CN110870842A - Application of haematococcus pluvialis extract - Google Patents
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- CN110870842A CN110870842A CN201810996943.2A CN201810996943A CN110870842A CN 110870842 A CN110870842 A CN 110870842A CN 201810996943 A CN201810996943 A CN 201810996943A CN 110870842 A CN110870842 A CN 110870842A
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- haematococcus pluvialis
- fibroblasts
- aging
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- skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9706—Algae
- A61K8/9722—Chlorophycota or Chlorophyta [green algae], e.g. Chlorella
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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Abstract
The invention belongs to the field of daily chemicals, and particularly relates to an application of haematococcus pluvialis extract, wherein the haematococcus pluvialis extract promotes fibroblasts to enter a G1/G0 stage in a dormant state on the premise of keeping the activity of the fibroblasts, prolongs the cell cycle of the fibroblasts, slows down the aging process of the fibroblasts, resists the aging of a dermis layer, and delays the aging of skin. No irritation to skin, low dosage, good safety and good therapeutic effect.
Description
Technical Field
The invention belongs to the field of daily chemicals, and particularly relates to an application of haematococcus pluvialis extract.
Background
Aging refers to the progressive process of functional and organic decline that all individuals will undergo over time. The skin is a good place for researching aging, and is beneficial to researching the aging of organisms on molecular and cellular levels due to superficial exposure of the skin. Skin aging occurs in two major forms, natural aging and photoaging. Natural aging is caused by intrinsic factors of the body and is often manifested by the appearance of wrinkles and skin laxity. Photoaging, also known as photoaging, is caused by the accumulation of ultraviolet damage, and is the result of the combined action of natural aging and ultraviolet radiation, manifested by rough skin exposed areas, increased wrinkles and elasticity of dermal fibers. Skin aging is mainly dermal aging, and is characterized in that the clearance of the dermis to foreign chemicals is reduced, the dermis layer is thinned, the synthesis of collagen and elastin is reduced, the decomposition is increased, the activity of catabolic enzymes is enhanced, the proportion of type I collagen and type III collagen is reduced, collagen fibers are thickened, and abnormal interlinkage is generated. Therefore, the skin fibroblast activity is improved, the collagen synthesis is promoted, the skin is enabled to tend to be young, and the skin aging process is delayed.
Aging of the skin appearance is reflected at the cellular level as cellular aging. In fact, the number of divisions of the somatic cells of the animal is limited, and after the cells divide for a limited number of times, the cells gradually lose the DNA synthesis ability or the mitotic ability is reduced and cannot continue to proliferate, and the phenomenon of senescence occurs. In aged skin, there are cases where cell proliferation is reduced due to Fb (fibroblast) aging, and degradation of extracellular matrix such as collagen is increased while synthesis is insufficient, resulting in aging of dermis. Therefore, prolonging the cell cycle can effectively slow down the cell aging process and delay the skin aging.
Haematococcus pluvialis is a unicellular green alga of the genus Haematococcus (Haematococcus) of the family Haematococcus of the order Volvocales of the class Chlorophyceae (Chlorophyta) of the phylum Chlorophyta (Chlorophyta). Haematococcus pluvialis is usually grown on rocks near the seaside, often in the form of a red blood crust attached to a pool or periodically watery shallow bay edge near the sea, and also grown in small puddles on rocks after rain, sporadically attached to hard, water-impermeable objects. The life process of haematococcus pluvialis is a green swimming stage after a red dormancy stage, and then a red dormancy stage. Haematococcus pluvialis is a microalgae which has high economic value after spirulina and chlorella and contains 1.5-3.0% of astaxanthin. Currently, there are 3 recognized biological sources of astaxanthin: waste of aquatic products such as shrimps and crabs, phaffia rhodozyma and haematococcus pluvialis. Wherein, the astaxanthin content in the waste of aquatic products such as shrimps, crabs and the like is low, and the extraction cost is high; the average astaxanthin content in the natural Phaffia rhodozyma is only 0.4%, and the content is low. Therefore, Haematococcus pluvialis is identified as the best biological source of natural astaxanthin in nature.
