Natural plant extract, preparation method and application of natural plant extract in medical beauty treatment or post-beauty repair

Abstract

According to the natural plant extract and the preparation method thereof provided by the invention, the gel taking the natural plant extract as a main component can effectively promote skin repair, has the beautifying effects of oxidation resistance, aging resistance and the like, is particularly suitable for skin repair after medical beauty treatment and repair after beautifying, and has relatively high application value and market prospect.

Description

Technical Field

[0001] The present invention relates to a natural plant extract, in particular to a natural plant extract with cosmetic effects, as well as a preparation method and application thereof in medical beauty treatment or post-cosmetic repair. The natural plant extract can effectively promote skin repair, improve skin quality, and has antioxidant, anti-aging, moisturizing and other effects. Background Art

[0002] With the improvement of living standards, people's attention to skin health and beauty has been increasing. Especially with the rapid development of modern medical beauty technology, laser, microneedle, radio frequency and other treatment methods have gradually become important means of skin beauty and anti-aging. However, although these treatments can improve skin problems such as fine lines, wrinkles, spots, etc. in the short term, they may also be accompanied by side effects such as damaged skin barrier, redness, swelling, sensitivity, etc. How to repair the skin after medical beauty treatment, reduce inflammatory response, and promote skin regeneration has become a difficult problem to be solved in the field of skin care. Therefore, the development of a natural, safe and effective plant extract to improve skin repair effect has become a research hotspot in the development of skin care products.

[0003] At present, plant extracts have been widely used as the main ingredients of skin care in the market. For example, aloe vera extract is widely used because of its anti-inflammatory, moisturizing and skin soothing effects; calendula extract is favored for its excellent antioxidant and wound healing properties; green tea extract is rich in tea polyphenols, has a strong antioxidant function, and can effectively slow down skin aging. However, most of the existing plant extracts are single, with limited effects on skin repair, and have problems such as low extraction efficiency and poor stability. In addition, these plant extracts are mostly used in the field of daily skin care, but their application in skin repair after medical beauty treatment is relatively scarce.

[0004] Therefore, the present invention aims to provide a more efficient and stable plant extract through the composite extraction of multiple plants and combined with modern efficient extraction technology, which plays an important role in skin repair after cosmetic treatment and meets the market's increasing demand for skin repair and care effects. Summary of the invention

[0005] The purpose of the present invention is to provide a natural plant extract that can effectively promote skin repair and has antioxidant, anti-aging, anti-inflammatory and other effects, and is particularly suitable for skin repair and care after medical beauty treatment.

[0006] Therefore, the present invention discloses a natural plant extract, which is a Bletilla striata extract, wherein the main components and contents of the Bletilla striata extract are: 50.2% Bletilla striata polysaccharide, 22.3% flavonoids, 15.6% polyphenols, and 12.1% organic acid.

[0007] In one aspect, the present invention also discloses a method for preparing the Bletilla striata extract, characterized in that the method comprises the following steps:

[0008] Step 1: Raw material preparation

[0009] (1) Plant material selection: Bletilla striata tubers grown at an altitude of more than 1,800 meters were selected to increase the content of active substances; after harvesting, they were washed with deionized water to remove sand and impurities;

[0010] (2) Pretreatment: Cut the tubers of Bletilla striata into 3 mm thick slices; freeze-dry at -40°C for 24 hours to retain the active ingredients to the maximum extent; and grind into 100 mesh powder using a high-speed grinder;

[0011] Step 2: Extraction

[0012] (1) Solvent selection: 70% ethanol and deionized water were mixed in a ratio of 7:3, 0.5% sodium acetate was added, and the pH was adjusted to 5.5;

[0013] (2) Ultrasound-assisted extraction combined with microwave-assisted extraction

[0014] Ultrasonic conditions: ultrasonic frequency 50 kHz, power 200 W, time 20 min, temperature 35 °C;

[0015] Microwave conditions: power 500W, time 5 minutes, 3 times of interval operation;

[0016] (3) Post-processing

[0017] Filtration: Use 0.22μm filter membrane to remove solid impurities;

[0018] Concentration: concentrated to 1 / 10 of the original volume by rotary evaporator (40°C);

[0019] Separation and purification: Use Sephadex G-100 column chromatography to separate polysaccharide components and collect active substances;

[0020] (4) Drying: vacuum freeze drying to prepare a powdered extract.

