CN104651349A - Method of preparing gynostemma pentaphyllum/towel gourd hybrid cell extractive and application - Google Patents
Method of preparing gynostemma pentaphyllum/towel gourd hybrid cell extractive and application Download PDFInfo
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- CN104651349A CN104651349A CN201510072719.0A CN201510072719A CN104651349A CN 104651349 A CN104651349 A CN 104651349A CN 201510072719 A CN201510072719 A CN 201510072719A CN 104651349 A CN104651349 A CN 104651349A
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Abstract
The invention discloses a method of preparing a gynostemma pentaphyllum/towel gourd hybrid cell extractive and an application. The method comprises the following steps: (1) acquiring a protoplast, namely, respectively selecting callus cells of gynostemma pentaphyllum and towel gourds, carrying out filtering and centrifugation by virtue of an enzyme solution, and carrying out suspension precipitation by virtue of a culture medium to obtain a gynostemma pentaphyllum protoplast or a towel gourd protoplast; and (2) fusing and screening the gynostemma pentaphyllum protoplast and the towel gourd protoplast, namely, fusing the gynostemma pentaphyllum protoplast and the towel gourd protoplast by adopting an electrofusion method, screening a hybrid protoplast, and carrying out subculture to prepare the gynostemma pentaphyllum/towel gourd hybrid cell extractive. The prepared gynostemma pentaphyllum/towel gourd hybrid cell extractive is controllable in quality and good in effect, is capable of synthesizing the efficacies of the two plants and is non-toxic.
Description
Technical field
The invention belongs to biological beauty field, be specifically related to the gynostemma pentaphylla/application of sponge gourd hybrid cell extract in beauty and skin care with beauty functions.
Background technology
Gynostemma pentaphylla has another name called Rhizome or herb of Fiveleaf Gynostemma, Pentapanax leschenaultii, gynostemma pentaphyllum makino etc., is Curcurbitaceae gynostemma pentaphyllum genus herbaceous perennial vine plant.Gynostemma Pentaphyllum nutrient enriches, and physiologically active is extensive, to body immunity moderation system, antitumor, reducing blood-fat, analgesia, anti-aging and promote longevity and all have good effect, is described as " southern ginseng ", " long-life health care product ".Containing various active composition in its cell extract, such as: gypenoside, flavonoid compound, polysaccharide, effects such as VITAMIN and various trace elements, have anti-ageing, anti-oxidant.
Sponge gourd, be a kind of common edible vegetable, it is in the origin from ancient times of effect cosmetically, domestic known collection towel gourd stem juice very early among the people, " Tian Luoshui " (sponge gourd is ancient also known as " sky sieve " " thing class platform wall ") is claimed to be used for putting flour nutrition skin on the skin, as beauty liquid.The same with gynostemma pentaphylla, it is also containing the ginsenoside identical with ginseng, and in addition, its cell extract is also containing many triterpene compounds, and polysaccharide etc., repair after having anti-inflammatory, Pesticidal and sterilizing, solarization, the effect such as skin whitening, moisturizing.
But the plant strain growth of gynostemma pentaphylla and sponge gourd needs the time for many years, and is subject to region, weather, the impact of season and disease and pest etc. and threat, its plant extract quality is unstable, and therefore effect is difficult to control.In addition, the gynostemma pentaphylla of single class or the extract skincare effect of sponge gourd single, difficulty or ease meet the demand in market.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, object of the present invention provides a kind of Gynostemma pentaphyllum Makino and sponge gourd carries out hybridizing the method obtaining hybrid cell line, not only quality controllable, and effect is better than the clone effect of single plant origin, and nontoxic to organism.
