CN105463052A - Method for detecting efficacies of compound Yishanhong extracts on HepG 2.2.15 cells - Google Patents
Method for detecting efficacies of compound Yishanhong extracts on HepG 2.2.15 cells Download PDFInfo
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Abstract
The invention discloses a method for detecting efficacies of compound Yishanhong extracts on HepG 2.2.15 cells. The detection method comprises the following steps: preparing compound Yishanhong extract solutions; inoculating and culturing the HepG 2.2.15 cells to an RPMll640 culture medium, so that a cell suspension is formed; testing light absorption value A of a solution b and light absorption value A1 of a supernatant a, as well as light absorption value A2 of a solution d and light absorption value A3 of a supernatant c, so as to calculate the survival rate of the HepG cells as well as inhibitory rates of compound Yishanhong extracts with different concentrations on HBsAg and HBeAg secreted by the HepG 2.2.15 cells; and determining the efficacies of the Yishanhong extracts with different concentrations on the HepG 2.2.15 cells. By virtue of the method, the efficacies of the compound Yishanhong can be objectively and sensitively responded; and the detection method disclosed by the invention is simple and easy to operate and is conducive to standard detection.
Description
Technical field
The present invention relates to the method detecting compound red drug effect near the mountain.More particularly, the present invention relates to a kind of detect compound near the mountain sage extract to the method for the drug effect of HepG2.2.15 cell
Background technology
Hepatitis B is by hepatitis B virus (hepatitisBvirus, HBV) disease of harm humans health in a kind of serious world wide caused, at China's infection rate up to 10%-20%, HBV persistent infection can cause the hepatic diseases such as liver cirrhosis and primary hepatocyte hepatocarcinoma, the curative effect of existing antiviral as α-interference rope and lamivudine still can not be satisfactory, and therefore finding Anti-HBV drugs has safely and effectively become current medicine sector urgent task.TCM Treatment for Chronic Hepatitis B has a long history in China, has nearly decades scholar to carry out Anti-HBV activity examination to some herbal medicine successively, finds really there is the herbal medicine that some are efficient, low toxicity suppresses HBV.Compound is red is near the mountain Empirical formula from Zhuang excavation among the people treatment hepatitis B out, has good result for the treatment of to chronic hepatitis B.Medicinal material is Rhododendron dauricum, Holotrichia trichophora, Japnanese St. John's wort Herb, Root of Beautiful Millettia etc. form by strengthening for the party, there is the effect (Li Changwei of eliminating evil toxin expelling, the loose stasis of blood of clear profit, dredging road, reinforcing body resistance, Li Bing, Lu Rumei, Pang Yuzhou. the Recent Advances of Chemical Constituents [J] of compound red prescription medicinal material near the mountain. Guangxi traditional Chinese medicine, 2014, (5): 7-8).But the pharmacological mechanism that at present compound is red is near the mountain also unclear, in order to promote the development of compound red preparation near the mountain better, this checking and appraising compound near the mountain sage extract to the drug effect of HepG2.2.15 cell.
Summary of the invention
An object of the present invention is to solve at least the problems referred to above, and a kind of method evaluating compound red drug effect is near the mountain provided.The red near the mountain pharmacological mechanism of current compound is also unclear, and in order to promote the development of compound red preparation near the mountain better, the present invention is by the test light absorption value A of described solution b and the light absorption value A of described supernatant liquor a
1, the light absorption value A of described solution d
2and the light absorption value A of described supernatant liquor c
3calculate the survival rate of HepG2.2.15 cell and different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis; Sage extract, can objective sensitive reaction compound red drug effect near the mountain to the drug effect of HepG2.2.15 cell near the mountain to determine different concns compound, and detection method of the present invention is simple to operation.
