A traditional Chinese medicine compound preparation for treating scald, extensive burn, deep chemical burn and gun wound, and a preparation method and application thereof

Abstract

This invention belongs to the field of traditional Chinese medicine (TCM) active ingredient extraction technology, and particularly relates to a TCM compound preparation for treating scalds, large-area burns, deep chemical burns, and gunshot wounds, as well as its preparation method and application. The TCM compound preparation for treating scalds, large-area burns, deep chemical burns, and gunshot wounds comprises, as raw materials, *Solanum lyratum* root extract and silkworm moth polypeptide. The invention also provides a corresponding preparation method for *Solanum lyratum* root extract and silkworm moth polypeptide. This invention further provides a pharmaceutical preparation containing the aforementioned TCM compound preparation. The *Solanum lyratum* root extract and silkworm moth polypeptide of this invention work synergistically, significantly shortening wound healing time while preventing wound inflammation. This provides inspiration for the development of drugs for burns, scalds, chemical burns, and other external injuries.

Description

Technical Field

[0001] This invention belongs to the field of extraction technology of effective components of traditional Chinese medicine, and in particular relates to a compound preparation of traditional Chinese medicine for treating scalds, large-area burns, deep chemical burns and gunshot wounds, as well as its preparation method and application. Background Technology

[0002] *Vitis thunbergii*, a plant belonging to the genus *Vitis* in the family Vitaceae, is distributed in southwestern China, including Shaanxi, Gansu, Hubei, and Hunan provinces. Medicinal plants in the *Vitis thunbergii* genus possess antibacterial, anti-inflammatory, analgesic, antiviral, antioxidant, antitumor, and hypoglycemic properties, and are commonly used to treat carbuncles, scabies, mumps, and erysipelas. Current research indicates that extracts from *Vitis thunbergii* plants have good antibacterial activity and can effectively prevent wound ulceration and inflammation. The current clinical approach to treating burns with topical medications primarily focuses on controlling infection, preventing wound deterioration, and accelerating the shedding of necrotic tissue, fibrous tissue reconstruction, and the regeneration of the epidermis and other tissues. Therefore, *Vitis thunbergii* shows promise for medicinal use in treating burns and scalds.

[0003] However, studies have shown that extracts from plants in the *Caulis Vigna* genus can affect the phosphorylation of AKT protein, thereby impacting fibroblast growth and ultimately skin wound healing, limiting their application in trauma treatment. Therefore, developing a drug combination that can both reduce inflammation and control infection while promoting skin wound healing is of practical significance. Summary of the Invention

[0004] In view of the above, the present invention provides the following technical solution:

[0005] A traditional Chinese medicine compound preparation for treating scalds, large-area burns, deep chemical burns, and gunshot wounds, the raw materials of which include Sow vine root extract and silkworm moth polypeptide.

[0006] The preparation method of the above-mentioned traditional Chinese medicine compound preparation, namely the sow vine root extract, includes extracting sow vine root powder with ethanol to obtain sow vine root extract.

[0007] Preferably, the ethanol is a 60-75% aqueous solution of ethanol.

[0008] Preferably, the extraction temperature is 50~75℃.

[0009] The above-mentioned traditional Chinese medicine compound preparation, wherein the preparation method of the silkworm moth polypeptide includes using a compound protease to enzymatically hydrolyze the defatted protein powder of male silkworm moths to obtain the silkworm moth polypeptide.

[0010] Preferably, the complex protease is a mixture of papain, bromelain, and neutral protease.

[0011] Preferably, the defatted protein powder of male silkworm moths is prepared by ultrasonic-assisted defatting of powder made from newly emerged male silkworm moths using n-hexane.

[0012] Preferably, the molecular weight of the silkworm moth polypeptide is 1~3 kDa.

[0013] A topical pharmaceutical preparation comprising the above-mentioned traditional Chinese medicine compound preparation; wherein the weight ratio of the sow vine root extract and the silkworm moth polypeptide is 20:(12.5~50).