Astaxanthin (Astaxanthin) belongs to carotenoid, is a natural pigment, usually has bright red or orange color, is commonly present in many plants, especially algae and phytoplankton, and is also present in a few bacteria and fungi, and marine animals such as shrimps, crabs, salmon, trout, and the like. Astaxanthin has effects of scavenging free radicals, protecting blood vessel, reducing cholesterol, whitening skin, preventing wrinkle, preventing cancer, protecting vision, and relieving arthralgia.
The Haematococcus pluvialis extract contains astaxanthin, and also contains other carotenoid, unsaturated fatty acid, stigmasterol, oil and fat, phenols, etc.
The research of the invention proves that the haematococcus pluvialis extract can effectively interfere the cell cycle of human fibroblasts, promote the fibroblasts to enter the G1/G0 stage of the dormant state on the premise of keeping the activity of the fibroblasts, slow down the aging process of the fibroblasts, resist the aging of dermis and delay the aging of the skin.
Disclosure of Invention
The invention aims to provide application of haematococcus pluvialis extract.
The haematococcus pluvialis extract can promote fibroblasts to enter a G1/G0 period in a dormant state on the premise of keeping the activity of the fibroblasts, prolong the cell cycle of the fibroblasts, slow down the aging process of the fibroblasts, resist the aging of dermis and delay the aging of the skin, can be used for preparing a skin external preparation, and can be used as an active ingredient for resisting the aging of the skin, particularly an active ingredient for resisting the aging of the dermis.
The Haematococcus pluvialis extract maintains the proliferative activity of fibroblasts, the collagen and/or elastin secretion activity, and the glycosaminoglycan and/or glycoprotein secretion activity by slowing the aging process of fibroblasts.
The collagen and/or elastin secreted by the fibroblasts can promote the generation of collagen fibers, and/or elastic fibers, and/or reticular fibers, and the secreted glycosaminoglycan and/or glycoprotein can promote the generation of matrix. Collagen fibers, and/or elastic fibers, and/or reticular fibers, and/or a matrix is generated, and/or fibroblasts are proliferated, the thickness, elasticity, plumpness, metabolic secretion and repair activity of the dermis layer are maintained, the structure and the function of the dermis layer are stabilized, the repair activity of the dermis layer is maintained, further the compactness, the saturation, the elasticity and the repair activity of the skin are maintained, and the skin aging is delayed.
Therefore, the haematococcus pluvialis extract can be used for preparing a skin external preparation, and can be used as an anti-skin-aging active ingredient in the skin external preparation, particularly as an anti-dermal-layer-aging active ingredient in the skin external preparation.
The haematococcus pluvialis extract is used as an active ingredient for stabilizing the structure and the function of a dermis and/or an active ingredient for repairing the dermis, so that the ageing of the dermis is resisted, the anti-skin ageing function is realized, and the skin ageing is delayed.
The haematococcus pluvialis extract is used as an active ingredient for slowing down the aging of fibroblasts, delays the aging of a dermis layer and realizes the anti-aging of skin.
The haematococcus pluvialis extract is used as an active ingredient for promoting fibroblasts to enter a G1/G0 stage of a dormant state, prolongs the cell cycle of the fibroblasts, maintains the proliferation activity of the fibroblasts and slows down the aging process of the fibroblasts by making the fibroblasts enter the G1/G0 stage of the dormant state.
Further, the haematococcus pluvialis extract maintains and/or promotes the activity of secreting collagen and/or elastin, the activity of secreting glycosaminoglycans and/or glycoproteins by slowing down the fibroblast senescence process.
The haematococcus pluvialis extract can realize the effects of stabilizing the structure and the function of the dermis by maintaining and/or promoting the activity of fibroblasts for secreting collagen and/or elastin, promoting the generation of collagen fibers, elastic fibers and/or reticular fibers, and/or maintaining the repairing activity of the dermis and/or resisting the aging of the dermis;
the composition can promote the generation of matrix, realize the stabilization of the structure and the function of the dermis by maintaining and/or promoting the activity of the fibroblast to secrete glycosaminoglycan and/or glycoprotein, and/or maintain the repair activity of the dermis, and/or resist the aging of the dermis.
