the four parts of the Chinese pharmacopoeia of 2020 edition.

5. Stability study of tendon-relaxing waist-strengthening pill (concentrated water-paste pill)

5.1 stability test protocol

Constant temperature and humidity accelerated test: the muscle-relaxing waist-strengthening pill (concentrated water pill) is packaged by a medical high-density polyethylene plastic bottle, is placed in a stability test box environment with the temperature adjusted to 40 +/-2 ℃ and the relative humidity of 75% +/-5% for storage for 6 months, and is respectively subjected to sampling inspection once at the end of 0 month, 1 month, 2 months, 3 months and 6 months of the placement. The manufacturer of the stability test box is Chongqing Chuangshi Co., Ltd, and the model is CSH-222 SD-C.

5.2 measurement of the content of specnuezhenide

The measurement is carried out according to high performance liquid chromatography (China pharmacopoeia 2015 edition of the general rules 0512 in four parts).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water (17:83) is used as a mobile phase; the detection wavelength was 224 nm. The number of theoretical plates is not less than 5000 calculated according to the specnuezhenide peak.

Preparation of reference substance solution A proper amount of specnuezhenide reference substance is precisely weighed, and 80% methanol is added to prepare a solution containing 65 μ g per 1 ml.

Preparing a test solution, taking a proper amount of the test solution, grinding, taking about 1g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 80% methanol, weighing, carrying out ultrasonic treatment (power is 250W, frequency is 37kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 80% methanol, shaking up, filtering, precisely taking 20ml of subsequent filtrate, drying by distillation, dissolving residues with 5ml of 80% methanol, adding on a neutral alumina column (100-200 meshes, 5g, inner diameter is about 1.5cm), eluting with 50ml of 80% methanol, collecting eluent, drying by distillation, dissolving residues with 80% methanol, transferring to a 10ml measuring flask, adding 80% methanol to dilute to scale, shaking up, filtering, and taking subsequent filtrate.

The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

The results are shown in Table 21.

TABLE 21 determination of specnuezhenide content

The muscle-relaxing waist-strengthening pill (concentrated water pill) is shown by a 6-month acceleration test: the content of fructus Ligustri Lucidi in the pill (concentrated watered pill) has stable trend and stable product quality.

Experimental example 1

Adopting positive and model control, Chinese patent medicine Shujin Jianyao pill (concentrated water pill) for experimental study of treating lumbar disc herniation.

1. Materials and methods

1.1 Experimental animal selection and grouping

4-week-old SD rats, male, animal weight range at the time of molding: 177-266 g, animal weight range when grouping: 220-339 g. Randomized into 5 groups: a: a sham operation group; b: a model group; c: positive control (fenmust) group; d: muscle-relaxing waist-strengthening pills (concentrated water pills) in the example group; e: original Shujin Jianyao Wan group. The negative control group had 21 individuals, and the remaining groups had 22 to 24 individuals. The source is as follows: the Hunan Slek Viewa laboratory animals Co., Ltd.

1.2 test methods

(1) Negative control, test substance, and positive control

Negative control-0.5% sodium carboxymethylcellulose (CMC-Na): weighing CMC-Na, adding pure water to dissolve, and preparing into 0.5% CMC-Na solution, wherein sodium carboxymethylcellulose (CMC-Na) is purchased from Dache chemical reagent factory of Tianjin.

Test article-example: weighing a certain amount of the muscle-relaxing waist-strengthening pills (concentrated water pills) prepared in the embodiment, crushing into fine powder, adding 0.5% CMC-Na solution, stirring and dissolving until the mixture is uniform and has no lumps, and preparing into a liquid medicine with the concentration of 0.027g/mL (equivalent to 2.19g crude drug/kg, which is about 1 time of 3g of the clinical daily dose of an adult).

③ Positive control-Fenbidus phenol currant: taking 1 tablet of fenbide phenolic currant tablet, grinding, adding 0.5% CMC-Na into the medicinal powder while stirring the solution until no lump exists, preparing the total amount to be 8.85mL, and preparing the mother solution with the concentration of 0.0565 g/mL; then, an appropriate amount of the mother liquor was taken up and diluted with 0.5% CMC-Na solution to a chemical solution with a concentration of 0.0051 g/mL. Wherein the Fenbide phenol currant tablet manufacturer is Zhongmei Tianjin Shike pharmacy Co.

Fourthly, the original muscle-relaxing waist-strengthening pill: weighing a certain amount of the muscle-relaxing waist-strengthening pill in the original dosage form, crushing into fine powder, adding 0.5% CMC-Na solution, stirring and dissolving until the mixture is uniform and has no lumps, and preparing into liquid medicine with the concentration of 0.135g/mL (equivalent to 2.19g of crude drug/kg, which is about 1 time of 15g of clinical daily dosage of adults).

(2) Primary reagent

Sodium pentobarbital: a solution with a concentration of 20mg/mL was prepared. The sodium pentobarbital is weighed to be 100mg, dissolved by a proper amount of 0.9 percent sodium chloride injection and added to be 5mL for preparation. The manufacturer is Germany Merck (split charging of Biotech technologies, Inc. of Huamei, Beijing).

