External application medicine for cerebral palsy muscle spasm and preparation method and application thereof

Abstract

The invention provides an external application medicine for cerebral palsy muscle spasm and a preparation method and application thereof. The external application medicine is prepared from 15-30 parts of lycopodium clavatum, 10-25 parts of radix paeoniae alba, 10-25 parts of garden balsam stem, 5-15 parts of angelica sinensis, 5-15 parts of ligusticum wallichii, 1-5 parts of borneol, 3-5 parts of cynanchum paniculatum, 1-5 parts of stiff silkworm, 5-15 parts of earthworm, 0.1-0.5 part of neurotrophic factor, 0.01-0.5 part of TRPV1 agonist and 0.01-0.05 part of liposome serving as a carrier system. The preparation method comprises the steps of traditional Chinese medicine alcohol extraction, superfine grinding, active ingredient loading, high-pressure homogenization, ultrasonic dispersion and the like, and dosage forms such as cataplasm and the like can be prepared. Through the multi-target synergistic effect of the traditional Chinese medicines, the neurotrophic factors and the TRPV1 agonist, and in combination with the liposome carrier, the transdermal and stability are enhanced, so that the cerebral palsy spasm is remarkably relieved, the muscular tension can be effectively reduced, the joint motion range is improved, and the use is safe and convenient.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine preparation, in particular to an external application medicine for muscle spasm of cerebral palsy and a preparation method and application thereof. BACKGROUND

[0002] Cerebral palsy, referred to as CP, is a group of permanent disorders of movement and posture, causing physical disabilities in children, resulting from non-progressive disturbances that occurred in the developing fetal or infant brain. It is often accompanied by sensory, cognitive, communication, behavioral and other disorders. Muscle spasm is the most common motor disorder of cerebral palsy, which is caused by the damage of upper motor neurons leading to hyperreflexia at the level of the spinal cord. Persistent muscle spasm can cause pain, joint contracture, abnormal posture, and seriously affect the motor function and quality of life of patients.

[0003] At present, the methods for treating muscle spasm of cerebral palsy mainly include physical therapy, oral anti-spasm drugs (such as baclofen and tizanidine), local injection (such as botulinum toxin injection) and surgical treatment. However, these methods have limitations: physical therapy has a slow effect and the effect is difficult to last; oral drugs can easily cause systemic side effects such as drowsiness and fatigue; botulinum toxin injection has a significant effect, but it is a invasive operation, has a limited effective period of about 3-6 months, and can produce antibodies leading to drug resistance; surgical treatment is traumatic and only suitable for severe cases.

[0004] External application medicine has certain application in the treatment of muscle spasm because it directly acts on the local lesion site and has the advantages of strong targeting and small adverse reactions. Traditional external application medicine is mostly made of traditional Chinese medicine, which can relieve spasm by promoting local blood circulation, but has problems such as low dissolution rate of effective components and single action, and is difficult to achieve nerve function repair. At the same time, the bioactive components are easy to be inactivated during the preparation process, and the transdermal absorption efficiency is low, which limits their application in external application medicine. In addition, the existing preparations lack precise formula design targeting the pathological mechanism of muscle spasm of cerebral palsy, and it is difficult to achieve the synergistic therapeutic effect of "spasm relief-nerve repair". Therefore, it is of great clinical significance to develop an external application medicine with coordinated components, stable preparation process and comprehensive curative effect. SUMMARY

[0005] In view of this, the present application provides a traditional Chinese medicine composition for treating diarrhea-predominant irritable bowel syndrome and a preparation method thereof, which solves the above problems.

[0006] The technical solution of the present application is as follows: an external application medicine for muscle spasm of cerebral palsy, comprising the following raw materials: 15-30 parts of rhizoma sinodendri, 10-25 parts of radix paeoniae alba, 10-25 parts of radix saposhnikoviae, 5-15 parts of radix angelicae sinensis, 5-15 parts of ligusticum wallichii, 1-5 parts of borneol, 3-5 parts of radix stephaniae tetrandrae, 1-5 parts of silkworm chrysalis, 5-15 parts of earthworm, 0.1-0.5 parts of neurotrophic factor, and 0.01-0.5 parts of TRPV1 agonist.