Technical scheme provided by the invention is:
Sage extract is to a method for the drug effect of HepG2.2.15 cell near the mountain to detect compound, and described detection method comprises the following steps:
1) the compound sage extract solution near the mountain of different concns, is configured;
2), HepG2.2.15 cell is inoculated in RPMl-1640 substratum to cultivate and forms cell suspending liquid;
3), successively by step 1) described in different concns compound near the mountain sage extract solution join in described cell suspending liquid respectively, be wherein provided with do not add compound near the mountain sage extract solution cell suspending liquid in contrast;
4), by step 3) in add different concns compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant a, add staining reagent, dye rear acetic acid wash, solution b is become, the light absorption value A of solution b described in last test and test the light absorption value A of described supernatant liquor a after adding alkali buffered soln again
1; By step 3) in do not add compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant c, add staining reagent, dye rear acetic acid wash, then become solution d after adding alkali buffered soln, tests the light absorption value A of described solution d
2and test the light absorption value A of described supernatant liquor c
3;
5), by the test light absorption value A of described solution b and the light absorption value A of described supernatant liquor a
1, the light absorption value A of described solution d
2and the light absorption value A of described supernatant liquor c
3calculate the survival rate of HepG2.2.15 cell and different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis; Determine different concns compound near the mountain sage extract to the drug effect of HepG2.2.15 cell, the survival rate=(A/A of wherein said HepG2.2.15 cell
2) × 100%, different concns compound near the mountain sage extract to the inhibiting rate=(A of HBsAg and the HBeAg of HepG2.2.15 emiocytosis
1/ A
3) × 100%.
Preferably, the compound of described configuration different concns near the mountain sage extract solution is: get compound sage extract near the mountain, be dissolved in aseptic deionized water, make the compound sage extract solution near the mountain that concentration is 0.00032mg/L, 0.0016mg/L, 0.08mg/L, 0.04mg/L, 0.2mg/L, 1mg/L.
Preferably, described compound is sage extract preparation method near the mountain: compound is the red strong medicinal material Rhododendron dauricum 20 by weight part near the mountain, Holotrichia trichophora 20, Root of Beautiful Millettia 10, Japnanese St. John's wort Herb 10, xanthorrhiza 5 forms, get 4 parts of described compounds respectively red near the mountain, every part of 65g, 95wt% ethanol is used respectively by red near the mountain for described 4 parts of compounds, 60wt% ethanol, 30wt% ethanol, water is as Extraction solvent, often kind of solvent refluxing extracts three times, united extraction liquid, four kinds of compounds sage extract is near the mountain obtained respectively after drying under reduced pressure, the alcohol extraction compound sage extract near the mountain of different concns, can better determine compound near the mountain sage extract to the efficient part of HepG2.2.15 cytosis.
Preferably, described RPMl1640 substratum contains that geneticin concentrations is 360-400mg/L, the weight percent of foetal calf serum is 8-12%, the concentration of glutamine is 1-3mmol/L, the weight percent of blue or green strepto-is 0.5-2% and NaHCO
3weight percent be 2-8%, the pH value of described RPMl1640 substratum is 7.4-7.6, be applicable to HepG2.2.15 cell cultures, can rapidly and efficiently turn out HepG2.2.15 cell, and viable count is greater than 90%.
Preferably, step 2) described in form cell suspension after calculate total cellular score, viable cell quantity is no less than 90% of total cellular score, and described HepG2.2.15 cell concn is 0.5 × 10
5individual/mL-1.5 × 10
5individual/mL.
Preferably, adopt automatic microplate reader under absorbing wavelength is 515nm condition, test the light absorption value A of described solution b and the light absorption value A of described solution d
2, under absorbing wavelength is 450nm condition, test the light absorption value A of described supernatant liquor a
1and the light absorption value A of described supernatant liquor c
3.
The present invention at least comprises following beneficial effect:
(1) the present invention by test and calculate the survival rate of HepG2.2.15 cell and different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis; Determine different concns compound near the mountain sage extract to the drug effect of HepG2.2.15 cell; The objective sensitive reaction compound of energy red drug effect near the mountain, and detection method of the present invention is simple to operation, is conducive to the standardization detected.
