CN109833334A - The preparation method and application of long shoot grape caulerpa extract - Google Patents
The preparation method and application of long shoot grape caulerpa extract Download PDFInfo
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- CN109833334A CN109833334A CN201811525384.3A CN201811525384A CN109833334A CN 109833334 A CN109833334 A CN 109833334A CN 201811525384 A CN201811525384 A CN 201811525384A CN 109833334 A CN109833334 A CN 109833334A
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Abstract
The present invention relates to a kind of preparation method and applications of long shoot grape caulerpa extract, the preparation method includes the following steps: for long shoot grape caulerpa sample to be dissolved in Extraction solvent, successively carry out grinding stirring, ultrasound, filtering or centrifugation, concentration, obtain extracting solution to get；The Extraction solvent is water or ethanol water, and the volumetric concentration of the ethanol water is 0.5-100%, the proportion of the long shoot grape caulerpa sample and the Extraction solvent are as follows: 1g:0.1-100ml.The long shoot grape caulerpa extract has the function of moisturizing moisturizing, anti-inflammatory, promotion hyperplasia and promotes wound healing.
Description
Technical field
The present invention relates to field of biological extraction, a kind of preparation method more particularly to long shoot grape caulerpa extract and answer
With.
Background technique
Long shoot grape caulerpa (scientific name: Caulerpa lentillifera) is a kind of edible algae that caulerpa belongs to, and is also cried
" sea grape " has the farm of this seeds algae in Philippine's Mactan Island and Okinawa County, Janpan.Currently, Chinese Guangdong, Shandong and sea
The ground such as south have cultivated success.Long shoot grape caulerpa is a kind of edible green alga full of nutrition, containing a large amount of amino acid,
ω -6 unsaturated fatty acid such as oleic acid, arachidonic acid etc., polysaccharide, minerals, vitamin are also rich in phosphorus, calcium, magnesium, copper, selenium
Equal microelements；Protein content 10.41%, the lower 1.6%-3.7% of total lipid content have purification blood, anti-oxidant, anti-
Cancer, antineoplastic action.
Sea grape contains important active substances, and such as caulerpa sequiterpene (Caulerpenyne), primary efficacy is, works as algae
After body is mechanically damaged, caulerpa sequiterpene can be converted to Oxytoxin-2, seal cellular damage position.In addition, caulerpa sesquialter
Terpene can also be used for treatment gout, and caulerpa sequiterpene has depression effect to xanthine oxidase (XOD) activity.The extract of sea grape
With antiviral and anti-tumor activity.The polysaccharide separated from long shoot grape caulerpa has immunostimulatory activity to macrophage,
In xylan hydrolysis at the oligosaccharides of average 5 monosaccharide molecules, can induce human breast cancer cell MCF-7 nuclear chromatin condensation,
And the sedimentation of Poly-ADP-ribose polymerase (adenosine diphosphate ribose polymerase), there is cancer cell specific induction of apoptosis
Effect.
But the effect of skin moisture-keeping moisturizing, anti-inflammatory and quickening wound healing, is not reported about the extract of sea grape
Road.
Summary of the invention
Based on this, the present invention provides the preparation methods and new opplication of a kind of long shoot grape caulerpa extract.
Specific technical solution is as follows:
A kind of preparation method of long shoot grape caulerpa extract, includes the following steps:
Long shoot grape caulerpa sample is dissolved in Extraction solvent, grinding stirring, ultrasound, filtering or centrifugation, dense are successively carried out
Contracting, obtain extracting solution to get；
The Extraction solvent is water or ethanol water, and the volumetric concentration of the ethanol water is 50-90%, the length
The proportion of stem grape caulerpa sample and the Extraction solvent is 1g:3-7ml.
In wherein some embodiments, the condition of the ultrasound is as follows: 175-200W, 30-50kHz, ultrasound 45-90min.
In wherein some embodiments, the condition of the ultrasound is as follows: 180-190W, 35-45kHz, ultrasound 55-65min.
In wherein some embodiments, the volume of the extracting solution after concentration and the volume ratio of the Extraction solvent are
0.25-0.5:1。
In wherein some embodiments, the volume of the extracting solution after concentration and the volume ratio of the Extraction solvent are
0.35-0.4:1.
In wherein some embodiments, the volumetric concentration of the ethanol water is 65-75%, the long shoot grape caulerpa
The proportion of sample and the Extraction solvent is 1g:4.8-5.2ml.
In wherein some embodiments, the condition of the centrifugation are as follows: 800-2300rcf/min is centrifuged 1-2 times.