[0014] The above-mentioned compound preparations of traditional Chinese medicine or the above-mentioned drug preparations are used in the preparation of drugs for treating scalds, large-area burns, deep chemical burns and gunshot wounds.

[0015] Beneficial effects:

[0016] This invention provides a traditional Chinese medicine compound preparation for treating burns, scalds, chemical burns, and gunshot wounds. The raw materials include *Solanum lyratum* root extract and silkworm moth polypeptide. The raw materials used are natural animal and plant extracts, without any chemical or other toxic side effects.

[0017] In this invention, the extract of *Solanum lyratum* root and silkworm moth polypeptide work synergistically to significantly shorten wound healing time while preventing inflammation. This indicates that the traditional Chinese medicine compound preparation in this invention has antibacterial and tissue-regenerating effects, and has a good effect on treating burns, providing new inspiration for the development of drugs for burns, scalds, chemical burns, and other external injuries. Attached Figure Description

[0018] Figure 1 Akt1 mRNA levels P<0.05, P<0.01, P<0.001, P<0.0001.

[0019] Figure 2 mTOR mRNA levels P<0.05, P<0.01, P<0.001, P<0.0001.

[0020] Figure 3 Changes in the healing rate of scalds in rats over time.

[0021] Figure 4 Comparison of the healing area of ​​scalded skin in rats at different time points, and comparison of each group with the control group. P<0.05, P<0.01, P<0.001, P<0.0001. Detailed Implementation

[0022] This invention provides a traditional Chinese medicine compound preparation for treating scalds, burns, chemical burns, and gunshot wounds. The raw materials include extracts from the roots of *Solanum tuberosum* and silkworm moth polypeptides.

[0023] In a preferred embodiment, the preparation method of the *Solanum lyratum* root extract includes extracting *Solanum lyratum* root powder with ethanol to obtain the *Solanum lyratum* root extract.

[0024] Preferably, the ethanol is a 60-75% aqueous solution of ethanol. More preferably, the ethanol is a 65% aqueous solution of ethanol.

[0025] Preferably, the extraction temperature is 50~75 °C. More preferably, the temperature is 70 °C. Optionally, the extraction method includes immersion extraction, percolation extraction, ultrasonic-assisted extraction, reflux extraction, and constant temperature water bath extraction.

[0026] Optionally, the sow vine root powder is prepared by air-drying the sow vine roots, grinding them into powder, and then passing them through a 50-200 mesh sieve. Preferably, it is passed through a 100 mesh sieve.

[0027] Optionally, the material-to-liquid ratio for extracting *Ilex chinensis* root powder with ethanol is 1g:25-75mL. A more preferred material-to-liquid ratio is 1g:50mL. As a preferred embodiment, the preparation method of the *Ilex chinensis* root extract includes extracting *Ilex chinensis* root powder with a 60-75% ethanol aqueous solution to obtain the extracted solution, filtering, collecting the supernatant, concentrating and drying to obtain the *Ilex chinensis* root extract.

[0028] Optionally, the method for preparing the silkworm moth polypeptide includes using a complex protease to enzymatically hydrolyze defatted protein powder from male silkworm moths to obtain the silkworm moth polypeptide.

[0029] Optionally, the complex protease is a mixture of papain, bromelain, and neutral protease.

[0030] Preferably, the enzyme activity ratio of papain:bromelain:neutral protease, measured by enzyme activity, is (0.5~2):(0.5~2):(1~4). More preferably, the enzyme activity ratio of papain:bromelain:neutral protease, measured by enzyme activity, is 1:1:2.

[0031] Preferably, the amount of the complex protease used relative to the defatted protein powder of male silkworm moths is 2000~6000 U / g. More preferably, the amount used is 4000 U / g.

[0032] Preferably, the enzymatic hydrolysis temperature is 40~50 ℃, more preferably, the enzymatic hydrolysis temperature is 45 ℃.

[0033] Preferably, the enzymatic hydrolysis time is 1 to 3 hours. More preferably, the enzymatic hydrolysis time is 2 hours.

