CN112656781A - Application of compound Terphynyllin in preparation of product for inhibiting melanin generation - Google Patents
Application of compound Terphynyllin in preparation of product for inhibiting melanin generation Download PDFInfo
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Abstract
The invention provides application of a compound Terphenyllin in preparation of a product for inhibiting melanin generation, wherein the compound Terphenyllin can obviously inhibit synthesis and accumulation of melanin on zebra fish and melanoma cell B16F10 models; the tyrosinase activity in B16F10 cells can be reduced, and the content of tyrosinase in B16F10 cells can be obviously reduced; and also has strong oxidation resistance, and can be developed into novel whitening products.
Description
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to application of a compound Terphynyllin in preparation of a product for inhibiting melanin generation.
Background
White skin is generally popular with the public, so that various so-called natural skin-beautifying products are in the market. Currently commercially used skin whitening agents are: placenta extract, hydroquinone, arbutin, fruit acid, vitamin C and its derivatives, etc. These substances have been proved to have certain whitening effects, and the inhibition of melanin generation is the main action principle of the whitening products. However, the whitening effect of these whitening agents is often unsatisfactory, and a high concentration of the whitening product is required for a long period of time to obtain a weak whitening effect. In addition, these whitening agents also present various health risks, and may cause allergy, desquamation, or even disease when applied to the human body. Long-term use of hydroquinone can cause exogenous leukoderma and brown-yellow disease. Abnormal accumulation of melanin may cause disease, and even severe cases may cause skin cancer, such as melanoma, which has a very high mortality rate. Therefore, safe and efficient novel melanin synthesis inhibitors are urgently needed in the whitening product market and the pharmaceutical market.
Disclosure of Invention
In order to solve one of the problems, the invention provides a new application of the compound Terphynyllin.
The structural formula of the compound Terphynyllin is:
in a first aspect of the invention, the invention proposes the use of the compound Terphenyllin for the preparation of a product inhibiting melanin production.
According to the embodiment of the invention, the incubation of the compound Terphenyllin with the concentration of 30 mu g/mL can obviously reduce the accumulation of melanin on the back skin of the young zebra fish, and does not influence the normal growth and development of the zebra fish. Meanwhile, the treatment of the compound Terphynyllin with the concentration of 2.5 mu g/mL can also reduce the melanin content of the mouse melanoma cells B16F 10. And can achieve the same melanin synthesis inhibiting effect as arbutin at a concentration lower than the drug treatment concentration of arbutin.
In a second aspect of the invention, the invention proposes the use of the compound Terphenyllin for the preparation of tyrosinase inhibitors.
According to the embodiment of the invention, the experimental result shows that the compound Terphenyllin can down-regulate the expression of tyrosinase and inhibit the activity of intracellular tyrosinase.
In a third aspect of the invention, the invention provides application of a compound Terphenyllin in preparation of a product for whitening skin.
According to the examples of the present invention, the results of in vitro experiments showed that 2.5. mu.g/mL of the compound Terphynyllin versus ABTS+The clearance rate of free radicals reaches more than 80 percent, which shows that the product has stronger oxidation resistance; and the effect of inhibiting melanin synthesis can be achieved by inhibiting the expression and activity of tyrosinase in cells and enhancing the antioxidant capacity, so that the skin whitening effect is achieved.
According to an embodiment of the invention, the product is a cosmetic or a pharmaceutical.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a graph showing the effect of the compound Terphenyllin on the distribution of melanin in zebrafish according to an embodiment of the present invention;
FIG. 2 is a graph showing the effect of the compound Terphenyllin on the melanin content of zebrafish according to an embodiment of the present invention;
FIG. 3 is a graph of the effect of the compound Terphenyllin on the proliferation of three melanoma cells according to an embodiment of the present invention;
FIG. 4 is an appearance of a cell pellet according to an embodiment of the present invention;
FIG. 5 is a graph showing the effect of the compound Terphenylin according to an embodiment of the present invention on the melanin content of cells B16F 10;
FIG. 6 shows the effect of the compound Terphenyllin on the tyrosinase activity of cell B16F10 according to an embodiment of the present invention;
FIG. 7 is a graph showing the effect of Terphenyllin on the expression level of tyrosinase in B16F10 cells, according to an embodiment of the present invention;
FIG. 8 shows the compound Terphenylin to ABTS according to an embodiment of the present invention+Radical scavenging effect.
