CN113230317A

CN113230317A - Transdermal drug delivery preparation for treating knee osteoarthritis and preparation method thereof

Info

Publication number: CN113230317A
Application number: CN202110521501.4A
Authority: CN
Inventors: 张永萍; 徐剑; 刘耀; 杨芳芳; 缪艳燕; 曹国琼; 成雪
Original assignee: Guizhou University of Traditional Chinese Medicine
Current assignee: Guizhou University of Traditional Chinese Medicine
Priority date: 2021-05-13
Filing date: 2021-05-13
Publication date: 2021-08-10
Anticipated expiration: 2041-05-13
Also published as: CN113230317B

Abstract

The invention discloses a transdermal drug delivery preparation for treating knee osteoarthritis and a preparation method thereof. The medicine has the effects of promoting blood circulation, relieving pain, removing dampness, removing blood stasis, relaxing tendons and activating collaterals, and can effectively treat knee osteoarthritis; the prepared microemulsion has small particle size, improved drug stability, promoted drug absorption, high biocompatibility, prolonged action time of the drug and prolonged drug effect.

Description

Transdermal drug delivery preparation for treating knee osteoarthritis and preparation method thereof

Technical Field

The invention relates to a transdermal drug delivery preparation (also called crossbow drug) for treating knee osteoarthritis and a preparation method thereof, belonging to the technical field of drugs.

Background

Crossbow medicine was first described in "the book of kanghai" (five elements) published in the western jin of china in 266 to 316 b.c.. In the process of hunting, the Miers of the Miao nationality often extract a crossbow drug from some highly toxic drugs so as to achieve the effects of meeting blood and sealing throat and poisoning and killing prey. In long-term production and living practices, the inventor of the Miao nationality finds that medicines for promoting blood circulation, dispelling wind, expelling toxin and expelling water are added in crossbow medicines to achieve the effect of treating diseases through hundreds of times of practice. Then, the attenuated medicine is added to act on human body. The crossbow medicine needle is called as a crossbow needle when appearing in history for the first time, and is evolved according to a medicine arrow method for mounting crossbow arrows, a Miao nationality obtains materials in situ, one end of the arrow hair (other wood sticks or chopsticks can be used) of the hedgehog is flattened, three or five cotton thread needles are taken, the ends with holes are inserted into the flattening place to form a product character or a plum blossom shape, the needle point is only exposed for about one minute (about 4mm), the crossbow medicine liquid is dipped, and the skin of an affected part is tapped to achieve the purposes of opening orifices, releasing pathogenic toxins and assisting medicine absorption. In the period of Han dynasty, there is a literature on the treatment of diseases by using a pricking therapy (consisting of crossbow drug and needle). In China, related documents also record in detail that people in Guanling county, Guizhou province use crossbow pharmacotherapy to treat diseases in recent times. The crossbow medicine needle therapy is to use a needle tool to dip and take crossbow medicine liquid, prick the pain part, and facilitate the transdermal absorption of the medicine through destroying the cuticle of the skin, so that the medicine can quickly reach the affected part. The crossbow drug needle can avoid the pain and side effect of oral administration, intravenous administration and intramuscular administration to the liver and kidney. The crossbow medicine needle is one of the original forms of the Miao nationality for treating diseases, and is popularized and used in the traditional Chinese medicine clinical at present.

In recent years, with the development of crossbow drug injection therapy and the intensive research on action mechanism, crossbow drug injection has good treatment effect on several diseases and is considered to be superior to other conventional Chinese and western medicines. Crossbow needles have been reported to be widely used in the treatment of: knee osteoarthritis, lumbar disc herniation, radiculocervical spondylosis, postherpetic neuralgia, rheumatoid arthritis.

The traditional crossbow liquid medicine is prepared by soaking the medicinal materials in edible white spirit for 14 days, combining crossbow medicine with a needle, dipping the medicine with the needle to prick an affected part when treating diseases, and then smearing the crossbow liquid medicine on the affected part. The steps are complicated when the crossbow medicine and the needle are used together for treating diseases, the compliance of a patient is poor, the medicine consumption is inaccurate, and the use is inconvenient. When the traditional crossbow medicine needle is used for treating diseases, 3-5 thin needles are tied at the upper end of a wood handle, and a patient is pricked at a pain position by dipping crossbow medicine liquid. The method has inaccurate dosage and cross infection, and although the crossbow medicine needle is clinically improved at present, the pain of the patient is still severe during treatment, and the compliance is poor.

Disclosure of Invention

The invention aims to provide a transdermal drug delivery preparation for treating knee osteoarthritis and a preparation method thereof. The medicine has the effects of promoting blood circulation, relieving pain, removing dampness, removing blood stasis, relaxing tendons and activating collaterals, and can effectively treat knee osteoarthritis; the prepared microemulsion has small particle size, improved drug stability, promoted drug absorption, high biocompatibility, prolonged action time of the drug and prolonged drug effect.

The technical scheme of the invention is as follows: a transdermal drug delivery preparation for treating knee osteoarthritis is prepared from 5-40 parts of raw kusnezoff monkshood root, 5-25 parts of white dragon beard, 5-40 parts of caulis periplocae, 30-70 parts of caulis perllae, and 30-70 parts of sargentgloryvine stem.

In the transdermal drug delivery preparation for treating knee osteoarthritis, the medicinal effective components of the drug are prepared from 10-30 parts of raw kusnezoff monkshood root, 10-20 parts of white dragon beard, 10-30 parts of periploca forrestii, 40-60 parts of lysimachia christinae hance and 40-60 parts of sargentgloryvine stem by weight.

In the transdermal drug delivery preparation for treating knee osteoarthritis, the medicinal effective components of the drug are prepared from 20 parts by weight of raw kusnezoff monkshood root, 15 parts by weight of white dragon beard, 30 parts by weight of caulis periplocae, 50 parts by weight of caulis periplocae and 50 parts by weight of sargentgloryvine stem.

The preparation method of the transdermal drug delivery preparation for treating knee osteoarthritis comprises the steps of combining the traditional Chinese medicines in the formula with auxiliary materials acceptable in the medicines, processing the mixture according to a conventional method, and preparing the corresponding preparation.

In the preparation method of the transdermal drug delivery preparation for treating knee osteoarthritis, the drug is an external preparation.

In the preparation method of the transdermal drug delivery preparation for treating knee osteoarthritis, the external preparation is microemulsion.

In the preparation method of the transdermal drug delivery preparation for treating knee osteoarthritis, the microemulsion is prepared by the following steps: weighing the traditional Chinese medicines according to the proportion, adding ethanol, refluxing and extracting for 3 times, filtering, recovering ethanol under reduced pressure, and drying in vacuum to obtain dry extract; adding isopropyl myristate, polyoxyethylene castor oil-35, and anhydrous alcohol into container, stirring, adding dry extract, stirring, adding water, and stirring to completely emulsify to obtain microemulsion.

