An extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet, its preparation method and application

Abstract

This invention provides an extract of *Elaeocarpus yunnanensis* from Yunnan and its preparation method and application. The preparation method includes the following steps: (1) crushing *Elaeocarpus yunnanensis*, mixing it with an enzyme, and then using a solvent for negative pressure cavitation extraction to obtain a crude extract of *Elaeocarpus yunnanensis*; (2) adsorbing and eluting the crude extract of *Elaeocarpus yunnanensis* obtained in step (1) using a macroporous resin to obtain the *Elaeocarpus yunnanensis* extract. The method provided by this invention can effectively extract active substances from *Elaeocarpus yunnanensis*. The obtained *Elaeocarpus yunnanensis* extract has high geranium content, high total flavonoid content, good antioxidant effect, high hyaluronidase inhibitory activity, good anti-inflammatory effect, and high collagenase activity inhibition rate. The preparation method is economical and environmentally friendly, and the reagents used can be recycled and reused. It can be used as an active substance in health care products and cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and preparation technology, specifically relating to a Yunnan-Tibet Elaeocarpus extract, its preparation method and application, and particularly to a Yunnan-Tibet Elaeocarpus extract with high geranium content, its preparation method and application. Background Technology

[0002] *Elaeocarpus yunnanensis* is a tall tree belonging to the genus *Elaeocarpus* in the family Elaeocarpaceae. It is found in Yunnan and Tibet, growing in evergreen forests at altitudes of 1300-3000 meters. Other plants in the genus *Elaeocarpus*, such as *Elaeocarpus shanensis* and *Elaeocarpus baldens*, have been studied to some extent. However, research on *Elaeocarpus yunnanensis*, both domestically and internationally, has largely focused on population distribution and species identification, with little research on its extracts and their activity.

[0003] CN114796172A discloses the active fraction of *Elaeocarpus decipiens* branch and leaf extract with acetylcholinesterase inhibitory activity and its application, belonging to the field of botanical medicine technology. This invention involves solvent extraction of dried *Elaeocarpus decipiens* branches and leaves to obtain a crude extract PXS48-1A. PXS48-1A is then further extracted to obtain an ethyl acetate extract PXS48-3A and a n-butanol extract PXS48-4A. These extracts are then further refined to obtain six active fractions: PXS48-23, PXS48-24, PXS48-25, PXS48-43, PXS48-44, and PXS48-45. The invention also provides a pharmaceutical composition composed of the active botanical fractions of *Elaeocarpus decipiens* branches and leaves, a method for preparing these active botanical fractions and their pharmaceutical compositions, and their application in the preparation of drugs for treating Alzheimer's disease.

[0004] CN114504602A discloses the preparation of H from *Elaeocarpus decipiens* extract. + -K + - The application of ATPase inhibitors and their application in the preparation of drugs for the prevention or treatment of gastric-related diseases, specifically the application of the extract of *Elaeocarpus tinctoria* in the preparation of drugs for the treatment of gastric ulcers, and the preparation method of the extract of *Elaeocarpus tinctoria*.

[0005] CN103467533A discloses a method for extracting eugenol from the leaves of *Elaeocarpus ovoidus*. The method is as follows: *Elaeocarpus ovoidus* leaves are pulverized, and an appropriate amount of water and biological enzymes are added for enzymatic hydrolysis for 5-10 hours. Then, 5-10 times the volume of 80-99% ethanol solution is added for reflux extraction 2-3 times. The ethanol is recovered under reduced pressure, the extract is concentrated, diluted with an appropriate amount of water, filtered, and then ultrafiltered using an ultrafiltration membrane. The filtrate is then concentrated using a nanofiltration membrane. The concentrated liquid is collected, and an appropriate amount of gelatin solution is added for complete precipitation. The precipitate is dissolved in acetone solution after centrifugation, and the acetone solution is collected to recover the reagent. The product is then vacuum dried to obtain eugenol. Using this invention to produce eugenol is a simple process with low energy consumption, low pollution, and is easily scalable for industrial production.

