Callus cell extract and its use in the preparation of anti-aging skin preparations

Abstract

The present invention belongs to the field of biotechnology. More specifically, it relates to a Rosa graciliflora callus cell extract, a preparation method thereof, and uses thereof in the preparation of anti-aging topical skin preparations. The preparation of the Rosa graciliflora callus cell extract includes the steps of first treating the Rosa graciliflora callus with an induction medium added with a low concentration of 2,4-dichlorophenoxyacetic acid and then treating it with an induction medium containing rare earth elements. The Rosa graciliflora callus cell extract prepared by the present invention has a high total polyphenol content, can effectively scavenge free radicals, has the effects of promoting the proliferation of HDF cells and promoting the secretion of COL-I, and can be used in the preparation of anti-aging topical skin 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 the chlorophyll content and photosynthesis of plants (Wei Youzhang, 2000; Wu Youheng, 1999), and also improve the stress resistance of plants (He Fenghua, 1994; Gao Fuhua, 2000). For example, in the callus of Rheum officinale with europium added to the culture medium, the contents of emodin and chrysophanol are lower than those in the crude drug, but are 10 times and 130 times respectively the contents in the control callus, indicating that an appropriate concentration of europium can increase the content of active ingredients in Rheum officinale (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 contents of berberine, jatrorrhizine and palmatine in the callus of Coptis chinensis. Among them, Yb 3+ inhibits the accumulation of berberine, jatrorrhizine and palmatine in the callus, while 0.1 mg / L of Eu 3+ is beneficial to the accumulation of jatrorrhizine and palmatine. At present, there is no study on the effect of rare earth elements on stimulating the production of secondary metabolites in Rosa plants.

[0006] [1] Li Huiling, Zhou Chunxia, Zhang Zhang. Exploration of the skin care effects of the extract of Rosa graciliflora callus on human dermal fibroblasts and 3D epidermal models [J]. China Surfactant Detergent & Cosmetics (Chinese & English Edition), 2023, 53(03): 300-307.

[0007] [2] Chen Ying. Research on the tissue and cell culture of Ginkgo biloba and its metabolic regulation for the production of flavonoids [D]. Nanjing Forestry University, 2005. SUMMARY OF THE INVENTION

[0008] Through research, the present invention has found that although 2,4-dichlorophenoxyacetic acid is often used to induce the formation of callus in Rosa plants, using only 2,4-dichlorophenoxyacetic acid for induction cannot make it produce a strong defense response. It is 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 the callus is first treated with a low concentration of 2,4-dichlorophenoxyacetic acid and then induced by specific rare earth elements. This strong defense response is manifested as a significantly increased polyphenol content, specifically about 1 time higher than that of the control group.

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

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

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

[0012] Generally, the effective concentration of 2,4-dichlorophenoxyacetic acid for inducing the formation of Rosa graciliflora callus is mostly above 4 μmol / L, but no strong defense response can be observed when combined with rare earth elements in this concentration range.

[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 the secondary metabolites of callus. In the present invention, the rare earth elements La and Nd were observed, and it was found that the induction effect of the combination of Nd and low-concentration 2,4-D is the best, especially when low-concentration 2,4-D is used for induction for a period of time first and then Nd is used for induction.

[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. In a higher concentration range, it shows an inhibitory effect on production, and this part of the results 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 Rosa graciliflora callus is treated with an induction medium supplemented with 2,4-dichlorophenoxyacetic acid for 5 - 15 days.

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

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

[0019] Another object of the present invention is to provide the use of the Rosa graciliflora callus extract in the preparation of an anti-aging skin topical preparation.