Detailed Description
The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The following reagents and cells were purchased from the market. Among them, the compound Terphenyllin (terphenyltrexin) was purchased from Shanghai pottery Biotech Ltd.
Wherein, the laboratory glassware has:
zebra fish mating tank, culture dish, come card sight glass, ultrasonic cleaner, -80 ℃ refrigerator, constant temperature metal bath, multifunctional enzyme mark instrument, high speed freezing centrifuge, electronic balance, freezing centrifuge, magnetic stirrer, acidimeter, micropipettor, DEAE-Sepharose ion exchange column, vertical plate electrophoresis tank, DC stabilized power supply, glass plate, 96 orifice plate, 6 orifice plate.
The experimental reagents are as follows:
embryo water, arbutin, dimethyl sulfoxide (DMSO), Triton X-100, MITF, PMSF, L-DOPA, MTT, DPPH, ABTS, 0.01mol/L Tri-HCl buffer, gel stock, gel isolation buffer, concentrated gel buffer, and electrode buffer (pH 8.3).
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
EXAMPLE 1 Effect of the Compound Terphenylin on the formation of melanin in Zebra Fish
Collecting high-quality zebra fish embryos, grouping and culturing at room temperature. Adding 0, 10, 20 and 30 mu g/mL of compound Terphenyllin solution and 0, 140, 150 and 160 mu g/mL of arbutin solution respectively 24h after fertilization, wherein arbutin is used as a positive control; after 48h of drug treatment, the film was observed under a body mirror and photographed and recorded. Then, the embryo is killed at-80 ℃, Triton X-100 containing 1mmoL/L PMSF in ice bath is added for ultrasonic crushing for 5min, the mixture is kept still on ice and centrifuged for 30min at 4 ℃ and 10000rpm, the centrifuged precipitate is resuspended and dissolved by 1mol/L NaOH, the solution is subjected to metal bath for 30min at 95 ℃, and the light absorption value is measured at 490nm after cooling, so that the melanin content is obtained.
As shown in FIGS. 1 and 2, after 48h of treatment with Terphenylin compound in the safe concentration range, the treated zebrafish eyes, back and yolk sac were less pigment synthesized, spotted and lighter in color. The control group had a particularly dark color, dense pigment distribution, and flaky distribution. In the highest concentration treatment group, the melanin content of the zebrafish was reduced to 55.37% of that of the blank control group. Meanwhile, the survival rate and the activity of zebra fish treated by the compound Terphynyllin are not influenced by obvious toxic and side effects of the medicine. The melanin formation inhibition effect of the compound Terphenylin of 30 mu g/mL is equivalent to 160 mu g/mL arbutin, and the compound Terphenylin is proved to have a better melanin formation inhibition effect, can inhibit the formation of the melanin of the zebra fish under a safer condition, and has the potential of being developed into a melanin formation inhibition drug.
EXAMPLE 2 Effect of the Compound Terpenylin on proliferation of melanoma cells B16F10, IgR3 and A375
B16F10, IgR3, and a375 cells were cultured in 96-well plates until the cells attached. A solution of 0, 1, 2, 3, 4, 5, 7, 9. mu.g/mL of the compound Terphenyllin was added, 4 replicates per concentration. After 36h of incubation, the old medium was aspirated off. Mu.l of 0.5mg/ml MTT was added to each well. The culture was continued for 4h and the medium was discarded. The cell viability was calculated by adding 200. mu.l DMSO to each well, measuring the absorbance at a wavelength of 570nm with a microplate reader immediately after shaking.
Cell viability (%) ═ 100a1/a0, where a0 represents absorbance of the drug-free blank; a1 represents the absorbance of the drug-added experimental group.
