TW202133871A - Application of Aquilaria malaccensis seeds extract to prepare skin anti-inflammatory composition capable of inhibiting inflammation factors in cells and releasing interleukin-1 to provide efficient anti-inflammatory effect - Google Patents

Abstract

The invention relates to an application of Aquilaria malaccensis seeds extract to prepare skin anti-inflammatory composition. A plurality of Aquilaria malaccensis seeds are crushed and then extracted by ultrasound with 90% ethanol. Each batch of crushed Aquilaria malaccensis seeds is extracted three times, and one extraction is performed per day. After the extractions are completed, all alcohol water dissolvent is removed by a vacuum concentration device and a vacuum pump so as to obtain an Aquilaria malaccensis seeds extract that can be used for skin anti-inflammatory purposes.

Description

Use of agarwood tree extract to prepare skin anti-inflammatory composition

The present invention is a use of Agarwood tree extract for preparing skin anti-inflammatory composition.

Since the skin is the human body directly exposed to the external environment, the skin not only serves as a protective layer to protect our vital organs, but also regulates the evaporation of water and protects the body from external infections. In addition, the epidermis of the skin can also prevent the evaporation of human water. From the outside, the epidermis is: stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. The stratum corneum cells of healthy people have high concentrations of natural moisturizing factors. (Natural moisturzing factor, NMF), so it effectively combines with moisture to prevent the skin's moisture from drying out.

However, excessive exposure of the skin to ultraviolet rays or pollutants can cause skin irritation and damage to skin cells. In particular, the stratum corneum is prone to skin inflammation, atopic dermatitis, psoriasis, and even skin damage due to cell damage. Cells produce lesions that lead to cancer or tumors.

The purpose of the present invention is to improve the above-mentioned deficiencies, and to provide an agarwood tree extract that can be used for skin anti-inflammatory purposes.

In order to achieve the above purpose, the Aquilaria tree extract which can be used to prepare the skin anti-inflammatory composition of the present invention is to wash a plurality of Aquilaria malaccensis sub-batch with reverse osmosis flowing water, each washing 10 minutes, each A batch of agarwood trees was washed three times repeatedly, then drained at room temperature, and spread flat on a water-absorbent material to dry in the shade for two days; then, use a grinder to crush the agarwood trees so that the particle size of the crushed agarwood trees is not larger than At the same time, control the crushing time of the crusher so that the continuous crushing time does not exceed 20 minutes. Then the crushed agarwood seeds are subjected to ultrasonic extraction in batches with 90% ethanol, and each batch of crushed agarwood seeds is extracted three times a day Extract once, after the extraction is completed, remove all the alcohol and water solvents with a vacuum concentrator and a vacuum pump to obtain agarwood tree extract;

In this way, the extract of Agarwood tree seed can be used to prepare skin anti-inflammatory composition.

Figure 1 is a flow chart of the steps of the present invention for extracting agarwood seed extract.

Figure 2 is a test diagram of the Agarwood tree extract of the present invention after one month of cold storage and dark storage.

Fig. 3 is the test diagram of the cell viability of the Agarwood tree extract of the present invention.

Fig. 4 is a microscope observation diagram of the cell type of the extract of Aquilaria sinensis according to the present invention.

Fig. 5 is an experimental diagram of the determination of the content of tumor necrosis factor alpha (TNF-alpha) in cells by the extract of Agarwood tree seeds of the present invention.

Fig. 6 is a test chart of the determination of the inhibition of intracellular leukocyte interleukin-1 (IL-1) content of the agarwood seed oil of the present invention.