[0021] In one aspect, the present invention further discloses a Bletilla striata polysaccharide gel, wherein the mass ratio of each component of the Bletilla striata polysaccharide gel is:

[0022] (1) Bletilla striata extract: 3%;

[0023] (2) Carbomer 940: 1.5%, thickener;

[0024] (3) Sodium hyaluronate: 0.2%, moisturizer;

[0025] (4) Glycerin: 3%, humectant;

[0026] (5) Anti-IL-6 monoclonal antibody: 2 μg / mL;

[0027] (6) Triethanolamine: appropriate amount, pH regulator, adjust the pH value to 6.8;

[0028] (7) Distilled water: appropriate amount.

[0029] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-IL-6 monoclonal antibody of the present invention are SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0030] In one aspect, the present invention also discloses a use of the anti-IL-6 monoclonal antibody in the preparation of Bletilla striata polysaccharide gel.

[0031] The beneficial effects of the present invention are as follows:

[0032] 1. Significant anti-inflammatory and repair effects: The bletilla striata polysaccharide gel prepared by the present invention combined with anti-IL-6 monoclonal antibodies shows significant anti-inflammatory and skin repair effects:

[0033] (1) Rapid reduction of inflammation: Compared with the use of Bletilla striata polysaccharide gel alone, the addition of anti-IL-6 reduced the IL-6 level by about 29% and the TNF-α level by about 33% on the third day after surgery.

[0034] (2) Accelerate wound healing: The healing area increased by about 20.3% on the third day, and the healing rate was close to 99% on the seventh day, shortening the healing time by about 2 times compared with the control group.

[0035] (3) Excellent tissue repair quality: Skin tissue recovered to near normal, collagen fibers were arranged regularly and tightly, there was no obvious fibrosis, and inflammatory cells almost completely disappeared.

[0036] 2. Innovative combination formula, superior to existing products: Compared with existing skin repair products (such as hyaluronic acid gel or other herbal extract preparations), the product of the present invention has advantages in many aspects:

[0037] (1) Optimization of anti-inflammatory mechanism: Through the synergistic effect of the natural anti-inflammatory properties of Bletilla striata polysaccharide and anti-IL-6 monoclonal antibodies, the level of inflammatory factors was significantly reduced, avoiding the occurrence of postoperative pigmentation and chronic inflammation.

[0038] (2) Higher repair efficiency: The duration of redness and swelling after laser treatment is shortened from an average of 3 days to 1.5 days, and the healing time is greatly shortened.

[0039] (3) Safety and naturalness: Bletilla striata polysaccharide is a natural plant extract, combined with low-concentration anti-IL-6 monoclonal antibodies, it is highly safe and has no obvious side effects.

[0040] 3. Wide range of applications: The product of the present invention is suitable for various medical and cosmetic treatments and daily cosmetic repair needs:

[0041] (1) Post-operative repair of medical aesthetics: rapid relief and repair of post-operative skin inflammation after laser treatment (such as 755nm picosecond laser), photorejuvenation, chemical peeling, etc., and microneedle and radiofrequency surgery.

[0042] (2) Beauty and skin care products: daily care products for damaged skin barrier (such as facial masks and essences), and special skin care products for repairing erythema, inflammation or sensitive skin.

[0043] (3) Auxiliary treatment of chronic inflammatory skin diseases: It can be used as an auxiliary therapy to treat chronic inflammatory skin diseases such as eczema and atopic dermatitis.

[0044] (4) Highly scalable technology platform: The technology route of the present invention is not limited to the combination of Bletilla striata polysaccharide and anti-IL-6, but can also be expanded to the joint development of other plant extracts and anti-inflammatory drugs to meet the needs of a wider range of medical aesthetics and skin disease treatment. By adjusting the gel concentration or adding other active substances, a series of products targeting different skin problems can be developed to form a complete medical aesthetics post-repair product line.

