A seabuckthorn bioconversion extract with anti-inflammatory and antioxidant capacity, a preparation method thereof and application thereof

Abstract

The present invention provides a method for obtaining a sea buckthorn bioconversion extract having improved anti-inflammatory and / or antioxidant capacity by bioconverting sea buckthorn using a mixture of broken microbial cells. The present invention also relates to a sea buckthorn bioconversion extract obtained by the bioconversion method and to pharmaceutical and cosmetic compositions comprising the sea buckthorn bioconversion extract. The present invention further relates to the use of the sea buckthorn bioconversion extract for the preparation of a pharmaceutical or cosmetic composition.

Description

Technical Field

[0001] This invention relates to a method for preparing a sea buckthorn biotransformation extract. The invention also relates to a sea buckthorn biotransformation extract with anti-inflammatory and / or antioxidant capabilities prepared by the said method, and pharmaceutical preparations or cosmetic compositions containing the sea buckthorn biotransformation extract. The invention further relates to the use of the sea buckthorn biotransformation extract in the preparation of pharmaceutical or cosmetic compositions. Background Technology

[0002] Sea buckthorn (Hippophae rhamnoides L.) is a deciduous shrub or tree belonging to the genus Hippophae in the family Elaeagnaceae. It is a cold- and drought-resistant perennial plant. China possesses 90% of the world's sea buckthorn resources, distributed in North China, Northwest China, and Southwest China. It typically grows at altitudes of 800-3600 meters in sunny mountain ridges, valleys, dry riverbeds, or slopes in temperate regions, often in gravelly, sandy, or loess soils. Sea buckthorn has a long history of medicinal use in China. Its fruit is a commonly used medicine in Tibetan and Mongolian medicine. Sea buckthorn is rich in various organic acids, vitamins, flavonoids, carotenoids, lipids, fatty acids, amino acids, sterols, and many other natural compounds. Among these, flavonoids are an important bioactive component, acting as a powerful antioxidant that effectively scavenge free radicals in the body, delaying cell degeneration and aging. Sea buckthorn flavonoids also possess anti-tumor, anti-inflammatory, and cardiovascular protective effects. Flavonoids are found in almost all parts of sea buckthorn, with the leaves being the richest in flavonoids. Compared to sea buckthorn berries, sea buckthorn leaves have advantages such as being easy to harvest, easy to preserve, and having a large storage capacity.

[0003] In recent years, the development speed and market popularity of plant fermentation extract products have been rising steadily. Plant fermentation is a process in which microorganisms ferment plants as substrates. Fermentation can bring a series of benefits, such as increased content of active substances, enhanced efficacy, reduced toxicity and side effects, and easier separation, purification, and application. These changes are due to the rich enzyme system and powerful biotransformation capabilities of microorganisms. However, fermentation may also consume the plant's effective components, leading to a decrease in efficacy, and the fermentation process can produce pigments and unpleasant odors.

[0004] The existing technology for extracting active ingredients from sea buckthorn for cosmetic applications has the following problems:

[0005] (1) In the existing technology for microbial fermentation of sea buckthorn, the growth and metabolism of microorganisms during the fermentation process will consume the components in sea buckthorn, which may lead to the loss of some active ingredients.

[0006] (2) Traditional fermentation processes can darken the color of sea buckthorn extract and produce an unpleasant odor;

[0007] (3) If an enzyme conversion method is used, such as using glycosidase to hydrolyze flavonoid glycosides in sea buckthorn to generate aglycones, this method is costly and has a single reaction.

[0008] Therefore, there is still a strong demand for sea buckthorn extract, its preparation method, and its application that have increased content of active ingredients, enhanced anti-inflammatory and / or antioxidant effects, and do not have the disadvantages of sea buckthorn fermentation extract such as darkening of color and unpleasant odor. Summary of the Invention

[0009] The first aspect of the present invention relates to a method for preparing a sea buckthorn biotransformation extract, wherein the method includes the following steps:

[0010] (1) Obtain a mixture of broken microbial cells;

[0011] (2) Obtain a crude extract of sea buckthorn in water;

[0012] (3) Add the broken microbial cell mixture obtained in step (1) to the crude extract of sea buckthorn in water in step (2) for biotransformation to obtain sea buckthorn biotransformation extract; optionally,

[0013] (4) Centrifuge and / or filter the seabuckthorn biotransformation extract obtained in step (3).

[0014] The second aspect of the present invention relates to a sea buckthorn biotransformation extract, which is prepared by the method described in the first aspect of the present invention.

[0015] A third aspect of the present invention relates to a cosmetic composition comprising the sea buckthorn biotransformation extract described in the second aspect of the present invention.

[0016] A fourth aspect of the present invention relates to a pharmaceutical preparation comprising the sea buckthorn biotransformation extract described in the second aspect of the present invention.

[0017] The fifth aspect of the invention relates to the use of the sea buckthorn biotransformation extract according to the second aspect of the invention in the preparation of pharmaceutical or cosmetic compositions having anti-inflammatory and / or antioxidant capabilities.

[0018] This invention provides a method for obtaining a sea buckthorn biotransformation extract by using a mixture of broken microbial cells to biotransform sea buckthorn. The sea buckthorn biotransformation extract obtained by this method has the following advantages:

[0019] (1) The total flavonoid content in sea buckthorn extract was significantly increased;

[0020] (2) The anti-inflammatory activity of sea buckthorn extract was significantly enhanced;

[0021] (3) The antioxidant activity of sea buckthorn extract was significantly enhanced;

[0022] (4) The sea buckthorn extract obtained through this biotransformation has a fresh smell and a light color, making it more suitable for use in the cosmetics industry.

[0023] In the technical solution of this invention, the various enzymes and metabolites (e.g., intracellular enzymes and membrane-bound enzymes) released from the broken microbial cell mixture act on sea buckthorn, not only converting flavonoid glycosides in sea buckthorn into flavonoid aglycones, but also catalyzing various biotransformation reactions, such as methylation and demethylation, dehydroxylation and hydroxylation, glycosylation, dehydrogenation and esterification, etc., thereby increasing the content of effective substances in sea buckthorn or introducing new active substances, providing more possibilities for enhancing the efficacy of sea buckthorn extract. Simultaneously, because the structure of the microbial cells themselves is destroyed in the broken microbial cell mixture, fermentation and growth metabolism cannot occur, thus avoiding the disadvantage of consuming components in sea buckthorn due to growth metabolism during the fermentation process. It also avoids the disadvantages of fermentation processes, such as darkening the color of sea buckthorn extract and producing unpleasant odors, making it unsuitable for cosmetic applications. Currently, there are no research reports on using this method to improve the efficacy of sea buckthorn extract. Attached Figure Description

[0024] Figure 1 The colors of the sea buckthorn biotransformation extract prepared according to Examples 1-3 of the present invention, the sea buckthorn extract prepared according to Comparative Example 1, and the sea buckthorn fermentation extract prepared according to Comparative Example 2 are shown. Invention Details

[0026] definition

[0027] In this article, the terms “s”, “min” and “h” stand for “second”, “minute” and “hour”, respectively.

