CN117414320A - Preparation method and application of sweet tea extract - Google Patents
Preparation method and application of sweet tea extract Download PDFInfo
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- CN117414320A CN117414320A CN202311399693.1A CN202311399693A CN117414320A CN 117414320 A CN117414320 A CN 117414320A CN 202311399693 A CN202311399693 A CN 202311399693A CN 117414320 A CN117414320 A CN 117414320A
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Abstract
The invention relates to the technical field of plant extraction, in particular to the field of IPC A61K8, and further relates to a preparation method and application of a sweet tea extract. The method comprises the following steps: s1, crushing sweet tea leaves into coarse powder; s2, carrying out ultrasonic leaching on the coarse powder in a solvent; s3, centrifuging after leaching, and filtering again to obtain filtrate; s4, adsorbing the filtrate by a macroporous adsorption resin column; s5, eluting with ethanol water solution after adsorption, and concentrating under reduced pressure. The sweet tea extract prepared by the invention can relieve skin inflammatory reaction, resist photodamage and oxidative stress reaction by repairing skin barrier damage caused by external stimulus, thereby achieving the effects of resisting skin stimulus and maintaining skin steady state.
Description
Technical Field
The invention relates to the technical field of plant extraction, in particular to the field of IPC A61K8, and further relates to a preparation method and application of a sweet tea extract.
Background
With the improvement of living standard, more and more people pay attention to skin health problems, and skin can generate different degrees of irritation reactions, such as barrier function damage, inflammatory reaction, oxidative stress reaction, skin accelerated aging and the like, due to excessive cleaning, excessive exfoliating, disordered use of an irritation product and other environmental irritation factors in daily life, and the symptoms of redness, stinging, itching, dryness, fine wrinkles and the like are displayed. These stimulus responses, in turn, promote accumulation and thus may be trapped in an unbalanced condition and poorly cycled skin, further exacerbating skin sensitivity and aging.
For example, environmental stimuli acting on the skin surface may first cause an impaired barrier function of the skin; the important factors responsible for the skin barrier are Filaggrin (FIG) and caspase-14 (caspase-14) down-regulation; when environmental stimuli penetrate further into the epidermis, they cause damage to skin cells, which produce and release various soluble inflammatory factors, such as the signaling molecules NO, pro-inflammatory factors (IL-1. Beta., PGE) 2 ) Tumor necrosis factor TNF-alpha, chemokine IL-8, causes an inflammatory response in the skin.
In addition to these problems, environmental stimuli like ultraviolet radiation can cause an imbalance in the antioxidant system of the skin in addition to directly causing inflammatory damage to the cells; this in turn results in:
(1) ROS (reactive oxygen species) are generated due to activated inflammatory cells and oxidative metabolites of various cells, etc.;
(2) Oxidative damage of ROS to cells triggers release of TNF- α, which binds to cell surface receptors activating nuclear transcription factor (nuclear transcription factor- κb, NF- κb) signaling pathways, up-regulation of ROS levels results in over-expression of other inflammatory factors, up-regulation of vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and inflammatory factor (IL-1β, IL-8, TNF- α, etc.) synthesis, contributing to chronic inflammation;
(3) Chronic inflammation further accelerates oxidative stress, and more ROS stimulates the cells to synthesize large amounts of matrix metalloproteinases (matrix metallo proteinase, MMPs), degrading the extracellular matrix (extracellular matrix, ECM) collagen and elastin, the most important of the dermis, causing damage to the connective tissue structure of the dermis, leading to skin aging.
The natural product has a wide application prospect for maintaining skin homeostasis due to the characteristics of the diversity structure and easy combination with biomacromolecules, but the research on the natural product which can resist skin irritation and maintain skin homeostasis is less at present.
Sweet tea (Rubus suavissimus S.Lee) is a medicinal and edible plant integrating medicine, tea and sugar. At present, the sweet tea is rich in various physiologically active components such as rubusoside, polyphenol, amino acid, flavonoid compounds and the like, and is mainly used in the fields of medicines and foods; can reduce blood sugar, blood lipid and blood pressure, promote metabolism, and regulate immunity. For example, chinese patent No. 116251160A discloses that sweet tea extract is used in combination in antibacterial and sterilizing products, and has the functions of sterilizing oral cavity, removing bad smell of oral cavity, cleaning and protecting teeth, etc.; chinese patent No. CN104351771a discloses a method for preparing antiallergic composite food containing sweet tea extract; none of the above technical solutions involves the use of sweet tea extract for skin protection against skin irritation and maintenance of skin homeostasis.
Disclosure of Invention
The first aspect of the invention provides a preparation method of sweet tea extract, comprising the following steps:
s1, crushing sweet tea leaves into coarse powder;
s2, carrying out ultrasonic leaching on the coarse powder in a solvent;
s3, centrifuging after leaching, and filtering again to obtain filtrate;
s4, adsorbing the filtrate by a macroporous adsorption resin column;
s5, eluting with ethanol water solution after adsorption, and concentrating under reduced pressure.
Preferably, the solvent is selected from water or an aqueous ethanol solution.
Preferably, the solvent is water.
Preferably, the liquor ratio (coarse powder: solvent) of the ultrasonic leaching is 1: (15-25) g/mL.
Preferably, the ratio of the feed liquid of the ultrasonic leaching is 1:20g/mL.
Preferably, the temperature of the ultrasonic leaching is 60-80 ℃.
