Preparation method of ottelia acuminata extract as well as product and application of ottelia acuminata extract

Abstract

The invention relates to a preparation method of ottelia acuminata extract as well as a product and application of the ottelia acuminata extract. The preparation method of the ottelia acuminata extract comprises the following steps: mixing an ottelia acuminata raw material with an ethanol aqueous solution, performing heating reflux extraction, and performing solid-liquid separation to obtain an ottelia acuminata extracting solution; performing membrane filtration on the ottelia acuminata extracting solution, and concentrating filtrate to obtain an ottelia acuminata concentrated solution; purifying and eluting the ottelia acuminata concentrated solution by adopting macroporous resin to obtain an ottelia acuminata eluent; and treating the ottelia acuminata eluent with a decolorizing agent to obtain the ottelia acuminata extract. The ottelia acuminata extraction product is prepared by sequentially adopting alcohol extraction, membrane filtration, resin purification and decolorizing agent treatment, the preparation process can extract active ingredients (such as polysaccharide, protein and polyphenol) in ottelia acuminata to the greatest extent, and a specific active ingredient proportion mode is formed; the final product has excellent anti-inflammatory, soothing, barrier repairing, moisturizing and oil control effects.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant extraction, and relates to a preparation method of Ottelia acuminata (Gagnep.) Dandy extract, a product thereof and application. BACKGROUND

[0002] Ottelia acuminata (Gagnep.) Dandy belongs to Ottelia of Hydrocharitaceae and is a kind of aquatic plant. Ottelia acuminata (Gagnep.) Dandy mainly grows in lakes, ponds, ditches and deep water fields below an altitude of 2700 m and is distributed in Lijiang Yongsheng, Ninglang and Dali Erhai in northwest Yunnan, Jianchuan and Eryuan, etc. Ottelia acuminata (Gagnep.) Dandy is mainly used as fresh vegetable and can be dried by air-drying or other methods for long-term preservation. Ottelia acuminata (Gagnep.) Dandy has the effects of clearing heat and reducing phlegm, detoxifying and diuresis and can be used to treat symptoms such as lung heat cough and asthma, dysuria and scalding.

[0003] At present, the research on Ottelia acuminata (Gagnep.) Dandy mainly focuses on planting and breeding, analysis of nutritional components, nutritional value and composition of Ottelia acuminata (Gagnep.) Dandy combined phenol. Liu Qing et al. developed a preparation method of Ottelia acuminata (Gagnep.) Dandy combined phenol. The method comprises the following steps: weighing samples of different parts of Ottelia acuminata (Gagnep.) Dandy, extracting the samples with methanol, hydrolyzing the residue with NaOH solution containing EDTA and ascorbic acid in the dark, adjusting the pH to 1 after the hydrolysis is completed, filtering, extracting with ethyl acetate, collecting the ethyl acetate phase, removing ethyl acetate by rotary evaporation, adsorbing the crude extract liquid with X-5 macroporous resin by water bath oscillation, filtering, collecting the macroporous resin, adding 70% ethanol, oscillating and desorbing under the same conditions, filtering, removing ethanol by rotary evaporation, and vacuum freeze-drying to obtain the purified combined phenol extract. The Ottelia acuminata (Gagnep.) Dandy combined phenol mainly contains five kinds of monomeric phenols, i.e. caffeic acid, quercetin-3-O-glucoside, luteolin, chlorogenic acid and ferulic acid.

[0004] At present, the efficacy development and application of Ottelia acuminata (Gagnep.) Dandy are rare, and there is no report on the efficacy application of Ottelia acuminata (Gagnep.) Dandy extract in anti-inflammatory soothing, barrier repair or oil control and moisturizing. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a preparation method of Ottelia acuminata (Gagnep.) Dandy extract, a product thereof and application, in particular to a preparation method of Ottelia acuminata (Gagnep.) Dandy extract with the effects of anti-inflammatory soothing, barrier repair, moisturizing and oil control and a product thereof and application.

[0006] To achieve the purpose of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a preparation method of Ottelia acuminata (Gagnep.) Dandy extract, which comprises the following steps:

[0008] (1) mixing sea cucumber flower raw materials with an ethanol aqueous solution and heating to reflux extraction, solid-liquid separation, to obtain sea cucumber flower extract;

[0009] (2) membrane filtration of the sea cucumber flower extract, concentration of the filtrate, to obtain sea cucumber flower concentrate;

[0010] (3) purification and elution of the sea cucumber flower concentrate using macroporous resin, first elution with pure water, then elution with an ethanol aqueous solution, collection of the combined water washing liquid and alcohol washing liquid, to obtain sea cucumber flower eluate;

[0011] (4) treatment of the sea cucumber flower eluate with a decolorizing agent, to obtain the sea cucumber flower extract.

