CN112694538A - Oriental beauty tea polysaccharide with whitening and moisturizing activities - Google Patents
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Abstract
The invention discloses oriental beauty tea polysaccharide with whitening and moisturizing activities. The relative molecular weight of TMP in the present invention is 3.4X 104Da, the monosaccharide composition comprises xylose, arabinose, mannose, glucose and galactose according to the molar ratio of 13.97:55.60:1.37:11.94:17.12, the extraction rate reaches more than 4%, and the purity reaches as high as 90%. The polysaccharide TMP of the oriental beauty tea has strong oxidation resistance, can effectively remove DPPH free radicals, and is proved to have remarkable tyrosinase inhibition capacity and strong water absorption and moisture retention activities by in vivo and in vitro experiments. TMP has effects in food and cosmeticHas great application prospect.
Description
One, the technical field
The invention relates to oriental beauty tea polysaccharide with whitening and moisturizing activities, and belongs to the technical related field of whitening and moisturizing cosmetics.
Second, background Art
Whitening and moisturizing have been the main appeal of female consumers focusing on skin care, and when skin is affected by environments such as ultraviolet rays, tyrosinase of pigment cells of the basal layer of the skin is activated and then catalyzes melanin to form skin problems such as macroscopic color spots and uneven skin color. There are two ways of whitening: reducing the generation of melanin from the source by inhibiting the activity of melanin synthesis rate-limiting enzyme; ② promoting the metabolism of melanin. The human skin has natural moisture retention performance and plays an irreplaceable role in keeping the moisture of the human body. Moisturizing and skin care can be similarly divided into two ways: firstly, prevent the evaporation loss of the skin moisture, secondly absorb the moisture from the external environment and keep the skin moist.
As the ceremony nation, China has the present of Guest tea since ancient times. Good tea is produced in the east, and the oriental beauty tea is produced in a tea garden with the altitude of about 1200 m in Taiwan, and is covered by a cloud cover in the morning and evening in a cold climate, so that the average sunshine is short, and the tea has unique natural environment, and the fragrance is pleasant and the taste is excellent. The oriental beauty tea can be used as a drink and contains various components beneficial to the body.
The existing moisturizing and whitening products are all used for inhibiting the generation of melanin by means of inhibiting the activity of tyrosinase, and popular whitening components in the market mainly comprise tartaric acid, arbutin, placenta extract and vitamin C, but the existing moisturizing and whitening products are not perfect. Burn can occur if the concentration of fruit acid is poorly controlled, and even if it is immediately stopped, the burn injury cannot be stopped immediately. Furthermore, the tartaric acid has antigenicity and can cause allergic reaction after being absorbed by skin. Arbutin has high photosensitivity, so a large amount of sunscreen agent is often added into the product, which is easy to cause burden on skin and accelerate skin aging. Moreover, the application of arbutin is easy to cause sensitive reaction on the skin. The placenta extract is unstable and easy to go bad, and a large amount of preservative needs to be added into the product, which can cause skin allergy. Vitamin C is a very specific, very classical antioxidant, having the action of reducing melanin, capturing free radicals, promoting collagen synthesis, and in particular of strengthening the skin against sun damage. However, vitamin C is not recommended for sensitive skin and dry skin, and the skin is dry due to excessive use of vitamin C. Therefore, it is important to find a safe and effective whitening and moisturizing product.
The flash extraction technology is high in yield, simple and easy to operate, and fast and efficient. The quaternary ammonium salt precipitation method is a polysaccharide purification method which is simple and convenient to operate, large in treatment capacity and free of other organic reagents. The membrane technology is a new green and energy-saving technology used for clarification, separation, sterilization, concentration and the like, has the advantages of simple operation, energy conservation, no pollution, continuous operation at normal temperature and the like, and is widely used in the fields of chemical industry, food, biology, pharmacy and the like.
