TWI702960B - Uses of osmanthus fragrans extracts for manufacturing a composition for wound healing - Google Patents

Abstract

This invention relates to uses of an Osmanthus fragrans extract for manufacturing a composition for wound healing, wherein the Osmanthus fragrans extract is obtained by extraction with water and further with ethanol solution.

Description

Use of Osmanthus fragrans extract for preparing wound healing composition

The present invention provides a use of Osmanthus fragrans extract for preparing hair growth promoting composition and wound healing composition.

Osmanthus ( Osmanthus fragrans (Thunb) Lour.) is an evergreen shrub of Oleaceae (Oleaceae) Osmanthus (Osmanthus). The flower is fragrant. Its essential oil can be extracted to prepare osmanthus extract. It can be used in the production of food and cosmetics. Above, it has the effects of warming the lung, dispelling cold and relieving pain. In medicinal use, its root or root bark can be used to treat stomach pain, toothache, rheumatism, numbness, and bone pain; its flower can be used to resolve phlegm, dispel blood stasis, treat phlegm, cough, intestinal wind and blood dysentery, hernia, toothache and bad breath ; And its fruit can be used to warm the stomach, calm the liver, invigorate the kidney, dispel cold and treat liver and stomach pain.

There has been a Republic of China (Taiwan) Patent Publication No. I341205 (Patent Application No. 096137207), which discloses a method of extracting anti-asthmatic substances from Osmanthus fragrans, and extracting substances with anti-asthmatic effects with ethanol, which can relieve the respiratory tract and lung The chronic inflammatory symptoms in the lower part of the body can relieve the symptoms of asthma, prevent and reduce the onset of asthma, and have obvious health care effects. The Republic of China (Taiwan) Patent Publication No. I442929 (Patent Application No. 101118954) discloses that a product extracted from Osmanthus fragrans water can be used to treat lung fibrosis. The Republic of China (Taiwan) Patent Publication No. I411440 (Patent Application No. 100119436) discloses the use of an Osmanthus fragrans extract in the preparation of drugs or health foods for the prevention or treatment of depression.

In addition, Chinese Patent Publication No. CN103768152 A (Patent Application No. 201410043501.8) discloses the application of an Osmanthus fragrans phenylethanol glycoside extract to prepare food, medicine or health care products for preventing and treating diabetes. Chinese Patent Publication No. CN103767975 A (Patent Application No. 201410043486.7) discloses the application of an Osmanthus fragrans phenylethanol glycoside extract to prepare whitening cosmetics. Chinese Patent Publication No. CN103768151 A (Patent Application No. 201410043488.6) discloses an application of an Osmanthus fragrans phenylethanol glycoside extract to prepare anti-aging drugs or health products. Chinese Patent Publication No. CN103223018 A (Patent Application No. 201310169455.1) discloses the application of an Osmanthus fragrans polyphenol extract in the preparation of anti-tumor drugs or health products.

However, there is no related literature that has reported that Osmanthus fragrans or its extract has the function of promoting hair growth and wound healing. The present invention unexpectedly finds a novel use of Osmanthus fragrans extract for promoting hair growth and wound healing.

One aspect of the present invention provides a use of Osmanthus fragrans extract for preparing a hair growth promoting composition.

Another aspect of the present invention provides a use of Osmanthus fragrans extract for preparing a composition for promoting wound healing.

In the embodiment of the present invention, the Osmanthus fragrans extract of the present invention can promote angiogenesis and promote collagen production.

In the embodiment of the present invention, the Osmanthus fragrans extract of the present invention is obtained by first extracting with pure water and then extracting with an ethanol solution.

These and other aspects of the present invention can be made clearer by the following descriptions and drawings of the preferred embodiments; even though there may be changes or modifications, they do not deviate from the novel concepts disclosed by the present invention Spirit and category.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those who are acquainted with in the field of the present invention.

Unless clearly specified in the text, the meanings of the singular forms "one", "one", and "the" used in this article all include the plural form of "at least one". Therefore, for example, when referring to "an ingredient", it includes a plurality of such ingredients and equivalents known to those with ordinary knowledge in the field.

The "extract" used here refers to the product obtained by extracting a material, usually a solution or concentrated preparation obtained by immersing or mixing the material to be extracted in a solvent. Typically, the extract is prepared from fresh plants or ground or dried plant samples.