[0012] The present application creatively uses alcohol extraction, membrane filtration, resin purification, and decolorizing agent treatment in sequence to prepare a sea cucumber flower extract product. This preparation process can maximize the extraction of active ingredients (such as polysaccharides, proteins, and polyphenols) in sea cucumber flowers and form a specific active ingredient ratio, so that the final product can inhibit the expression of inflammatory factors TNF-α, IL-6, and NO, promote the expression of FLG, LOR, and AQP-3 proteins, and inhibit the excessive secretion of sebum from sebaceous gland cells induced by 5α-dihydrotestosterone, thus having excellent anti-inflammatory, soothing, barrier repair, moisturizing, and oil control effects.

[0013] Preferably, the sea cucumber flower raw material is a dried and pulverized sea cucumber flower.

[0014] Preferably, the volume fraction of ethanol in the ethanol aqueous solution in step (1) is 60-80%, such as 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, etc.

[0015] Using 60-80% ethanol aqueous solution as the extraction solvent can make the final product have more excellent anti-inflammatory, soothing, barrier repair, moisturizing, and oil control effects compared to other concentrations of ethanol aqueous solution.

[0016] Preferably, the number of times of heating reflux extraction is 1-3 times, such as 1 time, 2 times, or 3 times; each time for 0.5-2 h, such as 0.5 h, 0.8 h, 1 h, 1.2 h, 1.5 h, 2 h, etc.

[0017] Preferably, the solid-liquid ratio of the sea cucumber flower raw material to the ethanol aqueous solution is 1:(5-20) g / mL, such as 1:5 g / mL, 1:7 g / mL, 1:8 g / mL, 1:10 g / mL, 1:12 g / mL, 1:15 g / mL, 1:20 g / mL, etc.

[0018] Preferably, the membrane filtration uses a membrane with a molecular weight cut-off of 10-30 kDa, such as 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, etc.

[0019] Before macroporous resin elution purification, the extract is first subjected to membrane filtration with a molecular weight cut-off of 10-30 kDa, which can make the final product have more excellent anti-inflammatory, soothing, barrier repair, moisturizing, and oil control effects.

[0020] Preferably, the filtrate concentration includes using a membrane with a molecular weight cut-off of 150-500 Da (such as 150 Da, 200 Da, 250 Da, 300 Da, 350 Da, 400 Da, 450 Da, 500 Da, etc.) for concentration, and then removing ethanol by vacuum concentration.

[0021] The above membrane concentration process can further remove small molecular weight impurities.

[0022] Preferably, the macroporous resin is selected from any one or a combination of at least two of AB-8 resin, D101 resin, SP20SS resin, and HP2MGL resin.

[0023] Preferably, the macroporous resin is a mixture of SP20SS resin and AB-8 resin, and the loading mode is to load SP20SS macroporous resin at the bottom and AB-8 macroporous resin at the top.

[0024] The present application creatively uses a mixture of SP20SS resin and AB-8 resin to purify the extraction product. Compared with other types of resin combinations or single resins, SP20SS resin and AB-8 resin can cooperate synergistically, and the final product obtained by separation and purification has more excellent effects in terms of anti-inflammatory, soothing, barrier repair, moisturizing, and oil control.

[0025] Preferably, the mass ratio of the SP20SS resin to the AB-8 resin is (1-3):1, such as 1:1, 3:2, 2:1, 5:2, 3:1, etc.

[0026] Preferably, the ethanol aqueous solution in the purification elution is a 20-30% ethanol aqueous solution, such as a 20%, 22%, 25%, 28%, 30%, etc. ethanol aqueous solution.

[0027] Preferably, the elution volume of the purified water is 3-8 BV, such as 3 BV, 3.5 BV, 4 BV, 4.5 BV, 5 BV, 5.5 BV, 6 BV, 6.5 BV, 7 BV, 7.5 BV, 8 BV, etc.