Preferably, the temperature of the ultrasonic leaching is 65-75 ℃.
Preferably, the temperature of the ultrasonic leaching is 70 ℃.
Preferably, the ultrasonic leaching time is 20-80min.
Preferably, the ultrasonic leaching time is 30-60min.
Preferably, the time of the ultrasonic leaching is 60 minutes.
In the prior art, an ethanol aqueous solution is mostly adopted for extracting sweet tea, for example, chinese patent CN01107015, but the applicant found in the experimental process that when the solvent used in the extraction process is water, the ratio of feed to liquid is controlled to be 1: (15-25) g/mL, the accumulation of active ingredients (such as polyphenol and rubusoside) in the sweet tea extract is more favorable than that of an ethanol aqueous solution, and the applicant speculates that the microstructure of the sweet tea coarse powder is also damaged to a certain extent under the action of ultrasound, so that the interaction force between water molecules and the sweet tea coarse powder molecules is larger, and further, the active ingredients in the sweet tea are more dissolved in water. But at the same time, the applicant also found that the sweet tea extract prepared only has the effect of resisting skin irritation when the temperature of ultrasonic extraction is 60-80 ℃, especially when the temperature of ultrasonic extraction is 70 ℃, and the applicant speculates that the extraction liquid can be prevented from containing a large amount of ineffective components to a certain extent because sweet tea leaves are extracted at the temperature; further, it was found that sweet tea extract capable of resisting skin irritation can be prepared when the time of ultrasonic extraction is 20-80min, especially when the time of ultrasonic extraction is 30-60min, but when the time of extraction exceeds 60min, not only the effective components of sweet tea extract are not increased, but also the effect of resisting skin irritation is affected; the applicant speculates that the difference in endo-solubles concentration between sweet tea and solution gradually decreases with the increase of leaching time, and the exchange rate of endo-solubles between the sweet tea and the solution becomes slow; at the same time, the ineffective ingredients are also increased, thereby causing the effect of resisting skin irritation to be affected.
Preferably, the adsorption time is 25-35min.
Preferably, the adsorption time is 30min.
Preferably, the volume fraction of ethanol in the ethanol aqueous solution is 30-90%.
Preferably, the volume fraction of ethanol in the ethanol aqueous solution is 40-80%.
Preferably, the volume fraction of ethanol in the aqueous ethanol solution is 80%.
In a second aspect, the invention provides the use of a sweet tea extract for anti-skin irritation by increasing expression of Caspase-14 and FLG inhibited after stimulation of HaCaT cells by SLS; reducing NO, IL-1 beta, IL-8, TNF-alpha and PGE after LPS stimulation of RAW264.7 cells 2 Is expressed by (a); the expression of MMP-1 and elastase after the HFF cells are stimulated by UVA is reduced, the expression of the suppressed Collagen I and elastin is improved, the clearance rate of DPPH and ABTS free radicals is improved, and the solid content of the sweet tea extract is more than 2wt%; the solid content is the dry matter content in the concentrated solution obtained after sweet tea extraction.
Preferably, the sweet tea extract has a solids content of 6-12.2wt%.
Further preferably, the sweet tea extract has a solids content of 12wt%.
Further preferably, the concentration of the sweet tea extract is 0.05-5mg/mL; the diluent is HaCaT cell culture solution or RAW264.7 cell culture solution or HFF cell culture solution.
Further preferably, the concentration of the sweet tea extract is 0.1-5mg/mL; the diluent is HaCaT cell culture solution or RAW264.7 cell culture solution or HFF cell culture solution.
Further preferably, the concentration of the sweet tea extract is 0.1-2mg/mL; the diluent is HaCaT cell culture solution or RAW264.7 cell culture solution or HFF cell culture solution.
The applicant is in experimentIn the process, the sweet tea extract with the solid content of more than 2 weight percent, particularly the sweet tea extract with the solid content of 12 percent, which is prepared by adopting the preparation method can be used for improving the expression of Caspase-14 and FLG which are inhibited after the SLS stimulates HaCaT cells; reducing NO, IL-1 beta, IL-8, TNF-alpha and PGE after LPS stimulation of RAW264.7 cells 2 Is expressed by (a); reduce the expression of MMP-1 and elastase after UVA stimulates HFF cells, improve the expression of restrained Collagen I and elastin, and promote the clearance rate of DPPH and ABTS free radicals, thereby realizing the effects of resisting skin irritation and maintaining skin steady state.
The beneficial effects are that:
1. the solvent used in the leaching process is water, and the control feed liquid ratio is 1: (15-25) g/mL, is more advantageous than aqueous ethanol for accumulation of active ingredients in sweet tea extract.
2. The sweet tea extract with high solid content and capability of resisting skin irritation can be prepared when the temperature of ultrasonic leaching is 60-80 ℃, especially when the temperature of ultrasonic leaching is 70 ℃.
3. The sweet tea extract with higher solid content and capability of resisting skin irritation can be prepared when the ultrasonic leaching time is 30-60min, especially when the ultrasonic leaching time is 60min.
4. According to the application, when the volume fraction of the ethanol in the ethanol water solution is 30-90%, particularly when the volume fraction of the ethanol in the ethanol water solution is 80%, the active ingredients which can resist skin irritation in the sweet tea extract can be effectively eluted, and meanwhile, the ineffective impurity content is also less, so that the solid content of the sweet tea extract is further improved, and the effect of the sweet tea extract on resisting skin irritation is improved.
