Pterocarpus santalinus heartwood extract, preparation method thereof and application of pterocarpus santalinus heartwood extract in preparation of products with mitochondrial function activation effect
By optimizing the preparation method of sandalwood heartwood extract, the problem of mitochondrial activator instability was solved, and significant activation of mitochondrial function was achieved. This can be applied in the fields of skin care products and pharmaceuticals to delay skin aging.
Patent Information
- Application Number
- CN202511088402.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-05
- Publication Date
- 2025-10-31
AI Technical Summary
There is a lack of stable and biosafe mitochondrial activators in the current technology, making it difficult to effectively activate mitochondrial function to delay cell aging.
The method for preparing extracts from the heartwood of sandalwood includes pulverization, solvent extraction, organic solvent extraction, and silica gel column chromatography purification. The solvent ratio and silica gel column elution conditions were optimized to obtain an extract that has a significant activating effect on mitochondrial function.
The prepared sandalwood heartwood extract can significantly increase the ATP release level of mitochondria and delay cell aging, making it suitable for use in skincare products, pharmaceuticals, and food.
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a sandalwood heartwood extract, its preparation method, and its application in the preparation of products that activate mitochondrial function. Background Technology
[0002] Skin aging can be divided into endogenous aging (natural aging) caused by internal factors and extrinsic aging secondary to external factors. Ultraviolet (UV) radiation is the main cause of extrinsic skin aging, leading to increased aging phenotypes and a higher incidence of skin cancer; this phenomenon is called photoaging. UV radiation is the most harmful external factor causing photoaging. UV radiation is classified into three types based on wavelength: long-wave UVA (λ = 320-400nm), medium-wave UVB (λ = 280-320nm), and short-wave UVC (λ = 100-280nm). Unlike UVA and UVB, UVC is almost completely absorbed by the ozone layer. UVA and UVB rays reaching Earth can damage skin structure, with UVB being the main cause of skin damage and closely related to both acute photodamage and long-term photoaging. Studies have shown that long-term UVB exposure is more likely to lead to dry skin, sensitivity, and increased wrinkles, among other signs of aging. It is well known that mitochondria are the site of ATP production within cells, providing energy for cellular metabolism. Mitochondrial dysfunction is a hallmark of aging. In photoaged cells, mitochondrial homeostasis is disrupted, mitochondrial permeability changes, leading to a decrease in membrane potential and even a drop in ATP levels. Therefore, reactivating mitochondrial function and restoring higher levels of ATP release and active substances can delay cellular aging, thus effectively reducing skin aging when used in skincare products.
[0003] When mitochondrial homeostasis is disrupted and function is impaired, certain cellular activators are expressed to reactivate mitochondrial function. These activators are also known as mitochondrial function activators, with ClpP being a common example of an activator that can alleviate mitochondrial damage. Human serine hydrolase ClpP is strictly localized to the mitochondrial matrix, promptly clearing erroneous or abnormal proteins generated by stress and other factors within the mitochondria, playing a crucial role in maintaining mitochondrial protein homeostasis. However, most of these activators are biological enzymes, which are unstable and easily inactivated, and are mostly found in theoretical research, making practical application in related technological fields difficult. Therefore, it is essential to find new, stable, and biosafe mitochondrial activators and apply them to technological products.
[0004] The heartwood of *Pterocarpus indicus* Willd., a plant in the legume family, is widely used in traditional medicine for its anti-inflammatory, antioxidant, and antibacterial properties. However, current technologies have not reported any activating effects of *Pterocarpus indicus* heartwood or its extracts on mitochondrial function. Therefore, developing a *Pterocarpus indicus* heartwood extract that can activate mitochondrial function has significant application value. Summary of the Invention
[0005] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides the following technical solution:
[0006] This invention first provides a method for preparing an extract from the heartwood of sandalwood, which includes the following steps:
[0007] (1) Take the heartwood of sandalwood, crush it to 80-120 mesh (preferably 100 mesh), extract it with solvent and separate the extract, concentrate and dry the extract to obtain sandalwood heartwood extract;
[0008] (2) Water is added to the sandalwood heartwood extract to suspend it, and sandalwood heartwood extract suspension is obtained; then an organic solvent is added to the sandalwood heartwood extract suspension for extraction, and the organic solvent extract is concentrated and dried to obtain organic solvent extract; the organic solvent extract is the sandalwood heartwood extract.