[0006] However, the methods described above only studied other species of *Elaeocarpus yunnanensis*, without addressing the extraction process and effects on *Elaeocarpus yunnanensis*. Therefore, developing a method for effectively extracting *Elaeocarpus yunnanensis* and studying the technical effects of related extracts has become an urgent problem to be solved. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a *Elaeocarpus yunnanensis* extract from Yunnan and Tibet, its preparation method, and its applications, particularly a *Elaeocarpus yunnanensis* extract with high geranium content, its preparation method, and its applications. The method provided by this invention can effectively extract active substances from *Elaeocarpus yunnanensis*, resulting in an extract with high geranium content, high total flavonoid content, and low content of macromolecular tannins such as *Elaeocarpus yunnanensis* tannins. It exhibits good antioxidant effects, high hyaluronidase inhibitory activity, good anti-inflammatory effects, and high collagenase activity inhibition rate. The preparation method is economical and environmentally friendly, and the reagents used are recyclable and reusable. It can be used as an active substance in health and wellness products.

[0008] To achieve this objective, the present invention employs the following technical solution:

[0009] On one hand, the present invention provides a method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet, the method comprising the following steps:

[0010] (1) The Yunnan-Tibet Elaeocarpus was crushed, mixed with enzymes, and then extracted by negative pressure cavitation with solvent to obtain crude extract of Yunnan-Tibet Elaeocarpus.

[0011] (2) The crude extract of *Elaeocarpus yunnanensis* obtained in step (1) is adsorbed and eluted using macroporous resin to obtain the *Elaeocarpus yunnanensis* extract.

[0012] The above extraction method first uses solvent extraction, and then uses macroporous resin for adsorption, which can effectively extract the active substances from *Elaeocarpus yunnanensis*. The obtained *Elaeocarpus yunnanensis* extract has high geranium content, high total flavonoid content, good antioxidant effect, high hyaluronidase inhibitory activity, good anti-inflammatory effect, high collagenase activity inhibition rate, and high product yield. It can be used as an active substance in health care products.

[0013] Geraniol is a polyphenolic tannin with strong antioxidant capabilities. Therefore, increasing the geraniol content in a product can effectively enhance its antioxidant effect. This invention utilizes a specific extraction method to effectively increase the geraniol content in the product. Combined with other active ingredients in the extract, this further enhances the product's antioxidant effect.

[0014] Preferably, the solvent in step (1) includes any one or a combination of at least two of water, ethanol or butanediol, and preferably a combination of ethanol and water.

[0015] The specific solvent combination mentioned above can further extract Elaeocarpus yunnanensis from Yunnan and Tibet, and can effectively improve the effect of the extract.

[0016] Preferably, the enzyme in step (1) includes pectinase and / or cellulase, with pectinase being preferred.

[0017] Preferably, the mass ratio of the enzyme to *Elaeocarpus yunnanensis* in step (1) is (1-3):10000, for example, 1:10000, 1.5:10000, 2:10000, 2.5:10000 or 3:10000, but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0018] Preferably, the temperature for mixing with the enzyme in step (1) is 40-55℃ and the time is 1-3h. The temperature can be 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃ or 55℃, etc., and the time can be 1h, 1.5h, 2h, 2.5h or 3h, etc., but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0019] Preferably, the ratio of the Yunnan Tibetan Elaeocarpus to the solvent in step (1) is 1:(8-12)g / mL, such as 1:8g / mL, 1:9g / mL, 1:10g / mL, 1:11g / mL or 1:12g / mL, but not limited to the values ​​listed above. Other values ​​not listed above are also applicable.

[0020] Preferably, the temperature of the negative pressure cavitation extraction in step (1) is 45-55℃ and the time is 1.5-2.5h. The temperature can be 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃ or 55℃, etc., and the time can be 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2h, 2.1h, 2.2h, 2.3h, 2.4h or 2.5h, etc., but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0021] Preferably, the negative pressure intensity extracted by negative pressure cavitation in step (1) is 70-100 mbar, such as 70 mbar, 75 mbar, 80 mbar, 85 mbar, 90 mbar, 95 mbar or 100 mbar, but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0022] The specific extraction parameters mentioned above can effectively improve the extraction effect, increase the geraniol content in the extract, and improve the product efficacy.

[0023] Preferably, the extraction in step (1) is performed at least twice.

[0024] Preferably, the macroporous resin in step (2) includes AB-8 or D101.

[0025] Preferably, the macroporous resin in step (2) is AB-8.

[0026] The aforementioned specific macroporous resins can effectively adsorb active ingredients, remove impurities, and improve the efficacy of extracts.