Tea polysaccharide is a natural polysaccharide with high biological activity, and has gradually become a hot spot in cosmetic development because the tea polysaccharide has good activities of resisting oxidation, preserving moisture, inhibiting neuraminidase and the like, is green and safe. However, at present, no whitening and moisturizing cosmetic taking oriental beauty tea polysaccharide as a raw material is reported.
Third, the invention
The invention aims to provide oriental beauty tea polysaccharide TMP with the effects of resisting oxidation, absorbing moisture, keeping moisture, inhibiting the activity of tyrosinase and the like and application thereof.
In order to realize the purpose of the invention, the following technical scheme is adopted: the oriental beauty tea polysaccharide with moisturizing and whitening activities is characterized in that: the oriental beauty tea polysaccharide TMP is obtained by taking oriental beauty tea as a raw material, extracting crude polysaccharide by adopting a flash extraction method with high yield and simple and easy operation, and purifying the polysaccharide by combining a quaternary ammonium salt precipitation method and an ultrafiltration technology. Molecular weight of oriental beauty tea polysaccharide TMP is 3.4 x 104Da, the sugar purity is higher than 90%, and the extraction rate reaches more than 4%.
The monosaccharide consists of xylose, arabinose, mannose, glucose and galactose according to the molar ratio
13.97:55.60:1.37:11.94: 17.12. The TMP can effectively remove DPPH free radicals and inhibit tyrosinase activity, and has good water absorption and moisture retention activities. The specific preparation method comprises the following steps:
A. the flash extraction technology comprises the steps of crushing commercially available Taiwan oriental beauty tea into 40-80 meshes, adding sterile water according to 10-50 times of the weight of the tea powder, extracting oriental beauty tea polysaccharide by a flash extraction method for 30-40 s at normal temperature, wherein the extraction voltage is 100-130V, performing centrifugal separation or suction filtration, collecting supernatant, and heating and concentrating until the solid content is 30-40%;
b, separating polysaccharide by a CTAB precipitation method, namely mixing the collected concentrated solution with a 1-10% hexadecyl trimethyl ammonium bromide (CTAB) solution according to a ratio of 1.5-2: 1, standing for 2-3 h, centrifugally collecting precipitate, and dissolving the obtained precipitate with a 10% NaCl solution; adding 3-4 times of anhydrous ethanol into the hydrosol, precipitating with ethanol completely, centrifuging, collecting precipitate, and dissolving the obtained precipitate with a small amount of distilled water;
C. desalting the solution obtained in the step B at 25-35 ℃ by using an ultrafiltration membrane with the molecular weight cutoff of 1000-1500 daltons, wherein the driving pressure of ultrafiltration is 0.10-0.18 MPa; and then carrying out ultrafiltration through an ultrafiltration membrane of 10 ten thousand daltons at the temperature of 25-35 ℃, collecting filtrate, and carrying out freeze drying at the temperature of-50 ℃ to obtain the oriental beauty tea polysaccharide TMP.
The sugar content of the oriental beauty tea polysaccharide TMP is more than 90 percent by a phenol-sulfuric acid method;
preparing the obtained polysaccharide component TMP into 5mg/mL solution, filtering with 0.22 μm microporous membrane, detecting by HPLC-ELSD, wherein the molecular weight of the polysaccharide TMP is 3.4 × 104Da, wherein the monosaccharide composition comprises xylose, arabinose, mannose, glucose and galactose according to the molar ratio of 13.97:55.60:1.37:11.94: 17.12.
