CN112353741A

CN112353741A - Anti-wrinkle composition and anti-wrinkle cream

Info

Publication number: CN112353741A
Application number: CN202011544525.3A
Authority: CN
Inventors: 潘兰英; 潘宏川
Original assignee: Ruizilan Hangzhou Biotechnology Co ltd
Current assignee: Ruizilan Hangzhou Biotechnology Co ltd
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-02-12
Anticipated expiration: 2040-12-24
Also published as: CN112353741B

Abstract

The invention provides an anti-wrinkle composition and an anti-wrinkle cream. The anti-wrinkle composition comprises a polygonatum sibiricum extract, a dogwood extract and a kumquat extract in a weight ratio of 5-7: 3-6: 1. The anti-wrinkle composition comprises a polygonatum extract, a dogwood extract and a kumquat extract, and can block voltage-dependent Nav1.4 channels on cell membranes in muscles, so that the release of ions is influenced, the action target is clear, and the effect is clear; and the use of the rhizoma polygonati extract and the dogwood extract promotes the absorption of the kumquat extract. The compounds in each extract of the present application have small molecular weights, are easily penetrated through the skin, can achieve the effect of significantly relaxing excessive single muscle tension, and can also alleviate and improve wrinkles that have already occurred.

Description

Anti-wrinkle composition and anti-wrinkle cream

Technical Field

The invention relates to the technical field of anti-wrinkle, in particular to an anti-wrinkle composition and an anti-wrinkle cream.

Background

With age, metabolism of the human body begins to slow down, moisture and subcutaneous fat are reduced, elastic fibers become thicker gradually, and the skin becomes wrinkled and glossy and fades. The aging of the skin is the prominent expression of the aging of the whole body, wherein the aging of the skin on the face is particularly obvious, the number of fibroblasts in the human body is gradually reduced, the secreted collagen and the elastic fibrin are gradually reduced and broken by oxidation, the supporting effect on the skin is eliminated, the inhomogeneous collapse is caused, the dermis layer begins to be thinned, the elasticity of the skin is deteriorated, and wrinkles begin to be gradually generated. Although skin aging is an irreversible physiological phenomenon, its progress can be slowed or slowed. For facial wrinkles, anti-wrinkle cosmetics are most commonly used.

At present, anti-wrinkle cosmetics mostly achieve an anti-oxidation function from the viewpoint of improving the anti-oxidation capacity of skin, the principle of anti-oxidation is basically to resist free radicals generated by a human body, and the main substances with the anti-oxidation function known at present comprise vitamin A/C/E, procyanidine PC, coenzyme Q10, astaxanthin, yeast extract and the like. However, since there are many types of radicals, it is difficult to include an effective component against various radicals in one product, and thus it is difficult to further improve the anti-wrinkle function of an antioxidant product.

Disclosure of Invention

The invention mainly aims to provide an anti-wrinkle composition and an anti-wrinkle cream, and aims to solve the problem that the anti-wrinkle function realized by an antioxidant technology in the prior art is limited.

In order to achieve the above object, according to one aspect of the present invention, an anti-wrinkle composition is provided, which includes polygonatum extract, cornus officinalis extract and kumquat extract in a weight ratio of 5-7: 3-6: 1.

Furthermore, the kumquat extract is a kumquat water extract and/or a kumquat alcohol extract.

Further, the preparation method of the kumquat aqueous extract comprises the following steps: heating and refluxing kumquat peel by adopting water to extract to obtain a water extracting solution; and carrying out solid-liquid separation and rotary evaporation on the water extracting solution to obtain a kumquat water extract, wherein the volume of the kumquat water extract is 1/8-1/3 of the volume of the water extracting solution, the weight ratio of water to kumquat peel is preferably 7-14: 1, and the time for heating reflux extraction is preferably 1-5 hours.

Further, the preparation method of the kumquat alcohol extract comprises the following steps: heating and refluxing the kumquat peel by using an ethanol solution to obtain an ethanol extract; and carrying out solid-liquid separation and rotary evaporation on the alcohol extract to obtain the kumquat alcohol extract, wherein the volume of the kumquat alcohol extract is 1/8-1/3 of the volume of the alcohol extract, preferably the weight ratio of the alcohol solution to kumquat peel is 7-14: 1 in terms of 20% of alcohol solution, and the time for heating reflux extraction is 1-5 h.

Further, the polygonatum sibiricum extract and the dogwood extract are respectively and independently alcohol extracts or water extracts.

Further, the preparation method of the polygonatum sibiricum extract or the dogwood extract comprises the following steps: mixing rhizoma polygonati or dogwood with water according to the proportion of 1 g: 20-30 mL, and leaching for 50-90 min at 50-90 ℃ to obtain a rhizoma polygonati extract or dogwood extract.

According to another aspect of the invention, the anti-wrinkle cream comprises an anti-wrinkle effective component and an auxiliary material, wherein the anti-wrinkle effective component is formed by mixing any one of the anti-wrinkle compositions.

Furthermore, the anti-wrinkle effective component in the anti-wrinkle cream is 3-10% by weight.

Further, the above adjuvants include stearic acid, glyceryl monostearate, liquid paraffin, white vaseline, glycerol, ultrapure water, triethanolamine and Tween 80.

Further, the ratio of the stearic acid, the glyceryl monostearate, the liquid paraffin, the white vaseline, the glycerol, the ultrapure water, the triethanolamine and the Tween 80 is 2-3 g: 1-1.5 g: 1.0-2.2 g: 0.2-1.0 g: 1.0-2.5 g: 2-8 mL: 0.05-0.1 mg: 0.14-0.2 mL.