[0028] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized in," and is inclusive or open-ended, not excluding additional, undescribed elements or method steps. However, in this document, each reference to "comprising" is intended to cover alternative embodiments of "consistently composed of" and "consisting of," wherein "consisting of" excludes any unspecified elements or steps, and "consisting of" allows the inclusion of other, undescribed elements or steps that do not materially affect the essential or essential characteristics and novelty of the composition or method discussed.

[0029] As used herein, the term “about” means ±10% of the value it modifies, more preferably ±5%, and most preferably ±2%, so that those skilled in the art can clearly determine the range of the term “about” based on the value it modifies.

[0030] The term “broken microbial cell mixture” as used in this article refers to microbial cells whose structure has been destroyed, in which various enzymes and metabolites in the cells can be released, but the cells themselves cannot ferment and grow due to the destruction of their structure.

[0031] As used in this article, "biotransformation extract" refers to an extract obtained by adding a mixture of broken microbial cells to a plant extract, thereby biotransforming the plant through the enzymes and metabolites released by the microorganisms.

[0032] Preparation method of sea buckthorn biotransformation extract

[0033] The first aspect of the present invention relates to a method for preparing a sea buckthorn biotransformation extract, wherein the method includes the following steps:

[0034] (1) Obtain a mixture of broken microbial cells;

[0035] (2) Obtain a crude extract of sea buckthorn in water;

[0036] (3) Add the broken microbial cell mixture obtained in step (1) to the crude extract of sea buckthorn in water in step (2) for biotransformation to obtain sea buckthorn biotransformation extract; optionally,

[0037] (4) Centrifuge and / or filter the seabuckthorn biotransformation extract obtained in step (3).

[0038] In some embodiments, the microorganism is a yeast. In some embodiments, the yeast is selected from *Saccharomycopsis fibuligera*, *Saccharomyces cerevisiae*, *Candida*, *Hansenula spora opuntiae*, *Pichia fermentans*, *Kluyveromyces*, and combinations thereof. In some embodiments, the yeast is *Saccharomycopsis fibuligera*.

[0039] In some embodiments, the sea buckthorn is selected from sea buckthorn roots, stems, leaves, fruits, and combinations thereof. In some embodiments, the sea buckthorn is sea buckthorn leaves.

[0040] In some implementations, the ruptured microbial cell mixture is obtained through the following steps:

[0041] (1.1) Obtain seed culture of microbial cells;

[0042] (1.2) Fermentation culture of microbial cells is carried out by fermentation culture of seed liquid of microbial cells to obtain fermentation broth of microbial cells;

[0043] (1.3) Clean microbial cells are obtained by repeatedly and alternately centrifuging and washing the fermentation broth of microbial cells;

[0044] (1.4) Break the clean microbial cells to obtain a mixture of broken microbial cells.

[0045] In some embodiments, a seed culture of microbial cells is obtained by inoculating a cryopreserved solution of microbial cells into a culture medium. In some embodiments, a seed culture of microbial cells is obtained by inoculating a cryopreserved solution of microbial cells into a YPD medium. In some embodiments, the YPD medium comprises tryptone, yeast extract, and glucose.

[0046] In some embodiments, the tryptone content in the YPD medium is 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L, 25-30 g / L, 30-35 g / L, 35-40 g / L, 40-45 g / L, or 45-50 g / L. In some embodiments, the tryptone content in the YPD medium is 15-25 g / L. In some embodiments, the tryptone content in the YPD medium is about 20 g / L.

[0047] In some embodiments, the yeast extract content in the YPD medium is 1-5 g / L, 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L, 25-30 g / L, 30-35 g / L, 35-40 g / L, 40-45 g / L, or 45-50 g / L. In some embodiments, the yeast extract content in the YPD medium is 5-15 g / L. In some embodiments, the yeast extract content in the YPD medium is about 10 g / L.

[0048] In some embodiments, the glucose content in the YPD medium is 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L, 25-30 g / L, 30-35 g / L, 35-40 g / L, 40-45 g / L, 45-50 g / L, or 50-100 g / L. In some embodiments, the glucose content in the YPD medium is 10-30 g / L. In some embodiments, the glucose content in the YPD medium is about 20 g / L.

[0049] In some embodiments, the pH of the YPD medium is 4.5-6.5. In some embodiments, the pH of the YPD medium is about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH of the YPD medium is 5.0-6.0.

[0050] In some embodiments, a seed culture of microbial cells is obtained by inoculating a cryopreserved solution of microbial cells into a culture medium, wherein the culture temperature is 20-40°C. In some embodiments, a seed culture of microbial cells is obtained by inoculating a cryopreserved solution of microbial cells into a culture medium, wherein the culture temperature is about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C.

[0051] In some embodiments, a seed culture of microbial cells is obtained by inoculating a cryopreserved solution of microbial cells into a culture medium, wherein the culture time is 5-10 h, 10-15 h, 15-20 h, 20-25 h, 25-30 h, 30-35 h, 35-40 h, 40-45 h, or 45-50 h. In some embodiments, a seed culture of microbial cells is obtained by inoculating a cryopreserved solution of microbial cells into a culture medium, wherein the culture time is approximately 15 h.

[0052] In some embodiments, a seed culture of microbial cells is obtained by inoculating a cryopreserved solution of microbial cells into a culture medium, wherein the volume ratio of the cryopreserved solution to the culture medium is 1:500 to 1:5000. In some embodiments, a seed culture of microbial cells is obtained by inoculating a cryopreserved solution of microbial cells into a culture medium, wherein the volume ratio of the cryopreserved solution to the culture medium is 1:1000.

[0053] In some embodiments, a seed culture of microbial cells is fermented in a fermentation medium to obtain a fermentation broth of microbial cells. In some embodiments, the fermentation medium comprises glucose, tryptone, yeast extract, KH₂PO₄, MgSO₄, and CaCl₂.

[0054] In some embodiments, the glucose content in the fermentation medium is 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L, 25-30 g / L, 30-35 g / L, 35-40 g / L, 40-45 g / L, 45-50 g / L, or 50-100 g / L. In some embodiments, the glucose content in the fermentation medium is 10-50 g / L. In some embodiments, the glucose content in the fermentation medium is 20-40 g / L.

[0055] In some embodiments, the trypsin content in the fermentation medium is 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L, 25-30 g / L, 30-35 g / L, 35-40 g / L, 40-45 g / L, or 45-50 g / L. In some embodiments, the trypsin content in the fermentation medium is 15-25 g / L. In some embodiments, the trypsin content in the fermentation medium is about 20 g / L.

[0056] In some embodiments, the yeast extract content in the fermentation medium is 1-5 g / L, 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L, 25-30 g / L, 30-35 g / L, 35-40 g / L, 40-45 g / L, or 45-50 g / L. In some embodiments, the yeast extract content in the fermentation medium is 5-15 g / L. In some embodiments, the yeast extract content in the fermentation medium is about 10 g / L.