Be achieved through the following technical solutions:
Prepare a method for gynostemma pentaphylla/sponge gourd hybrid cell extract, comprise the following steps:
(1) protoplastis is obtained:
1) enzyme liquid is prepared: choose that massfraction is 1.0-2.0% cellulase, 0.1-0.2% polygalacturonase, 6-12% N.F,USP MANNITOL and CPW washing lotion are mixed with enzyme liquid respectively;
2) callus cell choosing gynostemma pentaphylla and sponge gourd is respectively handled as follows: be (1-3) in callus cell and enzyme liquid mass ratio: the ratio of 10 will add in enzyme liquid in the callus cell of described gynostemma pentaphylla or sponge gourd, in 23 ~ 30 DEG C of dark surrounds, rotating speed is enzymolysis 8 ~ 12h under the condition of 50 ~ 100r/min, filter and collect filtrate and be placed in centrifuge tube, abandoning supernatant after the centrifugal 2-15min of 100g-300g; Suspend by CPW washing lotion and precipitate, get the centrifugal 2-15min of 100g-300g after mixing, abandoning supernatant; Then wash with containing the MS liquid culture medium of 0.2-0.4M N.F,USP MANNITOL and the protoplast culture medium of 0.2-0.4M N.F,USP MANNITOL; Then gynostemma pentaphylla protoplastis or sponge gourd protoplastis is obtained by the resuspended precipitation of protoplast culture medium containing 0.2M N.F,USP MANNITOL; And Gynostemma pentaphyllum Makino protoplastis, sponge gourd protoplastis mark respectively;
(2) gynostemma pentaphylla protoplastis and sponge gourd protoplast fusion and screening:
1) merge: gynostemma pentaphylla protoplastis and sponge gourd protoplastis equal-volume are mixed into after mixed solution and leave standstill, adopt cell fusion apparatus to merge, leave standstill after merging, and add protoplast culture medium and carry out centrifugal, will precipitate resuspended after centrifugal;
2) protoplastis screening is hybridized:
Select hybridization Protoplast cuhnre, after growing callus, carry out succeeding transfer culture;
(3) preparation of hybrid cell extract: by the gynostemma pentaphylla after succeeding transfer culture/sponge gourd hybrid cell ultrasonication, after filtration, lyophilize.
Described CPW washing lotion is by 101mg/L KNO
3, 27mg/LKH
2pO
4, 246mg/LMgSO
4.7H
20,1480mg/LCaC1
2.2H
2o is formulated.
Filter in described step (1) and adopt aperture to be not more than 70 μm of screen filtration; Repeat described CPW washing lotion suspend precipitation and centrifugal removing supernatant liquor is no less than 2 times; The consumption of protoplast culture medium be enzyme liquid long-pending 1/4 ~ 1/5.
In described step (1), Gynostemma pentaphyllum Makino protoplastis or sponge gourd protoplastis adopt the fluorescence dye of different colours to mark.
In described step (2), gynostemma pentaphylla protoplastis and sponge gourd protoplastis mixed solution leave standstill in fusion pond; Electro' asion liquid is 0.6moL/L N.F,USP MANNITOL and 0.1mmoL/L CaCl
2, be 7.0-7.5 by the pH of electro' asion liquid
20-30min is left standstill after gynostemma pentaphylla protoplastis described in described step (2) and sponge gourd protoplast fusion, then centrifuge tube is proceeded to, add 10 times to the protoplast culture medium of mixeding liquid volume, the centrifugal 5min of 200g, the resuspended dilution of precipitation protoplast culture medium after centrifugal, adopts the liquid culture method of shallow-layer to cultivate; After described hybridization protoplastis is screened, cultivate in the incubator of 25 DEG C ± 2 DEG C under dark surrounds; After described hybridization protoplastis grows callus, every three weeks succeeding transfer culture once.
By the gynostemma pentaphylla after described succeeding transfer culture/sponge gourd hybrid cell ultrasonication in step (3), and employing is not less than 0.22 μm of metre filter, lyophilize under (-100 DEG C)-(-60 DEG C) environment.
Gynostemma pentaphylla/sponge gourd hybrid cell extract that method of the present invention obtains is applied in skin care product.
Preferably, described skin care product are facial mask, toner, face cream, emulsion, eye cream, Cleansing Foam.