Compared with the prior art, the invention has the beneficial effects that:
the method takes the oriental beauty tea as a raw material, adopts a flash extraction method with high yield and simple and easy operation to extract crude polysaccharide, and combines a quaternary ammonium salt precipitation method and an ultrafiltration technology to purify the polysaccharide to obtain the oriental beauty tea polysaccharide TMP. The extraction rate of the polysaccharide TMP of the oriental beauty tea is more than 4%, and the purity of the product is as high as more than 90%. The compound is an active polysaccharide with a new source and a new structure. The polysaccharide TMP of the oriental beauty tea has stronger antioxidant capacity, and when the concentration of the TMP is 400ug/mL, the removal rate of DPPH free radicals reaches 59.22%. The TMP can obviously inhibit the tyrosinase activity in-vivo and in-vitro tests, and the tyrosinase inhibition rate reaches 67.2 percent when the concentration of the TMP is 2.5mg/mL in the determination of in-vitro inhibition of the tyrosinase activity. When the concentration of TMP in the determination of inhibiting the activity of the zebra fish embryo tyrosinase in vivo is 300ug/mL, the inhibition rate of the zebra fish embryo tyrosinase reaches 64.7 percent. Meanwhile, TMP also has good moisture absorption and moisture retention activity, so that the oriental beauty tea polysaccharide TMP has very wide market application prospect.
Description of the drawings:
FIG. 1 shows the result of the DPPH radical scavenging ability of eastern American tea polysaccharide TMP in example 3 of the present invention;
FIG. 2 is the results of an in vitro tyrosinase activity inhibition assay in example 4 of the present invention;
FIG. 3 shows the results of measurement of inhibitory activity of eastern American tea polysaccharide TMP on tyrosinase of zebrafish embryo in vivo in example 5 of the present invention;
FIG. 4 shows the result of observing the fluorescence image of the stained embryo by the laser confocal microscope in example 5 of the present invention;
FIG. 5 shows the moisture absorption rate of east American tea polysaccharide TMP at RH 43% in example 6 of the present invention;
FIG. 6 shows the moisture absorption rate of east American tea polysaccharide TMP at RH 81% in example 6 of the present invention;
FIG. 7 shows the measurement result of moisture retention of eastern American tea polysaccharide TMP at RH 43% in example 6 of the present invention;
FIG. 8 shows the measurement result of moisture retention of eastern American tea polysaccharide TMP at RH 81% in example 6 of the present invention;
fig. 9 is an HPLC diagram of a standard monosaccharide mixture (a) and an HPLC diagram of a monosaccharide composition (B) of oriental american tea polysaccharide TMP of the present invention, wherein: 1-rhamnose (Rha); 2-xylose (Xyl); 3-arabinose (Ara); 4-mannose (Man); 5-glucose (Glu); 6-galactose (Gal)
Example 1
A method for extracting, separating and purifying oriental beauty tea polysaccharide TMP with whitening and moisturizing activities comprises the following steps:
(1) pulverizing the oriental beauty tea fruit bodies by a tissue pulverizer, sieving by a 40-mesh sieve, and collecting powder;
(2) refluxing with 80% ethanol at 80 deg.C for 2 hr, cooling, centrifuging or filtering, and oven drying;
(3) weighing oriental beauty tea powder, putting into a flash extractor, adding 20 times of water, and extracting at normal temperature for 30 s. Extracting at 130V, centrifuging the mixture (5500r/min, 15min), and collecting supernatant;
(4) mixing the supernatant collected in the step (3) with a 2% hexadecyl trimethyl ammonium bromide (CTAB) solution according to a ratio of 1.5:1, standing for 2 hours, centrifuging (5500r/min, 15min) after complete precipitation, collecting the precipitate, and fully dissolving the obtained precipitate with a 10% NaCl solution;
(5) 4 volumes of absolute ethanol were added. Precipitating with ethanol completely, centrifuging (5500r/min, 5min), collecting precipitate, and dissolving the obtained precipitate with distilled water;
