Traditional Chinese medicine compound preparation for treating scalds, large-area burns, deep chemical burns and gun injuries as well as preparation method and application of traditional Chinese medicine compound preparation

Abstract

The invention belongs to the technical field of Chinese herbal medicine effective component extraction, and particularly relates to a traditional Chinese medicine compound preparation for treating scalds, large-area burns, deep chemical burns and gun injuries and a preparation method and application of the traditional Chinese medicine compound preparation. A traditional Chinese medicine compound preparation for treating scalds, large-area burns, deep chemical burns and gun wounds is prepared from the following raw materials: a solanum muricatum root extract and silkworm moth polypeptide. The invention further discloses a corresponding preparation method of the cayenne fargesii root extract and the silkworm moth polypeptide. The invention also provides a medicinal preparation containing the traditional Chinese medicine compound preparation. According to the invention, the solanum muricatum root extract and the silkworm moth polypeptide have a synergistic effect, so that the healing time of a wound is greatly shortened on the basis of preventing inflammation of the wound. And enlightenment is provided for research and development of medicines for trauma such as burn, scald and chemical burn.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction of effective components of traditional Chinese medicines, and in particular relates to a traditional Chinese medicine composite preparation for treating scalds, large-area burns, deep chemical burns and gunshot wounds, and a preparation method and application thereof. Background Art

[0002] Sow vine is a plant of the genus Vitaceae, distributed in the southwest and Shaanxi, Gansu, Hubei, Hunan and other places. Medicinal plants of the genus Vitaceae have pharmacological effects such as antibacterial, anti-inflammatory, analgesic, antiviral, antioxidant, anti-tumor, and hypoglycemic, and are often used to treat carbuncle, scabies, mumps, erysipelas, etc. Existing studies have shown that extracts of plants of the genus Vitaceae have good antibacterial activity and can effectively prevent wound ulceration and inflammation. At present, the concept of topical drugs for the treatment of burns in clinical practice is mainly to control infection, avoid the deterioration of trauma, and accelerate the shedding of necrotic tissue, reconstruction of fibrous tissue, and regeneration of epidermis and other tissues. It can be seen that sow vine has medicinal prospects for the treatment of burns and scalds.

[0003] However, studies have shown that extracts from the genus Raspberry can affect the phosphorylation of AKT protein, thereby affecting the growth of fibroblasts and thus affecting the healing of skin wounds, which limits its application in trauma treatment. Therefore, it is of practical significance to provide a drug combination that can both reduce inflammation and control infection and promote skin wound healing. Summary of the invention

[0004] In view of this, the present invention provides the following technical solutions:

[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 sow vine root extract of the above-mentioned traditional Chinese medicine composite preparation comprises extracting sow vine root powder with ethanol to obtain the sow vine root extract.

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

[0008] Preferably, the extraction temperature is 50-75°C.

[0009] The preparation method of the silkworm moth polypeptide in the above-mentioned traditional Chinese medicine composite preparation comprises the following steps: using a composite protease to enzymatically hydrolyze male silkworm moth defatted protein powder to obtain the silkworm moth polypeptide.

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

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

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

[0013] An external medicine preparation contains the above-mentioned traditional Chinese medicine compound preparation; wherein the weight ratio of the sow vine root extract to the silkworm moth polypeptide is 20:(12.5-50).

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

[0015] Beneficial effects:

[0016] The invention provides a Chinese medicine compound preparation for treating scalds, burns, chemical burns and gunshot injuries, and the raw materials include sow vine 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] The extract of sow vine root and silkworm moth polypeptide in the present invention work synergistically, and on the basis of preventing wound inflammation, the wound healing time is greatly shortened. This shows that the Chinese medicine compound preparation in the present invention has the effect of inhibiting bacteria and promoting tissue regeneration, has a good effect on treating scalds, and provides new inspiration for the research and development of drugs for burns, scalds, chemical burns and other traumas. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : mRNA level of Akt1, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0019] Figure 2 : mTOR mRNA level, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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

[0021] Figure 4 : Comparison of the healing area of ​​scalded skin in rats at different time periods, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the control group. DETAILED DESCRIPTION

[0022] The invention provides a traditional Chinese medicine composite preparation for treating scalds, burns, chemical burns and gunshot injuries. The raw materials include sow vine root extract and silkworm moth polypeptide.

[0023] Preferably, the method for preparing the sow vine root extract comprises extracting sow vine root powder with ethanol to obtain the sow vine root extract.

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

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

[0026] Optionally, the sow vine root powder is obtained by drying the sow vine root in the shade, grinding it into powder, and passing it through a 50-200 mesh sieve. Preferably, passing it through a 100 mesh sieve.