[0045] 5. Excellent market prospects

[0046] (1) Growing consumer demand: The medical beauty industry is developing rapidly, and there is a strong demand for postoperative repair products. The products of the present invention precisely meet the market demand for postoperative repair products such as laser and microneedle.

[0047] (2) Differentiated competitiveness: Through the innovative combination of Bletilla striata polysaccharide and anti-IL-6, the product of the present invention is significantly superior to similar products on the market in terms of repair efficiency, safety and anti-inflammatory mechanism, and has strong market competitiveness.

[0048] (3) Diversified application scenarios: This product can simultaneously cover multiple fields such as medical beauty restoration, high-end skin care, and treatment of inflammatory skin diseases, thus expanding market potential.

[0049] In summary, the present invention significantly improves the skin repair and anti-inflammatory properties by combining natural plant extracts (Bletilla striata polysaccharide) with biotechnology (anti-IL-6 monoclonal antibody). The product has shown excellent application effects in post-operative repair of medical aesthetics, beauty and skin care, and auxiliary treatment of inflammatory skin diseases, and has significant innovation and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1Photo of Bletilla striata.

[0051] Figure 2 Anti-IL-6 monoclonal antibody SDS-PAGE detection results. DETAILED DESCRIPTION

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0053] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0054] Example 1: Extraction and detection of natural plant extracts

[0055] 1. Raw material preparation

[0056] (1) Plant material selection: Bletilla striata tubers grown at an altitude of more than 1,800 meters were selected to increase the content of active substances; after harvesting, they were washed with deionized water to remove sand and impurities;

[0057] (2) Pretreatment: Cut the tubers of Bletilla striata into 3 mm thick slices; freeze-dry at -40°C for 24 hours to retain the active ingredients to the maximum extent; and grind into 100 mesh powder using a high-speed grinder;

[0058] 2. Extraction Steps

[0059] (1) Solvent selection: 70% ethanol and deionized water were mixed in a ratio of 7:3, 0.5% sodium acetate was added, and the pH was adjusted to 5.5;

[0060] (2) Ultrasound-assisted extraction combined with microwave-assisted extraction

[0061] Ultrasonic conditions: ultrasonic frequency 50 kHz, power 200 W, time 20 min, temperature 35 °C;

[0062] Microwave conditions: power 500W, time 5 minutes, 3 times of interval operation;

[0063] (3) Post-processing

[0064] Filtration: Use 0.22μm filter membrane to remove solid impurities;

[0065] Concentration: concentrated to 1 / 10 of the original volume by rotary evaporator (40°C);

[0066] Separation and purification: Use Sephadex G-100 column chromatography to separate polysaccharide components and collect active substances;

[0067] (4) Drying: vacuum freeze drying to prepare a powdered extract.

[0068] 3. Active ingredient detection

[0069] 3.1 Liquid chromatography-mass spectrometry (LC-MS) was used for detection, and the results showed that the main components and contents of the Bletilla striata extract extracted by the present invention were: 50.2% Bletilla striata polysaccharide, 22.3% flavonoids, 15.6% polyphenols, and 12.1% organic acids.

[0070] 3.2 Antioxidant testing

[0071] 3.2.1DPPH free radical scavenging experiment

[0072] Reagent preparation: Prepare 0.2mmol / LDPPH solution (dissolved in anhydrous ethanol). Prepare Bletilla striata extract solution (0.1, 0.2, 0.5, 1.0mg / mL).

[0073] Experimental procedure: Take 2 mL of each concentration of Bletilla striata extract solution and mix it with 2 mL of DPPH solution. Incubate the mixture in the dark at room temperature for 30 minutes. Measure the absorbance (A value) of the mixture at a wavelength of 517 nm.

[0074] Calculation formula:

[0075]

[0076] Among them, A0 is the absorbance of blank control, and A1 is the absorbance of sample.