Regarding the technical means and structure used by the present invention to achieve the purpose, I would like to re In conjunction with the embodiments shown in Figures 1 to 6, the detailed description is as follows:

As shown in Figure 1, the multiple Aquilaria malaccensis sub-groups are washed with reverse osmosis running water for 10 minutes each time. Each batch of Aquilaria malaccensis is washed three times and then drained at room temperature and laid flat. Dry in the shade on absorbent material for two days; then, use a grinder to crush the agarwood trees so that the particle size of the crushed agarwood trees is not greater than 2mm. At the same time, control the crushing time of the crusher so that the continuous crushing time does not exceed 20 minutes , In order to avoid changing the chemical composition of the agarwood tree due to overheating of the machine. Then, the crushed agarwood seeds were subjected to ultrasonic extraction in batches with 90% ethanol. Each batch of crushed agarwood seeds was extracted three times, only once a day. After the extraction, the vacuum concentrator and vacuum help Pu removes all the alcohol and water solvents, so that the agarwood tree extract (Crude-EtOH) is obtained, and the form of the agarwood tree extract (Crude-EtOH) is oily, so that agarwood seed oil is formed.

In accordance with the above, a preferred embodiment is to take 2.9 kg of Aquilaria malaccensis seeds, crush, drain and dry in the shade, and extract with 15 liters of 90% ethanol to obtain 102 grams of Aquilaria malaccensis seeds. Extract (Crude-EtOH), that is, 102 grams of agarwood oil.

Then, the above-mentioned agarwood oil was stored at 4°C and protected from light for one month. The physical properties of the agarwood oil, such as the smell, color, and pH value, did not change. At the same time, it was evaluated by the nuclear magnetic resonance instrument, such as As shown in Figure 2, the 1H NMR (CDCl3, 400MHz) on the first day and the same test sample after one month have the characteristic signals of phorbol esters and fatty acids, and there is no obvious change in the ratio of ingredients. The 1H NMR shows a stable agreement result. It can be seen that a 4°C refrigeration and light-proof environment is obviously a suitable storage method for the agarwood oil.

In addition, the specific examples of the following experiments can further prove that the present invention is used The scope of application of this route, but does not limit the scope of the present invention.

experiment one:

In this experiment, the stratum corneum of the skin is the most exposed to these products no matter what kind of primordial nourishment product is applied to the skin. Therefore, this experiment uses human skin keratinocytes for safety testing to evaluate the effects of agarwood oil on skin cells. Whether it is cytotoxic.

(1) This experiment uses MTT assay to detect. Culture skin keratinocytes (1×10 ⁴ /well) in a 96-well dish and incubate at 37°C and 5% CO _{2 in an} incubator for at least 24 hours. Add 10 μL of MTT (3-(4,5-cimethylthiazol- Reaction with 2-yl)-2,5-diphenyl tetrazolium bromide) solution. After reacting at 37℃ and 5% CO ₂ for 4 hours, remove the culture solution, add 100μL of DMSO to dissolve the formazan precipitate, and measure the absorbance at 570nm. (BioTek, SynergyTM2, USA).

(2) The test results are shown in Figure 3. The freeze-dried agarwood oil was dissolved in DMSO and prepared to 10 mg/mL for later use. After HaCaT cells were treated with agarwood oil for 24 hours, the results showed that 1-100μg/mL agarwood oil had no obvious toxicity to cells and showed obvious proliferation of cells at 10-100μg/mL.

Experiment 2:

This experiment uses a microscope to observe whether agarwood oil will change the shape of skin cells.

(1) Culture the human skin keratinized cells (1×104/well) in a 96-well dish and incubate at 37°C and 5% CO2 in an incubator for at least 24 hours. Add 1μL of agarwood oil and apply it for the specified time. After the reaction time is reached, observe the cell morphology under a microscope (Nikon, TE2000-U, Japan), and take pictures for recording.

(2) The test results are shown in Figure 4. The cell morphology was observed with a microscope, and there was no significant change in the cell morphology after the action of 1-100 μg/mL agarwood oil.

Experiment 3:

This experiment is because Proinflammatory cytokine is a general term for a series of cytokines that can promote inflammation. The more common proinflammatory cytokines include TNF-α and IL-1; tumor necrosis factor TNF-α (tumor necrosis) factor-α) is an inflammatory factor. When an inflammatory reaction occurs, its content in the serum will greatly increase. This is an indicator of an inflammatory reaction. The change in the content of TNF-α is used to evaluate whether agarwood oil has the ability to inhibit the inflammatory factor. freed.