In the preparation method of the transdermal drug delivery preparation for treating knee osteoarthritis, the dry extract is obtained by weighing the traditional Chinese medicines according to the proportion, adding 70% ethanol in an amount which is 12 times that of the traditional Chinese medicines, carrying out reflux extraction for 1 hour each time, carrying out extraction for 3 times in total, filtering, recovering ethanol under reduced pressure, and carrying out vacuum drying.

In the preparation method of the transdermal drug delivery preparation for treating knee osteoarthritis, the mass ratio of the isopropyl myristate to the polyoxyethylene castor oil-35 to the absolute ethyl alcohol to the water to the dry extract is 15:86.25:28.75:370: 15.

In the preparation method of the transdermal drug delivery preparation for treating knee osteoarthritis, the stirring temperature of the isopropyl myristate, the polyoxyethylene castor oil-35 and the absolute ethyl alcohol is 25 ℃, and the rotating speed is 300 r/min.

The invention is prepared by five traditional Chinese medicines of raw kusnezoff monkshood root, white dragon beard, black bone vine, Tou Gu Xiang and sargentgloryvine stem,

wherein the root tuber is selected from Aconitum carmichaeli Debx (wild species) of Ranunculaceae, Aconitum kusnezoffii nakai or other plants of the same genus; the functional indications are as follows: dispelling wind and eliminating dampness, dispelling cold and relieving pain, eliminating phlegm and relieving swelling; it is used to treat arthralgia due to wind-cold-dampness, paralysis due to apoplexy, tetanus, wind syndrome, abdominal psychroalgia, phlegm nodule, qi stagnation, cold dysentery, pharyngitis, carbuncle, furuncle, and lymphoid tuberculosis.

The white dragon beard is root, fibrous root or root bark of Alangium platanifolium or guava of Alangium platanifolium of Alangiaceae. The functional indications are as follows: dispelling pathogenic wind, dredging collaterals, removing blood stasis, relieving pain, and also has effects of anesthesia and relaxing muscle; can be used for treating rheumatalgia, numbness, paralysis, heart failure, lumbago due to strain, and traumatic injury.

Caulis et folium Periplocae Forrestii is root or whole plant of herba Polygoni Perfoliati of Asclepiadaceae plant. The functional indications are as follows: dredging channels and collaterals, dispelling pathogenic wind and dampness, promoting blood circulation, and relieving inflammation. It can be used for treating traumatic injury, rheumatic arthralgia, menoxenia, stomatitis, and mastitis.

Caulis et folium Gaultheriae Yunnanensis is stem and leaf of Yunnan Baizhu tree of Ericaceae. The functional indications are as follows: dispel wind and dampness, activate blood and dredge collaterals. It is used to treat rheumatic arthralgia, traumatic injury, toothache and eczema.

Caulis Sargentodoxae is rattan or root of Schisandra sphenanthera (Variegata) or Schisandra sphenanthera (Variegata) of Magnoliaceae. The functional indications are as follows: nourish blood and resolve stasis, regulate qi and resolve dampness. Can be used for treating blood vomiting due to tuberculosis, soreness and pain of limbs and joints, pain of heart and stomach, tinea pedis, flaccidity, arthralgia, menoxenia, and traumatic injury.

Square solution: in the formula, raw kusnezoff monkshood root and Tou Gu Xiang are used as the monarch drugs, raw kusnezoff monkshood root is a product with big pungent and big heat, which enters heart, liver and spleen channels and has the functions of dispelling wind and cold, removing dampness and relieving arthralgia, and especially has good effect of relieving pain; the Tou Gu is fragrant and pungent in flavor, warm in nature, enters lung, liver and kidney meridians, and has the effects of relieving exterior syndrome, dispelling cold, dispelling wind and removing dampness, and promoting blood circulation to arrest pain. The two medicines are used together to dispel wind and remove dampness to dredge vessels, remove stasis and relieve pain to disperse arthralgia, and enhance the functions of activating collaterals and relieving pain. The caulis et folium fici Tikouae and the white dragon beard are used as ministerial drugs, the caulis et folium fici Tikouae is pungent in flavor, warm in nature, capable of entering heart and kidney channels, good for dispelling wind-damp, dredging channels and collaterals and activating blood; the white dragon beard is pungent in flavor and warm in nature, enters heart and liver channels, has the effects of dispelling wind and removing dampness, removing blood stasis and relieving pain, and relaxing tendons and activating collaterals, and the combination of the two medicines assists the monarch medicines in dispelling cold, removing blood stasis and activating collaterals. The sargentgloryvine stem is used together with the sargentgloryvine stem, has mild nature, is good at detoxifying and eliminating carbuncle, activating blood and relieving pain, dispelling wind and eliminating dampness, and is used together with monarch drugs for treating high fever. The five medicines are combined to play the efficacies of promoting blood circulation, relieving pain, removing dampness, dissipating blood stasis, relaxing muscles and tendons and activating collaterals.

Examples of the experiments

First, pharmacodynamic study

1 Material

1.1 animals

SD rats, each half of male and female, with a body mass of 180-: SCXK (military) 2012-. Sufficient water and rat chow were given, water was allowed to freely drink and fed acclimatically for 1 week.

1.2 drugs and reagents

Raw kusnezoff monkshood root, caulis periplocae, sargentgloryvine stem, caulis perllae, and white dragon beard (all purchased from Taisheng medicinal material market in Guiyang city orchard); chloral hydrate (batch number: 2015050501, new corson chemical reagent factory, metropolis); 75% Disinfection alcohol (batch No. 180826, Kongtaili Disinfection products, Inc., Guizhou); 4% paraformaldehyde (batch No. 1804895, biosharp Life science); hematoxylin (batch No. 180634, Nanjing Saibo Biotech Co., Ltd.); sitagliptin (batch number: VP1235, Beijing Nowa pharmaceutical Co., Ltd.); IL-1 beta, IL-6, TNF-alpha enzyme-linked immunosorbent assay (Elisa) kit (batch No.: 1218E18, Nanjing Saibo Biotech Co., Ltd.); papain (batch No.: BCCB8238, Sigma, USA).

1.3 instruments

BCD-201STPA refrigerator (Qingdao Haier Co., Ltd.); a microplate reader (Multlskan Mk3, manufactured by Sammer Feishel instruments Co., Ltd.); -80 ℃ refrigerator (Qingdao Haier Special appliances Co., Ltd.); rotary wheel slicers (come-2016; germany); TSJ-II type fully automatic closed tissue dehydrator (Wien electronics Co., Ltd., Changzhou city); BMJ-III type embedding machine (Wei electronics, Inc. in the suburbs of Changzhou); PHY-III type bleaching and baking instrument for pathological tissues (Wei electronic instruments, Inc. in Changzhou city); digital trinocular video microscopes (BA400Digital, mcondi industries group ltd); image analysis software Image-Pro Plus6.0(Media Cybernetics; USA).

