Callus cell extract and application thereof in preparation of anti-aging skin preparation

Abstract

The invention belongs to the technical field of biology. More specifically, the invention relates to a rosa multiflora callus cell extract, a preparation method thereof, and an application of the rosa multiflora callus cell extract in preparation of an anti-aging skin external preparation. The preparation method of the rosa multiflora callus cell extract comprises the following steps: firstly, treating rosa multiflora callus by using an induction culture medium added with low-concentration 2, 4-dichlorphenoxyacetic acid, and then treating the rosa multiflora callus by using an induction culture medium containing rare earth elements. The rosa multiflora callus cell extract prepared by the invention is high in total polyphenol content, can effectively scavenge free radicals, has the effects of promoting proliferation of HDF cells and promoting secretion of COL-I, and can be used for preparing anti-aging skin external preparations.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a callus cell extract, a preparation method thereof, and use thereof in preparing an anti-aging skin external preparation. Background Art

[0002] Studies have shown that Rosa graciliflora callus extract (RCE) obtained by pure water ultrasonic extraction and vacuum freeze drying contains a variety of active ingredients such as amino acids, vitamins, organic acids and polyphenols. In vitro skin care test results show that RCE has a certain proliferation effect on human dermal fibroblasts, indicating that RCE has the potential to delay skin aging and improve skin barrier function, and is expected to be used as a green raw material in cosmetics. [1] .

[0003] When plants are subjected to external adverse factors such as microbial invasion and trauma, plant cells tend to turn to the direction of secondary product synthesis and produce phytoalexin. Such stress factors that can cause plants to produce phytoalexin are collectively called elicitors (Song Jingyuan, 1999). It can induce the formation of new enzymes by changing the enzymatic activity of catalytic enzymes in secondary metabolic pathways or activating specific enzyme genes in secondary metabolic pathways, causing changes in the flux and reaction rate of secondary metabolic pathways, thereby increasing the production of secondary metabolites. According to the defense response, it can be divided into endogenous elicitors and exogenous elicitors. The former refers to elicitors from plant cells, and the latter refers to factors other than plant cells that can induce defense responses (Weinberger F, 2000); according to specificity, it can be divided into specific elicitors and nonspecific elicitors (WFL, Wunderlin R, 1999) [2] .

[0004] 2,4-Dichlorophenoxyacetic acid (2,4-D) can be used to induce and promote the formation and proliferation of callus. Previous studies have also shown that the use of 2,4-D alone can induce a large number of callus in rose plants. Liu Jun et al. reported that 2,4-D plays a major role in the process of inducing callus. The culture medium with 2,4-D added is more efficient and has a larger number of callus formation than the culture medium with other hormones. However, current research has not shown that 2,4-D can induce defense responses in rose plants.

[0005] Rare earth elements can promote the absorption of nutrients by plant roots, increase plant chlorophyll content and photosynthesis (Wei Youzhang, 2000, Wu Youheng, 1999), and can also improve plant stress resistance (He Fenghua, 1994, Gao Fuhua, 2000). For example, although the content of rhubarb callus and chrysophanol in the culture medium with europium added was lower than that in the crude drug, they were 10 times and 130 times that in the control callus, respectively, indicating that an appropriate concentration of europium can increase the content of the effective components of rhubarb (Hu Guowu, 2000). However, different rare earth elements have different effects on the production of plant secondary metabolites. For example, Lu Ping et al. (1999) pointed out that rare earths have a great influence on the content of berberine, jatrorrhizine and bamipine in Coptis chinensis callus, among which Yb 3+ Inhibits the accumulation of berberine, jatrorrhizine and palmatine in callus tissue, while 0.1 mg / L Eu 3+ It is beneficial to the accumulation of jatrorrhizine and bamipine. At present, there is no research on the effect of rare earth elements on the production of secondary metabolites in Rosa plants.

