CN112300257A - Sericin with anti-inflammatory function and preparation method and application thereof - Google Patents
Sericin with anti-inflammatory function and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses sericin with anti-inflammatory function and a preparation method and application thereof, which is characterized in that cocoon sheets are pretreated at high temperature and then extracted by water to obtain sericin; the obtained sericin can inhibit the synthesis of inflammatory factors IL-6, IL-1 beta and TNF-alpha at the same time, so that the sericin can be used for preparing anti-inflammatory preparations.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to sericin with an anti-inflammatory function, and also relates to a preparation method and application of sericin.
Background
Sericin is originally a waste material of the reeling industry, and its application has been receiving attention in recent years. Firstly, the safety of sericin has been approved by a series of researches, which has a precondition for application in tissue engineering materials and daily chemical skin care. Secondly, the antioxidant, whitening and moisturizing effects of the composition are widely applied. However, due to the limitations of the conventional extraction process of sericin, a sufficiently clean protein cannot be obtained, resulting in that the efficacy of sericin has not been fully developed, particularly the anti-inflammatory function of sericin. The sericin recovered from industrial silk reeling waste water or extracted from sufficient pretreated silkworm cocoons has no anti-inflammatory function and even promotes inflammation. This is also a reason why the safety of fibroin has been controversial. Through technological innovation, the anti-inflammatory sericin can be extracted quickly and efficiently, so that the application scene of the sericin is expanded, and the multiple application of the silkworm mulberry resource is realized.
LPS is a marker structure on the cell wall of gram-negative bacteria, and the inhibition of inflammatory response caused by LPS means that the inflammatory response generated by the action of bacteria and sterilized bacterial fragments on human bodies can be inhibited. In vitro experiments, RAW264.7 cells were stimulated with LPS, which was converted from an aggregated form to a lipopolysaccharide binding protein (LPB), then to a monomeric form and transferred to CD14 in an albumin-dependent manner. Next, LPS is transferred to the TLR4/MD2 complex to form a TLR4/MD2/LPS activity complex, which signals and activates a series of inflammatory responses. Using this model, it is possible to assess whether the efficacy agent has anti-inflammatory activity.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide sericin having an anti-inflammatory function; the second purpose of the invention is to provide a preparation method of sericin with an anti-inflammatory function, and the third purpose of the invention is to provide application of sericin in preparation of an anti-inflammatory preparation.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the sericin with the anti-inflammatory function is prepared by pretreating cleaned silkworm cocoon sheets at 220-250 ℃ for 2.5-3.5 h, then adding water to extract at 120 ℃ for 1-2 h, collecting protein extract, drying and pulverizing to obtain the sericin.
In the present invention, the extraction is performed at 120 ℃ for 1 hour.
In the invention, the protein ID number of the major component of sericin is as follows: ref | NP _001037506.1|, BGIBMGA002689-PA, ref | XP _021208623.1|, ref | XP _021208622.1|, BGIBMGA009393-PA, ref | NP _001106733.1|, BGIBMGA000013-PA, ref | NP _001166287.1|, and BGIBMGA 009953-PA.
In the present invention, the extraction is performed at 120 ℃ for 2 hours.
In the invention, the major component of sericin has the following protein ID number: ref | NP _001037506.1|, BGIBMGA002689-PA, ref | XP _021208623.1|, ref | XP _021208622.1|, BGIBMGA009393-PA, ref | NP _001106733.1|, BGIBMGA000013-PA, ref | NP _001166287.1 |.
2. The preparation method of the sericin with the anti-inflammatory function comprises the following steps of pretreating a cleaned silkworm cocoon sheet at 220-250 ℃ for 2.5-3.5 h, then adding water, extracting at 120 ℃ for 1-2 h, collecting a protein extracting solution, and drying and pulverizing to obtain the sericin.
Preferably, the extraction is at 120 ℃ for 2 hours.
3. The application of the sericin in preparing an anti-inflammatory preparation.
The invention has the beneficial effects that: the invention discloses sericin with an anti-inflammatory function, which is obtained by pretreating cocoon sheets and then extracting with water; the obtained sericin can inhibit the synthesis of inflammatory factors IL-6, IL-1 beta and TNF-alpha at the same time, so that the sericin can be used for preparing anti-inflammatory preparations.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the results of ELISA assay for inflammatory factors.
FIG. 2 shows the results of real-time quantitative PCR detection of inflammatory factors.
FIG. 3 shows the anti-inflammatory effect of sericin extracted in the comparative example.