[0009] (3) The mixed extract was purified by silica gel column chromatography, and impurities were removed by chloroform-acetone at a volume ratio of 94:6, followed by elution of the target component with chloroform-acetone at a volume ratio of 82:18.
[0010] The inventors were surprised to discover during their research that the sandalwood heartwood extract prepared by the method described in this invention has an activating effect on mitochondrial function.
[0011] Preferably, in step (1), the ratio of the amount of sandalwood heartwood to the amount of solvent is 1 kg: 5 to 15 L.
[0012] Most preferably, in step (1), the ratio of the amount of sandalwood heartwood to the amount of solvent is 1 kg: 10 L.
[0013] Preferably, in step (1), the solvent is an aqueous ethanol solution with a volume fraction of 70-95%.
[0014] Preferably, the extraction in step (1) is a heating reflux extraction; the extraction time is 1 to 2 hours.
[0015] Preferably, the organic solvent in step (2) is petroleum ether and / or ethyl acetate.
[0016] Preferably, the ratio of sandalwood heartwood extract to water and organic solvent is 1 kg: 3-8 L: 3-8 L.
[0017] Most preferably, the ratio of sandalwood heartwood extract to water and organic solvent is 1 kg: 3 L: 6 L.
[0018] Preferably, the specific method in step (2) is as follows:
[0019] Water was added to the heartwood extract of sandalwood to suspend it, and a sandalwood heartwood extract suspension was obtained. Then, petroleum ether and ethyl acetate were added to the sandalwood heartwood extract suspension in sequence for extraction. The petroleum ether extract was concentrated and dried to obtain the petroleum ether extract, and the ethyl acetate extract was concentrated and dried to obtain the ethyl acetate extract.
[0020] The petroleum ether extract and the ethyl acetate extract are mixed evenly to obtain a mixed extract, which is then used to obtain the sandalwood heartwood extract.
[0021] Further research by the inventors revealed that the mixed extract obtained by uniformly mixing the petroleum ether extract and the ethyl acetate extract had a significantly higher mitochondrial function activation effect than either the petroleum ether extract alone or the ethyl acetate extract alone. Furthermore, the mixing of the petroleum ether extract and the ethyl acetate extract could produce a certain synergistic effect in enhancing mitochondrial function activation.
[0022] Preferably, the weight ratio of petroleum ether extract to ethyl acetate extract is 2 to 4:1.
[0023] Most preferably, the weight ratio of petroleum ether extract to ethyl acetate extract is 3:1.
[0024] Preferably, the method for preparing the sandalwood heartwood extract further includes the following steps:
[0025] The mixed extract was loaded onto a silica gel column and eluted with chloroform and acetone in a volume ratio of 93-95:7-5 (3-5 column volumes) to remove impurities. Then, it was eluted again with chloroform and acetone in a volume ratio of 81-84:19-16 (3-5 column volumes). The eluent obtained by elution with chloroform and acetone in a volume ratio of 81-84:19-16 was collected, concentrated, and dried to obtain the sandalwood heartwood extract.
[0026] Further research by the inventors revealed that the sandalwood heartwood extract obtained by eluting the mixed extract under the silica column elution conditions described in this invention exhibited a significantly higher mitochondrial function activation effect than the mixed extract. Furthermore, the inventors also found that the silica column elution conditions of this invention are crucial; only the sandalwood heartwood extract obtained by eluting the mixed extract under the silica column elution conditions described in this invention showed a significantly higher mitochondrial function activation effect than the mixed extract. However, sandalwood heartwood extracts obtained by eluting the mixed extract under other silica column elution conditions did not show a significantly higher mitochondrial function activation effect than the mixed extract.
[0027] The present invention also provides a sandalwood heartwood extract prepared by the above preparation method.
[0028] The present invention also provides the application of the above-mentioned sandalwood heartwood extract in the preparation of products that activate mitochondrial function.
[0029] The present invention also provides an application of the above-mentioned sandalwood heartwood extract in the preparation of mitochondrial function activators.