By applying the technical scheme, the anti-wrinkle composition comprises the polygonatum extract, the dogwood extract and the kumquat extract, and can block voltage-dependent Nav1.4 channels on cell membranes in muscles, so that the release of ions is influenced, the action target is clear, and the effect is clear; and the use of the rhizoma polygonati extract and the dogwood extract promotes the absorption of the kumquat extract. The compounds in each extract of the present application have small molecular weights, are easily penetrated through the skin, can achieve the effect of significantly relaxing excessive single muscle tension, and can also alleviate and improve wrinkles that have already occurred.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows a cell and patch clamp tip approaching cell membrane under microscope according to example 1 of the present invention;

FIG. 2 shows the current transformation before and after addition of the mixture according to example 1 of the present invention; and

FIG. 3 shows the perfusion inhibiting effect of the mixture according to example 1 of the invention on Nav1.4 in current pA.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

As analyzed in the background of the present application, it is difficult for the prior art to include effective ingredients against various free radicals in a product due to the large number of free radical types, which results in the difficulty in further improving the anti-wrinkle function of the antioxidant product. To address this problem, the present application attempts other anti-wrinkle pathways. The present application found that facial skin produces numerous expression lines, such as a head-up line, a fishtail line, and vertical folds between eyes, due to strong muscle contraction as the skin adheres to the muscle surface. Therefore, the anti-wrinkle effect should be achieved by relieving the degree of muscle contraction. Based on the thought, the factors influencing muscle contraction are researched, and the Nav1.4 channel is found to have close relation with muscle contraction.

To facilitate understanding of the technical principles of the present application by those skilled in the art, Nav sodium ions are illustrated below:

sodium ions are composed of an alpha subunit responsible for sensing voltage and ion selection, and 1-2 beta subunits that regulate it, and in humans there are 9 different subtypes of the alpha subunit, named Nav1.1-1.9, respectively, and 4 subtypes of the beta subunit, named beta 1-beta 4, respectively.

The alpha subunit typically comprises about 2000 amino acids and includes four structurally similar transmembrane domains I-IV, each containing 6 transmembrane helices, in close sequence. The first four transmembrane helices S1-S4 in each domain form a Voltage Sensor (VSD), the four groups of S5-S6 transmembrane helices jointly form a pore domain responsible for ion permeation in the center of the whole structure, and the two semi-transmembrane short helices connecting the transmembrane helices S5-S6 jointly support a small stretch sequence to form a selection screen responsible for ion permeation specificity. The relatively short sequence connecting the third and fourth domains has proven to be critical for rapid inactivation of sodium channels, but the specific mechanism is not clear.

The rapid inactivation mechanism allows the sodium channel to be rapidly closed after the sodium channel is stimulated to cause action potential, thereby preventing the continuous discharge of neuromuscular cells. Meanwhile, the sodium-potassium pump rebuilds the membrane potential to prepare for the next action potential. Rapid inactivation of the sodium channel is therefore critical for its normal physiological function.

Voltage-gated sodium channels are transmembrane glycoprotein complexes consisting of a large alpha subunit with 24 transmembrane domains and one or more regulatory beta subunits, encoded by the SCN4A gene in humans. Mutations in the coding SCN4A gene are associated with low potassium periodic paralysis, high potassium periodic paralysis, paramyosis and potassium-aggravated myotonia. Different sodium ion subtypes produce a variety of pharmacological effects, while transmembrane glycoprotein complexes are responsible for the generation and spread of action potentials in neurons and muscles. The gene encodes a member of the sodium channel alpha subunit gene family. It is expressed in skeletal muscle, and mutations in this gene have been associated with several myotonic and periodic paralytic disorders.

If the voltage-dependent Nav1.4 channel on the cell membrane in the muscle can be blocked, the release of ions is influenced, and then the contracted muscle is quickly recovered, so that the anti-wrinkle effect is realized. Further research in the application finds that the extracts of kumquat and the like have obvious effect on blocking voltage-dependent Nav1.4 channels on cell membranes in muscles, so the application provides an anti-wrinkle composition and an anti-wrinkle cream.

In an exemplary embodiment of the present application, an anti-wrinkle composition is provided, which includes an extract of polygonatum sibiricum, an extract of cornus officinalis and an extract of kumquat in a weight ratio of 5-7: 3-6: 1.

The anti-wrinkle composition comprises a polygonatum sibiricum extract, a dogwood extract and a kumquat extract, and can block voltage-dependent Nav1.4 channels on cell membranes in muscles, so that release of ions is influenced, an action target is clear, and an effect is clear. The compounds in each extract of the present application have small molecular weights, are easily penetrated through the skin, can achieve the effect of significantly relaxing excessive single muscle tension, and can also alleviate and improve wrinkles that have already occurred. Of course, it can also be applied to symptoms such as excessive neuromuscular tension.

Although it is not clear which specific component or which components of the above extracts act synergistically to block the voltage-dependent Nav1.4 channel on the cell membrane in the muscle, it can be determined that either kumquat aqueous extract or kumquat alcohol extract can achieve the above effects when applied to the above anti-wrinkle composition, and thus the kumquat extracts of the present application include, but are not limited to, kumquat aqueous extract and/or kumquat alcohol extract.

In order to facilitate the preparation of the anti-wrinkle composition of the present application, and to stabilize the discovery of the anti-wrinkle effect of the anti-wrinkle composition of the present application, some embodiments of the present application also provide a method for preparing an aqueous kumquat extract comprising: heating and refluxing kumquat peel by adopting water to extract to obtain a water extracting solution; and carrying out solid-liquid separation and rotary evaporation on the water extracting solution to obtain a kumquat water extract, wherein the volume of the kumquat water extract is 1/8-1/3 of the volume of the water extracting solution, the weight ratio of water to kumquat peel is preferably 7-14: 1, and the time for heating reflux extraction is preferably 1-5 hours. In the prior art, kumquat aqueous extracts obtained by other extraction methods also have an anti-wrinkle effect to a certain extent, but the preparation method is simple and easy to operate, and the obtained kumquat aqueous extracts have a more remarkable effect on blocking voltage-dependent Nav1.4 channels on cell membranes in muscles when being applied to the anti-wrinkle composition.

Similarly, some embodiments of the present application further provide a preparation method of the kumquat alcohol extract, including: heating and refluxing the kumquat peel by using an ethanol solution to obtain an ethanol extract; and carrying out solid-liquid separation and rotary evaporation on the alcohol extract to obtain the kumquat alcohol extract, wherein the volume of the kumquat alcohol extract is 1/8-1/3 of the volume of the alcohol extract, preferably the weight ratio of the alcohol solution to kumquat peel is 7-14: 1 in terms of 20% of alcohol solution, and the time for heating reflux extraction is 1-5 h. The kumquat alcohol extract obtained by other extraction methods in the prior art also has an anti-wrinkle effect to a certain extent, but the preparation method is simple and easy to operate, and the obtained kumquat alcohol extract has a more significant effect on blocking voltage-dependent Nav1.4 channels on cell membranes in muscles when being applied to the anti-wrinkle composition.