[0006] [1] Li Huiling, Zhou Chunxia, ​​Zhang Zhang. Study on the skin care efficacy of Rosa tenuifolia callus extract on human dermal fibroblasts and 3D epidermal model[J]. Journal of Surfactant Chemical Industry, 2023, 53(03): 300-307.

[0007] [2] Chen Ying. Study on metabolic regulation of Ginkgo biloba tissue and cell culture and its flavonoid production[D]. Nanjing Forestry University, 2005. Summary of the invention

[0008] The present invention has been found through research that although 2,4-dichlorophenoxyacetic acid is often used to induce the formation of callus tissue of rose plants, induction with 2,4-dichlorophenoxyacetic acid alone cannot produce a strong defense response. It was unexpectedly found that a strong induced defense response can be observed when 2,4-dichlorophenoxyacetic acid and rare earth elements are used in combination, and the best effect is achieved when low concentration of 2,4-dichlorophenoxyacetic acid is used first and then induced by specific rare earth elements. This strong defense response is manifested as a significantly increased polyphenol content, specifically, an increase of about 1 times compared with the control group.

[0009] The present invention aims to provide a method for increasing the polyphenol content in a callus extract of Rosa tenuipedunculata, comprising the steps of first treating the callus with an induction medium added with low concentration of 2,4-dichlorophenoxyacetic acid and then treating the callus with an induction medium containing rare earth elements.

[0010] The order of adding inducers also has a great influence on the production of secondary metabolites. Inducing with low concentration 2,4-D for a period of time and then inducing with Nd can promote the callus tissue to synthesize more total polyphenols, while changing the order cannot achieve the above effect.

[0011] In one embodiment of the present invention, the concentration of 2,4-dichlorophenoxyacetic acid in the induction medium is less than 5 μmol / L. More preferably, the concentration of 2,4-dichlorophenoxyacetic acid in the induction medium is 1 to 3 μmol / L, preferably 1 μmol / L, 2 μmol / L or 3 μmol / L, more preferably 2 μmol / L, and this concentration of 2,4-dichlorophenoxyacetic acid combined with the rare earth element Nd can produce the most total polyphenols.

[0012] Generally speaking, the effective concentration of 2,4-dichlorophenoxyacetic acid used to induce callus formation in Rosa tenuissima is mostly above 4 μmol / L, but no strong defense response was observed when this concentration range was combined with rare earth elements.

[0013] In one embodiment of the present invention, the rare earth element in the induction medium is Nd. Different rare earth elements have different stimulating effects on callus secondary metabolites. The present invention observed the rare earth elements La and Nd and found that the induction effect of Nd combined with low concentration of 2,4-D was the best, especially after induction with low concentration of 2,4-D for a period of time and then with Nd.

[0014] In one embodiment of the present invention, the concentration of the rare earth element is 0.02-0.05 mmol / L. Different concentrations of Nd also have a certain effect on the synthesis of total flavonoids in callus tissue, and in a higher concentration range, it shows an inhibitory effect on production, which is not shown in this application.

[0015] In one embodiment of the present invention, the induction medium is MS medium.

[0016] In one embodiment of the present invention, the callus of Rosa tenuissima is treated with an induction medium supplemented with 2,4-dichlorophenoxyacetic acid for 5 to 15 days.

[0017] In one embodiment of the present invention, the callus of Rosa tenuissima is treated with an induction medium containing rare earth elements for 5 to 10 days.

[0018] Another object of the present invention is to provide a Rosa tenuissima callus extract prepared by the method.

[0019] Another object of the present invention is to provide the use of the Rosa tenuissima callus extract in preparing anti-aging skin external preparations.