In wherein some embodiments, freeze-drying is further included the steps that after the concentration step, obtains freeze-dried powder, i.e.,
Obtain the long shoot grape caulerpa extract.
In wherein some embodiments, the condition of the freeze-drying are as follows: temperature is -75-85 DEG C, time 2.5-3.5
It.
In wherein some embodiments, when include thing steps that freeze-drying after the concentration step, the long shoot
The preparation method of grape caulerpa extract further includes the steps that the freeze-dried powder being dissolved in water, obtains aqueous solution to get the long shoot
Grape caulerpa extract.
In wherein some embodiments, the concentration of the aqueous solution is 0.01-1.5mg/mL.
In wherein some embodiments, the concentration of the aqueous solution is 0.1-1.2mg/mL.
In wherein some embodiments, the concentration of the aqueous solution is 0.25-1.0mg/mL.
In wherein some embodiments, the concentration of the aqueous solution is 0.7-1.0mg/mL.
In wherein some embodiments, the long shoot grape caulerpa sample includes: the stem of long shoot grape caulerpa, long shoot grape
The spherical thallus of caulerpa or whole strain long shoot grape caulerpa.
Long shoot grape caulerpa or long shoot grape caulerpa extract are thin with moisturizing moisturizing or anti-inflammatory or promotion in preparation
Application in the skin products of the effect of born of the same parents' hyperplasia or promotion wound healing.
In wherein some embodiments, the skin products are skin care item, cosmetics, health care product, food or drug.
In wherein some embodiments, the long shoot grape caulerpa extract is prepared by above-mentioned preparation method.
In wherein some embodiments, the use concentration of the long shoot grape caulerpa extract is 0.01-1.5mg/mL.
In wherein some embodiments, the use concentration of the long shoot grape caulerpa extract is 0.1-1.2mg/mL.
In wherein some embodiments, the use concentration of the long shoot grape caulerpa extract is 0.25-1.0mg/mL.
In wherein some embodiments, the use concentration of the long shoot grape caulerpa extract is 0.7-1.0mg/mL.
There is moisturizing moisturizing or anti-inflammatory or promotion hyperplasia the invention also discloses one kind or wound is promoted to be cured
The skin products of the effect of conjunction.
Specific technical solution is as follows:
A kind of skin products, the skin products have moisturizing moisturizing or anti-inflammatory or promotion hyperplasia or promotion
The effect of wound healing, the active constituent in the skin products includes long shoot grape caulerpa extract.
In wherein some embodiments, the skin products are skin care item, cosmetics, health care product, food or drug.
In wherein some embodiments, the long shoot grape caulerpa extract is prepared by above-mentioned preparation method.
In wherein some embodiments, the concentration of the long shoot grape caulerpa extract is 0.01-1.5mg/mL.
In wherein some embodiments, the concentration of the long shoot grape caulerpa extract is 0.1-1.2mg/mL.
In wherein some embodiments, the concentration of the long shoot grape caulerpa extract is 0.25-1.0mg/mL.
In wherein some embodiments, the concentration of the long shoot grape caulerpa extract is 0.7-1.0mg/mL.
The present inventor carries out the experiment of hyperplasia and wound healing with Skin Cell HaCaT, finds sea grape
Extract not only has the effect of promoting HaCaT hyperplasia, but also plays the role of promoting wound healing.With Skin Cell HaCaT
Intracellular moisturizing moisturizing factor FLG2mRNA expression quantity level experiment is carried out, discovery sea grape extract has Skin Cell bright
The effect of aobvious moisturizing moisturizing, the effect of especially sea grape stem and the water extract of complete stool are more preferable.Skin is induced with lipopolysaccharides (LPS)
Skin cell HaCaT inflammatory factor TNFalpha and IL-6 establish cellular inflammation model, test the anti-inflammatory experiment of sea grape extract, hair
Existing sea grape extract has preferable anti-inflammatory effect.Therefore, the application is to sea grape extract for skin moisture-keeping moisturizing, anti-
Scorching and wound healing, skin regeneration provide substantive experimental data.It was found from experimental data: sea grape extract can be used for skin
Skin moisturizing moisturizing, anti-inflammatory and wound healing, skin regeneration；Also it can be used for wound inflammation, healing or skin regeneration, skin moisture-keeping is mended
The preparation of the medical cares such as water and cosmetic product.
The preparation method of the sea grape extract of the application has yield high and is easier to the absolute predominance of the marketization, and
It is sea grape extract its skin moisture-keeping moisturizing for being obtained using the preparation method of the application, anti-inflammatory and promote hyperplasia, accelerate
The effect of wound healing is more significant, and moisturizing moisturizing effect is also more stable, and the production cost of sea grape extract is low, and can be wide
It is used for drug, health care product, food and beauty product generally, there is huge market potential.
