CN114152584A - Method for detecting quality of periplaneta americana ethanol extract based on biological activity detection - Google Patents
Method for detecting quality of periplaneta americana ethanol extract based on biological activity detection Download PDFInfo
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
Abstract
The invention provides a method for detecting the quality of an ethanol extract of periplaneta americana based on biological activity detection, which can effectively solve the problem of quality control of the ethanol extract of periplaneta americana. The method is based on the 'function and indication' of the periplaneta americana ethanol extract, and can realize good evaluation on the quality control and the quality of the biological activity of the periplaneta americana ethanol extract through the established human immortalized epidermal cell (HaCAT cell) cell proliferation method/MTT colorimetric method. The method is good and visual, can be used for measuring the bioactivity of the ethanol extract of the periplaneta Americana so as to achieve the aim of comprehensively evaluating and controlling the product quality, and is an innovation in the quality control and evaluation method of the traditional Chinese medicine.
Description
Technical Field
The invention relates to a method for measuring and evaluating in-vitro bioactivity, in particular to an ethanol extract of periplaneta americana.
Background
At present, the quality standard of traditional Chinese medicine is mainly to control the quality of traditional Chinese medicine by measuring the content of one or more active ingredients, and the index ingredient is not necessarily the effective ingredient or the main effective ingredient of the medicine to a great extent, even if the index ingredient is the main effective ingredient, the measurement of the index ingredient does not necessarily represent the total biological activity because the content is low and the clinical effective dose is lack of correlation. The most important point is that for traditional Chinese medicine preparations with more and more components and complex components, the drug effect is difficult to be explained by using a certain chemical component. Secondly, although the Chinese patent medicine is proved to have effectiveness through main pharmacodynamics and clinical research, the representativeness and the reliability of samples and the variability of active ingredients of the Chinese medicine during the pharmacodynamics research and the clinical research can not ensure whether each batch of products still have the pharmacodynamic action, and the reproducibility is not verified generally. Finally, when the raw material sources of the traditional Chinese medicine preparation are changed in the production process, the production process is improved, and when the stability of the product is checked, the traditional Chinese medicine preparation is considered to meet the current medicine standard, and the change of the biological activity is rarely considered. Thus, there are certain drugs whose biological activities have changed greatly, while still meeting current drug standards. In this regard, the chinese pharmacopoeia 2010, 2015 and 2020 edition proposes guidelines for determination of biological activity of traditional Chinese medicines (9105). The guiding principle points out that the biological activity determination method is a method for determining the effectiveness of the medicine by using the biological effect of the medicine as the basis and biological statistics as a tool and applying specific experimental design, thereby achieving the effect of controlling the quality of the medicine.
The periplaneta americana ethanol extract/rehabilitation new liquid is a traditional Chinese medicine solution prepared from periplaneta americana dry insect body ethanol extract, and the functional main indications are as follows: promoting blood circulation, nourishing yin and promoting granulation. The periplaneta americana ethanol extract/new healing liquid is mainly applied to diseases with skin injury and mucous membrane injury as pathological characteristics, and has obvious curative effects in other aspects of resisting inflammation, eliminating inflammatory edema, promoting the growth of new granulation tissues, quickly repairing ulcer surfaces, shortening wound healing time and the like. At present, the national drug administration national drug standards (WS 3-B3674-2000 (Z)) American cockroach ethanol extract quality standards load the contents of characters, identification, inspection, content measurement and the like, and the quality of the American cockroach ethanol extract is difficult to be comprehensively evaluated according to the existing quality standards.
The invention establishes a method for controlling the quality of the bioactivity of the ethanol extract of the periplaneta americana by using a human immortalized epidermal cell (HaCAT cell) cell proliferation method/an MTT colorimetric method for quality evaluation on the periplaneta americana based on the guiding principle (9105) of the determination of the biological activity of the traditional Chinese medicine in Chinese pharmacopoeia, can embody the efficacy characteristics of the ethanol extract of the periplaneta americana/a rehabilitation new liquid medicine, supplement and improve the defects of the quality control mode of the prior ethanol extract of the periplaneta americana/the rehabilitation new liquid, improve the prior quality control standard and ensure that the near-production medicine is safer and more effective.
Disclosure of Invention
The invention aims to provide a method for determining and evaluating the in-vitro biological activity of an ethanol extract of periplaneta americana.