2 method

2.1 grouping and modeling of rats

SD rats after being adaptively fed for one week are randomly divided into a blank group, a model group, a crossbow drug high dose group, a crossbow drug medium dose group, a crossbow drug low dose group, a positive group and 8 rats in each group. Rats were anesthetized with 10% chloral hydrate (0.3mL/100g), fixed on an operating table, and left hind leg rat hair was carefully removed with scissors without cutting the rat skin and exposing the entire knee joint. Except for the blank group, 0.2mL of 4% papain solution was slowly injected into the left knee joint cavity from the inferior-medial aspect of the patella at 45 degrees, while 0.2mL of physiological saline was injected into the blank group at 1st, 4 th and 7 th days, and after 3 injections, the blank group was kept for 1 week as usual.

2.2 preparation of the reagent

According to the clinical prescription of the crossbow medicine, raw kusnezoff monkshood root 20g, caulis periplocae 30g, white dragon beard 15g, sargentgloryvine stem 50g and caulis et folium gaultheriae yunnanensis 50g are 165g in total. Soaking the crossbow in 1000mL of 50% ethanol for 14 days to obtain crossbow liquid medicine. The effective dose of the rat is converted according to the clinical dosage of the crossbow drug, namely the dosage of the high, middle and low groups of the crossbow drug is 2.25mL, 1.1mL and 0.56mL respectively.

2.3 rat dosing

1d before administration, the hair of the rat at the joint of the left rear leg and knee of the rat is carefully cut off by scissors, and administration is started after 24h observation without damage. Applying the medicinal liquid on the knee joint and the joint periphery of the left hind leg, gently kneading the joint position until the medicinal liquid is administered for 1 time/d each time, and continuously administering for 30 days, wherein the dosages of the high, middle and low groups of crossbow medicines are respectively 2.25 mL/piece, 1.1 mL/piece and 0.56 mL/piece. The positive group had a concentration of 0.46 cm/nataline ointment, about the size of soybean.

2.4 taking materials

After the last administration, fasting is not forbidden for 12h, a small opening is cut above the patella after abdominal anesthesia with 10% chloral hydrate, lavage is carried out for 5 times by using 0.6mL of physiological saline, and joint fluid is collected. Centrifuging the obtained joint fluid at 3500r/min for 10min, collecting supernatant, storing in a refrigerator at-80 deg.C, and detecting the contents of interleukin IL-1 beta, matrix metalloproteinase MMP-3 and tumor necrosis factor TNF-alpha. After the joint fluid is taken out, the knee joint is taken down immediately, the muscle and fat at the joint part are removed, and the joint is fixed in 4% paraformaldehyde solution for later stage pathological tissue HE staining.

2.5 preparation and HE staining of pathological section of cartilage tissue of knee joint

Washing the fixed tissue with running water, dehydrating with gradient alcohol, sequentially adding xylene I and xylene II until the tissue is transparent, embedding the tissue with paraffin, slicing, oven drying, dewaxing, performing hematoxylin-eosin staining and sealing, and observing pathological tissue change under microscope.

2.6 Knee cartilage tissue Mankin, s score

The cartilage histology grading standard of Mankin, s was followed by scoring cartilage tissue structure, cells and tide status, taking 100 Xpictures with image software and performing statistical analysis on the data obtained. The scoring criteria are shown in table 1.

TABLE 1 Mankin's Scoring criteria

2.7 detection of IL-1 beta, MMP-3, and TNF-alpha content in synovial fluid

And (3) detecting the contents of IL-1, MMP-3 and TNF-alpha in the supernatant of the joint fluid by using an ELISA kit. The obtained synovial fluid is subjected to content determination according to the procedures of an IL-1 beta kit, an MMP-3 kit and a TNF-alpha kit.

2.8 statistical methods

2.8.1 analysis of variance

Performing one-factor variance analysis on the inflammatory factor data and Mankin, s score by SPSS 17.0 statistical software, performing LSD analysis on pairwise comparison between groups, and performing mean value plus or minus standard deviation on data results

Is represented by P<0.05 indicates that the difference is statistically significant.

3 results

3.1 histopathological changes of cartilage tissue of knee joint of rats in each group

Morphological changes of articular cartilage tissues: the tissue nuclei were stained blue and the cytoplasm red. The blank group has complete and clear bone layer, smooth surface and uniform distribution of chondrocytes, and does not have clustering and obvious tide lines; the cartilage surface of the model group is rough, the chondrocytes are reduced or increased, the arrangement is disordered, and the tide line moves upwards; the positive group has clear bone layer structure, relatively rough surface, normal number and arrangement mode of chondrocytes and complete tide line. The cartilage structures of the high, medium and low dose groups of the crossbow drug are basically complete, the tide lines are complete, the surfaces of the occasional cartilage are rough, and the cell arrangement is disordered. As shown in FIGS. 1-6, HE staining of rat knee cartilage (. times.100).

Compared with a blank group, the damage of cartilage histiocyte of knee joint of the rat in the model group is obviously enhanced, compared with the model group, the pathological change degrees of the rat in the positive group and the crossbow drug high, medium and low dose groups are relatively lighter, and the rat in the positive group and the crossbow drug high, medium and low dose group has better treatment effect on the rat in the knee osteoarthritis model.

3.2 Knee Joint cartilage tissue Mankin, s score results

And (3) processing and displaying according to the data of the joint cartilage tissue Mankin's scoring result: the model group had significant differences compared to the blank group (P < 0.05); compared with the model group, the positive group, the crossbow drug high, medium and dosage group can obviously reduce the Mankin's score, have statistical difference (P <0.05), and the score is still reduced although the crossbow drug low dosage group has no significant difference. See table 2.

TABLE 2 results of scoring of articular cartilage mankin's of rats in each group

Comparison of representative model groups with each group P < 0.05; comparison P between the triangle representative model group and the blank group is less than 0.05;

3.3 IL-1 beta, MMP-3, and TNF-alpha content in articular fluids of rats of each group

Compared with the blank group, the level of expression of IL-1 beta, MMP-3 and TNF-alpha in the joint fluid of the rat in the model group is obviously increased on average, and the difference has statistical significance (P is less than 0.01); compared with the model group, the expression levels of IL-1 beta, MMP-3 and TNF-alpha in the joint fluid of the positive group and the crossbow drug high, medium and low dose groups are obviously reduced, and the difference has statistical significance (P is less than 0.05). The results are shown in Table 3.

TABLE 3 detection results of IL-1 beta, TNF-alpha, MMP-3 in synovial fluid

Represents comparison with model group, represents P < 0.01; represents P < 0.05; the delta represents the comparison with the blank group,

DELTA.DELTA.represents P < 0.01; delta represents P < 0.05.

4 small knot

Through the result of the experiment, the crossbow prescription can improve knee cartilage pathological changes of papain-induced rat knee osteoarthritis, reduce knee cartilage tissue Mankin, score s, and inhibit the expression of IL-1 beta, TNF-alpha and MMP-3 in knee joint cavity fluid, thereby demonstrating that the crossbow medicine can effectively treat papain-induced rat knee osteoarthritis.

