Tobacco extract for promoting wound healing and preparation method and application thereof

Abstract

The invention discloses a tobacco extract for promoting wound healing, the tobacco extract comprises a tobacco acid water layer extract and a tobacco alcohol layer extract, the tobacco acid water layer extract and the tobacco alcohol layer extract are both mixtures of nicotine, chlorogenic acid and solanesol, and a preparation method of the tobacco extract comprises the steps of tobacco extraction, extraction treatment, purification pretreatment and purification treatment. The invention also discloses an application of the tobacco extract in promoting wound healing. The traditional Chinese medicine has the advantages of being convenient to use and small in side effect, wound healing can be promoted, meanwhile, the effects of local bacteriostasis, anti-inflammation, angiogenesis promotion and the like are achieved, complications such as wound infection are avoided, and the traditional Chinese medicine comprehensively plays a role in different stages of wound healing.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco extraction, and in particular to a tobacco extract for promoting wound healing, and a preparation method and application thereof. Background Art

[0002] Tobacco is often used to treat various diseases, such as colds, headaches, toothaches, trauma, burns, abscesses and ulcers. It can also be used for anesthesia, whitening teeth, and resisting fatigue. At the same time, it has the effect of removing dead tissue and promoting tissue regeneration and promoting wound healing in the treatment of burns, scalds, and wound ulcers.

[0003] Wound healing is an important and very complex dynamic process for humans or animals, including hemostasis, inflammation, proliferation and remodeling stages; the hemostasis stage begins immediately after the body is injured, endothelial damage activates platelets and initiates the coagulation cascade to promote thrombosis and fibrin clot formation, control bleeding and limit the spread of microorganisms; after the wound bleeding stops, inflammatory cells migrate to the wound and infiltrate, during which neutrophils, macrophages and lymphocytes play a role, releasing proinflammatory cytokines, cationic peptides, proteases, reactive oxygen species and growth factors, etc., to encourage inflammatory response, inhibit the penetration of exogenous microorganisms, kill microorganisms, and stimulate keratinocytes, fibroblasts and angiogenesis.

[0004] Tobacco contains nicotine, solanesol, chlorogenic acid and other polyphenols, flavonoids, sugars and terpenoids. Domestic and foreign studies have shown that nicotine can effectively promote angiogenesis both in vivo and in vitro and accelerate wound healing in diabetic mice. Chlorogenic acid has antioxidant, anti-inflammatory, antibacterial, antiviral and other effects, and can regulate the immune system, and has a high clinical application value. Solanesol has also been reported to have antioxidant, anti-inflammatory, neuroprotective and antibacterial activities.

[0005] Currently, there is no effective method for mixed extraction of compounds in tobacco. Therefore, the present invention proposes a tobacco extract for promoting wound healing and a preparation method and application thereof to solve the problems existing in the prior art. Summary of the invention

[0006] In view of the above problems, the purpose of the present invention is to propose a tobacco extract for promoting wound healing, and a preparation method and application thereof. The tobacco extract for promoting wound healing is easy to use and has few side effects. It can not only promote wound healing, but also play a role in local antibacterial, anti-inflammatory, and angiogenesis promotion, avoid the occurrence of complications such as wound infection, and play a comprehensive role in different stages of wound healing.

[0007] To achieve the purpose of the present invention, the present invention is implemented through the following technical scheme: a tobacco extract for promoting wound healing, including a tobacco acid aqueous layer extract and a tobacco alcohol layer extract, wherein the main components of the tobacco acid aqueous layer extract and the tobacco alcohol layer extract are a mixture mainly composed of nicotine, chlorogenic acid and solanesol.