The polygonatum sibiricum extract and the cornus officinalis extract used in the present application are not particularly limited, and those skilled in the art may select respective alcohol extracts or water extracts as the polygonatum sibiricum extract and the cornus officinalis extract.

In a preferred embodiment, the preparation method of the polygonatum sibiricum extract or the dogwood extract comprises the following steps: mixing rhizoma polygonati or dogwood with water according to the proportion of 1 g: 20-30 mL, and leaching for 50-90 min at 50-90 ℃ to obtain a rhizoma polygonati extract or dogwood extract. The preparation method is easy to realize, and the cooperation effect of the obtained extract and the kumquat extract is more proposed, so that the kumquat extract can be absorbed more easily.

For use of the anti-wrinkle composition of the present application as an anti-wrinkle product, in another exemplary embodiment of the present application, there is provided an anti-wrinkle cream comprising an anti-wrinkle active ingredient mixed with any of the anti-wrinkle compositions described above and an adjuvant. The anti-wrinkle cream is formed by mixing the anti-wrinkle composition with auxiliary materials, so that the convenience of use of consumers is realized.

In one embodiment, the anti-wrinkle cream comprises 3-10 wt% of anti-wrinkle active ingredients. The anti-wrinkle cream has sufficient content of anti-wrinkle effective components, and the dosage of the auxiliary materials is enough to keep the stability of the anti-wrinkle cream under the conventional use and storage conditions.

The auxiliary materials used for the application can adopt the auxiliary materials commonly used in the current conventional skin cream, and in order to ensure the stability of the anti-wrinkle cream under the conditions of normal temperature, high temperature and low temperature, the preferable auxiliary materials comprise stearic acid, glyceryl monostearate, liquid paraffin, white vaseline, glycerol, ultrapure water, triethanolamine and tween 80. Further preferably stearic acid, glyceryl monostearate, liquid paraffin, white vaseline, glycerol, ultrapure water, triethanolamine and Tween 80 in a ratio of 2-3 g: 1-1.5g g: 1.0-2.2 g: 0.2-1.0 g: 1.0-2.5 g: 2-8 mL: 0.05-0.1 mL: 0.14-0.2 mL

Of course, the anti-wrinkle composition of the present application may be prepared not only as an anti-wrinkle of the anti-wrinkle cream, but also as a single use or as an emulsion, a spray, etc., and the specific combination thereof may refer to the prior art and is not described herein again.

The following provides a description of advantageous effects of the present application with reference to examples and comparative examples.

Example 1

1. Raw material preparation

Kumquat alcohol extract: heating and refluxing 50g of fresh kumquat peel by using 500mL of 20% ethanol solution for 3 times, wherein each time is 1h, combining 3 extracting solutions, performing suction filtration, and performing rotary evaporation to obtain about 50mL of ethanol extract.

Kumquat aqueous extract: extracting 50g fresh kumquat peel with 500mL water under heating and refluxing for 3 times, each time for 1h, mixing the 3 extractive solutions, suction filtering, and rotary evaporating to obtain about 50mL water extract.

And (3) extracting rhizoma polygonati: leaching 20g of dogwood by using 500mL of water at 50-90 ℃ for 50-90 min to obtain a polygonatum sibiricum extract;

and (3) extracting dogwood fruit: leaching 20g of dogwood by 500mL of water at 50-90 ℃ for 50-90 min to obtain the dogwood extract.

Mixing the rhizoma Polygonati extract, Corni fructus extract and Fortunella margarita (lour.) Swingle water extract at a weight ratio of 5: 1 to form a mixture, and performing cell patch clamp experiment of the mixture on Nav1.4 channel blocking effect to determine the Nav1.4 channel blocking effect of the extracts.

2. Test cells

The CHO cell line stably transfected with Na1.4 Na ion channel was awarded by Olifu Biotech, Inc., Del.

2.1 assay-related reagent preparation

One) extracellular fluid (1L):

adding 8.01g of NaCl, 0.30g of KCl, 1.8ml of CaCl2(1m mol/L), 1ml of MgCl2(1m mol/L), 1.80g of Glucose and 2.39g of HEPES reagents into a beaker of 800ml of sterile water, uniformly stirring, adding 200ml of sterile water, and keeping the volume to 1L; adjusting the pH value to 7.35-7.40 with NaOH according to the display operation of a pH meter; filtering with a suction filtration device, filling the prepared reagent into a glass bottle, tightly covering, and storing in a refrigerator at 4 ℃ for later use.

II) intracellular fluid (1L):

2.4457g of KASP, 0.2383g of HEPES, 0.0407g of MgCl2 and 0.481g of EGTA are weighed and added into a beaker of 800ml of sterile water, the mixture is stirred and mixed evenly, and 200ml of sterile water is added to the beaker until the volume is 1L; adjusting the pH value to 7.35-7.40 with NaOH according to the display operation of a pH meter; filtering with suction filtration device, subpackaging into 1.5ml PE tube, and storing in-20 deg.C refrigerator for use.