[0077] 3.2.2 ABTS free radical scavenging experiment

[0078] Reagent preparation: Prepare 7mmol / L ABTS solution and 2.45mmol / L potassium persulfate solution, mix and let stand in the dark for 12 hours to form ABTS free radical solution. Dilute the ABTS free radical solution with PBS to an absorbance of 0.70±0.02 (734nm).

[0079] Experimental procedure: Take 2 mL of each concentration of Bletilla striata extract solution and mix it with 2 mL of ABTS solution. After reacting at room temperature for 6 minutes, measure the absorbance at 734 nm.

[0080] Calculation formula:

[0081]

[0082] Among them, A0 is the absorbance of blank control, and A1 is the absorbance of sample.

[0083] 3.2.3 FRAP reduction ability assay

[0084] Reagent preparation: FRAP working solution: 300mmol / L acetic acid buffer, 10mmol / L TPTZ solution (dissolved in 40mmol / L hydrochloric acid), 20mmol / L ferric chloride solution are mixed in a ratio of 10:1:1. Prepare Bletilla striata extract solution (same as above).

[0085] Experimental procedure: Take 0.2 mL of each concentration of Bletilla striata extract solution and mix it with 1.8 mL of FRAP working solution. Incubate at room temperature for 10 minutes and measure the absorbance at 593 nm.

[0086] Standard curve: Use known concentrations of FeSO 4 The standard curve was drawn by the solution, and the sample absorbance was substituted into the calculation of the FRAP value (μmol Fe 2+ / g).

[0087] 3.2.4 The results of the antioxidant experiment are shown in Table 1. The results show that when the extract concentration is 1.0 mg / mL, it has a good application effect.

[0088] Table 1 Summary of antioxidant test results

[0089]

[0090] 3.3 Cell repair experiment

[0091] 3.3.1 Cell culture

[0092] Cell line: Human dermal fibroblasts (HDFs).

[0093] Culture medium: DMEM high glucose medium containing 10% fetal bovine serum (FBS) and 1% double antibody.

[0094] Culture conditions: 37°C, 5% CO 2 Saturated humidity environment.

[0095] 3.3.2 Cell viability assay

[0096] Induction of oxidative damage: 300 μM HO 2 O 2 Cells were treated for 2 hours to simulate oxidative damage.

[0097] Intervention with Bletilla striata extract: The culture medium was replaced with the culture medium containing different concentrations of the extract and incubated for 24 hours.

[0098] CCK-8 assay: Add 10 μL CCK-8 reagent to each well and incubate for 2 hours. Use an ELISA reader to measure the absorbance at 450 nm. The calculation formula is as follows:

[0099]

[0100] 3.3.3ROS generation detection

[0101] Probe staining: cells were labeled with 10 μM DCFH-DA probe and incubated at 37°C for 30 min.

[0102] Fluorescence intensity measurement: Observe with a fluorescence microscope and measure the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm using a fluorescence spectrometer. The calculation formula is as follows:

[0103]

[0104] 3.3.4 Cell migration assay

[0105] Scratch test: Use a sterile pipette tip to scratch a straight line in the middle of the cell monolayer. Add medium containing different concentrations of extracts.

[0106] Imaging records: Take photos of the scratch area at 0 hours and 24 hours, and use ImageJ software to measure the scratch healing area. The calculation formula is as follows:

[0107]

[0108] 3.3.4 Experimental results are shown in Table 2. The results show that when the extract concentration is 1.0 mg / mL, it has a good application effect.

[0109] Table 2 Summary of cell repair experiment results

[0110]

[0111] 3.4 Animal experiments

[0112] 3.4.1 Animal models

[0113] Animal selection: SD rats (6-8 weeks old, body weight 180-220 g).

[0114] Wound model preparation: Full-thickness skin with a diameter of 10 mm was excised from the back to expose the dermis.

[0115] 3.4.2 Treatment with Bletilla striata extract

[0116] Divided into 3 groups, as follows

[0117] Control group: normal saline.

[0118] Low-dose group: 50 μg / mL Bletilla striata extract gel.

[0119] High-dose group: 200 μg / mL Bletilla striata extract gel.