Detailed description of the invention
Fig. 1 is the proliferative effect of sea grape each section water extract and alcohol extract to Skin Cell HaCaT.
Fig. 2 is the influence of the spherical thallus water extract of sea grape and alcohol extracting thing to wound healing rate, and T0 is starting
Time, T24 are the variation after 24 hours.
Fig. 3 is the influence of sea grape stem water extract and alcohol extracting thing to wound healing rate, and T0 is initial time, T24
For the variation after 24 hours.
Fig. 4 is the influence of sea grape herb water extract and alcohol extracting thing to wound healing rate, and T0 is initial time,
T24 is the variation after 24 hours.
Fig. 5 is the moisturizing moisturizing effect of sea grape each section water extract and alcohol extract to tested Skin Cell.
Fig. 6 is the inhibiting effect of sea grape each section water extract and alcohol extract to TNF α inflammatory factor.
Fig. 7 is the inhibiting effect of sea grape each section water extract and alcohol extract to IL-6 inflammatory factor
Specific embodiment
It does below in conjunction with preparation method and application of the specific embodiment to long shoot grape caulerpa extract of the invention into one
Step detailed description.
Term " sea grape " is identical as the meaning of " long shoot grape caulerpa " in the application, " sea grape extract " and " long shoot
The meaning of grape caulerpa extract " is identical.
Embodiment 1
The preparation method of long shoot grape caulerpa extract
The present embodiment is exemplary to provide the preparation method of long shoot grape caulerpa extract, comprising the following steps:
A) stem, spherical thallus and whole strain will be divided into after sea grape sample clean.
B) the sea grape sample of 8 grams of different pieces is dissolved in 40 milliliters of water or 70% (volumetric concentration) ethanol water,
It is stirred, grinds.
C) ultrasound 60 minutes at 185W, 40kHz so that sea grape is sufficiently dissolved, obtain sea grape ethanol extract or
Aqueous extract.
D) after sea grape extracting solution is filtered (or be centrifuged: 800-2300rcf/min and be centrifuged 1-2 times), concentrated extracting solution
To 15 milliliters, to obtain high concentration sea grape extracting solution.
E) freeze-drying 3 days is carried out to sea grape extracting solution at -80 degrees Celsius, obtains sea grape extract lyophilized products
0.08g, yield 1.0%.
F) dissolution sea grape extract lyophilized products obtain the long shoot grape caulerpa extract made of sea grape in water, point
The long shoot grape caulerpa extract solution that concentration is 0.25mg/mL, 0.5mg/mL, 0.75mg/mL, 1.0mg/mL etc. is not obtained.
Embodiment 2
Experiment of the sea grape extract to HaCaT Skin Cell proliferative effect
The present embodiment carries out HaCaT Skin Cell hyperplasia to sea grape extract prepared according to the method for embodiment 1
Effect experiment.
Specifically, carried out HaCaT Skin Cell proliferative effect experiment the following steps are included:
A) sea grape extract is made referring to the preparation method of embodiment 1.
B) prepared sea grape extracting solution is added to people and immortalizes epidermal cell (HaCaT), with HaCaT Skin Cell
It cultivates 48 hours together, collects that sea grape extract is added and the HaCaT Skin Cell of sea grape extract is not added and ground
Study carefully.
C) MTT experimental test cell survival rate is applied, is made comparisons with blank control, to assess sea grape different parts system
Proliferative effect of the standby sea grape extract to Skin Cell.
As a result sea grape extract prepared by the sea grape of different parts does not have Skin Cell HaCaT as shown in Figure 1:
It is toxic, and in 0.75mg/mL concentration (final concentration of the freeze-dried powder of sea grape extract in culture solution) or more, it is right
There is apparent proliferative effect in Skin Cell HaCaT.
Embodiment 3
Sea grape extract tests the experiment that wound healing acts on
The present embodiment carries out wound healing assay test to sea grape extract prepared according to the method for embodiment 1.
Specifically, the wound healing assay that is carried out the following steps are included:
A) sea grape extract is made referring to the preparation method of embodiment 1.
B) by HaCaT cell kind in 6 orifice plates plate hole until complete Mi Pu.
C) upper cross is drawn in each 6 orifice plates plate hole bottom center with suction pipette head；
D) blank control is added in 6 orifice plates respectively, (sea grape mentions the extract 0.25mg/mL of sea grape different parts
Take final concentration of the freeze-dried powder of object in culture solution)；Co-incubation 24 hours；
E) it shoots cellular layer coverage rate under 50 times of magnifying power microscopes to note down, sea grape extract is added in observation comparison
The cellular layer coverage rate of front and back.T0 is the cytological map that sea grape extract is not added, and T24 is to be added sea grape extract 24 hours
Cytological map.