5. The sweet tea extract with the solid content of more than 2 weight percent, particularly the sweet tea extract with the solid content of 12 percent, can improve the expression of Caspase-14 and FLG which are inhibited after SLS stimulates HaCaT cells; reducing NO, IL-1 beta, IL-8, TNF-alpha and PGE after LPS stimulation of RAW264.7 cells 2 Is expressed by (a); reduce MMP-1 and elastase expression after UVA stimulates HFF cells, and improve the surface of inhibited Collagen I and elastinThe scavenging rate of DPPH and ABTS free radicals is improved, so that the skin barrier injury caused by external stimulation is repaired, the skin inflammatory reaction is relieved, the photodamage and the oxidative stress reaction are resisted, and the effects of resisting skin stimulation and maintaining the skin steady state are achieved.
Drawings
FIG. 1 is a graph of the dilution of sweet tea extract at half a concentration of 0.1mg/mL versus DPPH radical scavenging test;
in the figure: a is 0.1mg// mL VC sesquidilution; b is 0.1mg/mL of sweet tea extract sesquidilution;
FIG. 2 is a graph of sweet tea extract versus ABTS radical clearance test;
in the figure: a is VC; b is sweet tea extract.
Detailed Description
The sweet tea leaves used in examples 1 to 3 were purchased from Yao mountain, a major autonomous area of Guangxi Zhuang nationality, china.
Example 1: a preparation method of sweet tea extract comprises the following steps: pulverizing folium hydrangeae strigosae into coarse powder, adding water at a feed liquid ratio of 1:20g/mL, ultrasonic extracting at 70deg.C for 30min, filtering, centrifuging, filtering again, adsorbing filtrate with macroporous adsorbent resin column for 30min, eluting with 40% ethanol, and concentrating under reduced pressure to obtain folium hydrangeae strigosae extract.
The solid content of the sweet tea extract is 5wt%.
Example 2: a preparation method of sweet tea extract comprises the following steps: pulverizing folium hydrangeae strigosae into coarse powder, adding water at a feed liquid ratio of 1:20g/mL, ultrasonic extracting at 70deg.C for 60min, filtering, centrifuging, filtering again, adsorbing filtrate with macroporous adsorbent resin column for 30min, eluting with 60% ethanol, and concentrating under reduced pressure to obtain folium hydrangeae strigosae extract.
The solid content of the sweet tea extract is 8wt%.
Example 3: a preparation method of sweet tea extract comprises the following steps: pulverizing folium hydrangeae strigosae into coarse powder, adding water at a feed liquid ratio of 1:20g/mL, ultrasonic extracting at 70deg.C for 60min, filtering, centrifuging, filtering again, adsorbing filtrate with macroporous adsorbent resin column for 30min, eluting with 80% ethanol, and concentrating under reduced pressure to obtain folium hydrangeae strigosae extract.
The solid content of the sweet tea extract is 12wt%.
Example 4: effect of sweet tea extract on SLS-stimulated HaCaT cell barrier-related index
The sweet tea extract prepared in example 3 was tested as follows:
1. effect of sweet tea extract on the viability of HaCaT cells:
the CCK-8 method is a high sensitivity, non-radioactive colorimetric assay for determining the number of living cells in a cell proliferation or toxicity assay. The orange yellow formazan dye generated after the CCK-8 reagent is oxidized and reduced by the intracellular dehydrogenase can be dissolved in a tissue culture medium, can be directly measured, and has smaller experimental error.
HaCaT cells are human immortalized epidermal cells, are human normal skin immortalized keratinocyte strains derived from non-tumor sources, have similar differentiation characteristics to normal human keratinocytes, can synthesize biomolecules such as Filaggrin (FLG), caspase-14 (Caspase-14) and the like which are related to skin barriers, and are in vitro research models commonly used for researching skin barrier functions.
A culture flask with good growth of HaCaT cells was selected, and after digestion with trypsin for 3min, 100. Mu.L of cell suspension per well (2-3X 10 5 cells/mL) were inoculated into 96-well plates and incubated in an incubator (37 ℃ C., 5% CO) 2 ) For 24h, 100. Mu.L of the sample of the example with solid content of 0.1-5mg/mL diluted by the culture solution is added into each hole of the dosing group, 100. Mu.L of the culture solution is directly added into each hole of the control group, 6 compound holes are designed in each group, and the culture is carried out in an incubator for 24h. After 24h, the CCK-8 reagent and the serum-free medium are mixed uniformly according to the proportion of 1:10, 100 mu L of the serum-free medium containing the CCK-8 reagent is added into each hole, and an incubator (37 ℃ C., 5% CO) 2 ) Incubation was performed for 30min, absorbance values were measured at 450nm using a microplate reader and the results are shown in Table 1.
TABLE 1 Effect of sweet tea extract on HaCaT cell viability
Remarks: # #: p <0.0001, compared to BC; # # # #: p <0.001, compared to BC; #: p <0.05, compared to BC.
As can be seen from table 2: compared with the BC in the blank group, the activity of HaCaT cells treated by the sweet tea extract with the concentration range of 0.1-2mg/mL is over 90 percent, and no obvious cytotoxicity exists. However, the cell viability was reduced to 0.65% at a sweet tea extract concentration of 5mg/mL, with very significant cytotoxicity (p < 0.0001). According to the experimental result, selecting the sample concentration in the range of 0.1-2mg/mL for subsequent experiments.