[0020] The present invention has the following beneficial effects:

[0021] The present invention first uses low concentration of 2,4-dichlorophenoxyacetic acid induction and then uses Nd induction to significantly promote the synthesis of total polyphenols in Rosa tenuifolia callus tissue, and the total polyphenol content is increased by about 1 times compared with the prior art. In the HDF cell test, the Rosa tenuifolia callus tissue extract prepared by the present invention shows obvious effects of promoting HDF proliferation activity and secreting COL-I. DETAILED DESCRIPTION

[0022] The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0023] Example 1: Preparation of Rosa serrata Callus Extract

[0024] 1) Use leaves of sterile seedlings of Rosa serrata to induce callus on MS medium containing 0.2 mg / mL 6-BA, 0.1 mg / mL α-NAA, 30 g / L sucrose and 6 g / L agar;

[0025] 2) 40 mL of MS medium supplemented with 2 μmol / L 2,4-dichlorophenoxyacetic acid was placed in a 100 mL culture bottle, and 2 g of Rosa serrata callus was inoculated into the culture bottle and cultured for 15 days;

[0026] 3) taking the Rosa serrata callus obtained in step 2), inoculating 2 g of the callus per bottle into a culture bottle containing MS medium supplemented with 0.03 mmol / L NdCl3 and culturing for 10 days;

[0027] 4) Collecting Rosa tenuissima callus, measuring the fresh weight and dry weight, adding deionized water at a solid-liquid ratio of 1:10, crushing and homogenizing, ultrasonic extraction at 500W twice, filtering, combining the filtrate and vacuum freeze-drying to obtain Rosa tenuissima callus extract (RCE).

[0028] The culture conditions of steps 1) to 3) are: temperature 22±2°C, humidity 60-75%, light intensity 1500Lx, and light 12h / d.

[0029] Example 2: Preparation of Rosa serrata Callus Extract

[0030] The difference from Example 1 is that the concentration of 2,4-dichlorophenoxyacetic acid in the MS medium in step 2) is 1 μmol / L, the concentration of Ndcl3 in the MS medium in step 3) is 0.05 mmol / L, and the other parameters are the same.

[0031] Example 3: Preparation of Rosa serrata Callus Extract

[0032] The difference from Example 1 is that the concentration of 2,4-dichlorophenoxyacetic acid in the MS medium in step 2) is 3 μmol / L, the concentration of Ndcl3 in the MS medium in step 3) is 0.02 mmol / L, and the other parameters are the same.

[0033] Comparative Example 1 is different from Example 1 in that step 2) is omitted, and the other parameters are the same.

[0034] Comparative Example 2 is different from Example 1 in that step 3) is omitted, and the other parameters are the same.

[0035] Comparative Example 3 is different from Example 1 in that the concentration of 2,4-dichlorophenoxyacetic acid in step 2) is 5 μmol / L, and the other parameters are the same.

[0036] Comparative Example 4 is different from Example 1 in that La2O3 of the same concentration is used to replace NdCl3 in step 3), and the other parameters are the same.

[0037] Comparative Example 5 is different from Example 1 in that step 3) is performed first and then step 2), and the other parameters are the same.

[0038] 1. Callus growth detection

[0039] The growth rate was expressed as the fresh weight growth rate of callus tissue. When measuring the fresh weight, the callus tissue was taken out from the culture bottle, the surface water was absorbed and then weighed to obtain the fresh weight. The results are shown in Table 1.

[0040] Fresh weight growth rate = (harvest fresh weight - inoculation fresh weight) / inoculation fresh weight × 100%

[0041] Table 1 Comparison of callus growth in each treatment group

[0042]

[0043]

[0044] As can be seen from the table above, 2,4-dichlorophenoxyacetic acid added alone or with rare earth element Nd 3+ The combination of these two methods can promote the growth of callus cells of Rosa tenuissima. The effect of 2,4-dichlorophenoxyacetic acid added alone is the most significant. However, the addition of Nd 3+ It showed the effect of inhibiting the growth of callus cells of Rosa tenuifolia.