"Wound" as used herein includes infectious wounds or non-infectious wounds, and refers to injuries to the epidermis (such as the dermis layer of the skin), including skin tears, cuts, or punctures. Wounds include but are not limited to open wounds, such as incisions, cuts, cuts, abrasions, stab wounds, traumatic skin injuries, penetrating wounds, burns; wounds caused by disease or other chronic tissue damage; caused by external injuries or surgical operations Wounds; wounds caused by medical procedures, such as dermatological surgery. Wounds can also cause skin symptoms such as allergic dermatitis due to corticosteroid reactions.

According to the present invention, it has been found in animal experiments that Osmanthus fragrans extract has the effect of promoting hair growth. Therefore, the present invention provides a use of Osmanthus fragrans extract for preparing hair growth promoting composition. The hair growth promoting composition can be made into the form of medicine, shampoo, conditioner, hair conditioner, hair dye, hair oil or spray.

On the other hand, the present invention also provides an Osmanthus fragrans extract to promote wound healing. Therefore, the present invention provides a use of Osmanthus fragrans extract for preparing a composition for promoting wound healing. The wound healing promoting composition can be made into the form of medicine, skin care agent, insect repellent or spray.

According to the present invention, the wound may be an infectious wound or a non-infectious wound. In a specific embodiment of the present invention, the infectious wound is infected by Staphylococcus aureus.

As described in the examples of the present invention, the Osmanthus fragrans extract of the present invention can promote angiogenesis and promote collagen production.

According to an embodiment of the present invention, the osmanthus extract is obtained by first extracting with pure water and then extracting with an ethanol solution. In one embodiment, the Osmanthus fragrans extract is obtained by extracting and filtering Osmanthus fragrans with pure water, then extracting with an ethanol solution, and then filtering and concentrating.

To implement the present invention, the Osmanthus fragrans extract can be used in the form of various types of compositions. The composition may be in the form of a solution, hydrogel, spray, drops or ointment. In addition, it may further contain other antibacterial agents, anti-inflammatory agents, cleansing or skin care components.

According to the present invention, the pharmaceutical form can be combined with commonly used excipients or fillers according to any conventional technology. It can also be formulated into medicines according to common techniques or methods commonly used in medicine, containing a medically effective amount of the Osmanthus fragrans extract of the present invention, together with a pharmaceutically acceptable carrier.

The above description of the invention and the following examples illustrate the present invention, but are not intended to limit the scope of the present invention.

Example

Example 1: Preparation and treatment of Osmanthus fragrans extract

Take 1.0 kg of osmanthus ( Osmanthus fragrans ) into 10-20 times the volume of pure water, heat to extract and filter, then concentrate the extract, then extract with ethanol solution, filter and concentrate, you can get osmanthus extract (about 250.4 g).

Example 2: Evaluation of hair growth patterns

The experimental animal of the present invention is C57BL/6 mice purchased from the National Laboratory Animal Center, and the age is 6 weeks. The experimental animal was raised in the Animal Center of the National Defense Medical College. The temperature of the animal room was maintained at 18~26°C, the relative humidity was 30%~70%, the photoperiod was 12 hours of light and 12 hours of night, and sufficient feed and water were provided.

In order to confirm the hair growth-promoting activity of the Osmanthus fragrans extract, group treatments were carried out. Among them, the positive control group was treated with the known hair growth ingredient Minoxidil. The mice were randomly divided into three groups as follows: blank control group (BC; 10% ethanol as a carrier), positive control group (PC; 3% minosidic) and osmanthus extract (CE; 1% with 10% ethanol) in). Five mice in each group were tested. The hair growth-promoting activity of Osmanthus fragrans extract was partially modified by Choi et al. (Choi et al., Biol Pharm Bull 37:44-53, 2014). In short, the mouse carefully shaved a 2 cm×3 cm area of hair on the dorsal side with electric scissors. The test substance (100 μL) was locally applied to the skin of the back of the mouse once a day for 4 weeks. The hair growth enhancing activity of the test substance is based on the darkening of the dorsal skin as an evaluation standard, which means that the hair follicle of the hair is in the growth phase.