[0028] Preferably, the elution volume of the aqueous ethanol solution is 3-8 BV, such as 3 BV, 3.5 BV, 4 BV, 4.5 BV, 5 BV, 5.5 BV, 6 BV, 6.5 BV, 7 BV, 7.5 BV, 8 BV, etc.

[0029] Preferably, the decoloring agent is a combination of alumina and zeolite molecular sieve.

[0030] The present application creatively uses a combination of alumina and zeolite molecular sieve to further purify the eluted product, and compared with other types of decoloring agents or single decoloring agents, alumina and zeolite molecular sieve can synergize, and the final product of separation and purification has better effects in terms of anti-inflammatory, soothing, barrier repair, moisturizing, and oil control.

[0031] Preferably, the mass ratio of the alumina to the zeolite molecular sieve is (2-4):1, such as 2:1, 5:2, 3:1, 7:2, 4:1, etc.

[0032] Preferably, the amount of the decoloring agent is 0.1-0.5% of the mass of the sea flower eluate, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, etc.

[0033] Preferably, the treatment condition of the decoloring agent is stirring at 40-60℃ (such as 40℃, 45℃, 50℃, 55℃, 60℃, etc.) for 20-60 min (such as 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 60 min, etc.).

[0034] Preferably, after the treatment with the decoloring agent, concentration and / or drying treatment is further performed.

[0035] All other specific point values not listed in the above-mentioned numerical ranges can be selected, and are within the protection scope of the present application. Considering the brevity of expression, they will not be described one by one here.

[0036] In a second aspect, the sea flower extract prepared by the preparation method of the first aspect.

[0037] In a third aspect, the present application provides the use of the sea flower extract of the second aspect in the preparation of a product having any one of the following effects or a combination of at least two effects:

[0038] 1) anti-inflammatory; 2) soothing; 3) barrier repair; 4) moisturizing; 5) oil control.

[0039] In a fourth aspect, the present application provides the use of the sea flower extract of the second aspect in the preparation of an inflammation factor expression inhibitor, wherein the inflammation factor includes any one of TNF-α, IL-6, NO or a combination of at least two.

[0040] In a fifth aspect, the present application provides use of the sea flower extract of the second aspect in the preparation of a filaggrin activator and / or a lorcrin activator.

[0041] In a sixth aspect, the present application provides use of the sea flower extract of the second aspect in the preparation of an aquaporin-3 activator.

[0042] Compared with the prior art, the present application has the following beneficial effects:

[0043] The present application creatively uses alcohol extraction, membrane filtration, resin purification and decolorizing agent treatment in sequence to prepare a sea flower extract product. The preparation process can extract the active ingredients (such as polysaccharides, proteins and polyphenols) in sea flowers to the greatest extent and form a specific proportion of active ingredients, so that the final product can inhibit the expression of inflammatory factors TNF-α, IL-6 and NO, promote the expression of FLG, LOR and AQP-3 proteins, and inhibit the excessive secretion of sebum from sebaceous gland cells induced by 5α-dihydrotestosterone, and has excellent anti-inflammatory, soothing, barrier repair, moisturizing and oil control effects. DETAILED DESCRIPTION

[0044] In order to further illustrate the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below in combination with preferred embodiments of the present application, but the present application is not limited in the scope of the embodiments.

[0045] The 10 kDa, 20 kDa and 30 kDa ultrafiltration membranes referred to in the following content are purchased from PALL Co., Ltd.; the 150 Da, 300 Da and 500 Da nanofiltration membranes are purchased from China Kailun Membrane Technology (Beijing) Co., Ltd.; the aluminum oxide is purchased from Luoen Chemical Co., Ltd.; and the zeolite molecular sieve is purchased from Zhuo Ran Environmental Protection Technology (Dalian) Co., Ltd.

[0046] Preparation Example 1

[0047] The present preparation example provides a sea flower extract prepared by the following preparation method:

[0048] (1) After the sea flower is dried and crushed through a 40-mesh sieve, it is mixed with 70% ethanol aqueous solution at a solid-liquid ratio of 1:10 g / mL for heating reflux extraction twice, 1 h each time, and then solid-liquid separation is performed. The filtrates are combined to obtain a sea flower extract;

[0049] (2) The sea flower extract is filtered by an ultrafiltration membrane with a molecular weight cut-off of 20 kDa, and the filtrate is collected. The filtrate is filtered by a nanofiltration membrane with a molecular weight cut-off of 300 Da, and the cut-off liquid is collected. The sea flower concentrate is obtained by reducing pressure concentration.