[0027] Preferably, the eluent used in step (2) comprises water and ethanol;

[0028] Preferably, the specific process of elution in step (2) is as follows:

[0029] First, elute with water for 1.5-2.5 column volumes, then elute with 15-25% (v / v) ethanol aqueous solution for 4-6 column volumes, and finally elute with 45-55% (v / v) ethanol aqueous solution for 8-12 column volumes.

[0030] The aforementioned specific eluent can effectively elute the active ingredients adsorbed by macroporous resin, thereby effectively improving the extract's efficacy.

[0031] Secondly, the present invention also provides an extract of *Elaeocarpus yunnanensis* prepared by the preparation method described above.

[0032] Thirdly, the present invention also provides the application of the above-mentioned extract of *Elaeocarpus yunnanensis* in the preparation of food, health products or cosmetics.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention provides a method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet. By first using solvent extraction and then using macroporous resin for adsorption, the active substances in *Elaeocarpus yunnanensis* from Yunnan and Tibet can be effectively extracted. The obtained extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet has high geranium content, high total flavonoid content, good antioxidant effect, high hyaluronidase inhibitory activity, good anti-inflammatory effect, and high collagenase activity inhibition rate. It can be used as an active substance in health care products. Detailed Implementation

[0035] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0036] In the following examples, the pectinase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and the enzyme activity was 50,000 U / g;

[0037] Cellulase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 50 U / g.

[0038] Example 1

[0039] This embodiment provides a method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet, with the following specific steps:

[0040] (1) Crush 100g of pitted Yunnan Tibetan Elaeocarpus fruit to 20 mesh, mix with 200g of water, then add 10mg of pectinase and react for 2 hours. After that, add an ethanol aqueous solution with a material-to-liquid ratio of 1:10g / mL and an ethanol concentration of 50%. Adjust the vacuum of the extraction tank to 70mbar, heat to 50℃ and extract for 2 hours. Repeat once, combine the extracts and concentrate under reduced pressure to obtain crude extract of Yunnan Tibetan Elaeocarpus.

[0041] (2) The crude extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet was adsorbed with 200g of AB-8 macroporous resin, and then eluted with two column volumes of water and five column volumes of 20% ethanol solution to remove insoluble matter. Then, it was eluted with 10 column volumes of 50% ethanol. The eluent was concentrated and dried under reduced pressure to obtain the extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet.

[0042] Example 2

[0043] This embodiment provides a method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet. The difference between this method and Example 1 is that step (1) is changed to: 100g of pitted *Elaeocarpus yunnanensis* fruit is crushed to 20 mesh, mixed with 200g of water, then 10mg of pectinase is added and reacted for 2 hours. After that, an ethanol aqueous solution is added to make the material-liquid ratio 1:8g / mL and the ethanol concentration 55%. The vacuum degree of the extraction tank is adjusted to 100mbar, and the extraction is carried out at 55℃ for 2 hours. This process is repeated once. The extracts are combined and concentrated under reduced pressure to obtain crude extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet.

[0044] Example 3

[0045] This embodiment provides a method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet. The difference between this method and Example 1 is that step (1) is changed to: 100g of pitted *Elaeocarpus yunnanensis* fruit is crushed to 20 mesh, mixed with 200g of water, then 10mg of pectinase is added and reacted for 2 hours. After that, an ethanol aqueous solution is added to make the material-liquid ratio 1:20g / mL and the ethanol concentration 85%. The vacuum degree of the extraction tank is adjusted to 70mbar, and the extraction is carried out at 50℃ for 2 hours. This process is repeated once. The extracts are combined and concentrated under reduced pressure to obtain crude extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet.

[0046] Example 4

[0047] This embodiment provides a method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet. The difference between this method and Example 1 is that step (1) is changed to: 100g of pitted *Elaeocarpus yunnanensis* fruit is crushed to 20 mesh, mixed with 200g of water, then 10mg of pectinase is added and reacted for 2 hours. After that, an ethanol aqueous solution is added to make the material-liquid ratio 1:10g / mL and the ethanol concentration 45%. The vacuum degree of the extraction tank is adjusted to 70mbar, and the extraction is carried out at 45℃ for 2 hours. This process is repeated once. The extracts are combined and concentrated under reduced pressure to obtain crude extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet.