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

[0021] The present invention first uses low - concentration 2,4 - dichlorophenoxyacetic acid for induction and then uses Nd for induction, which significantly promotes the synthesis of total polyphenols in the callus of Rosa graciliflora. The content of total polyphenols is increased by about 1 time compared with the prior art. In the HDF cell experiment, the extract of Rosa graciliflora callus prepared by the present invention shows an obvious effect of promoting the proliferation activity of HDF and secreting COL - I. Detailed implementation mode

[0022] The present invention can be further described through the following examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0023] Example 1: Preparation of Rosa graciliflora callus extract

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

[0025] 2) Load 40mL of MS medium added with 2μmol / L 2,4 - dichlorophenoxyacetic acid into a 100mL culture flask, inoculate Rosa graciliflora callus into the culture flask at a rate of 2g per bottle, and culture for 15 days;

[0026] 3) Take the Rosa graciliflora callus obtained in step 2), inoculate it into a culture flask containing MS medium added with 0.03mmol / L Ndcl3 at a rate of 2g per bottle, and culture for 10 days;

[0027] 4) Collect the Rosa graciliflora callus, measure the fresh weight and dry weight, then add deionized water according to the solid - liquid ratio of 1∶10, crush and homogenize, extract twice by ultrasonic wave at 500W, filter, and after combining the filtrates, perform vacuum freeze - drying to obtain the extract of Rosa graciliflora callus (RCE).

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

[0029] Example 2: Preparation of Rosa graciliflora callus extract

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

[0031] Example 3: Preparation of Rosa graciliflora callus extract

[0032] The difference from Example 1 is that in step 2), the concentration of 2,4-dichlorophenoxyacetic acid in the MS medium is 3 μmol / L, and in step 3), the concentration of NdCl3 in the MS medium is 0.02 mmol / L, with the remaining parameters being the same.

[0033] Comparative Example 1: The difference from Example 1 is that step 2) is omitted, with the remaining parameters being the same.

[0034] Comparative Example 2: The difference from Example 1 is that step 3) is omitted, with the remaining parameters being the same.

[0035] Comparative Example 3: The difference from Example 1 is that in step 2), the concentration of 2,4-dichlorophenoxyacetic acid is 5 μmol / L, with the remaining parameters being the same.

[0036] Comparative Example 4: The difference from Example 1 is that in step 3), La2O3 with the same concentration is used to replace NdCl3, with the remaining parameters being the same.

[0037] Comparative Example 5: The difference from Example 1 is that step 3) is carried out first, and then step 2), with the remaining parameters being the same.

[0038] I. Detection of the growth amount of callus

[0039] The growth amount is represented by the fresh weight growth rate of the callus. When measuring the fresh weight, the callus is taken out from the culture bottle, and after absorbing the surface moisture, it is weighed to obtain the fresh weight. The results are shown in Table 1.

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

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

[0042]

[0043]

[0044] As can be seen from the above table, the addition of 2,4-dichlorophenoxyacetic acid alone or in combination with the rare earth element Nd 3+ can promote the growth of Rosa filipes callus cells. Among them, the promotion effect is most significant when 2,4-dichlorophenoxyacetic acid is added alone, but the addition of Nd alone 3+ shows an inhibitory effect on the growth of Rosa filipes callus cells.

[0045] II. Detection of the total polyphenol content

[0046] The total polyphenol content was detected by the Folin-Ciocalteu method: gallic acid was used as a reference substance. A 0.445 mg / mL solution of gallic acid was prepared with water and diluted successively into 7 mass concentration gradients. 0.1 mL of the gallic acid standard solution and the sample solution to be tested were taken, in parallel for 3 times, 0.5 mL of 10% Folin-Ciocalteu reagent was added, mixed, left at room temperature for 5 min, then 0.4 mL of 7.5% Na2CO3 solution was added, and after mixing, left at room temperature for 60 min, and the OD value at 765 nm was measured. From the OD of each tube of the standard solution 520 A standard curve was made for the gallic acid content, and the polyphenol content in the sample was calculated according to the standard curve. The results are shown in Table 2 below.

[0047] Table 2 Comparison of total polyphenol content in Rosa acicularis Lindl. callus of each treatment group

[0048] Processing number Total polyphenol content (%) Example 1 2.46 Example 2 1.89 Example 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 obtained in step 1) of the example as the sample.