[0034] Optionally, the defatted protein powder of male silkworm moths is prepared by ultrasonic-assisted defatting of powder made from newly emerged male silkworm moths using n-hexane.

[0035] Preferably, the molecular weight of the silkworm moth polypeptide is 1~3 kDa.

[0036] The present invention also provides a topical pharmaceutical preparation containing the above-mentioned traditional Chinese medicine compound preparation; wherein the weight ratio is 20:(12.5~50). Preferably, the weight ratio is 20:(20~40).

[0037] Optionally, the formulation includes ointments, solutions, and powders. Preferably, the ointment comprises the above-mentioned *Solanum lyratum* root extract and the above-mentioned silkworm moth polypeptide formulated into an ointment using sesame oil.

[0038] The above-mentioned traditional Chinese medicine compound preparations or drug preparations are used in the preparation of drugs for treating scalds, large-area burns, deep chemical burns and gunshot wounds.

[0039] In this invention, the drug dosage can be determined by factors such as the type and severity of the disease, the route of administration, the patient's age, gender, and health status. Unless otherwise specified, all raw material components in this invention are commercially available products well-known to those skilled in the art. The technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0040] Example 1

[0041] Preparation of silkworm moth polypeptides:

[0042] (1) Take newly emerged male silkworm moths, remove their wings and scales, wash and drain them, dry them at 55~60 ℃, and grind them into powder.

[0043] (2) Ultrasonic-assisted degreasing was performed using n-hexane as solvent. The ultrasonic conditions were: material-to-liquid ratio 1g:5mL, temperature 55~60℃, time 1 h, and power 500 W. After drying, defatted protein powder from male silkworm moths was obtained for use in enzymatic hydrolysis experiments.

[0044] (3) Dissolve the defatted protein of male silkworm moth in water at a ratio of 1:10 (w / v), and add 4000 U / g of compound protease (the enzyme activity ratio of papain: bromelain: neutral protease is 1:1:2, that is, the enzyme activities of papain, bromelain and neutral protease are 1000 U / g, 1000 U / g and 2000 U / g respectively) according to the mass of the defatted protein powder of male silkworm moth. Under the condition of pH 7, enzymatic hydrolysis is carried out at 45 ℃ for 2 h, and the enzymatic hydrolysis is completed.

[0045] (4) The enzyme was inactivated by water bath at 80 ℃ for 20 min, and the supernatant was filtered through an ultrafiltration membrane to separate an aqueous solution of silkworm moth polypeptide with a molecular weight of 1~3 KDa. The solution was concentrated under reduced pressure and spray-dried into powder at low temperature.

[0046] Example 2

[0047] Preparation of silkworm moth polypeptides:

[0048] The defatted protein powder of male silkworm moths prepared by the method in Example 1 was used for enzymatic hydrolysis experiments.

[0049] The defatted protein from male silkworm moths in Example 1 (2) was dissolved in water at a ratio of 1:10 (w / v). 2000 U / g of a complex protease (the enzyme activity ratio of papain: bromelain: neutral protease was 0.5:1:1, i.e., the enzyme activities of papain, bromelain and neutral protease were 400 U / g, 800 U / g and 800 U / g, respectively) was added according to the mass of the defatted protein powder. Enzymatic hydrolysis was carried out at 40 °C for 3 h under pH 7 conditions.

[0050] Enzymes were inactivated in an 80 ℃ water bath for 20 min, and the mixture was filtered multiple times. The supernatant was filtered through an ultrafiltration membrane to separate an aqueous solution of silkworm moth polypeptides with a molecular weight of 1~3 KDa. The solution was then concentrated under reduced pressure and spray-dried at low temperature into powder.

[0051] Example 3

[0052] Preparation of silkworm moth polypeptides:

[0053] The defatted protein powder of male silkworm moths prepared by the method in Example 1 was used for enzymatic hydrolysis experiments.