Secondly, preparing crossbow drug microemulsion and evaluating the quality of the crossbow drug microemulsion

5 materials

5.1 instruments

Agilent 1260 high performance liquid chromatograph (Agilent technologies, Inc., USA), UV-9000S ultraviolet visible spectrophotometer (Shanghai Meta instruments, Inc.), Wondasil C18 (Issuzu Engler, Shanghai), DKZ-2 type electrothermal constant temperature oscillatory water tank (Taicang precision instruments, Inc.), ten thousand analytical balance (Shanghai Shuyu Sci instruments, Inc.), HY-2 thunder gyromagnetic instrument (Shanghai Sci Ltd.), Marwin Zetasizer nano ZS90 (Marwin Meyer Instrument, U.K.), DJ-A high power constant temperature magnetic stirrer (Ware instruments, Inc.), ten thousand balance (Shanghai Yu Sci instruments, Inc.), TGL-16B centrifuge (Shanghai Seimitsu scientific instruments, Inc.), PHS-3B precision pH (Shanghai Rainbi instruments, effective instruments).

5.2 reagents

Benzoylhypaconine (lot number: DST200620-055, purity not less than 98%), benzoylmesaconine (lot number: DST191023-056, purity not less than 98%), mesaconine (lot number: DST191015-025, purity not less than 98%), periplocin (lot number: DST191106-041, purity not less than 98%), methyl salicylate (lot number: DST190930-125, purity not less than 98%) were purchased from WUDDES Biotech Limited; chinese alangium, raw kusnezoff monkshood root, sargentgloryvine stem and other medicinal materials (purchased in Taisheng medicinal material market in Guiyang city orchard); 50% ethanol (homemade in the laboratory); isopropyl myristate (lot number: Y17D9S77792), ethyl oleate (lot number: Z09J9Y63472), polyethylene glycol glyceryl oleate (lot number: D13J8L37819), and polyethylene glycol glyceryl caprylate decanoate (lot number: T06J9R62846) were purchased from Shanghai-sourced leaf Biotech Ltd; polyoxyethylene castor oil EL-35 (lot: R0200056), glacial acetic acid (lot: R009609), polyoxyethylene hydrogenated castor oil-40 (lot: R032953), polyethylene glycol-400 (lot: R010818) were purchased from Shanghai Yi En chemical technology, Inc.; labrasol (batch No. N1810120014, Dulai Bio Inc.); tween-80 (batch No. 20190722) and glycerol (batch No. 20190913) were purchased from Chongqing Chuanjiang chemical reagent works; 1, 2-propanediol (batch No. 20191120, Xiong chemical Co., Ltd.), isopropanol (batch No. 20190712, Shanghai Shenbo chemical Co., Ltd.); chloroform (batch No. 20190423, Kun Long science Co., Ltd.), acetonitrile (batch No. 20200722), glacial acetic acid (batch No. 20200822), and absolute ethanol (batch No. 20180712) were purchased from the national drug group chemical reagent Co., Ltd.

6 methods and results

6.1 determination of the content of the principal ingredient in the microemulsion of crossbow drug

6.1.1 chromatographic conditions

A chromatographic column: wondasil C18 (4.6X 250 mm); mobile phase: using 0.1moL/L ammonium acetate solution (1000ml ammonium acetate solution added with 1ml glacial acetic acid) as water phase, acetonitrile-tetrahydrofuran (25:15) as organic phase, gradient elution (0-10 min, 5% B → 15% B; 10-50 min, 15% B → 15.5% B; 50-85 min, 15.5% B → 17.5% B; 85-105 min, 17.5% B → 18% B; 105-125 min, 18% B → 22% B; 125-145 min, 22% B → 22% B; 145-150 min, 22% B → 5% B) to detect wavelength: 235nm, column temperature: 30 ℃, flow rate: 1ml/min, sample size: 10 ul. Under the chromatographic condition, the separation degrees are all larger than 1.5, the number of theoretical plates is not lower than 3000, and negative samples do not interfere with the measurement.

6.1.2 preparation of Mixed control solutions

Accurately weighing appropriate amount of benzoylmesaconine, benzoylhypaconine, mesaconine, periplocin, and methyl salicylate, and diluting with 0.05% hydrochloric acid methanol to obtain mixed reference substances with concentrations of 60 μ g/mL, 782 μ g/mL, 234 μ g/mL, 2586 μ g/mL, and 115 μ g/mL.

6.1.3 preparation of crossbow drug microemulsion

The Chinese medicinal materials of the prescription are taken, namely 20g of raw kusnezoff monkshood root, 15g of white dragon beard, 30g of periploca forrestii schltr, 50g of caulis perllae, and 50g of sargentgloryvine stem. Adding 70% ethanol in an amount which is 12 times that of the medicinal materials, performing reflux extraction for 1 hour each time, performing extraction for 3 times in total, filtering, recovering ethanol under reduced pressure, and performing vacuum drying to obtain a crossbow drug dry extract. Taking 0.15g of isopropyl myristate, 0.8625g of polyoxyethylene castor oil-35 and 0.2875g of absolute ethyl alcohol into a 15mL penicillin bottle, setting a magnetic stirrer at the temperature of 25 ℃ and the rotating speed of 300r/min, stirring under the conditions, adding 0.15g of dry extract of the crossbow drug after stirring uniformly, continuing stirring, adding 3.7g of water, and continuing stirring until complete emulsification, thus preparing 5.15g of microemulsion.

6.1.4 preparation of test solutions

Transferring the crossbow drug micro emulsion in a penicillin bottle to a conical flask, adding 5mL of ammonia water for wetting, adding 40mL of isopropanol-ethyl acetate (1:1), carrying out ultrasonic treatment at 500w, 35kHz and 25 ℃ for 30min, transferring all the components to an evaporation dish, evaporating in a40 ℃ water bath, dissolving in a 10mL volumetric flask with 0.05% of hydrochloric acid methanol, filtering, and taking a subsequent filtrate to obtain the crossbow drug micro emulsion.

6.1.5 preparation of negative sample solution

Preparing a negative sample without crossbow drug according to the preparation process of the crossbow drug microemulsion, and preparing a test sample solution according to the method under the item 6.1.3.

6.1.6 Special Property test

And respectively taking 10 mu L of the mixed reference substance solution, the sample solution and the negative reference substance solution, injecting into a 1260Agilent high performance liquid chromatograph under the condition of '6.1.1', and obtaining a chromatogram. See FIGS. 7-9, HPLC chromatograms of blank solvent (A), mixed control (B), and sample (C).