2. Effect of sweet tea extract on SLS stimulation of HaCaT cell secretion barrier associated factors
Sodium dodecyl sulfate (Sodium lauryl sulfate, SLS) is a surfactant that is widely used in cleaning products, cosmetics and personal care products. SLS is an anionic surfactant that distorts cell membrane proteins. It is well known that irritation is caused when 0.5% sls is used, and skin irritation is usually caused in this way in studies of the efficacy of cosmetics.
A culture flask with good growth of HaCaT cells was selected, and after digestion with trypsin for 3min, 100. Mu.L of cell suspension per well (2-3X 10 5 cells/mL) were inoculated into 96-well plates and incubated in an incubator (37 ℃ C., 5% CO) 2 ) After 24h of medium culture, the cells were cultured for 24h, the remaining groups were added with a medium containing 60. Mu.g/mL SLS, the blank group was added with complete medium, and the 96-well plate was placed in an incubator (37 ℃, 5% CO) 2 ) Culturing for 24h; after 24h, the old culture medium of the pore plate is discarded, the pore plate is washed once by PBS, a positive control group PC is added with a VE solution of 100 mug/mL, a sample group is added with a sweet tea extract solution of 0.1-2mg/mL, a blank group BC is added with a complete culture medium, the culture is carried out in an incubator for 24h, after 24h of dosing culture, the supernatant of the pore plate is collected, and absorbance values are measured by an enzyme-labeled instrument at corresponding wavelengths according to the operation steps of an ELISA kit instruction corresponding to barrier factors FLG and Caspase-14, and the liquid preparation of the SLS-induced HaCaT cell model is shown in Table 2.
TABLE 2 SLS-induced HaCaT cell model fluid formulation
Remarks: BC is blank, NC is model, PC is positive control.
2.1 Effect of sweet tea extract on FLG content, the results are shown in Table 3.
TABLE 3 Effect of sweet tea extract on HaCaT cell FLG content
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC; * **: p <0.001, compared to NC; * *: p <0.01, compared to NC.
As can be seen from Table 3, the SLS treated HaCaT cells in NC group showed a decrease in the amount of FLG secreted by 1500pg/mL, which is significantly lower than that of BC in blank group. After VE (PC group) action, SLS-HaCaT cell barrier stimulation model showed an increase in the level of FLG secreted, resulting in a very significant difference (P < 0.0001). After synchronous treatment of sweet tea extract with concentration of 0.1-2mg/mL, the FLG content increases from 3300pg/mL to 4000pg/mL with the increase of the sample concentration, and the FLG content is concentration-dependent. It can be seen that sweet tea extract has promoting effect on FLG synthesis of SLS-HaCaT cell barrier stimulation model, similar to VE effect.
2.2 Effect of sweet tea extract on Caspase-14 content the results are shown in Table 4.
TABLE 4 Effect of sweet tea extract on HaCaT cell Caspase-14 content
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC; * **: p <0.001, compared to NC; * *: p <0.01, compared to NC.
As can be seen from table 4: the Caspase-14 content of HaCaT cells after SLS treatment in NC group was reduced by 5pmol/L (P < 0.0001), which was significantly lower than that in blank group BC. After VE (PC group) was applied, the SLS-HaCaT cell barrier stimulation model showed an increase in Caspase-14 content, resulting in a very significant difference (P < 0.0001). After synchronous treatment of sweet tea extract with the concentration of 0.1-2mg/mL, the content of Caspase-14 increases from 1.78pmol/L to 3.31pmol/L along with the increase of the sample concentration, and the concentration dependence is shown, wherein 0.1mg/mL of sweet tea extract has no obvious influence on the content of Caspase-14 synthesized by an SLS-HaCaT cell barrier stimulation model. Taken together, the sweet tea extract has a promoting effect on Caspase-14 synthesis of SLS-HaCaT cell barrier stimulation model, and is similar to VE effect.
Example 5: effects of sweet tea extract on related indicators of LPS-stimulated RAW264.7 cell inflammation:
the sweet tea extract prepared in example 3 was tested as follows:
1. effect of sweet tea extract on RAW264.7 cell viability
RAW264.7 is a monocyte/macrophage-like cell line derived from the Abelson leukemia virus transformed cell line of BALB/c microribonucleic acid. RAW264.7 is the in vitro study model most commonly used to screen anti-inflammatory actives and study inflammation. RAW264.7 cells mimic inflammatory response and release or up-regulate various inflammatory mediators such as Nitric Oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1 beta (IL-1 beta) and the like under the action of an inducer (such as lipopolysaccharide LPS).
Culture flasks with good growth of RAW264.7 cells were selected, digested with trypsin for 20s, and after termination of digestion, 100. Mu.L of cell suspension per well (3-5X 10 5 cells/mL) were inoculated into 96-well plates and incubated in an incubator (37 ℃ C., 5% CO) 2 ) For 24h, 100 mu L of the test substance with the solid content of 0.1-20mg/mL diluted by the culture solution is added into each hole of the dosing group, 100 mu L of the culture solution is directly added into each hole of the control group, 6 compound holes are designed in each group, and the culture is carried out in an incubator for 24h. After 24 hours, the CCK-8 reagent and the serum-free culture medium are uniformly mixed according to the proportion of 1:10, and C-containing substances are added into each hole100. Mu.L of CK-8 reagent serum-free medium, incubator (37 ℃, 5% CO) 2 ) Incubation was performed for 30min, absorbance values were measured at 450nm using a microplate reader and the results are shown in Table 5.
