Camellia oleifera leaf extract and preparation method thereof

Abstract

The invention discloses a camellia oleifera leaf extract and a preparation method thereof, and relates to the technical field of plant extracts.The preparation method of the camellia oleifera leaf extract comprises the following steps that S1, camellia oleifera leaf powder is prepared, camellia oleifera leaves are collected, dried and smashed, and the camellia oleifera leaf powder is obtained; s2, preparing the camellia oleifera leaf extract: S201, adding a solvent into camellia oleifera leaf powder, and performing ultrasonic extraction to obtain a solution A; s202, the solution A is filtered, filtrate is taken and subjected to centrifugal operation, and an extracting solution is obtained; s203, performing rotary evaporation concentration on the extracting solution by using a rotary evaporator to obtain a concentrated extract, and performing freeze drying on the concentrated extract to obtain an oil tea leaf extract; the camellia oleifera leaf extract has activity inhibition effect on tyrosinase, has proliferative activity effect on HaCaT cells and HFF-1 cells, and is beneficial to skin repair and regeneration, so that the camellia oleifera leaf has wide application potential in the development of beauty and skin care products, health care products and medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extracts, and particularly relates to an oil tea leaf extract and a preparation method thereof. Background Art

[0002] Camellia oleifera has a 4,000-year history in China. Today, its cultivation is accelerating due to its superior oil quality, high yield and rapid growth. With the central government's overall policy continuing to refine the development trend of the camellia oleifera industry, and the continuous improvement of regional policies and subsidies, it is expected that the scale of my country's camellia oleifera industry will accelerate its expansion in the next three years.

[0003] Camellia oleifera is an oil-bearing shrub or small tree of the genus Camellia in the family Theaceae. It is an evergreen plant. Studies have shown that camellia oleifera contains a variety of chemical components, including polyphenols, flavonoids, terpenes, saponins, alkaloids and other components, which make it have certain effects in anti-oxidation, anti-inflammatory and antibacterial. For example, studies have found that camellia oleifera can be used as a natural oil antioxidant, and its antioxidant activity is similar to that of gallic acid; camellia oleifera flavonoids have good antibacterial effects, and under experimental conditions, their antibacterial effects are better than camellia oleifera flavonoids; and the MTT cytotoxicity test has confirmed that camellia oleifera extract has anti-tumor effects.

[0004] Modern pharmacological research has proven that camellia oil has anti-cancer and antibacterial effects. More studies have shown that camellia oil extract has an inhibitory effect on caspase and can prolong cell life. Combined with its antioxidant properties and free radical scavenging properties, it shows that camellia oil extract is an anti-wrinkle and anti-aging additive; it also has anti-inflammatory and weight loss effects. At the same time, camellia oil extract has been included in the "Catalogue of Used Cosmetic Raw Materials (2021 Edition)", which lays the foundation for the research and application of camellia oil in cosmetic raw materials.

[0005] Camellia oleifera is pruned in spring and autumn, which will produce a large number of waste tea leaves, all of which will be discarded and incinerated, resulting in a large waste of resources. Currently, most of the pharmacological activities of camellia oleifera seeds and shells have been reported in society, while there is a lack of research on camellia oleifera, especially its application in cosmetic raw materials. Its development and utilization has not been well developed. If camellia oleifera can be developed and utilized at a deeper level, it will greatly improve its comprehensive utilization value. Summary of the invention

[0006] The purpose of the present invention is to provide a tea leaf extract and a preparation method thereof in order to solve the above problems.

[0007] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0008] In one aspect, the present invention provides a method for preparing an oil tea leaf extract, which specifically comprises the following steps:

[0009] S1. Preparation of oil tea leaf powder: collecting oil tea leaves, drying them, and crushing them to obtain oil tea leaf powder;

[0010] S2. Preparation of Camellia oleifera extract:

[0011] S201, adding a solvent to the oil tea leaf powder, and performing ultrasonic extraction to obtain a solution A;

[0012] S202, filtering solution A, and centrifuging the filtrate to obtain an extract;

[0013] S203, using a rotary evaporator to concentrate the extract to obtain a concentrated extract, and freeze-drying the concentrated extract to obtain an oil tea leaf extract.