[0120] Application: Apply once daily for 7 consecutive days.

[0121] 3.4.3 Data Recording

[0122] Wound healing area: Wound photos were taken daily, and the healing area was measured using ImageJ software.

[0123] Collagen fiber density: Skin tissue was collected on the 7th day and observed under a microscope after HE staining and Masson staining. The OD value was measured using Image-Pro Plus software.

[0124] Epidermal thickness: Epidermal thickness was measured in HE-stained sections, and the recovery rate was calculated.

[0125] 3.4.4 Experimental results are shown in Table 3. The results show that when the extract concentration is 1.0 mg / mL, it has a good application effect.

[0126] Table 3 Summary of animal experiment results

[0127]

[0128] 4. At the same time, we compared different extraction methods, and the results are shown in Table 4. The extract extracted by the optimized extraction method of the present invention has the best effect.

[0129] Table 4 Comparison results of different extraction methods

[0130]

[0131] 4. In addition, we also compared other extracts, and the results are shown in Table 5. The extract of the present invention has the best effect.

[0132] Table 5 Summary of the comparison results of the application effects of different extracts

[0133]

[0134] 5. Summary

[0135] The invention combines the optimized process of ultrasound-assisted extraction with microwave-assisted extraction to significantly improve the extraction efficiency and the content of active ingredients, and greatly shorten the extraction time.

[0136] The bletilla striata extract of the present invention is significantly superior to existing extraction methods and commercially available products in terms of anti-oxidation, cell repair and collagen fiber density restoration. Therefore, the bletilla striata extract of the present invention can be used as a core ingredient in medical aesthetic post-repair, laser post-operative care and anti-aging skin care products, and has obvious market competitiveness.

[0137] Example 2: Preparation of Bletilla striata polysaccharide gel

[0138] 1. Material preparation and its mass ratio

[0139] (1) Bletilla striata extract (prepared in Example 1): 3%;

[0140] (2) Carbomer 940: 1.5%, thickener;

[0141] (3) Sodium hyaluronate: 0.2%, moisturizer;

[0142] (4) Glycerin: 3%, humectant;

[0143] (5) Anti-IL-6 monoclonal antibody (prepared in Example 5): 2 μg / mL;

[0144] (6) Triethanolamine: appropriate amount, pH regulator, adjust the pH value to 6.8;

[0145] (7) Distilled water: appropriate amount.

[0146] 2. Preparation Steps

[0147] (1) Add 300 mL of distilled water to a 500 mL beaker, gradually add Carbomer 940, and stir to dissolve into a uniform, transparent colloid.

[0148] (2) Add Bletilla striata extract and / or anti-IL-6 monoclonal antibody and stir well.

[0149] (3) Add sodium hyaluronate and glycerin and stir until thoroughly mixed.

[0150] (4) Adjust the pH to 6.8 using triethanolamine.

[0151] (5) After vacuum degassing, package into sterile containers for later use.

[0152] Example 3: Application of Bletilla striata polysaccharide gel (mouse test)

[0153] 1. Model establishment: 8-week-old male BALB / c mice (20±2g) were selected, and the backs were shaved and skin damage was caused by 755nm laser.

[0154] 2. Experimental grouping: The model mice were divided into the following groups, with 10 mice in each group, as follows:

[0155] (1) Experimental group 1 (gel group containing only Bletilla striata extract): Bletilla striata polysaccharide gel (3% Bletilla striata extract) was applied twice a day.

[0156] (2) Experimental group 2 (gel group containing both Bletilla striata extract and anti-IL-6 monoclonal antibody): Bletilla striata polysaccharide gel containing anti-IL-6 monoclonal antibody (3% Bletilla striata extract + 2 μg / mL anti-IL-6 monoclonal antibody) was applied twice a day.

[0157] (3) Control group 1 (inactive matrix group): Apply inactive matrix gel.

[0158] (4) Control group 2 (blank group): no treatment was performed.

[0159] 3. Treat the rats according to the experimental design and record the wound healing area, inflammation level and tissue repair status every day. Treat the rats for 7 consecutive days, record the indicators every day, and collect tissue samples for further analysis after the experiment.