TABLE 5 Effect of sweet tea extract on RAW264.7 cell viability
Remarks: # #: p <0.0001, compared to BC; #: p <0.05, compared to BC; # #. p <0.01, compared to BC.
As can be seen from table 5: compared with BC, the cell viability of the sample in the concentration range of 5-20mg/mL is lower than 0.32%, and the sample has extremely remarkable cytotoxicity (p < 0.0001). The RAW264.7 cells treated by the sample with the concentration range of 0.1-1mg/mL have the activity of more than 103.44 percent and have obvious proliferation promoting effect (p is less than 0.01), and the sample concentration in the range of 0.1-2mg/mL is selected for subsequent testing according to comprehensive analysis of experimental results.
2. Effects of sweet tea extract on LPS stimulation of RAW264.7 cells to secrete inflammatory factors:
lipopolysaccharide (LPS) is an endotoxin, commonly used in experimental models of inflammation. Lipopolysaccharide stimulates signaling pathways in macrophages (RAW 264.7) and activates nuclear transcription factors such as Mitogen Activated Protein Kinases (MAPKs) and nuclear factor κb (NF- κb). Lipopolysaccharide also activates macrophage-initiating pro-inflammatory mediators including signal molecules NO, pro-inflammatory factors (IL-1 beta, PGE) 2 ) Tumor necrosis factor TNF-alpha, chemokine IL-8, causes an inflammatory response in the skin.
Culture flask with good growth state of RAW264.7 cells was selected, and after digestion was completed with trypsin for 20s, 1mL of cell suspension per well (1.8-2X 10 5 cells/mL) were inoculated into a 12-well plate, and incubated in an incubator (37 ℃ C., 5% CO) 2 ) After 24h of medium culture and 24h of cell culture, the rest groups are added with a culture medium containing 0.4 mug/mL LPS, the blank groups are added with a complete culture medium, and a 12-well plate is placed in an incubator (37 ℃ C., 5% CO) 2 ) Culturing for 24h; after 24h, the old medium of the well plate was discarded and washed once with PBSAdding dexamethasone solution 100 μg/mL into positive control group PC, adding sweet tea extract solution 0.1-2mg/mL into sample group, adding complete culture medium into blank group BC, culturing in incubator for 24 hr, collecting supernatant of pore plate after administration culture for 24 hr, and mixing with inflammatory factors such as NO, IL-1 beta, IL-8, TNF-alpha, and PGE 2 The corresponding enzyme-linked immunosorbent assay kit instruction manual operation steps are that absorbance values are measured by using an enzyme-labeled instrument under corresponding wavelengths, and the LPS-induced RAW264.7 cell model liquid is shown in Table 6.
TABLE 6 LPS-induced RAW264.7 cell model fluid formulation
2.1 Effect of sweet tea extract on NO content the results are shown in Table 7.
TABLE 7 Effect of sweet tea extract on NO content of RAW264.7 cells
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC.
As can be seen from table 7: after induction by LPS, the NO content released by RAW264.7 cells in NC group increased from 0.70 μm to 26.43 μm with a very significant difference (P < 0.0001) relative to that of the blank BC group. After dexamethasone (PC group) had acted, the amount of NO released by LPS-RAW264.7 inflammatory stimulus model was reduced from 26.43. Mu.M to 8.76. Mu.M, resulting in very significant differences (P < 0.0001). The dry state of the sweet tea extract with the concentration of 0.1-2mg/mL is added into the model, the dry state has extremely remarkable inhibition effect (P < 0.0001) on NO release, the NO release amount is reduced in a dose-dependent manner relative to the concentration of a sample, and when the concentration of the sweet tea extract is increased to 2mg/mL, the NO content is reduced to 1.92 mu M, the sweet tea extract can effectively play a role of relieving stimulation, and the effect is remarkably superior to that of dexamethasone.
2.2 Effect of sweet tea extract on IL-1 beta content the results are shown in Table 8.
TABLE 8 Effect of sweet tea extract on IL-1 beta content of RAW264.7 cells
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC.
As can be seen from table 8: IL-1 beta concentration of RAW264.7 cells after LPS treatment in NC group increased by 63pg/mL, which is significantly higher than that in blank group BC (P < 0.0001). Following dexamethasone (PC) action, the IL-1β content released by the LPS-RAW264.7 inflammatory stimulus model was reduced from 73pg/mL to 27pg/mL (P < 0.0001). After synchronous treatment of the sweet tea extract with the concentration of 0.1-2mg/mL, the content of IL-1 beta is reduced from 66pg/mL to 25pg/mL along with the increase of the sample concentration, and the concentration dependence is shown, wherein the effect of inhibiting the release of IL-1 beta by the 2mg/mL sweet tea extract is better than that of dexamethasone.
2.3 Effect of sweet tea extract on IL-8 content the results are shown in Table 9.
TABLE 9 Effect of sweet tea extract on RAW264.7 cell IL-8 content
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC.