Preferably, the described gynostemma pentaphylla/addition of sponge gourd hybrid cell extract in skin care product is 10-50 μ g/ml.
Beneficial effect:
1, quality controllable.Adopt cell line extract, than using plant extract, the quality of production is more controlled.Because can not be subject to region, season, weather, disease and pest affects.
2, effect is better and nontoxic.Adopt the legal Gynostemma pentaphyllum Makino of electric smelting and sponge gourd protoplast fusion to obtain the clone of hybridizing, experiment shows that effect is better and nontoxic to organism than its effect of effect of comprehensive two plants of clone energy of single plant origin.
Accompanying drawing explanation
The antioxidant effect figure of each cell extract of Fig. 1
The each cell extract of Fig. 2 is to the effect diagram of photoaging human skin fibrocyte apoptosis
Fig. 3 each cell OD value schematic diagram
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.
Embodiment 1
The preparation of preparation gynostemma pentaphylla/sponge gourd hybrid cell extract, gynostemma pentaphylla cell extract, sponge gourd cell extract
1, gynostemma pentaphylla/sponge gourd hybrid cell extract is prepared
(1) gynostemma pentaphylla and sponge gourd protoplastis is obtained
1) enzyme liquid is prepared: choosing massfraction is respectively 1.0% cellulase, 0.1% polygalacturonase, 8% N.F,USP MANNITOL and CPW washing lotion (101mg/L KNO
3, 27mg/LKH
2pO
4, 246mg/LMgSO
4.7H
20,1480mg/LCaC1
2.2H
2o) enzyme liquid is mixed with, filtration sterilization.
2) callus cell choosing gynostemma pentaphylla and sponge gourd is respectively handled as follows: in the callus cell of gynostemma pentaphylla or sponge gourd described in every 1g, add 10ml enzyme liquid, in 27 DEG C and dark surrounds, and rotating speed controls enzymolysis 8h under the condition of 50r/min, by the disposable screen filtration of the protoplastis after enzymolysis by 70 μm of apertures, to remove non-enzymolysis cell mass completely.Collect filtrate and be placed in centrifuge tube, the supernatant liquor discarded after the centrifugal 5min of 200g; Suspend by CPW washing lotion and precipitate, get the centrifugal 5min of 200g after mixing, abandoning supernatant, repeats this step 3 time; Then 1 time is washed with MS liquid nutrient medium; Then gynostemma pentaphylla protoplastis or sponge gourd protoplastis is obtained by 2mL protoplast culture medium suspension precipitation;
In gynostemma pentaphylla protoplastis, mark red fluorescence dyestuff, in sponge gourd protoplastis, mark Green fluorescent dye.
(2) gynostemma pentaphylla protoplastis and sponge gourd protoplast fusion and screening:
Electro fusion method is adopted to carry out gynostemma pentaphylla and sponge gourd protoplast fusion.Electro' asion liquid is 0.6moL/L N.F,USP MANNITOL and 0.1mmoL/L CaCL
2, pH is 7.0, and carries out autoclaving to electro' asion liquid, and after protoplastis electro' asion liquid is resuspended, the centrifugal 5min of 200g, then suspends for subsequent use with electro' asion liquid.
1) merge: gynostemma pentaphylla protoplastis and sponge gourd protoplastis equal-volume are mixed into mixed solution, liquid will be merged and leave standstill about 10min to merging in pond, after protoplast pellet is stablized ac electric field strength 100V/cm, alternating-electric field action time be 35s, DC pulse intensity is 1000V/cm, DC pulse action time is 50 μ s, DC pulse number of times is 3 times, DC pulse interval time be the condition of 2s under merge; Leave standstill 30min after merging, then proceed to 15mL centrifuge tube, add protoplast culture medium to the centrifugal 6min of 8mL, 800r/min.Precipitation protoplast culture medium is diluted to low density (5x10
4individual/mL), draw lmL in the culture dish of diameter 3.5cm.