(6) desalting the solution obtained in the step (5) at 25 ℃ by an ultrafiltration membrane with the molecular weight cutoff of 1500Da, wherein the ultrafiltration driving pressure is 0.10 MPa; ultrafiltering with 10 ten thousand Da ultrafiltration membrane at 25 deg.C, collecting filtrate, and freeze drying at-50 deg.C to obtain Oriental beauty tea polysaccharide TMP;
example 2
A method for extracting, separating and purifying oriental beauty tea polysaccharide TMP with whitening and moisturizing activities comprises the following steps:
(1) pulverizing the oriental beauty tea fruit bodies by a tissue pulverizer, sieving by a 40-mesh sieve, and collecting powder;
(2) refluxing with 80% ethanol at 80 deg.C for 3 hr, cooling, centrifuging or filtering, and oven drying;
(3) weighing oriental beauty tea powder, putting the oriental beauty tea powder into a flash extractor, adding 25 times of water, and extracting for 40s at normal temperature. Extracting at 100V, centrifuging the mixture (5500r/min, 15min), and collecting supernatant;
(4) mixing the supernatant collected in the step (3) with a 3% hexadecyl trimethyl ammonium bromide (CTAB) solution according to a ratio of 1.5:1, standing for 3 hours, centrifuging (5500r/min, 15min) after complete precipitation, collecting the precipitate, and fully dissolving the obtained precipitate with a 10% NaCl solution;
(5) 4 volumes of absolute ethanol were added. Precipitating with ethanol completely, centrifuging (5500r/min, 5min), collecting precipitate, and dissolving the obtained precipitate with distilled water;
(6) desalting the solution obtained in the step (5) at 25 ℃ by an ultrafiltration membrane with the molecular weight cutoff of 1000Da, wherein the ultrafiltration driving pressure is 0.10 MPa; ultrafiltering with 10 ten thousand Da ultrafiltration membrane at 25 deg.C, collecting filtrate, and freeze drying at-50 deg.C to obtain Oriental beauty tea polysaccharide TMP;
example 3
Example 1 determination of DPPH radical scavenging by polysaccharide TMP of oriental beauty tea obtained:
adding 1.3X 10 concentration into the test tube-4Adding 2.5mL of DPPH solution of mol/L, adding different amounts (100ug/mL, 200 ug/mL, 300ug/mL, 400ug/mL and 500ug/mL) of polysaccharide TMP of the oriental beauty tea, diluting to 5mL with distilled water, shaking, mixing, standing at room temperature for 30min, placing in a cuvette, and measuring absorbance As under ultraviolet photometer wavelength of 517 nm; the blank control was distilled water and its absorbance was A0(ii) a The control system uses absolute ethyl alcohol instead of DPPH, and the absorbance of the control system is AT. The clearance is calculated according to the formula:
the test result is shown in figure 1, the oriental beauty tea polysaccharide TMP has stronger capability of removing free radicals, the removal rate is gradually increased along with the increase of the concentration, and the oriental beauty tea polysaccharide TMP tends to be stable after 400 ug/mL. When the concentration of TMP is 400ug/mL, the scavenging rate of DPPH free radicals reaches 59.22%.
Example 4
Example 1 assay of the in vitro tyrosinase inhibitory activity of polysaccharide TMP of oriental beauty tea obtained:
1. accurately weighing 0.1g of oriental beauty tea polysaccharide TMP0, adding 20mL of dimethyl sulfoxide mock, fully dissolving to obtain 5mg/mL of sample mother liquor to be detected, and diluting to 2.5, 1.25, 0.625, 0.312 and 0.156mg/mL in a double way to obtain a sample solution.
2. Preparing positive control arbutin solution according to the steps.
3. According to the experimental system design shown in the following table, phosphate buffer solution, sample solutions with different gradients and enzyme solution are sequentially added into a test tube, water bath is carried out at 37 ℃ for 10min, then substrate L-tyrosine is added, and timing is immediately started. The absorbance at a wavelength of 475nm at 20min of the reaction was determined. The corresponding negative control is used as reference during the determination (sample group, blank group, arbutin control group are respectively negative control group 1#、2#And 3#Pipe adjusterZero), the inhibition rate of the sample solution and the positive control on tyrosinase was calculated by the following formula.