(1) Coating buffer (0.2M sodium phosphate buffer): Weigh _{11.8g of Na 2} HPO ₄ and 16.1g of NaH ₂ PO ₄ , add an appropriate amount of ddH ₂ O, adjust the pH to 6.5 and then quantify to 1L.

Assay diluent (PBS+10% FBS): Add 100mL FBS to 900mL PBS and store at 2~8℃. It must be used up within three days of preparation.

Wash buffer (0.05% PBST): 999.5Ml of PBS added 0.5mL of tween-20, stored at 2 ~ 8 ℃, it must be used within three days of preparation.

Stop solution: 1M H ₃ PO ₄ or 2N H ₂ SO ₄ . The cell number of 3×10 ⁵ /mL was cultured in 24well, and placed in an _{incubator at 37°C and 5% CO 2} for 24 hours. After adding agarwood oil to react for 30 minutes, add 1μg/mL LPS to react. After the reaction time, the supernatant was collected, centrifuged at 1200 rpm for 5 minutes, and then added to a new 1.5 ml centrifuge tube and stored at -20°C.

(2) In this experiment, BD OptEIATMSet was used to determine the intracellular TNF-α content. First, add 100μL capture Ab (1:250) to 96-well, seal with plastic wrap After the plate was stored at 4°C until overnight. Then wash the plate three times with wash buffer, and place the plate upside down on the filter paper for the last time to ensure that the remaining liquid is completely removed. Then add 200μL of Assay diluent and react for 1 hour at room temperature, and then repeat the washing step. Then add 100μL of supernatant, seal the plate and react at room temperature for 2 hours, then wash five times, then add 100μL of working detector (1:500 detection antibody and 1:250 enzyme reagent), seal the plate and leave at room temperature React for 1 hour, then wash 7 times, then add 50μL of substrate solution, and react at room temperature for 30 minutes, and finally add 50μL of Stop solution to measure the absorbance at 450nm (BioTek, SynergyTM2, USA).

(3) Test results: After the human skin keratinocytes are treated with 1μg/mL LPS, the content of TNF-α in the supernatant is 20.2pg/mL, and the agarwood oil (0.1-100μg/mL) After the action, as shown in Figure 5, the contents of TNF-α in the supernatant were 19.3, 18.3, 16.2, 17.2, and 14.5 pg/mL, respectively, which showed that agarwood oil has the ability to inhibit the release of the intracellular inflammatory factor TNF-α.

Experiment 4:

This experiment is to determine the inhibitory cell interleukin-1 (IL-1) content of agarwood oil.

(1) Coating buffer (0.2M sodium phosphate buffer): Weigh _{11.8g of Na 2} HPO ₄ and 16.1g of NaH ₂ PO _4, add an appropriate amount of ddH ₂ O, adjust the pH to 6.5, and then quantify to 1L.

Assay diluent (PBS+10% FBS): 900mL of PBS is added to 100mL of FBS, stored at 2~8℃, and it must be used up within three days of preparation.

Wash buffer (0.05% PBST): 999.5mL of PBS is added to 0.5mL tween-20, stored at 2~8℃, it must be used up within three days of preparation.

Stop solution: 1M H ₃ PO ₄ or 2N H ₂ SO ₄ . The cell number of 3×10 ⁵ /mL was cultured in 24well, and placed in an _{incubator at 37°C and 5% CO 2} for 24 hours. After adding agarwood oil to react for 30 minutes, add 1μg/mL LPS to react. After the reaction time, the supernatant was collected, centrifuged at 1200 rpm for 5 minutes, and then added to a new 1.5 ml centrifuge tube and stored at -20°C.