[0050] (3) The sea flower concentrate is purified and eluted by using a macroporous resin, and the macroporous resin is SP20SS resin. The process of purification and elution is as follows: first, eluted by using pure water for 5 BV, and then eluted by using 25% ethanol aqueous solution for 5 BV. The water eluate and the alcohol eluate are collected to obtain a sea flower eluate;

[0051] (4) In the sea flower eluate, alumina and zeolite molecular sieves are added in a mass ratio of 3:1, and the total amount is 0.25% of the mass of the sea flower eluate. Stirring is performed at 50°C for 30 min. After decolorization is completed, concentration and freeze-drying are performed to obtain the sea flower extract.

[0052] Preparation Example 2

[0053] The preparation example provides a sea flower extract prepared by the following preparation method.

[0054] (1) After the sea flower is dried and crushed to pass through a 40-mesh sieve, the sea flower is mixed with 60% ethanol aqueous solution at a solid-liquid ratio of 1:15 g / mL for heating reflux extraction twice, each time for 1 h. Solid-liquid separation is performed, and the filtrates are collected to obtain a sea flower extract;

[0055] (2) The sea flower extract is filtered by using an ultrafiltration membrane with a molecular weight cut-off of 30 kDa. The filtrate is filtered by using a nanofiltration membrane with a molecular weight cut-off of 150 Da. The cut-off liquid is collected, and concentration is performed under reduced pressure to obtain a sea flower concentrate;

[0056] (3) The sea flower concentrate is purified and eluted by using a macroporous resin, and the macroporous resin is AB-8 resin. The process of purification and elution is as follows: first, eluted by using pure water for 5 BV, and then eluted by using 20% ethanol aqueous solution for 5 BV. The water eluate and the alcohol eluate are collected to obtain a sea flower eluate;

[0057] (4) In the sea flower eluate, alumina and zeolite molecular sieves are added in a mass ratio of 2:1, and the total amount is 0.1% of the mass of the sea flower eluate. Stirring is performed at 60°C for 20 min. After decolorization is completed, concentration and freeze-drying are performed to obtain the sea flower extract.

[0058] Preparation Example 3

[0059] The preparation example provides a sea flower extract prepared by the following preparation method.

[0060] (1) After the sea flower is dried and crushed to pass through a 40-mesh sieve, the sea flower is mixed with 80% ethanol aqueous solution at a solid-liquid ratio of 1:8 g / mL for heating reflux extraction once for 2 h. Solid-liquid separation is performed to obtain a sea flower extract;

[0061] (2) The sea cucumber flower extract solution is filtered by using an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, the filtrate is collected, the filtrate is filtered by using a nanofiltration membrane with a molecular weight cut-off of 500 Da, the cut-off liquid is collected, and the sea cucumber flower concentrate is obtained by concentration under reduced pressure;

[0062] (3) The sea cucumber flower concentrate is purified and eluted by using a macroporous resin, the macroporous resin is HP2MGL resin, and the process of purification and elution is as follows: 5 BV of pure water is eluted first, and then 5 BV of 30% ethanol aqueous solution is eluted, the water washing liquid and the alcohol washing liquid are collected, and the sea cucumber flower eluate is obtained;

[0063] (4) The sea cucumber flower eluate is added with alumina and zeolite molecular sieve with a mass ratio of 4:1, the total amount is 0.5% of the mass of the sea cucumber flower eluate, stirring is carried out at 40°C for 40 min, after decolorization is completed, concentration and freeze-drying are carried out, and the sea cucumber flower extract is obtained.

[0064] Preparation Example 4

[0065] The preparation example provides a sea cucumber flower extract, and the difference between the preparation method thereof and that of the preparation example 1 is that 70% ethanol aqueous solution is replaced by 50% ethanol aqueous solution in step (1), and other steps remain unchanged.

[0066] Preparation Example 5

[0067] The preparation example provides a sea cucumber flower extract, and the difference between the preparation method thereof and that of the preparation example 1 is that 70% ethanol aqueous solution is replaced by 90% ethanol aqueous solution in step (1), and other steps remain unchanged.

[0068] Preparation Example 6

[0069] The preparation example provides a sea cucumber flower extract, and the difference between the preparation method thereof and that of the preparation example 1 is that the macroporous resin is replaced by AB-8 resin in the same column volume in step (3), and other steps remain unchanged.