[0007] Preferably, the external application medicine for muscle spasm of cerebral palsy comprises the following raw materials: 25 parts of rhizoma sinodendri, 18 parts of radix paeoniae alba, 15 parts of radix saposhnikoviae, 10 parts of radix angelicae sinensis, 10 parts of rhizoma chuanxiong, 3 parts of borneolum syntheticum, 4 parts of radix valerianae, 3 parts of cantharis, 10 parts of lumbricus, 0.3 parts of neurotrophic factor, and 0.2 parts of TRPV1 agonist.

[0008] Preferably, the external application medicine further comprises 0.01-0.05 parts of a carrier system, which is liposome, and the weight ratio of soybean phospholipid, cholesterol and octadecylamine is 10:(2-4):(1-3), and the average particle size is 100-150 nm.

[0009] Preferably, the neurotrophic factor is basic fibroblast growth factor (bFGF) or brain-derived neurotrophic factor (BDNF).

[0010] Preferably, the TRPV1 agonist is capsaicin.

[0011] Preferably, the dosage form of the external application medicine is cataplasm, gel, plaster or liniment.

[0012] Preferably, the preparation method of the external application medicine for muscle spasm of cerebral palsy comprises the following steps:

[0013] S1, the rhizoma sinodendri, radix paeoniae alba, radix saposhnikoviae, radix angelicae sinensis and rhizoma chuanxiong are weighed by weight parts, crushed and passed through a 60-80 mesh sieve, 8-12 times the weight of 70-80% ethanol solution is added, and reflux extraction is carried out at 50-60°C for 1-3 times, each time for 1.5-2.5 hours, the extract is combined and filtered, and concentrated under reduced pressure to an alcohol extract with a relative density of 1.10-1.15;

[0014] S2, the radix valerianae, cantharis and lumbricus are weighed by weight parts, and are treated by super micro-powdering technology to obtain super micro-powder with a particle size of 10-20 μm for standby use;

[0015] S3, the borneolum syntheticum is heated to 45-50°C to melt, the super micro-powder obtained in step S2 is added, and stirring is carried out to disperse uniformly to obtain a mixed powder;

[0016] S4, the carrier system and the neurotrophic factor are mixed and incubated at a temperature of 2-8°C for 5-20 minutes, and then are mixed with the alcohol extract of S1, the mixed powder of S3 and the TRPV1 agonist, and are treated by high-pressure homogenization for 1-3 times under a pressure of 100-150 MPa;

[0017] S5, according to the target dosage form, the corresponding excipients are added, stirring is carried out to disperse uniformly, and then the processes of molding, drying and cutting are carried out to obtain the external application medicine.

[0018] Preferably, the vacuum degree of the reduced pressure concentration in S1 is 0.07-0.09 MPa, and the temperature is 55-65°C.

[0019] Preferably, after the homogenization in S4, ultrasonic-assisted dispersion is adopted, the ultrasonic power is 250-350 W, and the time is 10-15 minutes.

[0020] Preferably, the composite external preparation is applied in the preparation of a medicine for reducing muscle tension and improving joint range of motion of a patient with cerebral palsy.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] The present application adopts the synergistic collocation of traditional Chinese medicine raw materials, bioactive components and TRPV1 agonists, the traditional Chinese medicine components have the effects of relieving muscle spasm and activating collaterals (such as Herba Lophatheri and Herba Lycopodii), nourishing blood (such as Radix Paeoniae Alba, Angelica sinensis and Rhizoma Chuanxiong), relieving convulsion and relieving pain (such as Radix et Rhizoma Veratrini, Bombyx Batryticatus and Lumbricus), nerve trophic factors promote nerve function repair, TRPV1 agonists improve transdermal absorption efficiency, and the carrier system guarantees the stability and targeting of bioactive components, so that the multiple components achieve synergistic effect.

[0023] The present application adopts the process of 'alcohol extraction + ultrafine grinding' to improve the dissolution rate of effective components of traditional Chinese medicines, and adopts the combined technology of low-temperature incubation, high-pressure homogenization and ultrasonic dispersion to solve the problems of easy inactivation and uneven dispersion of bioactive components, so as to guarantee the stability of the quality of the preparation.

[0024] The external preparation of the present application not only can effectively reduce the muscle tension and improve the joint range of motion of a patient with cerebral palsy, but also can promote nerve function repair, break through the limitation of traditional external preparations which only focus on relieving symptoms, and has strong targeting and small adverse reactions in the external application mode, so that the present application is suitable for a wide range of people. DETAILED DESCRIPTION

[0025] In order to better understand the technical content of the present application, the following specific examples are provided to further illustrate the present application.