[0008] A method for preparing a tobacco extract for promoting wound healing comprises the following steps:

[0009] Step 1: Extracting tobacco: drying the tobacco and grinding it into powder, then weighing the tobacco powder and adding 95% ethanol for extraction;

[0010] Step 2, extraction treatment, the extract in step 1 is filtered, the tobacco powder is filtered out, and then the extract is rotary evaporated to a small volume, and then the extract is spread on a tray and dried to form an extract. When the extract is viscous, it is stirred at intervals of 30 minutes, and then the extract is dissolved in acid water with pH = 4, and finally ethyl acetate is added for extraction;

[0011] Step 3: Pre-purification treatment: the ethyl acetate layer after extraction is back-extracted with an equal volume of alkaline water with a pH value of 10, the acid water and alkaline water obtained in step 2 are mixed and rotary evaporated to a small volume, which is marked as a sample, and the ethyl acetate layer is then rotary evaporated and dried to complete the pre-treatment;

[0012] Step 4, purification treatment, load AB-8 macroporous adsorption resin onto the column and calculate the column bed volume, rinse when the liquid level is close to the material surface, then load the marked sample in step 3, and when the sample liquid level is close to the material surface, add three times the column bed volume of acid water, four times the column bed volume of 30% ethanol, and three times the column bed volume of 95% ethanol for elution, finally collect the acid water layer and the 30% ethanol layer for drying to complete the purification.

[0013] A further improvement is that: in the step 1, the tobacco drying temperature is 75° C.; when adding ethanol for extraction, the extraction is repeated three times, each time for 2 hours, and the temperature is kept constant during the extraction.

[0014] Further improvements are: in step 2, the rotary evaporation temperature is 75° C. and the rotation speed is 60 rpm; the temperature for spreading and drying is also 75° C.; and the extraction is repeated three times with each extraction lasting 30 minutes.

[0015] Further improvements are: in step 3, the temperature of rotary evaporation of the acid water and alkaline water mixture is 85-95°C and the rotation speed is 60rpm; the temperature of rotary evaporation and drying of the ethyl acetate layer is 75°C and the rotation speed is 60rpm.

[0016] The further improvement is that: in the step 4, the macroporous adsorption resin needs to be soaked in 100-200 ml of 95% ethanol overnight before being loaded onto the column; the column bed volume after loading is equal to the product of the bottom area and the height; when flushing, add acid water or pure water three times the volume of the column bed for flushing until the chromatography column has no alcohol taste.

[0017] A further improvement is that if the elution column is not white at the end of elution in step 4, 95% ethanol is used for repeated elution, and the flow rate is reduced until the color of the material is completely eluted.

[0018] The invention discloses an application of a tobacco extract for promoting wound healing in promoting wound healing, and an application of a preparation added with the tobacco extract in promoting wound healing, inhibiting bacteria, resisting inflammation, and promoting angiogenesis.

[0019] The beneficial effects of the present invention are as follows: the present invention increases the utilization of tobacco resources by designing the extraction process of tobacco extract, and applies tobacco to the medical field; substances such as nicotine in the extract can promote platelet activation and angiogenesis, thereby promoting wound hemostasis and healing process; components such as chlorogenic acid and solanesol have antibacterial effects, can prevent bacterial infection, inhibit inflammatory response, and thus promote wound healing;

[0020] At the same time, tobacco extract is weakly acidic, which can lower the weakly alkaline wound pH, achieve dynamic acid-base regulation of the wound pH, and promote adipocytes and macrophages to eliminate necrotic tissue and cells in a low pH environment. At the same time, it promotes fibroblasts to synthesize extracellular matrix and accelerate cell proliferation, thereby promoting skin healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a flow chart of tobacco extraction in Example 1 of the present invention.

[0022] Figure 2 This is a flow chart of the purification of tobacco extract in Example 1 of the present invention.

[0023] Figure 3 This is a flow chart of the efficacy evaluation of Example 2 of the present invention. DETAILED DESCRIPTION

[0024] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0025] This embodiment provides a tobacco extract for promoting wound healing, including a tobacco acid aqueous layer extract and a tobacco alcohol layer extract, wherein the main components of the tobacco acid aqueous layer extract and the tobacco alcohol layer extract are a mixture mainly composed of nicotine, chlorogenic acid and solanesol.

[0026] Example 1

[0027] according to Figure 1 and Figure 2 As shown, this embodiment provides a method for preparing a tobacco extract for promoting wound healing, comprising the following steps:

[0028] Step 1: Dry the tobacco at 75°C and grind it into powder. Then weigh 5 kg of tobacco powder and add 20 L of 95% ethanol. Extract it three times at 78-80°C reflux temperature, each time for 2 hours.