And thirdly, preparing a (10% FBS + P/S) DMEM culture medium, a (10% FBS) DMEM non-resistant culture medium, a DMEM (20% FBS + P/S) cell freezing medium and a 20% DMSO cell freezing medium:

reagent: calf serum (FBS) solution, 100 XP/S (penicillin + streptomycin) solution; DMEM medium (containing L-glutamine), DMSO

The method comprises the following steps: 1. preparing an ultra-clean bench: putting a pipette, a gun head and the like into a super clean bench in advance, opening ultraviolet sterilization for 0.5h, ventilating for 15min, and igniting an alcohol lamp;

2. taking out 45ml of FBS solution and 5ml of 100 XP/S solution which are already separately prepared for use from a refrigerator at the temperature of-20 ℃; taking out a bottle of DMEM solution at 4 ℃ in a refrigerator; putting all the materials into a constant-temperature water bath box at 37 ℃ until the temperature reaches the room temperature for later use;

3. and when the FBS, DMEM and P/S solution reaches the room temperature, taking out the solution from the water bath box, and wiping water drops in the bottle body with a paper towel. Spraying 70% sterilized alcohol to the bottom and neck of the bottle, wiping with paper towel, and transferring to a sterile operating table with lighted alcohol lamp;

4. opening a DMEM culture bottle, placing the bottle cap with the bottle mouth facing downwards, enabling the bottle mouth to pass through flame on an alcohol lamp, taking a 50ml centrifuge tube filled with 45ml of FBS, opening a cover, enabling the bottle mouth to pass through flame, immediately pouring into 500ml of DMEM, enabling the content of FBS in the DMEM to almost reach 10%, and covering the cover;

5-1 DMEM (10% FBS + P/S): another 15ml centrifuge tube containing 5ml P/S (penicillin + streptomycin) was poured into 500ml DMEM containing FBS (10%) prepared in the same way;

5-2 DMEM (10% FBS): preparing nonreactive DMEM only by the fourth step, well shaking and subpackaging for storage.

5-3 DMEM (20% FBS + P/S): cell cryopreservation solution: injecting 18ml of DMEM (10% FBS + P/S) into a 50ml centrifuge tube, and adding 2ml of FBS (fetal bovine serum);

5-420% DMSO cell lysates: injecting 8ml of prepared DMEM (20% FBS + P/S) into a 15ml centrifuge tube, transferring 2ml of DMSO by using a liquid transfer gun, slowly adding dropwise, and continuously mixing uniformly;

6. and (3) timely sticking a label on the prepared reagent bottle, marking the name of the reagent and the preparation time, and storing in a common refrigerator for later use.

Fourthly) preparation of plant extract solution: the plant extract is extracted independently in a laboratory. Dissolving the plant extract with DMSO as solvent to obtain 50mmol/L stock concentration, sequentially diluting to 9mmol/L, 3mmol/L, 0.9mmol/L, and 0.3mmol/L, and storing in PE tubule at-20 deg.C in 167 μ L according to test concentration. In the test, one of each concentration was diluted into 50ml of extracellular fluid, and the concentrations were 30. mu. mol/L, 10. mu. mol/L, 3. mu. mol/L, and 1. mu. mol/L, respectively, in this order.

The cell culture process is as follows:

one) recovery and liquid change of cells

Reagents and materials: a 10ml pipette, pipette gun (10 microliters, 200 microliters, 1000 microliters), tip (10 microliters, 200 microliters, 1000 microliters), centrifuge tube (15ml), petri dish (100mm), alcohol (75%), alcohol lamp, lighter, scissors, waste liquid jar, napkin;

the instrument comprises the following steps: a 37 ℃ constant-temperature water bath kettle, a super clean bench (100 grade) and a 37 ℃ cell culture box;

reagent: DMEM (10% FBS + P/S);

1. putting the experimental material to be used on a super clean bench, turning on ultraviolet, sterilizing for 0.5h, and turning off an ultraviolet turning-on fan to blow for about 15 min;

2. DMEM (10% FBS + P/S) is put into a constant-temperature water bath kettle at 37 ℃ for preheating for standby;

3. after the first step is finished, spraying a proper amount of alcohol (75%) on the super clean bench for sterilizing again, wiping the super clean bench by using paper, igniting an alcohol lamp, taking out the preheated DMEM, wiping the DMEM dry, sterilizing by using a 75% alcohol spray pipe body, and transferring the DMEM to the super clean bench;

4. adding 8ml and 9ml of DMEM into two 15ml centrifuge tubes respectively for later use;

5. preparing a-80 ice box, taking out the frozen cells from the liquid nitrogen tank, placing the ice box at-80 ℃ and quickly transferring the ice box into a 37 ℃ water bath kettle to shake for 1-2min so that ice cubes in the frozen cells are nearly completely melted, and quickly moving the ice cubes into a super clean bench when only ice cubes with rice grain sizes exist in the frozen cells (when the ice cubes are moved out, water is firstly wiped off, and then the ice cubes are moved into the super clean bench after the outer walls of the frozen cells are disinfected by alcohol);

6. adding 1ml of DMEM into a freezing tube, blowing and uniformly mixing by using a pipette gun, transferring all cells in the freezing tube into a pre-prepared centrifuge tube filled with 8ml of DMEM (10% FBS + P/S), then placing the centrifuge tube into a centrifuge for balancing, centrifuging for 5min at 1500 rmp, and removing supernatant;

7. adding 1ml of DMEM into a centrifuge tube to suspend the centrifuged cell sediment, then completely transferring the cell sediment into a prepared DMEM centrifuge tube filled with 9ml of DMEM, centrifuging the cell sediment to 1500 rmp for 5 min;

8. suspending the cell pellet with 1ml of DMEM culture solution;

9. uniformly adding the cell suspension into a culture dish containing 10ml of DMEM (10% FBS + P/S), slightly shaking up and down, left and right on a table board to ensure that the cells are uniformly distributed, then putting the recovered cells into an incubator, culturing under the conditions of 37 ℃, 5% CO2 and saturated humidity, and allowing the cells to grow adherent;

10. the next day, the growth of the cells was observed and the medium was changed when the cells died more or the medium was yellow, as follows: preparing a super clean bench according to the method in the early stage preparation work; and taking the cells out of the incubator, putting the cells into a super clean bench top for operation, gently sucking all waste liquid by using a liquid transfer gun, adding 10ml of complete culture medium, putting the cells back into the incubator for continuous culture and observation, and increasing the liquid change times if the cells die more.