[0026] The experimental methods used in the embodiments of the present application are all conventional methods unless otherwise specified.

[0027] The materials, reagents and the like used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified.

[0028] The basic conditions of the main raw materials are introduced below according to 'Chinese Pharmacopoeia', 'Chinese Herbal Medicine Dictionary' and 'Chinese Herbs'.

[0029] Herba Lophatheri: nature and taste and meridian entry: slightly bitter and pungent, warm. Belongs to liver, spleen and kidney meridians. Function and indication: dispelling wind and dampness, relieving muscle spasm. Used for joint pain and difficulty in flexing and extending. The product is specially used for relieving muscle spasm and activating collaterals, and has special effect on muscle spasm and difficulty in joint movement, so it is one of the monarch drugs in the prescription.

[0030] White Peony Root: Nature, flavor, and meridian tropism: Bitter, sour, slightly cold. It enters the liver and spleen meridians. Functions and indications: Tonifying blood and regulating menstruation, astringing yin and stopping sweating, soothing the liver and relieving pain, suppressing hyperactive liver yang. It is used for sallow complexion due to blood deficiency, spasms of the limbs, and hyperactive liver yang. In this formula, it is mainly used for its function of soothing the liver, alleviating spasm, and relieving pain to relieve muscle spasm.

[0031] Chinese Speranskia Root: Nature, flavor, and meridian tropism: Pungent, warm. It enters the liver and kidney meridians. Functions and indications: Expelling wind and removing dampness, relaxing muscles and activating collaterals, promoting blood circulation and relieving pain. It is used for rheumatic arthralgia and cramped tendons. Its name "penetrating bone" means that the medicinal power can penetrate deep into the tendons and bones, guiding all the herbs to reach the affected area and enhancing the permeability of the whole formula.

[0032] Angelica Sinensis: Nature, flavor, and meridian tropism: Sweet, pungent, warm. It enters the liver, heart, and spleen meridians. Functions and indications: Tonifying blood and promoting blood circulation, regulating menstruation and relieving pain, moistening the intestines and relaxing bowel movements. It is used for sallow complexion due to blood deficiency, rheumatic arthralgia, and injuries from falls and bruises. In this formula, its property of promoting blood circulation and nourishing blood is utilized, following the principle of "treating wind by first treating blood, and when blood circulates smoothly, wind will disappear", to improve local blood circulation and nourish the tendons and vessels.

[0033] Chuanxiong: Nature, flavor, and meridian tropism: Pungent, warm. It enters the liver, gallbladder, and pericardium meridians. Functions and indications: Promoting blood circulation and regulating qi, expelling wind and relieving pain. It is used for pain syndromes due to qi stagnation and blood stasis, and rheumatic arthralgia. This herb is known as "the herb of qi in blood". It can promote blood circulation and regulate qi simultaneously. When qi moves, blood will circulate smoothly, and when combined with Angelica Sinensis, it enhances the power of promoting blood circulation and dredging collaterals.

[0034] Borneol: Nature, flavor, and meridian tropism: Pungent, bitter, slightly cold. It enters the heart, spleen, and lung meridians. Functions and indications: Resuscitating the consciousness and opening the orifices, clearing heat and relieving pain. This herb has extremely strong penetrating power and can significantly promote the transdermal absorption of other drug components. It is often used as a "guiding herb" in topical formulas to lead the effective components of the medicine deep into the body.

[0035] Pyrola: Nature, flavor, and meridian tropism: Pungent, warm. It enters the liver and stomach meridians. Functions and indications: Expelling wind and relieving pain, promoting blood circulation and activating collaterals. It is widely used for rheumatic arthralgia and injuries from falls and bruises, with a significant pain-relieving effect and a safe property of the medicine.

[0036] Bombyx Batryticatus: Nature, flavor, and meridian tropism: Salty, pungent, flat. It enters the liver, lung, and stomach meridians. Functions and indications: Dispelling wind and arresting convulsions, expelling wind and relieving pain, resolving phlegm and dissipating nodules. It is a commonly used herb for treating infantile convulsions, epilepsy, facial paralysis due to stroke, and headache due to wind-heat, with a good antispasmodic effect and no toxicity.

[0037] Pheretima: Nature, flavor, and meridian tropism: Salty, cold. It enters the liver, spleen, and bladder meridians.