(2) the invention provides a kind of detect compound near the mountain sage extract to the method for the drug effect of HepG2.2.15 cell, by compound near the mountain sage extract to the drug effect of HepG2.2.15 cell, can determine objectively compound near the mountain sage extract quality and by the compound of different concns ethanol-extracted near the mountain sage extract test its drug effect to HepG2.2.15 cell, can better determine compound near the mountain sage extract to the efficient part of HepG2.2.15 cytosis.
Part is embodied by explanation below by other advantage of the present invention, target and feature, part also will by research and practice of the present invention by those skilled in the art is understood.
Embodiment
In conjunction with embodiment below, the present invention is described in further detail, can implement according to this with reference to specification sheets word to make those skilled in the art.
Embodiment 1
Sage extract is to a method for the drug effect of HepG2.2.15 cell near the mountain to detect compound, and described detection method comprises the following steps:
1) the compound sage extract solution near the mountain of different concns, is configured;
2), HepG2.2.15 cell is inoculated in RPMl-1640 substratum to cultivate and forms cell suspending liquid;
3), successively by step 1) described in different concns compound near the mountain sage extract solution join in described cell suspending liquid respectively, be wherein provided with do not add compound near the mountain sage extract solution cell suspending liquid in contrast;
4), by step 3) in add different concns compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant a, add staining reagent, dye rear acetic acid wash, solution b is become, the light absorption value A of solution b described in last test and test the light absorption value A of described supernatant liquor a after adding alkali buffered soln again
1; By step 3) in do not add compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant c, add staining reagent, dye rear acetic acid wash, then become solution d after adding alkali buffered soln, tests the light absorption value A of described solution d
2and test the light absorption value A of described supernatant liquor c
3;
5), by the test light absorption value A of described solution b and the light absorption value A of described supernatant liquor a
1, the light absorption value A of described solution d
2and the light absorption value A of described supernatant liquor c
3calculate the survival rate of HepG2.2.15 cell and different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis; Determine different concns compound near the mountain sage extract to the drug effect of HepG2.2.15 cell, the survival rate=(A/A of wherein said HepG2.2.15 cell
2) × 100%, different concns compound near the mountain sage extract to the inhibiting rate=(A of HBsAg and the HBeAg of HepG2.2.15 emiocytosis
1/ A
3) × 100%.
TC
50be that to test survivaling cell in 50% toxic concentration and embodiment 1 be drug level when contrasting 50% in embodiment 1, ID50 is infective dose 50.
Table 1 different concns compound near the mountain sage extract to HepG2.2.15 cytotoxic effect
Table 2 different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis
Embodiment 2
Sage extract is to a method for the drug effect of HepG2.2.15 cell near the mountain to detect compound, and described detection method comprises the following steps:
1), the compound sage extract solution near the mountain of configuration different concns, the compound of described configuration different concns near the mountain sage extract solution is: get compound sage extract near the mountain, be dissolved in aseptic deionized water, making concentration is 0.00032mg/L, 0.0016mg/L, 0.08mg/L, 0.04mg/L, 0.2mg/L, the compound sage extract solution near the mountain of 1mg/L, described compound is sage extract preparation method near the mountain: compound is the red strong medicinal material Rhododendron dauricum 20 by weight part near the mountain, Holotrichia trichophora 20, Root of Beautiful Millettia 10, Japnanese St. John's wort Herb 10, xanthorrhiza 5 forms, and gets 4 parts of described compounds respectively red near the mountain, every part of 65g, uses 95wt% ethanol respectively by red near the mountain for described 4 parts of compounds, 60wt% ethanol, 30wt% ethanol, water is as Extraction solvent, often kind of solvent refluxing extracts three times, united extraction liquid, obtains four kinds of compounds sage extract near the mountain respectively after drying under reduced pressure, the alcohol extraction compound sage extract near the mountain of different concns, can better determine compound near the mountain sage extract to the efficient part of HepG2.2.15 cytosis,