6.1.7 inspection of linear relationship

Precisely measuring a proper amount of mixed reference substance under item '6.1.2', diluting benzoylmesaconine, benzoylhypaconine, mesaconine, periplocin, and methyl salicylate with 0.05% hydrochloric acid methanol to obtain benzoylmesaconine with different concentrations of 3 μ g/mL, 4.5 μ g/mL, 6 μ g/mL, 7.5 μ g/mL, and 9 μ g/mL respectively; 39.1 mu g/mL, 58.65 mu g/mL, 78.2 mu g/mL, 97.75 mu g/mL, 117.3 mu g/mL of benzoylhypaconitine; mesaconitine 11.7 μ g/mL, 17.55 μ g/mL, 23.4 μ g/mL, 29.25 μ g/mL, 35.1 μ g/mL; periplocin 129.3 μ g/mL, 193.95 μ g/mL, 258.6 μ g/mL, 323.25 μ g/mL, 387.9 μ g/mL; 5.75 mu g/mL, 8.63 mu g/mL, 11.5 mu g/mL, 14.38 mu g/mL, 17.25 mu g/mL of methyl salicylate; 10 mu L of mixed reference substance solution with different concentrations is injected into 1260Agilent high performance liquid chromatograph to obtain different peak areas. The concentration is used as the abscissa and the peak area is used as the ordinate to draw a standard curve, so as to obtain the regression equation and the linear range of the 5 components, and the result is shown in table 4. The results showed that the respective index components were well correlated in a predetermined linear range.

Table 45 regression equation and linear range for index constituents (n ═ 5)

Table 4regression equation and linear range offive index components(n＝5)

6.1.8 precision test

Taking a proper amount of the mixed reference solution under the item of 6.1.2, and continuously injecting sample for 6 times according to the chromatographic condition under the item of 6.1.1. As a result, the average peak areas of benzoylmesaconine, benzoylhypaconine, mesaconine, periplocin and methyl salicylate were 50.72 + -0.70, 839.78 + -14.87, 277.12 + -5.07, 1001.92 + -15.42 and 287.27 + -3.26, respectively, the RSD of the peak areas of the respective components were 1.39%, 1.77%, 1.83%, 1.54% and 1.13%, and the RSD of the peak areas of the respective control products was less than 2% (n: 6), indicating good precision of the instrument.

6.1.9 stability test

Taking a proper amount of the sample solution under the item of 6.1.4, respectively standing at room temperature for 0, 2, 4, 8, 10, and 12h, and performing sample injection determination under the chromatographic condition under the item of 6.1.1. As a result, the average peak areas of benzoylmesaconine, benzoylhypaconine, mesaconine, periplocin and methyl salicylate were 83.53 + -1.36, 648.03 + -8.28, 255.60 + -2.79, 1553.73 + -21.56 and 342.95 + -5.06, respectively, and the RSD values of the peak areas of the respective components were 1.6%, 1.28%, 1.09%, 1.39% and 1.47%, respectively, which were less than 2%. The results show good stability over 12h for 5 components. The test solution is proved to have good stability within 12 hours when being placed at room temperature.

6.1.10 repeatability test

Taking a proper amount of crossbow drug micro-emulsion, taking 6 parts in total, preparing a sample solution according to the method under the item of 6.1.4, then carrying out sample injection measurement under the chromatographic condition under the item of 6.1.1, and calculating the contents of benzoylmesaconine, benzoylhypoaconitine, mesaconine, periplocin and methyl salicylate in the sample by using the peak area according to a regression equation. As a result, the average contents of benzoylmesaconine, benzoylhypaconine, mesaconine, periplocin and methyl salicylate are 479.33 +/-13.93 mu g/g, 3912.80 +/-106.67 mu g/g, 1517.11 +/-39.94 mu g/g, 21606.78 +/-557.37 mu g/g and 990.67 +/-15.82 mu g/g respectively, and the RSD is 2.91%, 2.73%, 2.63%, 2.58% and 1.59% (n is 6), which indicates that the method has good repeatability.

2.1.11 sample recovery test

Preparing 6 parts of sample solution according to the item of 6.1.4, adding a certain amount of mixed reference solution, carrying out sample injection measurement according to the chromatographic condition under the item of 6.1.1, and calculating the sample injection recovery rate according to the regression equation by using the peak area. As a result, the average recovery rates of benzoylmesaconine, benzoylhypaconine, mesaconine, periplocin and methyl salicylate were 101.34 + -1.84%, 100.16 + -2.62%, 98.46 + -2.91%, 100.59 + -1.98%, 101.27 + -2.84%, and the RSD values were 1.82%, 2.62%, 2.96%, 2.97% and 2.81%, respectively.

6.2 prescription screening of crossbow drug microemulsion

6.2.1 determination of solubility of oil phase, emulsifier and Co-emulsifier in extracts

Respectively taking 3g of polyglycolized glyceride oleate, ethyl oleate, isopropyl laurate, isopropyl myristate, polyoxyethylene castor oil-35, polyoxyethylene hydrogenated castor oil-40, Labrosol, polyethylene glycol-400, glycerol, absolute ethyl alcohol, isopropanol and tween-80 in a 15mL test tube with a plug, adding excessive crossbow prescription extract, vortex for 5min, ultrasonically vibrating for 30min at 37 ℃ and 120r/min for 24h at constant temperature, taking out, centrifuging for 10min at 13000r/min, taking 1mL of supernatant in a separating funnel, and determining according to a method for determining the content of total alkaloids by UV determination. The results are shown in Table 5. From the results in table 5, the solubility of the oil versus the extract was found to be: isopropyl myristate > isopropyl laurate > ethyl oleate > macrogol oleate; the solubility of the emulsifier to the extract is as follows: polyoxyethylene castor oil-35 > tween-80 > polyoxyethylene hydrogenated castor oil-40 > Labrosol; the solubility of the auxiliary emulsifier to the extract is as follows: absolute ethyl alcohol > glycerol > isopropanol > polyethylene glycol-400. Therefore, isopropyl myristate is selected as an oil phase, tween-80 and polyoxyethylene castor oil-35 are selected as surfactants, and absolute ethyl alcohol and glycerol are selected as cosurfactants for continuing the investigation.

TABLE 5 solubility determination of oil phase, emulsifier and co-emulsifier for extracts

Table 5solubility test results ofoil phase,emulsifier and co emulsifier to extract

6.2.2 pseudo-ternary phase diagram screening of surfactants

Taking isopropyl myristate as an oil phase, precisely weighing Tween-80 and polyoxyethylene castor oil-35 as a Surfactant (SF), taking absolute ethyl alcohol as a cosurfactant (CoSF), uniformly mixing the mixture according to a mass ratio of 2:1 to serve as a mixed emulsifier, precisely weighing the mixed emulsifier and the oil phase in a penicillin bottle according to a mass ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1, uniformly mixing the mixture at a constant temperature of 25 ℃, titrating the mixture with distilled water, observing phase change, simultaneously irradiating the mixture with incident light of 600nm, observing whether a Tyndall phenomenon exists, recording a critical change point of a system, and determining an O/W type microemulsion region by using a pseudo-ternary phase diagram drawn by Origin2019 software microemulsion, wherein the result is shown in figures 10-11. As a result, the area of a micro-emulsion area formed by the emulsifier polyoxyethylene castor oil-35 is larger, and the polyoxyethylene castor oil-35 is selected as the emulsifier.