[0045] 2. Total polyphenol content detection

[0046] The total polyphenol content was tested using the Folin-Ciocalteu method: Gallic acid was used as the reference substance for detection. Gallic acid was prepared into a 0.445 mg / mL solution with water, and diluted into 7 mass concentration gradients in sequence. 0.1 mL of gallic acid standard solution and the sample solution to be tested were taken in parallel 3 times. 0.5 mL of 10% Folin-Ciocalteu reagent was added, mixed, and placed at room temperature for 5 minutes. After that, 0.4 mL of 7.5% NaCO3 solution was added. After mixing, it was placed at room temperature for 60 minutes, and the OD value at 765 nm was measured. The OD value of each tube of the standard solution was 0.1 mL. 520 A standard curve was drawn for the gallic acid content, and the polyphenol content in the sample was calculated based on the standard curve. The results are shown in Table 2 below.

[0047] Table 2 Comparison of total polyphenol content in callus tissue of Rosa serrata in each treatment group

[0048] Processing number Total polyphenol content (%) Embodiment 1 2.46 Embodiment 2 1.89 Embodiment 3 2.17 Comparative Example 1 1.19 Comparative Example 2 1.31 Comparative Example 3 1.29 Comparative Example 4 1.37 Comparative Example 5 1.15 Blank control group 1.26

[0049] The blank control group used the callus tissue obtained in step 1) of Example 1 as a sample.

[0050] The results in Table 2 show that, compared with the blank control group, the addition of 2,4-dichlorophenoxyacetic acid alone can slightly increase the content of total polyphenols, while the addition of Nd 3+ showed an inhibitory effect on synthesis; however, low concentrations of 2,4-dichlorophenoxyacetic acid and Nd 3+ The combined use can promote the accumulation of polyphenolic compounds, and the total polyphenol content increased by about 1 times. However, no similar effect was observed when the concentration of 2,4-dichlorophenoxyacetic acid was increased.

[0051] 3. Free Radical Scavenging Test

[0052] The DPPH method was used to determine the antioxidant activity of the Rosa tenuissima callus extract: 0.1 mL of sample was taken into a test tube, 0.1 mL of DPPH ethanol solution was added, mixed, and reacted in a dark place at room temperature for 30 min. The OD value was measured at 525 nm, and the clearance rate was calculated according to the following formula. The results are shown in Table 3.

[0053] Clearance rate (%) = [1-(T-T0) / (C-C0)] × 100%,

[0054] In the formula: T0: absorbance of 0.1ml sample solution plus 0.1mL 95% ethanol; T: absorbance of 0.1ml sample solution plus 0.1ml DPPH solution; C0: absorbance of 0.1ml water plus 0.1ml 95% ethanol; C: absorbance of 0.1ml water plus 0.1ml DPPH solution.

[0055] Table 3 DPPH removal rate of each treatment group

[0056] Processing number Test concentration (mg / mL) DPPH clearance Embodiment 1 1.0 86.37％ Embodiment 2 1.0 79.44％ Embodiment 3 1.0 83.21％ Comparative Example 1 1.0 39.24％ Comparative Example 2 1.0 50.37％ Comparative Example 3 1.0 47.46％ Comparative Example 4 1.0 59.21％ Comparative Example 5 1.0 40.53％

[0057] 4. Experiment on human dermal fibroblasts (HDF)

[0058] (1) Effect of RCE on HDF cell viability: HDF cells in the logarithmic phase were taken at 1×10 5 The cells were inoculated at a density of 10% (V / V) FBSDMEM culture medium in a 96-well plate and cultured in an incubator containing 5% (V / V) CO2 at 37°C. After the cells adhered to the wall, they were replaced with serum-free DMEM culture medium and continued to be cultured for 16 hours. The culture medium was removed and DMEM culture medium containing 0.05% RCE was added. The group without RCE was used as the blank control group (NT). Six replicates were made for each group and treated for 24 hours. After removing the culture medium, 200 μL 0.5 mg / mL MTT was added. After reacting at 37°C for 3 to 4 hours, MTT was removed and 100 μL DMSO was added. The absorbance at 550 nm was measured. The cell viability of the blank control was 100%. The blank control was used as a reference to evaluate the effect of RCE on the viability of HDF cells.