The hair growth scoring system was performed by two independent laboratory members who were not aware of the implementation plan. The hair growth line is measured once a week for 4 consecutive weeks. The hair growth score is defined as follows: 0 points = no growth is observed; 1 point = 20% growth; 2 points = 20-40% growth; 3 points = 40- 60% growth; 4 points = achieve 60-80% growth; and 5 points = achieve 80-100% growth. The digital image of total hair growth for 4 weeks was taken with Nikon Cool Pix P100 (Tokyo, Japan). The average of the two individual scores is used as the hair growth index for evaluating hair growth.

Observing the results after treatment of the mice, on the 16th day, it was found that the Osmanthus fragrans extract group had a more complete wound appearance, the epidermal layer of the wound tissue was completely restored, and the dermal layer structure was more similar to that of normal mice. The wound photos of mice in each group from 0-28 days after treatment are shown in Figure 1A. Compared with the blank control group and the positive control group, the osmanthus extract group has grown hair on the back skin of the mice on the 14th day , Its hair follicle development, hair growth area and hair volume were significantly higher than other groups from day 16 to day 28. The quantitative results are presented as the hair growth index. As shown in Figure 1B, the hair growth treated with Osmanthus fragrans extract is significantly different from other groups (see Figure 1B for details). The stained photos of the skin tissues of the mice in each group after wound treatment are shown in Figure 1C. Compared with the blank control group and the positive control group, the osmanthus extract group has obvious hair follicle development and hair growth from the skin tissue staining. The above results show that Osmanthus fragrans extract has the effect of promoting hair growth.

It can be seen from the results of the examples that the Osmanthus fragrans extract provided by the present invention can significantly promote hair growth.

Example 3: Evaluation of experimental animal model for wound healing

1. Experimental animals and models

The experimental animals used in this experiment were purchased from the C57BL/6J Narl strain mice of the National Laboratory Animal Center, weighing 40-45 g, and aged 8 weeks. The experimental animal was raised in the Animal Center of the National Defense Medical College. The temperature of the animal room was maintained at 18~26°C, the relative humidity was 30%~70%, the photoperiod was 12 hours of light and 12 hours of night, and sufficient feed and drinking water were provided.

其中，正對照組為敏諾西迪（Minoxidil）。The groups of each experimental mode are shown in the table below:

Among them, the positive control group is Minoxidil.

2. Wound treatment

Before the operation, mice were given 0.6 g/kg Avertin by intraperitoneal injection, where the Avertin was 0.6 g 2,2,2-Tribromoethanol (2,2,2-Tribromoethanol) and 1.5 ml 2-methyl-2-butanol. After mixing the alcohol (2-Methyl-2-butanol), add physiological saline to 50 ml, filter with 0.2μm filter paper, and store at 4°C. After the mice were anesthetized, their back hair was shaved. Fix the paper card on the back of the mouse to determine the location of the wound, and draw an area of 1.2 x 1.2 cm2 with a marker. Use surgical scissors to cut off the skin in this area to form a wound. In the infectious wound mode, dilute Staphylococcus aureus to 10 ⁵ CFU/150 μl, and add 150 μl bacterial solution to each mouse to make the bacterial solution completely penetrate into the skin for about 5 minutes (non-infectious wound mode does not This step is required). Use a cotton swab to dip an appropriate amount of the drug to be tested and apply it evenly on the wound. Use waterproof and breathable dressing (Tegaderm, 3M) to cover the wound, and fix the waterproof and breathable dressing with breathable tape to prevent the waterproof and breathable dressing from falling off and other wound infections.

3. Cultivation and calculation of Staphylococcus aureus

Pick a single colony of Staphylococcus aureus with an inoculating loop, and inoculate it on an LB agar plate by dividing and drawing lines, and incubate it in an incubator at 37°C for about 16 hours. Pick a single colony of Staphylococcus aureus with an inoculating loop, and place it in a bacterial culture test tube containing LB medium for about 16 hours. Take out the bacterial solution from the test tube and centrifuge at 4°C and 7,000 rpm for 20 minutes. Remove the supernatant, and dilute the Staphylococcus aureus cells obtained by centrifugation with LB medium to 1 x 107 CFU/ml for later use.