6.2.3 pseudo-ternary phase diagram screening cosurfactants

Taking isopropyl myristate as an oil phase, taking polyoxyethylene castor oil-35 as an emulsifier, respectively taking absolute ethyl alcohol and glycerol as cosurfactants, comparing the emulsion forming conditions taking the absolute ethyl alcohol and the glycerol as the cosurfactants, weighing the polyoxyethylene castor oil-35, the absolute ethyl alcohol and the glycerol according to the ratio of 2:1, and uniformly mixing. According to the method under the item of 6.2.2, a pseudo-ternary phase diagram of the microemulsion is drawn, an O/W type microemulsion area is determined, the area of the microemulsion formed by the absolute ethyl alcohol is larger than that of the glycerol, and the absolute ethyl alcohol is finally selected as the co-emulsifier, and the result is shown in the figure of 12-13.

6.2.4 Effect of the mass ratio (Km) of emulsifier to Co-emulsifier on microemulsion formation

Polyoxyethylene castor oil-35 is used as a surfactant, absolute ethyl alcohol is used as a cosurfactant, the components are uniformly mixed according to the mass ratio of 1:3, 1:2, 1:1, 2:1 and 3:1, then a pseudo-ternary phase diagram of the microemulsion is drawn according to the method under the item '6.2.2', an O/W type microemulsion area is determined, the area of a formed microemulsion area is larger when Km is 3:1, and finally the Km is 3:1, and the result is shown in a figure 14.

6.2.5D-optimized mixing design optimized crossbow drug micro-emulsion prescription

Through investigation of single factor and pseudo ternary phase diagram, isopropyl myristate is finally selected as an oil phase, polyoxyethylene castor oil-35 is selected as an emulsifier, absolute ethyl alcohol is selected as a co-emulsifier, and Km is 3: 1. the isopropyl myristate is 0.03-0.07 obtained by pre-experiment and pseudo ternary phase diagram investigation; the mixed emulsifier is 0.21-0.35; 0.62-0.74 of water. Taking the oil phase as A, the mixed emulsifier as B and the water as C as investigation factors, taking the particle size, PDI and solubility as evaluation indexes, and adopting Design Expert 8.0.6 statistical software to carry out D-optimal mixed material Design and optimize the prescription. The experimental arrangement and results are shown in table 6. According to the table 6, the oil phase is weighed, the emulsifier is mixed, stirred and mixed evenly at the constant temperature of 25 ℃ and 300r/min, then the water phase is added, and stirring is continued. Stirring to form emulsion, taking 2mL blank microemulsion at each level, adding excessive crossbow drug extract, vortexing for 5min, oscillating at 37 deg.C for 24h, centrifuging at 13000r/min, and collecting supernatant and injecting into UV to detect content of aconite alkaloid. And another 2mL of the solution is used for detecting the particle size and PDI of the microemulsion.

TABLE 6D-optimal compounding design test arrangement and results

Table 6D-optimal mix design test arrangement and results

As can be seen from table 7, the particle size and PDI software suggested a quartic model and a cubic model, respectively, without significant differences in solubility and therefore no conforming model. The regression equation of the finally obtained particle size is as follows: y is_{Particle size}-6.3446.13 a + 20.36B + 19.16C +1.147E +005 a B +1.097E +005 a C + 162.30B C-1.065E +005 a B C +56275 a B (a-B) +47312.25 a C (a-C) + 331.85B C (B-C), the model P < 0.0068, degree of mismatching 0.1858; regression equation of PDI: log10(PDI) +27034.61343 a-482.78287B-0.57293C-35117.50455 a B-44642.29727 a C + 933.85521B C +24451.32447 a B C-8247.05275 a B (a-B) -18477.77848 a C (a-C) + 610.82275B C (B-C), model P0.0028, degree of mismatching 0.8767, indicating that this model can accurately predict the results of the experiment and that the equation is good. The effect of the relative particle size and PDI of the oil phase, mixed emulsifier and water is shown in fig. 15 and 16, and the results show that the lower the oil phase content, the higher the mixed emulsifier content, the smaller the particle size; the PDI is mainly influenced by the mixed emulsifier, and the higher the content of the mixed emulsifier is, the smaller the PDI is.

TABLE 7 comparison of the drug loading, particle size and PDI equations

Table 7Comparison ofdrug loading,particle size and PDI fitting equations

In the preparation of microemulsion, a large amount of surfactant is often added, but the large amount of surfactant has influence on the hydration of epidermis and the result, and on the premise of ensuring the particle size, a prescription with a small amount of surfactant is selected as much as possible, so that the prescription with small particle size and few surfactant is finally selected: 3% isopropyl myristate, 17.25% cremophor oil-35, 5.75% absolute ethanol and 74% water. The formula is verified, the result is shown in table 8, the particle size difference of the optimized microemulsion is small, the PDI is smaller than the predicted value, the stability is higher, and the experimental result can be well predicted by the model.

TABLE 8 optimization of predicted and actual values of prescription indicators

Table 8predicted and actual values ofoptimized prescription indexes

6.2.6 determination of drug load of microemulsion of crossbow

Prescription changes are found to have insignificant effect on the solubility of the crossbow extract through prescription optimization of the crossbow microemulsion, so the drug loading is further determined by particle size. Weighing IPM, EL-35 and absolute ethyl alcohol according to the prescription amount, uniformly mixing, respectively adding 1.0%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3% and 3.5% of crossbow drug extract, stirring for 30min at a constant temperature of 25 ℃ for 300r/min to fully dissolve, adding the water phase, and continuously stirring for 30 min. The prepared drug-loaded microemulsion is measured for particle size and PDI, and the drug-loaded amount is screened by taking the appearance, the particle size and the PDI of the microemulsion as evaluation indexes.

TABLE 9 drug Loading screening results

Table 9drug loading screening results

From table 9, it can be seen that the larger the drug loading rate is, the larger the particle size and PDI are, the particle size still meets the requirement of the microemulsion particle size when the drug loading rate is 3.5%, and finally, the stability of the microemulsion is examined by placing the microemulsion at 4 ℃, 25 ℃ and 40 ℃, and it is found that the microemulsion with the drug loading rate of 3.5% can precipitate after being placed, so that the stability is ensured while the drug loading rate is high, and the drug loading rate is finally selected to be 3%.

Quality evaluation of 7 crossbow drug microemulsion

7.1 appearance Properties

3 batches of the crossbow drug micro-emulsion are prepared according to the preparation process under the item of 6.1, and the crossbow drug micro-emulsion is a yellow brown transparent liquid with low viscosity and uniform dispersion and the tyndall effect.

7.2pH value

From each of the 3 samples, 1mL was diluted to 10mL at room temperature, and the pH of the sample was measured with a pH meter. As a result, the average pH of the sample was found to be 5.17. + -. 0.021.