[0160] 4. Detection indicators

[0161] (1) Wound healing area: Pictures were taken daily using a digital camera and analyzed using ImageJ software.

[0162] (2) Inflammation level: Serum was collected on the 3rd and 7th days after surgery, and ELISA was used to detect IL-6 and TNF-α levels.

[0163] 5. Experimental Results

[0164] (1) The results of wound healing area are shown in Table 6.

[0165] Table 6 Wound healing area results

[0166]

[0167] (2) The results of the inflammation level (IL-6 and TNF-α) tests are shown in Table 7.

[0168] Table 7 Inflammatory factor detection results

[0169]

[0170] (3) The above results show that the addition of anti-IL-6 monoclonal antibody significantly enhanced the anti-inflammatory effect of Bletilla striata polysaccharide gel and further accelerated wound healing. Compared with the use of Bletilla striata polysaccharide gel alone:

[0171] Healing speed: On the third day, the healing area increased by about 20.3%.

[0172] Inflammation levels: IL-6 and TNF-α levels were further reduced (by 29% and 33%, respectively).

[0173] Example 4: Application of Bletilla striata polysaccharide gel

[0174] 1. Subject selection

[0175] Number: 30 patients undergoing laser treatment were randomly divided into two groups, experimental group and control group, with 10 patients in each group.

[0176] Experimental group 1: After surgery, the Bletilla striata polysaccharide gel of the present invention (gel containing both Bletilla striata extract and anti-IL-6 monoclonal antibody) was applied 3 times a day for 7 consecutive days.

[0177] Experimental group 2: After surgery, commercial gel (YAMAN hyaluronic acid essence gel) was applied 3 times a day for 7 consecutive days.

[0178] Control group: inactive matrix gel was used.

[0179] 2. Evaluation indicators and steps

[0180] Determination of duration of redness and swelling: Use the grading standard to record the degree of redness and swelling after surgery until the redness and swelling completely subside.

[0181] Pigmentation measurement: The pigment value (L* value) was measured using a skin spectrophotometer 2 weeks after surgery.

[0182] Skin barrier repair evaluation: Use TEWL (transepidermal water loss) meter to record the skin barrier repair status on the 7th day after surgery.

[0183] 3. The experimental results are shown in Table 8. The gel prepared by the present invention has a good application effect and is better than the application effect of experimental group 2 (commercial gel).

[0184] Table 8 Summary of experimental results

[0185]

[0186] Example 5: Preparation of anti-IL-6 monoclonal antibodies

[0187] 1. Preparation of anti-IL-6 monoclonal antibody

[0188] 1.1 Immunization of mice: IL-6 recombinant protein (ab259381) was selected as the immunogen. BALB / c mice were immunized with 50-100 μg of IL-6 protein injected every 14 days, for a total of 3 immunizations. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent immunizations. The immune response of the mice was monitored after injection to ensure the immunization effect.

[0189] 1.2 Fusion of spleen cells and myeloma cells: The spleen was removed from the immunized mouse, spleen lymphocytes were separated and washed and counted with RPMI-1640 medium. Red blood cells were removed using red blood cell lysis buffer. SP2 / 0 myeloma cell line was cultured to the logarithmic growth phase after aseptic operation. Spleen cells and SP2 / 0 cells were mixed in a ratio of 5:1, PEG (molecular weight 1500-2000) was added for fusion, and then the fused cells were transferred to a medium containing HAT selection factor to screen out hybridoma cells.

[0190] 1.3 Screening and culture of hybridoma cells: The culture fluid of the fused cells was screened by ELISA to find positive hybridoma clones that can secrete highly effective anti-IL-6 antibodies. After selecting the positive clones, monoclonal amplification was performed and antibody titer tests were performed step by step. The amplified cells were continuously cultured in RPMI-1640 medium and the antibody production was tested regularly.