II) cell passage and cryopreservation

When the cells in the dish grow to 80% (adherent coverage), they should be passaged or cryopreserved. The method comprises the following steps:

1. preparing a clean bench and a culture medium according to the previous method;

2. taking out the grown cells from the incubator, sucking away all original culture media, washing the cells once by using 10ml of PBS, and discarding;

3. slowly adding 1ml of 0.25% pancreatin digestive juice, and inclining the culture dish up and down, left and right to ensure that the cell surface is completely covered with pancreatin;

4. placing the culture dish in a CO2 constant temperature incubator at 37 ℃ for digestion for about 5min, wherein if the digestion is difficult, the digestion time can be properly prolonged in the digestion process (taking the observation of whether the cells are separated from the bottom of the culture dish as a standard, the culture dish does not need to be shaken forcefully to prevent the cells from adhering to pieces and being not beneficial to complete digestion);

5. after digestion is completed, taking out the culture dish, adding 1ml of complete culture medium containing serum, and stopping digestion;

6. gently blow the cells with a 1ml gun to disperse them into single cells;

7-1. cell passage: after the step 6 is finished, sucking 200 microliters of cells, adding the cells into a 100mm culture dish with 10ml of culture medium, slightly shaking up and down, left and right along a table board to enable the cells to be uniformly distributed, marking time and generation number on the culture dish, and putting the culture dish into an incubator for culture and observation after the cell types are marked; if needing the diaphragm clamp experiment cell then prepare 35mm culture dish, place 3 aseptic slide in every culture dish, add 90 mul in the cell that disappears in the 6 th step on every slide with the pipette, 5min of stewing, after most cell pastes the slide, add the culture medium that does not contain the antibiotic gently with the pipette, can take out after 12h and carry out the diaphragm clamp experiment.

7-1. cell cryopreservation: after the step 6 is finished, sucking 10ml of DMEM (10% FBS + P/S) culture solution by using a sterile pipette, adding the DMEM culture solution along the bottom of the culture dish with proper force, slightly blowing and beating the DMEM culture solution, completely moving the DMEM culture solution into a 15ml centrifuge tube, putting the centrifuge tube into a centrifuge, and centrifuging the centrifuge tube at 1500rpm for 5 min; placing the centrifuged centrifuge tube on an operation table, removing the supernatant, pouring the rest liquid drops on a paper towel, adding 0.5ml of DMEM (20% FBS + p/s), lightly and uniformly blowing, quickly moving the liquid drops into a 2ml cryopreservation tube, and then dropwise adding 0.5ml of 20% DMSO to store the cells in the cryopreservation tube in 1ml of liquid (20% FBS + 10% DMSO); marking the name, the generation number and the preservation time of the cells on the cryopreservation tube, sticking a label on the outer wall of the cryopreservation tube, recording the name, the generation number and the preservation time of the cells on label paper again, and placing the tube in a refrigerator at 4 ℃ for 30 min; standing overnight in a refrigerator at-80 deg.C; and finally, moving to a liquid nitrogen tank for preservation.

The specific whole-cell patch clamp experiment is as follows:

preparation before experiment: 1. respectively taking out extracellular fluid from a refrigerator at 4 deg.C, taking out the subpackaged intracellular fluid and plant extract from a refrigerator at-20 deg.C, and cooling to room temperature for use; the perfusion flushing device is installed, and extracellular fluid containing plant extracts with different concentrations are respectively installed according to concentration gradients as required, so that the whole device is ensured to be watertight; 2. opening a digital-to-analog converter, a patch clamp amplifier, a three-dimensional microelectrode manipulator and an inverted microscope, opening a computer host and a display screen, clicking patch clamp software, confirming that the mode is a Digitata 1550 mode, entering a patch page, and clicking a bath key; 3. opening a microelectrode drawing instrument, putting a hard neutral glass tube, and adjusting a program to enable the drawn electrode to reach a proper size;

nav1.4 high expression cells were cultured in this laboratory.

Patch clamp experiments: taking out the experimental cells which are packed in the incubator for the first day after the whole system is prepared, clamping the climbing sheet in the culture dish, placing the climbing sheet into the bath with the surface facing upwards, opening the perfusion device, and continuously supplementing extracellular fluid (closing an extracellular fluid valve and opening the extracellular fluid containing plant extracts as required in the experimental process);

adjusting the multiple under a microscope, finding out a cell in a better state, adjusting the position of the cell to the center (the CHO cell for patch clamp needs to find a spherical cell with larger size, smooth surface and no impurities, and the cell in the state is easier to patch and can survive for a longer time), and adjusting the multiple of the microscope to the lowest; injecting intracellular fluid into the drawn electrode, popping out internal bubbles, installing the bubbles on a holder, and applying certain positive pressure before water is injected to prevent dust and the like from entering; after entering water, a base line with square waves can be seen on a display screen, and the micro-manipulator is slowly adjusted under a microscope to enable the recording electrode to be close to the cell; when the electrode touches the cell and the square wave is reduced to about 1/2, a certain negative pressure is given to enable the electrode to form sealing with the cell, when the sealing resistance reaches more than 100G omega, the gear is adjusted to a patch gear, the holding voltage is-80 mV, and at the moment, the baseline square wave is continuously reduced and the sealing resistance is gradually increased; when the base line is nearly horizontal and a stable sealing resistance of G omega is formed between the electrode and the cell, the cell membrane is broken by negative pressure to form a whole cell mode; and (3) setting a related protocol program according to needs under a whole-cell current clamp mode by using a 'cell' key of an electrode interface after membrane rupture, and recording voltage-gated sodium current.

Data processing: according to the experimental requirement, the current signals of the recording points are copied into the Execl for processing, the Origin and the PPT are used for drawing, the difference between the data is analyzed by using the T test of the SSPS, and the significant difference is judged if the analysis result between the two data is P < 0.05.

FIG. 1 shows a microscopic approach of a cell and patch clamp tip to the cell membrane; FIG. 2 shows the current change before and after the addition of the aforementioned mixture, the curve of the peak heights being the current change without the addition of the mixture and the curve of the peak heights being the current change after the addition of the mixture, demonstrating that the mixture of the present application has an inhibitory effect on Nav1.4, macroscopically manifested as a stretching effect on skin wrinkles, wherein the corresponding content of plant extract in the extracellular fluid is 8 mg/ml. Figure 3 shows the perfusion inhibition effect of the mixture on Nav1.4, the dosing phase showing a significantly smaller current after addition of the mixture, but the recovery of the current after washing away the mixture, also macroscopically manifested as a stretching effect on skin wrinkles, with a corresponding plant extract content in the extracellular fluid of 8 mg/ml.