[0048] Example 5

[0049] This embodiment provides a method for preparing the extract of Eupatorium fortunei from Yunnan and Tibet. The only difference between this method and Example 1 is that the ethanol concentration in step (1) is 35%, while all other preparation conditions remain unchanged.

[0050] Example 6

[0051] This embodiment provides a method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet. The only difference between this method and Example 1 is that the extraction solvent in step (1) is water, while all other preparation conditions remain unchanged.

[0052] Example 7

[0053] This embodiment provides a method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet. The only difference between this method and Example 1 is that the AB-8 macroporous resin in step (2) is replaced with an equal amount of D101 macroporous resin.

[0054] Example 8

[0055] This embodiment provides a method for preparing the extract of Eupatorium fortunei from Yunnan and Tibet. The only difference between the preparation method and that in Example 1 is that the elution method in step (2) is changed to: elution with 5 column volumes of 20% ethanol solution to remove insoluble matter; then elution with 10 column volumes of 50% ethanol, and the eluent is concentrated to dryness under reduced pressure.

[0056] Example 9

[0057] This embodiment provides a method for preparing the extract of Eupatorium fortunei from Yunnan and Tibet. The difference between the preparation method and that in Example 1 is that the elution method in step (2) is changed to: elution with 2 column volumes of water to remove insoluble matter; then elution with 10 column volumes of 50% ethanol, and the eluent is concentrated and dried under reduced pressure.

[0058] Example 10

[0059] This embodiment provides a method for preparing the extract of Eupatorium fortunei from Yunnan and Tibet. The only difference between this method and that of Example 1 is that the elution method in step (2) is changed to: elution with 10 column volumes of 50% ethanol, and the eluent is concentrated and dried under reduced pressure.

[0060] Example 11

[0061] This embodiment provides a method for preparing the extract of Elaeocarpus decipiens from Yunnan and Tibet. The only difference from Example 1 is that the enzymatic hydrolysis part in step (1) is changed to mixing with 200g of water and then adding 5mg of pectinase and reacting for 2 hours.

[0062] Example 12

[0063] This embodiment provides a method for preparing the extract of Elaeocarpus decipiens from Yunnan and Tibet. The only difference from Example 1 is that the enzymatic hydrolysis part in step (1) is changed to mixing with 200g of water and then adding 10mg of cellulase and reacting for 2 hours.

[0064] Comparative Example 1

[0065] This comparative example provides a method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet. The specific steps are the same as in Example 1, except that enzymatic hydrolysis and negative pressure cavitation extraction are not used, and the following extraction method is used instead.

[0066] 100g of pitted Yunnan-Tibet Elaeocarpus fruit was crushed to 20 mesh, and an ethanol aqueous solution was added to make the material-liquid ratio 1:10g / mL and the ethanol concentration 50%. The mixture was heated and refluxed for 2 hours. The extraction was repeated once. The extracts were combined and concentrated under reduced pressure to obtain crude extract of Yunnan-Tibet Elaeocarpus.

[0067] Comparative Example 2

[0068] This comparative example provides a method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet. The specific steps are the same as in Example 1, except that pectinase hydrolysis is not used, and extraction is performed using the following method.

[0069] 100g of pitted *Elaeocarpus yunnanensis* fruit from Yunnan and Tibet was crushed to 20 mesh and added to an ethanol-water solution to make a material-to-liquid ratio of 1:10 g / mL and an ethanol concentration of 50%. The vacuum degree of the extraction tank was adjusted to 70 mbar, and the extraction was carried out at 50℃ for 2 hours. The process was repeated once. The extracts were combined and concentrated under reduced pressure to obtain crude extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet.

[0070] Effect test:

[0071] ① The extracts of *Elaeocarpus yunnanensis* obtained in Examples 1-12 and Comparative Examples 1-2 were detected by liquid chromatography. The detection parameters are as follows:

[0072] Chromatographic column: Agilent ZORBAX SB-Aq 3.5µm, 3.0×100mm, 0.2% formic acid aqueous solution / acetonitrile;

[0073] Detection wavelength: 254nm;

[0074] Flow rate: 0.5 mL / min;

[0075] Column oven: 35℃;

[0076] Chromatographic conditions:

[0077] Time / min 0.2% formic acid aqueous solution Acetonitrile Flow rate 0 95 5 0.5 mL / min 2 95 5 0.5 mL / min 20 85 15 0.5 mL / min 21 10 90 0.5 mL / min 24 5 95 0.5 mL / min 25 90 5 0.5 mL / min 30 90 5 0.5 mL / min

[0078] The results are as follows:

[0079]

[0080] The data above shows that by using a combination of specific enzymes, extraction solvents, macroporous resins and eluents, and by employing specific extraction processes, methods and parameters, the present invention can effectively increase the geraniol content in the product, thereby improving the product's antioxidant effect.