F) cellular layer coverage rate is analyzed and compared using TScratch software.
As a result as shown in Figure 2, Figure 3 and Figure 4: sea grape extract can accelerate the healing rate of wound, especially sea grape
The water extract of stem and herb (strain) promotes the effect of wound healing best.
Embodiment 4
The moisturizing moisturizing efficacy test of sea grape extract:
The present embodiment carries out the intracellular moisturizing of HaCaT to sea grape extract prepared according to the method for embodiment 1 and mends
The experiment of water mrna expression amount level.The effect of cutting from RNA level, be risen to cell transcription with Test extraction object, to obtain
The influence expressed skin protein and generated tests cell to understand extract to the effectiveness of skin moisture-keeping thus
FLG2 gene expression.FLG2 gene is located at the stratum granulosum of skin epidermis, parallel with skin surface, discovery FLG2 gene in 2009
In the middle role of skin: can control the expression of profilaggrin, profilaggrin can be converted into small peptide, most
Afterwards in skin surface breaks down into amino acids, become skin natural moisturizing factor；In addition, FLG2 gene helps point of horn cell
Change, horn cell is the natural barrier of skin, prevents the invasion of chemistry, microorganism, water and ultraviolet light.
Specifically carried out HaCaT Skin Cell moisturizing moisturizing experiment the following steps are included:
A) sea grape extract is made referring to the preparation method of embodiment 1.
B) HaCaT Skin Cell is added in prepared sea grape extract to cultivate together, the end of sea grape extract is dense
Degree is 0.25mg/mL, collects the HaCaT Skin Cell that sea grape extract is added and sea grape extract is not added.
C) total serum IgE for extracting HaCaT cell is carried out reverse transcription and obtains cDNA.
D) real time aggregation enzyme chain reaction (RT-PCR) is used, HaCaT cell mRNA expression quantity is quantified, is made with blank control
Compare, to assess effect of the sea grape extract to skin moisture-keeping moisturizing.Using fluorescent label, so as to quantify sample
Target gene (FLG2 gene) quantity in this, this technology can detect multiple reactions the same time.In each reaction, it is added
CDNA sample, upstream primer, downstream primer and SybrGreen fluorescein stain recycle480 RT-PCR
Instrument detects HaCaT Skin Cell under different pharmaceutical, the variation of cell FLG2 gene expression.
As a result as shown in Figure 5: sea grape extract plays the role of obvious moisturizing moisturizing for Skin Cell, especially extra large Portugal
The water extract of grape stem and complete stool.
Embodiment 5
The anti-inflammatory efficacy of sea grape extract is tested
The present embodiment establishes cellular inflammation model, utilization is enzyme-linked with lipopolysaccharides (LPS) inducing skin cells HaCaT inflammation
Immunization (ELISA) measures the TNF α and IL-6 Inflammatory Factors Contents being secreted into culture medium.Inflammatory factor TNFalpha and IL-6 contain
Amount is higher, and inflammation is more serious.When sea grape extract is added into HaCaT cell culture medium, if Inflammatory Factors Contents are lower,
The antiphlogistic effects for then representing sea grape extract are better.
(a) HaCaT cell is with 5x104The concentration kind of/ml changes low blood serum medium into 96 orifice plates before administration.
(b) extract is diluted with low blood serum medium, lipopolysaccharides LPS0.5 μ g/mL, culture 12 is added after administration 3 hours
Culture medium is collected after hour, is measured for ELISA.Using no test result that any control is added as experimental comparison group,
Simultaneously only to have added lipopolysaccharides LPS group as a control group.Each sample repeats 3 holes, is averaged and is calculated.
(c) according to ELISA kit the step of, measures containing for the TNF α and IL-6 inflammatory factor being secreted into culture medium
Amount.
(d) data are indicated with mean ± SD (n=3), are counted using Student ' st-test.It is carried out with control group
Compare, p < 0.01 * p < 0.05, * *.
As a result as shown in Figure 6 and Figure 7 (the final concentration of 0.25mg/mL of sea grape extract in the present embodiment): sea grape
Spherical thallophytic alcohol extract and water extract extract inhibit the effect of inflammatory factor TNFalpha most strong；The same position of sea grape, alcohol
Extract is slightly better than the effect of water extract.The alcohol extract of sea grape stem inhibits the effect of IL-6 inflammatory factor best, other positions
Extract has the effect of inhibiting IL-6 inflammatory factor.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of preparation method of long shoot grape caulerpa extract, which comprises the steps of:
Long shoot grape caulerpa sample is dissolved in Extraction solvent, grinding stirring, ultrasound, filtering or centrifugation, concentration is successively carried out, obtains
Extracting solution to get；
The Extraction solvent is water or ethanol water, and the volumetric concentration of the ethanol water is 50-90%, the long shoot Portugal
The proportion of grape caulerpa sample and the Extraction solvent is 1g:3-7ml.