Inhibition ratio (%) - (A-B)/Ax 100
Wherein A is the absorbance of the standard control, and B is the absorbance of the sample fluid (or positive control)
Table 1 shows the design of the in vitro tyrosinase activity test system,
the test result is shown in figure 2, the oriental beauty tea polysaccharide TMP can effectively inhibit the tyrosinase activity in vitro, the tyrosinase inhibition rate is gradually increased along with the increase of the concentration, and when the concentration of the TMP is 2.5mg/mL, the tyrosinase inhibition rate reaches 67.2%.
Example 5
Example 1 determination of in vivo inhibition of tyrosinase activity of zebrafish embryo by polysaccharide TMP of oriental beauty tea obtained:
6-7hpf zebrafish embryos were collected and cultured in 24-well plates with different concentrations of TMP polysaccharide (75, 150 and 300. mu.g/mL), kojic acid (300. mu.g/mL) was used as a positive control, and when cultured to 48hpf, fresh medium was replaced, 24hpf culture was continued, and then embryos were removed, and fluorescence images of the stained embryos were observed using a laser confocal microscope (FV 1000; Olympus, Japan). Then placing the embryo in a tissue homogenizer, repeatedly extracting by using a tissue protein extraction kit, centrifuging 5426g for 20min, carefully absorbing supernatant, measuring the content of each group of proteins by using a BCA protein kit, buffering by using phosphate to prepare consistent concentration, absorbing 100 mu L of total protein in each group, placing the total protein in a 96-well plate, adding 100 mu L of 5 mmol/LL-tyrosine solution (phosphate buffer solution, pH6.8) as a substrate, standing and reacting for 30min at 28 ℃ in an incubator, measuring the light absorption value at 475nm, setting 3 more wells for each concentration, and averaging. The blank control contained 100. mu.L of buffer and 100. mu. L L-tyrosine solution, and the tyrosinase activity inhibition ratio was calculated as follows:
tyrosinase activity inhibition rate (1-A1/A0). times.100%
Wherein A1 is expressed as sample absorbance and A0 is expressed as blank control absorbance.
The test result is shown in figures 3 and 4, the eastern beauty tea polysaccharide TMP can effectively inhibit the activity of the tyrosinase of the zebra fish embryo in vivo, the tyrosinase inhibition rate is gradually increased along with the increase of the concentration, and when the concentration of the TMP is 300ug/mL, the inhibition rate of the tyrosinase of the zebra fish embryo reaches 64.7%. This trend can be seen more intuitively in fig. 4.
Example 6
Example 1 measurement of moisture absorption and retention properties of polysaccharide TMP of oriental beauty tea obtained:
the saturated calcium chloride solution and the saturated ammonium chloride solution are placed in a dryer for presaturation for 12 hours at room temperature, and environments with relative humidity of 43% and 81% are formed respectively. Accurately weighing 2g of polysaccharide TMP and sodium alginate of the oriental beauty tea respectively in a culture dish, preparing 3 parts of samples in parallel, placing the samples in the driers with different humidity, taking out the samples after 1, 3, 6, 9, 12, 18, 24, 36 and 48 hours, weighing the samples, and calculating the moisture absorption rate.
Moisture absorption rate [ (Wn-W)0)/W0]×100％
Wherein, W0And Wn is the mass of the sample before and after placement, respectively.
The saturated calcium chloride solution was placed in a desiccator at room temperature for 12h to presaturate to an environment with a relative humidity of 43% and 81%. The oriental beauty tea polysaccharide TMP and sodium alginate each 2g are precisely weighed to prepare 1% solution respectively, 3 parts of samples are prepared in parallel and placed in a drier with the humidity of 81% and 43% respectively, and the temperature is controlled at 20 ℃. Weighing the mass at 1, 3, 6, 9, 12, 18, 24, 36 and 48h, and calculating the moisture retention rate.