2), by HepG2.2.15 cell be inoculated in RPMl-1640 substratum to cultivate and form cell suspending liquid, calculate total cellular score after described formation cell suspension, viable cell quantity is no less than 90% of total cellular score, and described HepG2.2.15 cell concn is 0.5 × 10
5individual/mL/mL; Described RPMl1640 substratum contains that geneticin concentrations is 360-400mg/L, the weight percent of foetal calf serum is 8%, the concentration of glutamine is 1mmol/L, the weight percent of blue or green strepto-is 0.5% and NaHCO
3weight percent be 2%, the pH value of described RPMl1640 substratum is 7.4, be applicable to HepG2.2.15 cell cultures, can rapidly and efficiently turn out HepG2.2.15 cell, and viable count is greater than 90%;
3), successively by step 1) described in different concns compound near the mountain sage extract solution join in described cell suspending liquid respectively, be wherein provided with do not add compound near the mountain sage extract solution cell suspending liquid in contrast;
4), by step 3) in add different concns compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant a, add staining reagent, dye rear acetic acid wash, solution b is become, the light absorption value A of solution b described in last test and test the light absorption value A of described supernatant liquor a after adding alkali buffered soln again
1; By step 3) in do not add compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant c, add staining reagent, dye rear acetic acid wash, then become solution d after adding alkali buffered soln, tests the light absorption value A of described solution d
2and test the light absorption value A of described supernatant liquor c
3, adopt automatic microplate reader under absorbing wavelength is 515nm condition, test the light absorption value A of described solution b and the light absorption value A of described solution d
2, under absorbing wavelength is 450nm condition, test the light absorption value A of described supernatant liquor a
1and the light absorption value A of described supernatant liquor c
3;
5), by the test light absorption value A of described solution b and the light absorption value A of described supernatant liquor a
1, the light absorption value A of described solution d
2and the light absorption value A of described supernatant liquor c
3calculate the survival rate of HepG2.2.15 cell and different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis; Determine different concns compound near the mountain sage extract to the drug effect of HepG2.2.15 cell, the survival rate=(A/A of wherein said HepG2.2.15 cell
2) × 100%, different concns compound near the mountain sage extract to the inhibiting rate=(A of HBsAg and the HBeAg of HepG2.2.15 emiocytosis
1/ A
3) × 100%.
TC
50be that to test survivaling cell in 50% toxic concentration and embodiment 2 be drug level when contrasting 50% in embodiment 2, ID50 is infective dose 50.
In table 1 embodiment 2 different concns compound near the mountain sage extract to HepG2.2.15 cytotoxic effect
In table 2 embodiment 2 different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis
Embodiment 3
Sage extract is to a method for the drug effect of HepG2.2.15 cell near the mountain to detect compound, and described detection method comprises the following steps:
1), the compound sage extract solution near the mountain of configuration different concns, the compound of described configuration different concns near the mountain sage extract solution is: get compound sage extract near the mountain, be dissolved in aseptic deionized water, making concentration is 0.00032mg/L, 0.0016mg/L, 0.08mg/L, 0.04mg/L, 0.2mg/L, the compound sage extract solution near the mountain of 1mg/L, described compound is sage extract preparation method near the mountain: compound is the red strong medicinal material Rhododendron dauricum 20 by weight part near the mountain, Holotrichia trichophora 20, Root of Beautiful Millettia 10, Japnanese St. John's wort Herb 10, xanthorrhiza 5 forms, and gets 4 parts of described compounds respectively red near the mountain, every part of 65g, uses 95wt% ethanol respectively by red near the mountain for described 4 parts of compounds, 60wt% ethanol, 30wt% ethanol, water is as Extraction solvent, often kind of solvent refluxing extracts three times, united extraction liquid, obtains four kinds of compounds sage extract near the mountain respectively after drying under reduced pressure, the alcohol extraction compound sage extract near the mountain of different concns, can better determine compound near the mountain sage extract to the efficient part of HepG2.2.15 cytosis,
2), by HepG2.2.15 cell be inoculated in RPMl-1640 substratum to cultivate and form cell suspending liquid, calculate total cellular score after described formation cell suspension, viable cell quantity is no less than 90% of total cellular score, and described HepG2.2.15 cell concn is 1.5 × 10