The results are shown in fig. 3, the survival rates of three melanoma cells (B16F10, IgR3 and A375) after the compound Terphynyllin is treated for 36 hours are detected by adopting an MTT method, the compound Terphynyllin has no obvious influence on the proliferation activity of the three cells in the range of 0-9 mu g/mL, and the compound Terphynyllin is non-toxic to the three cells in the range.
EXAMPLE 3 Effect of the Compound Terphenylin on melanogenesis in melanoma cells B16F10
High-sugar culture medium with DMEM at 37 deg.C and CO2B16F10 cells were cultured under the conditions of a concentration of 5% (volume fraction) and saturation humidity, and after the cells were grown to a confluent state, they were digested with 25% (mass fraction) trypsin and rapidly transferred to a 96-well plate for culture. Adding 20 mu L of single cell suspension into each well, sucking off the original culture medium after the growth and adherence, respectively adding 0, 0.5, 1.0, 1.5, 2.0 and 2.5ug/mL of compound Terphenylin solution and 0 and 10 mu g/mL of arbutin solution, and taking arbutin as positive control. After culturing for 36 hours, cell pellets were collected by centrifugation. Washing the precipitate with 0.01mol/L PBS (pH 7.2-7.4) for three times, and centrifuging at low speed of 2000rpm after the first two times of washingAfter the last washing, the cells were centrifuged at 10000rpm, and the cell pellet was retained. Adding 200 μ L of 1mol/L NaOH into the precipitate, performing water bath at 95 deg.C for 30min to dissolve cell disruption pigment, cooling, and measuring OD at 490nm to obtain melanin content.
As a result, as shown in FIGS. 4 and 5, the effect of various concentrations of the compound Terphenyllin on the amount of melanin in B16F10 cells was examined. As can be seen from the apparent image (FIG. 4) of the collected cell pellet, the cells in the blank group became darker and the cells in the treated group became lighter. The melanin content is characterized by the absorbance value of the cell extract in the visible light range of 490nm, and the B16F10 cell is treated by the compound Terpenylin, so that the melanin content is obviously reduced, and the concentration effect is shown, and when the concentration is 2.5 mu g/mL, the melanin content is 62.48 percent of that of a control group.
EXAMPLE 4 Effect of the Compound Terphenylin on the intracellular tyrosinase Activity of melanoma cells B16F10
High-sugar culture medium with DMEM at 37 deg.C and CO2B16F10 cells were cultured under the conditions of a concentration of 5% (volume fraction) and saturation humidity, and after the cells were grown to a confluent state, they were digested with 25% (mass fraction) trypsin and rapidly transferred to a 96-well plate for culture. Adding 20 mul of single cell suspension into each well, sucking off the original culture medium after the growth and adherence, and respectively adding 0, 0.5, 1.0, 1.5, 2.0 and 2.5ug/mL of compound Terphenylin solution. After culturing for 36 hours, cell pellets were collected by centrifugation. To the cell pellet, 200. mu.L of 0.01mol/L PBS containing 1mmol/L PMSF and 1% TritonX-100 was added, and freeze-thawing was repeated 3 times. Centrifuging at 4 deg.C and 10000rpm for 30min to obtain supernatant as crude enzyme solution. To 200. mu.l of the test organism line were added 150. mu.l of 1mg/mL crude enzyme solution in PBS at pH 6.8, and 50. mu.l of cell tyrosinase solution. The reaction system is put in a constant temperature water bath kettle at 30 ℃ for half an hour, and the light absorption value at 475nm is measured.
The result is shown in fig. 6, the tyrosinase activity in B16F10 cells decreased with the increase of the treated concentration of the compound Terphenyllin, showing obvious concentration dependence, and when the concentration of the compound Terphenyllin is 2.5 μ g/mL, the intracellular tyrosinase activity decreased to 50.89% of that of the blank control group, which indicates that the compound Terphenyllin can significantly decrease the intracellular tyrosinase activity.