[0038] Functions and indications: Clearing heat and calming fright, dredging collaterals, relieving asthma, promoting diuresis. It is used for high fever with delirium, numbness of limbs, and hemiplegia. This herb is good at dredging collaterals and is often used for treating obstruction of meridians and difficulty in flexion and extension of joints.

[0039] In this formula, Herba Lycopodii is specialized in relaxing tendons, Herba Speranskiae Tuberculatae is good at penetrating, and Radix Cynanchi Paniculati is effective in relieving pain. The three herbs work together to play a core role in dispelling wind and dampness, dredging collaterals and relieving pain for the main disease and syndrome of "blocked meridians and cramped tendons and vessels". Bombyx Batryticatus and Pheretima are both insect drugs, which can search for wind and dredge collaterals, assisting the monarch drug in relieving spasm, especially for convulsions caused by wind pathogen. Paeoniae Radix Alba nourishes the liver and softens the tendons, nourishing the tendons and vessels from the inside and relieving spasm, complementing with the collaterals dredging drugs to treat both the symptoms and root causes. Angelicae Sinensis Radix tonifies blood and promotes blood circulation, and Chuanxiong Rhizoma promotes blood circulation and regulates qi. The combination of the two ensures smooth flow of qi and blood, providing power and basis for the collaterals dredging and spasm relieving effects of the monarch and ministerial drugs, embodying the treatment concept of "when blood circulates smoothly, wind will disappear". Using its extremely strong penetrability, it guides all pharmacological components in the formula to penetrate through the skin and directly reach the affected area (spastic muscles and meridians), playing a key role as the guiding herb to conduct the medicinal properties to the affected part.

[0040] Example 1: Preparation of External Application Cataplasm

[0041] Prepare according to the following ratio:

[0042] 15 parts of Herba Lycopodii, 10 parts of Paeoniae Radix Alba, 10 parts of Herba Speranskiae Tuberculatae, 5 parts of Angelicae Sinensis Radix, 5 parts of Chuanxiong Rhizoma, 1 part of Borneolum Syntheticum, 3 parts of Radix Cynanchi Paniculati, 1 part of Bombyx Batryticatus, 5 parts of Pheretima, 0.1 part of neurotrophic factor, 0.01 part of TRPV1 agonist, 0.01 part of carrier system.

[0043] The processing method of Radix Cynanchi Paniculati is as follows: Take raw Strychnos Semen, add sand and stir-fry until the surface bulges and the color turns brownish-yellow, remove the sand and then crush and sieve through a 80-mesh sieve to obtain Radix Cynanchi Paniculati powder.

[0044] The neurotrophic factor is basic fibroblast growth factor.

[0045] The carrier system is soybean phospholipid, cholesterol and octadecylamine with a weight ratio of 10:2:1.

[0046] Preparation steps:

[0047] S1. Weigh Herba Lycopodii, Paeoniae Radix Alba, Herba Speranskiae Tuberculatae, Angelicae Sinensis Radix and Chuanxiong Rhizoma according to parts by weight, crush them and sieve through a 60-mesh sieve, add 8 times the weight of 70% ethanol solution, reflux and extract at 50 °C for 1 time, 1.5 hours each time, combine the extraction solutions and filter, and concentrate under reduced pressure at 0.07 MPa and a temperature of 55 °C to an ethanol extract with a relative density of 1.10;

[0048] S2. Weigh Radix Cynanchi Paniculati, Bombyx Batryticatus and Pheretima according to parts by weight, and process them by ultrafine comminution technology to an ultrafine powder with a particle size of 10 μm for standby;

[0049] S3. Heat Borneolum Syntheticum to melt at 45 °C, add the ultrafine powder obtained in step S2, stir and disperse evenly to obtain a mixed powder;

[0050] S4. First, mix and incubate the carrier system with the neurotrophic factor at 2°C for 5 minutes, then mix with the alcohol extract of S1, the mixed powder of S3 and the TRPV1 agonist, and homogenize once with a high-pressure homogenizer at 100MPa pressure, followed by ultrasonic treatment at 250W for 10 minutes.

[0051] S5. Mix the homogeneous mixture obtained in S4 with the poultice matrix. The weight ratio of the poultice matrix to the homogeneous mixture is 1.5:1. Stir in a mixer at 20 rpm for 15 minutes. After stirring and degassing, coat the mixture onto a nonwoven fabric with a coating machine, cover with a protective film, and cut into 10cm×14cm patches to obtain the final product.