[0081] ② The antioxidant activity of the *Elaeocarpus yunnanensis* extracts obtained in Examples 1-12 was tested and compared with that of the 95% ethanol extract (obtained by reflux extraction of the pitted *Elaeocarpus yunnanensis* fruit in Example 1 using 10 times the amount of 95% ethanol, followed by vacuum concentration and drying) and vitamin C. Prepare solutions of 95% ethanol extract and vitamin C with concentrations of 0.8 mg / mL, 0.4 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, and 0.013 mg / mL, and take 1 mL of each solution for later use. Prepare solutions of extracts from Examples 1-12 with concentrations of 0.8 mg / mL, 0.4 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, and 0.013 mg / mL, and take 1 mL of each solution for later use. Prepare a 0.51 mg / mL solution of DPPH, and add 1 mL to each of the test sample and the control sample. After reacting for 30 minutes, measure the absorbance. Plot a curve between absorbance and sample concentration, and calculate the IC50 value. The results are as follows:

[0082]

[0083]

[0084] The data above shows that by using specific enzymes, extraction solvents, macroporous resins and eluents, and employing specific extraction processes, methods and parameters, this invention can effectively improve the antioxidant effect of the product, and has superior technical effects compared to other extraction processes and existing antioxidants.

[0085] ③ The total flavonoid content in the *Elaeocarpus yunnanensis* extracts obtained in Examples 1-12 and Comparative Examples 1-2 was detected using the following methods: Rutin was used as a reference standard, and the total flavonoid content of each sample was determined by the aluminum nitrate colorimetric method. Rutin standard was prepared into solutions with concentrations of 0.8 mg / mL, 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL; each sample was prepared into a 2 mg / mL solution. The standard and the test samples were respectively subjected to the total flavonoid colorimetric reaction using the aluminum nitrate colorimetric method, and their absorbance was measured. A standard curve of the rutin standard was plotted using absorbance and flavonoid concentration, and the results are as follows:

[0086]

[0087] The data above shows that by using specific enzymes, extraction solvents, macroporous resins and eluents, and by employing specific extraction processes, methods and parameters, the present invention can effectively increase the total flavonoid content of the product.

[0088] ④ The hyaluronidase inhibitory activity of the *Elaeocarpus yunnanensis* extracts obtained in Examples 1-12 and Comparative Examples 1-2 was detected using the following method: 50 μL of hyaluronidase solution and 50 μL of the 0.3 mg / mL test sample were added to each high-throughput tube. Acetic acid buffer was used instead of hyaluronidase solution for the control group. A blank control group was also included. The tubes were incubated at 37°C for 20 min. Then, 100 μL of calcium chloride solution was added to each tube, and the tubes were incubated at 37°C for 20 min. Next, 50 μL of sodium hyaluronate was added, and acetate buffer was used instead of hyaluronidase solution for the control group. The tubes were incubated at 37°C for 40 min. After incubation, 100 μL of sodium hydroxide solution and potassium borate solution were added to each tube. The tubes were incubated in a water bath at 85°C for 5 min, an ice bath for 2 min, and then at room temperature for 5 min. 100 μL of the reaction solution was then transferred to a 96-well plate, and 100 μL of colorimetric reagent was added to each well. The 96-well plate was quickly placed in a microplate reader and shaken at 37°C for 1 min. After adding the chromogenic solution and 10 min later, the absorbance was measured at 585 nm. The results are as follows:

[0089]

[0090] The data above shows that by using specific extraction solvents, macroporous resins, and eluents, and employing specific extraction processes, methods, and parameters, this invention can effectively improve the product's inhibitory effect on hyaluronidase.