[0029] Step 2: Filter the three extracts in step 1, filter out the tobacco powder, and then perform rotary evaporation to a small volume at a temperature of 75° C. and a rotation speed of 60 rpm, then transfer it to a tray and spread it out, and dry it at a temperature of 75° C. to form an extract. When the extract becomes viscous, stir it at intervals of 30 minutes, then take 5g of the extract and stir and dissolve it in 100ml of acid water with pH=4, and finally add 50ml of ethyl acetate to extract at room temperature, extract it three times for 30 minutes each time, and collect the ethyl acetate layer for later use.

[0030] Step 3: back-extract the ethyl acetate layer with an equal volume of alkaline water with a pH value of 10. After completion, discard the ethyl acetate layer, mix the acid water obtained in step 2 with the alkaline water and rotary evaporate to a small volume of concentrate at a temperature of 75°C and a rotation speed of 60rpm, mark it as a sample, and then rotary evaporate and dry the ethyl acetate layer at a temperature of 75°C and a rotation speed of 60rpm.

[0031] Step 4: Place 6L of AB-8 macroporous adsorption resin soaked overnight in 100-200ml of 95% ethanol on the column and calculate the column bed volume BV. The column bed volume after loading is equal to the product of the bottom area and the height. When the liquid level is close to the material surface, add three times the column bed volume of pure water for washing until the chromatography column has no alcohol taste;

[0032] Then, the marked sample in step 3 was loaded at a loading speed of 1.5 BV / h. When the sample liquid level was close to the material surface, 3 BV of acid water, 4 BV of 30% ethanol, and 1 BV of 95% ethanol were added in sequence for elution at a desorption rate of 1.5 BV / h. The 3 BV acid water layer and 4 BV of 30% ethanol layer samples were collected and rotary evaporated at a rotary evaporation temperature of 75°C and a rotary evaporation speed of 60 rpm. After rotary evaporation to a small volume of 4 L, the samples were transferred to an oven, dried at 85°C for 48 hours, and then crushed and packaged.

[0033] If the elution column is not white at the end of elution, it is necessary to use 95% ethanol to elute repeatedly and reduce the flow rate until the color of the material is completely eluted.

[0034] Example 2

[0035] according to Figure 3 As shown, this embodiment provides an application of a tobacco extract for promoting wound healing in promoting wound healing, and an application of a preparation added with the tobacco extract in promoting wound healing, inhibiting bacteria, anti-inflammation, and promoting angiogenesis.

[0036] Specifically, 48 male ICR mice were randomly divided into 4 groups, with 12 mice in each group, namely, blank control group: given 0.9% normal saline; positive control group: given 5% Yunnan Baiyao powder suspension; tobacco extract group A: 5% acid water layer extract gel; tobacco extract group B: 5% ethanol layer extract gel.

[0037] After the mice were anesthetized, the hair on the back of the mice near the tail was removed with a depilatory cream, and a 6mm skin puncher was used to punch holes at the same position on both sides of the mouse spine to create a full-thickness skin defect model. The mice were kept in a single cage after waking up, and the day of modeling was regarded as the 0th day of the experiment. Starting from the next day of modeling, the wound was administered at a specific time every day, and the drug was evenly applied to the entire wound area for 12 consecutive days. The wound healing of the mice was observed, and a ruler with a unit scale of 1mm was used as a ruler when taking wound photos every day. The size of the wound area on the 3rd, 6th, 9th, and 12th days was analyzed using ImageJ software. The effect of the drug on promoting wound healing was evaluated by calculating the wound healing rate. Wound healing rate (%) = (initial wound area-wound area after administration) / initial wound area × 100. On the 3rd, 6th, 9th, and 12th days after modeling, 3 mice were randomly selected from each group, and the full-thickness skin tissue of 3.0mm at the wound edge on both sides was cut, and paraffin sections were made after being fixed with 4% paraformaldehyde for 24 hours.

[0038] HE staining was used to observe inflammatory cell infiltration, epidermal connection status, epithelial tissue regeneration and dermal tissue integrity to analyze wound healing; MASSON staining was used to observe the formation of local new blood vessels and granulation tissue in the wound; immunohistochemistry was used to analyze the expression of IL-6, TNF-α and CD31 in the wound tissue to evaluate the level of inflammatory factors and the amount of angiogenesis, in order to discover the relevant mechanism by which tobacco extract promotes wound healing.

