A method for preparing and applying an agarwood extract
By preparing extracts from agarwood leaves and rhizomes, the problems of promoting fibroblast growth and resisting oxidative damage in agarwood extracts were solved, achieving wound healing effects, promoting wound healing and reducing scar formation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- 科奕美(广州)健康产业有限公司
- Filing Date
- 2025-04-09
- Publication Date
- 2026-06-30
Smart Images
Figure BDA0005350638180000041 
Figure BDA0005350638180000051 
Figure BDA0005350638180000061
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant extract technology, and in particular to a method for preparing and applying agarwood extract. Background Technology
[0002] Agarwood is a commonly used medicine in Traditional Chinese Medicine. It comes from the resinous wood of Aquilaria agallocha Roxb. and A. sinensis (Lour.) Gilg, both belonging to the Thymelaeaceae family. Agarwood has a pungent and bitter taste, and is slightly warm in nature. It has the effects of promoting qi circulation and relieving pain, warming the stomach and stopping vomiting, and calming asthma.
[0003] Various traumas cause varying degrees of cell degeneration, necrosis, and tissue loss, necessitating tissue repair through cell proliferation and the formation of the intercellular matrix. Fibroblasts, the main cellular component of loose connective tissue, are the most important functional cells in the wound repair process. Under the regulation of factors such as cytokines, fibroblasts proliferate, migrate, and synthesize and secrete collagen, extracellular matrix components, and collagenases, participating in granulation tissue formation, wound contraction, scar formation, and tissue reconstruction. Protecting and promoting fibroblast growth is fundamental and crucial for accelerating wound healing. Simultaneously, an increased number of fibroblasts contributes to hair follicle formation during wound healing, thereby reducing the likelihood of scarring after skin healing.
[0004] There are currently no studies on the application of agarwood extract in promoting fibroblast growth. Summary of the Invention
[0005] Therefore, this invention proposes a method for preparing and applying agarwood extract.
[0006] The technical solution of this invention is implemented as follows:
[0007] Application of an agarwood extract in the preparation of products that promote fibroblast growth.
[0008] Furthermore, the product is a wound healing medication or skincare product.
[0009] Furthermore, the concentration of agarwood extract in the product is 1-10 wt%.
[0010] Furthermore, the agarwood extract is an alcoholic extract of agarwood leaves and / or an ethyl acetate extract of agarwood rhizomes.
[0011] Furthermore, the preparation method of the alcohol extract of agarwood leaves includes: ultra-finely pulverizing fresh agarwood leaves to form agarwood leaf powder, adding it to a 75%-85% v / v ethanol solution, ultrasonically treating it, and concentrating the extract under reduced pressure to obtain the alcohol extract of agarwood leaves.
[0012] Furthermore, the particle size of the ultrafine pulverizer is 5-10 μm; the ratio of agarwood leaf powder to ethanol is 1:10-15 g / mL; and the ultrasonic treatment is performed at 20-30 kHz and 50-70 °C for 2-4 hours.
[0013] Furthermore, the preparation method of the ethyl acetate extract of agarwood rhizome includes: crushing fresh agarwood rhizomes, soaking them in petroleum ether, percolating, recovering the residue, evaporating the petroleum ether from the residue, then adding 60%-70% v / v ethanol for ultrasonic extraction 2-3 times, combining the extracts, concentrating to form an ethanol extract, dissolving in water, extracting with an equal volume of ethyl acetate, collecting the supernatant, concentrating, and vacuum drying to obtain the ethyl acetate extract of agarwood rhizome.
[0014] Furthermore, the ratio of agarwood rhizome to petroleum ether is 1:3-5 g / mL; the soaking time is 10-12 h at a temperature of 25-35℃; the ratio of residue to ethanol is 1:5-7 g / mL; the ultrasonic extraction is performed at 20-30 kHz and 60-75℃ for 1-3 h; and the volume of water is 5-10 times the volume of the ethanol extract.
[0015] Furthermore, when the product is a wound healing medicine, it is a topical preparation made from agarwood extract and pharmaceutically acceptable excipients and carriers.
[0016] Furthermore, when the product is a skincare product, it is a water, lotion, cream, or ointment made from agarwood extract and skincare additives or raw materials.
[0017] Compared with the prior art, the beneficial effects of the present invention are:
[0018] The agarwood extract obtained by the extraction method of this invention exhibits significant biological activity and is virtually non-toxic to cells, promoting fibroblast proliferation and growth, and enhancing cell viability, thereby accelerating wound healing. Examples of this invention demonstrate that as the concentration of the agarwood extract increases, fibroblast cell viability first increases and then slightly decreases, reaching its maximum at a 5 wt% concentration, which is 41.1%-49.4% higher than the control group. Therefore, the agarwood extract of this invention can be subsequently used as an active ingredient in pharmaceuticals or skincare products to promote wound healing, showing promising application prospects. Detailed Implementation
[0019] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.
[0020] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.
[0021] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.