[0052] Example 2

[0053] Prepare according to the following proportions:

[0054] 30 parts of *Shenjincao*, 25 parts of *Bai Shao*, 25 parts of *Tougucao*, 15 parts of *Danggui*, 15 parts of *Chuanxiong*, 5 parts of borneol, 5 parts of *Xu Changqing*, 5 parts of *Jiangcan*, 15 parts of earthworm, 0.5 parts of neurotrophic factor, 0.5 parts of TRPV1 agonist, and 0.05 parts of carrier system.

[0055] The method for processing Xu Changqing is as follows: Take raw nux vomica seeds, add sand and stir-fry until the surface bulges and the color turns brownish-brown. After removing the sand, crush the seeds and pass them through a 100-mesh sieve to obtain Xu Changqing powder.

[0056] The neurotrophic factor is basic fibroblast growth factor.

[0057] The carrier system consists of soybean phospholipids, cholesterol, and octadecylamine in a weight ratio of 10:4:3.

[0058] Preparation steps:

[0059] S1. Weigh out the following herbs by weight: *Lycopodium clavatum*, *Paeonia lactiflora*, *Clematis chinensis*, *Angelica sinensis*, and *Ligusticum chuanxiong*. Crush them and pass them through an 80-mesh sieve. Add 12 times the weight of 80% ethanol solution and reflux extract at 60℃ for 2.5 hours each time. Combine the extracts and filter. Concentrate under reduced pressure at 0.09 MPa and 65℃ to obtain an alcohol extract with a relative density of 1.15.

[0060] S2. Weigh out Xu Changqing, Bombyx mori, and Pheretima aspergillum according to the weight parts, and process them into ultrafine powder with a particle size of 20μm using ultrafine pulverization technology, and set aside for later use.

[0061] S3. Heat the borneol to 50°C to melt it, add the ultrafine powder obtained in step S2, stir and disperse it evenly to obtain a mixed powder.

[0062] S4. First, mix and incubate the carrier system with the neurotrophic factor at 8°C for 20 minutes. Then, mix it with the alcohol extract of S1, the mixed powder of S3 and the TRPV1 agonist. Homogenize it three times at 150MPa using a high-pressure homogenizer. Then, sonicate it for 15 minutes at 350W.

[0063] S5. Mix the homogeneous mixture obtained in S4 with the poultice matrix. The weight ratio of the poultice matrix to the homogeneous mixture is 1.5:1. Stir in a mixer at 40 rpm for 30 minutes. After stirring and degassing, coat the mixture onto a nonwoven fabric with a coating machine, cover with a protective film, and cut into 10cm×14cm patches to obtain the final product.

[0064] Example 3

[0065] Prepare according to the following proportions:

[0066] 25 parts of *Shenjincao*, 18 parts of *Bai Shao*, 15 parts of *Tougucao*, 10 parts of *Danggui*, 10 parts of *Chuanxiong*, 3 parts of borneol, 4 parts of *Xu Changqing*, 3 parts of *Jiangcan*, 10 parts of earthworm, 0.3 parts of neurotrophic factor, 0.2 parts of TRPV1 agonist, and 0.03 parts of carrier system.

[0067] The method for processing Xu Changqing is as follows: Take raw nux vomica seeds, add sand and stir-fry until the surface bulges and the color turns brownish-brown. After removing the sand, crush the seeds and pass them through a 90-mesh sieve to obtain Xu Changqing powder.

[0068] The neurotrophic factor is basic fibroblast growth factor.

[0069] The carrier system consists of soybean phospholipids, cholesterol, and octadecylamine in a weight ratio of 10:3:2.

[0070] Preparation steps:

[0071] S1. Weigh out the following ingredients by weight: *Lycopodium clavatum*, *Paeonia lactiflora*, *Clematis chinensis*, *Angelica sinensis*, and *Ligusticum chuanxiong*. Crush them and pass them through a 60-80 mesh sieve. Add 10 times the weight of 75% ethanol solution and reflux extract twice at 55℃ for 2 hours each time. Combine the extracts and filter them. Concentrate under reduced pressure at 0.08 MPa and 60℃ to obtain an alcohol extract with a relative density of 1.12.