[0030] Preferably, the sandalwood heartwood extract is used as a mitochondrial function activator in the preparation of anti-skin aging products.
[0031] Preferably, the product is a skin care product or a medicine.
[0032] Beneficial Effects: This invention provides a novel method for preparing a sandalwood heartwood extract. Studies have shown that the sandalwood heartwood extract prepared by the method of this invention has an activating effect on mitochondrial function, restoring ATP release levels to a higher level of active substances, which can delay cell aging and thus effectively reduce skin aging when used in skincare products. Therefore, the sandalwood heartwood extract of this invention can be used as an active ingredient to prepare products that activate mitochondrial function; or further, the sandalwood heartwood extract of this invention has certain application value in the fields of cosmetics, food, and pharmaceuticals. Detailed Implementation
[0033] The present invention will be further explained below with reference to specific embodiments, but the embodiments do not limit the present invention in any way.
[0034] Example 1: Preparation of Sandalwood Heartwood Extract
[0035] (1) Take sandalwood heartwood, crush it and pass it through a 100-mesh sieve. Then, heat it with solvent to 70°C and reflux for 1.5 hours. After the extraction is completed, separate the extract and concentrate and dry the extract to obtain sandalwood heartwood extract. The ratio of sandalwood heartwood to solvent is 1 kg: 10 L. The solvent is an aqueous ethanol solution with a volume fraction of 95%.
[0036] (2) Add water to the sandalwood heartwood extract to suspend it, and obtain a sandalwood heartwood extract suspension; then add petroleum ether and ethyl acetate to the sandalwood heartwood extract suspension in sequence for extraction, take the petroleum ether extract, concentrate and dry it to obtain petroleum ether extract, take the ethyl acetate extract, concentrate and dry it to obtain ethyl acetate extract; wherein, the ratio of sandalwood heartwood extract to water and petroleum ether and ethyl acetate is 1kg:3L:3L:3L.
[0037] The extract of sandalwood heartwood is obtained by taking the petroleum ether extract.
[0038] Example 2: Preparation of Sandalwood Heartwood Extract
[0039] (1) Take sandalwood heartwood, crush it and pass it through a 100-mesh sieve. Then, heat it with solvent to 70°C and reflux for 1.5 hours. After the extraction is completed, separate the extract and concentrate and dry the extract to obtain sandalwood heartwood extract. The ratio of sandalwood heartwood to solvent is 1 kg: 10 L. The solvent is an aqueous ethanol solution with a volume fraction of 95%.
[0040] (2) Add water to the sandalwood heartwood extract to suspend it, and obtain a sandalwood heartwood extract suspension; then add petroleum ether and ethyl acetate to the sandalwood heartwood extract suspension in sequence for extraction, take the petroleum ether extract, concentrate and dry it to obtain petroleum ether extract, take the ethyl acetate extract, concentrate and dry it to obtain ethyl acetate extract; wherein, the ratio of sandalwood heartwood extract to water and petroleum ether and ethyl acetate is 1kg:3L:3L:3L.
[0041] The ethyl acetate extract yields the sandalwood heartwood extract.
[0042] Example 3: Preparation of Sandalwood Heartwood Extract
[0043] (1) Take sandalwood heartwood, crush it and pass it through a 100-mesh sieve. Then, heat it with solvent to 70°C and reflux for 1.5 hours. After the extraction is completed, separate the extract and concentrate and dry the extract to obtain sandalwood heartwood extract. The ratio of sandalwood heartwood to solvent is 1 kg: 10 L. The solvent is an aqueous ethanol solution with a volume fraction of 95%.
[0044] (2) Add water to the sandalwood heartwood extract to suspend it, and obtain a sandalwood heartwood extract suspension; then add petroleum ether and ethyl acetate to the sandalwood heartwood extract suspension in sequence for extraction, take the petroleum ether extract, concentrate and dry it to obtain petroleum ether extract, take the ethyl acetate extract, concentrate and dry it to obtain ethyl acetate extract; wherein, the ratio of sandalwood heartwood extract to water and petroleum ether and ethyl acetate is 1kg:3L:3L:3L.