[0022] Example 1
[0023] The agarwood extract in this embodiment is an alcoholic extract of agarwood leaves. The preparation method is as follows: fresh agarwood leaves are ultra-finely pulverized into a particle size of 5-10 μm to form agarwood leaf powder. The agarwood leaf powder is added to a 75%-85% v / v ethanol solution at a material-to-liquid ratio of 1:10-15 g / mL. The solution is ultrasonically treated at 20-30 kHz and 50-70℃ for 2-4 hours to obtain an extract. The extract is then concentrated under reduced pressure to obtain the alcoholic extract of agarwood leaves.
[0024] Example 2
[0025] The agarwood extract in this embodiment is an ethyl acetate extract of agarwood rhizomes. The preparation method is as follows: Fresh agarwood rhizomes are crushed and soaked in petroleum ether at 25-35℃ for 10-12 hours at a material-to-liquid ratio of 1:3-5 g / mL. After percolation, the residue is collected and the petroleum ether in the residue is evaporated. Then, the residue is added to 60%-70% v / v ethanol at a material-to-liquid ratio of 1:5-7 g / mL for ultrasonic extraction 2-3 times, each time at 20-30 kHz, and treated at 60-75℃ for 1-3 hours. The extracts are combined, concentrated, and an ethanol extract is formed. Water is added to dissolve the ethanol extract at 5-10 times its volume. An equal volume of ethyl acetate is used for extraction, the supernatant is collected, concentrated, and vacuum dried to obtain the ethyl acetate extract of agarwood rhizomes.
[0026] Example 3
[0027] The agarwood extract in this embodiment is an alcoholic extract of agarwood leaves and an ethyl acetate extract of agarwood rhizomes, with a mass ratio of 2:1. The preparation method is as follows:
[0028] (1) Fresh agarwood leaves are ultra-finely pulverized into agarwood leaf powder with a particle size of 5-10 μm. The powder is added to a 75%-85% v / v ethanol solution at a material-liquid ratio of 1:10-15 g / mL and ultrasonically treated at 20-30 KHz and 50-70℃ for 2-4 h to obtain an extract. The extract is concentrated under reduced pressure to obtain an alcohol extract of agarwood leaves.
[0029] (2) Crush fresh agarwood rhizomes and soak them in petroleum ether at 25-35℃ for 10-12 hours at a material-to-liquid ratio of 1:3-5 g / mL. After percolation, the residue is recovered and the petroleum ether in the residue is evaporated. Then, the residue is added to 60%-70% v / v ethanol at a material-to-liquid ratio of 1:5-7 g / mL and ultrasonically extracted 2-3 times at 20-30 kHz each time at 60-75℃ for 1-3 hours. The extracts are combined, concentrated, and ethanol extract is formed. 5-10 times the volume of water is added to dissolve the ethanol extract. Equal volume of ethyl acetate is used for extraction. The supernatant is collected, concentrated, and vacuum dried to obtain ethyl acetate extract of agarwood rhizomes.
[0030] (3) Mix the alcohol extract of agarwood leaves and the ethyl acetate extract of agarwood rhizome evenly to obtain agarwood extract.
[0031] Experiment 1 - Cell Proliferation Experiment
[0032] Experimental methods: NIH-3T3 cells in good growth condition and in the logarithmic growth phase were selected and subjected to 1.0 × 10⁻⁶ cells. 4 Cells were seeded per well in 96-well culture plates and incubated at 37°C with 5% CO2 for 24 h. They were then randomly divided into a control group and an experimental group (Examples 1-3), with 6 replicates per group. The supernatant was discarded. The control group was treated with DMEM complete culture medium (purchased from Shanghai Yiji Industrial Co., Ltd.). The experimental groups (Examples 1-3) were set up in triplicate, with 100 μL of agarwood extract at different concentrations (1 wt%, 5 wt%, and 10 wt%) corresponding to the examples added to each well. After culturing for 24 h, 20 μL of MTT (5 mg / ml) was added to each well, and the cells were incubated for 4 h. The supernatant was discarded, and 150 μL of DMSO complete culture medium was added to completely dissolve the extract. The cells were then placed on a microplate reader, and the absorbance was measured at 490 nm. Cell viability was determined by the MTT assay to compare the effect of agarwood extract on fibroblast proliferation. Each experiment was repeated 3 times. The experimental results are shown in Table 1.
[0033] Table 1
[0034]
[0035] Note: ※ p < 0.05 compared to the control group.
[0036] As can be seen from Table 1, the agarwood extracts of Examples 1-3 of the present invention can effectively promote fibroblast proliferation. As the concentration increases, cell viability first increases and then decreases slightly. The cell viability value is the highest at a concentration of 5 wt%, which is 41.1%-49.4% higher than that of the control group. Although the cell viability shows a decreasing trend at a concentration of 10 wt%, it is still higher than that of the control group.