[0070] Preparation Example 7

[0071] The preparation example provides a sea cucumber flower extract, and the difference between the preparation method thereof and that of the preparation example 1 is that the macroporous resin is replaced by a mixture of SP20SS resin and AB-8 resin with a mass ratio of 2:1 in the same column volume in step (3), the SP20SS resin is arranged at the lower part of the resin column, and the AB-8 resin is arranged at the lower part of the resin column, and other steps remain unchanged.

[0072] Preparation Example 8

[0073] The preparation example provides a sea flower extract, and the preparation method is only different from that of the preparation example 1 in that the mass ratio of the alumina and the zeolite molecular sieve is replaced by single alumina in step (4), and the total amount is still 0.25% of the mass of the sea flower eluent.

[0074] Preparation example 9

[0075] The preparation example provides a sea flower extract, and the preparation method is only different from that of the preparation example 1 in that the mass ratio of the alumina and the zeolite molecular sieve is replaced by single zeolite molecular sieve in step (4), and the total amount is still 0.25% of the mass of the sea flower eluent.

[0076] Comparative preparation example 1

[0077] The comparative preparation example provides a sea flower extract, and the preparation method is only different from that of the preparation example 1 in that step (1) is that the sea flower is dried, crushed and passed through a 40-mesh sieve, then mixed with pure water at a solid-liquid ratio of 1:10 g / mL to perform heating reflux extraction twice, 1 h each time, and the solid-liquid separation is performed, and the filtrate is combined to obtain a sea flower extract. Other steps remain unchanged.

[0078] Comparative preparation example 2

[0079] The comparative preparation example provides a sea flower extract, and the preparation method is only different from that of the preparation example 1 in that step (2) is omitted, and the sea flower extract of step (1) is directly subjected to step (3) operation after distillation and concentration. Other steps remain unchanged.

[0080] Comparative preparation example 3

[0081] The comparative preparation example provides a sea flower extract, and the preparation method is only different from that of the preparation example 1 in that step (3) is omitted: the sea flower concentrated solution of step (2) is directly subjected to step (4) operation. Other steps remain unchanged.

[0082] Comparative preparation example 4

[0083] The comparative preparation example provides a sea flower extract, and the preparation method is only different from that of the preparation example 1 in that step (4) is omitted, and the sea flower eluent is directly concentrated and freeze-dried to obtain the sea flower extract.

[0084] Comparative preparation example 5

[0085] The comparative preparation example provides a sea flower extract, and the preparation method is only different from that of the preparation example 1 in that the purification elution process in step (3) is that 5 BV of pure water is used for elution, and the water washing liquid is collected to obtain a sea flower eluent, and other steps remain unchanged.

[0086] Comparative preparation example 6

[0087] The present comparative preparation example provides a sea flower extract, the preparation method of which is only different from that of preparation example 1 in that the process of purifying and eluting in step (3) is that 5 BV of pure water is first eluted, and then 5 BV of 25% ethanol aqueous solution is eluted, and only the 25% alcohol eluate is collected to obtain a sea flower eluate, and other steps remain unchanged.

[0088] Test example 1

[0089] Anti-inflammatory soothing efficacy evaluation:

[0090] (1) Preparation of the test sample:

[0091] The sea flower extracts prepared in preparation examples 1-9 and comparative preparation example 1-6 are dissolved in a cell culture medium to prepare a test sample solution with a concentration of 120 μg / mL.

[0092] (2) The anti-inflammatory efficacy is tested by using LPS-induced mouse macrophage RAW264.7 as a test model, and the specific operation is as follows: the logarithmic growth phase RAW264.7 cells are inoculated in a 96-well plate at a concentration of 8×10 4

[0093] After incubation at 37°C in a 5% CO2 incubator for 24 h, the cell supernatants of each group are collected, and then the contents of inflammatory factors TNF-α, IL-6 and NO in the collected cell supernatants are detected according to the ELISA kit detection method. The experimental results are analyzed by data processing software to evaluate the inhibition rate of the sample on the expression of inflammatory factors. The inhibition rate calculation formula is: inhibition rate (%) = ((model group relative expression amount-sample group relative expression amount) / model group relative expression amount) × 100%, and the results are shown in Table 1.

[0094] Table 1

[0095]

[0096] As can be seen from the data results in Table 1, the sea flower extract prepared by the preparation method disclosed in the present application has excellent inflammatory factor inhibition activity, i.e., excellent anti-inflammatory and soothing potential.