Moisture retention rate Hn/H0×100％
Wherein H0And Hn is the moisture mass of the sample before and after placement.
The test results are shown in fig. 5-8, the oriental beauty tea polysaccharide TMP has good moisture absorption and retention activity. It can be seen from fig. 5 that the moisture absorption rate of the oriental beauty tea polysaccharide TMP at RH 43% is gradually increased and becomes gentle after 36h as time goes on. It can be seen from fig. 6 that the moisture absorption rate of the oriental beauty tea polysaccharide TMP at RH 81% is gradually increased with the lapse of time, and at 48h, the moisture absorption rate reaches 40.1%. As can be seen from fig. 7, the moisture absorption rate of the oriental beauty tea polysaccharide TMP at RH 34% was gradually decreased with the lapse of time, but the moisture retention rate was maintained at 73.1% even at 48 h. As can be seen from fig. 8, the moisture absorption rate of the oriental beauty tea polysaccharide TMP at RH 81% was gradually decreased, but the moisture retention rate was maintained at 93.67% even at 48h as time passed.
Claims (4)
1. The oriental beauty tea polysaccharide with moisturizing and whitening activities is characterized in that: the oriental beauty tea polysaccharide TMP is obtained by taking oriental beauty tea as a raw material, extracting crude polysaccharide by adopting a flash extraction method with high yield and simple and easy operation, and purifying the polysaccharide by combining a quaternary ammonium salt precipitation method and a membrane separation technology.
2. The oriental beauty tea polysaccharide with moisturizing and whitening activities according to claim 1, characterized in that: the molecular weight of the oriental beauty tea polysaccharide TMP is 3.4 multiplied by 104Da, the sugar purity is higher than 90%, and the extraction rate is more than 4%. The monosaccharide composition comprises xylose, arabinose, mannose, glucose and galactose according to the molar ratio of 13.97:55.60:1.37:11.94: 17.12.
3. The oriental beauty tea polysaccharide with moisturizing and whitening activities according to claim 1 or 2, characterized in that: the oriental beauty tea polysaccharide TMP can effectively remove DPPH free radicals and inhibit tyrosinase activity, and has good water absorption and moisture retention activities.
4. A preparation method of oriental beauty tea polysaccharide TMP with moisturizing and whitening activities is characterized in that: the method comprises the following steps:
A. the method comprises the following steps of crushing commercially available Taiwan oriental beauty tea into 40-80 meshes by a flash extraction technology, adding sterile water according to 10-50 times of the weight of the tea powder, extracting oriental beauty tea polysaccharide by a flash extraction method for 30-40 s at normal temperature, wherein the extraction voltage is 100-130V, carrying out centrifugal separation or suction filtration, collecting supernatant, and heating and concentrating until the solid content is 30-40%;
B. separating polysaccharide by quaternary ammonium salt precipitation method, mixing the collected concentrated solution with 1-10% hexadecyl trimethyl ammonium bromide (CTAB) solution according to the proportion of 1.5-2: 1, standing for 2-3 h, centrifuging and collecting precipitate, and dissolving the obtained precipitate with 10% NaCl solution; adding 3-4 times of anhydrous ethanol into the hydrosol, precipitating with ethanol completely, centrifuging, collecting precipitate, and dissolving the obtained precipitate with a small amount of distilled water;
C. desalting the solution obtained in the step B at 25-35 ℃ by using an ultrafiltration membrane with the molecular weight cutoff of 1000-1500 daltons, wherein the driving pressure of ultrafiltration is 0.10-0.18 MPa; and then carrying out ultrafiltration through an ultrafiltration membrane of 10 ten thousand daltons at the temperature of 25-35 ℃, collecting filtrate, and carrying out freeze drying at the temperature of-50 ℃ to obtain the oriental beauty tea polysaccharide TMP.
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