As can be seen from table 9: the IL-8 content of RAW264.7 cells after LPS treatment in NC group increased by 846pg/mL, which is obviously higher than that in blank group BC (P < 0.0001). After dexamethasone (PC group) had acted, the IL-8 content released by LPS-RAW264.7 inflammatory stimulus model was reduced from 900pg/mL to 276pg/mL (P < 0.0001). After synchronous treatment of sweet tea extract with the concentration of 0.1-2mg/mL, the content of IL-8 is reduced from 560pg/mL to 132pg/mL along with the increase of the sample concentration, and the concentration of 2mg/mL of sweet tea extract is concentration-dependent, wherein the effect of inhibiting IL-8 release of an LPS-RAW264.7 model is superior to that of dexamethasone.
2.4 Effect of sweet tea extract on TNF- α content, results are shown in Table 10.
TABLE 10 Effect of sweet tea extract on TNF- α content of RAW264.7 cells
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC.
As can be seen from table 10: TNF- α release from RAW264.7 cells after LPS treatment in NC group increased by 3.76pg/mL, significantly higher than that in the blank group BC (P < 0.0001). After dexamethasone (PC) action, the TNF- α content released by LPS-RAW264.7 inflammatory stimulation model was reduced from 3.9pg/mL to 0.67pg/mL (P < 0.0001). After synchronous treatment of the sweet tea extract with the concentration of 0.1-2mg/mL, the TNF-alpha content is reduced relative to that of NC groups, and the sweet tea extract has an inhibitory effect on TNF-alpha release of an LPS-RAW264.7 inflammation stimulation model, and has a similar effect to dexamethasone.
2.5 sweet tea extract vs PGE 2 The effect of the content is shown in Table 11.
Table 11 sweet tea extract versus RAW264.7 cell PGE 2 Influence of the content
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC.
As can be seen from table 11: PGE of RAW264.7 cells treated with LPS in NC group 2 The release amount of (C) is increased by 220pg/mL, which is obviously higher than that of the BC (P)<0.0001). LPS-RAW264.7 inflammatory stimulus model released PGE after dexamethasone (PC group) action 2 The content was reduced from 240pg/mL to 75pg/mL (P<0.0001). Synchronous treatment of sweet tea extract with concentration of 0.1-2mg/mL, and PGE 2 The content of the sweet tea extract is reduced relative to NC group, and the PGE of the sweet tea extract on the LPS-RAW264.7 cell inflammation stimulus model can be seen 2 Has an inhibitory effect similar to that of dexamethasone.
Example 6: evaluation of related index results of UVA-stimulated HFF cell aging by sweet tea extract
The sweet tea extract prepared in example 3 was tested as follows:
1. effect of sweet tea extract on HFF cell viability
Fibroblasts (HFFs) exist in the dermis and are the main cellular component of loose connective tissue, and cells are in the shape of a spindle or a flat star with protrusions, larger cells and nuclei, clear outline and large and obvious nuclei. Fibroblasts synthesize and secrete not only collagen and elastin, but also matrix components such as collagen fibers, reticular fibers and elastin, and glycosaminoglycans and glycoproteins.
A culture flask with good growth of HFF cells was selected, and after digestion with trypsin for 40s, 100. Mu.L of the cell suspension per well (1-1.5X10 5 cells/mL) were inoculated into 96-well plates and incubated in an incubator (37 ℃ C., 5% CO) 2 ) For 24h, 100 μl of 0.1-5mg/mL solid content sample of the example diluted with culture solution is added to each well of the dosing group, 100 μl of culture solution is directly added to each well of the control group, 6 compound wells are designed for each group, and the culture is carried out in an incubator for 24h. After 24h, the CCK-8 reagent and the serum-free medium are mixed uniformly according to the proportion of 1:10, 100 mu L of the serum-free medium containing the CCK-8 reagent is added into each hole, and an incubator (37 ℃ C., 5% CO) 2 ) Incubation was performed for 30min, absorbance values were measured at 450nm using a microplate reader and the results are shown in Table 12.
TABLE 12 Effect of sweet tea extract on HFF cell viability
Remarks: # #: p <0.0001, compared to BC; # # # #: p <0.001, compared to BC.
As can be seen from table 12: compared with the BC in the blank group, the HFF cell activity treated by the sweet tea extract with the concentration range of 0.1-1mg/mL is above 90%, and no obvious cytotoxicity exists. However, when the concentration of the sweet tea extract is in the range of 1-5mg/mL, the cell viability is obviously reduced along with the increase of the concentration, and when the concentration of the sweet tea extract is in the range of 5mg/mL, the HFF cell viability is reduced to 1.07%, and the sweet tea extract has extremely obvious cytotoxicity (p < 0.0001). And (3) according to comprehensive analysis of experimental results, selecting the concentration of the sample within the range of 0.1-2mg/mL for subsequent experiments.
2. Effect of sweet tea extract on UVA-stimulated HFF cells secreting senescence-associated factors
A culture flask with good growth of HFF cells was selected, and after digestion with trypsin for 40s, 500. Mu.L of the cell suspension per well (0.8-1X 10) 5 cells/mL) were inoculated into 24-well plates and incubated in an incubator (37 ℃ C., 5% CO) 2 ) Is cultured for 24 hours. After cell culture for 24h, the medium in the wells was discarded, PBS was added at 5J/cm 2 UVA irradiation under intensity, adding positive control and sample group with certain concentration, adding complete culture medium, placing 24-well plate in incubator (37deg.C, 5% CO) 2 ) Culturing for 24h; after 24h, the old culture medium of the well plate is discarded, the well plate is washed once by PBS, 100ng/mL of TGF-beta (transforming growth factor beta) solution is added into a positive control group PC, 0.1-2mg/mL of sweet tea extract solution is added into a sample group, the complete culture medium is added into a culture box for culturing for 24h, after 24h of dosing culture, the supernatant of the well plate is collected, and absorbance values are measured by an enzyme-labeled instrument at corresponding wavelengths according to the operation steps of an enzyme-linked immunosorbent kit specification corresponding to the Collagen I, elastin, elastase and MMP-1, and the UVA-induced HFF cell model preparation is shown in a table 13.