The haematococcus pluvialis extract is prepared by performing wall breaking, extraction, drying and other processes on haematococcus pluvialis spores. The wall breaking is carried out at 3 ℃ to 8 ℃, preferably at 4 ℃. Extracting with extraction solvent, removing solvent, and drying. The extraction solvent is an organic solvent, preferably chloroform, ethanol or a mixture thereof; more preferred is a mixture of chloroform and ethanol; further, the volume ratio of chloroform to ethanol was 1: 0.8 to 1.5. The extraction temperature is 35-50 deg.C, preferably 40 deg.C.
In a preferred embodiment of the present invention, the volume ratio of chloroform to ethanol is 1: 1. Specifically, haematococcus pluvialis is uniformly ground in a cold room to break the wall, and then is extracted by using a mixed solvent of chloroform and ethanol to obtain the haematococcus pluvialis extract. The temperature of the cold chamber is 3 ℃ to 8 ℃, and the preferred temperature is 4 ℃. The volume ratio of chloroform to ethanol is 1: 0.8 to 1.5, preferably 1: 1. the extraction temperature is 35 ℃ to 50 ℃, preferably 40 ℃.
Also comprises drying treatment after extraction, wherein the drying treatment method comprises vacuum drying or freeze drying.
Haematococcus pluvialis is produced in Tibet regions and is a spore of Haematococcus pluvialis. Preferably, Haematococcus pluvialis with the strain preservation number of CGMCC NO.15297 (preservation unit: China general microbiological culture Collection center, address: No.1 Xilu-Beijing university, Chaoyang district, No. 3, institute of microbiology, China academy of sciences; preservation date: 2018, 2 months and 5 days)
The haematococcus pluvialis extract has no toxic or side effect on fibroblast when the dosage of the haematococcus pluvialis extract is not more than 0.002 wt%. Within the dosage range of 0.0005 wt% -0.002 wt%, the cell cycle of pre-formed fibroblasts can be effectively dried, more fibroblasts are promoted to enter the G1/G0 stage of the dormant state on the premise of keeping the activity of the fibroblasts, the cell cycle of the fibroblasts is prolonged, the aging process of the fibroblasts is slowed down, the anti-dermal aging is realized, and the skin aging is delayed.
The haematococcus pluvialis extract can be used for preparing a skin external preparation, and is used as an anti-aging active ingredient in the skin external preparation, particularly one or more of a dermis structure and function stabilizing active ingredient, a dermis repairing active ingredient and an anti-dermis aging active ingredient. The amount of Haematococcus pluvialis extract in the skin external preparation is 0.0001 to 1 wt%, preferably 0.0005 to 0.02 wt%, and more preferably 0.0005 to 0.002 wt%.
The haematococcus pluvialis extract promotes fibroblasts to enter a G1/G0 stage of a dormant state, prolongs the period of the fibroblasts, and slows down the aging process of the fibroblasts; maintaining fibroblast proliferation activity, promoting secretion of collagen and/or elastin in fibroblast, and synthesizing collagen fiber, and/or elastic fiber, and/or reticular fiber; promoting secretion of glycosaminoglycan and/or glycoprotein to produce matrix; firming the structure and function of the dermis layer, and/or maintaining the repair activity of the dermis layer, and/or resisting the aging of the dermis layer.
Haematococcus pluvialis extract is used for preparing skin external preparation for promoting fibroblast to enter dormant state in G1/G0 stage.
The Haematococcus pluvialis extract can be used for preparing skin external preparation for prolonging fibroblast cycle and slowing down fibroblast aging process, and can maintain fibroblast proliferation activity.
The Haematococcus pluvialis extract can be used for preparing skin external preparation for maintaining fibroblast proliferation activity, promoting secretion of collagen, and/or elastin, and/or glycosaminoglycan, and/or glycoprotein, stabilizing dermis structure and function, maintaining dermis repair activity, and resisting aging of dermis.
The Haematococcus pluvialis extract can be used for preparing skin external preparation for promoting collagen and/or elastin secretion, synthesizing collagen fiber, and/or elastic fiber, and/or reticular fiber, stabilizing dermis structure and function, maintaining dermis repairing activity, and resisting dermis aging.