[0059] Cell viability = (absorbance of RCE group / absorbance of blank group) × 100%.

[0060] (2) Effect of RCE on the secretion of type I collagen (COL-I) by HDF cells

[0061] HDF cells were incubated in a 6-well plate for 24 h, then replaced with serum-free DMEM medium for 4-5 h, the culture medium was removed and treated with 0.05% RCE for 48 h. The group without RCE was used as the blank control group (NT). Three replicates were used for each treatment. The content of type I collagen was detected according to the enzyme-linked immunosorbent assay kit (Biyuntian Elisa kit) and calculated according to the standard curve. The results are shown in Table 4 below.

[0062] Table 4 Effects of each treatment group on HDF cell viability and COL-I secretion

[0063] Processing number Cell viability (%) COL-I (ng / mL) NT 100.00 0.72±0.12 Embodiment 1 <![CDATA[225.67±8.15 *** ]]> <![CDATA[1.89±0.24 *** ]]> Embodiment 2 <![CDATA[201.59±5.32 *** ]]> <![CDATA[1.56±0.20 *** ]]> Embodiment 3 <![CDATA[221.83±9.06 *** ]]> <![CDATA[1.72±0.16 *** ]]> Comparative Example 1 <![CDATA[105.48±5.29 ### ]]> <![CDATA[0.79±0.01 ### ]]> Comparative Example 2 <![CDATA[136.52±6.37 **## ]]> <![CDATA[1.08±0.06 **## ]]> Comparative Example 3 <![CDATA[124.39±5.42 **## ]]> <![CDATA[0.95±0.04 *## <!-- 5 -->]]> Comparative Example 4 <![CDATA[162.18±4.63 **## ]]> <![CDATA[1.17±0.15 **## ]]> Comparative Example 5 <![CDATA[108.37±6.02 ### ]]> <![CDATA[0.82±0.02 ### ]]>

[0064] Note: Compared with the NT group, * P<0.05, ** P<0.01, *** P<0.001; compared with the embodiment group, # P<0.05, ## P<0.01, ### P<0.001.

[0065] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for increasing the polyphenol content in a callus extract of Rosa gracilis, characterized in that: The method comprises the steps of first treating the callus of Rosa serrata with an induction medium added with low concentration of 2,4-dichlorophenoxyacetic acid and then treating the callus of Rosa serrata with an induction medium containing rare earth elements.

2. The method for increasing the polyphenol content in the Rosa gracilipes callus extract according to claim 1, characterized in that: In the induction medium, the concentration of 2,4-dichlorophenoxyacetic acid is less than 5 μmol / L.

3. The method for increasing the polyphenol content in the callus extract of Rosa gracilis according to claim 2, characterized in that: In the induction medium, the concentration of 2,4-dichlorophenoxyacetic acid is 1-3 μmol / L.

4. The method for increasing the polyphenol content in the Rosa gracilipes callus extract according to claim 1, characterized in that: In the induction medium, the rare earth element is Nd.

5. The method for increasing the polyphenol content in the callus extract of Rosa gracilis according to claim 4, characterized in that: The concentration of the rare earth element is 0.02-0.05 mmol / L.

6. The method for increasing the polyphenol content in the Rosa tenuissima callus extract according to any one of claims 1 to 5, characterized in that: The induction medium is MS medium.

7. The method for increasing the polyphenol content in the callus extract of Rosa gracilipes according to any one of claims 1 to 5, characterized in that: The callus of Rosa tenuissima was treated with an induction medium supplemented with 2,4-dichlorophenoxyacetic acid for 5 to 15 days.

8. The method for increasing the polyphenol content in the callus extract of Rosa gracilis according to claim 7, characterized in that: The callus of Rosa tenuissima was treated with an induction medium containing rare earth elements for 5 to 10 days.

9. The Rosa tenuipedunculata callus extract prepared according to the method according to any one of claims 1 to 8.

10. Use of the Rosa tenuissima callus extract according to claim 9 in preparing an anti-aging skin external preparation.

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