TABLE 13 UVA-induced HFF cell model formulation
2.1 Effect of sweet tea extract on Collagen I content the results are shown in Table 14.
TABLE 14 Effect of sweet tea extract on HFF cell Collagen I content
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC; * *: p <0.01, compared to NC; * : p <0.05, compared to NC.
As can be seen from table 14: the content of Collagen I in HFF cells after UVA treatment was reduced by 5ng/mL in NC group compared to BC group. After TGF-beta (PC) action, the Collagen I content of the UVA-HFF aging stimulation model increased from 6ng/mL to 8ng/mL (P < 0.0001). After synchronous treatment of the sweet tea extract with the concentration of 0.1-2mg/mL, the content of the Collagen I is increased relative to that of NC groups, and the sweet tea extract has a promotion effect on the Collagen I secreted by a UVA-HFF aging stimulation model and is similar to the TGF-beta effect.
2.2 influence of sweet tea extract on elastin content, the results are shown in Table 15.
TABLE 15 Effect of sweet tea extract on the elastin content of HFF cells
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC; * *: p <0.01, compared to NC; * : p <0.05, compared to NC.
As can be seen from table 15: the content of Collagen I in HFF cells after UVA treatment was reduced by 2.6ng/mL in NC group compared to BC group. After TGF-beta (PC) action, the elastin content of the UVA-HFF aging stimulation model increased from 0.2ng/mL to 1.8ng/mL (P < 0.0001). After synchronous treatment of sweet tea extract with concentration of 0.1-2mg/mL, the elastin content increases from 0.46ng/mL to 1.6ng/mL along with the increase of the sample concentration, and the concentration dependence is shown. It can be seen that the sweet tea extract has an effect of promoting the secretion of elastin by UVA-HFF cell senescence-stimulated models, similar to the TGF-beta effect.
2.3 Effect of sweet tea extract on elastase content the results are shown in Table 16.
TABLE 16 Effect of sweet tea extract on the elastase content of HFF cells
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC; * **: p <0.001, compared to NC; * : p <0.05, compared to NC.
As can be seen from table 16: the elastase content of HFF cells after UVA treatment in NC group increased by 79.5ng/mL, which is significantly higher than that in blank group BC (P < 0.0001). After TGF- β (PCgroup) action, the elastase content of the UVA-HFF aging stimulation model was reduced from 112.5ng/mL to 56.25ng/mL (P < 0.0001). After synchronous treatment of sweet tea extract with concentration of 0.1-2mg/mL, elastase content decreases from 84.8ng/mL to 63ng/mL with increasing sample concentration, and the concentration is dependent. Taken together, the sweet tea extract has an inhibitory effect on the secretion of elastase by UVA-HFF cell senescence-stimulated models, and is similar to TGF-beta effect.
2.4 Effect of sweet tea extract on MMP-1 content the results are shown in Table 17.
TABLE 17 Effect of sweet tea extract on MMP-1 content of HFF cells
Remarks: # #: p <0.0001, compared to BC; * ***: p <0.0001, compared to NC; * **: p <0.001, compared to NC; * *: p <0.01, compared to NC.
As can be seen from table 17: MMP-1 levels of HFF cells after UVA treatment were increased by 38ng/mL in NC group, significantly higher than in blank group BC (P < 0.0001). After TGF- β (PCgroup) action, MMP-1 content of UVA-HFF aging stimulation model was reduced from 57ng/mL to 24ng/mL (P < 0.0001). MMP-1 content was reduced relative to NC group after synchronous treatment with sweet tea extract at concentration of 0.1-2 mg/mL. Taken together, the sweet tea extract has an inhibitory effect on the secretion of MMP-1 by UVA-HFF cell aging stimulation models, and is similar to TGF-beta effect.
Example 7: evaluation of in vitro biochemical antioxidant efficacy of sweet tea extract
The sweet tea extract prepared in example 3 was subjected to the following test
1. DPPH free radical scavenging ability of sweet tea extract
20mg DPPH is weighed, dissolved in absolute ethyl alcohol and fixed in a 250mL volumetric flask, and stored in a dark place at 0-4 ℃. Transferring equal volumes of 2mL of sweet tea extract solution with different concentrations and 2mL of VC solution with the same concentrations into test tubes, taking VC as positive control, adding 2mL of PPH solution (A) 1 ) The method comprises the steps of carrying out a first treatment on the surface of the The blank group was taken from 2mL of absolute ethanol, and 2mL of LDPPH solution (A) 2 ) The method comprises the steps of carrying out a first treatment on the surface of the And 2mL of the sample was measured, and 2mL of absolute ethanol (A 3 ) The method comprises the steps of carrying out a first treatment on the surface of the After mixing, the mixture was reacted in the dark for 30min, and A was measured at 517nm 1 、A 2 、A 3 Tube absorbance values. DPPH free radical scavenging ability of sweet tea extract solution DPPH scavenging rate was calculated from the following formula:
clearance (%) = [ (a) 2 +A 3 )-A 1 ]/A 2
Wherein: a is that 1 Absorbance of DPPH solution containing sample; a is that 2 Absorbance of absolute ethanol containing DPPH; a is that 3 The absorbance of absolute ethanol containing the sample was shown in Table 18.