5individual/mL; Described RPMl1640 substratum contains that geneticin concentrations is 360-400mg/L, the weight percent of foetal calf serum is 12%, the concentration of glutamine is 3mmo1/L, the weight percent of blue or green strepto-is 2% and NaHCO
3weight percent be 8%, the pH value of described RPMl1640 substratum is 7.6, be applicable to HepG2.2.15 cell cultures, can rapidly and efficiently turn out HepG2.2.15 cell, and viable count is greater than 90%;
3), successively by step 1) described in different concns compound near the mountain sage extract solution join in described cell suspending liquid respectively, be wherein provided with do not add compound near the mountain sage extract solution cell suspending liquid in contrast;
4), by step 3) in add different concns compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant a, add staining reagent, dye rear acetic acid wash, solution b is become, the light absorption value A of solution b described in last test and test the light absorption value A of described supernatant liquor a after adding alkali buffered soln again
1; By step 3) in do not add compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant c, add staining reagent, dye rear acetic acid wash, then become solution d after adding alkali buffered soln, tests the light absorption value A of described solution d
2and test the light absorption value A of described supernatant liquor c
3, adopt automatic microplate reader under absorbing wavelength is 515nm condition, test the light absorption value A of described solution b and the light absorption value A of described solution d
2, under absorbing wavelength is 450nm condition, test the light absorption value A of described supernatant liquor a
1and the light absorption value A of described supernatant liquor c
3;
5), by the test light absorption value A of described solution b and the light absorption value A of described supernatant liquor a
1, the light absorption value A of described solution d
2and the light absorption value A of described supernatant liquor c
3calculate the survival rate of HepG2.2.15 cell and different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis; Determine different concns compound near the mountain sage extract to the drug effect of HepG2.2.15 cell, the survival rate=(A/A of wherein said HepG2.2.15 cell
2) × 100%, different concns compound near the mountain sage extract to the inhibiting rate=(A of HBsAg and the HBeAg of HepG2.2.15 emiocytosis
1/ A
3) × 100%.
TC
50be that to test survivaling cell in 50% toxic concentration and embodiment 3 be drug level when contrasting 50% in embodiment 3, ID50 is infective dose 50.
In table 1 embodiment 3 different concns compound near the mountain sage extract to HepG2.2.15 cytotoxic effect
In table 2 embodiment 3 different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis
Embodiment 4
Sage extract is to a method for the drug effect of HepG2.2.15 cell near the mountain to detect compound, and described detection method comprises the following steps:
1), the compound sage extract solution near the mountain of configuration different concns, the compound of described configuration different concns near the mountain sage extract solution is: get compound sage extract near the mountain, be dissolved in aseptic deionized water, making concentration is 0.00032mg/L, 0.0016mg/L, 0.08mg/L, 0.04mg/L, 0.2mg/L, the compound sage extract solution near the mountain of 1mg/L, described compound is sage extract preparation method near the mountain: compound is the red strong medicinal material Rhododendron dauricum 20 by weight part near the mountain, Holotrichia trichophora 20, Root of Beautiful Millettia 10, Japnanese St. John's wort Herb 10, xanthorrhiza 5 forms, and gets 4 parts of described compounds respectively red near the mountain, every part of 65g, uses 95wt% ethanol respectively by red near the mountain for described 4 parts of compounds, 60wt% ethanol, 30wt% ethanol, water is as Extraction solvent, often kind of solvent refluxing extracts three times, united extraction liquid, obtains four kinds of compounds sage extract near the mountain respectively after drying under reduced pressure, the alcohol extraction compound sage extract near the mountain of different concns, can better determine compound near the mountain sage extract to the efficient part of HepG2.2.15 cytosis,
2), by HepG2.2.15 cell be inoculated in RPMl-1640 substratum to cultivate and form cell suspending liquid, calculate total cellular score after described formation cell suspension, viable cell quantity is no less than 90% of total cellular score, and described HepG2.2.15 cell concn is 1.0 × 10
5individual/mL; Described RPMl1640 substratum contains that geneticin concentrations is 360-400mg/L, the weight percent of foetal calf serum is 10%, the concentration of glutamine is 2mmol/L, the weight percent of blue or green strepto-is 1% and NaHCO