The Haematococcus pluvialis extract can be used for preparing skin external preparation for promoting secretion of glycosaminoglycan and/or glycoprotein and generating matrix, stabilizing structure and function of dermis, maintaining repairing activity of dermis, and resisting aging of dermis.
A skin external preparation has effects in stabilizing dermal structure and function, and/or repairing dermal layer, and/or resisting dermal layer aging, and/or resisting skin aging, and contains Haematococcus pluvialis extract. Wherein the content of Haematococcus pluvialis extract is 0.0001 wt% to 1 wt%, preferably 0.0005 wt% to 0.02 wt%, more preferably 0.0005 wt% to 0.002 wt%, and still more preferably 0.0008 wt% to 0.0012 wt%.
The skin external preparation containing Haematococcus pluvialis extract may further contain other active ingredients, including one or more of moisturizing active ingredients, whitening active ingredients, moisturizing active ingredients, antioxidant active ingredients, anti-wrinkle active ingredients, freckle-removing active ingredients, acne-removing active ingredients, sunscreen active ingredients, acne-removing/acne active ingredients, dandruff-removing active ingredients, antiallergic active ingredients, or sebaceous gland-inhibiting active ingredients.
The formulation of the skin external preparation is not particularly limited, and may be determined according to the use condition, such as cleansing cream, essence, lotion, cream, lotion (such as smoothing toner, toner), mask, makeup remover, gel, aerosol or spray. All can be applied to any part of the surface of human body by smearing, spraying or other similar methods to clean, maintain, beautify, modify, change appearance or modify human body odor, and can be used as basic cosmetics, facial cosmetics, head care products and body care products.
The skin external preparation further comprises one or more of thickener, solubilizer, cosolvent, stabilizer, softener, aerosol solvent, antiseptic, surfactant, controlled release agent, sustained release agent, aromatic, toner, penetration enhancer, humectant, emulsifier, shaping agent or pearling agent.
Compared with the prior art, the invention has the following advantages:
(1) the haematococcus pluvialis extract can effectively intervene in the cell cycle of fibroblasts, prolong the cell cycle of the fibroblasts on the premise of keeping the activity of the fibroblasts, slow down the aging process of the fibroblasts, resist the aging of dermis and delay the aging of skin.
(2) The haematococcus pluvialis extract has high activity, can generate obvious effect under the condition of extremely low content, does not irritate skin, and is natural, safe and effective.
Drawings
FIG. 1 is a graph showing a comparison of the activities of fibroblasts cultured in the medium of blank (NT), SDS control, red-1, red-2, red-3, red-4 and red-5 groups, respectively, in example 1.
FIG. 2 shows the results of cell cycle measurements of fibroblasts of example 2 after 24 hours of culture in blank NT (D), red-1 (A), red-6 (B) and red-7 (C) media, respectively.
FIG. 3 is a diagram showing the state of fibroblasts after 24 hours of culture in blank group NT (D), red-1 group (A), red-6 group (B) and red-7 group (C), respectively, in example 2.
FIG. 4 shows the results of cell cycle measurements of fibroblasts of example 2 cultured in blank NT (D), red-1 (A), red-6 (B) and red-7 (C) media for 24 hours and then transferred to normal media for 24 hours.
FIG. 5 is a diagram showing the state of fibroblasts obtained in example 2 after culturing fibroblasts in the blank NT (D), red-1 (A), red-6 (B) and red-7 (C) media for 24 hours and then culturing the fibroblasts in the normal complete medium for 24 hours.
FIG. 6 is a graph showing a comparison of activities of fibroblasts obtained in example 2 after culturing fibroblasts in the blank NT (D), red-1 (A), red-6 (B) and red-7 (C) media for 24 hours and then culturing the fibroblasts in the normal complete medium for 24 hours.
FIG. 7 shows the results of cell cycle measurements of fibroblasts of example 2 cultured in blank NT (D), red-1 (A), red-6 (B) and red-7 (C) media for 24 hours and then transferred to normal media for 48 hours.