[0057] In some embodiments, the KH₂PO₄ content in the fermentation medium is 1-3 g / L, 3-6 g / L, 6-9 g / L, 9-12 g / L, 12-15 g / L, 15-18 g / L, 18-21 g / L, 21-24 g / L, 24-27 g / L, or 27-30 g / L. In some embodiments, the KH₂PO₄ content in the fermentation medium is about 6 g / L.

[0058] In some embodiments, the MgSO4 content in the fermentation medium is 1-5 g / L, 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L, or 25-30 g / L. In some embodiments, the MgSO4 content in the fermentation medium is about 5 g / L.

[0059] In some embodiments, the CaCl2 content in the fermentation medium is 0.01-0.05 g / L, 0.05-0.10 g / L, 0.10-0.15 g / L, 0.15-0.20 g / L, 0.20-0.25 g / L, 0.25-0.30 g / L, or 0.30-0.50 g / L. In some embodiments, the CaCl2 content in the fermentation medium is about 0.05 g / L.

[0060] In some embodiments, a seed culture of microbial cells is fermented in a fermentation medium to obtain a fermentation broth of microbial cells, wherein the culture temperature is 20-40°C. In some embodiments, a fermentation broth of microbial cells is obtained by fermenting a seed culture of microbial cells in a fermentation medium, wherein the culture temperature is about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C.

[0061] In some embodiments, a fermentation broth of microbial cells is obtained by fermenting a seed culture of microbial cells in a fermentation medium for 5-10 h, 10-15 h, 15-20 h, 20-25 h, 25-30 h, 30-35 h, 35-40 h, 40-45 h, or 45-50 h. In some embodiments, a seed culture of microbial cells is obtained by inoculating a cryopreserved solution of microbial cells into a culture medium for approximately 20 h.

[0062] In some embodiments, clean microbial cells are obtained by repeatedly and alternately centrifuging and washing the fermentation broth of the microbial cells, wherein the supernatant is discarded after centrifugation, the precipitate is retained, and the precipitate is washed. In some embodiments, the centrifugation speed is 5000-10000×g, 10000-15000×g, 15000-20000×g, 20000-25000×g, 25000-30000×g, 30000-35000×g, 35000-40000×g, 40000-45000×g, or 45000-50000×g. In some embodiments, the centrifugation speed is approximately 10000×g.

[0063] In some embodiments, clean microbial cells are obtained by repeatedly and alternately centrifuging and washing the fermentation broth of the microbial cells. In some embodiments, the centrifugation time is 5-10 min, 10-15 min, 15-20 min, 20-25 min, 25-30 min, or 30-35 min. In some embodiments, the centrifugation time is approximately 10 min.

[0064] In some embodiments, clean microbial cells are obtained by repeatedly and alternately centrifuging and washing the fermentation broth of the microbial cells, wherein the washing is performed using a phosphate buffer solution. In some embodiments, the molar concentration of the phosphate buffer solution is 0.01-0.05 mol / L, 0.05-0.10 mol / L, 0.10-0.15 mol / L, 0.15-0.20 mol / L, 0.20-0.25 mol / L, 0.25-0.30 mol / L, 0.30-0.35 mol / L, 0.35-0.40 mol / L, or 0.40-0.50 mol / L. In some embodiments, the molar concentration of the phosphate buffer solution is about 0.05 mol / L.

[0065] In some embodiments, clean microbial cells are obtained by repeatedly and alternately centrifuging and washing the fermentation broth of the microbial cells, wherein the washing is performed using a phosphate buffer solution. In some embodiments, the pH of the phosphate buffer solution is 6.0-7.0. In some embodiments, the pH of the phosphate buffer solution is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. In some embodiments, the pH of the phosphate buffer solution is about 6.5.

[0066] In some embodiments, clean microbial cells are obtained by repeatedly and alternately centrifuging and washing the fermentation broth of microbial cells, wherein centrifugation and washing are performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times each. In some embodiments, clean microbial cells are obtained by repeatedly and alternately centrifuging and washing the fermentation broth of microbial cells, wherein centrifugation is performed 3 times and washing is performed 3 times.

[0067] In some embodiments, a mixture of broken microbial cells is obtained by ultrasonically disrupting cleaned microbial cells. In some embodiments, a mixture of broken microbial cells is obtained by ultrasonically disrupting cleaned microbial cells in an ice bath. In some embodiments, the power of the ultrasonic disruption is 200-250W, 250-300W, 300-350W, 350-400W, 400-450W, 450-500W, 500-550W, 550-600W, 600-650W, 650-700W, 700-750W, 750-800W, 800-850W, 850-900W, 900-950W, 950-1000W, or 1000-1500W. In some embodiments, the power of the ultrasonic disruption is approximately 650W.

[0068] In some embodiments, the ultrasonic ablation is performed in an intermittent mode with a 3-10 second operation followed by a 3-10 second pause. In some embodiments, the ultrasonic ablation is performed in an intermittent mode with an operation time of 3, 4, 5, 6, 7, 8, 9, or 10 seconds followed by a 3, 4, 5, 6, 7, 8, 9, or 10 seconds pause. In some embodiments, the ultrasonic ablation is performed in an intermittent mode with an operation time of 5 seconds followed by a 5-second pause.

[0069] In some embodiments, the ultrasonic fragmentation time is 1-5 min, 5-10 min, 10-15 min, 15-20 min, 20-25 min, 25-30 min, 30-35 min, 35-40 min, or 40-50 min. In some embodiments, the ultrasonic fragmentation time is approximately 20 min.

[0070] In some embodiments, before ultrasonic disruption, the cleaned microbial cells are resuspended in phosphate buffer and NaCl is added. In some embodiments, the concentration of NaCl added is 1-5 mM, 5-10 mM, 10-15 mM, 15-20 mM, 20-25 mM, 25-30 mM, 30-35 mM, 35-40 mM, 40-45 mM, or 45-50 mM. In some embodiments, the concentration of NaCl added is approximately 20 mM.

[0071] In some embodiments, the steps for obtaining a crude extract of sea buckthorn in water include: first mixing sea buckthorn with water, then adding cellulase, and after enzymatic hydrolysis for a period of time, inactivating the cellulase to obtain a crude extract of sea buckthorn. In some embodiments, sea buckthorn is dried, pulverized, and sieved to obtain sea buckthorn powder before being mixed with water.

[0072] In some embodiments, the concentration of sea buckthorn in water after mixing with water is 1-5 g / L, 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L, 25-30 g / L, 30-35 g / L, 35-40 g / L, 40-45 g / L, or 45-50 g / L. In some embodiments, the concentration of sea buckthorn in water after mixing with water is approximately 20 g / L.