[0039] The experimental results showed that tobacco alcohol extract gel had a significant wound healing function compared with the blank group, and the healing effect was slightly better than that of tobacco acid water gel, and its effect was equivalent to that of Yunnan Baiyao. Its mechanism of action mainly involves the following points: 1. Tobacco extract can promote the remodeling process of epithelial tissue on the wound surface; 2. Tobacco extract can promote the formation of collagen tissue and angiogenesis on the wound surface; 3. Tobacco extract reduces local inflammatory cell infiltration in the wound and the production of inflammatory factors IL-6 and TNF-α.

[0040] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A tobacco extract for promoting wound healing, characterized in that: The invention comprises a tobacco acid aqueous layer extract and a tobacco alcohol layer extract, wherein the main components of the tobacco acid aqueous layer extract and the tobacco alcohol layer extract are a mixture mainly composed of nicotine, chlorogenic acid and solanesol.

2. A method for preparing a tobacco extract for promoting wound healing, characterized in that: The following steps are involved: Step 1: Extraction of tobacco: drying the tobacco and grinding it into powder, then weighing the tobacco powder and adding 95% ethanol for extraction; Step 2: Extraction treatment, the extract in step 1 is filtered, the tobacco powder is filtered out, and then the extract is rotary evaporated to a small volume, and then the extract is spread on a tray and dried to form an extract. When the extract is viscous, it is stirred at intervals of 30 minutes, and then the extract is dissolved in acid water with pH = 4, and finally ethyl acetate is added for extraction; Step 3: Purification pretreatment, the ethyl acetate layer after extraction is back-extracted with an equal volume of alkaline water with pH = 10, the acid water and alkaline water obtained in step 2 are mixed and rotary evaporated to a small volume, which is marked as a sample, and the ethyl acetate layer is rotary evaporated and dried to complete the pretreatment; Step 4: Purification treatment, load AB-8 macroporous adsorption resin onto the column and calculate the column bed volume, rinse when the liquid level is close to the material surface, then load the marked sample in step 3, and when the sample liquid level is close to the material surface, add three times the column bed volume of acid water, four times the column bed volume of 30% ethanol, and three times the column bed volume of 95% ethanol for elution, finally collect the acid water layer and the 30% ethanol layer for drying to complete the purification.

3. The method for preparing a tobacco extract for promoting wound healing according to claim 2, characterized in that: In the step 1, the tobacco drying temperature is 75° C., and the extraction temperature is 78-80° C.; when ethanol is added for extraction, the extraction is repeated three times, each time for 2 hours, and the temperature is kept constant during the extraction.

4. The method for preparing a tobacco extract for promoting wound healing according to claim 2, characterized in that: In the step 2, the rotary evaporation temperature is 75° C. and the rotation speed is 60 rpm; the temperature of spreading and drying is also 75° C.; and the extraction is repeated three times with each time lasting 30 minutes.

5. The method for preparing a tobacco extract for promoting wound healing according to claim 2, characterized in that: In the step 3, the temperature of rotary evaporation of the mixture of acid water and alkaline water is 85-95° C. and the rotation speed is 60 rpm; the temperature of rotary evaporation and drying of the ethyl acetate layer is 75° C. and the rotation speed is 60 rpm.

6. The method for preparing a tobacco extract for promoting wound healing according to claim 2, characterized in that: In the step 4, the macroporous adsorption resin needs to be soaked in 100-200 ml of 95% ethanol overnight before being loaded onto the column; the column bed volume after loading is equal to the product of the bottom area and the height; when flushing, add acid water or pure water three times the volume of the column bed to flush until the chromatography column has no alcohol taste.

7. The method for preparing a tobacco extract for promoting wound healing according to claim 2, characterized in that: If the elution column is not white at the end of elution in step 4, it is necessary to use 95% ethanol to elute repeatedly and reduce the flow rate until the color of the material is completely eluted.

8. Use of a tobacco extract for promoting wound healing, characterized in that: The preparation added with the tobacco extract is used in promoting wound healing, inhibiting bacteria, resisting inflammation and promoting angiogenesis.

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