2) protoplastis screening is hybridized:
Under fluorescent microscope, red green protoplastis that is alternate or brown is marked, cultivates under putting into the incubator dark condition of temperature 25 DEG C.After growing callus, every three weeks subcultures once.
(3) preparation of hybrid cell extract: get the gynostemma pentaphylla after amplification/sponge gourd hybrid cell, after adding appropriate ultrapure water, ultrasonication, after the centrifugal 5min of 300g, crosses 0.22 μm of filter membrane, after BCA kit measurement protein concentration, for subsequent use.
2, gynostemma pentaphylla cell extract is prepared: preparation method is with the method preparing gynostemma pentaphylla/sponge gourd hybrid cell extract.
3, the preparation of sponge gourd cell extract: preparation method is with the method preparing gynostemma pentaphylla/sponge gourd hybrid cell extract.
Embodiment 2: the anti-oxidant experiment of gynostemma pentaphylla/sponge gourd hybrid cell extract, gynostemma pentaphylla cell extract, sponge gourd cell extract
The ability of the gynostemma pentaphylla/sponge gourd hybrid cell extract adopting DPPH method mensuration example one to prepare, gynostemma pentaphylla cell extract, sponge gourd cell extract scavenging free radicals.
It is 0.2mg/mL solution that gynostemma pentaphylla/sponge gourd hybrid cell extract, gynostemma pentaphylla cell extract, sponge gourd cell extract are mixed with concentration, respectively as experimental group.Adopt the xitix (VC) of 0.2mg/ml as positive controls.Gynostemma pentaphylla/sponge gourd hybrid cell extract, gynostemma pentaphylla cell extract, sponge gourd cell extract are reacted completely with the DPPH equal-volume of 0.2mg/ml respectively and after 30min, records final absorbancy.
Wherein, free radical scavenging activity (%)=[absorbancy of 1-(sample adds the absorbancy of the Xi Guang Du – sample of DPPH)/DPPH] × 100%
As shown in Figure 1, gynostemma pentaphylla/sponge gourd hybrid cell extract, gynostemma pentaphylla cell extract, sponge gourd cell extract three kinds of samples all have in various degree to anti-radical action, the best results of wherein gynostemma pentaphylla/sponge gourd hybrid cell extract sample.
Embodiment 3: gynostemma pentaphylla/sponge gourd hybrid cell extract, gynostemma pentaphylla cell extract, sponge gourd cell extract are on the impact of photoaging human skin fibrocyte apoptosis
The preparation of preparation gynostemma pentaphylla/sponge gourd hybrid cell extract, gynostemma pentaphylla cell extract, sponge gourd cell extract
1, gynostemma pentaphylla/sponge gourd hybrid cell extract is prepared
(1) gynostemma pentaphylla and sponge gourd protoplastis is obtained
1) enzyme liquid is prepared: choosing massfraction is respectively 2.0% cellulase, 0.2% polygalacturonase, 12% N.F,USP MANNITOL and CPW washing lotion (101mg/L KNO
3, 27mg/LKH
2pO
4, 246mg/LMgSO
4.7H
20,1480mg/LCaC1
2.2H
2o) enzyme liquid is mixed with, filtration sterilization.
2) callus cell choosing gynostemma pentaphylla and sponge gourd is respectively handled as follows: in the callus cell of gynostemma pentaphylla or sponge gourd described in every 3g, add 10ml enzyme liquid, in 30 DEG C and dark surrounds, and rotating speed controls enzymolysis 12h under the condition of 100r/min, by the disposable screen filtration of the protoplastis after enzymolysis by 70 μm of apertures, to remove non-enzymolysis cell mass completely.Collect filtrate and be placed in centrifuge tube, the supernatant liquor discarded after centrifugal 5min under centrifugal force is 200g; With CPW washing lotion suspend precipitation, after mixing under centrifugal force 200g centrifugal 5min, abandoning supernatant, repeats this step 3 time; Then 1 time is washed with MS liquid nutrient medium; Then gynostemma pentaphylla protoplastis or sponge gourd protoplastis is obtained by 2mL protoplast culture medium suspension precipitation; In gynostemma pentaphylla protoplastis, mark red fluorescence dyestuff, in sponge gourd protoplastis, mark Green fluorescent dye.