FIG. 8 is a state diagram of fibroblasts obtained in example 2, which were cultured in a medium of blank group NT (D), red-1 group (A), red-6 group (B) and red-7 group (C) for 24 hours and then cultured in a normal complete medium for 48 hours.
FIG. 9 is a graph showing a comparison of activities of fibroblasts obtained in example 2 after culturing fibroblasts in the blank NT (D), red-1 (A), red-6 (B) and red-7 (C) media for 24 hours and then culturing the fibroblasts in the normal complete medium for 48 hours.
Detailed Description
Fibroblasts are mainly located in the dermis layer of the skin, and the growth condition of the fibroblasts is an important representative for responding to skin aging. The application adopts the fibroblast as a research object to evaluate the anti-aging effect of the haematococcus pluvialis extract. The evaluation method consists of two separate tests, the first one being a cytotoxicity test, screening for suitable additive concentrations of Haematococcus pluvialis extract. The second part is cell cycle experiment, which includes culturing fibroblast in culture medium with added haematococcus pluvialis extract in proper concentration, measuring cell cycle, culturing the fibroblast in normal culture medium, measuring cell cycle and cell activity and observing whether the fibroblast is damaged. The change of the length of the cell cycle is observed through a cell cycle experiment.
The existing opinion shows that the active ingredients have the effect of delaying senescence if the active ingredients can prolong the cell cycle of fibroblasts or avoid the phenomenon that telomeres in the fibroblasts accelerate to shorten due to external oxidative stimulation and further shorten the cell cycle.
The haematococcus pluvialis extract is prepared from haematococcus pluvialis spores through wall breaking, extraction, drying and other processes, and is prepared from astaxanthin serving as a main component and substances such as carotenoid, unsaturated fatty acid, stigmasterol, grease, phenols and the like. Haematococcus pluvialis extracts are also known as Haematococcus pluvialis stress-resistance factors (HPEs), and the Haematococcus pluvialis stress-resistance factors (HPEs) of the following examples refer to Haematococcus pluvialis extracts.
The preparation method of the haematococcus pluvialis extract comprises the following steps: uniformly grinding Haematococcus pluvialis (produced in Tibet region, with the strain preservation number of CGMCC NO.15297, preservation unit: China general microbiological culture Collection center) in a cold room at 4 ℃ for 2 minutes to break the wall, transferring the crushed Haematococcus pluvialis into an extraction bottle, extracting with chloroform: extracting with ethanol (1:1, V/V) as extractant at 40 deg.C for 45 min, wherein the ratio of haematococcus pluvialis to extractant is 1 g: 10mL, removing the extractant and drying to obtain Haematococcus pluvialis extract for cytotoxicity and activity detection in the following examples.
EXAMPLE 1 cytotoxicity test
Cell culture: at 37 ℃ with 5% CO2Human fibroblasts AL15012LF-P2 were cultured in complete medium, and when 90% confluent, the plates were digested.
Cytotoxicity test methods: on an AL15012 LF-P35000/well 96-well plate, 5 sample groups (Red-1, Red-2, Red-3, Red-4, Red-5), a blank group and an SDS control group of Table 1 were divided into 3 parts in parallel, and a stock solution of Haematococcus extract of each sample group of Table 1 was diluted 100-fold with a medium (DMEM, 2% FCS, 1% P/S) and added to the wells (final concentration), the blank group was added with a normal medium (DMEM, 2% FCS, 1% P/S), and the SDS control group was added with a medium (DMEM, 2% FCS, 1% P/S) having an SDS content of 2.5%, and then the wells of each group were inoculated with human fibroblasts cultured in a cell culture stage at 37 ℃ with 5% CO2After culturing for 24 hours in the cell culture box, removing the culture medium, adding 0.5mg/ml of MTT, carefully removing the supernatant after 3 hours, adding DMSO, and detecting at 550 nm; with SDS (0.25%) as reference, sample wells were defined as T, untreated wells (blank) as NT, and the ratio of T to NT (T/NT) ≥ 90% was considered as non-cytotoxic<90% were considered cytotoxic. Fibroblast activity is shown in figure 1.