3weight percent be 4%, the pH value of described RPMl1640 substratum is 7.5, be applicable to HepG2.2.15 cell cultures, can rapidly and efficiently turn out HepG2.2.15 cell, and viable count is greater than 90%;
3), successively by step 1) described in different concns compound near the mountain sage extract solution join in described cell suspending liquid respectively, be wherein provided with do not add compound near the mountain sage extract solution cell suspending liquid in contrast;
4), by step 3) in add different concns compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant a, add staining reagent, dye rear acetic acid wash, solution b is become, the light absorption value A of solution b described in last test and test the light absorption value A of described supernatant liquor a after adding alkali buffered soln again
1; By step 3) in do not add compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant c, add staining reagent, dye rear acetic acid wash, then become solution d after adding alkali buffered soln, tests the light absorption value A of described solution d
2and test the light absorption value A of described supernatant liquor c
3, adopt automatic microplate reader under absorbing wavelength is 515nm condition, test the light absorption value A of described solution b and the light absorption value A of described solution d
2, under absorbing wavelength is 450nm condition, test the light absorption value A of described supernatant liquor a
1and the light absorption value A of described supernatant liquor c
3;
5), by the test light absorption value A of described solution b and the light absorption value A of described supernatant liquor a
1, the light absorption value A of described solution d
2and the light absorption value A3 of described supernatant liquor c calculate the survival rate of HepG2.2.15 cell and different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis; Determine different concns compound near the mountain sage extract to the drug effect of HepG2.2.15 cell, the survival rate=(A/A of wherein said HepG2.2.15 cell
2) × 100%, different concns compound near the mountain sage extract to the inhibiting rate=(A of HBsAg and the HBeAg of HepG2.2.15 emiocytosis
1/ A
3) × 100%.
TC
50be that to test survivaling cell in 50% toxic concentration and embodiment 4 be drug level when contrasting 50% in embodiment 4, ID50 is infective dose 50.
In table 1 embodiment 4 different concns compound near the mountain sage extract to HepG2.2.15 cytotoxic effect
In table 2 embodiment 4 different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the embodiment described.
Claims (6)
1. detect compound near the mountain sage extract to a method for the drug effect of HepG2.2.15 cell, it is characterized in that, described detection method comprises the following steps:
1) the compound sage extract solution near the mountain of different concns, is configured;
2), HepG2.2.15 cell is inoculated in RPM1-1640 substratum to cultivate and forms cell suspending liquid;
3), successively by step 1) described in different concns compound near the mountain sage extract solution join in described cell suspending liquid respectively, be wherein provided with do not add compound near the mountain sage extract solution cell suspending liquid in contrast;
4), by step 3) in add different concns compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant a, add staining reagent, dye rear acetic acid wash, solution b is become, the light absorption value A of solution b described in last test and test the light absorption value A of described supernatant liquor a after adding alkali buffered soln again
1; By step 3) in do not add compound sage extract near the mountain cell suspending liquid carry out centrifugal, after separation of supernatant c, add staining reagent, dye rear acetic acid wash, then become solution d after adding alkali buffered soln, tests the light absorption value A of described solution d
2and test the light absorption value A of described supernatant liquor c
3;
5), by the test light absorption value A of described solution b and the light absorption value A of described supernatant liquor a
1, the light absorption value A2 of described solution d and the light absorption value A3 of described supernatant liquor c calculate the survival rate of HepG2.2.15 cell and different concns compound near the mountain sage extract to the inhibiting rate of HBsAg and the HBeAg of HepG2.2.15 emiocytosis; Determine different concns compound near the mountain sage extract to the drug effect of HepG2.2.15 cell, the survival rate=(A/A of wherein said HepG2.2.15 cell
2) × 100%, different concns compound near the mountain sage extract to the inhibiting rate=(A of HBsAg and the HBeAg of HepG2.2.15 emiocytosis
1/ A
3) × 100%.