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 extraction of sericin
The extraction method of sericin comprises the following steps:
(1) cleaning the silkworm cocoons;
(2) cutting into cocoon sheets with the size of 2x 2cm, and treating at the high temperature of 220-250 ℃ for 2.5-3.5 h;
(3) adding Milli-Q water according to a bath ratio of 1:30, and extracting at 120 deg.C for 1 hr and 2 hr respectively;
(4) filtering cocoon sheet residues, and collecting a protein extracting solution;
(5) freeze drying the protein extractive solution to obtain powder, and storing in refrigerator at-20 deg.C.
The sericin obtained by high-temperature extraction for 1h is numbered as HS1, and the sericin obtained by high-temperature extraction for 2h is numbered as HS 2.
Example 2 anti-inflammatory Effect of sericin
1. The cell culture method comprises the following steps:
culturing the cells at 37 deg.C with 5% CO2The NIH3T3 and HaCaT cell lines were cultured in DMEM medium containing 10% fetal calf serum, and the RAW264.7 cell lines were cultured in 1640s medium containing 10% fetal calf serum. All cell lines were fluid changed daily and passaged every other day.
Three cells were treated with HS1 and HS2 prepared in example 1, while three commercially available sericins (nos. S1, S2, S3) were used as controls, and dexamethasone (DMX was a positive control) was used, and then inflammatory factors were detected by real-time quantitative PCR and ELISA.
2. ELISA detection
(1) After the cells are treated, sucking cell culture fluid, centrifuging at 13500rpm for 10min, and taking supernatant; used or stored at-20 deg.C for use.
(2) Adding 100 microliter of cell culture sample, standard substance and contrast into a micropore, and repeating 3 samples; sealed and incubated for 2h at room temperature.
(3) The sample was removed and the wash repeated 3 times with wash buffer.
(4) Add 200. mu.L of detection antibody to each well, seal, and incubate at room temperature for 2 h.
(5) And (4) repeating the operation of the step (3).
(6) 200uL of HRP-labeled secondary antibody was added to each well and incubated at room temperature in the dark for 20 min.
(7) And (4) avoiding light, and repeating the operation of the step (3).
(8) Adding 100 mu L of TMB substrate, sealing, and incubating at room temperature for 20-30 min.
(9) Add 50. mu.L STOP solution and shake gently.
(10) Absorbance at 450nm was measured within 30min after the addition of the STOP solution, and the results are shown in FIG. 1.
3. Fluorescent quantitative PCR detection
The experiment comprises three parts of RNA extraction, reverse transcription and fluorescence quantitative PCR. The method comprises the following specific steps:
RNA extraction:
(1) the cells were treated and washed twice with PBS after the treatment.
(2) Every 10 th6The cells were lysed by adding 1mL TRIZOL, gently pipetting, mixing well, and then centrifuging at 12000rpm for 10 min.
(3) 200uL of chloroform was added to each 1ml of the TRIZOL reagent lysed samples and incubated at room temperature for 20 min.
(4) Centrifuge at 12000rpm for 15min at 4 ℃. The upper aqueous phase was transferred to a clean rnase-free centrifuge tube.
(5) Adding isopropanol with equal volume, mixing, and incubating for 10 min; centrifuge at 12000rpm for 10min at 4 ℃.
(6) The supernatant was removed, 1mL of 75% ethanol was added to each 1mL of TRIZOL lysed sample, mixed well, and centrifuged at 7000rpm for 5min at 4 ℃. Repeating for 1-2 times.
(7) Carefully removing the ethanol solution by suction, and drying at room temperature for 5-10 min.
(8) Adding about 40uL of RNase-free water, repeatedly beating to dissolve RNA completely, and storing the obtained RNA solution at-80 ℃ for later use.
Reverse transcription:
(1) the purity and concentration of RNA was determined using Nano-drop.
(2) The DNA in the system was removed as shown in Table 1:
TABLE 1 DNA removal System
Digesting in metal bath at 42 deg.C for 2 min.
(3) 10uL 2x super RT Mix (containing gDNA digesterter inhibitor) was added.
(4) Reverse transcription was performed in a PCR instrument under the conditions shown in Table 2:
TABLE 2 reverse transcription conditions
A fluorescent quantitative PCR method:
(1) the quantitative primers are shown in Table 3.
TABLE 3 quantitative PCR primers
(2) The reagents are shown in Table 4:
TABLE 4 fluorescent quantitative PCR reaction System
The PCR reaction process is shown in Table 5:
TABLE 5 PCR reaction procedure
The results of the detection are shown in FIG. 2. The results show that both HS1 and HS2 prepared in example 1 have the ability to resist LPS-induced inflammatory factors and are not significantly different. And the anti-inflammatory abilities of S1, S2 and S3 are different.