TABLE 1
As can be seen from FIG. 1, the SDS (0.25 wt%) control group had significant cytotoxicity, none of the sample groups (Red-1, Red-2, Red-3, Red-4, Red-5) had cytotoxicity (> 90%), and the Haematococcus extract was used in the range of 0.0000002 wt% to 0.002 wt% (about 2ppb to 20ppm) and had no cytotoxicity; that is, when the amount of Haematococcus extract is not more than 0.002 wt%, it can be regarded as non-toxic to cells, and the subsequent cell cycle test can be continued in this concentration range.
Example 2 cell cycle test and cell Activity recovery MTT assay
When the amount of the haematococcus extract without cytotoxicity is in the range of 0.0000002-0.002 wt%, the haematococcus extract stock solution of haematococcus-1 is sequentially diluted for 2 times by taking 0.20 wt% as the highest concentration, and two groups of the haematococcus extract stock solutions of 0.10 wt% and 0.05 wt% are obtained by twice dilution each time, as shown in red-6 and red-7 of Table 1. Cell cycle experiments were performed using the sample groups red-1, red-6 and red-7 shown in Table 1.
2.1. Adding sample and culturing for 24 hours
Experimental groups were divided into blank group (NT), red-1 group, red-6 group and red-7 group, each group was divided into 3 portions, fibroblasts cultured in the cell culture stage of example 1 were inoculated into AL15012 LF-P30.1M/bottle T25 cell culture flasks used in each group, after 12 hours (synchronization) treatment with serum-free medium, the blank group was added with normal medium (DMEM, 2% FCS, 1% P/S), and the sample groups (red-1, red-6 and red-7) were added with the medium (DMEM, 2% FCS, 1% P/S) diluted 100-fold in each group of Rhodococcus extract stock solution shown in Table 1 (final concentration), at 37 ℃ with 5% CO2After culturing for 24 hours in the cell culture box, digesting, centrifuging, collecting cells, washing the cells once by PBS, fixing the cells by precooled 70% ethanol, and standing and fixing the cells overnight at 4 ℃ for dyeing; before staining, the cells are taken out from a refrigerator at 4 ℃, washed by centrifugal PBS once, then added with 500 mul of staining solution (PI 50 mu g/ml, RNase 100 mu g/ml) per tube, fully mixed and protected from light for 30min, and the detection can be carried out by an up-flow cytometer.
The cell cycle measurements are shown in table 2 and fig. 2, the cell status diagram is shown in fig. 3, the cell ratio of the blank group (NT) in the G1 phase is 81.60%, the cell ratio of the red-1 group (0.002 wt%) in the G1 phase is 92.51%, the cell ratio of the red-6 group (0.001 wt%) in the G1 phase is 87.30%, and the cell ratio of the red-7 group (0.0005 wt%) in the G1 phase is 85.78%, and as the mass concentration of the haematococcus pluvialis extract (haematococcus erythropolis factor) increases, the ratio of the fibroblasts to the G1 phase gradually increases and is higher than that of the blank group (NT), indicating that the haematococcus pluvialis extract (haematococcus pluvialis anti-adversis factor) interferes with the cell cycle distribution, promotes the cells to enter the G0/G1 resting phase, and as the mass concentration of the haematococcus pluvialis anti-adversis factor (HPEs) increases, the ratio of the cells to the G1 phase increases.
TABLE 2
2.2 after 24 hours of incubation, the cells were transferred to normal medium and incubated for 24 hours
The experimental groups and methods were the same as 2.1 except that after 24 hours of incubation with the sample, the original medium was removed and the medium was incubated with normal complete medium (DMEM, 2% FCS, 1% P/S) for an additional 24 hours.