[0097] As can be seen from the data results of preparation example 1 and comparative preparation example 1, the alcohol extraction method used in the present application can make the extracted product have more excellent inflammatory factor inhibition activity than the water extraction method. ​

[0098] From the data results of Preparation Example 1 and Comparative Preparation Examples 2, it can be seen that the present application can ensure that the extracted product has more excellent inflammatory factor inhibitory activity by performing a membrane filtration operation before resin elution.

[0099] From the data results of Preparation Example 1 and Comparative Preparation Examples 3-4, it can be seen that the present application can ensure that the extracted product has more excellent inflammatory factor inhibitory activity by performing resin elution treatment operation after extraction and performing decolorizing agent treatment after resin elution.

[0100] From the data results of Preparation Example 1 and Comparative Preparation Examples 5-6, it can be seen that the present application can ensure that the extracted product has more excellent inflammatory factor inhibitory activity by collecting the combined water washing liquid and alcohol washing liquid as the basis for subsequent decolorization when performing macroporous resin elution.

[0101] Test Example 2

[0102] Barrier repair efficacy evaluation:

[0103] (1) Preparation of the test sample:

[0104] The sea flower extracts prepared in Preparation Examples 1-9 and Comparative Preparation Examples 1-6 were dissolved in cell culture medium to prepare a test sample solution with a concentration of 120 μg / mL.

[0105] (2) HaCaT cells in the logarithmic growth phase and with good morphology were inoculated in a 24-well plate at 1×10 5 After 24 h of incubation in an incubator, the supernatant was removed, and then the cells were treated in groups. A blank control group and a test sample group were set up. The blank control group was added with an equal volume of cell culture medium, and the test sample group was added with 120 μg / mL of each test sample. Then, the cells were incubated for another 24 h in a 37°C, 5% CO2 incubator. RNA was extracted according to the operation instructions of the cell RNA extraction kit, and reverse transcription was performed into cDNA using a reverse transcription kit. Finally, the mRNA expression of barrier repair-related factors filaggrin (FLG) and loricrin (LOR) was detected by real-time fluorescent quantitative PCR. The activation rate was calculated according to the formula: activation rate (%) = ((relative expression of the sample group - relative expression of the blank control group) / relative expression of the blank control group) × 100%. The mRNA primer sequences were as follows:

[0106] ① FLG (forward: 5'-TGAAGCCTATGACACCACTGA-3', reverse: 5'-TCCCCTACGCTTTCTTGTCCT-3');

[0107] ② LOR (forward: 5'-GCCGTCCAAATAGATCCCCC-3', reverse: 5'-GCAAACCTCGGGTAGCATCA-3').

[0108] The results are shown in Table 2.

[0109] Table 2

[0110]

[0111] From the data results of Table 2, it can be seen that the sea flower extract prepared by the preparation method of the present application has excellent effects of promoting the expression of filaggrin (FLG) and loricrin (LOR), i.e. excellent barrier repair potential.

[0112] From the data results of Preparation Example 1 and Comparative Preparation Example 1, it can be seen that the alcohol extraction of the present application compared with water extraction can make the extraction product have more excellent effects of promoting the expression of filaggrin and loricrin.

[0113] From the data results of Preparation Example 1 and Comparative Preparation Example 2, it can be seen that the membrane filtration before resin elution of the present application can ensure that the extraction product has more excellent effects of promoting the expression of filaggrin and loricrin.

[0114] From the data results of Preparation Example 1 and Comparative Preparation Examples 3-4, it can be seen that the resin elution treatment operation after extraction and the decolorizing agent treatment after resin elution of the present application can ensure that the extraction product has more excellent barrier repair potential.

[0115] From the data results of Preparation Example 1 and Comparative Preparation Examples 5-6, it can be seen that when the macroporous resin elution is performed, the present application collects the combined water washing liquid and alcohol washing liquid as the basis for subsequent decolorization, which can ensure that the extraction product has more excellent barrier repair potential.

[0116] Test Example 3

[0117] Moisturizing effect evaluation:

[0118] (1) Preparation of the sample to be tested:

[0119] The sea flower extracts prepared by Preparation Examples 1-9 and Comparative Preparation Examples 1-6 were prepared into sample solutions to be tested with a concentration of 120 μg / mL using cell culture medium as the solvent.