[0045] The petroleum ether extract and ethyl acetate extract were mixed evenly at a weight ratio of 3:1 to obtain a mixed extract. The mixed extract was then used to obtain the sandalwood heartwood extract.
[0046] Example 4: Preparation of Sandalwood Heartwood Extract
[0047] (1) Take sandalwood heartwood, crush it and pass it through a 100-mesh sieve. Then, heat it with solvent to 70°C and reflux for 1.5 hours. After the extraction is completed, separate the extract and concentrate and dry the extract to obtain sandalwood heartwood extract. The ratio of sandalwood heartwood to solvent is 1 kg: 10 L. The solvent is an aqueous ethanol solution with a volume fraction of 95%.
[0048] (2) Water was added to the sandalwood heartwood extract to suspend it, and a sandalwood heartwood extract suspension was obtained. Then, petroleum ether and ethyl acetate were added to the sandalwood heartwood extract suspension in sequence for extraction. The petroleum ether extract was concentrated and dried to obtain the petroleum ether extract. The ethyl acetate extract was concentrated and dried to obtain the ethyl acetate extract. The petroleum ether extract and the ethyl acetate extract were mixed evenly at a weight ratio of 3:1 to obtain the mixed extract. The ratio of sandalwood heartwood extract to water, petroleum ether and ethyl acetate was 1 kg: 3 L: 3 L: 3 L.
[0049] (3) The mixed extract was loaded onto a silica gel column (the filler in the silica gel column was 200-300 mesh silica gel; the weight of silica gel was 30 times the weight of the mixed extract). First, impurities were removed by elution with chloroform and acetone in a volume ratio of 94:6 for 4 column volumes. Then, it was eluted with chloroform and acetone in a volume ratio of 82:18 for 5 column volumes. The eluent obtained by elution with chloroform and acetone in a volume ratio of 82:18 was collected, concentrated and dried to obtain the sandalwood heartwood extract.
[0050] Comparative Example 1: Preparation of Sandalwood Heartwood Extract
[0051] (1) Take sandalwood heartwood, crush it and pass it through a 100-mesh sieve. Then, heat it with solvent to 70°C and reflux for 1.5 hours. After the extraction is completed, separate the extract and concentrate and dry the extract to obtain sandalwood heartwood extract. The ratio of sandalwood heartwood to solvent is 1 kg: 10 L. The solvent is an aqueous ethanol solution with a volume fraction of 95%.
[0052] (2) Water was added to the sandalwood heartwood extract to suspend it, and a sandalwood heartwood extract suspension was obtained. Then, petroleum ether and ethyl acetate were added to the sandalwood heartwood extract suspension in sequence for extraction. The petroleum ether extract was concentrated and dried to obtain the petroleum ether extract. The ethyl acetate extract was concentrated and dried to obtain the ethyl acetate extract. The petroleum ether extract and the ethyl acetate extract were mixed evenly at a weight ratio of 3:1 to obtain the mixed extract. The ratio of sandalwood heartwood extract to water, petroleum ether and ethyl acetate was 1 kg: 3 L: 3 L: 3 L.
[0053] (3) Load the mixed extract onto a silica gel column (the filler in the silica gel column is 200-300 mesh silica gel; the weight of silica gel is 30 times the weight of the mixed extract), first elute with chloroform and acetone at a volume ratio of 85:15 (4 column volumes) to remove impurities; then elute with chloroform and acetone at a volume ratio of 70:30 (5 column volumes). Collect the eluent eluted with chloroform and acetone at a volume ratio of 70:30, concentrate and dry it to obtain the sandalwood heartwood extract.
[0054] Comparative Example 2: Preparation of Sandalwood Heartwood Extract
[0055] (1) Take sandalwood heartwood, crush it and pass it through a 100-mesh sieve. Then, heat it with solvent to 70°C and reflux for 1.5 hours. After the extraction is completed, separate the extract and concentrate and dry the extract to obtain sandalwood heartwood extract. The ratio of sandalwood heartwood to solvent is 1 kg: 10 L. The solvent is an aqueous ethanol solution with a volume fraction of 95%.