[0014] Furthermore, the oil tea leaves come from Lu'an "Dabie Mountain No. 1" oil tea.

[0015] Furthermore, in step S201, the solution is pure water, and the material-liquid ratio of the oil tea leaf powder to pure water is 1:20-50.

[0016] Further, in step S201, the temperature of ultrasonic extraction is 20-50°C, the ultrasonic power is 500W, and the extraction time is 20-40min.

[0017] Furthermore, in step S202, the centrifugal speed is 6000-8000 rpm, and the centrifugal time is 5-10 min.

[0018] Furthermore, in step S203, vacuum freeze drying technology is used, and the drying time is 24-72 hours.

[0019] Another aspect of the present invention further provides a camellia oleifera extract, which is prepared by the above-mentioned camellia oleifera extract preparation method.

[0020] Furthermore, the oil tea leaf extract contains flavonoids and polyphenols, the flavonoids content is 11.99% to 12.11%, and the polyphenols content is 14.43% to 14.79%.

[0021] Furthermore, the oil tea leaf extract has an IC of 120.74 μg / mL to 152.98 μg / mL for inhibiting tyrosinase. 50 value.

[0022] Furthermore, the oil tea leaf extract has an IC of (33.33±0.41) μg / mL to scavenge DPPH free radicals. 50 value.

[0023] Furthermore, the ORAC antioxidant capacity index of the oil tea leaf extract is 2314.14 μmol TE / g-2475.86 μmol TE / g.

[0024] Furthermore, the maximum proliferation rate of the oil tea leaf extract on HaCaT cells is 158.83%-164.97%.

[0025] Furthermore, the maximum proliferation rate of the oil tea leaf extract on HFF-1 cells is 113.02%-13.46%.

[0026] The present invention has at least the following beneficial effects: the present invention utilizes ultrasonic water extraction and freeze-drying technology to obtain a tea leaf extract with stable ingredients, and the preparation cost is low. The prepared tea leaf extract has active inhibition on tyrosinase, has a positive effect on skin whitening and anti-aging, and has antioxidant activity, slows down the cell aging process, protects cells from oxidative damage, and plays an important role in preventing various diseases and promoting skin health. In addition, it has a proliferation activity effect on HaCaT cells and HFF-1 cells, helps to repair and regenerate the skin, and improves the overall health of the skin, so that the tea leaf has a wide range of application potential in the development of beauty and skin care products, health products and drugs, and realizes the recycling of waste, avoiding the waste of tea leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a flow chart for preparing the oil tea leaf extract of the present invention;

[0028] Figure 2 is a standard curve diagram of rutin of the present invention;

[0029] Figure 3 is a gallic acid standard curve diagram of the present invention;

[0030] Figure 4 It is a DPPH free radical scavenging curve diagram of VC in the DPPH free radical scavenging test of the oil tea leaf extract of the present invention;

[0031] Figure 5 is a DPPH free radical scavenging curve of the oil tea leaf water extract of the present invention;

[0032] Figure 6 It is a curve diagram of the fluorescence intensity decay over time of the Trol ox series standard solutions of the present invention;

[0033] Figure 7 It is a standard curve diagram of the Trol ox series standard solutions of the present invention;

[0034] Figure 8 is a kojic acid standard curve diagram of the present invention;

[0035] Fig. 9 It is a bar chart showing the proliferation rate of the tea leaf water extract of the present invention on HaCaT cells;

[0036] Fig.10 It is a bar chart showing the proliferation rate of HFF-1 cells by the oil tea leaf water extract of the present invention. DETAILED DESCRIPTION

[0037] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0038] like Figure 1 As shown, a method for preparing an oil tea leaf extract comprises the following specific steps: taking fresh Lu'an "Dabie Mountain No. 1" oil tea leaves, drying and crushing them to obtain oil tea leaf powder;

[0039] Add pure water to the tea leaf powder, with a solid-liquid ratio of 1:20, perform ultrasonic extraction, the extraction temperature is 30°C, the ultrasonic power is 500W, the extraction time is 30min, and the extract is filtered;

[0040] The mixture was centrifuged at a centrifugal speed of 8000 rpm for 5 minutes; the supernatant was concentrated by rotary evaporation and freeze-dried for 48 hours to obtain the oil tea leaf extract.