[0072] S2. Weigh out Xu Changqing, Bombyx mori, and Pheretima aspergillum according to the weight parts, and process them into ultrafine powder with a particle size of 15μm using ultrafine pulverization technology, and set aside for later use;

[0073] S3. Heat the borneol to 48°C to melt it, add the ultrafine powder obtained in step S2, stir and disperse it evenly to obtain a mixed powder;

[0074] S4. First, mix and incubate the carrier system with the neurotrophic factor at 5°C for 15 minutes, then mix with the alcohol extract of S1, the mixed powder of S3 and the TRPV1 agonist, and homogenize 1-3 times with a high pressure homogenizer at 100-150MPa pressure, followed by ultrasonic treatment at 300W for 12 minutes.

[0075] S5. Mix the homogeneous mixture obtained in S4 with the poultice matrix. The weight ratio of the poultice matrix to the homogeneous mixture is 1.5:1. Stir in a mixer at 30 rpm for 25 minutes. After stirring and degassing, coat the mixture onto nonwoven fabric with a coating machine, cover with a protective film, and cut into 10cm×14cm patches to obtain the final product.

[0076] Comparative Example 1

[0077] The formulation and preparation process are the same as in Example 3, but without the addition of bFGF, capsaicin, and carrier.

[0078] Comparative Example 2

[0079] The formulation and preparation process are the same as in Example 3, but bFGF is not added.

[0080] Comparative Example 3

[0081] The formulation and preparation process are the same as in Example 3, but TRPV1 agonist is not added, and bFGF is added in free form without using a carrier system.

[0082] I. Pharmacodynamic Experiments (Animal Experiments)

[0083] 1. Model establishment: SD rats were selected, and a spasticity model was established by injecting colchicine into the motor cortex of the rat brain.

[0084] 2. Grouping and Administration: Rats with successfully established models were randomly divided into 8 groups: model control group (applied blank poultice), positive control group (orally administered baclofen), Examples 1-3 of this invention, and Comparative Examples 1-3. Each group consisted of 8 rats. The poultice was applied once daily for 4 consecutive weeks.

[0085] Evaluation indicators: Passive range of motion (PROM) and muscle tone of the hind limbs of rats were measured weekly using a dystonia meter.

[0086]

[0087] Results: As shown in Table 1, compared with the model control group, all treatment groups improved PROM and reduced muscle tone to varying degrees. Among them, the effect of the group in this invention was significantly better than that of the positive control group and the control group, indicating that the preparation of this invention has excellent antispasmodic effect, and the combination of traditional Chinese medicine and biological factors produced a significant synergistic effect.

[0088] Example 3, compared with Comparative Example 2, demonstrates that the addition of bFGF and capsaicin to the traditional Chinese medicine base produced a synergistic effect, rather than a simple addition.

[0089] Compared with Comparative Example 3, Example 3 demonstrates that the absence of bFGF and its carrier, although having a certain short-term effect, results in insufficient long-term nerve repair function and significantly lower efficacy than the whole formula.

[0090] II. Skin Irritation Test

[0091] After hair removal from the backs of rabbits, poultices prepared according to Example 3 of this invention and poultices with a blank matrix were applied, respectively. These were removed after 24 hours, and skin reactions were observed. The results showed that the formulation of this invention did not cause irritation to either intact or damaged skin in rabbits.

[0092] III. Transdermal Transdermal Test

[0093] 1. Experimental materials and equipment

[0094] 1.1 Skin model: Ex vivo pig skin, 0.5-1.0 mm thick, stored at -20°C, thawed and inspected for integrity before use.

[0095] 1.2 Formulation preparation:

[0096] Group A: Embodiment 3 of the present invention;

[0097] Group B: Same as Group A, but without TRPV1 agonist (replaced with an equal volume of physiological saline).

[0098] Group C (Comparative Example 3): Contains active ingredients of traditional Chinese medicine and bFGF, but does not contain TRPV1 agonist and liposome carrier (bFGF is added in free form);

[0099] Group D: Contains active ingredients of traditional Chinese medicine and bFGF, but the TRPV1 agonist is replaced with 1% azone.

[0100] All formulations were adjusted to the same pH and viscosity to ensure comparability.

[0101] 1.3 Detection markers

[0102] Active components of traditional Chinese medicine: Paeoniflorin from white peony root was selected as a representative marker.

[0103] bFGF: Recombinant human bFGF was used and detected by immunological methods.

[0104] 1.4 Equipment and Reagents

[0105] Franz diffusion cell (effective diffusion area approximately 1.77 cm²) 2 (Receptor pool volume 12 mL).