By comparing the results of real-time quantitative PCR and inflammatory factor ELISA, the results show that sericin can reduce the inflammatory reaction of RAW264.7 cells to a certain extent: s1, S3, HS1, HS2 showed the ability to inhibit IL-6 and IL-1 β production (jp <0.05,/pp <0.01,/pp < 0.001). S1 slightly inhibited TNF-. alpha.synthesis at the protein level, S3 slightly inhibited TNF-. alpha.synthesis at the mRNA and protein levels, but showed no significant difference (P > 0.05). HS1 and HS2 showed the ability to inhibit TNF-alpha synthesis at both mRNA level (P <0.05) and protein level (P < 0.01). S2 increased IL-6 and IL-1 β synthesis at the mRNA and protein levels (# P <0.05, # P <0.01), and also slightly increased TNF- α synthesis (P > 0.05). Overall, only HS1 and HS2 inhibited the production of three inflammatory factors. The sericin extracted by the method has the effect of inhibiting inflammatory factors, but other brands of sericin do not have the effect.
The sericin proteins HS1 and HS2 extracted in the present invention and the sericin proteins S1, S2 and S3 purchased from the present invention were subjected to composition analysis, and the results are shown in Table 6:
TABLE 6 sericin composition analysis
Therefore, the sericin extracted by the method has different types and contents, and the protein ID numbers of the main components of HS1 are as follows: ref | NP _001037506.1|, BGIBMGA002689-PA, ref | XP _021208623.1|, ref | XP _021208622.1|, BGIBMGA009393-PA, ref | NP _001106733.1|, BGIBMGA000013-PA, ref | NP _001166287.1|, and BGIBMGA 009953-PA; ref | NP _001037506.1| of HS2, BGIBMGA002689-PA, ref | XP _021208623.1|, ref | XP _021208622.1|, BGIBMGA009393-PA, ref | NP _001106733.1|, BGIBMGA000013-PA, ref | NP _001166287.1 |.
Comparative examples
According to the method of the embodiment 1, the method is characterized in that sericin is not extracted by high-temperature treatment at 220-250 ℃ for 2.5-3.5 hours, and then the extracted sericin is subjected to an inflammatory reaction experiment. The results are shown in FIG. 3. The result shows that the anti-inflammatory effect of sericin which is not extracted by high-temperature treatment at 220-250 ℃ for 2.5-3.5 h is poorer than that of sericin which is extracted by high-temperature treatment. The high temperature treatment can enhance the anti-inflammatory effect of sericin.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (8)
1. Sericin having an anti-inflammatory function, which is characterized in that: the sericin is prepared by pretreating a clean silkworm cocoon sheet at 220-250 ℃ for 2.5-3.5 h, then adding water to extract at 120 ℃ for 1-2 h, collecting a protein extract, and drying and pulverizing to obtain the sericin.
2. Sericin with anti-inflammatory function according to claim 1, characterized in that: the extraction is carried out at 120 ℃ for 1 hour.
3. Sericin with an anti-inflammatory function according to claim 2, wherein: the protein ID number of the major component of sericin is as follows: ref | NP _001037506.1|, BGIBMGA002689-PA, ref | XP _021208623.1|, ref | XP _021208622.1|, BGIBMGA009393-PA, ref | NP _001106733.1|, BGIBMGA000013-PA, ref | NP _001166287.1|, and BGIBMGA 009953-PA.
4. Sericin with anti-inflammatory function according to claim 1, characterized in that: the extraction is carried out at 120 ℃ for 2 hours.
5. Sericin with an anti-inflammatory function according to claim 4, characterized in that: the major component of sericin has the following protein ID number: ref | NP _001037506.1|, BGIBMGA002689-PA, ref | XP _021208623.1|, ref | XP _021208622.1|, BGIBMGA009393-PA, ref | NP _001106733.1|, BGIBMGA000013-PA, ref | NP _001166287.1 |.
6. The preparation method of sericin with anti-inflammatory function is characterized in that: the method comprises the following steps of pretreating cleaned silkworm cocoon sheets for 2.5-3.5 hours at 220-250 ℃, adding water, extracting for 1-2 hours at 120 ℃, collecting protein extracting solution, drying and pulverizing to obtain the sericin.
7. The method of claim 6, wherein: the extraction is carried out at 120 ℃ for 2 hours.
8. Use of the sericin according to any one of claims 1 to 5 for preparing an anti-inflammatory agent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115724937A (en) * | 2022-08-26 | 2023-03-03 | 西南大学 | Repetitive motif of Sericin3 protein with antibacterial activity and application thereof |
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| CN115724937B (en) * | 2022-08-26 | 2023-09-15 | 西南大学 | Repeated motif of Sericiin 3 protein with antibacterial activity and application thereof |
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