[0073] In some implementations, the added cellulase concentration is 0.1-0.2 mg / ml, 0.2-0.3 mg / ml, 0.3-0.4 mg / ml, 0.4-0.5 mg / ml, 0.5-0.6 mg / ml, 0.6-0.7 mg / ml, 0.7-0.8 mg / ml, 0.8-0.9 mg / ml, 0.9-1.0 mg / ml, 1.0-1.1 mg / ml, 1.1-1.2 mg / ml, or 1.2-1.3 mg / ml. The dosages are 1.3-1.4 mg / ml, 1.4-1.5 mg / ml, 1.5-1.6 mg / ml, 1.6-1.7 mg / ml, 1.7-1.8 mg / ml, 1.8-1.9 mg / ml, 1.9-2.0 mg / ml, 2.0-2.5 mg / ml, 2.5-3.0 mg / ml, 3.0-3.5 mg / ml, 3.5-4.0 mg / ml, 4.0-4.5 mg / ml, or 4.5-5.0 mg / ml. In some embodiments, the added cellulase concentration is approximately 1.0 mg / mL.

[0074] In some embodiments, the enzymatic hydrolysis of sea buckthorn with cellulase is carried out at a temperature of 30-60°C. In some embodiments, the enzymatic hydrolysis of sea buckthorn with cellulase is carried out at temperatures of approximately 30°C, approximately 31°C, approximately 32°C, approximately 33°C, approximately 34°C, approximately 35°C, approximately 36°C, approximately 37°C, approximately 38°C, approximately 39°C, approximately 40°C, approximately 41°C, approximately 42°C, approximately 43°C, approximately 44°C, approximately 45°C, approximately 46°C, approximately 47°C, approximately 48°C, approximately 49°C, approximately 50°C, approximately 51°C, approximately 52°C, approximately 53°C, approximately 54°C, approximately 55°C, approximately 56°C, approximately 57°C, approximately 58°C, approximately 59°C, or approximately 60°C. In some embodiments, the enzymatic hydrolysis of sea buckthorn with cellulase is carried out at a temperature of approximately 45°C.

[0075] In some embodiments, the enzymatic hydrolysis time of sea buckthorn with cellulase is 0.1-0.5h, 0.5-1h, 1-1.5h, 1.5-2h, 2-2.5h, 2.5-3h, 3-3.5h, 3.5-4h, 4-4.5h, 4.5-5h, or 5-10h. In some embodiments, the enzymatic hydrolysis time of sea buckthorn with cellulase is approximately 2h.

[0076] In some implementations, cellulase is inactivated at a temperature of 80-120°C. In some implementations, cellulase is inactivated at temperatures of approximately 80°C, approximately 81°C, approximately 82°C, approximately 83°C, approximately 84°C, approximately 85°C, approximately 86°C, approximately 87°C, approximately 88°C, approximately 89°C, approximately 90°C, approximately 91°C, approximately 92°C, approximately 93°C, approximately 94°C, approximately 95°C, approximately 96°C, approximately 97°C, approximately 98°C, approximately 99°C, approximately 100°C, approximately 101°C, approximately 102°C, approximately 103°C, approximately 104°C, approximately 105°C, approximately 106°C, approximately 107°C, approximately 108°C, approximately 109°C, approximately 110°C, approximately 111°C, approximately 112°C, approximately 113°C, approximately 114°C, approximately 115°C, approximately 116°C, approximately 117°C, approximately 118°C, approximately 119°C, or approximately 120°C. In some implementations, cellulase is inactivated at a temperature of approximately 100°C.

[0077] In some embodiments, the inactivation time of cellulase is 1-5 min, 5-10 min, 10-15 min, 15-20 min, 20-25 min, 25-30 min, 30-35 min, 35-40 min, 40-45 min, 45-50 min, 50-55 min, or 55-60 min. In some embodiments, the inactivation time of cellulase is approximately 20 min.

[0078] In some embodiments, 0.1%-0.5%, 0.5%-1%, 1%-1.5%, 1.5%-2%, 2%-2.5%, 2.5%-3%, 3%-3.5%, 3.5%-4%, 4%-4.5%, 4.5%-5%, 5%-5.5%, 5.5%-6%, 6%-6.5%, 6.5%-7%, 7%-7.5%, 7.5%-8%, 8%-8.5%, 8.5%-9%, 9%-9.5% or 9.5%-10% by volume of the ruptured microbial cell mixture obtained in step (1) is added to the crude extract of sea buckthorn in water in step (2) for biotransformation to obtain sea buckthorn biotransformation extract. In some embodiments, about 2% by volume of the ruptured microbial cell mixture obtained in step (1) is added to the crude extract of sea buckthorn in water in step (2) to carry out biotransformation and obtain sea buckthorn biotransformation extract.

[0079] In some embodiments, the temperature for carrying out the biotransformation reaction is 20-60°C. In some embodiments, the temperature for carrying out the biotransformation reaction is about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, or about 60°C. In some implementations, the temperature for carrying out the biotransformation reaction is approximately 35°C.

[0080] In some embodiments, the reaction time of the biotransformation reaction is 6-8 h, 8-10 h, 10-12 h, 12-14 h, 14-16 h, 16-18 h, 18-20 h, 20-22 h, 22-24 h, 24-26 h, 26-28 h, 28-30 h, 30-32 h, 32-34 h, 34-36 h, 36-38 h, 38-40 h, 40-42 h, 42-44 h, 44-46 h, 46-48 h, or 48-50 h. In some embodiments, the reaction time of the biotransformation reaction is approximately 18 h. In some embodiments, the reaction time of the biotransformation reaction is approximately 24 h.

[0081] In some implementation schemes, the enzymes in the system are inactivated at a temperature of 80-120°C after biotransformation. In some implementations, after biotransformation, the enzymes in the system are inactivated at temperatures of approximately 80°C, approximately 81°C, approximately 82°C, approximately 83°C, approximately 84°C, approximately 85°C, approximately 86°C, approximately 87°C, approximately 88°C, approximately 89°C, approximately 90°C, approximately 91°C, approximately 92°C, approximately 93°C, approximately 94°C, approximately 95°C, approximately 96°C, approximately 97°C, approximately 98°C, approximately 99°C, approximately 100°C, approximately 101°C, approximately 102°C, approximately 103°C, approximately 104°C, approximately 105°C, approximately 106°C, approximately 107°C, approximately 108°C, approximately 109°C, approximately 110°C, approximately 111°C, approximately 112°C, approximately 113°C, approximately 114°C, approximately 115°C, approximately 116°C, approximately 117°C, approximately 118°C, approximately 119°C, or approximately 120°C. In some implementations, the enzymes in the system are inactivated at a temperature of approximately 100°C after biotransformation.

[0082] In some implementations, after biotransformation, the enzymes in the system are inactivated for 1-5 min, 5-10 min, 10-15 min, 15-20 min, 20-25 min, 25-30 min, 30-35 min, 35-40 min, 40-45 min, 45-50 min, 50-55 min, or 55-60 min. In some implementations, after biotransformation, the enzymes in the system are inactivated for approximately 20 min.