[0027] Optionally, the solid-liquid ratio of the sow vine root powder extracted with ethanol is 1g:25-75mL. A more optimal solid-liquid ratio is 1g:50mL. As a preferred embodiment, the preparation method of the sow vine root extract comprises extracting the sow vine root powder with a 60-75% ethanol aqueous solution to obtain the extracted extract, filtering, collecting the supernatant, concentrating and drying, and obtaining the sow vine root extract.

[0028] Alternatively, the method for preparing the silkworm moth polypeptide comprises using a composite protease to enzymatically hydrolyze defatted protein powder of male silkworm moths to obtain the silkworm moth polypeptide.

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

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

[0031] Preferably, the amount of the composite protease used is 2000-6000 U / g relative to the male silkworm moth defatted protein powder, and more preferably, the amount used is 4000 U / g.

[0032] Preferably, the enzymatic hydrolysis temperature is 40-50°C, more preferably, the enzymatic hydrolysis temperature is 45°C.

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

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

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

[0036] The present invention also provides an external medicine preparation, comprising the above-mentioned Chinese medicine compound preparation, wherein the weight ratio is 20:(12.5-50). Preferably, the weight ratio is 20:(20-40).

[0037] Optionally, the preparation includes an ointment, a solution and a powder. Preferably, the ointment includes mixing the sow vine root extract and the silkworm moth polypeptide into an ointment with sesame oil.

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

[0039] In the present invention, the dosage of the drug of the present invention can be determined by factors such as the type of disease to be treated, the severity of the disease, the route of administration, the patient's age, gender, and health conditions. In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art. The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians of the art without making creative work belong to the scope of protection of the present invention.

[0040] Example 1

[0041] Preparation of silkworm moth polypeptide:

[0042] (1) taking a newly emerged male silkworm moth, removing its wings and scales, washing and draining, drying at 55-60° C., and grinding into powder;

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

[0044] (3) dissolving the male silkworm defatted protein in (2) in water at a ratio of 1:10 (w / v), adding 4000U / g of composite protease (the enzymatic activity ratio of papain: bromelain: neutral protease is 1:1:2, i.e., the enzymatic activities of papain, bromelain and neutral protease are 1000U / g, 1000U / g and 2000U / g, respectively) according to the mass of the male silkworm defatted protein powder, and performing enzymolysis at 45°C for 2h under the condition of pH 7 to complete the enzymolysis;

[0045] (4) Inactivate the enzyme in a water bath at 80° C. for 20 min, perform multiple filtration, and filter the obtained supernatant through an ultrafiltration membrane to separate an aqueous solution of a silkworm moth polypeptide with a molecular weight of 1 to 3 KDa, concentrate under reduced pressure, and spray-dry at low temperature to obtain a powder.

[0046] Example 2

[0047] Preparation of silkworm moth polypeptide:

[0048] The male silkworm moth defatted protein powder prepared by the method of Example 1 was used for enzymatic hydrolysis experiment;

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

[0050] The enzyme was inactivated in a water bath at 80°C for 20 minutes, and the mixture was filtered multiple times. The obtained supernatant was filtered and retained by an ultrafiltration membrane to separate an aqueous solution of a silkworm moth polypeptide with a molecular weight of 1 to 3 KDa, which was concentrated under reduced pressure and spray-dried at low temperature into powder.

[0051] Example 3

[0052] Preparation of silkworm moth polypeptide:

[0053] The male silkworm moth defatted protein powder prepared by the method of Example 1 was used for enzymatic hydrolysis experiment;

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

[0055] The enzyme was inactivated in a water bath at 80°C for 20 minutes, and the mixture was filtered multiple times. The obtained supernatant was filtered and retained by an ultrafiltration membrane to separate an aqueous solution of a silkworm moth polypeptide with a molecular weight of 1 to 3 KDa, which was concentrated under reduced pressure and spray-dried at low temperature into powder.

[0056] Example 4

[0057] Preparation of silkworm moth polypeptide:

[0058] The male silkworm moth defatted protein powder prepared by the method of Example 1 was used for enzymatic hydrolysis experiment;

[0059] The male silkworm moth defatted protein in 2) of Example 1 was dissolved in water at a ratio of 1:10 (w / v), and 4000U / g of composite protease (the enzymatic activity ratio of papain: bromelain: neutral protease was 2:2:1, i.e., the enzymatic activities of papain, bromelain and neutral protease were 1600U / g, 1600U / g and 800U / g, respectively) was added according to the mass of the male silkworm moth defatted protein powder, and enzymolysis was performed at 45°C for 2h under the condition of pH 7 to complete the enzymolysis.

[0060] The enzyme was inactivated in a water bath at 80°C for 20 minutes, and the mixture was filtered multiple times. The obtained supernatant was filtered and retained by an ultrafiltration membrane to separate an aqueous solution of a silkworm moth polypeptide with a molecular weight of 1 to 3 KDa, which was concentrated under reduced pressure and spray-dried at low temperature into powder.