[0056] (2) Water was added to the sandalwood heartwood extract to suspend it, and a sandalwood heartwood extract suspension was obtained. Then, petroleum ether and ethyl acetate were added to the sandalwood heartwood extract suspension in sequence for extraction. The petroleum ether extract was concentrated and dried to obtain the petroleum ether extract. The ethyl acetate extract was concentrated and dried to obtain the ethyl acetate extract. The petroleum ether extract and the ethyl acetate extract were mixed evenly at a weight ratio of 3:1 to obtain the mixed extract. The ratio of sandalwood heartwood extract to water, petroleum ether and ethyl acetate was 1 kg: 3 L: 3 L: 3 L.
[0057] (3) Load the mixed extract onto a silica gel column (the filler in the silica gel column is 200-300 mesh silica gel; the weight of silica gel is 30 times the weight of the mixed extract), first elute with chloroform and acetone at a volume ratio of 70:30 (4 column volumes) to remove impurities; then elute with chloroform and acetone at a volume ratio of 50:50 (5 column volumes), collect the eluent eluted with chloroform and acetone at a volume ratio of 50:50, concentrate and dry to obtain the sandalwood heartwood extract.
[0058] Experiment Example 1: Detection of mitochondrial ATP levels in UVB-induced photoaging HSF cells
[0059] The specific steps are as follows: (1) Collect HSF cells and seed them into 12-well plates. After the cells adhere to the plate, discard the old culture medium and add an equal amount of PBS to each well. Cover the blank control group cells with aluminum foil to avoid irradiation. Use vitamin C (VC) as a positive control. Then place the 12-well plate under a UVB lamp for 3 days and irradiate with a cumulative irradiation dose of 400 mJ / cm2. (2) After irradiation, replace the blank control group and the model group with complete culture medium and incubate for 48 h. Replace the experimental group with complete culture medium containing 3.13 μg / mL of sandalwood heartwood extract and incubate for 48 h. Replace the positive control with complete culture medium containing 50 μM vitamin C and incubate for 48 h. (3) Discard the culture medium, wash twice with PBS, add 100 μL of lysis buffer to each well, and lyse on ice for 10 min. After lysis, centrifuge at 12000g for 5 min at 4℃ and take the supernatant. (4) Prepare the standard solution according to the ratio and prepare the detection solution. Add 100 μL of ATP detection working solution to the detection well or detection tube. (5) Add 20 μL of sample to each well and measure the RLU value using a chemiluminescence analyzer. (6) Calculate the ATP release in the mitochondria of HSF cells. The results are shown below. The higher the ATP release, the stronger the mitochondrial functional activity.
[0060] Table 1. Results of the effect of sandalwood heartwood extract on mitochondrial ATP release in UVB-induced photoaged HSF cells.
[0061] Extracts to be tested <![CDATA[ATP release amount (μmol / 10 9 )]]> Blank group - 1.0 Model group - 0.52 Positive control group - 0.83 Experimental group 1 Sandalwood heartwood extract prepared in Example 1 0.69 Experimental group 2 Sandalwood heartwood extract prepared in Example 2 0.75 Experimental group 3 Sandalwood heartwood extract prepared in Example 3 0.84 Experimental group 4 Sandalwood heartwood extract prepared in Example 4 0.99 Experimental group 5 Sandalwood heartwood extract prepared in Comparative Example 1 0.88 Experimental group 6 Sandalwood heartwood extract prepared in Comparative Example 2 0.85
[0062] UVB can induce senescence in HSF cells. As shown in Table 1, the amount of ATP released from the mitochondria in the model group HSF cells was significantly lower than that in the control group. This indicates that mitochondrial function in UVB-induced senescent HSF cells is reduced.
[0063] The experimental results in Table 1 also show that the ATP release in the mitochondria of experimental groups 1 and 2 HSF cells was significantly higher than that in the model group. This indicates that the sandalwood heartwood extract prepared by the method described in this invention has an activating effect on mitochondrial function.
[0064] The experimental results in Table 1 also show that the ATP release in the mitochondria of experimental group 3HSF cells was significantly higher than that in experimental groups 1 and 2. This indicates that the mixed extract obtained by mixing petroleum ether extract and ethyl acetate extract has a significantly higher activating effect on mitochondrial function than either petroleum ether extract or ethyl acetate extract alone. Mixing petroleum ether extract and ethyl acetate extract can produce a certain synergistic effect in enhancing the activation of mitochondrial function.