2. according to claim 1 detect compound near the mountain sage extract to the method for the drug effect of HepG2.2.15 cell, it is characterized in that: the compound of described configuration different concns near the mountain sage extract solution is: get compound sage extract near the mountain, be dissolved in aseptic deionized water, make the compound sage extract solution near the mountain that concentration is 0.00032mg/L, 0.0016mg/L, 0.08mg/L, 0.04mg/L, 0.2mg/L, 1mg/L.
3. detect according to claim 1 or 2 compound near the mountain sage extract to the method for the drug effect of HepG2.2.15 cell, it is characterized in that, described compound is sage extract preparation method near the mountain: compound is the red strong medicinal material Rhododendron dauricum 20 by weight part near the mountain, Holotrichia trichophora 20, Root of Beautiful Millettia 10, Japnanese St. John's wort Herb 10, xanthorrhiza 5 forms, get 4 parts of described compounds respectively red near the mountain, every part of 65g, 95wt% ethanol is used respectively by red near the mountain for described 4 parts of compounds, 60wt% ethanol, 30wt% ethanol, water is as Extraction solvent, often kind of solvent refluxing extracts three times, united extraction liquid, four kinds of compounds sage extract is near the mountain obtained respectively after drying under reduced pressure.
4. according to claim 1 detect compound near the mountain sage extract to the method for the drug effect of HepG2.2.15 cell, it is characterized in that, described RPM11640 substratum contains that geneticin concentrations is 360-400mg/L, the weight percent of foetal calf serum is 8-12%, the concentration of glutamine is 1-3mmol/L, the weight percent of blue or green strepto-is 0.5-2% and NaHC0
3weight percent be 2-8%, the pH value of described RPM11640 substratum is 7.4-7.6.
5. according to claim 1 detect compound near the mountain sage extract to the method for the drug effect of HepG2.2.15 cell, it is characterized in that, step 2) described in form cell suspension after calculate total cellular score, viable cell quantity is no less than 90% of total cellular score, and described HepG2.2.15 cell concn is 0.5 × 10
5individual/mL-1.5 × 10
5individual/mL.
6. according to claim 1 detect compound near the mountain sage extract to the method for the drug effect of HepG2.2.15 cell, it is characterized in that, adopt automatic microplate reader under absorbing wavelength is 515nm condition, test the light absorption value A of described solution b and the light absorption value A of described solution d
2, under absorbing wavelength is 450nm condition, test the light absorption value A of described supernatant liquor a
1and the light absorption value A of described supernatant liquor c
3.
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Citations (2)
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| CN104370871A (en) * | 2013-08-12 | 2015-02-25 | 北京大学 | Xanthine separated from swertia punicea and application thereof in inhibiting hepatitis B virus |
| CN105017376A (en) * | 2015-05-30 | 2015-11-04 | 福建农林大学 | Sarcopyramis nepalensis wall steroidal saponin and application thereof |
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| CN104370871A (en) * | 2013-08-12 | 2015-02-25 | 北京大学 | Xanthine separated from swertia punicea and application thereof in inhibiting hepatitis B virus |
| CN105017376A (en) * | 2015-05-30 | 2015-11-04 | 福建农林大学 | Sarcopyramis nepalensis wall steroidal saponin and application thereof |
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