[0120] (2) HaCaT cells in logarithmic growth phase and good morphology were selected and inoculated in 24-well plates at 1×10 5Incubate the cells / cone in the incubator for 24 h, remove the supernatant after culture, and then perform grouping treatment, set up a blank control group, a positive control group and a sample group to be tested, add the same volume of cell culture medium to the blank control group, add 0.3 mM CaCl2 solution to the positive control group, and add 120 μg / mL of each sample to be tested in the sample group to be tested, and continue to incubate at 37°C, 5% CO2 incubator for 24 h. According to the operation instruction of the cell RNA extraction kit, extract RNA, and use the reverse transcription kit to reverse transcribe into cDNA, and finally detect the mRNA expression of water channel protein-3 (AQP-3) by real-time fluorescent quantitative PCR method.

[0121] The relative expression amount calculation formula is: relative expression amount = sample group expression amount / blank control group expression amount, and the primer sequence of the measured mRNA is: AQP3 (forward: 5'-GGGGAGATGCTCCACATCC-3', reverse: 5'-AAAGGCCAGGTTGATGGTGAG-3'), and the results are shown in Table 3:

[0122] Table 3

[0123]

[0124] From the data results in Table 3, it can be seen that the sea flower extract prepared by the preparation method of the present application has excellent water channel protein-3 (AQP-3) expression effect, i.e. excellent moisturizing potential.

[0125] From the data results of Preparation Example 1 and Comparative Preparation Example 1, it can be seen that the alcohol extraction used in the present application compared with water extraction can make the extraction product have more excellent moisturizing potential.

[0126] From the data results of Preparation Example 1 and Comparative Preparation Example 2, it can be seen that the membrane filtration before resin elution in the present application can ensure that the extraction product has more excellent moisturizing potential.

[0127] From the data results of Preparation Example 1 and Comparative Preparation Example 3-4, it can be seen that the resin elution treatment operation after extraction and the decolorizing agent treatment after resin elution in the present application can ensure that the extraction product has more excellent moisturizing potential.

[0128] From the data results of Preparation Example 1 and Comparative Preparation Example 5-6, it can be seen that when the macroporous resin elution is performed, the combined water washing liquid and alcohol washing liquid are collected as the basis for subsequent decolorization in the present application, which can ensure that the extraction product has more excellent moisturizing potential.

[0129] Test Example 4

[0130] (1) Preparation of the sample to be tested:

[0131] The extracts of *Ottelia acuminata* obtained in Preparation Examples 1-9 and Comparative Preparation Examples 1-6 were used to prepare a 120 μg / mL sample solution using cell culture medium as a solvent.

[0132] (2) SZ95 human sebaceous gland cells were used as the research object, at a concentration of 1×10 5 SZ95 cells were seeded into 24-well plates at a seeding density of cells / well and incubated overnight in an incubator (37℃, 5% CO2). When the cell deposition rate in the 24-well plates reached 40-60%, the culture medium was discarded, and the cells were divided into groups: the blank group was cultured with cell culture medium only, the model group was supplemented with 1 μM 5α-dihydrotestosterone (DHT), the positive control group was supplemented with 1 μM DHT and 3 μg / mL isotretinoin, and the sample group was supplemented with 1 μM DHT and 120 μg / mL of the sample to be tested. The cells were incubated for another 24 h in an incubator (37℃, 5% CO2). The culture medium was discarded, and the cells were rinsed with PBS. After fixation, Nile red staining was added for 15 min, and after rinsing, the fluorescence intensity was recorded using a fluorescence microscope. The fluorescence intensity was quantitatively analyzed using Image Pro Plus software. The lipid inhibition rate (%) = ((fluorescence value of model group - fluorescence value of sample group) / fluorescence value of model group) × 100%. The results are shown in Table 4.

[0133] Table 4

[0134]

[0135] As shown in Table 4, the extract of *Ottelia acuminata* prepared by the method of this invention has excellent effect in inhibiting the secretion of sebum by sebaceous gland cells, that is, excellent oil control potential.

[0136] The data from Preparation Example 1 and Comparative Preparation Example 1 show that the alcohol extraction method used in this invention, compared with water extraction, can make the extracted product have better oil control potential.

[0137] The data from Preparation Example 1 and Comparative Preparation Example 2 show that the membrane filtration performed before resin elution in this invention can ensure that the extracted product has better oil control potential.