(2) gynostemma pentaphylla protoplastis and sponge gourd protoplast fusion and screening:
Electro fusion method is adopted to carry out gynostemma pentaphylla and sponge gourd protoplast fusion.Electro' asion liquid is 0.6moL/L N.F,USP MANNITOL and 0.1mmoL/L CaCL
2, pH is 7.5, and carries out autoclaving to electro' asion liquid, and after protoplastis electro' asion liquid is resuspended, centrifugal 5min under centrifugal force is 200g, then suspends for subsequent use with electro' asion liquid.
1) merge: gynostemma pentaphylla protoplastis and sponge gourd protoplastis equal-volume are mixed into mixed solution, liquid will be merged and leave standstill about 10min to merging in pond, after protoplast pellet is stablized ac electric field strength 100V/cm, alternating-electric field action time be 35s, DC pulse intensity is 1000V/cm, DC pulse action time is 50 μ s, DC pulse number of times is 3 times, DC pulse interval time be the condition of 2s under merge; Leave standstill 30min after merging, then proceed to 15mL centrifuge tube, add protoplast culture medium to the centrifugal 6min of 8mL, 800r/min.Precipitation protoplast culture medium is diluted to low density (5x10
4individual/mL), draw lmL in the culture dish of diameter 3.5cm.
2) protoplastis screening is hybridized:
Under fluorescent microscope, red green protoplastis that is alternate or brown is marked, cultivates under putting into the incubator dark condition of temperature 25 DEG C.After growing callus, every three weeks subcultures once.
(3) preparation of hybrid cell extract: get the gynostemma pentaphylla after amplification/sponge gourd hybrid cell for subsequent use.
2, gynostemma pentaphylla cell extract is prepared: preparation method is with the method preparing gynostemma pentaphylla/sponge gourd hybrid cell extract.
3, the preparation of sponge gourd cell extract: preparation method is with the method preparing gynostemma pentaphylla/sponge gourd hybrid cell extract.
Gynostemma pentaphylla/sponge gourd hybrid cell the extract adopting TUNEL method mensuration embodiment three to prepare, gynostemma pentaphylla cell extract, sponge gourd cell extract are on the impact of human dermis's layer fibroblast growth.
Detect sample: gynostemma pentaphylla/sponge gourd hybrid cell extract, gynostemma pentaphylla cell extract, sponge gourd cell extract prepared by embodiment 3.
Biological subject body: HSF cell.
Concrete steps are as follows:
(1) employing volume ratio is DMEM/F12 substratum (penicillin 200U/ml, the Streptomycin sulphate 250U/ml of 10% foetal calf serum (FBS), pH7.2) HSF cell is cultivated, when cell density reaches fusion 80%, utilize 0.25% trypsin digestion cell, with PBS so that 1x104/ml is inoculated in T25 bottle, photoaging HSF cell model is set up, irradiation dose 36J/cm with employing long wave ultraviolet (UVA) illuminating method
2.
(2) respectively with perfect medium dilute hybrid cell, sponge gourd cell, gynostemma pentaphylla cell extract to final concentration be 50 μ g/ml, by 0.22 μm of disposable screen filtration.HSF, after irradiating, after abandoning PBS, adds appeal substratum respectively.37 DEG C, 5%CO2, saturated humidity cell culture incubator in continue cultivate; To add the perfect medium of not application of sample for model group, with the HSF without uv irradiating for control group.
(3), after 12h, apoptosis number is detected by the method for TUNEL apoptosis in-situ detection reagent box.
As shown in Figure 2, three sample standard deviations have anti-light aging effect, and wherein gynostemma pentaphylla/sponge gourd hybrid cell extract obviously can suppress the apoptosis of photoaging HSF.