[0054] The defatted protein from male silkworm moths in Example 1 (2) was dissolved in water at a ratio of 1:10 (w / v). 6000 U / g of a complex protease (the enzyme activity ratio of papain: bromelain: neutral protease was 0.5:0.5:4, i.e., the enzyme activities of papain, bromelain and neutral protease were 600 U / g, 600 U / g and 4800 U / g, respectively) was added according to the mass of the defatted protein powder. Enzymatic hydrolysis was carried out at 50 °C for 1 h under pH 7 conditions, and the hydrolysis was completed.

[0055] Enzymes were inactivated in an 80 ℃ water bath for 20 min, and the mixture was filtered multiple times. The supernatant was filtered through an ultrafiltration membrane to separate an aqueous solution of silkworm moth polypeptides with a molecular weight of 1~3 KDa. The solution was then concentrated under reduced pressure and spray-dried at low temperature into powder.

[0056] Example 4

[0057] Preparation of silkworm moth polypeptides:

[0058] The defatted protein powder of male silkworm moths prepared by the method in Example 1 was used for enzymatic hydrolysis experiments.

[0059] The defatted protein from male silkworm moths in Example 1(2) was dissolved in water at a ratio of 1:10 (w / v). 4000 U / g of a complex protease (with an enzyme activity ratio of papain: bromelain: neutral protease of 2:2:1, i.e., the enzyme activities of papain, bromelain, and neutral protease were 1600 U / g, 1600 U / g, and 800 U / g, respectively) was added according to the mass of the defatted protein powder. Enzymatic hydrolysis was performed at 45 °C for 2 h at pH 7, and the hydrolysis was completed.

[0060] Enzymes were inactivated in an 80 ℃ water bath for 20 min, and the mixture was filtered multiple times. The supernatant was filtered through an ultrafiltration membrane to separate an aqueous solution of silkworm moth polypeptides with a molecular weight of 1~3 KDa. The solution was then concentrated under reduced pressure and spray-dried at low temperature into powder.

[0061] Example 5

[0062] Preparation of Sow Vine Root Extract:

[0063] (1) Take the roots of the sow vine, dry them in the shade, grind them into powder, and pass them through a 100-mesh sieve to obtain sow vine root powder;

[0064] (2) Using 65% ethanol solution as the extraction solution, the sow vine root powder in (1) was suspended at a ratio of 1g:50mL and extracted in an electric thermostatic water bath at 70℃ for 2 h to obtain the extracted solution.

[0065] (3) Filter the extract after leaching, collect the supernatant, concentrate the supernatant by rotary evaporation at 80 °C, and then freeze dry to obtain the root extract powder of Sow vine.

[0066] Example 6

[0067] Preparation of Sow Vine Root Extract:

[0068] (1) Take the root of the sow vine, dry it in the shade, grind it into powder, and pass it through a 50-mesh sieve to obtain the powder of the sow vine root;

[0069] (2) Using 60% ethanol solution as the extraction solution, the sow vine root powder in (1) was suspended at a ratio of 1g:25mL and extracted in an electric thermostatic water bath at 50℃ for 3 h to obtain the extracted solution.

[0070] (3) Filter the extract after leaching, collect the supernatant, concentrate the supernatant by rotary evaporation at 80 °C, and then freeze dry to obtain the root extract powder of Sow vine.

[0071] Example 7

[0072] Preparation of Sow Vine Root Extract:

[0073] (1) Take the roots of the sow vine, dry them in the shade, grind them into powder, and pass them through a 200-mesh sieve to obtain sow vine root powder;

[0074] (2) Using 75% ethanol solution as the extraction solution, the sow vine root powder in (1) was suspended in a ratio of 1g:75mL and extracted in an electric thermostatic water bath at 75℃ for 1 h to obtain the extracted solution.

[0075] (3) Filter the extract after leaching, collect the supernatant, concentrate the supernatant by rotary evaporation at 80 °C, and then freeze dry to obtain the root extract powder of Sow vine.

[0076] Example 8

[0077] Cellular level experiments:

[0078] (1) Human fibroblast HFF-1 cells were cultured in complete DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin). When the cell confluence was 80%~90%, the cells were seeded in 12-well cell culture plates (cell number 1×10⁶). 5 (1), continue culturing for 24 hours to allow the cells to fully adhere to the culture vessel.