[0083] In some embodiments, the sea buckthorn biotransformation extract obtained in step (3) is centrifuged and / or filtered, wherein the centrifugation is performed at 3000-4000×g, 4000-5000×g, 5000-6000×g, 6000-7000×g, 7000-8000×g, 8000-9000×g, 9000-10000×g, 10000-11000×g, The extraction is carried out at 11000-12000×g, 12000-13000×g, 13000-14000×g, 14000-15000×g, 15000-16000×g, 16000-17000×g, 17000-18000×g, 18000-19000×g, 19000-20000×g, or 20000-50000×g. In some embodiments, the sea buckthorn biotransformation extract obtained in step (3) is centrifuged and / or filtered, said centrifugation being carried out at approximately 10000×g.

[0084] In some embodiments, the sea buckthorn biotransformation extract obtained in step (3) is centrifuged and / or filtered for 1-5 min, 5-10 min, 10-15 min, 15-20 min, 20-25 min, 25-30 min, 30-35 min, 35-40 min, 40-45 min, 45-50 min, 50-55 min, or 55-60 min. In some embodiments, the sea buckthorn biotransformation extract obtained in step (3) is centrifuged and / or filtered for approximately 10 min.

[0085] In some embodiments, the sea buckthorn biotransformation extract obtained in step (3) is centrifuged and / or filtered, the filtration being performed through a 0.22 μm membrane.

[0086] Seabuckthorn biotransformation extract

[0087] The second aspect of the present invention relates to a sea buckthorn biotransformation extract, which is prepared by the method according to the first aspect of the present invention.

[0088] In some embodiments, the sea buckthorn biotransformation extract has a higher total flavonoid content than sea buckthorn extract subjected to biotransformation without the addition of a mixture of broken microbial cells under the same conditions. In some embodiments, the sea buckthorn biotransformation extract has a higher total flavonoid content than sea buckthorn fermentation broth fermented with microbial cells under the same conditions.

[0089] In some embodiments, the sea buckthorn biotransformation extract exhibits stronger anti-inflammatory properties than sea buckthorn extract that undergoes biotransformation under the same conditions without the addition of a mixture of broken microbial cells.

[0090] In some embodiments, the sea buckthorn biotransformation extract exhibits stronger antioxidant capacity than sea buckthorn extract subjected to biotransformation without the addition of a mixture of broken microbial cells under the same conditions. In some embodiments, the sea buckthorn biotransformation extract exhibits stronger antioxidant capacity than sea buckthorn fermentation broth fermented with microbial cells under the same conditions.

[0091] In some embodiments, the sea buckthorn biotransformation extract has a fresher odor than sea buckthorn fermentation liquid fermented by microbial cells under the same conditions.

[0092] In some embodiments, the sea buckthorn biotransformation extract is lighter in color than sea buckthorn fermentation broth fermented by microbial cells under the same conditions.

[0093] Pharmaceutical formulations and cosmetic compositions

[0094] A third aspect of the invention relates to a cosmetic composition comprising a sea buckthorn biotransformation extract according to a second aspect of the invention.

[0095] The fourth aspect of the present invention relates to a pharmaceutical preparation comprising a sea buckthorn biotransformation extract according to the second aspect of the present invention.

[0096] In some embodiments, the pharmaceutical formulation or cosmetic composition is used for skin care. In some embodiments, the pharmaceutical formulation or cosmetic composition is used for anti-inflammatory and / or antioxidant purposes. In some embodiments, the pharmaceutical formulation or cosmetic composition comprises sea buckthorn biotransformation extract as an active ingredient and one or more pharmaceutically or cosmetically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical formulation or cosmetic composition is a cream, lotion, paste, ointment, mask, gel, lotion, or serum. In some embodiments, the pharmaceutical formulation or cosmetic composition of the present invention may also contain one or more other ingredients, such as plant extracts, nutritional additives, surfactants, fragrances and flavors, pigments, preservatives, antioxidants, moisturizers, UV absorbers, astringents, penetration enhancers, pH adjusters, etc. Those skilled in the art can make selections based on their common knowledge and specific needs.

[0097] Uses of sea buckthorn biotransformation extract in the preparation of pharmaceutical or cosmetic compositions. The fifth aspect of the invention relates to the use of sea buckthorn biotransformation extract according to the fourth aspect of the invention in the preparation of pharmaceutical or cosmetic compositions having anti-inflammatory and / or antioxidant capabilities. Detailed Implementation

[0098] The present invention can be implemented through the following embodiments, but the present invention is not limited thereto.

[0099] The instruments and equipment used in the embodiments of the present invention are all conventional instruments and equipment in the field and can be replaced by instruments and equipment that conform to the corresponding standards.

[0100] Unless otherwise specified, all Chinese herbal medicine raw materials used in the embodiments of the present invention are commercially available, and all reagents used in the embodiments of the present invention are commercially available analytical chemical reagents unless otherwise specified.

[0101] The raw materials and instruments mentioned in this invention are all commonly used in the art and are merely examples, not intended to limit the scope of protection of this invention. Those skilled in the art can select equivalent raw materials and related instruments based on the disclosure of this invention.

[0102] Experimental materials, reagents, equipment and statistical analysis methods

[0103] The strain source in the examples: *Saccharomyces cerevisiae* was purchased from the China General Microbiological Culture Collection Center, accession number CGMCC 2.560.

[0104] Sea buckthorn leaf raw material: Sea buckthorn leaves, purchased from Ningxia Xiangcao Biotechnology Co., Ltd.

[0105] Experimental equipment: (1) Constant temperature oscillator, Shanghai Zhicheng ZWY-2102C; (2) Clean workbench, Sujing Antai HVS-1300-U; (3) High-speed pulverizer, Zhejiang Hongjingtian Industry and Trade Co., Ltd. DE-300g; (4) Ultrasonic cell pulverizer, Xinzhi JY92-IIDN.

[0106] Experimental reagents: Yeast extract and tryptone were purchased from OXOID (UK), 500g / bottle; cellulase was purchased from Adamas-life, enzyme activity 50U / mg, 25g / bottle; the remaining reagents were purchased from Shanghai Titan Technology Co., Ltd. under the General-reagent brand, packaged in 500g / bottles.

[0107] Statistical methods: The experimental results are the mean ± standard deviation of the data from three parallel experiments. A paired t-test was used to test whether the differences between groups were statistically significant. SigmaPlot 14.0 was used as the statistical software.

[0108] Example 1 - Preparation of Seabuckthorn Biotransformation Extract

[0109] Sea buckthorn biotransformation extract was prepared using a mixture of broken capsule-coated yeast cells via the following steps (Example 1):

[0110] A. On a clean bench, inoculate 30 μL of the cryopreserved yeast culture into a 250 ml Erlenmeyer flask containing 30 ml of YPD medium. Incubate at 28°C and 180 rpm for 15 h in a constant temperature shaker until the cells are in the logarithmic growth phase. Obtain the yeast seed culture. The YPD medium consists of: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, and pH 5.5.