[0041] Determination of flavonoids content in tea leaf extract:

[0042] The present invention adopts sodium nitrite-aluminum nitrate colorimetry to determine the flavonoid content of the obtained oil tea leaf extract, which specifically includes the following steps:

[0043] 1. Preparation of reagents:

[0044] a. Rutin standard solution: Accurately weigh 6.0 mg of rutin standard, dissolve it in a small amount of methanol, and then dilute it to a 25 mL volumetric flask. Shake well to obtain a rutin standard solution with a concentration of 0.024 mg / mL;

[0045] b. Test solution: Accurately weigh the aqueous extract of tea leaves and prepare it with pure water to a mass concentration of 1.00 mg / mL.

[0046] 2. Determination steps:

[0047] The entire reaction process was carried out in a 96-well plate with a total reaction system of 200 μL, and the absorbance was measured at 510 nm using an enzyme reader.

[0048] First, add 0μL, 4μL, 8μL, 16μL, 32μL, and 48μL of rutin standard solution to a 96-well plate, then add 48μL of deionized water and 8μL of 5% NaNO2 reagent, shake evenly, and place in the dark for 6 minutes;

[0049] Then add 8 μL of 10% Al(NO3)3 reagent, shake evenly and let stand in the dark for 6 min;

[0050] Then add 80 μL of 1 mol / L NaOH solution to terminate the reaction, and finally dilute to 200 μL with deionized water, and repeat three groups in parallel. The blank control group is the one without adding rutin standard solution, and the absorbance value is measured at a wavelength of 510 nm. The regression equation between the concentration of rutin standard solution and the absorbance value is calculated based on this value;

[0051] The sample group took 4 μL of the test solution and performed the above method in three parallel groups to measure the absorbance values. The average absorbance value was substituted into the regression equation of the rutin standard curve to obtain the total flavonoids content in each group of samples.

[0052] 3. Measurement results:

[0053] like Figure 2 As shown in Figure 2, the regression equation between the concentration of rutin standard solution and the absorbance value is y = 7.5685x-0.0021, R 2 =0.999 (where R 2 is the determination coefficient, which indicates the goodness of model fitting. A value close to 1 means that the regression equation can well explain the data variation). The flavonoid content of tea leaf extract is (120.52±0.62) mg / g.

[0054] Determination of polyphenol content in tea leaf extract:

[0055] The present invention adopts the Folin-Ciocalteu colorimetric method to measure the polyphenol content of the obtained oil tea leaf extract, which specifically includes the following steps:

[0056] 1. Preparation of reagents:

[0057] a. Gallic acid standard solution: accurately weigh 6.5 mg of gallic acid standard, dissolve it in a small amount of methanol, and then dilute it to a 25 mL volumetric flask. Shake well to obtain a gallic acid standard solution with a concentration of 0.026 mg / mL.

[0058] b. Test solution: Accurately weigh the aqueous extract of tea leaves and prepare it with pure water to a mass concentration of 1.00 mg / mL.

[0059] 2. Determination steps:

[0060] The entire reaction process was carried out in a 96-well plate with a total reaction system of 200 μL, and the absorbance was measured at 765 nm using an ELISA reader.