[0079] (2) Divide into 5 groups on average:

[0080] ① The control group was given complete DMEM medium;

[0081] ②The sow vine root extract group was added to DMEM medium containing the sow vine root extract (20 mg / L) prepared in Example 5;

[0082] ③ Group L was given DMEM medium containing the sow vine root extract (20 mg / L) prepared in Example 5 and the silkworm moth polypeptide (12.5 mg / mL) prepared in Example 1;

[0083] ④ Group M was given DMEM medium containing the sow vine root extract (20 mg / L) prepared in Example 5 and the silkworm moth polypeptide (25 mg / mL) prepared in Example 1;

[0084] ⑤ Group H was given DMEM medium containing the root extract of Sow vine (20 mg / L) prepared in Example 5 and the silkworm moth polypeptide (50 mg / mL) prepared in Example 1;

[0085] Cells were collected after 72 h of culture, total RNA was extracted, and RNA concentration was measured by UV spectrophotometer. 2 μg of RNA from each group was transcribed into cDNA using a reverse transcription premix kit.

[0086] (3) Take the cDNA from (2) for qPCR detection. The primer sequences are shown in Table 1.

[0087] Table 1 qPCR primer set

[0088]

[0089] (4) Calculate the relative expression level of mRNA (2) -ΔΔCT ).

[0090] The results are as follows Figure 1 and Figure 2 As shown, *Solanum lyratum* root extract significantly reduced mTOR transcription levels in human epidermal cells, while the addition of silkworm moth polypeptide significantly slowed this trend. *Solanum lyratum* root extract did not significantly affect Akt1 transcription levels in human epidermal cells, but the addition of silkworm moth polypeptide significantly promoted Akt1 transcription. Since mTOR is downstream of the PI3K / Akt signaling pathway, *Solanum lyratum* root extract had no effect on Akt transcription levels but significantly inhibited mTOR transcription, indicating that *Solanum lyratum* root extract affects downstream pathways by inhibiting Akt activation. The addition of silkworm moth polypeptide can alleviate the inhibitory effect of *Solanum lyratum* root extract on downstream pathways by activating Akt transcription, thereby promoting cell migration and proliferation.

[0091] Example 9

[0092] Animal model experiments of burns:

[0093] Experimental animals: 28 healthy 6-week-old SD rats were acclimatized for one week and weighed 240±20 g. The rats were divided into 5 groups: control group (n=5), model group (n=5), and 3 experimental groups (n=6 each).

[0094] Establishment of a rat scald model: 24 hours before the experiment, apply 10% sodium sulfide to the back of the rats using absorbent cotton. After approximately 15 seconds, gently wash away the solution and rat fur with warm water, then remove the rat's back fur, ensuring the affected area is slightly larger than 3.0 × 3.0 cm. 2 During the experiment, a 10×10 cm sample was taken. 2 A piece of cardboard, with a 3.0 x 3.0 cm area cut out in the center with a scalpel. 2 To create a small hole, take a 1 L beaker and fill it with 450 mL of distilled water. Seal the mouth of the beaker with paper and heat it on an electric stove. Once steam is emitted from the hole in the cardboard, begin the experiment. After anesthetizing the rat, fix it with oval forceps, place the shaved area of ​​the rat directly over the small hole, and cauterize the rat's back with steam for 3 seconds, creating a 3.0 × 3.0 cm lesion. 2 The burn area was successfully modeled.

[0095] Criteria for judging the results: Healed, the scab on the burn falls off completely, and the surface of the repaired tissue is fresh and relatively flat; Basically healed, the scab falls off intermittently, the surface of the new tissue is not flat and there is a small amount of exudate in a small area, but there is no obvious infection focus; Infected, obvious redness and swelling appear around the scab, and there is pus or ulceration under the scab.