Dilute the bacterial solution to 10-9 in sequence. Take out 100 μl of bacterial solution from the test tube of different dilution multiples and add it to the LB agar dish and spread evenly. Incubate in a 37°C incubator for about 16 hours and record the number of colonies. The number of bacteria per milliliter in the original bacterial solution (CFU/ml) = the number of colonies * the dilution factor * 10.

4. Antibacterial ring test

Take out 100 μl of Staphylococcus aureus liquid from the test tubes of different dilution multiples, drop them on the agar plate and apply evenly. After the bacterial liquid is sucked into the agar, use tweezers to flatten the filter paper on the agar, and drop each filter paper into 50 μl Osmanthus extract. After culturing in a 37°C incubator for about 16 hours, measure the diameter of the liquid bacteria and the range of inhibition ring = diameter of inhibition ring (mm)-diameter of filter paper (mm).

5. Bacteria content test in wound tissue

After the seventh day of the infectious wound model mouse experiment, the purulent tissue from the back wound of the wounded mouse was collected. Cut the tissue into pieces and break the tissue with an ultrasonic cell crusher. After centrifugation at 1,500 rpm for 8 minutes at 4°C, the supernatant was collected. The supernatant was serially diluted, the bacterial solution was evenly smeared on the agar, and then incubated in a 37°C incubator for about 16 hours, and the number of colonies was counted. The number of bacteria per gram of tissue (CFU/g) = (number of colonies * dilution factor * 10) / grams of tissue.

6. Skin tissue section and Hematoxylin & Eosin (H&E) staining

Add an appropriate amount of OCT (optimum cutting temperature) embedding agent into the aluminum foil tissue embedding box, put the freshly removed skin tissue into the embedding box, and then add OCT embedding agent to cover the entire tissue and place it in liquid nitrogen After being quickly frozen, store at -70°C. Add an appropriate amount of OCT embedding agent to the aluminum alloy tissue specimen table, put the pretreated tissue on the specimen table, then place the specimen table on the microtome freezing table for freezing, and place the specimen table on the microtome and lock it tightly . Next, use the rough cut mode (20 μm) to flatten the tissue block, and then use the fine cut mode (5-7 μm) to start sectioning. Attach the glass slide to the tissue slice, and soak the glass slide in acetone at -20°C. After 30 minutes, store the glass slide at -70°C.

After immersing the tissue slides in PBS buffer for about 3 to 4 minutes, stain the slides with hematoxylin for 10 minutes. After washing the slides with pure water three times, soak them in pure water for 5, 5, 5, and 10 minutes respectively. Stain the slide with eosin for 15 seconds. Soak the slides in 95%, 95%, 100%, and 100% alcohol, respectively, for 1 minute. Soak in xylene for 1 minute. Wipe the back of the slide with 70% alcohol and wait for the sample to dry. Finally, mount the slide with a cover glass (mounting tablet: xylene=2:1).

7. Results

In the non-infectious wound model, changes in the appearance of wounds in mice were evaluated. First, after creating a wound on the back skin of C57BL/6 mice, the wound was cleaned and the medicine was changed every two days. As shown in Figure 2A, compared with other groups, non-infectious wounds treated with Osmanthus fragrans extract have better wound healing efficiency. As shown in Fig. 2B, on the 4th to 8th day, the wound healing of the blank control group and the petrolatum group was not obvious. In contrast, the appearance of the wound in the Osmanthus fragrans extract group has obviously begun to heal. The results show that after treatment with Osmanthus fragrans extract, non-infectious wounds can heal about 4 to 8 days earlier.

In the infectious wound mode, the wound is infected with Staphylococcus aureus. After the seventh day of infection, the wound is cleaned and the medicine is changed every two days. As shown in Figure 3A, compared with other groups, the infected wounds have a significant shrinking trend after being treated with Osmanthus fragrans extract. On the 6th day, the wounds treated with Osmanthus fragrans extract had reached 60% wound healing. As shown in Figure 3B, on the 6th day, the wounds of the blank control group and the petrolatum group had not yet begun to heal. In contrast, the osmanthus extract group had a significant appearance of wounds and began to heal. The results show that after treatment with Osmanthus fragrans extract in the infectious wound mode, the wound can heal about 8 to 12 days earlier.