The method for determining and evaluating the in vitro biological activity of the ethanol extract of the periplaneta americana comprises the following steps:
a. preparing a standard solution and a standard diluent: resuspending the standard lyophilized powder to 500 μ g/mL with sterile PBS solution; then diluting the solution to a standard substance diluent containing 200 ng/mL per 1mL of the solution by using a maintenance culture medium;
b. preparing a test solution and a test diluent: the concentration of the periplaneta americana ethanol extract sample is firstly determined by a BCA method, and the periplaneta americana ethanol extract sample is diluted to be within the range of 31.25ug/mL to 125 ug/mL by using a maintenance culture medium according to the initial concentration of the sample.
c. Measurement of
The experimental procedure was as follows:
(1) the HaCAT cell strain is cultured at 37 ℃ and 5% CO2Culturing under the condition of controlling cell concentration to be 1.0 × 105 ~5.0×10 5Carrying out passage on the cells/mL, and detecting the biological activity 24-36 hours after passage;
(2) discard the supernatant, digest and collect the cells, prepare 5.0X 10 with complete medium4cell/mL;
(3) seeded in 96-well cell culture plates at 100. mu.L per well at 37 ℃ in 5% CO2Culturing overnight under the condition;
(4) removing maintenance liquid from the prepared cell culture plate, adding 200 μ L of standard solution and sample of test solution with different concentrations, and culturing at 37 deg.C under 5% CO 2 to maintain cells cultured in culture medium as blank control;
(5) when the culture is carried out for 48 hours, adding 20 mu L MTT into each hole, continuing to culture for 4 hours until purple precipitates appear, stopping the culture, carefully abandoning the supernatant, adding 100 mu L DMSO into each hole, and uniformly blowing and stirring to ensure that crystals are fully dissolved;
(6) and measuring the light absorption value by using a microplate reader, and drawing a cell growth curve by taking time as an abscissa and the light absorption value as an ordinate according to the measured light absorption value.
d. Determination of results
Compared with the prior art, the biological potency detection method provided by the application is based on the 'function and indication' of the periplaneta Americana ethanol extract, and the biological activity of the periplaneta Americana ethanol extract is visually determined and evaluated on the cellular level on the whole.
Specifically, compared with the existing quality evaluation method of the periplaneta americana ethanol extract, the method for testing the biological potency of the periplaneta americana ethanol extract has the following advantages:
(1) a biological evaluation method is added on the basis of content measurement and evaluation to supplement and perfect the deficiency of a quality control mode and simultaneously meet the requirements of production and clinical application on a reliable and effective medicine quality evaluation method.
(2) The Chinese medicine quality control mode and method taking biological evaluation as the core are more closely related to clinical efficacy, and the inherent quality and clinical curative effect can be effectively reflected.
(3) The biological activity detection method has more superiority for traditional Chinese medicines with complex chemical components of the periplaneta americana ethanol extract, the content of the periplaneta americana ethanol extract cannot be represented by a physicochemical method, and the biological activity and the clinical curative effect cannot be reflected by physicochemical measurement.
In a word, the HaCAT cell strain/MTT colorimetric method provided by the invention is based on the 'function and indication' of the periplaneta americana ethanol extract, can accurately and systematically detect the clinical efficacy of the periplaneta americana ethanol extract, marks the uniform biological potency of the periplaneta americana extract process, and is used as a quality control means in the production process of the periplaneta americana ethanol extract.
Drawings
FIG. 1 shows the cell proliferation of HaCAT cell line under different serum concentrations;
FIG. 21% serum concentration effect of EGF and FGF on HaCAT cell proliferation;
FIG. 32% serum concentration effect of EGF and FGF on HaCAT cell proliferation;
FIG. 4 influence of ethanol extract of Periplaneta americana on HaCAT cell proliferation
FIG. 5 the effect of different batches of ethanol extract of Periplaneta americana on HaCAT cell proliferation
Detailed Description
EXAMPLE 1 Effect of different concentrations of serum on the proliferation of HaCAT cell lines
(1) Preparation of related experimental reagent and serum with different concentrations
(a) RPMI 1640 medium;
(b) complete medium: adding 100 mL fetal bovine serum of Hyclone company into 900 mL RPMI 1640 medium to obtain 10% final serum concentration;
(c) MTT solution: purchased from Sigma, cat #: m2128, preparing MTT with the concentration of 5 mg/mL by using PBS, filtering and sterilizing at 0.22 mu m, and freezing and storing at-20 ℃;
(d) home-made DMSO;
(e) preparation of media with different serum concentrations: cell culture media were prepared with serum concentrations of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 10%, respectively. In 96-well cell culture plates, dilutions were made at different serum concentrations for 10 dilutions, with 3 duplicate wells for each dilution.