[0111] B. Inoculate the seed culture obtained in step A into a 500ml Erlenmeyer flask containing 120ml of fermentation medium, and culture it in a constant temperature shaker at 28℃ and 200rpm for 20h to obtain yeast fermentation broth. The fermentation medium consists of: glucose 20g / L, tryptone 20g / L, yeast extract 10g / L, KH2PO4 6g / L, MgSO4 5g / L, and CaCl2 0.05g / L.

[0112] C. Centrifuge the yeast fermentation broth obtained in step B at 10000×g for 10 min, discard the supernatant, and the precipitate is the yeast cells. Wash with phosphate buffer, then centrifuge again, repeating the washing process three times to obtain clean yeast cells. The phosphate buffer has a molar concentration of 0.05 mol / L and a pH of 6.5.

[0113] D. Resuspend the wet yeast cells obtained in step C in 1.5 times the mass of the phosphate buffer described in step C, and add 20 mM NaCl. Disrupt the cells using an ultrasonic cell disruptor in an ice-water bath to obtain a mixture of disrupted cells, which is the crude enzyme solution from the yeast cells. Ultrasonic disruption conditions: ultrasonic power 650 W, 5-second pause every 5 seconds, disruption time 20 minutes.

[0114] E. Dry and pulverize the sea buckthorn leaves, pass them through a 60-mesh sieve, and mix them with water. The sea buckthorn leaf powder content is 20 g / L. Then add cellulase at 1 mg / ml, enzymatically hydrolyze at 45℃ for 2 hours, then raise the temperature to 100℃ and keep it at that temperature for 20 minutes to inactivate the enzyme, thus obtaining the sea buckthorn leaf extract.

[0115] F. Add the crude yeast cell enzyme solution obtained in step D to the sea buckthorn leaf extract obtained in step E to carry out the biotransformation reaction. The crude enzyme solution was added at a ratio of 2% by volume, and the reaction was carried out in a constant temperature shaker at 35℃ and 120 rpm for 18 h. After the transformation was completed, the temperature was raised to 100℃ and held for 10 min to inactivate the enzyme. After cooling, the extract was centrifuged at 10000×g for 10 min and filtered through a 0.22 μm membrane to obtain the sea buckthorn leaf biotransformation extract filtrate.

[0116] Example 2 - Preparation of Seabuckthorn Biotransformation Extract

[0117] Seabuckthorn biotransformation extract was prepared using a mixture of broken cytosolic yeast cells in a manner similar to that in Example 1 (Example 2), the only difference being the composition of the fermentation medium in step B. The fermentation medium in step B for preparing the seabuckthorn biotransformation extract in Example 2 consisted of: 40 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract, 6 g / L KH2PO4, 5 g / L MgSO4, and 0.05 g / L CaCl2.

[0118] Example 3 - Preparation of Seabuckthorn Biotransformation Extract

[0119] Seabuckthorn biotransformation extract was prepared using a mixture of broken capsule-coated yeast cells in a manner similar to that in Example 1 (Example 3), the only difference being the reaction time of biotransformation in step F, where the reaction time of biotransformation in step F for preparing the seabuckthorn biotransformation extract of Example 3 was 24 h.

[0120] Comparative Example 1 - Preparation of sea buckthorn extract without fermentation and biotransformation

[0121] Sea buckthorn extract that has not undergone fermentation or biotransformation was prepared using the following method:

[0122] The sea buckthorn leaves were dried, pulverized, and passed through a 60-mesh sieve. They were then mixed with water to obtain a sea buckthorn leaf powder content of 20 g / L. Cellulase was added at a concentration of 1 mg / ml, and the mixture was incubated at 45°C for 2 hours. The temperature was then raised to 100°C and held for 20 minutes to inactivate the enzyme (same as step E in Example 1). The resulting sea buckthorn leaf extract was then centrifuged at 10000 × g for 10 minutes and filtered through a 0.22 μm membrane to obtain the sea buckthorn leaf extract filtrate.

[0123] Comparative Example 2 - Preparation of Seabuckthorn Fermentation Broth

[0124] Sea buckthorn fermentation broth was prepared using the following method:

[0125] A. On a clean bench, inoculate 30 μL of the cryopreserved yeast culture into a 250 ml Erlenmeyer flask containing 30 ml of YPD medium. Incubate at 28°C and 180 rpm for 15 h in a constant temperature shaker until the cells are in the logarithmic growth phase. Obtain the yeast seed culture. The YPD medium consists of: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, and pH 5.5.

[0126] B. Dry and pulverize the sea buckthorn leaves, pass them through a 60-mesh sieve, and mix them with water. The sea buckthorn leaf powder content is 20g / L. Then add cellulase at 1mg / ml, enzymatically hydrolyze at 45℃ for 2h, then raise the temperature to 100℃ and keep it at that temperature for 20min to inactivate the enzyme, and obtain the sea buckthorn leaf extract.

[0127] C. Inoculate the yeast seed culture obtained in step A into the sea buckthorn leaf extract obtained in step B for fermentation. The inoculation amount is 2% by volume, and fermentation is carried out for 18 hours in a constant temperature shaker at 35℃ and 120 rpm. After fermentation, the temperature is raised to 100℃ and incubated for 10 minutes to inactivate the yeast. After cooling, the fermentation broth is centrifuged at 10000×g for 10 minutes and filtered through a 0.22μm membrane to obtain the fermentation broth filtrate of sea buckthorn leaf extract.

[0128] Effect Example

[0129] 1. Total flavonoid content

[0130] 1) Measurement method

[0131] Standard curve creation:

[0132] Accurately weigh 10.8 mg of dried, constant-weight rutin standard, dissolve it in 60% ethanol, and dilute to a 100 mL volumetric flask to obtain a standard solution of 0.108 mg / mL. Accurately pipette 0 μL, 500 μL, 1000 μL, 1500 μL, 2000 μL, and 2500 μL of the standard solution into six 10 mL test tubes, and add 30% ethanol solution to each tube to a final volume of 2.5 mL. Then, accurately add 150 μL of 5% NaNO₂ solution, shake well, and let stand for 6 min. Next, add 150 μL of 10% Al(NO₃)₃ solution, shake well, and let stand for 6 min. Then, add 2 mL of 1 mol / L NaOH solution and 200 μL of water. Shake well and let stand for 10 min. Using the first tube as a blank, measure the absorbance at a wavelength of 510 nm. Plot the absorbance against the standard concentration to create a standard curve.

[0133] Sample content determination:

[0134] Accurately pipette 1 mL of sample, add 1.5 mL of 30% ethanol solution, then accurately add 150 μL of 5% NaNO2 solution, shake well and let stand for 6 min. Next, add 150 μL of 10% Al(NO3)3 solution, shake well and let stand for 6 min. Then add 2 mL of 1 mol / L NaOH solution and 200 μL of water. Shake well and let stand for 10 min, then measure the absorbance at 510 nm. Calculate the total flavonoid content based on the standard curve.