The cell cycle measurement results are shown in table 3 and fig. 4, and the cell status map is shown in fig. 5, where the cell occupancy rate in G1 phase of blank group (NT) is 73.84%, the cell occupancy rate in G1 phase of red-1 group (0.002 wt%) is 79.65%, the cell occupancy rate in G1 phase of red-6 group (0.001 wt%) is 76.53%, and the cell occupancy rate in G1 phase of red-7 group (0.0005 wt%) is 73.84%; the G1 phase ratio of each sample group to which Haematococcus pluvialis extract was added was approaching the blank group (NT), and the G1 phase ratio of the red-7 group (0.0005 wt%) was consistent with NT, both 73.84%. After the haematococcus pluvialis extract (haematococcus pluvialis anti-adversity factor) is removed, the cell cycle of the fibroblasts of each sample group is gradually recovered to the cell cycle distribution level of normal fibroblasts, the cells begin to divide and proliferate, and the cell ratio in the G1 phase is reduced; and the lower the mass concentration of haematococcus pluvialis extract (haematococcus pluvialis stress-resistant factor), the faster the cell cycle of the fibroblasts is restored to the normal distribution level.
TABLE 3
And (3) determining the activity of the fibroblasts which are transferred into a normal culture medium and cultured for 24 hours after sample-adding culture for 24 hours by adopting an MTT method, defining the sample hole as T, and an untreated hole (blank group) as NT, and regarding the ratio (T/NT) of the T to the NT to be more than or equal to 90 percent as normal cell activity. The results of the cell viability assay are shown in FIG. 6.
As can be seen from Table 3 and FIG. 6, since the fibroblast occupancy rates of the sample groups (Red-1 group, Red-6 group and Red-7 group) at G1 were increased compared with the blank group (NT) and the proliferation of the fibroblast cells was slow when the Haematococcus pluvialis extract (Haematococcus adversis resistance factor) was added before the culture, the numbers of fibroblast cells of the sample groups (Red-1 group, Red-6 group and Red-7 group) were decreased compared with the blank group (NT), and the activity of the Haematococcus pluvialis extract (Haematococcus adversis resistance factor) in the amount of 0.0005 wt% of the Red-7 group was greater than 90%, indicating that the fibroblast cells began to recover after being transplanted into the normal medium for 24 hours.
2.3. Adding sample, culturing for 24 hr, transferring into normal culture medium, and culturing for 48 hr
The experimental group and procedure were the same as 2.1 except that after 24 hours of incubation with the sample, the original medium was removed and the medium was incubated with normal complete medium (DMEM, 2% FCS, 1% P/S) for an additional 48 hours.
The cell cycle measurement results are shown in Table 4 and FIG. 7, the cell state diagram is shown in FIG. 8, and the cell occupancy rates in the G1 phases were around 83% in both the blank group (NT) and the sample groups (Red-1 group, Red-6 group, and Red-7 group). After 48 hours of culture in normal medium, the sample groups (red-1, red-6, and red-7) all returned to a cell cycle distribution similar to that of the blank (NT). After removing haematococcus pluvialis extract (haematococcus pluvialis anti-adversity factor), the cells begin to divide and proliferate, and the proportion of cells in the G1 stage is reduced; with the increase in cells, cell contact was inhibited, the rate of G1 phase increased again, and finally, the G1 phase was 83%. As can be seen from FIG. 8, the morphology of the cells was normal.
TABLE 4
And (3) determining the activity of the fibroblasts transferred into a normal culture medium for 48 hours after sample-adding culture for 24 hours by adopting an MTT method, defining the sample wells as T, untreated wells (blank groups) as NT, and regarding the ratio of the T to the NT (T/NT) of more than or equal to 90% as normal cell activity. The results of the cell viability assay are shown in FIG. 9.
As can be seen from FIG. 9, after the samples (red-1, red-6, and red-7) were cultured in the normal medium for 48 hours, the cell activities were all greater than 90% and normal compared to the blank group of NTs.
From the above test results, it can be seen that the fibroblast cells intervened by Haematococcus anti-adversity factors (HPEs) are not affected in cell activity after normal culture is resumed; it can be known from the cell cycle test structure and the MTT test result in the recovery stage that after the normal culture medium is recovered, the cell cycle of the fibroblasts interfered by haematococcus stress-resistant factors (HPEs) can be recovered to the level consistent with that of the blank group NT, and the cell activity is normal and is not influenced by the haematococcus stress-resistant factors (HPEs).
In conclusion, haematococcus anti-adversity factors (HPEs) can effectively intervene in the human skin fibroblast cell cycle in vitro, promote more cells to enter the G1/G0 stage of the dormant state on the premise of keeping the cell activity, prolong the cell cycle of healthy cells, reduce the division times in the same time, avoid the premature consumption of the cells and the division times and enter the programmed senescence in advance.