2. the preparation method of long shoot grape caulerpa extract according to claim 1, which is characterized in that the item of the ultrasound
Part is as follows: 175-200W, 30-50kHz, ultrasound 45-90min；And/or
The volume of the extracting solution after concentration and the volume ratio of the Extraction solvent are 0.25-0.5:1；And/or
The volumetric concentration of the ethanol water is 65-75%, and the long shoot grape caulerpa sample is matched with the Extraction solvent
Than for 1g:4.8-5.2ml.
3. the preparation method of long shoot grape caulerpa extract according to claim 1, which is characterized in that walked in the concentration
Further include the steps that freeze-drying after rapid, obtains freeze-dried powder to get the long shoot grape caulerpa extract.
4. the preparation method of long shoot grape caulerpa extract according to claim 3, which is characterized in that the freeze-drying
Condition are as follows: temperature be -75-85 DEG C, the time be 2.5-3.5 days.
5. the preparation method of long shoot grape caulerpa extract according to claim 1-4, which is characterized in that described
Long shoot grape caulerpa sample includes: the stem of long shoot grape caulerpa, the spherical thallus of long shoot grape caulerpa or Zheng Zhu long shoot Portugal
Grape caulerpa.
6. long shoot grape caulerpa or long shoot grape caulerpa extract have moisturizing moisturizing or anti-inflammatory or promotion cell in preparation
Application in the skin products of the effect of hyperplasia or promotion wound healing.
7. application according to claim 6, which is characterized in that the long shoot grape caulerpa extract is by claim 1-5
Described in any item preparation methods are prepared.
8. a kind of skin products, the skin products have moisturizing moisturizing or anti-inflammatory or promotion hyperplasia or promote wound
The effect of mouth healing, which is characterized in that the active constituent in the skin products includes long shoot grape caulerpa extract.
9. skin products according to claim 8, which is characterized in that the concentration of the long shoot grape caulerpa extract is
0.01-1.5mg/mL。
10. skin products according to claim 9, which is characterized in that the concentration of the long shoot grape caulerpa extract is
0.25-1.0mg/mL。
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113274490A (en) * | 2021-05-13 | 2021-08-20 | 华南农业大学 | Preparation method and application of botryococcus longipediculus polysaccharide disease-resistant inducer |
| CN113412890A (en) * | 2021-06-25 | 2021-09-21 | 厦门海洋职业技术学院 | Preparation method of grape caulerpa extract and application of grape caulerpa extract in formula feed for ornamental fish |
| CN113491718A (en) * | 2020-04-01 | 2021-10-12 | 台湾海洋大学 | Application of vitis amurensis extract in preparing pharmaceutical composition or health food for relieving diabetes and preventing or treating reproductive disorders |
| CN117159424A (en) * | 2023-09-25 | 2023-12-05 | 揭阳市嘉胜日用精细化工有限公司 | Moisturizing and whitening composition and application thereof |
| CN117323350A (en) * | 2023-10-27 | 2024-01-02 | 江苏海洋大学 | Application of pteridophyta longifolia polyphenol in preparation of medicines for treating thrombus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113491718A (en) * | 2020-04-01 | 2021-10-12 | 台湾海洋大学 | Application of vitis amurensis extract in preparing pharmaceutical composition or health food for relieving diabetes and preventing or treating reproductive disorders |
| CN113491718B (en) * | 2020-04-01 | 2022-12-23 | 台湾海洋大学 | Application of vitis amurensis extract in preparing pharmaceutical composition or health food for relieving diabetes and preventing or treating reproductive disorders |
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| CN113412890A (en) * | 2021-06-25 | 2021-09-21 | 厦门海洋职业技术学院 | Preparation method of grape caulerpa extract and application of grape caulerpa extract in formula feed for ornamental fish |
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| CN117323350A (en) * | 2023-10-27 | 2024-01-02 | 江苏海洋大学 | Application of pteridophyta longifolia polyphenol in preparation of medicines for treating thrombus |
| CN117323350B (en) * | 2023-10-27 | 2024-03-05 | 江苏海洋大学 | Application of pteridophyta longifolia polyphenol in preparation of medicines for treating thrombus |
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