[0091] ⑤ The collagenase inhibitory activity of the extracts of *Elaeocarpus yunnanensis* obtained in Examples 1-12 and Comparative Examples 1-2 was detected. N-[3-(2-furanyl)acryloyl]-L-leucine-glycine-L-proline-L-alanine (FALGPA), also known as FALGPA, was used as the substrate. Type I collagenase derived from *Bacillus histolyticus* was selected as the reactive enzyme. The collagenase inhibitory activity of different *Elaeocarpus yunnanensis* extracts was detected at 330 nm. The results are as follows:

[0092]

[0093] The data above shows that by using specific enzymes, extraction solvents, macroporous resins and eluents, and employing specific extraction processes, methods and parameters, this invention can effectively improve the product's inhibitory effect on collagenase, and has more significant advantages compared to existing products.

[0094] ⑥ The anti-inflammatory and soothing activities of the *Elaeocarpus yunnanensis* extracts obtained in Examples 1-12 and Comparative Examples 1-2 were tested. The anti-inflammatory and soothing effects of the extracts were verified using a mouse AW264.7 cell model. The detection method is as follows: LPS-induced mouse macrophages RAW264.7 were used as the soothing model. A blank group and dexamethasone were set up as the positive control. Specifically, lipopolysaccharide (LPS)-induced mouse macrophages RAW264.7 were used as the soothing model. The positive control group was induced with dexamethasone (DEX) at 50 μg / mL; the sample group was induced with LPS and each group was treated with *Elaeocarpus yunnanensis* extract solution (concentration added at 120 μg / mL). The inhibitory effect of *Elaeocarpus yunnanensis* extract on NO was detected using a NO kit. The experimental results were analyzed using data processing software. The results are as follows:

[0095]

[0096] The data above shows that by using specific enzymes, extraction solvents, macroporous resins and eluents, and by employing specific extraction processes, methods and parameters, this invention can effectively improve the anti-inflammatory and soothing effects of the product.

[0097] ⑦ The human cell line activation test (h-CLAT) was performed on the extract of *Elaeocarpus yunnanensis* obtained in Example 1. The THF-1 human acute monocytic leukemia cell line was used, and H-CLAT cells were used as the experimental model. According to the in vitro skin allergy detection method of OECD Guideline 442E, the results showed that the product did not have sensitizing properties at 60 μg / mL, indicating that the product provided by the present invention has high safety.

[0098] The applicant declares that this invention illustrates the Yunnan-Tibet Elaeocarpus extract, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0099] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0100] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet, characterized in that, The preparation method includes the following steps: (1) The Yunnan-Tibet Elaeocarpus was crushed, mixed with enzymes, and then extracted by negative pressure cavitation with solvent to obtain crude extract of Yunnan-Tibet Elaeocarpus. (2) The crude extract of Elaeocarpus yunnanensis obtained in step (1) is adsorbed and eluted with macroporous resin to obtain the Elaeocarpus yunnanensis extract. The enzyme mentioned in step (1) is pectinase, and the mass ratio of the enzyme to *Elaeocarpus yunnanensis* is (1-3):10000; In step (1), the ratio of *Elaeocarpus yunnanensis* to solvent is 1:(8-12) g / mL; The solvent is a combination of ethanol and water, wherein the concentration of ethanol is 55% or 50%; The specific process of elution in step (2) is as follows: First, elute with water for 1.5-2.5 column volumes, then elute with 15-25% (v / v) ethanol aqueous solution for 4-6 column volumes, and finally elute with 45-55% (v / v) ethanol aqueous solution for 8-12 column volumes.

2. The preparation method according to claim 1, characterized in that, The temperature for mixing with the enzyme in step (1) is 40-55℃, and the time is 1-3 h.

3. The preparation method according to claim 1, characterized in that, The temperature of the negative pressure cavitation extraction in step (1) is 45-55℃ and the time is 1.5-2.5 h.

4. The preparation method according to claim 1, characterized in that, The negative pressure intensity of the negative pressure cavitation extraction in step (1) is 70-100 mbar.

5. The preparation method according to claim 1, characterized in that, The extraction in step (1) is performed at least twice.

6. The preparation method according to claim 1, characterized in that, The macroporous resin in step (2) includes AB-8 or D101.

7. The preparation method according to claim 6, characterized in that, The macroporous resin used in step (2) is AB-8.

8. An extract of *Elaeocarpus yunnanensis* prepared by any one of claims 1-7.

9. The application of the *Elaeocarpus yunnanensis* extract according to claim 8 in the preparation of food, health products or cosmetics.

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