The haematococcus pluvialis stress resistance factors (HPEs) are within the dosage range of 0.002 wt% -0.0005 wt%, and the effect of promoting cells to enter the dormant state G1/G0 is more obvious along with the increase of the dosage.
After the haematococcus stress-resistant factors (HPEs) are used for treatment, normal culture solution culture is resumed, the cell cycle of fibroblasts is restored to the level consistent with that of normal fibroblasts which are not treated by the haematococcus stress-resistant factors, meanwhile, the cell activity is kept normal, and the cell activity is proved to be normal and still has normal proliferation capacity.
Claims (10)
1. Use of Haematococcus pluvialis extract in the preparation of a skin external preparation, characterized in that the Haematococcus pluvialis extract is used as an anti-skin-aging active ingredient in the skin external preparation.
2. The use of claim 1, wherein the haematococcus pluvialis extract achieves anti-aging of skin by slowing the aging process of fibroblasts and delaying the aging of the dermis layer.
3. The use as claimed in claim 2, wherein the extract of Haematococcus pluvialis is effective in prolonging the cell cycle of fibroblasts, maintaining the proliferative activity of fibroblasts and slowing down the aging process of fibroblasts by promoting the fibroblasts to enter the dormant G1/G0 phase.
4. Use according to claim 2 or 3, wherein the Haematococcus pluvialis extract maintains and/or promotes collagen and/or elastin secreting activity, glycosaminoglycan and/or glycoprotein secreting activity by slowing down the fibroblast senescence process.
5. The use of claim 4, wherein the Haematococcus pluvialis extract achieves firm dermis structure and function, and/or maintains dermis repair activity, and/or resists dermis aging by maintaining and/or promoting collagen and/or elastin secretion activity of fibroblasts, promoting collagen fibers, and/or elastic fibers, and/or reticular fibers;
the composition can promote the generation of matrix, realize the stabilization of the structure and the function of the dermis by maintaining and/or promoting the activity of the fibroblast to secrete glycosaminoglycan and/or glycoprotein, and/or maintain the repair activity of the dermis, and/or resist the aging of the dermis.
6. The use of claim 1, wherein the haematococcus pluvialis extract is prepared by breaking walls of haematococcus pluvialis spores, extracting and drying.
7. The use as claimed in claim 6, wherein the Haematococcus pluvialis extract is prepared from Haematococcus pluvialis with a strain preservation number of CGMCC NO. 15297.
8. A skin external preparation having dermal structure stabilizing and functional activities, and/or dermal layer repairing activities, and/or dermal layer aging resisting activities, and/or skin aging resisting activities, which comprises Haematococcus pluvialis extract.
9. The external preparation for skin as claimed in claim 8, wherein the content of Haematococcus pluvialis extract is 0.0005 wt% to 1 wt%.
10. The external preparation for skin according to claim 8, further comprising one or more of a moisturizing active ingredient, a whitening active ingredient, a moisturizing active ingredient, an antioxidant active ingredient, an anti-wrinkle active ingredient, a spot-removing active ingredient, an acne-removing active ingredient, a sunscreen active ingredient, an acne/acne-removing active ingredient, a dandruff-removing active ingredient, an antiallergic active ingredient, or a sebaceous gland-inhibiting active ingredient.
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| CN107375148A (en) * | 2017-08-16 | 2017-11-24 | 佐登妮丝（广州）美容化妆品有限公司 | A kind of skin repair composition |
| CN107497133A (en) * | 2017-07-12 | 2017-12-22 | 上海伽誉生物科技有限公司 | The degeneration-resistant factor HPEs of haematococcus preparation method |
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| WO1999013855A1 (en) * | 1997-09-12 | 1999-03-25 | Sederma S.A. | Composition for cosmetic or dermopharmaceutical use containing a combination of algae extract and exopolysaccharides |
| KR20050116455A (en) * | 2004-06-07 | 2005-12-13 | 정세영 | Compositions for skin wrinkle improvement containing astaxanthin |
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