[0061] First, add 0 μL, 3.2 μL, 4.0 μL, 4.8 μL, 6.4 μL, and 8.0 μL of gallic acid standard solution to a 96-well plate, then add 80 μL of deionized water and 16 μL of folin phenol colorimetric agent, shake evenly, and place in the dark for 8 minutes;

[0062] Then add 16 μL of 15% Na2CO3 reagent, shake evenly and let stand in the dark for 2 h;

[0063] Finally, the volume was adjusted to 200 μL with deionized water, and the experiment was repeated three times in parallel. The blank control group was a control group without adding gallic acid standard solution, and the absorbance value was measured at a wavelength of 765 nm. The regression equation between the concentration of gallic acid standard solution and the absorbance value was calculated based on this value.

[0064] The sample group took 8 μL of the test solution and repeated the above method in three groups in parallel to measure the absorbance values. The average absorbance value was substituted into the regression equation of the gallic acid standard curve to obtain the total polyphenol content in each group of samples.

[0065] 3. Measurement results:

[0066] like Figure 3 As shown in Figure 2, the regression equation between the concentration of gallic acid standard solution and the absorbance value is y = 63.828x-0.0038, R 2 =0.9998, the polyphenol content of camellia oleifera extract is (146.16±1.88) mg / g.

[0067] DPPH free radical scavenging test of tea leaf extract:

[0068] 1. Reagent preparation:

[0069] a. DPPH solution: Accurately weigh DPPH, fully dissolve it in anhydrous ethanol, prepare a 0.06 mg / mL DPPH solution, and store it away from light;

[0070] b. Positive control: Accurately weigh the ascorbic acid standard, fully dissolve it in pure water, and prepare a 1 mg / mL standard solution;

[0071] c. Test solution: Accurately weigh the aqueous extract of tea leaves and prepare it with pure water to a mass concentration of 1.00 mg / mL.

[0072] 2. Experimental steps:

[0073] The entire reaction was carried out in a 96-well plate with a total reaction system of 200 μL. The absorbance was measured at 517 nm using an ELISA reader. The specific design is shown in Table 1.

[0074] Table 1

[0075]

[0076] In this system:

[0077] The sample solutions were the test substance (water extract of tea leaf) and the positive control (V C ).

[0078] The final concentrations of the tea leaf water extract were 3.91 μg / mL, 7.81 μg / mL, 15.62 μg / mL, 31.25 μg / mL, and 62.50 μg / mL;

[0079] V C The final concentrations were 1.95 μg / mL, 3.91 μg / mL, 7.81 μg / mL, 15.62 μg / mL, and 31.25 μg / mL;

[0080] During the test, the sample solution was added to the 96-well plate in sequence, and the DPPH solution was added in the dark, mixed thoroughly, and incubated in a 37°C constant temperature environment for 30 minutes in the dark. The plate was measured at a wavelength of 517 nm using an ELISA reader, and three parallel wells were made for each group. The scavenging rate of the test substance or kojic acid on DPPH free radicals was calculated using the following formula, and the half inhibition concentration (IC) was calculated based on the concentration-scavenging rate curve. 50 ).

[0081] DPPH free radical scavenging rate = (1-(A1-A d ) / A0)×100%

[0082] in,

[0083] A1—average absorbance of sample group;

[0084] A d —The average absorbance of the sample background group;

[0085] A0—average absorbance of DPPH group.

[0086] 3. Test results:

[0087] like Figure 4 , Figure 5 As shown in Figure 2, the IC of scavenging DPPH free radicals by tea leaf extract 50 The value was (33.33±0.41)μg / mL; the positive control V C ICs that scavenge DPPH free radicals 50 The value is (5.65±0.56)μg / mL.