[0138] As can be seen from the data results of Preparation Example 1 and Comparative Preparation Examples 3-4, the resin elution treatment after extraction and the decolorizing agent treatment after resin elution in this invention can ensure that the extracted product has better oil control potential.

[0139] As can be seen from the data results of Preparation Example 1 and Comparative Preparation Examples 5-6, when the present invention performs macroporous resin elution, collecting and combining the water washing liquid and alcohol washing liquid as the basis for subsequent decolorization can ensure that the extracted product has better oil control potential.

[0140] Test Example 5

[0141] Safety evaluation:

[0142] The safety indexes such as skin irritation and eye irritation of the sea flower extract prepared from Preparation Example 1-9 were determined according to the Cosmetic Safety Technical Specifications 2015 edition, and the results are shown in Table 5.

[0143] Table 5

[0144]

[0145] As can be seen from Table 5, the sea flower extract prepared by the present application has no skin irritation, eye irritation and the like, and has good safety.

[0146] The applicant declares that the technical solutions of the present application are illustrated by the above examples, but the present application is not limited to the above examples, that is, it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement on the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes and the like all fall within the protection scope and disclosure scope of the present application.

[0147] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0148] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A method for preparing an extract from *Ottelia acuminata*, characterized in that, The preparation method of the seaweed extract includes the following steps: (1) Mix the raw material of seaweed with an ethanol aqueous solution and heat and reflux to extract it. Separate the solid and liquid to obtain seaweed extract. (2) The extract of seaweed flower was subjected to membrane filtration and the filtrate was concentrated to obtain seaweed flower concentrate; (3) The concentrated water lily extract was purified and eluted using macroporous resin. First, it was eluted with pure water, and then with an ethanol aqueous solution. The water wash and the ethanol wash were collected and combined to obtain the water lily eluent. (4) The water lily eluent was treated with a decolorizing agent to obtain the water lily extract.

2. The preparation method according to claim 1, characterized in that, The volume fraction of ethanol in the aqueous ethanol solution described in step (1) is 60-80%; Preferably, the heating and reflux extraction is performed 1-3 times, each time for 0.5-2 hours; Preferably, the raw material of the seaweed is dried and pulverized seaweed; Preferably, the ratio of the raw material of *Ottelia acuminata* to the aqueous ethanol solution is 1:(5-20) g / mL; Preferably, the membrane used for membrane filtration has a molecular weight cutoff of 10-30 kDa; Preferably, the filtrate concentration includes concentration by membrane filtration with a molecular weight cutoff of 150-500 Da, followed by concentration under reduced pressure to remove ethanol.

3. The preparation method according to claim 1 or 2, characterized in that, The macroporous resin is selected from any one or a combination of at least two of AB-8 resin, D101 resin, SP20SS resin, and HP2MGL resin. Preferably, the macroporous resin is a mixture of SP20SS resin and AB-8 resin; Preferably, the mass ratio of SP20SS resin to AB-8 resin is (1-3):

1.

4. The preparation method according to any one of claims 1-3, characterized in that, The ethanol-water solution used in the purification and elution process is a 20-30% ethanol-water solution; Preferably, the elution volume of the pure water is 3-8 BV; Preferably, the elution volume of the ethanol-water solution is 3-8 BV.

5. The preparation method according to any one of claims 1-4, characterized in that, The decolorizing agent is a combination of alumina and zeolite molecular sieve; Preferably, the mass ratio of alumina to zeolite molecular sieve is (2-4):1; Preferably, the amount of the decolorizing agent is 0.1-0.5% of the mass of the seaweed eluent; Preferably, the decolorizing agent treatment is performed by stirring at 40-60°C for 20-60 min; Preferably, the treatment with the decolorizing agent is followed by concentration and / or drying.

6. The seaweed extract prepared by any one of claims 1-5.

7. The use of the *Ottelia acuminata* extract according to claim 6 in the preparation of a product having any one of the following effects or a combination of at least two of the following effects: 1) Anti-inflammatory; 2) Soothing; 3) Barrier repair; 4) Moisturizing; 5) Oil control.

8. The use of the seaweed extract according to claim 6 in the preparation of inflammatory factor expression inhibitors, wherein the inflammatory factors include any one or a combination of at least two of TNF-α, IL-6, and NO.

9. The use of the seaweed extract according to claim 6 in the preparation of filaggrin activator and / or pachyphyll activator.

10. The use of the *Ottelia acuminata* extract according to claim 6 in the preparation of aquaporin-3 activators.

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