In addition, the plant extracts formed in the following table in each ratio were subjected to cell patch clamp experiments in the manner described above to clarify the blocking effect of the extracts on the nav1.4 channel, wherein the current changes before and after dosing (| maximum before dosing-minimum after dosing |) are recorded in the following table.

EXAMPLE 2 adjuvant screening

Weighing glyceryl monostearate, stearic acid, white vaseline, and liquid paraffin, heating to melt, and keeping the temperature at 86 deg.C to obtain oil phase. Weighing glycerol, triethanolamine, sodium dodecyl sulfate and ultrapure water, heating to dissolve to 86 deg.C, slowly adding oil phase, stirring, adding Fortunella margarita (lour.) Swingle extract, stirring, and condensing for preliminary experiment.

The auxiliary materials obtained in the preliminary experiment result have good appearance, are uniform, fine and smooth, have no granular feeling, have good coating property and are easy to clean. And through the test of volunteers, no allergic phenomenon occurs, which indicates that the auxiliary material is feasible and can be used for the next experiment.

Screening of optimal formulation of adjuvants

Carrying out experimental research according to a conventional emulsion preparation method, putting stearic acid, glyceryl monostearate, liquid paraffin and white vaseline into a beaker as an oil phase, and heating in water bath at 86 ℃; placing the other samples of each formula into another beaker, and heating with water; and slowly pouring the water phase into the oil phase, uniformly stirring, and cooling.

The stability of the auxiliary materials directly determines the stability of the product, and the auxiliary material prescription needs to be screened in the experiment. The experiment decided to use the type of O/W emulsion-type matrix for experimental study. The aqueous phase matrix adopts ultrapure water and glycerol; the oil phase matrix is selected from stearic acid, white vaseline, lanolin, liquid paraffin, and glyceryl monostearate; the emulsifier is selected from triethanolamine, sodium dodecyl sulfate or Tween 80 or their mixture. The choice of emulsifier is determined by the single factor level and the choice of oil phase is determined by orthogonal experiments on a single factor level basis.

The auxiliary materials are selected from the following 5 formulas:

formula 1: 1-1.5g of glycerin monostearate, 2-3g of stearic acid, 2.2g of liquid paraffin, 0.7g of white vaseline, 2.3g of glycerol, 50-70mg of triethanolamine, 7.2g of ultrapure water and 0.14g of lanolin.

And (2) formula: 1-1.5g of glycerin monostearate, 2-3g of stearic acid, 2.2g of liquid paraffin, 0.7g of white vaseline, 2.3g of glycerol, 50-70mg of triethanolamine, 7.2g of ultrapure water, 800.14 g of tween and 0.14g of sodium dodecyl sulfate.

And (3) formula: 1-1.5g of glycerin monostearate, 2-3g of stearic acid, 2.2g of liquid paraffin, 0.7g of white vaseline, 2.3g of glycerol, 50-70mg of triethanolamine, 7.2g of ultrapure water, 800.14 g of tween and 80(0.14ml) of tween.

And (4) formula: 1-1.5g of glycerin monostearate, 2-3g of stearic acid, 2.2g of liquid paraffin, 0.7g of white vaseline, 2.3g of glycerol, 50-70mg of triethanolamine, 7.2g of ultrapure water, 800.14 g of tween and lanolin (0.14 g).

And (5) formula: 1-1.5g of glycerin monostearate, 2-3g of stearic acid, 2.2g of liquid paraffin, 0.7g of white vaseline, 2.3g of glycerol, 50-70mg of triethanolamine, 7.2g of ultrapure water, 800.14 g of tween and 0.14g of sodium dodecyl sulfate.

After the preparation of the 5 auxiliary materials is finished, the appearances of the 5 auxiliary materials are inspected, a plurality of volunteers are selected for skin allergy experiments, each auxiliary material is respectively filled into 3 centrifuge tubes, a high-temperature stability experiment is respectively carried out in a constant oven at 60 ℃ for more than 6h, a low-temperature stability experiment is carried out in a refrigerator at-25 ℃ to-27 ℃ for 24h, and a centrifugal stability experiment is carried out in a centrifugal machine.

Results and analysis of the experiments

(1) Appearance and properties

The five emulsions have good appearance and properties, no granular feeling and no pungent smell, and can be used as any one of the five emulsions.

(2) Centrifugal stability test

Recipe number	1	2	3	4	5
						30min 4000r/min	Without change	Without change	Without change	Without change	Without change
45min 4000r/min	Without change	Without change	Without change	Without change	Without change
						60min 4000r/min	Without change	Without change	Without change	Is not changedTransforming	Without change
30min 7000r/min	Without change	Without change	Without change	Layering	Layering
						60min 7000r/min	Without change	Layering	Without change	-	-

And (3) analysis: at 4000r/min, no oil separation was observed, and emulsification was considered to be preferable. At 7000r/min for 30min, layers 4 and 5 are separated, discarded, and non-separated layers 1, 2 and 3 are retained. At 60min7000r/min, 2 was also layered and discarded, leaving 1, 3 still non-layered. Indicating that the emulsification of 1, 3 is higher and thus less likely to separate

(3) High temperature stability test

And (3) analysis: no. 1 has slight water-oil stratification, No. 5 has obvious water-oil stratification, No. 2, No. 3 and No. 4 have no change in hardness, and the water-oil stratification condition does not occur, so that the result is better.

(4) Low temperature stability test

And (3) analysis: the hardness of No. 1 and No. 5 is slightly hardened, the hardness of No. 2, No. 3 and No. 4 is unchanged, and the water-oil stratification is not generated, so that the result is better.

(5) The five formulas are tested by volunteers, and have no anaphylactic reaction and can be considered to be qualified.