(2) Experimental procedure
(a) HaCAT cell line was cultured at 37 ℃ in complete medium with 5% CO2Culturing under the condition of controlling cell concentration to be 1.0 × 105~5.0×105Carrying out passage on the cells/ml, and detecting the biological activity 24-36 hours after passage;
(b) discard the supernatant, digest and collect the cells, prepare 5X 10 with complete medium4cell/mL;
(c) seeded in 96-well cell culture plates at 100. mu.L/well, i.e. 5000 cells/well, 5% CO at 37 ℃2Culturing under the condition;
(d) discarding the cell complete medium, changing to cell culture medium with different serum concentrations, and culturing at 37 deg.C with 5% CO2Culturing under the condition, and observing the growth condition of the cells at 24 hours and 48 hours;
(e) the proliferation of the HaCAT cell line was measured 48 hours after cell culture.
(3) Results and analysis of the experiments
The experimental results show that: the HaCAT cell strain can keep certain activity under the condition that the serum content is 0.5 percent and 1 percent. The HaCAT cell strain keeps better activity under the condition that the serum content is more than or equal to 2 percent, which indicates that the morphology and the normal growth of the cells can be maintained under the condition that the serum content is more than or equal to 2 percent. The HaCAT cell line was cultured under different serum concentrations, and the cell proliferation was shown in FIG. 1.
Example 2 Effect of recombinant human epidermal growth factor and recombinant human basic fibroblast growth factor on HaCAT cell proliferation
(1) Preparation of related experimental reagent and related solution
(a) RPMI 1640 medium
(b) Maintenance medium: adding 0.1 mL fetal bovine serum of Hyclone company into 9.9 mL RPMI 1640 medium to obtain 1% final serum concentration; adding 0.4 mL fetal bovine serum of Hyclone company into 19.6 mL RPMI 1640 medium to obtain a final serum concentration of 2%;
(c) complete medium: adding 100 mL fetal bovine serum of Hyclone company into 900 mL RPMI 1640 medium to obtain 10% final serum concentration;
(d) MTT solution: purchased from Sigma, cat #: m2128, preparing MTT with the concentration of 5 mg/mL by using PBS, filtering and sterilizing at 0.22 mu m, and freezing and storing at-20 ℃;
(e) domestic DMSO (dimethyl sulfoxide)
(f) Recombinant human basic fibroblast growth factor: purchased from R & D SYSTEMS, cargo number: 233-FB-025/CF, 25ug specification, 800 IU/ug activity.
Preparation of standard solution: taking the recombinant human basic fibroblast growth factor, and resuspending the recombinant human basic fibroblast growth factor to 100 mu g/mL by using sterile PBS, namely adding 250 mu L of sterile PBS into 25 mu g of freeze-dried powder, wherein the activity is 80 IU/mu L. Subpackaging at 5. mu.L/piece, and freezing at-70 ℃.
The suspension was diluted with 1% serum concentration maintenance medium to 0.75. mu.L of the protein resuspension of b-FGF described above per 1mL of maintenance medium containing 50 ng/mL of b-FGF, i.e., 1.5 mL of 1% serum concentration. In 96-well cell culture plates, 2-fold serial dilutions were made for 2 dilutions, each dilution making 3 wells.
The suspension was diluted with 2% serum concentration maintenance medium to 1mL of 50 ng/mL b-FGF, i.e., 3mL of 2% serum concentration maintenance medium, to which 1.5. mu.L of the protein resuspension of the above b-FGF was added. In 96-well cell culture plates, 2-fold serial dilutions were made for 2 dilutions, each dilution making 3 wells.
(g) Recombinant human epidermal growth factor: purchased from R & D SYSTEMS, cargo number: 236-EG-200 with the specification of 200 mug and the vitality of 1400 IU/mug.
Preparation of hrEGF standard solution: the recombinant human epidermal growth factor is resuspended to 500 mug/mL by sterile PBS, namely 400 mug of sterile PBS is added into 200 mug of freeze-dried powder, and the activity is 700 IU/mug. Subpackaging at 5. mu.L/piece, and freezing at-70 ℃.