Table 18 sweet tea extract concentration and DPPH radical scavenging Rate
As can be seen from table 18 and fig. 1: when the concentration of the sweet tea extract solution is 0.00625-0.1mg/mL, the clearance rate of DPPH is reduced and then increased along with the gradual increase of the concentration of the sweet tea extract solution. When the concentration of the sweet tea extract solution is equal to 0.1mg/mL, the efficiency of the sweet tea extract solution for removing DPPH free radicals is highest and reaches 24.9%. Compared with the positive reference VC, the sweet tea extract solution has weaker DPPH free radical removing capability and a certain antioxidant effect.
2. ABTS radical scavenging ability of sweet tea extract
0.03841g of ABTS powder is accurately weighed, dissolved in distilled water and fixed in a 10mL volumetric flask to prepare solution A. Accurately weighing 0.0662g K 2 S 2 O 8 The powder was dissolved in distilled water to a constant volume in a 100mL volumetric flask to prepare solution B. And mixing the obtained solution A and solution B in equal volume to obtain the ABTS reagent, namely mixing 10mLA solution and 10mLB solution, and carrying out light-shielding reaction for 12-15h. Continuously diluting the ABTS reagent stock solution with absolute ethyl alcohol, and obtaining the working solution when the absorbance of the reagent at 734nm is 0.7+/-0.02.
Preparing a liquid to be tested: 20mg of sweet tea extract powder is taken and dissolved in 10mL of distilled water, and the concentration is 2mg/mL, 1.8mg/mL, 1.6mg/mL, 1.4mg/mL, 1.2mg/mL, 1mg/mL, 0.8mg/mL, 0.6mg/mL, 0.5mg/mL, 0.4mg/mL, 0.2mg/mL, 0.1mg/mL are sequentially prepared.
Positive control: 0.2mg/mL VC aqueous solution: accurately weighing 20mg of VC powder in a beaker, adding 100mL of distilled water for full dissolution, and preparing for use as a positive control.
Negative control: distilled water
Blank group: absolute ethyl alcohol
The formulation of each set of reaction systems is shown in Table 19.
TABLE 19 Table of the formulation of the reaction systems of the respective groups
3 groups of the samples and the control groups are repeated, reagents are sequentially added according to the reaction system, the reaction is carried out for half an hour in the dark, the absorbance is measured at 734nm, and the results are shown in Table 20.
The ability of the sweet tea extract solution to scavenge ABTS free radicals is calculated by the following formula:
ABTS radical clearance: i (%) = [1- (A-B)/C ]. Times.100%
TABLE 20 sweet tea extract concentration and ABTS radical scavenging Rate
As can be seen from table 20 and fig. 2: when the concentration of the sweet tea extract solution is 0.1-1mg/mL, the clearance rate of the sweet tea extract solution to ABTS free radicals is above 86.96%, and the effect is obviously better than that of a VC control group, which shows that the sweet tea extract has better antioxidation effect.
Claims (10)
1. A preparation method of sweet tea extract is characterized by comprising the following steps:
s1, crushing sweet tea leaves into coarse powder;
s2, carrying out ultrasonic leaching on the coarse powder in a solvent;
s3, centrifuging after leaching, and filtering again to obtain filtrate;
s4, adsorbing the filtrate by a macroporous adsorption resin column;
s5, eluting with ethanol water solution after adsorption, and concentrating under reduced pressure.
2. The method for preparing a sweet tea extract according to claim 1, wherein the solvent is selected from water or an aqueous ethanol solution.
3. The method for preparing sweet tea extract according to claim 1 or 2, wherein the ratio of feed liquid for ultrasonic leaching is 1: (15-25).
4. A method of preparing a sweet tea extract according to claim 3 wherein the temperature of the ultrasonic extraction is 60-80 ℃.
5. The method of producing sweet tea extract according to claim 4, wherein the time of ultrasonic extraction is 20-80min.
6. The method of preparing a sweet tea extract according to claim 5, wherein the time of ultrasonic extraction is 30-60min.
7. The method for preparing sweet tea extract according to claim 6, wherein the adsorption time is 25-35min.
8. The method for preparing a sweet tea extract according to claim 1, wherein the volume fraction of ethanol in the aqueous ethanol solution is 30-90%.
9. Use of a sweet tea extract according to any one of claims 1 to 8 for combating skin irritation by increasing expression of Caspase-14 and FLG inhibited after SLS stimulation of HaCaT cells; reducing NO, IL-1 beta, IL-8, TNF-alpha and PGE after LPS stimulation of RAW264.7 cells 2 Is expressed by (a); reduce the expression of MMP-1 and elastase after UVA stimulates HFF cells, improve the expression of restrained Collagen I and elastin, and promote the clearance of DPPH and ABTS free radicals, wherein the solid content of the sweet tea extract is more than 2wt%.
10. Use of a sweet tea extract according to claim 9 for combating skin irritation, wherein the concentration of sweet tea extract is between 0.05 and 5mg/mL; the diluent is HaCaT cell culture solution or RAW264.7 cell culture solution or HFF cell culture solution.
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