Embodiment 4: the toxicity assessment of gynostemma pentaphylla/sponge gourd hybrid cell extract
The present embodiment adopts to be evaluated the toxicity of gynostemma pentaphylla/sponge gourd hybrid cell extract prepared by embodiment 1.Mainly be divided into three part tests, i.e. the rabbit skin phototoxicity test of chmice acute Oral toxicity test (method of once limiting the quantity) of gynostemma pentaphylla/sponge gourd hybrid cell extract, the rat acute percutaneous toxicity test (method of once limiting the quantity) of gynostemma pentaphylla/sponge gourd hybrid cell extract and gynostemma pentaphylla/sponge gourd hybrid cell extract.
Chmice acute Oral toxicity test (method of once limiting the quantity) of one, gynostemma pentaphylla/sponge gourd hybrid cell extract
Sample: adopt gynostemma pentaphylla/sponge gourd hybrid cell extract prepared by embodiment 1.
Biological subject body: kunming mice.
Test method:
Preliminary in vitro is tested: hybrid cell extract (i.e. gynostemma pentaphylla/sponge gourd hybrid cell extract) is configured to 50 μ g/ml concentration, add in substratum, its multiplication capacity is detected: get HSF cell by CCK-8 method, be inoculated in 96 orifice plates with 2000, every hole cell, get first, three, five, the cell of seven days detects, and adds 10 μ l staining agents, at cultivation 2h.Detect OD value by microplate reader at 450nm place, compare with the culture medium culturing of not adding hybrid cell extract, often organize the repetition of 6, work, average, its experiment the results are shown in Figure 3.
As can be known from Fig. 3, hybrid cell extract does not have toxic effect to HSF, and Growth of Cells is normal, even has certain promoter action.
Therefore, the result of preliminary in vitro experiments shows, gynostemma pentaphylla/sponge gourd hybrid cell extract is substantially nontoxic, therefore method of once limiting the quantity is adopted to confirm its security to organism, namely once maximum given low adopts its 10 times of actual effects to indicate the tested material of doses (50.0 ± 0.5 μ l/ prop up × 10) to 10 kunming mice (male and female half and half, body weight is 20.0 ± 0.2g) carry out gavage, observe its toxic reaction, when not causing animal dead, then no longer carry out the acute oral toxicity test of multiple dosage.
Observe the time limit: 14 days.
Experimental result is observed: not observing experimental animal has any untoward reaction, does not also have animal dead.Illustrate that gynostemma pentaphylla of the present invention/sponge gourd hybrid cell extract freeze-drying powder is without acute oral toxicity effect.
The rat acute percutaneous toxicity test (method of once limiting the quantity) of two, gynostemma pentaphylla/sponge gourd hybrid cell extract
Sample: gynostemma pentaphylla/sponge gourd hybrid cell extract prepared by embodiment 1.
Biological subject body: SD rat (Guangdong Province's animal experimental center).
Test method:
The result of preliminary in vitro experiments shows, gynostemma pentaphylla/sponge gourd hybrid cell extract is substantially nontoxic, therefore method of once limiting the quantity is adopted to confirm its security to organism further, namely 10 animal (male and female half and half are used, body weight is 149.1 ± 12.4g), gynostemma pentaphylla/sponge gourd hybrid cell the extract of dermal application 2000mg/kg body weight dose, when not causing animal dead, then no longer carries out the acute dermal toxicity test of multiple dosage.
Observe the time limit: 14 days.
Observations: not observing experimental animal has any untoward reaction, does not also have animal dead.Illustrate that gynostemma pentaphylla of the present invention/sponge gourd hybrid cell extract freeze-drying powder is without skin toxic action.
The rabbit skin phototoxicity test of three, gynostemma pentaphylla/sponge gourd hybrid cell extract
Sample: gynostemma pentaphylla/sponge gourd hybrid cell extract prepared by embodiment 1.