[0096] Administration methods: The model group received no treatment; the control group received sesame oil; Experimental group 1 received a drug mixture with sesame oil as the base, mixed with 0.5 mg / kg of *Sophora japonica* root extract powder prepared in Example 5; Experimental group 2 received a drug mixture with sesame oil as the base, mixed with 0.5 mg / kg of *Sophora japonica* root extract powder prepared in Example 5 + 0.5 mg / kg of silkworm moth polypeptide powder prepared in Example 1; Experimental group 3 received a drug mixture with sesame oil as the base, mixed with 0.5 mg / kg of *Sophora japonica* root extract powder prepared in Example 5 + 1 mg / kg of silkworm moth polypeptide powder prepared in Example 1. Drug administration was performed every 12 hours. The skin healing rate of the burn wound was observed and recorded at 3, 6, 9, 12, 15, 18, 21, and 24 days post-injury.

[0097] ;

[0098] .

[0099] Table 2 Average Healing Area

[0100]

[0101] Experimental Results: It should be noted that during the experiment, two rats in the model group and one rat in the control group showed signs of infection, with their wound healing time being significantly prolonged. Therefore, these rats were excluded from the statistical analysis. This phenomenon did not occur in the experimental group, indicating that the extract of *Solanum lyratum* root has anti-inflammatory and antibacterial effects.

[0102] like Figure 3 As shown, the mice's wounds gradually healed over time. The wounds of rats in experimental group 3 were basically healed by day 18, and the wounds of rats in experimental group 2 were basically healed by day 21.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a traditional Chinese medicine compound preparation in the preparation of a medicine for treating burns, characterized in that, The raw materials of the traditional Chinese medicine compound preparation are composed of Sow vine root extract and silkworm moth polypeptide; The preparation method of the sow vine root extract is to extract the sow vine root powder with ethanol to obtain the sow vine root extract; The ethanol is a 60-75% aqueous solution of ethanol; The extraction temperature is 50~75℃; The preparation method of the silkworm moth polypeptide is to obtain the silkworm moth polypeptide by enzymatic hydrolysis of defatted protein powder from male silkworm moths using a complex protease. The complex protease is a mixture of papain, bromelain, and neutral protease; Based on enzyme activity, the enzyme activity ratio of papain: bromelain: neutral protease is (0.5~2):(0.5~2):(1~4); The amount of the complex protease used relative to the defatted protein powder of male silkworm moths is 2000~6000 U / g; The enzymatic hydrolysis temperature is 40~50℃; The enzymatic hydrolysis time is 1-3 hours; The molecular weight of the silkworm moth polypeptide is 1~3 kDa; The weight ratio of the sow vine root extract to the silkworm moth polypeptide is 20:(12.5~50).

2. The application according to claim 1, characterized in that, The defatted protein powder from male silkworm moths is prepared by ultrasonic-assisted defatting of powder made from newly emerged male silkworm moths using n-hexane.

3. A compound preparation of traditional Chinese medicine, characterized in that, The raw materials of the traditional Chinese medicine compound preparation are composed of Sow vine root extract and silkworm moth polypeptide; The preparation method of the sow vine root extract is to extract the sow vine root powder with ethanol to obtain the sow vine root extract; The ethanol is a 60-75% aqueous solution of ethanol; The extraction temperature is 50~75℃; The preparation method of the silkworm moth polypeptide is to obtain the silkworm moth polypeptide by enzymatic hydrolysis of defatted protein powder from male silkworm moths using a complex protease. The complex protease is a mixture of papain, bromelain, and neutral protease; Based on enzyme activity, the enzyme activity ratio of papain: bromelain: neutral protease is (0.5~2):(0.5~2):(1~4); The amount of the complex protease used relative to the defatted protein powder of male silkworm moths is 2000~6000 U / g; The enzymatic hydrolysis temperature is 40~50℃; The enzymatic hydrolysis time is 1-3 hours; The molecular weight of the silkworm moth polypeptide is 1~3 kDa; The weight ratio of the sow vine root extract to the silkworm moth polypeptide is 20:(12.5~50).

4. A topical pharmaceutical preparation, characterized in that, It contains the traditional Chinese medicine compound preparation as described in claim 1 or 2.

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