[0050] As can be seen from the results in Table 2, compared with the blank control group, only adding 2,4-dichlorophenoxyacetic acid could slightly increase the total polyphenol content, while only adding Nd 3+ showed an inhibitory effect on synthesis; but the combined use of low concentrations of 2,4-dichlorophenoxyacetic acid and Nd 3+ could promote the accumulation of polyphenolic compounds, and the total polyphenol content increased by about 1 time. However, when the concentration of 2,4-dichlorophenoxyacetic acid increased, no similar effect was observed.

[0051] III. Free radical scavenging test

[0052] The antioxidant property of the extract of Rosa acicularis Lindl. callus was determined by the DPPH method: 0.1 mL of the sample was taken in a test tube, 0.1 mL of DPPH ethanol solution was added, mixed well, and reacted in the dark at room temperature for 30 min, and the OD value was measured at 525 nm. The scavenging rate was calculated according to the following formula, and the results are shown in Table 3 below.

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

[0054] Where: T0: Absorbance of 0.1 ml of sample solution plus 0.1 mL of 95% ethanol; T: Absorbance of 0.1 ml of sample solution plus 0.1 ml of DPPH solution; C0: Absorbance of 0.1 ml of water plus 0.1 mL of 95% ethanol; C: Absorbance of 0.1 ml of water plus 0.1 ml of DPPH solution.

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

[0056] Processing number Test concentration (mg / mL) DPPH scavenging rate Example 1 1.0 86.37％ Example 2 1.0 79.44％ Example 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] IV. Test on Human Dermal Fibroblasts (HDF)

[0058] (1) Effect of RCE on the viability of HDF cells: HDF cells in the logarithmic growth phase were seeded into a 96-well plate containing DMEM medium with 10% (V / V) FBS at a density of 1×10 5 per well, and cultured in an incubator with 5% (V / V) CO2 at 37°C. After the cells adhered, they were switched to serum-free DMEM medium and cultured for another 16 h. Then, the medium was removed, and DMEM medium containing 0.05% RCE was added respectively. The group without RCE addition was used as the blank control group (NT), with 6 replicates in each group, and treated for 24 h. After removing the medium, 200 μL of 0.5 mg / mL MTT was added, and after reacting at 37°C for 3 - 4 h, the MTT was removed and 100 μL of DMSO was added. The absorbance at 550 nm was measured. The cell viability of the blank control was 100%. Referring to the blank control, the effect of RCE on the viability of HDF cells was evaluated.

[0059] Cell viability = (Absorbance of the RCE group / Absorbance of the 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 switched to serum-free DMEM medium and treated for 4 - 5 h. After removing the culture medium, they were treated with 0.05% RCE for 48 h. The group without RCE addition was used as the blank control group (NT), with 3 replicates for each treatment. The content of type I collagen was detected according to the method of the enzyme-linked immunosorbent assay kit (Beyotime 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 the viability of HDF cells and the secretion of COL-I

[0063] Processing number Cell viability (%) COL-I (ng / mL) NT 100.00 0.72±0.12 Example 1 <![CDATA[225.67±8.15 *** > <![CDATA[1.89±0.24 *** > Example 2 <![CDATA[201.59±5.32 *** > <![CDATA[1.56±0.20 *** > Example 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 Example 1 group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0065] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all 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 callus tissue of Rosa serrata with an induction medium added with low concentration of 2,4-dichlorophenoxyacetic acid and then treating callus tissue of Rosa serrata with an induction medium containing rare earth elements; in the induction medium, the concentration of 2,4-dichlorophenoxyacetic acid is 1-3 μmol / L; in the induction medium, the rare earth element is Nd; the concentration of the rare earth element is 0.02-0.05 mmol / L; and the induction medium is MS medium.

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

3. The method for increasing the polyphenol content in the Rosa gracilipes callus extract according to claim 1, characterized in that: The callus of Rosa tenuissima was treated with induction medium containing rare earth elements for 5 to 10 days.

Images