Biological subject body: white rabbit (Guangdong Province's animal experimental center) 50 of growing up, female half and half.
UV light source: wavelength is the UVA of 320nm ~ 400nm.
Irradiation time: 1.5h.
Testing sequence:
Carry out the front 18 ~ 24h of formal light poison test, by rabbit backbone both sides skin unhairing, test area skin needs intact, can't harm and injures exception; Standby 4 pieces of unhairing districts, every block unhairing area is about 2cm × 2cm, and the test arrangement of unhaired hide is as shown in table 1; Animal is fixed, according to following table, applies 1m gynostemma pentaphylla/sponge gourd hybrid cell extract respectively in animal unhairing district 1 and 2; After 30min, left side (unhairing district 1 and 3) is topped with aluminium foil, and adhesive tape is fixed, right side UVA irradiates, observe skin reaction respectively at 1,24,48 and 72h after end, judge whether there is irritant reaction to skin according to skin wound repair grade form, result is as shown in table 2.
The test arrangement in table 1 animal unhairing district
| Unhairing district numbers | Test process |
| 1 | Be coated with tested material, do not irradiate |
| 2 | Be coated with tested material, irradiate |
| 3 | Be not coated with tested material, do not irradiate |
| 4 | Be not coated with tested material, irradiate |
Reaction grade form is commented in table 2 skin irritation
Test-results shows: 60 tested rabbit after treatment 1h, 24h, 48h and 72h all do not observe skin wound repair, show that gynostemma pentaphylla of the present invention/sponge gourd hybrid cell extract freeze-drying powder is without phototoxic action.
The announcement of book and instruction according to the above description, those skilled in the art in the invention can also change above-mentioned embodiment and revise.Therefore, the present invention is not limited to embodiment disclosed and described above, also should fall in the protection domain of claim of the present invention modifications and changes more of the present invention.In addition, although employ some specific terms in this specification sheets, these terms just for convenience of description, do not form any restriction to the present invention.
Claims (10)
1. prepare a method for gynostemma pentaphylla/sponge gourd hybrid cell extract, it is characterized in that, comprise the following steps:
(1) protoplastis is obtained:
1) enzyme liquid is prepared: choose that massfraction is 1.0-2.0% cellulase, 0.1-0.2% polygalacturonase, 6-12% N.F,USP MANNITOL and CPW washing lotion are mixed with enzyme liquid respectively;
2) callus cell choosing gynostemma pentaphylla and sponge gourd is respectively handled as follows: be (1-3) in callus cell and enzyme liquid mass ratio: the ratio of 10 will add in enzyme liquid in the callus cell of described gynostemma pentaphylla or sponge gourd, in 23-30 DEG C of dark surrounds, rotating speed is enzymolysis 8-12h under the condition of 50-100r/min, filter and collect filtrate and be placed in centrifuge tube, abandoning supernatant after the centrifugal 2-15min of 100g-300g; Suspend by CPW washing lotion and precipitate, get the centrifugal 2-15min of 100g-300g after mixing, abandoning supernatant; Then wash with containing the MS liquid culture medium of 0.2-0.4M N.F,USP MANNITOL and the protoplast culture medium of 0.2-0.4M N.F,USP MANNITOL; Then gynostemma pentaphylla protoplastis or sponge gourd protoplastis is obtained by the resuspended precipitation of protoplast culture medium containing 0.2M N.F,USP MANNITOL; And Gynostemma pentaphyllum Makino protoplastis, sponge gourd protoplastis mark respectively;
(2) gynostemma pentaphylla protoplastis and sponge gourd protoplast fusion and screening:
1) merge: leave standstill after gynostemma pentaphylla protoplastis and sponge gourd protoplastis equal-volume being mixed into mixed solution, adopt cell fusion apparatus to merge, electro' asion liquid is 0.6moL/L N.F,USP MANNITOL and 0.1mmoL/L CaCl
2, leave standstill after merging, and add protoplast culture medium and carry out centrifugal, will precipitate resuspended after centrifugal;
2) protoplastis screening is hybridized:
Select hybridization Protoplast cuhnre, after growing callus, carry out succeeding transfer culture;
(3) preparation of hybrid cell extract: the gynostemma pentaphylla after succeeding transfer culture/sponge gourd hybrid cell is dry.