Diluting with 1% serum concentration maintaining medium to obtain a solution containing 100 ng/mL hrEGF in 1mL maintaining medium, i.e. 1.5 mL 1% serum concentration maintaining medium, and adding 0.3 μ L of the above protein resuspension. In 96-well cell culture plates, 2-fold serial dilutions were made for 2 dilutions, each dilution making 3 wells.
The suspension was diluted with 2% serum maintenance medium to a concentration of 0.6. mu.L of the protein resuspension of hrEGF per 1mL of maintenance medium containing 100 ng/mL of hrEGF, i.e., 3mL of 2% serum maintenance medium. In 96-well cell culture plates, 2-fold serial dilutions were made for 2 dilutions, each dilution making 3 wells.
(2) Experimental procedure
(a) HaCAT cell line was cultured at 37 ℃ in complete medium with 5% CO2Culturing under the condition of controlling cell concentration to be 1.0 × 105~5.0×105Carrying out passage on the cells/ml, and detecting the biological activity 24-36 hours after passage;
(b) discard the supernatant, digest and collect the cells, prepare 5.0X 10 with complete medium4Cells/ml;
(c) seeded in 96-well cell culture plates at 100. mu.L/well, i.e. 5000 cells/well, 5% CO at 37 ℃2Culturing overnight under the condition;
(d) the prepared cell culture plate was discarded from the complete medium, 200. mu.L of standard solutions hrEGF and b-FGF at different concentrations were added, respectively, at 37 ℃ and 5% CO2Culturing under the condition for 24 hours and 48 hours, and taking cells cultured in maintenance medium with different serum concentrations as blank controls;
(e) and detecting the growth condition of the cells.
(3) Results and analysis of the experiments
Under the maintenance culture medium with the serum concentration of 1% and 2%, 100 ng/mL and 200 ng/mL recombinant human epidermal growth factor and 50 ng/mL and 100 ng/mL b-FGF b-FGF can promote HaCAT cell growth; specific cell proliferation is shown in FIGS. 2 and 3.
Example 3 Effect of ethanol extract intermediate of Periplaneta americana on HaCAT cell line proliferation
(1) Preparation of related experimental reagent and related solution
(a) RPMI 1640 medium;
(b) maintenance medium: adding 3mL of RPMI 1640 medium containing 10% fetal calf serum into 27 mL of RPMI 1640 medium, wherein the final concentration of the serum is 1%;
(c) complete medium: adding 100 mL fetal bovine serum into 900 mL RPMI 1640 culture medium, wherein the final concentration of the serum is 10%;
(d) MTT solution: purchased from Sigma, cat #: m2128, preparing MTT with the concentration of 5 mg/mL by using PBS, filtering and sterilizing at 0.22 mu m, and freezing and storing at-20 ℃;
(e) home-made DMSO;
(f) recombinant human epidermal growth factor: purchased from R & D SYSTEMS, cargo number: 236-EG-200 with the specification of 200 mug and the vitality of 1400 IU/mug. The hrEGF standard solution was prepared as in example 2.
(g) The ethanol extract of periplaneta americana (lot No. B190311, labeled KFX-B190311) was from the responsible company, related to the chargey group of the good doctor of sichuan province;
preparation of KFX-B190311 solutions of varying concentrations: dilute to 1mL of maintenance medium containing 0.5 mg/mL KFX-B190311 intermediate at 1% serum concentration, i.e., 3.69 mL of maintenance medium at 1% serum concentration, to which 310. mu.L of KFX-B190311 described above was added. In 96-well cell culture plates, 2-fold serial dilutions were made for 8 dilutions, 4 wells for each dilution (three wells were finally selected for statistical analysis).
(2) Experimental procedure
(a) HaCAT cell line was cultured at 37 ℃ in complete medium with 5% CO2Culturing under the condition of controlling cell concentration to be 1.0 × 105~5.0×105Carrying out passage on the cells/ml, and detecting the biological activity 24-36 hours after passage;
(b) discard the supernatant, digest and collect the cells, prepare 5.0X 10 with complete medium4Cells/ml;
(c) seeded in 96-well cell culture plates at 100. mu.L/well, i.e. 5000 cells/well, 5% CO at 37 ℃2Culturing overnight under the condition;
(d) the prepared cell culture plate was discarded from the complete medium, and 200. mu.L of standard solutions hrEGF and KFX-B190311 were added, respectively, at 37 ℃ with 5% CO2Culturing under the condition for 24 hours and 48 hours, and taking cells cultured in maintenance medium with different serum concentrations as blank controls;
(e) and detecting the growth condition of the cells.