[0135] 2) Experimental Results

[0136] The results of determining the total flavonoid content in the sea buckthorn extracts of Examples 1-3 and Comparative Examples 1-2 of this application according to the above determination method are shown in Table 1 below:

[0137] Table 1

[0138]

[0139] Note: Different letter superscripts indicate significant differences in flavonoid content (P < 0.05), that is, there are significant differences (P < 0.05) between the following groups: Comparative Example 1 and Example 1, Comparative Example 1 and Example 2, Comparative Example 1 and Example 3, Comparative Example 2 and Example 1, Comparative Example 2 and Example 2, Comparative Example 2 and Example 3, Example 1 and Example 2, and Example 2 and Example 3.

[0140] The data in the table show that, compared with the sea buckthorn extract of Comparative Example 1 (which was neither fermented with live yeast nor biotransformed with broken yeast), the total flavonoid content in the sea buckthorn fermentation extract of Comparative Example 2 (fermented with live yeast) decreased slightly. This may be because some flavonoids were consumed during the growth and metabolism of the yeast. In contrast, the total flavonoid content in the sea buckthorn biotransformation extracts of Examples 1-3 (which underwent biotransformation with broken yeast) was significantly increased.

[0141] 2. Evaluation of antioxidant capacity

[0142] 1) Measurement method - Intracellular reactive oxygen species (ROS) scavenging capacity

[0143] Add 100 μL of a solution with a density of 1.5 × 10⁻⁶ to a 96-well plate. 5HaCaT cells were incubated overnight at 37°C with 5% CO2. 11 μL of sample was added to each well in the sample group, and 11 μL of sample solvent was added to each well in the control group. Cells were incubated for 16 h. Cells were washed once with PBS, and 100 μL of DMEM containing 20 μM DCFH-DA solution and 5 μg / mL Hoechst was added to each well. Cells were incubated for 20 min. Cells were washed once with PBS, and 100 μL of 100 μM H2O2 prepared with PBS was added to each well. Cells were incubated for 20 min. Intracellular ROS oxidize non-fluorescent DCFH to generate fluorescent DCF, and the fluorescence intensity is directly proportional to the ROS level. Hoechst was used to stain cell nuclei to detect viable cells and determine the appropriate sample concentration.

[0144]

[0145] 2) Experimental Results

[0146] Based on the results of Hoechst staining of cell nuclei to detect viable cells, the cell viability was above 95% at a 1% sample concentration. Therefore, a 1% sample concentration was selected to compare the ROS scavenging rates of each group of experiments. The results are shown in Table 2 below:

[0147] Table 2

[0148]

[0149] Note: Different letter superscripts indicate significant differences in ROS clearance rates (P < 0.05), i.e., significant differences (P < 0.05) between the following groups: Comparative Example 1 and Comparative Example 2, Comparative Example 1 and Example 1, Comparative Example 1 and Example 2, Comparative Example 1 and Example 3, Comparative Example 2 and Example 1, Comparative Example 2 and Example 2, Comparative Example 2 and Example 3, Example 1 and Example 2, and Example 2 and Example 3.

[0150] The data in the table show that, compared with the sea buckthorn extract of Comparative Example 1 (which was neither fermented with live yeast nor biotransformed with broken yeast), the sea buckthorn fermented extract of Comparative Example 2 (fermented with live yeast) exhibited a significant decrease in its ability to scavenge intracellular ROS. In contrast, the sea buckthorn biotransformed extracts of Examples 1-3 (biotransformed with broken yeast) showed a significant increase in their ability to scavenge intracellular ROS, reflecting the significantly enhanced antioxidant capacity of the sea buckthorn biotransformed extract obtained through broken yeast biotransformation in this invention.

[0151] 3. Evaluation of anti-inflammatory ability

[0152] 1) Measurement Method - Detection of Inflammatory Factors Based on RAW264.7 Cells

[0153] Overexpression of inflammatory factors can induce cellular senescence and inhibit skin barrier repair and wound healing. This assay utilizes lipopolysaccharide (LPS) to induce RAW264.7 cells to establish an inflammation model and generate excessive inflammatory factors. The anti-inflammatory effect is evaluated by detecting the inhibitory effect of the sample on these inflammatory factors.

[0154] Procedure: Add 100 μL of a solution with a density of 2.5 × 10⁻⁶ to a 96-well plate. 5 RAW264.7 cells per mL were incubated at 37°C with 5% CO2 for 24 h. 10 μL of sample was added to each well of the sample group, while 10 μL of sample solvent was added to each well of the blank and control groups. The cells were incubated overnight. 10 μL of LPS (final concentration 1 μg / mL) prepared with DMEM was added to each well of the control and sample groups, while 10 μL of DMEM was added to each well of the blank group. The cells were incubated for 6 h. Cell culture medium was collected, and relevant inflammatory factors were detected using an ELISA kit. Cell viability was also assessed to determine the appropriate sample concentration. Assay method: Cells were washed once with PBS, and 100 μL of CCK-8 solution (enhanced CCK-8 and DMEM mixed at a 1:10 ratio) was added to each well. After incubation for 1–2 h, absorbance was measured at 450 nm.

[0155] Cell viability % = As / Ac × 100%

[0156] As: Absorbance of the sample group; Ac: Absorbance of the control group.

[0157] 2) Experimental Results

[0158] Based on the cell viability test results, the cell viability was above 95% at a 1% sample concentration. Therefore, a 1% sample concentration was selected to compare the anti-inflammatory capabilities of each group of experimental samples.

[0159] After sample solution treatment, compared with the untreated LPS model group, the secretion of the inflammatory factor IL-6 was significantly reduced, indicating that the sea buckthorn leaf extract has a significant inhibitory effect on IL-6. The results of IL-6 detection based on RAW264.7 cells are shown in Table 3 below:

[0160] Table 3

[0161] IL-6 concentration pg / ml IL-6 inhibition rate % LPS model group <![CDATA[2374.97±12.63 a ]]> 0 Comparative Example 1 <![CDATA[719.71±49.93 b ]]> 69.70 Comparative Example 2 <![CDATA[440.49±28.18 c ]]> 81.45 Example 1 <![CDATA[425.45±25.28 c ]]> 82.09 Example 2 <![CDATA[433.80±2.32 c ]]> 81.73 Example 3 <![CDATA[440.91±17.73 c ]]> 81.44

[0162] Note: Different letter superscripts indicate significant differences in ROS clearance rates (P < 0.05), i.e., significant differences (P < 0.05) between the following groups: LPS model group and Comparative Example 1, LPS model group and Comparative Example 2, LPS model group and Example 1, LPS model group and Example 2, LPS model group and Example 3, Comparative Example 1 and Comparative Example 2, Comparative Example 1 and Example 1, Comparative Example 1 and Example 2, Comparative Example 1 and Example 3.