② sodium penicillin for injection: the solution with the concentration of 16 ten thousand units/mL is prepared. Taking 1 bottle of penicillin sodium for injection each time, dissolving the penicillin sodium in a proper amount of 0.9% sodium chloride injection, and adding the dissolved penicillin sodium into 10mL of the solution to prepare the solution. The manufacturer is Hebei Yuanzhen pharmaceutical Co.

(3) Solvent

The solvent for preparing the test substance and the fenbifide paracetamol and caffeine tablet is 0.5 percent of sodium carboxymethyl cellulose (CMC-Na) (analytically pure), and the manufacturer is Daoqin chemical reagent factory in Tianjin.

② the menstruum used for preparing the sodium pentobarbital and the sodium penicillin for injection is 0.9 percent sodium chloride injection with the specification of 100 mL/bottle, and the manufacturer is Guangdong Konlun pharmaceutical Co.

(4) The test steps are as follows:

before molding (T0), animals qualified for quarantine are selected for molding, the number of the false operation groups is 21, and the remaining qualified animals are molded in 5 batches. When the model is made, animals qualified in quarantine are selected to be subjected to abdominal cavity anesthesia, 2% pentobarbital sodium (3mL/kg) is given to each animal for anesthesia, the animal is fixed in a prone position, iodine tincture and alcohol are sequentially disinfected, an incision about 4cm is made in the middle of the back by taking the L5/L6 and the L5/L6 spinous process gap as the center, the skin and subcutaneous tissues are cut layer by layer, the lumbar paraspinal muscles are separated, the vertebral lamina is deeply reached, and the transverse process and the intervertebral foramen of the L5 are exposed. An L-shaped titanium rod 0.8mm in diameter and 4mm in length was introduced into the foramen at L5 at an angle of about 4mm at the dorsal midline of about 30-40 ° and-10 ° below the horizontal line of the vertebral body by 15 °. When the steel rod presses against the ganglion, the ipsilateral hind limb flesh usually exhibits one or two slight twitches. In addition, the middle of the back of the rat is incised by taking lumbar vertebrae L5-L6 as a center, a scalpel is used for making a longitudinal incision with the length of about 2cm, tissue forceps pull skin and subcutaneous tissues towards the two sides of the incision, and blood vessel forceps are used for separating and exposing 2 segments of intervertebral fibrous rings at the tail of the rat. When the tip of the scalpel is used to make an incision on the annulus fibrosus, the tail intervertebral of the rat is squeezed, and the clear gelatinous nucleus pulposus tissue protrudes from the incision of the annulus fibrosus. The 2-segment nucleus was scraped with a curette, and the wound cover was removed from the back of the rat, and the nucleus tissue was carefully transplanted into the intervertebral hole of L5 with forceps. After placement, the back muscles and skin were sutured layer by layer, and the tail incision was sutured as well. The animals in the sham operation group were not removed, placed the nucleus pulposus of the tail intervertebral disc and not placed the L-shaped steel rod. The molding was completed and the right muscle of each animal was injected continuously with 3 days of penicillin to prevent infection. The model is continuously molded for 5 days, each batch of molded animals are randomly and evenly divided into other 4 groups according to the animal motion function score and the body weight on the 8 th day of molding (T8), and each group comprises 22-24 animals which are all male. And performing intragastric administration after grouping. After the febuxostat group is continuously administrated for 5 days, the administration is carried out once every 2 days, and the administration is carried out for 29 days continuously for other animals in each group 1 time per day. After 1h of total anesthesia on day 29 after the last dose, blood was collected for biochemical examinations.

TABLE 22 summary of group and dosage design

2. Test results

TABLE 23 influence of SHUJINJIANYA pill on serum IgG, IgM, IL-1, IL-6, COX-2, and PGE-2 of rat with prolapse of lumbar intervertebral disc

Note: compared with the false operation group, the operation table has the advantages that,^*P＜0.05，^**P＜0.01，^***p is less than 0.001; in comparison to the set of models,^#P＜0.05，^##P＜0.01，^###p is less than 0.001, the pill with functions of relieving rigidity of muscles and strengthening waist has influence on pathological changes of CGRP, SP, IL-1, NO, PGE2 and COX-2 of dorsal root ganglion of rat with lumbar intervertebral disc protrusion