(3) Results of the experiment
The experimental results show that: under a maintenance culture medium with 1% of serum concentration, the recombinant human epidermal growth factor can remarkably promote the growth of HaCAT cells; in the maintenance medium with 1% serum concentration, low concentration KFX-B190311 (less than or equal to 62.5 μ g/mL) has the effect of promoting HaCAT cell proliferation at 48 hours, and is shown in figure 4.
Example 4 Effect of different batches of ethanol extract intermediate of Periplaneta americana on HaCAT cell line proliferation
(1) Preparation of related experimental reagent and related solution
The relevant experimental reagents are from responsible companies of Sichuan good doctors climbing the Western pharmaceutical industry group in the same way as example 3, 4 different batches of ethanol extracts of periplaneta americana (B190303, B190307, B190310 and B190502); different concentrations of ethanol extract solutions of periplaneta americana were prepared as in example 3.
(2) The experimental steps are as follows: same procedure as in example 3
(3) Results of the experiment
The experimental results show that: under a maintenance culture medium with 1% of serum concentration, different batches of periplaneta americana ethanol extracts have the effect of promoting HaCAT cell proliferation under the condition of concentration within a certain range (31.25 ug/mL-125 ug/mL) within 48 hours, and the specific figure is shown in FIG. 5.
Although the invention has been described in detail above with reference to a general description and specific embodiments, it can be modified or improved on the basis of the invention, for example by using different cell lines such as: it will be apparent to those skilled in the art that mouse embryonic fibroblast line BALB/c 3T3, rat normal gastric mucosal epithelial cells RGM1, human gastric mucosal epithelial cells GES-1, and the like are similarly studied. Accordingly, such modifications and improvements are intended to be within the scope of this invention without departing from the spirit thereof.
Claims (3)
1. An in vitro biological activity evaluation method of an ethanol extract of periplaneta americana comprises the following steps:
a. preparing a standard solution and a standard diluent:
resuspending the standard lyophilized powder to 500 μ g/mL with sterile PBS solution; then diluting the solution to a standard substance diluent containing 200 ng/mL per 1mL of the solution by using a maintenance culture medium;
b. preparing a test solution and a test diluent:
firstly, determining the concentration of a periplaneta americana ethanol extract sample by using a BCA method; diluting the sample to be within the range of 31.25 ug/mL-125 ug/mL by using a maintenance culture medium according to the initial concentration of the sample;
c. measurement of
The experimental procedure was as follows:
(1) the HaCAT cell strain is cultured at 37 ℃ and 5% CO2Culturing under the condition of controlling cell concentration to be 1.0 × 105~5.0×10 5Carrying out passage on the cells/mL, and detecting the biological activity 24-36 hours after passage;
(2) discard the supernatant, digest and collect the cells, prepare 5.0X 10 with complete medium4cell/mL;
(3) seeded in 96-well cell culture plates at 100. mu.L per well at 37 ℃ in 5% CO2Culturing overnight under the condition;
(4) removing maintenance liquid from the prepared cell culture plate, adding 200 μ L of standard solution and sample of test solution with different concentrations, and culturing at 37 deg.C under 5% CO 2 to maintain cells cultured in culture medium as blank control;
(5) when the culture is carried out for 48 hours, adding 20 mu L MTT into each hole, continuing to culture for 4 hours until purple precipitates appear, stopping the culture, carefully abandoning the supernatant, adding 100 mu L DMSO into each hole, and uniformly blowing and stirring to ensure that crystals are fully dissolved;
(6) measuring a light absorption value by using an enzyme-labeling instrument, and drawing a cell growth curve by taking time as an abscissa and the light absorption value as an ordinate according to the measured light absorption value;
d. and (6) judging the result.
2. The bioassay method as set forth in claim 1, wherein the positive control standard is recombinant human epidermal growth factor hEGF.
3. The bioassay method as set forth in claim 1, wherein the cells are human normal skin immortalized keratinocyte cell line HaCAT cells of non-tumor origin.
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