[0106] HPLC system (for the quantification of paeoniflorin).

[0107] ELISA kit (for bFGF quantification).

[0108] A constant temperature water bath (maintained at 37±0.5°C).

[0109] Magnetic stirrer.

[0110] Physiological saline or PBS (pH 7.4) is used as the receptor solution.

[0111] 2. Experimental Procedure:

[0112] 2.1 Skin preparation: Cut the excised skin into appropriate sizes and fix it on the Franz diffusion cell, with the epidermis facing the donor cell and the dermis facing the recipient cell. Fill the recipient cell with preheated physiological saline and remove air bubbles.

[0113] 2.2 Balancing: Turn on the water bath and stir for 30 minutes to balance and ensure that the skin temperature is stable at 37°C.

[0114] 2.3 Administration: Apply 0.2 g of the formulation (equivalent to the unit area dose of the poultice in the patent) evenly to the skin surface in the donor pool. Six parallel samples were set up in each group (n=6).

[0115] 2.4 Sampling: At time points of 1, 2, 4, 6, 8, 12, and 24 hours, all liquid was taken from the receptor pool (and immediately replenished with an equal amount of fresh receptor liquid) and stored at -80°C for testing.

[0116] 2.5 Sample Analysis:

[0117] (1) Detection of paeoniflorin permeation rate (HPLC method)

[0118] HPLC chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-0.1% phosphoric acid aqueous solution (30:70, v / v); detection wavelength: 230 nm; column temperature: 30 ℃; flow rate: 1.0 mL / min; injection volume: 20 μL.

[0119] Standard curve construction: Prepare paeoniflorin standard solutions (concentrations of 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL), inject the solutions, and measure the peak areas. Plot a standard curve with concentration on the x-axis and peak area on the y-axis to obtain the regression equation (R²). 2 ≥0.999).

[0120] Sample determination: The collected sample from the receiving cell was injected and measured. The concentration of paeoniflorin at each time point was calculated based on the standard curve, and then the cumulative permeate (Q) and permeate rate (J, unit: μg / cm³) were calculated. 2 ·h, where J is the slope of the linear segment of the Qt curve.

[0121] (2) bFGF osmotic rate detection (ELISA method)

[0122] Kit selection: bFGF enzyme-linked immunosorbent assay kit (sensitivity 0.1 pg / mL, specificity ≥98%).

[0123] Operating procedures: According to the kit instructions, add the standard and the sample from the receiving pool to the wells of the ELISA plate and incubate at 37°C for 30 minutes; after washing the plate, add the enzyme-labeled antibody and incubate at 37°C for 30 minutes; after washing the plate, add the substrate chromogenic solution and incubate at 37°C in the dark for 15 minutes; add the stop solution and measure the absorbance (OD value) at 450 nm using an ELISA reader.

[0124] Standard curve plotting: A standard curve was plotted with the concentrations of bFGF standards (0 pg / mL, 1 pg / mL, 2 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL) on the x-axis and the OD value on the y-axis. 2 ≥0.998).

[0125] Sample determination: Based on the sample OD value, the bFGF concentration at each time point was calculated from the standard curve, and the cumulative permeability (Q) and permeability rate (J, unit: pg / cm³) were converted. 2 ·h).

[0126] 2.6 Determination of skin retention: After the experiment, the skin surface was scraped, the residual drug was extracted with solvent, and the content of Chinese medicine markers and bFGF in the skin was measured to assess local accumulation.

[0127] 3. Data Analysis

[0128] 3.1 Cumulative Transmission (Q): Calculate the cumulative transmission rate (μg / cm³) at each time point. 2 ).

[0129]

[0130] Among them, C n Let V be the concentration of the nth sampling, and V be the volume of the acceptor pool. i This represents the sampling volume.

[0131] 3.2 Transdermal transdermal rate (J): calculated from the steady-state slope of the Qt curve (μg / cm). 2 / h).

[0132] 3.3 Lag time: Obtained by extrapolating the Qt curve to the time axis.

[0133] 4. Experimental Results Data

[0134]

[0135]

[0136] Results analysis:

[0137] Group A, containing TRPV1 agonists, showed significantly higher cumulative permeation rates of active ingredients and bFGF compared to Groups B and C (p < 0.01), indicating that TRPV1 agonists can effectively promote transdermal absorption.