Note: compared with the false operation group, the operation table has the advantages that,^*P＜0.05，^**P＜0.01，^***p is less than 0.001; in comparison to the set of models,^#P＜0.05，^##P＜0.01，^###p is less than 0.001; compared with the original pill group for relaxing muscles and tendons and strengthening waist,^△P＜0.05，^△△P＜0.01，^△△△P＜0.001

under the test condition, the muscle-relaxing and waist-strengthening pill for the test can reduce the levels of inflammatory factors IL-1 and oxidation and pain related factors (NO, PGE2) at the serum and lesion positions of rats and reduce the concentration of peptide neurotransmitter (CGRP, SP) at the lesion positions of dorsal root ganglia, and preliminarily prompts that the muscle-relaxing and waist-strengthening pill for the test possibly plays a role in inhibiting inflammatory reaction, oxidative stress, improving nerve root pain and other targets. The concentrations of inflammatory factors IL-1, oxidation and pain related factors (NO, PGE2) and dorsal root ganglion lesion site peptide neurotransmitter (CGRP and SP) of rat serum and lesion sites in the test substance tendon-relaxing and waist-strengthening pill (concentrated water pill) embodiment group are lower than the level of the original tendon-relaxing and waist-strengthening pill group, and the difference of CGRP of lesion sites has statistical significance, which preliminarily suggests that the tendon-relaxing and waist-strengthening pill (concentrated water pill) of the invention has better effect on improving dorsal root neuropathic pain than the original tendon-relaxing and waist-strengthening pill.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The preparation method of the Chinese patent medicine for treating the lumbar intervertebral disc protrusion is characterized by comprising the following raw materials in parts by weight:

preparing thick paste 1: extracting 800 parts of rhizoma Cibotii 400 with water under reflux, filtering to obtain extractive solution, adsorbing the extractive solution with macroporous adsorbent resin column, washing the resin with water until the water eluate is colorless, eluting with 50-80% ethanol, collecting ethanol eluate, and concentrating to obtain soft extract;

preparing thick paste 2: soaking 350 parts of beautiful millettia root, 100 parts of cherokee rose fruit, 300 parts of suberect spatholobus stem, 100 parts of philippine flemingia root, 500 parts of Japanese ardisia herb, 10-60 parts of shinyleaf pricklyash root and 300 parts of Chinese taxillus twig in water, decocting, filtering, collecting filtrate, and concentrating into thick paste;

preparing a volatile oil clathrate compound: crushing the bark of the black tiger, sieving the crushed bark with a 10-20-mesh sieve, soaking the crushed bark in water, heating and extracting, collecting the volatile oil of the black tiger, soaking 10-30 parts of prepared frankincense and 4-30 parts of prepared myrrh in water, heating and extracting, and collecting the volatile oil of the frankincense and the myrrh; mixing the volatile oil of Kadsura coccinea with the volatile oil of Olibanum and Myrrha, and clathrating to obtain volatile oil clathrate;

2. The preparation method according to claim 1, wherein the step of preparing the thick paste 1 comprises the following steps: reflux-extracting rhizoma Cibotii for 1-3 times for 1-3 hr, filtering, mixing filtrates to obtain extractive solution, adsorbing with macroporous adsorbent resin column, washing with water until the water eluate is colorless, eluting with 3-5 times column volume of 50-80% ethanol, collecting ethanol eluate, and concentrating to obtain soft extract.

3. The preparation method according to claim 1, wherein the step of preparing the thick paste 2 comprises the following steps: crushing radix Millettiae Speciosae, sieving with 5-12 mesh sieve, soaking with fructus Rosae Laevigatae, caulis Spatholobi, radix Flemingiae Philippinensis, caulis Kadsurae Coccineae, herba Taxilli, and radix Zanthoxyli in water for 0.5-3 hr, with the water amount being 8-12 times of solid mass, decocting for 1-4 hr for 1-3 times, filtering, mixing filtrates, and concentrating into soft extract.

4. The preparation method according to claim 1, wherein in the step of preparing the volatile oil inclusion compound, the bark of the black tiger is crushed and sieved by a 10-20 mesh sieve, water is added for soaking for 0.5-1h, heating and extracting are carried out for 4-10h, and the volatile oil is collected.

5. The preparation method of claim 1, wherein the Olibanum and Myrrha are soaked in water for 0.5-1 hr, heated and extracted for 6-15 hr, and the volatile oil is collected.

6. The preparation method according to claim 1, wherein volatile oils of Kadsura coccinea, Olibanum and Myrrha are mixed, and the beta-cyclodextrin is included by saturated aqueous solution method or grinding method.

7. The preparation method of claim 6, wherein in the step of preparing the volatile oil inclusion compound, the proportion of the mixed volatile oil of the black tiger, the frankincense and the myrrh to the beta-cyclodextrin is 1:6-10, the inclusion temperature is 20-50 ℃, and the inclusion time is 10min-3 h.

8. The method of claim 1, further comprising the step of: and preparing the mixture of the Chinese patent medicines into pills, capsules or tablets.

9. The method of preparation according to claim 8, wherein the preparation of the pellets is obtained by: mixing the soft extract 1, soft extract 2, soft extract 3, fine powder, volatile oil clathrate, appropriate amount of starch and microcrystalline cellulose, making into pill, coating with medicinal active carbon, drying, and polishing to obtain concentrated watered pill.

10. A Chinese patent medicine obtained by the preparation method of any one of claims 1 to 9.

11. Use of a Chinese patent medicine obtained by the preparation method of any one of claims 1-9 in the preparation of a medicament for treating lumbar intervertebral disc protrusion.