Optimization of oil phase matrix proportion of prescription through orthogonal experimental design

Dosing of adjuvants

After the optimal formula is screened out to be formula 3, the formula amounts of oil phase glyceryl monostearate (A), stearic acid (B) and triethanolamine (C) are determined, each factor is set to 3 levels, and an L9(34) orthogonal table is selected for testing. Through appearance character, centrifugal stability, high and low temperature stability, allergy test and variance analysis of scoring results, the optimal proportion of the auxiliary materials prepared from the mixture taking the kumquat alcohol extract as the raw material is 1-1.3g of glyceryl monostearate, 2-2.6g of stearic acid and 70-100mg of triethanolamine; the optimal proportion of the auxiliary materials prepared from the mixture taking kumquat aqueous extract as raw materials is 1-1.5g of glyceryl monostearate, 2-3g of stearic acid and 50-70mg of triethanolamine.

Preparation of anti-wrinkle cream

The total dosage of the anti-wrinkle cream is 20g, and the ratio of the main component mixed extract is as follows: the sealwort extract, the dogwood extract and the kumquat extract account for 5 percent of the total weight of the anti-wrinkle cream, namely 5 to 1.

(1) Taking the polygonatum extract, the dogwood extract and the kumquat water extract prepared in the example 1 as raw materials, preparing the anti-wrinkle cream: weighing 1-1.5g of glyceryl monostearate, 2-3g of stearic acid, 2.2g of liquid paraffin and 0.7g of white vaseline, heating to melt, stirring uniformly, keeping the temperature to 85-87 ℃ to be used as an oil phase, weighing 2.3g of glycerol, 50-70mg of triethanolamine, 7.2g of ultrapure water and 800.14 g of Tween, heating to dissolve, stirring uniformly to 85-87 ℃, slowly adding the oil phase, stirring uniformly, adding 1ml of extract, stirring uniformly to condense.

(2) Taking the polygonatum extract, the dogwood extract and the kumquat alcohol extract prepared in the example 1 as anti-wrinkle cream, preparing an emulsion: 1-1.3g of glycerin monostearate, 2-2.6g of stearic acid, 2.2g of liquid paraffin and 0.7g of white vaseline are heated and melted, the mixture is stirred uniformly and then is kept at the temperature of 85-87 ℃ to be used as an oil phase, 2.3g of glycerin, 70-100mg of triethanolamine, 7.2g of ultrapure water and 800.14 g of Tween are weighed, the mixture is heated and dissolved, the mixture is stirred uniformly to the temperature of 85-87 ℃, the oil phase is slowly added, the mixture is stirred uniformly, 1ml of extract is added, and the mixture is stirred uniformly to be condensed.

In summary, the auxiliary materials of formula 3 are determined to be the optimal formula, and specifically: stearic acid (2-3 g), glyceryl monostearate (1-1.3 g), liquid paraffin (1.0-2.2 g), white vaseline (0.2-1.0 g), glycerol (1.0-2.5 g), ultrapure water (2-8 mL), triethanolamine (0.050-0.1 mL), and Tween 80 (0.14-0.2 mL). Under the conditions of good appearance and property and no anaphylactic reaction, the stability of the preparation formulation is optimal, the water-oil demixing condition after centrifugation is avoided, the hardness change and the water-oil demixing under high and low temperature tests are avoided, and the comprehensive score is highest.

Evaluation of anti-wrinkle cream quality

(1) Appearance and properties

The anti-wrinkle cream is milky white, free of pungent smell, easy to apply, good in ductility and free of granular sensation.

(2) Centrifugal stability test

Respectively weighing 1g of the anti-wrinkle cream, placing the anti-wrinkle cream in a centrifuge tube, and centrifuging the cream for 30min at the rotating speed of 4000r/min to avoid the oil-water layered demulsification phenomenon.

(3) Thermal, low constant temperature stability test

Respectively putting the anti-wrinkle cream into a centrifugal tube, sealing, placing in a thermostat at +50 ℃ (placing in a refrigerator at-20 ℃), and standing for 6h (after standing for 24 h), wherein the anti-wrinkle cream has no oil-water layering demulsification and softening phenomenon (has no oil-water layering demulsification and hardening phenomenon).

(4) Skin allergy test

20 healthy volunteers selected skin of the arm, behind the ear and the like as test areas, and each person was smeared with the prepared anti-wrinkle cream. The skin condition of the test area was observed, and then the administration was continued for 3 consecutive days. No erythema, edema, pruritus, allergy and the like were observed in 20 volunteers.

Example 3

The polygonatum extract, the dogwood extract and the kumquat aqueous extract prepared in the example 1 are mixed to form anti-wrinkle effective components, and the weight ratio of the polygonatum extract, the dogwood extract and the kumquat aqueous extract is 6: 4: 1.

The auxiliary materials comprise: stearic acid (1g), glyceryl monostearate (0.5g), liquid paraffin (1.5g), white petrolatum (0.5g), glycerol (1.6g), ultrapure water (5ml), triethanolamine (0.02ml) Tween 80(0.02 ml). The anti-wrinkle active ingredient and the auxiliary materials are mixed according to the process of the embodiment 2 to form the anti-wrinkle cream of the embodiment 3, wherein the weight percentage of the anti-wrinkle active ingredient in the anti-wrinkle cream is 5%.

Example 4

The difference from the example 3 is that the weight ratio of the polygonatum rhizome extract, the cornus officinalis extract and the kumquat aqueous extract is 7: 3: 1.

Example 5

The difference from the example 3 is that the weight ratio of the polygonatum rhizome extract, the cornus officinalis extract and the kumquat aqueous extract is 5: 6: 1.

Example 6

The difference from the embodiment 3 is that the anti-wrinkle effective component content in the anti-wrinkle cream is 10% by weight.

Example 7

The difference from the embodiment 3 is that the anti-wrinkle effective component content in the anti-wrinkle cream is 3% by weight.

Example 8

The difference from the embodiment 3 is that the anti-wrinkle effective component content in the anti-wrinkle cream is 1 percent by weight.

Example 9

The difference from the embodiment 3 is that the anti-wrinkle effective component content in the anti-wrinkle cream is 15% by weight.

Example 10

The difference from the embodiment 3 is that the polygonatum extract, the dogwood extract and the kumquat alcohol extract prepared in the embodiment 1 are mixed to form the anti-wrinkle effective component, and the weight ratio of the polygonatum extract, the dogwood extract and the kumquat water extract is 6: 4: 1.