EXAMPLE 5 Effect of the Compound Terpenylin on the B16F10 tyrosinase content of melanoma cells
High-sugar culture medium with DMEM at 37 deg.C and CO2B16F10 cells were cultured under the conditions of a concentration of 5% (volume fraction) and saturation humidity, and after the cells were grown to a confluent state, they were digested with 25% (mass fraction) trypsin and rapidly transferred to a 96-well plate for culture. Adding 20 μ L of single cell suspension into each well, sucking off the original culture medium after the growth and adherence, and adding 0, 0.5, 1.0, 1.5 and 2.0ug/mL of compound Terphynyllin solution respectively. After culturing for 36 hours, cell pellets were collected by centrifugation. Adding 200 μ L RIPA lysate containing 1mmol/L PMSF into the cell precipitate, suspending the cells thoroughly, placing on ice for lysis for 1h, centrifuging at 10000rpm for 30min, and collecting the supernatant. The obtained supernatant was analyzed for the expression level of TYR by the western blot method.
The results are shown in FIG. 7, with β -actin as the internal reference. From FIG. 7, it can be seen that the compound Terpenyllin has a significant inhibitory effect on the expression of intracellular tyrosinase.
EXAMPLE 6 antioxidant Effect of Terphenylin Compound
Using ABTS+The antioxidant capacity of the compound Terphynyllin was tested by the free radical scavenging method.
ABTS + free radical scavenging method, preparing ComP10 and positive control Vc (10 μ l) with concentration of 0, 0.5, 1, 1.5, 2 μ g/mL, respectively adding into prepared ABTS + working solution (190 μ l), mixing thoroughly, reacting at room temperature for 6min, and measuring absorbance at 714 nm.
The free radical scavenging ability of the medicine, namely the antioxidant ability, is calculated by the formula of (1-A1/A0) × 100; wherein A0 represents the absorbance of the drug-free blank; a1 represents the absorbance of the drug-added experimental group.
The results are shown in FIG. 8, in which the compound Terphenylin has a strong antioxidant activity, and ABTS of 1.5. mu.g/mL of the compound Terphenylin+The clearance rate of free radicals reaches more than 80 percent, which is equivalent to that of positive control strong oxidant Vc.
In conclusion, the compound Terphenylin in the embodiment of the invention can obviously inhibit the generation and accumulation of melanin in zebra fish and melanoma cell B16F10 models, and can achieve the same melanin generation inhibition effect as arbutin at a concentration lower than the drug treatment concentration of arbutin. And experimental data show that the compound Terpenyllin can reduce the activity of tyrosinase in B16F10 cells, and can also obviously reduce the content of tyrosinase in B16F10 cells. In addition, the compound Terphynyllin also has strong antioxidant capacity. In combination with the above experimental results, it is speculated that the compound Terpenyllin can inhibit melanin synthesis through various pathways, including reduction of intracellular tyrosinase activity, inhibition of cell tyrosinase synthesis or promotion of tyrosinase decomposition, scavenging of ROS produced by cells, and the like.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (5)
1. Application of compound Terphynyllin in preparing product for inhibiting melanin generation.
2. Application of compound Terphynyllin in preparation of tyrosinase inhibitor.
3. Application of compound Terphynyllin in preparation of skin whitening products.
4. Use according to claim 1, wherein the product is a cosmetic or pharmaceutical product.
5. Use according to claim 3, wherein the product is a cosmetic or pharmaceutical product.
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| CN114894923A (en) * | 2022-04-20 | 2022-08-12 | 广州大学 | Method for inhibiting melanin in organism based on active oxygen free radical scavenging by antioxidant |
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| 刘芬等: "亮白曲霉代谢物中两种联苯化合物的分离鉴定", 《厦门大学学报（自然科学版）》 * |
| 吕霞霞: "《实用临床医学基础与进展》", 31 March 2019, 吉林科学技术出版社 * |
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| CN114894923A (en) * | 2022-04-20 | 2022-08-12 | 广州大学 | Method for inhibiting melanin in organism based on active oxygen free radical scavenging by antioxidant |
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