[0061] Example 5

[0062] Preparation of Sow Vine Root Extract:

[0063] (1) taking the root of Sow Rattan, drying it in the shade, grinding it into powder, and passing it through a 100-mesh sieve to obtain Sow Rattan root powder;

[0064] (2) using 65% ethanol solution as an extracting solution, suspending the sow vine root powder in (1) at a ratio of 1 g:50 mL, and extracting in an electric constant temperature water bath at 70° C. for 2 h to obtain an extracting solution;

[0065] (3) Filter the extracted solution, collect the supernatant, concentrate the supernatant by rotary evaporation at 80° C., and then freeze-dry to obtain the sow vine root extract powder.

[0066] Example 6

[0067] Preparation of Sow Vine Root Extract:

[0068] (1) taking the root of sow vine, drying it in the shade, grinding it into powder, and passing it through a 50-mesh sieve to obtain sow vine root powder;

[0069] (2) using 60% ethanol solution as an extracting solution, suspending the sow vine root powder in (1) at a ratio of 1 g:25 mL, and extracting in an electric constant temperature water bath at 50° C. for 3 h to obtain an extracting solution;

[0070] (3) Filter the extracted solution, collect the supernatant, concentrate the supernatant by rotary evaporation at 80° C., and then freeze-dry to obtain the sow vine root extract powder.

[0071] Example 7

[0072] Preparation of Sow Vine Root Extract:

[0073] (1) taking the root of Sow Rattan, drying it in the shade, grinding it into powder, and passing it through a 200-mesh sieve to obtain Sow Rattan root powder;

[0074] (2) using 75% ethanol solution as an extracting solution, suspending the sow vine root powder in (1) at a ratio of 1 g:75 mL, and extracting in an electric constant temperature water bath at 75° C. for 1 h to obtain an extracting solution;

[0075] (3) Filter the extracted solution, collect the supernatant, concentrate the supernatant by rotary evaporation at 80° C., and then freeze-dry to obtain the sow vine root extract powder.

[0076] Example 8

[0077] Cell 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 reached 80% to 90%, the cells were seeded in a 12-well cell culture plate (cell number was 1×10 5 The cells were cultured for 24 h to allow them to fully adhere to the wall.

[0079] (2) Divided into 5 groups:

[0080] ①Complete DMEM medium was added to the control group;

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

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

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

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

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

[0086] (3) The cDNA obtained in (2) was subjected to qPCR detection. The primer sequences are shown in Table 1.

[0087] Table 1 qPCR primer sets

[0088]

[0089]

[0090] (4) Calculate the relative expression of mRNA (2 -ΔΔCT ).

[0091] The results are as follows Figure 1 and Figure 2 As shown: Sow vine root extract significantly reduces the transcription level of mTOR in human epidermal cells, and the addition of silkworm moth polypeptide can significantly slow down this trend. Sow vine root extract has no significant effect on the transcription level of Akt1 in human epidermal cells, and the addition of silkworm moth polypeptide significantly promotes the transcription level of Akt1. Since mTOR is located downstream of the PI3K / Akt signaling pathway, sow vine root extract has no effect on the transcription level of Akt, but significantly inhibits the transcription of mTOR, indicating that sow vine root extract affects the downstream pathway by inhibiting the activation of Akt. The addition of silkworm moth polypeptide can promote the activation of downstream pathways by activating the transcription level of Akt, thereby alleviating the inhibitory effect of sow vine root extract on downstream pathways, thereby promoting cell migration and proliferation.

[0092] Example 9

[0093] Scald animal model experiment:

[0094] Experimental animals: After one week of adaptive feeding, 28 healthy SD rats aged 6 weeks weighed 240±20 g and were divided into 5 groups: a control group of 5 rats, a model group of 5 rats and 3 experimental groups of 6 rats each.

[0095] Construction of rat scald model: 24 hours before the experiment, 10% sodium sulfide was applied to the back of the rat with cotton wool. After about 15 seconds, the liquid and the rat hair were gently washed off with warm water. The rat's back hair was removed, and the area slightly larger than 3.0×3.0cm 2 During the experiment, take a 10×10cm 2 Use a scalpel to cut a 3.0×3.0cm square in the middle of the cardboard. 2 Take a 1L beaker and fill it with 450mL of distilled water. Seal the beaker with paper and heat it on an electric stove. When water vapor comes out of the hole in the cardboard, start the experiment. After anesthetizing the rat, fix the rat with oval forceps, place the depilatory part of the rat facing the small hole, and use water vapor to burn the rat's back for 3 seconds, creating a 3.0×3.0cm 2 The area of ​​burns was successfully modeled.