[0088] In vitro antioxidant capacity index test of tea leaf extract:

[0089] 1. Reagent preparation:

[0090] a. Potassium phosphate buffer (PBS): pH = 7.4, 0.2 mol / mL;

[0091] b. Fluorescein sodium solution: Accurately weigh 23.8 mg of fluorescein sodium, add PBS and mix thoroughly, make up to 100 mL, store the solution at 4°C for later use, and dilute 1000 times with buffer solution when used;

[0092] c. Free radical AAPH solution: accurately weigh 495.938 mg of AAPH, add PBS and mix thoroughly, and dilute to 100 mL;

[0093] d. Water-soluble V E Standard solution (Tro-lox): Accurately weigh 25.03 mg of Tro-lox, add PBS and mix thoroughly, make up to 10 mL, and dilute 1000 times with PBS before use;

[0094] e. Test solution: Accurately weigh the aqueous extract of tea leaves and prepare it with pure water to a mass concentration of 1.00 mg / mL.

[0095] 2. Test steps:

[0096] The entire reaction was carried out in a 96-well plate with a total reaction system of 200 μL. The fluorescence intensity was continuously measured at a constant temperature of 37°C using an ELISA reader with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The measurement time was set until the fluorescence decayed to the baseline. The specific design is shown in Table 2:

[0097] Table 2

[0098]

[0099] In this system:

[0100] The sample solution is an aqueous extract of tea leaves; the Trol ox control group is the positive control group; in addition, two other controls need to be set, namely the FL fluorescence natural attenuation control without the addition of free radicals (solvent control group) and the free radical action control in the absence of antioxidants (AAPH control group).

[0101] The final concentrations of the tea leaf water extract were 0.09 μg / mL, 0.18 μg / mL, 0.39 μg / mL, 0.78 μg / mL, and 1.56 μg / mL;

[0102] The final concentrations of Tro l ox were 0.03 μg / mL, 0.06 μg / mL, 0.12 μg / mL, 0.25 μg / mL, 0.50 μg / mL, and 1.00 μg / mL;

[0103] During the test, the sample solution (or Trol ox solution), PBS, and sodium fluorescein solution were added to the 96-well plate in sequence and pre-set at a constant temperature of 37°C for 5 minutes. Then, 140 μL of AAPH solution was quickly added to each well to start the reaction. The reaction was repeated in three groups in parallel. The 96-well plate was placed in a microplate reader and continuously measured at a constant temperature of 37°C with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The fluorescence intensity of each well was measured every 2 minutes, and the measurement time was generally set until the fluorescence decayed to the baseline.

[0104] like Figure 7 As shown, the absolute fluorescence intensity of each microwell at different time points obtained in the experiment is compared with the fluorescence intensity at the initial time, and converted into relative fluorescence intensity f. The area under the fluorescence decay curve (AUC) is calculated using the relative fluorescence intensity using the approximate integration method. The formula is:

[0105] AUC = 0.5 × [2 × (f0 + f1 + ... + f n-1 +f n )-f0-f n ]×△t

[0106] in,

[0107] f n represents the relative fluorescence intensity of the nth measurement point;

[0108] △t represents the time interval between two adjacent time points (i.e., 2 min);

[0109] Then the above formula can be simplified to:

[0110] AUC=2×(f0+f1+…+f n-1 +f n )-f0-f n

[0111] like Figure 6 As shown in the figure, the difference between the area under the fluorescence decay curve under the action of antioxidants and the area under the fluorescence decay curve under the action of free radicals in the absence of antioxidants, that is, the delayed part area of ​​the fluorescence quenching curve NetAUC is the protection area of ​​the antioxidant. The ORAC value of the antioxidant's oxygen free radical scavenging ability, also known as the antioxidant capacity index of the antioxidant, is obtained by comparing the protection area of ​​the fluorescence decay curve with the protection area of ​​the standard antioxidant substance. The calculation formula is:

[0112] ORAC value = [(AUC sample -AUC+AAPH ) / (AUC Trolox -AUC +AAPH )]×C1 / C2

[0113] Wherein, C1 is the Trol ox concentration and C2 is the sample concentration.

[0114] 3. Test results:

[0115] The ORAC value of the oil tea leaf extract was (2395±80.86) μmol TE / g.