7.3 particle size distribution, PDI and zeta potential determination

From each of the 3 batches, 0.2mL was diluted to 2mL at room temperature, and the particle size, PDI and zeta potential were measured using a Malvern laser scattering particle sizer. The results showed an average particle diameter of 19.83. + -. 0.777, an average PDI of 0.147. + -. 0.020 and an average zeta potential of-0.51. + -. 0.097. The particle size distribution of the crossbow drug microemulsion is uniform, and the whole system is stable.

7.4 stability

3 batches of samples were centrifuged in a high speed centrifuge at 15000 r.min-1 for 10min without separation of the solution. Three batches of samples were placed at-4 deg.C, 25 deg.C for one month without solution stratification. The crossbow drug micro emulsion has better stability.

7.5 drug load determination

3 batches of samples were taken, and sample solutions were prepared under the "6.1.4" condition, and the sample composition measurement was performed according to the "6.1.1" condition. As a result, the average contents of benzoylmesaconine, benzoylhypaconine, mesaconine, periplocin and methyl salicylate in the sample were 457.87 + -25.39 μ g/g, 3881.27 + -98.62 μ g/g, 1491.73 + -34.74 μ g/g, 21570.67 + -563.06 μ g/g and 959.06 + -32.36 μ g/g, respectively.

Compared with the prior art, the invention optimizes the prescription composition of the crossbow drug microemulsion by combining D-optimal mixture design on the basis of single-factor and pseudo-ternary phase diagram, and evaluates the final prescription through the contents of particle size, PDI, zeta potential, benzoylmesaconine, mesaconine, periplocin and methyl salicylate; in addition, the microemulsion is used as a novel transdermal drug delivery system, the interfacial tension is extremely low, the particle size of a dispersed phase (drug-containing phase) is small, the microemulsion can quickly permeate into a stratum corneum and be mixed with a skin lipid layer in the stratum corneum, and the drug embedded in the microemulsion is not required to be released from the microemulsion and can enter the stratum corneum together with the microemulsion in a very short time. The crossbow drug is prepared into microemulsion, so that the transdermal rate of the crossbow drug can be increased, the slow release effect can be achieved, and the administration times can be reduced. The inventor adopts a high performance liquid chromatography method to determine the content of benzoylmesaconine, benzoylhypaconine, mesaconine, periplocin and methyl salicylate in the microemulsion of the crossbow drug, and the method has strong specificity, high sensitivity and good accuracy. Determining that isopropyl myristate is an oil phase, tween-80 and polyoxyethylene castor oil-35 are surfactants, and absolute ethyl alcohol and glycerol are cosurfactants through the solubility of total alkaloids in the crossbow medicine in different oil phases, the surfactants and the cosurfactants; the size of an emulsion forming area of a pseudo-ternary phase diagram determines that the polyoxyethylene castor oil-35 is used as a surfactant and absolute ethyl alcohol is used as a cosurfactant, and the maximum microemulsion forming area is determined when Km is 3: 1. The optimal formula is determined by D-optimal mixed material design to be that the surfactant is polyoxyethylene castor oil-35, the cosurfactant is absolute ethyl alcohol, the oil phase is isopropyl myristate, the water phase is water, and the mass ratio of the surfactant to the cosurfactant is 17.25:5.75:3:74. The microemulsion prepared by the method has transparent and clear appearance and uniform particle size.

In conclusion, the medicine has the effects of promoting blood circulation, relieving pain, removing dampness, removing blood stasis, relaxing tendons and activating collaterals, and can effectively treat knee osteoarthritis; the prepared microemulsion has small particle size, improved drug stability, promoted drug absorption, high biocompatibility, prolonged action time of the drug and prolonged drug effect.

Drawings

FIG. 1 is a graph (X100) showing HE staining of cartilage of knee joint of 3.1 part of blank rat in experimental example;

FIG. 2 is a graph (X100) showing HE staining of cartilage of knee joint of rat in 3.1 part of model group in experimental example;

FIG. 3 is a graph (X100) showing HE staining of cartilage of knee joint of 3.1 part of positive rats in experimental example;

FIG. 4 is a graph of HE staining (X100) of cartilage in knee joint of 3.1 part of crossbow drug high dose group rats in experimental example;

FIG. 5 is a graph of HE staining (x 100) of cartilage in knee joint of rats in the dose group of 3.1 part of crossbow drug in the experimental example;

FIG. 6 is a graph of HE staining (X100) of cartilage in knee joint of 3.1 part of crossbow drug low dose group rats in experimental example;

FIG. 7 is an HPLC chromatogram of 6.1.6 part of the blank solvent (A) in the experimental example;

FIG. 8 is an HPLC chromatogram of 6.1.6 part of the mixed control (B) in the experimental example;

FIG. 9 is an HPLC chromatogram of 6.1.6 fraction (C) in the experimental example;

FIG. 10 is a pseudo-ternary phase diagram of 6.2.2 portions of Tween-80 in the experimental example;

FIG. 11 is a pseudo-ternary phase diagram of 6.2.2 parts of polyoxyethylated castor oil-3 in the experimental example;

FIG. 12 is a pseudo-ternary phase diagram of 6.2.3 parts of glycerol in the experimental example;

FIG. 13 is a pseudo-ternary phase diagram of 6.2.3 portions of anhydrous ethanol in the experimental example;

FIG. 14 is a pseudo-ternary phase diagram of part 6.2.4 of the experimental example;

FIG. 15 is a graph of 6.2.5 partial PDI response plots in the experimental example;

FIG. 16 is a graph showing 6.2.5 partial particle size response in the experimental example.

Detailed Description

The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.

Example 1. a microemulsion for treating knee osteoarthritis, which is prepared from 20g of raw kusnezoff monkshood root, 15g of white dragon beard, 30g of caulis periplocae, 50g of caulis perllae and 50g of sargentgloryvine stem;

adding 70% ethanol 12 times the amount of the above medicinal materials, reflux extracting for 1 hr each time, extracting for 3 times, filtering, recovering ethanol under reduced pressure, and vacuum drying to obtain dry extract. Taking 1.5g of isopropyl myristate, 8.625g of polyoxyethylene castor oil-35 and 2.875g of absolute ethyl alcohol in a penicillin bottle, setting a magnetic stirrer at the temperature of 25 ℃ and the rotating speed of 300r/min, stirring under the condition, adding 1.5g of dry extract after stirring uniformly, continuing stirring, adding 37g of water, and continuing stirring until complete emulsification, thus obtaining the microemulsion.

The usage and dosage are as follows: the crossbow medicinal liquor is clinically used for treating knee osteoarthritis, a needle is used for dipping a medicinal liquid to prick knee joints, and each knee joint takes 5mL of the medicinal liquid every time and is used once a day. The crossbow drug microemulsion was applied to 4% papain-induced knee osteoarthritis rats, 0.72g per leg, once a day, according to a clinical dosing switch.