No matter in the non-infectious trauma model (Figure 4A) or infectious trauma model (Figure 4B), the appearance of the wound skin can be found. Compared with the blank control group and the petroleum jelly group, the Osmanthus fragrans extract group has a significant promotion of blood vessel area, The length and branch development show that the extract of Osmanthus fragrans has a significant effect of promoting angiogenesis.

As the synthesis of collagen helps wound healing, further examine the effect of osmanthus extract vinegar in promoting collagen synthesis during wound healing. For the non-infectious trauma model, on the 21st day of the mouse trauma, and for the infectious trauma model, on the 27th day, take the skin tissue around the wound and observe the synthesis of collagen in the skin tissue by Masson’s trichrom staining method. The protein is dyed blue to appear. Observing the experimental results, it was found that compared with normal mice in non-infectious or infected trauma mode, in the blank control group and petrolatum group, the amount of collagen synthesis in the tissue was less and the structure was loose; while in the osmanthus extract treated In the group, the amount of collagen synthesis and the tightness of the structure within the tissue are similar to those of normal mice. The above results show that Osmanthus fragrans extract has the effect of promoting collagen synthesis.

Wound infection is one of the reasons why the wound cannot heal effectively. Therefore, the antibacterial circle test is used to test whether the Osmanthus fragrans extract has antibacterial activity. The test results showed that after Staphylococcus aureus was treated with drugs, there was no antibacterial phenomenon after comparison with the control group, indicating that the Osmanthus fragrans extract had no direct antibacterial effect.

After the wound was created on the back skin of the mouse, the wound was infected with Staphylococcus aureus and treated with drugs. Seven days after the infection, the purulent tissue around the wound was collected and the bacterial content was analyzed. As shown in Figure 5, compared with the blank control group and the petrolatum group, the number of pus on the wound surface was significantly reduced after treatment with Osmanthus fragrans extract. As a result, it was found that Osmanthus fragrans extract has indirect antibacterial activity.

From the above results, it can be seen that the Osmanthus fragrans extract of the present invention can promote wound healing, has the effects of promoting wound angiogenesis and promoting collagen production.

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The illustrations of preferred specific embodiments presented in the present invention are for the purpose of illustrating the present invention. It should be understood that the present invention is not limited to the preferred embodiments shown.

Figure 1 shows the results of the Osmanthus fragrans extract of the present invention in promoting hair growth. Figure 1A shows the hair growth of each group from day 0 to day 28. Figure 1B shows the hair growth scores from day 0 to day 28 (error bars, SE; *, P <0.05). Figure 1C shows the results of skin tissue staining in each group.

Figure 2 shows the results of wound healing in the non-infectious wound mode of the Osmanthus fragrans extract of the present invention. Figure 2A shows the wound healing efficiency from day 0 to day 18 (error bars, SE; *, P <0.05). Figure 2B shows the wound healing from day 0 to day 18.

Figure 3 shows the healing results of the Osmanthus fragrans extract of the present invention in the infectious wound mode. Figure 3A shows the wound healing efficiency from day 0 to day 18 (error bars, SE; *, P <0.05). Figure 3B shows the wound healing from day 0 to day 18.

Figure 4 shows the effect of the Osmanthus fragrans extract of the present invention in promoting angiogenesis in a non-infectious wound mode or an infectious wound mode. Figure 4A shows the effect of each group on angiogenesis in the non-infectious trauma model. Figure 4B shows the influence of each group on angiogenesis in the infectious trauma model.

Figure 5 shows the bacteriostatic effect of the Osmanthus fragrans extract of the present invention; in the non-infectious wound mode or the infectious wound mode, the wound is infected with Staphylococcus aureus, and the purulent tissue around the wound after treatment of each group contains bacteria number.

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Claims (4)

An osmanthus extract is used for preparing a composition for promoting wound healing, wherein the osmanthus extract is first extracted with pure water and then extracted with an ethanol solution. The use according to claim 1, wherein the composition is prepared in the form of medicine, skin care agent, insect repellent or spray. The use according to claim 1, wherein the composition is in the form of a solution, hydrogel, spray, drops or ointment. The use according to claim 1, wherein the composition may further comprise other antibacterial agents, anti-inflammatory drugs, cleansing or skin care components.

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