[0116] Tyrosinase activity inhibition test of tea leaf extract:

[0117] 1. Preparation of reagents:

[0118] a. Phosphate buffer solution (PBS): pH = 6.8, 0.1 mol / mL;

[0119] b L-tyrosine solution: weigh 0.025 g L-tyrosine, dissolve it in PBS buffer, sonicate, and dilute to 50 mL. Prepare and use immediately.

[0120] c. Tyrosinase solution: Prepare tyrosinase to 500 U / mL with PBS buffer, aliquot and store at -20℃ to avoid secondary freezing and thawing;

[0121] d. Positive control: accurately weigh kojic acid and dissolve it in PBS buffer and dilute it into a series of mass concentration gradient solutions: 0.010 mg / mL, 0.008 mg / mL, 0.005 mg / mL, 0.002 mg / mL, 0.001 mg / mL;

[0122] e. Test solution: Accurately weigh the water extract of tea leaf oil and dissolve it in PBS buffer and dilute it into a series of mass concentration gradients: 1.00 mg / mL, 0.80 mg / mL, 0.50 mg / mL, 0.25 mg / mL, 0.10 mg / mL.

[0123] 2. Test steps:

[0124] The entire reaction process was carried out in a 96-well plate, the total reaction system was 130 μL, and the absorbance was measured at 475 nm using an ELISA reader. The specific design is shown in Table 3:

[0125] Table 3

[0126]

[0127]

[0128] In this system:

[0129] The sample solutions were the test substance (water extract of tea leaf oil) and the positive control (kojic acid).

[0130] The final concentrations of the oil tea water extract were 307.69 μg / mL, 246.15 μg / mL, 153.85 μg / mL, 76.92 μg / mL, and 30.77 μg / mL;

[0131] The final concentrations of kojic acid were 3.08 μg / mL, 2.46 μg / mL, 1.54 μg / mL, 0.62 μg / mL, and 0.31 μg / mL;

[0132] During the test, L-tyrosine solution, different concentrations of test substance solution or kojic acid solution were added to the 96-well plate in sequence, and then PBS solution was added to make the reaction system reach 110 μL, and the mixture was fully mixed. After incubation at 37°C for 10 minutes, 20 μL of tyrosinase solution was added to each well in sequence, and the mixture was mixed at 37°C for 5 minutes. The measurement was performed at a wavelength of 475 nm using an enzyme reader, and three parallel wells were made for each group. The inhibition rate of the test substance or kojic acid on tyrosinase was calculated using the following formula, and the half inhibition concentration (IC) was calculated based on the concentration-inhibition rate curve. 50 ), the calculation formula is as follows:

[0133] Y=(1-(A d -A c ) / (A b -A a ))×100%

[0134] in

[0135] Y—tyrosinase activity inhibition rate (%);

[0136] A d —Average absorbance of sample reaction wells;

[0137] A c —Average absorbance of sample background wells;

[0138] A b —Average absorbance of solvent reaction wells;

[0139] A a —Average absorbance of solvent background wells.

[0140] 3. Test results:

[0141] like Figure 8 As shown in Figure 2, the IC value of the inhibition rate of tea leaf extract on tyrosinase is 50 The value is (136.86±16.12)μg / mL; the IC value of the positive control kojic acid on tyrosinase activity inhibition 50The value is (1.68±0.06)μg / mL.

[0142] It should be noted that tyrosinase is the rate-limiting enzyme in melanin synthesis, which can catalyze the oxidation of L-tyrosine into dopaquinone, and then generate melanin. Overexpression of melanin can lead to pigmentation, freckles, spots and even melanoma. Inhibiting the activity of tyrosinase can reduce the production of melanin, achieving the effect of removing spots and whitening. In recent years, many reagents have been found to inhibit the activity of tyrosinase, such as arbutin, kojic acid, ascorbic acid, etc., but these all have certain limitations and safety hazards. Therefore, finding tyrosinase inhibitors from natural plants has become the key research direction of whitening cosmetics.

[0143] HaCaT cell proliferation activity test of tea leaf extract:

[0144] 1. Preparation of reagents:

[0145] DMEM culture medium; fetal bovine serum (FBS); double antibody; trypsin; PBS buffer (PBS, 0.01 mol / L, pH 7.4); CCK-8.