[0163] As shown in the table above, compared with the sea buckthorn extract of Comparative Example 1, which was neither fermented with live yeast nor biotransformed with broken yeast, the sea buckthorn biotransformed extracts of Examples 1-3, which were biotransformed with broken yeast, showed a significant improvement in their inhibitory ability on the inflammatory factor IL-6. This reflects that the sea buckthorn biotransformed extracts obtained by biotransformation with broken yeast in this invention have significantly improved anti-inflammatory capabilities.

[0164] 4. Evaluation of the color and odor of sea buckthorn extract

[0165] 1) Visual observation and evaluation of the color of sea buckthorn extract

[0166] like Figure 1 As shown, compared with the sea buckthorn extract of Comparative Example 1, which was neither fermented with live yeast nor biotransformed with broken yeast, the sea buckthorn biotransformation extracts of Examples 1-3, which underwent biotransformation with broken yeast, showed no significant color change. However, the sea buckthorn fermentation extract of Comparative Example 2, which underwent fermentation with live yeast, was significantly darker in color.

[0167] 2) Evaluation of subjective pleasantness of the aroma of sea buckthorn extract

[0168] The sea buckthorn extract of Comparative Example 1, which was neither fermented with live yeast nor biotransformed with broken yeast, had a fresh aroma of sea buckthorn leaves. The sea buckthorn biotransformed extracts of Examples 1-3, which were biotransformed with broken yeast, retained this fresh aroma. However, the sea buckthorn fermented extract of Comparative Example 2, which was fermented with live yeast, had a significantly different aroma.

[0169] Subjective pleasantness evaluation method: Eight non-laboratory personnel evaluated the odor pleasantness of the extract in the sample vials using a 9-point scale (as shown in Table 4). They opened the vials, smelled the extract from 1.5-2.5 cm away from their noses, and provided their evaluation results. The final pleasantness score was the average of all results.

[0170] Table 4

[0171]

[0172] As shown in Table 5, the odor pleasantness rating of the sea buckthorn biotransformation extracts from Examples 1-3, which underwent biotransformation with broken yeast, ranged between slightly pleasant and moderately pleasant. In contrast, the odor pleasantness rating of the sea buckthorn fermentation extract from Comparative Example 2, which underwent fermentation with live yeast, was -0.88, close to slightly unpleasant. The sea buckthorn extract obtained using the biotransformation method of this invention has a pleasant odor, overcoming the drawback of unpleasant odors produced during fermentation.

[0173] Table 5

[0174]

[0175] This invention provides a method for biotransforming and extracting sea buckthorn (e.g., sea buckthorn leaves) using various enzymes and metabolites released after microbial (e.g., yeast) rupture to obtain a sea buckthorn biotransformation extract. The biotransformed sea buckthorn leaf extract exhibits significantly increased total flavonoid content, as well as significantly enhanced anti-inflammatory and antioxidant activities. In comparison, conventional fermentation methods only improve the anti-inflammatory capacity of sea buckthorn leaf extract, while decreasing the total flavonoid content and antioxidant capacity. The biotransformation method of this invention is simple and easy to control, and is more conducive to improving the efficacy (e.g., anti-inflammatory and antioxidant capacity) of sea buckthorn extract in cosmetic applications compared to conventional fermentation methods. Furthermore, the extract has a lighter color and a refreshing, pleasant aroma. This biotransformation extraction method can be applied to the production of cosmetic raw materials, is low-cost, and can bring significant economic value to the cosmetic industry.

Claims

1. A method for preparing sea buckthorn biotransformation extract, wherein the method comprises the following steps: (1) Obtain a mixture of broken microbial cells; (2) Obtain a crude extract of sea buckthorn in water; (3) Add the broken microbial cell mixture obtained in step (1) to the crude extract of sea buckthorn in water in step (2) for biotransformation to obtain sea buckthorn biotransformation extract; optionally, (4) The sea buckthorn biotransformation extract obtained in step (3) is centrifuged and / or filtered, wherein: The following steps are used to obtain a mixture of broken microbial cells: (1.1) Obtain seed culture of microbial cells; (1.2) Fermentation culture of microbial cells is carried out by fermentation culture of seed culture of microbial cells to obtain fermentation broth of microbial cells; (1.3) Clean microbial cells are obtained by repeatedly and alternately centrifuging and washing the fermentation broth of microbial cells; (1.4) The clean microbial cells are broken to obtain a mixture of broken microbial cells. The microorganisms mentioned therein are yeasts, specifically *Saccharomyces cerevisiae*. The sea buckthorn mentioned above refers to sea buckthorn leaves; In step (2), sea buckthorn is first mixed with water, and then cellulase is added. After enzymatic hydrolysis for a period of time, the cellulase is then inactivated to obtain crude sea buckthorn extract. The concentration of cellulase added is 0.2-5 mg / ml; enzymatic hydrolysis is carried out at 30-60℃ for 1-3 hours; and cellulase is inactivated at 80-120℃ for 10-40 minutes. In step (3), the amount of broken microbial cells added is 0.5%-10% by volume; The biotransformation reaction was carried out at a temperature of 20-50℃ for 10-40 hours. After the biotransformation was completed, the enzymes in the system were inactivated at a temperature of 80-120℃ for 5-20 minutes. In step (4), the centrifugation is carried out at 3000-20000×g for 5-20 min, and the filtration is performed through a 0.22μm membrane.

2. The method of claim 1, wherein the ruptured microbial cell mixture is obtained by ultrasonic disruption of the microbial cells, the ultrasonic disruption optionally being performed under ice bath conditions.

3. The method according to claim 2, wherein the power of the ultrasonic crushing is 200-900W, the ultrasonic crushing is performed in an intermittent mode of working for 3-10 seconds and pausing for 3-10 seconds, and the ultrasonic crushing time is 10-40 minutes.

4. The method according to claim 3, wherein the ultrasonic crushing power is 600-700W and the ultrasonic crushing time is 15-25min.

5. The method according to any one of claims 1-4, wherein the concentration of the added cellulase is 0.5-2 mg / ml; enzymatic hydrolysis is carried out at 40-50°C for 1.5-2.5 h; and the cellulase is inactivated at 90-110°C for 15-30 min.

6. The method according to any one of claims 1-4, wherein in step (3), the amount of broken microbial cells added is 1%-5% by volume.

7. The method according to any one of claims 1-4, wherein the temperature for the biotransformation reaction is 30-40°C and the reaction time is 15-30 h; after the biotransformation is completed, the enzymes in the system are inactivated at a temperature of 90-110°C.

8. A sea buckthorn biotransformation extract, wherein the sea buckthorn biotransformation extract is prepared by the method according to any one of claims 1-7.

9. A pharmaceutical preparation or cosmetic composition comprising the sea buckthorn biotransformation extract of claim 8.

10. Use of the sea buckthorn biotransformation extract according to claim 8 in the preparation of pharmaceutical or cosmetic compositions having anti-inflammatory and / or antioxidant capabilities.

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