Example 2. a microemulsion for treating knee osteoarthritis, which is prepared from 50g of raw kusnezoff monkshood root, 50g of white dragon beard, 50g of caulis periplocae, 300g of caulis perllae and 30g of sargentgloryvine stem; adding 70% ethanol 12 times the amount of the above medicinal materials, reflux extracting for 1 hr each time, extracting for 3 times, filtering, recovering ethanol under reduced pressure, and vacuum drying to obtain dry extract. Taking 1.5g of isopropyl myristate, 8.625g of polyoxyethylene castor oil-35 and 2.875g of absolute ethyl alcohol in a penicillin bottle, setting a magnetic stirrer at the temperature of 25 ℃ and the rotating speed of 300r/min, stirring under the condition, adding 1.5g of dry extract after stirring uniformly, continuing stirring, adding 37g of water, and continuing stirring until complete emulsification, thus obtaining the microemulsion.

The usage amount is the same as that of the example 1.

Example 3. a microemulsion for treating knee osteoarthritis, which is prepared from 40g of raw kusnezoff monkshood root, 25g of white dragon beard, 40g of caulis periplocae, 70g of caulis perllae and 70g of sargentgloryvine stem; adding 70% ethanol 12 times the amount of the above medicinal materials, reflux extracting for 1 hr each time, extracting for 3 times, filtering, recovering ethanol under reduced pressure, and vacuum drying to obtain dry extract. Taking 1.5g of isopropyl myristate, 8.625g of polyoxyethylene castor oil-35 and 2.875g of absolute ethyl alcohol in a penicillin bottle, setting a magnetic stirrer at the temperature of 25 ℃ and the rotating speed of 300r/min, stirring under the condition, adding 1.5g of dry extract after stirring uniformly, continuing stirring, adding 37g of water, and continuing stirring until complete emulsification, thus obtaining the microemulsion.

The usage amount is the same as that of the example 1.

Example 4. a microemulsion for treating knee osteoarthritis, which is prepared from raw wild aconite root 10g, white dragon beard g, black vine 10g, Tou Gu Xiang 40g and sargentgloryvine stem 40 g; adding 70% ethanol 12 times the amount of the above medicinal materials, reflux extracting for 1 hr each time, extracting for 3 times, filtering, recovering ethanol under reduced pressure, and vacuum drying to obtain dry extract. Taking 1.5g of isopropyl myristate, 8.625g of polyoxyethylene castor oil-35 and 2.875g of absolute ethyl alcohol in a penicillin bottle, setting a magnetic stirrer at the temperature of 25 ℃ and the rotating speed of 300r/min, stirring under the condition, adding 1.5g of dry extract after stirring uniformly, continuing stirring, adding 37g of water, and continuing stirring until complete emulsification, thus obtaining the microemulsion.

The usage amount is the same as that of the example 1.

Example 5. a microemulsion for treating knee osteoarthritis, which is prepared from 30g of raw kusnezoff monkshood root, 20g of white dragon beard, 30g of caulis periplocae, 60g of caulis perllae, and 60g of sargentgloryvine stem; adding 70% ethanol 12 times the amount of the above medicinal materials, reflux extracting for 1 hr each time, extracting for 3 times, filtering, recovering ethanol under reduced pressure, and vacuum drying to obtain dry extract. Taking 1.5g of isopropyl myristate, 8.625g of polyoxyethylene castor oil-35 and 2.875g of absolute ethyl alcohol in a penicillin bottle, setting a magnetic stirrer at the temperature of 25 ℃ and the rotating speed of 300r/min, stirring under the condition, adding 1.5g of dry extract after stirring uniformly, continuing stirring, adding 37g of water, and continuing stirring until complete emulsification, thus obtaining the microemulsion.

The usage amount is the same as that of the example 1.

Claims

1. A transdermal drug delivery preparation for treating knee osteoarthritis is characterized in that: the medicinal effective components are prepared from 5-40 parts of raw kusnezoff monkshood root, 5-25 parts of white dragon beard, 5-40 parts of periploca forrestii, 30-70 parts of caulis perllae, and 30-70 parts of sargentgloryvine stem by weight.

2. The transdermal drug delivery formulation for the treatment of knee osteoarthritis according to claim 1, wherein: the medicinal effective components of the medicine are calculated according to the weight parts and are prepared from 10-30 parts of raw kusnezoff monkshood root, 10-20 parts of white dragon beard, 10-30 parts of periploca forrestii, 40-60 parts of caulis perllae and 40-60 parts of sargentgloryvine stem.

3. The transdermal drug delivery formulation for the treatment of knee osteoarthritis according to claim 2, wherein: the medicinal effective components of the medicine are calculated according to the weight parts and are prepared by 20 parts of raw kusnezoff monkshood root, 15 parts of white dragon beard, 30 parts of caulis periplocae, 50 parts of caulis perllae and 50 parts of sargentgloryvine stem.

4. A method for preparing a transdermal drug delivery formulation for the treatment of knee osteoarthritis according to any one of claims 1 to 3, characterized in that: the Chinese medicaments in the formula are combined with the auxiliary materials acceptable in the medicaments, and the medicaments are processed according to a conventional method to prepare the corresponding preparation.

5. The method for preparing a transdermal drug delivery formulation for the treatment of knee osteoarthritis according to claim 4, wherein: the medicine is an external preparation.

6. The method for preparing the transdermal drug delivery preparation for treating knee osteoarthritis according to claim 5, wherein: the external preparation is microemulsion.

7. The method for preparing the transdermal drug delivery preparation for treating knee osteoarthritis according to claim 6, wherein the transdermal drug delivery preparation comprises the following components: the microemulsion is prepared by: weighing the traditional Chinese medicines according to the proportion, adding ethanol, refluxing and extracting for 3 times, filtering, recovering ethanol under reduced pressure, and drying in vacuum to obtain dry extract; adding isopropyl myristate, polyoxyethylene castor oil-35, and anhydrous alcohol into container, stirring, adding dry extract, stirring, adding water, and stirring to completely emulsify to obtain microemulsion.

8. The method for preparing the transdermal drug delivery preparation for treating knee osteoarthritis according to claim 7, wherein the transdermal drug delivery preparation comprises the following components: the dry extract is prepared by weighing the traditional Chinese medicines according to the proportion, adding 70% ethanol in an amount which is 12 times that of the traditional Chinese medicines, performing reflux extraction for 3 times, extracting for 1 hour each time, filtering, recovering ethanol under reduced pressure, and performing vacuum drying.

9. The method for preparing the transdermal drug delivery preparation for treating knee osteoarthritis according to claim 7, wherein the transdermal drug delivery preparation comprises the following components: the mass ratio of the isopropyl myristate to the polyoxyethylene castor oil-35 to the absolute ethyl alcohol to the water to the extract is 15:86.25:28.75:370: 15.

10. The method for preparing the transdermal drug delivery preparation for treating knee osteoarthritis according to claim 7, wherein the transdermal drug delivery preparation comprises the following components: the stirring temperature of the isopropyl myristate, the polyoxyethylene castor oil-35 and the absolute ethyl alcohol is 25 ℃, and the rotating speed is 300 r/min.