(2) In this experiment, BD OptEIATMSet was used to determine the intracellular IL-1 content. First, add 100 μL capture Ab (1:250) to 96-well, seal the plate with plastic wrap and store at 4°C until overnight. Then wash the plate three times with wash buffer, and place the plate upside down on the filter paper for the last time to ensure that the remaining liquid is completely removed. Then add 200μL of 5 Assay diluent and react for 1 hour at room temperature, and then repeat the washing step. Add 100μL of supernatant, seal the plate and react at room temperature for 2 hours, then wash five times, then add 100μL of working detector (1:500 detection antibody and 1:250 enzyme reagent), seal the plate and leave at room temperature React for 1 hour, then wash 7 times, then add 50μL of substrate solution and react for 30 minutes at room temperature, and finally add 50μL of Stop solution to measure the absorbance at 450nm (BioTek, SynergyTM2, USA).

(3) The results of the test are shown in Figure 6. After the human skin keratinized cells were treated with 1μg/mL LPS, the IL-1 content in the supernatant was 14.0pg/mL, and the content of IL-1 in the supernatant was 14.0pg/mL. -100μg/mL), the content of IL-1 in the supernatant was 9.4, 9.6, 10.5, 12.1, 13.8 pg/mL, indicating that agarwood oil has a slight ability to inhibit the release of the intracellular inflammatory factor IL-1.

From the above experiments, it can be seen that the agarwood seed extract of the present invention (that is, the above-mentioned agarwood oil) does have the ability to inhibit the release of intracellular inflammatory factors TNF-α and interleukin-1, and the cell type after the action has proliferation The phenomenon means that the Agarwood tree extract of the present invention (ie The above-mentioned agarwood oil) is applied to the skin for anti-inflammatory purposes, and it does have obvious effects.

In addition, in order to enhance the application range of the agarwood tree extract (i.e., the above-mentioned agarwood oil) of the present invention, the highest concentration of the agarwood tree extract (i.e., the above-mentioned agarwood oil) in the above experiment of the present invention can be 50-100 μg/mL ( 0.005~0.01%) magnify 100 times, add the effective concentration (0.5~1%) of agarwood tree extract (i.e. the above-mentioned agarwood oil) in the oil phase to antioxidant vitamin E and moisturizing olive oil , Rosehip oil, can also be added to the water phase to add hyaluronic acid for moisturizing and rejuvenating skin, Sanxian gum for moisturizing and thickening, seaweed gum for smoothing and thickening, and chlorphenesin for improving preservation ( Chlorphenesin), ... and other ingredients, and then obtain products such as agarwood compound oil, agarwood oil essence cream, ...; therefore, the agarwood tree extract of the present invention can effectively anti-inflammatory the skin, and the present invention can be used to prepare skin anti-inflammatory purposes. The composition.

Therefore, it can be seen from the above that the agarwood tree extract of the present invention is indeed novel and progressive, and sincerely it has met the requirements of a patent for invention. I filed a patent application in accordance with the law and prayed for the patent. Poop.

However, the foregoing is only a feasible embodiment of the present invention. This embodiment is mainly used to illustrate the technical means and structure used by the present invention to achieve the purpose, and therefore cannot be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the specification of the present invention and the scope of the patent application shall still fall within the scope of protection covered by the present invention.

Claims (2)

A use of agarwood tree extract to prepare skin anti-inflammatory composition, which is washed with reverse osmosis flowing water in batches from multiple Aquilaria malaccensis sub-groups, each wash is 10 minutes, and each batch of agarwood tree is washed three times after repeated washing. Drain at room temperature and spread it flat on the absorbent material to dry in the shade for two days; then, use a grinder to crush the agarwood trees so that the particle size of the crushed agarwood trees is not greater than 2mm, and at the same time control the crushing of the crusher Time, so that the continuous crushing time does not exceed 20 minutes, and then the crushed agarwood seeds are subjected to ultrasonic extraction in batches with 90% ethanol. Each batch of crushed agarwood seeds is extracted three times, once a day, after the extraction is completed, A decompression concentrator and a vacuum pump are used to remove all alcohol and water solvents to obtain agarwood tree extract, and the form of the agarwood tree extract is oily to form agarwood oil. The use of agarwood tree extract for preparing an anti-inflammatory composition according to claim 1, wherein the effective concentration of the agarwood tree extract is 50-100 μg/mL.

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