[0065] The experimental results in Table 1 also show that the ATP release in the mitochondria of experimental group 4HSF cells was significantly higher than that of experimental group 3. This indicates that the sandalwood heartwood extract obtained by eluting the mixed extract under the silica column elution conditions described above has a significantly higher mitochondrial function activation effect than the mixed extract.
[0066] The experimental results in Table 1 also show that, although the ATP release in the mitochondria of HSF cells in experimental groups 5 and 6 was increased compared to experimental group 3, the increase was not significant and was much smaller than that in experimental group 4. This indicates that the elution conditions of the silica gel column in this invention are crucial; only the sandalwood heartwood extract obtained by eluting the mixed extract under the silica gel column elution conditions described above can have a significantly higher mitochondrial activation effect than the mixed extract. However, the sandalwood heartwood extract obtained by eluting the mixed extract under other silica gel column elution conditions does not have a significantly higher mitochondrial activation effect than the mixed extract.
Claims
1. A method for preparing an extract from the heartwood of sandalwood, characterized in that, It includes the following steps: (1) Take the heartwood of sandalwood, extract it with solvent and separate the extract. Concentrate and dry the extract to obtain sandalwood heartwood extract. (2) Water is added to the sandalwood heartwood extract to suspend it, and sandalwood heartwood extract suspension is obtained; then an organic solvent is added to the sandalwood heartwood extract suspension for extraction, and the organic solvent extract is concentrated and dried to obtain organic solvent extract; the organic solvent extract is the sandalwood heartwood extract. (3) The mixed extract was purified by silica gel column chromatography, and impurities were removed by chloroform-acetone at a volume ratio of 94:
6. The target component was then eluted by chloroform-acetone at a volume ratio of 82:
18.
2. The method for preparing the sandalwood heartwood extract according to claim 1, characterized in that, In step (1), the ratio of sandalwood heartwood to solvent is 1kg:5-15L; Most preferably, in step (1), the ratio of the amount of sandalwood heartwood to the amount of solvent is 1 kg: 10 L.
3. The method for preparing the sandalwood heartwood extract according to claim 1, characterized in that, In step (1), the solvent is an aqueous ethanol solution with a volume fraction of 70-95%; Preferably, the extraction in step (1) is a heating reflux extraction; the extraction time is 1 to 2 hours.
4. The method for preparing the sandalwood heartwood extract according to claim 1, characterized in that, The organic solvent mentioned in step (2) is petroleum ether and / or ethyl acetate.
5. The method for preparing the sandalwood heartwood extract according to claim 1, characterized in that, The ratio of sandalwood heartwood extract to water and organic solvent is 1 kg: 3-8 L: 3-8 L; Most preferably, the ratio of sandalwood heartwood extract to water and organic solvent is 1 kg: 3 L: 6 L.
6. The method for preparing the sandalwood heartwood extract according to claim 4, characterized in that, The specific method in step (2) is as follows: Water was added to the heartwood extract of sandalwood to suspend it, and a sandalwood heartwood extract suspension was obtained. Then, petroleum ether and ethyl acetate were added to the sandalwood heartwood extract suspension in sequence for extraction. The petroleum ether extract was concentrated and dried to obtain the petroleum ether extract, and the ethyl acetate extract was concentrated and dried to obtain the ethyl acetate extract. The petroleum ether extract and the ethyl acetate extract are mixed evenly to obtain a mixed extract, which is then used to obtain the sandalwood heartwood extract.
7. The method for preparing the sandalwood heartwood extract according to claim 5, characterized in that, The weight ratio of petroleum ether extract to ethyl acetate extract is 2–4:1; Most preferably, the weight ratio of petroleum ether extract to ethyl acetate extract is 3:
1.
8. The sandalwood heartwood extract prepared by the preparation method according to any one of claims 1 to 7.
9. The use of the sandalwood heartwood extract according to claim 8 in the preparation of products that activate mitochondrial function.
10. The use of the sandalwood heartwood extract according to claim 9 in the preparation of mitochondrial function activators.