2. a kind of method preparing gynostemma pentaphylla/sponge gourd hybrid cell extract according to claim 1, it is characterized in that, described CPW washing lotion is by 101mg/L KNO
3, 27mg/L KH
2pO
4, 246mg/L MgSO
4.7H
20,1480mg/L CaC1
2.2H
2o is formulated.
3. a kind of method preparing gynostemma pentaphylla/sponge gourd hybrid cell extract according to claim 1, is characterized in that, filters and adopt aperture to be not more than 70 μm of screen filtration in described step (1); Repeat described CPW washing lotion suspend precipitation and centrifugal removing supernatant liquor is no less than 2 times; The consumption of protoplast culture medium be enzyme liquid long-pending 1/4 ~ 1/5.
4. a kind of method preparing gynostemma pentaphylla/sponge gourd hybrid cell extract according to claim 1, is characterized in that, in described step (1), Gynostemma pentaphyllum Makino protoplastis or sponge gourd protoplastis adopt the fluorescence dye of different colours to mark.
5. a kind of method preparing gynostemma pentaphylla/sponge gourd hybrid cell extract according to claim 1, is characterized in that, in described step (2), gynostemma pentaphylla protoplastis and sponge gourd protoplastis mixed solution leave standstill in fusion pond; The pH of described electro' asion liquid is 7.0-7.5.
6. a kind of method preparing gynostemma pentaphylla/sponge gourd hybrid cell extract according to claim 1, it is characterized in that, 20-30min is left standstill after gynostemma pentaphylla protoplastis described in step (2) and sponge gourd protoplast fusion, then centrifuge tube is proceeded to, add 10 times to the protoplast culture medium of mixeding liquid volume, the centrifugal 5min of 200g, the resuspended dilution of precipitation protoplast culture medium after centrifugal, adopts the liquid culture method of shallow-layer to cultivate; After described hybridization protoplastis is screened, cultivate in the incubator of 25 DEG C ± 2 DEG C under dark surrounds; After described hybridization protoplastis grows callus, every three weeks succeeding transfer culture once.
7. a kind of method preparing gynostemma pentaphylla/sponge gourd hybrid cell extract according to claim 1, it is characterized in that, in step (3), the gynostemma pentaphylla after described succeeding transfer culture/sponge gourd hybrid cell is freezing under (-100 DEG C)-(-60 DEG C) environment, grind into powder after dry, and employing is not less than 60 object screen clothes screenings.
8. gynostemma pentaphylla/sponge gourd hybrid cell extract that method according to claim 1 is obtained is applied in skin care product.
9. apply as claimed in claim 8, it is characterized in that: described skin care product are facial mask, toner, face cream, emulsion, eye cream, Cleansing Foam.
10. apply as claimed in claim 8, it is characterized in that: the described gynostemma pentaphylla/addition of sponge gourd hybrid cell extract in skin care product is 10-50 μ g/ml.
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| CN110215490A (en) * | 2019-07-05 | 2019-09-10 | 任辛 | A kind of preparation method of plant cell enzymatic hydrolyzed extract that treating women HPV viruse |
| CN110279836A (en) * | 2019-07-05 | 2019-09-27 | 任辛 | A kind of preparation method of plant cell enzymatic hydrolyzed extract that treating helicobacter pylori |
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| JPH02222676A (en) * | 1989-02-23 | 1990-09-05 | Nonogawa Shoji:Kk | Crossbred cell, production thereof and cosmetic |
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| CN110279836A (en) * | 2019-07-05 | 2019-09-27 | 任辛 | A kind of preparation method of plant cell enzymatic hydrolyzed extract that treating helicobacter pylori |
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Application publication date: 20150527 |