[0096] Result judgment criteria: Healed, the scab on the burn area falls off completely, and the surface of the repaired tissue is fresh and relatively flat; basically healed, the scab falls off intermittently at the burn area, the surface of the new tissue is not flat enough and there is a small amount of exudate in a small area, but there is no obvious infection focus; infected, there is obvious redness and swelling around the scab, and there is pus or ulceration under the scab.

[0097] Administration: The model group was not treated; the control group was smeared with sesame oil; the experimental group 1 was smeared with a drug with sesame oil as the base and mixed with the powder of the extract of the root of the sow vine prepared in Example 5 (0.5 mg / kg); the experimental group 2 was smeared with a drug with sesame oil as the base and mixed with the powder of the extract of the root of the sow vine prepared in Example 5 (0.5 mg / kg) + the powder of the silkworm moth polypeptide prepared in Example 1 (0.5 mg / kg); the experimental group 3 was smeared with a drug with sesame oil as the base and mixed with the powder of the extract of the root of the sow vine prepared in Example 5 (0.5 mg / kg) + the powder of the silkworm moth polypeptide prepared in Example 1 (1 mg / kg). The drug was administered every 12 hours. The skin healing rate of the burn wound was observed and recorded on days 3, 6, 9, 12, 15, 18, 21, and 24 after injury.

[0098]

[0099] Table 2 Average healing area

[0100] Group 3d 6d 9d 12d 15d 18d 21d 24d Model 0.78 1.71 2.67 3.40 4.31 5.32 6.17 7.33 Comparison 0.66 1.30 2.05 2.56 3.60 4.74 5.58 7.01 Experimental Group 1 0.93 1.89 2.89 3.56 4.70 6.00 6.95 7.87 Experimental Group 2 1.11 2.50 4.14 5.74 6.98 7.93 8.58 8.99 Experimental Group 3 1.62 3.12 4.86 6.35 7.63 8.69 8.98 9.00

[0101] Experimental results: It should be noted that during the experiment, two rats in the model group and one rat in the control group developed infection, and their wound healing time was greatly prolonged, so they were eliminated during statistics. This phenomenon did not occur in the experimental group, which shows that the sow vine root extract has anti-inflammatory and antibacterial effects.

[0102] like Figure 3 As shown, the wounds of the mice gradually healed over time. The wounds of the rats in experimental group 3 were basically healed on the 18th day, and the wounds of the rats in experimental group 2 were basically healed on the 21st day. Figure 4 As shown, there was a significant difference between the control group and the model group on the 9th day, indicating that the sow vine root extract had an inhibitory effect on wound healing. On the 24th day, the wound healing of the control group was still better than that of the model group, but this advantage was not significant, which indicates that the inhibitory effect of the sow vine root extract on the wound gradually weakened as the wound healed.

[0103] In general, as shown in Table 2, Figure 3 and Figure 4 The experimental results show that the extract of sow vine root has a certain inhibitory effect on wound healing. The addition of silkworm moth polypeptide can offset this inhibitory effect and promote wound healing, which is consistent with the experimental results at the cellular level.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicine compound preparation for treating scalds, large-area burns, deep chemical burns and gunshot wounds, characterized in that: The raw materials include sow vine root extract and silkworm moth polypeptide.

2. The Chinese medicine composite preparation according to claim 1, characterized in that: The preparation method of the sow vine root extract comprises extracting sow vine root powder with ethanol to obtain the sow vine root extract.

3. The Chinese medicine composite preparation according to claim 2, characterized in that: The ethanol is a 60-75% ethanol aqueous solution.

4. The Chinese medicine composite preparation according to claim 2, characterized in that: The extraction temperature is 50-75°C.

5. The Chinese medicine composite preparation according to claim 1, characterized in that: The method for preparing the silkworm moth polypeptide comprises the steps of using a composite protease to enzymatically hydrolyze defatted protein powder of male silkworm moths to obtain the silkworm moth polypeptide.

6. The Chinese medicine composite preparation according to claim 5, characterized in that: The composite protease is a mixture of papain, bromelain and neutral protease.

7. The Chinese medicine composite preparation according to claim 5, characterized in that: The male silkworm moth defatted protein powder is prepared by using n-hexane to perform ultrasonic-assisted defatting on powder made from newly emerged male silkworm moths.

8. The Chinese medicine composite preparation according to claim 5, characterized in that: The molecular weight of the silkworm moth polypeptide is 1-3 KDa.

9. A pharmaceutical preparation for external use, characterized in that: A Chinese medicine composite preparation containing any one of claims 1 to 8, wherein the weight ratio of the sow vine root extract to the silkworm moth polypeptide is 20:(12.5-50).

10. Use of the Chinese medicine compound preparation according to any one of claims 1 to 8 or the pharmaceutical preparation according to claim 9 in the preparation of medicines for treating scalds, large-area burns, deep chemical burns and gunshot wounds.

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