Example 11

The difference from the embodiment 10 is that the polygonatum extract, the dogwood extract and the kumquat alcohol extract prepared in the embodiment 1 are mixed to form the anti-wrinkle effective component, and the weight ratio of the polygonatum extract, the dogwood extract and the kumquat water extract is 7: 3: 1.

Example 12

The difference from example 10 is that the polygonatum extract, the dogwood extract and the kumquat alcohol extract prepared in example 1 are mixed to form the anti-wrinkle effective component, and the weight ratio of the polygonatum extract, the dogwood extract and the kumquat water extract is 5: 6: 1.

Example 13

The difference from the example 10 is that the anti-wrinkle effective component content in the anti-wrinkle cream is 10% by weight.

Example 14

The difference from the example 10 is that the anti-wrinkle effective component content in the anti-wrinkle cream is 3% by weight.

Example 15

The difference from the example 10 is that the anti-wrinkle effective component content in the anti-wrinkle cream is 1% by weight.

Example 16

The difference from the example 10 is that the anti-wrinkle effective component content in the anti-wrinkle cream is 15% by weight.

Comparative example 1

The difference from the example 3 is that the weight ratio of the polygonatum rhizome extract, the cornus officinalis extract and the kumquat aqueous extract is 1: 1.

Comparative example 2

The difference from example 3 is that, without using kumquat aqueous extract, the weight ratio of polygonatum rhizome extract to cornus officinalis extract was 6: 5.

Comparative example 3

The difference from example 3 is that the polygonatum extract is not used and the weight ratio of the dogwood extract to the kumquat aqueous extract is 10: 1.

Comparative example 4

The difference from example 3 is that, without using the cornus officinalis extract, the weight ratio of the polygonatum sibiricum extract to the kumquat aqueous extract is 10: 1.

Anti-wrinkle experiment

The testing personnel: 180 volunteers aged 23-60 years; the anti-wrinkle creams of examples 3 to 16 and comparative examples 1 to 4 above were tested for anti-wrinkle ability by dividing the volunteers into 18 groups of 10 individuals each having initial skin wrinkle conditions as close as possible in each group.

And (3) inclusion standard: the skin of volunteers with skin problems such as wrinkles and the like appears, but no history of skin and systemic diseases exists, and hormone medicines are not used in the last month;

the product use rule is as follows: the skin care products were not changed 2-3 days before the test and throughout the test period, 1 time each day, morning and evening. The forehead skin is selected as a tested area, the left half part of the forehead of each person is coated with the prepared anti-wrinkle cream, the right half part of the forehead of each person is coated with a blank cream without medicines, the skin condition of the tested area is observed, the product is continuously used, and the continuous observation is carried out for 3 months. Wrinkle improvement was compared before and after one week and three months of use, 3 times per area, averaged, and then averaged within the group.

Evaluation criteria:

10 minutes, no wrinkles;

7-9 minutes, 1-2 wrinkles, and the length is less than 1.5 cm;

4-6 minutes, 3-5 wrinkles, and the length is less than 3 cm;

1-3 minutes, and has a plurality of main wrinkles, and the length is more than 3 cm.

The final scores are given in the following table:

the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-wrinkle composition is characterized by comprising a polygonatum extract, a dogwood extract and a kumquat extract in a weight ratio of 5-7: 3-6: 1.

2. The anti-wrinkle composition according to claim 1, wherein the kumquat extract is a kumquat aqueous extract and/or a kumquat alcohol extract.

3. The anti-wrinkle composition according to claim 2, wherein the kumquat aqueous extract is prepared by a method comprising:

heating and refluxing kumquat peel by adopting water to extract to obtain a water extracting solution;

performing solid-liquid separation and rotary evaporation on the water extracting solution to obtain the kumquat aqueous extract, wherein the volume of the kumquat aqueous extract is 1/8-1/3 of the volume of the water extracting solution,

preferably, the weight ratio of the water to the kumquat peel is 7-14: 1, and the time for heating reflux extraction is 1-5 hours.

4. The anti-wrinkle composition according to claim 2, wherein the kumquat alcohol extract is prepared by a method comprising:

heating and refluxing the kumquat peel by using an ethanol solution to obtain an ethanol extract;

carrying out solid-liquid separation and rotary evaporation on the alcohol extract to obtain the kumquat alcohol extract, wherein the volume of the kumquat alcohol extract is 1/8-1/3 of the volume of the alcohol extract,

preferably, the weight ratio of the ethanol solution to the kumquat peel is 7-14: 1 calculated by 20% ethanol solution, and the time of heating reflux extraction is 1-5 h.

5. The anti-wrinkle composition according to claim 1, wherein the polygonatum sibiricum extract and the cornus officinalis extract are each independently an alcohol extract or an aqueous extract.

6. The anti-wrinkle composition according to claim 5, wherein the preparation method of the Polygonatum sibiricum extract or the Cornus officinalis extract comprises:

mixing rhizoma polygonati or dogwood with water according to the proportion of 1 g: 20-30 mL, and leaching for 50-90 min at 50-90 ℃ to obtain the rhizoma polygonati extract or the dogwood extract.

7. An anti-wrinkle cream comprises an anti-wrinkle active ingredient and an auxiliary material, and is characterized in that the anti-wrinkle active ingredient is formed by mixing the anti-wrinkle composition as claimed in any one of claims to 6.

8. The anti-wrinkle cream according to claim 7, wherein the anti-wrinkle effective component is contained in the anti-wrinkle cream in an amount of 3 to 10% by weight.

9. The anti-wrinkle cream according to claim 8, wherein the auxiliary materials include stearic acid, glyceryl monostearate, liquid paraffin, white vaseline, glycerol, ultrapure water, triethanolamine and tween 80.

10. The anti-wrinkle cream according to claim 9, wherein the ratio of stearic acid, the glyceryl monostearate, the liquid paraffin, the white petrolatum, the glycerin, the ultrapure water, the triethanolamine and the tween 80 is 2 to 3 g: 1 to 1.5 g: 1.0 to 2.2 g: 0.2 to 1.0 g: 1.0 to 2.5 g: 2 to 8 mL: 0.05 to 0.1 mg: 0.14 to 0.2 mL.