[0037] Experiment Example 2 - Anti-NIH-3T3 Cell Injury Experiment
[0038] Experimental method: NIH-3T3 cells in the logarithmic growth phase were subjected to a reaction at a concentration of 1.0 × 10⁶ cells / year. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. Cells were then randomly divided into control, model, and experimental groups (Examples 1-3, with 1 wt%, 5 wt%, and 10 wt% cells per group, respectively), for a total of 11 groups, with 6 replicates per group. The control group received 100 μL of DMEM complete culture medium per well, while the model and experimental groups were induced with 100 μL of DMEM complete culture medium containing 0.8 mmol / L H2O2. Cells were then incubated in a CO2 incubator for another 24 h. The OD values of each group were measured at 490 nm using the MTT assay to calculate cell viability, thus evaluating the ability of agarwood extract to resist oxidative damage in NIH-3T3 cells. The experimental results are shown in Table 2.
[0039] Table 2
[0040]
[0041] Note: * Compared with the control group, p < 0.05; § Compared with the model group, p < 0.05
[0042] As can be seen from Table 2, the agarwood extracts prepared in Examples 1-3 have good anti-oxidative damage effects on NIH-3T3 cells. The anti-damage effect first increases and then decreases with increasing concentration. The anti-damage effect is most significant at a concentration of 5 wt%, which can significantly improve the survival rate of NIH-3T3 cells, increasing the cell survival rate by 24.4%-35.8% compared with the model group.
[0043] Experimental Example 3 - Wound Healing Experiment
[0044] Experimental methods: 110 mice were randomly divided into 11 groups: model group, positive control group and experimental group (Examples 1-3, each group was set at 1wt%, 5wt% and 10wt%, respectively), with 10 mice in each group, half male and half female;
[0045] Mice in each group were shaved. Twenty-four hours later, a deep second-degree burn model was created on the mouse back using a burn instrument. Drug administration began one hour after model creation. The model group received no treatment for the wounds, while the positive control group received topical burn ointment at a concentration of 100 mg / cm². 2 The experimental groups were given different concentrations of agarwood extract corresponding to the examples, administered continuously. In the first week, the dressing was changed every 4-6 hours; in the second and third weeks, every 6-8 hours; and in the fourth week, twice a day. The wound diameter of each mouse was measured 1 hour after modeling, at week 1, week 3, and week 4. The wound area was calculated, and the wound area 1 hour after the burn was used as the burn area to calculate the wound healing rate.
[0046] Wound healing rate (%) = (Scalded area - Wound area) / Scalded area × 100%
[0047] The experimental results are shown in Table 3.
[0048] Table 3
[0049]
[0050]
[0051] Note: a) Compared with the model group, p<0.05; b) Compared with the positive control group, p<0.05
[0052] As shown in Table 3, the agarwood extracts of Examples 1-3 of the present invention can promote wound healing to varying degrees. Among them, the medium-dose group of Example 3 showed the most significant effect, with a wound healing rate of over 96% at week 4, which was not significantly different from the positive control group. Therefore, the agarwood extract of the present invention can significantly promote wound healing.
[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. The application of an agarwood extract in the preparation of wound healing drugs, characterized in that, The concentration of agarwood extract in the wound healing medicine is 1-10 wt%; the agarwood extract is an alcoholic extract of agarwood leaves and an ethyl acetate extract of agarwood rhizomes in a mass ratio of 2:
1. The preparation method of the ethyl acetate extract of agarwood rhizome includes: crushing fresh agarwood rhizome, soaking it in petroleum ether, percolating, recovering the residue, evaporating the petroleum ether in the residue, then adding it to 60%-70% v / v ethanol for ultrasonic extraction 2-3 times, combining the extracts, concentrating them to form an ethanol extract, dissolving it in water, extracting it with an equal volume of ethyl acetate, collecting the supernatant, concentrating it, and vacuum drying it to obtain the ethyl acetate extract of agarwood rhizome. The method for preparing the alcohol extract of agarwood leaves includes: ultra-finely pulverizing fresh agarwood leaves to form agarwood leaf powder, adding it to a 75%-85% v / v ethanol solution, ultrasonically treating it, and concentrating the extract under reduced pressure to obtain the alcohol extract of agarwood leaves.
2. The application as described in claim 1, characterized in that, In the preparation method of the alcohol extract of agarwood leaves, the particle size of the ultrafine pulverization is 5-10µm; the ratio of agarwood leaf powder to ethanol is 1:10-15g / mL; and the ultrasonic treatment is performed at 20-30KHz and 50-70℃ for 2-4h.
3. The application as described in claim 1, characterized in that, In the preparation method of ethyl acetate extract of agarwood rhizome, the ratio of agarwood rhizome to petroleum ether is 1:3-5 g / mL; the soaking time is 10-12 h at a temperature of 25-35℃; the ratio of residue to ethanol is 1:5-7 g / mL; the ultrasonic extraction is performed at 20-30 kHz and 60-75℃ for 1-3 h; and the volume of water is 5-10 times the volume of the ethanol extract.
4. The application as described in claim 1, characterized in that, The wound healing medicine is a topical preparation made from agarwood extract and pharmaceutically acceptable excipients and carriers.