[0146] 2. Test steps:

[0147] Place HaCaT cells in a cell culture flask, add 4 mL of cell culture fluid (90% DMEM medium + 10% fetal bovine serum), and culture in a 37°C, 5% CO2 incubator. After a period of time, place the culture flask under an inverted microscope to observe the cell growth. When the cells grow to 80%-90% of the culture flask, plate the cells;

[0148] Take logarithmic phase cells and inoculate them into 96-well plates, with about 2000 cells per well. After culturing for 24 hours, aspirate the cell culture medium, add 200 μL of tea leaf extract sample solution to each well according to the concentration gradient, continue culturing for 24 hours, and then half-change the medium. After another 24 hours of culturing, aspirate the sample solution, add 100 μL of DMEM medium containing 10% CCK-8 to each well, incubate in a 37°C, 5% CO2 incubator for 2 hours, and use an enzyme marker to measure the absorbance value at 450nm. The experiment set up three groups, namely the experimental group, the control group, and the blank group. Three parallel wells were made in each group, and the proliferation activity of the test substance on HaCaT cells was calculated using the following formula:

[0149] Cell survival rate = [(A s -A b ) / (A c -A b )]×100%

[0150] in

[0151] As is the absorbance value of the experimental well (containing cells, culture medium, CCK-8 solution and sample solution);

[0152] A c is the absorbance value of the control well (containing cells, culture medium, and CCK-8 solution, but not sample solution);

[0153] A b The absorbance value of the blank well (containing culture medium and CCK-8 solution, but not cells or sample solution)

[0154] 3. Test results:

[0155] like Fig. 9 As shown, the optimum concentration of tea leaf extract on the proliferation activity of HaCaT cells was 125.00 μg / mL, and the cell proliferation rate reached (161.89±6.53)%.

[0156] HaCaT cells are human immortalized keratinocytes, which are immortalized keratinocytes of non-tumor origin from normal human skin. They are epidermal cells, have an epithelial cell-like morphology, and generally grow adherently. HaCaT cells can cause melanin to accumulate in the skin, prevent and reduce the formation of blood spots, and play a protective role on the skin. Because of their immortality, they can continue to divide and proliferate under culture conditions without undergoing natural apoptosis, and retain the characteristics of normal epidermal cells, and are relatively easy to culture, thus providing convenient experimental conditions suitable for various laboratory studies. Currently, HaCaT cells are widely used in the study of skin-related diseases, such as psoriasis, eczema, etc., as well as toxicity testing of drugs, cosmetics, and compounds.

[0157] Camellia oleifera extract HFF-1 cell proliferation activity test:

[0158] 1. Preparation of reagents:

[0159] DMEM medium; fetal bovine serum (FBS); double antibody; trypsin; PBS buffer (PBS, 0.01 mol / L, pH 7.4); CCK-8

[0160] 2. Experimental steps:

[0161] Place HFF-1 cells in a cell culture flask, add 4 mL of cell culture fluid (90% DMEM medium + 10% fetal bovine serum), and culture in a 37°C, 5% CO2 incubator. After a period of time, place the culture flask under an inverted microscope to observe cell growth. When the cells grow to 80%-90% of the culture flask, plate the cells.

[0162] Take the logarithmic phase cells and inoculate them into a 96-well plate, with each well containing about 2000 cells. After culturing for 24 hours, the cell culture medium was aspirated, and 200 μL of tea leaf extract sample solution was added to each well according to the concentration gradient. After continuing to culture for 24 hours, half of the medium was replaced. After another 24 hours of culture, the sample solution was aspirated, and 100 μL of DMEM culture medium containing 10% CCK-8 was added to each well. The plates were incubated in a 37°C, 5% CO2 incubator for 2 hours. The absorbance value was measured at 450nm using an enzyme marker. Three groups were set up in the experiment, namely, the experimental group, the control group, and the blank group. Three parallel wells were made in each group. The proliferation activity of the test substance on HFF-1 cells was calculated using the following formula:

[0163] Cell survival rate = [(A s -A b ) / (A c -A b )]×100%

[0164] in

[0165] A s is the absorbance value of the experimental well (containing cells, culture medium, CCK-8 solution and sample solution);

[0166] A c is the absorbance value of the control well (containing cells, culture medium, and CCK-8 solution, but not sample solution);

[0167] A b The absorbance value of the blank well (containing culture medium and CCK-8 solution, but not cells or sample solution)

[0168] 3. Test results:

[0169] like Fig.10 As shown, the optimum concentration of tea leaf extract on the proliferation activity of HFF-1 cells was 62.50 μg / mL, and the cell proliferation rate reached (113.24±0.22)%.

[0170] HFF-1 cells are human skin fibroblasts, belonging to the dermis. They are isolated and established from the foreskin of newborns and can be used as feeder cells to support the growth of embryonic stem cells or maintain the undifferentiated state of embryonic stem cells. HFF-1 cells are a limited passage cell line that plays an important role in the process of skin aging. When the skin ages, fibroblasts will undergo a series of changes such as irreversible growth inhibition, decreased secretion and synthesis functions, and reduced cell self-repair ability. Fibroblasts are one of the typical cell model cells in the field of skin research. Recent studies have shown that human skin fibroblasts are widely used in skin wound healing and signal transduction mechanisms. In addition, human skin fibroblasts are stable in state and have strong proliferation ability, and are also widely used in experimental studies on the antioxidant efficacy of natural plant extracts.

[0171] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing an oil tea leaf extract, characterized in that: The following steps are involved: S1. Preparation of oil tea leaf powder: collecting oil tea leaves, drying them, and crushing them to obtain oil tea leaf powder; S2. Preparation of Camellia oleifera extract: S201, adding a solvent to the oil tea leaf powder, and performing ultrasonic extraction to obtain a solution A; S202, filtering solution A, and centrifuging the filtrate to obtain an extract; S203, using a rotary evaporator to concentrate the extract to obtain a concentrated extract, and freeze-drying the concentrated extract to obtain an oil tea leaf extract.

2. The method for preparing a tea leaf extract according to claim 1, characterized in that: In step S201, the solution is pure water, and the material-liquid ratio of the oil tea leaf powder to pure water is 1:20-50.

3. The method for preparing a tea leaf extract according to claim 2, characterized in that: In step S201, the temperature of ultrasonic extraction is 20-50°C, the ultrasonic power is 500W, and the extraction time is 20-40min.

4. The method for preparing the tea leaf extract according to claim 3, characterized in that: In step S202, the centrifugal speed is 6000-8000 rpm, and the centrifugal time is 5-10 min.

5. The method for preparing the tea leaf extract according to claim 4, characterized in that: In step S203, vacuum freeze drying technology is used, and the drying time is 24-72 hours.

6. A tea leaf extract, characterized in that The oil tea leaf extract is prepared according to the method for preparing the oil tea leaf extract according to any one of claims 1 to 5.

7. The oil tea leaf extract according to claim 6, characterized in that The oil tea leaf extract contains flavonoids and polyphenols, wherein the flavonoid content is 11.99% to 12.11%, and the polyphenol content is 14.43% to 14.79%.

8. The oil tea leaf extract according to claim 7, characterized in that The oil tea leaf extract has an IC of 120.74 μg / mL to 152.98 μg / mL for inhibiting tyrosinase. 50 value.

9. The oil tea leaf extract according to claim 7, characterized in that: The oil tea leaf extract has an IC of (33.33±0.41) μg / mL to scavenge DPPH free radicals. 50 value.

10. The oil tea leaf extract according to claim 9, characterized in that: The ORAC antioxidant capacity index of the oil tea leaf extract is 2314.14 μmol TE / g-2475.86 μmol TE / g.

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