[0191] 1.4 Purification of monoclonal antibodies: Protein A / G affinity chromatography columns were used to purify antibodies, remove impurities and obtain high-purity antibodies. After purification, the antibodies were sterilized by a 220 nm filter and aliquoted and stored at -80 °C. The antibody concentration was determined using the BCA method, and the final antibody concentration was 4.65 mg / mL.

[0192] 2. Testing of anti-IL-6 monoclonal antibodies

[0193] 2.1 SDS-PAGE detection: The purified monoclonal antibody was subjected to SDS-PAGE detection. The results are as follows Figure 2 As shown, it shows that the antibody purification effect is good and has a high molecular weight consistency, and the purity of the monoclonal antibody is very high, reaching more than 90%.

[0194] 2.2 Sequence analysis: mRNA was extracted from hybridoma cells and sequenced after specific amplification. Through sequence analysis, it was confirmed that the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody were SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0195] 2.3ELISA detection: 0.5 μg / mL recombinant IL-6 protein was coated on a 96-well plate and incubated at 4°C overnight. A 5% skim milk powder PBS solution was added to block at room temperature for 1 hour. Different concentrations (5 μg / mL, 2 μg / mL, 1 μg / mL) of anti-IL-6 monoclonal antibodies were added and incubated at 37°C for 1 hour. Then, HRP-labeled secondary antibodies were added and incubated at room temperature for another 30 minutes. Finally, TMB substrate was added for color development, and 2M H 2 SO 4The reaction was stopped. By measuring the OD value, it was determined that the antibody had a higher affinity at a low concentration (see Table 9).

[0196] Table 9 Monoclonal antibody ELISA test results

[0197]

[0198] 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 natural plant extract, characterized in that: The natural plant extract is bletilla striata extract, wherein the main components and contents of the bletilla striata extract are: 50.2% bletilla striata polysaccharide, 22.3% flavonoids, 15.6% polyphenols, and 12.1% organic acid.

2. A method for preparing the Bletilla striata extract according to claim 1, characterized in that: The method comprises the following steps: Step 1: Raw material preparation (1) Plant material selection: Bletilla striata tubers grown at an altitude of more than 1,800 meters were selected to increase the content of active substances; after harvesting, they were washed with deionized water to remove sand and impurities; (2) Pretreatment: Cut the tubers of Bletilla striata into 3 mm thick slices; freeze-dry at -40°C for 24 hours to retain the active ingredients to the maximum extent; and grind into 100 mesh powder using a high-speed grinder; Step 2: Extraction (1) Solvent selection: 70% ethanol and deionized water were mixed in a ratio of 7:3, 0.5% sodium acetate was added, and the pH was adjusted to 5.5; (2) Ultrasound-assisted extraction combined with microwave-assisted extraction Ultrasonic conditions: ultrasonic frequency 50 kHz, power 200 W, time 20 min, temperature 35 °C; Microwave conditions: power 500W, time 5 minutes, 3 times of interval operation; (3) Post-processing Filtration: Use 0.22μm filter membrane to remove solid impurities; Concentration: concentrated to 1 / 10 of the original volume by rotary evaporator (40°C); Separation and purification: Use Sephadex G-100 column chromatography to separate polysaccharide components and collect active substances; (4) Drying: vacuum freeze drying to prepare a powdered extract.

3. A Bletilla striata polysaccharide gel, characterized in that: The mass ratios of the components of the bletilla striata polysaccharide gel are: (1) the Bletilla striata extract according to claim 1: 3%; (2) Carbomer 940: 1.5%, thickener; (3) Sodium hyaluronate: 0.2%, moisturizer; (4) Glycerin: 3%, humectant; (5) Anti-IL-6 monoclonal antibody: 2 μg / mL; (6) Triethanolamine: appropriate amount, pH regulator, adjust the pH value to 6.8; (7) Distilled water: appropriate amount.

4. The Bletilla striata polysaccharide gel according to claim 3, characterized in that: The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-IL-6 monoclonal antibody are SEQ ID NO.1 and SEQ ID NO.2, respectively.

5. Use of the anti-IL-6 monoclonal antibody according to claim 4 in the preparation of Bletilla striata polysaccharide gel.

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