[0138] There was no significant difference between group A and group D (containing azone) (p > 0.05), indicating that the penetration-enhancing effect of TRPV1 agonist is comparable to that of the known penetration enhancer azone, thus verifying its effectiveness.

[0139] Group C, lacking both agonists and carriers, performed poorly, indicating that the carrier system also has some effect on transdermal absorption.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A topical medication for muscle spasms in cerebral palsy, characterized in that, The ingredients include: 15-30 parts of *Lycopodium clavatum*, 10-25 parts of *Paeonia lactiflora*, 10-25 parts of *Herba Clematis chinensis*, 5-15 parts of *Radix Angelicae Sinensis*, 5-15 parts of *Rhizoma Ligustici Chuanxiong*, 1-5 parts of borneol, 3-5 parts of *Radix Cynanchi Paniculati*, 1-5 parts of *Bombyx Batryticatus*, 5-15 parts of earthworm, 0.1-0.5 parts of neurotrophic factor, and 0.01-0.5 parts of TRPV1 agonist.

2. The topical medication for muscle spasms in cerebral palsy as described in claim 1, characterized in that, The ingredients include: 25 parts of *Lycopodium clavatum*, 18 parts of *Paeonia lactiflora*, 15 parts of *Clematis chinensis*, 10 parts of *Angelica sinensis*, 10 parts of *Ligusticum chuanxiong*, 3 parts of borneol, 4 parts of *Cynanchum paniculatum*, 3 parts of *Bombyx mori*, 10 parts of earthworm, 0.3 parts of neurotrophic factor, and 0.2 parts of TRPV1 agonist.

3. The topical medication for muscle spasms in cerebral palsy as described in claim 1, characterized in that, It also includes 0.01-0.05 parts of a carrier system, wherein the carrier system is a liposome containing soybean phospholipids, cholesterol and octadecylamine in a weight ratio of 10:(2-4):(1-3) with an average particle size of 100-150 nm.

4. The topical medication for muscle spasms in cerebral palsy as described in claim 1, characterized in that, The neurotrophic factor is basic fibroblast growth factor or brain-derived neurotrophic factor.

5. The topical medication for muscle spasms in cerebral palsy as described in claim 1, characterized in that, The TRPV1 agonist is capsaicin.

6. The topical medication for muscle spasms in cerebral palsy as described in claim 1, characterized in that, The dosage form of the topical medication is a poultice, gel, plaster, or liniment.

7. The method for preparing the topical medicine for muscle spasms in cerebral palsy as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Weigh out the following herbs by weight: *Lycopodium clavatum*, *Paeonia lactiflora*, *Clematis chinensis*, *Angelica sinensis*, and *Ligusticum chuanxiong*. Crush them and pass them through a 60-80 mesh sieve. Add 8-12 times the weight of 70-80% ethanol solution and reflux extract at 50-60℃ for 1-3 times, each time for 1.5-2.5 hours. Combine the extracts and filter them. Concentrate under reduced pressure to obtain an alcohol extract with a relative density of 1.10-1.

15. S2. Weigh out Xu Changqing, Bombyx mori, and Pheretima aspergillum according to the weight parts, and process them into ultrafine powder with a particle size of 10-20μm using ultrafine pulverization technology, and set aside for later use; S3. Heat the borneol to 45-50℃ to melt it, add the ultrafine powder obtained in step S2, stir and disperse it evenly to obtain a mixed powder; S4. First, mix and incubate the carrier system with the neurotrophic factor at a temperature of 2-8℃ for 5-20 minutes, then mix with the alcohol extract of S1, the mixed powder of S3 and the TRPV1 agonist, and homogenize 1-3 times using a high-pressure homogenizer at a pressure of 100-150MPa. S5. Add appropriate excipients according to the target dosage form, stir evenly, and then proceed with molding, drying, cutting and other processes to obtain the topical medicine.

8. The method for preparing the topical medicine for muscle spasms in cerebral palsy as described in claim 7, characterized in that, The vacuum degree of the reduced pressure concentration in S1 is 0.07-0.09 MPa, and the temperature is 55-65℃.

9. The method for preparing the topical medicine for muscle spasms in cerebral palsy as described in claim 7, characterized in that, In step S4, after homogenization, ultrasonic-assisted dispersion is used with an ultrasonic power of 250-350W for 10-15 minutes.

10. The use of a compound topical preparation as described in any one of claims 1-6 in the preparation of a medicament for reducing muscle tone and improving joint mobility in patients with cerebral palsy.

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