CN103048373A - Method for detecting salable pinellia ternata - Google Patents
Method for detecting salable pinellia ternata Download PDFInfo
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Abstract
The invention discloses a method for detecting salable pinellia ternate. The method comprises the steps of (1) taking a sample to be detected, adding 1-3 times of water according to weight, wherein the water temperature is 0-5 DEG C, homogenating and centrifuging to obtain supernate; (2) detecting the supernate obtained in the step (1) by using an SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) electrophoresis method, wherein spectrum zones are available at Rm values of 0.208, 0.232, 0.312, 0.335, 0.383, 0.405, 0.429, 0.491, 0.615, 0.85 and/or 0.897; the spacer gel concentration of the electrophoresis is 5%; and the separation gel concentration is 12%. The method for detecting the salable pinellia ternate is high in accuracy, convenient to operate, low in cost and good in market application prospect.
Description
Technical field
The present invention relates to the detection method of the certified products tuber of pinellia, belong to the field of Chinese medicines.
Background technology
The tuber of pinellia derives from aroid tuber of pinellia Pinellia ternata(Thunb.) dry tuber of breit., be the class sphere, the slightly deflection minority that has is long-range sphere, diameter 1 ~ 1.5cm; Surface white or light yellow, there is the stem trace of depression on the top, and pit shape root trace on every side gathers; Below blunt circle, more smooth; Matter is solid, and section is pure white, rich mealiness, the nose of choking that powder is smelt; Gas is little, distinguishes the flavor of pungent, numb tongue and stings larynx.
The market demand of the tuber of pinellia continued to increase in recent years, and wild resource reduces year by year, and the artificial cultivation slower development is so that tuber of pinellia resource reserves and output are all declining to a great extent.Cause having occurred on the market a lot of adulterants, brought very large impact for the quality of Pinellia Ternate, the safety and efficacy of medication.
Find that by the investigation to Chinese Medicinal Materials Markets the adulterant of the tuber of pinellia is mainly RHIZOMA TYPHONII FLAGELLIFORMIS, RHIZOMA ARISAEMATIS and rhizoma thyponii.
RHIZOMA TYPHONII FLAGELLIFORMIS is the dry tuber of aroid whip eaves.Be subcircular, ellipse, taper shape or obovate, diameter 0.5 ~ 1.5cm, high 0.8 ~ 3cm.Surface off-white color or faint yellow, unsmooth, all over the body shape root trace that mays be seen indistinctly; The upper end similar round, commonly used have deflection and leaf scar or the bud trace of projection, yellowish-brown, the lower end is point a bit slightly.Matter is solid, section white, mealiness.Gas is little, and it is pungent to distinguish the flavor of, numb tongue and sting larynx.
RHIZOMA ARISAEMATIS is the dry tuber of aroid pinellia pedatisecta Schott.Be subsphaeroidal, diameter 3 ~ 4cm, crust is coarse, brown, peelling off crust is off-white color or light brown yellow, and there is the stem trace that gets deeply stuck on the top, and the periphery that has has several oblate spheroid shape stem tubers, and shape such as tiger slap, and gas is little, and it is pungent to distinguish the flavor of.
Rhizoma thyponii is the dry tuber of aroid Typhonium giganteum.Ovalize or oval, diameter 1 ~ 2cm, high 2 ~ 5cm.Surface white or yellow-white, slightly coarse, ring grain and root trace are arranged, top tool depression stem trace, the blunt circle in lower end.Matter is hard, and difficulty fractures, section off-white color, mealiness.Gas is little, and is lightly seasoned, the spicy thorn tongue of chewing.
At present, effective detection method of the certified products tuber of pinellia is the discrimination method of traditional form feature and physicochemical property, and it is very complicated and have certain limitation.
Summary of the invention
In order to address the above problem, the invention provides a kind of detection method of the certified products tuber of pinellia.
The detection method of the certified products tuber of pinellia of the present invention comprises the steps:
(1) get sample to be checked, add the water of 1 ~ 3 times of weight, water temperature is 0 ℃ ~ 5 ℃, and homogenate is centrifugal, gets supernatant;
(2) supernatant of usefulness SDS-PAGE electrophoresis method detecting step (1), it has bands of a spectrum in Rm value 0.208,0.232,0.312,0.335,0.383,0.405,0.429,0.491,0.615,0.85 and/or 0.897, the concentrated gum concentration of described electrophoresis is 5%, resolving gel concentration 12%.
Wherein, in the step (1), described frozen water weight is 2 times of example weight to be checked, described centrifugal be the centrifugal 2min of 2000r/min.
Wherein, in the step (2), described supernatant has bands of a spectrum in Rm value 0.208,0.232,0.312,0.335,0.383,0.405,0.429,0.491,0.615,0.85 and 0.897.
SDS-PAGE electrophoresis: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Rm value: relative mobility or the relative absolute migration distance of swimming rate=albumen/bromophenol blue migration distance.
Wherein, described step (2) comprises the steps:
1) get supernatant, add 0.1 ~ 1 times of volume sample damping fluid, mixing, centrifugal, get sample solution, described sample buffer is 1mol/l Tris-hydrochloride buffer, and is added with 0.15mol/l SDS and 0.03mol/l bromophenol blue;
2) get the sample solution loading of step 1), electrophoresis, fixing, dyeing, wash-out gets final product, and described electrophoretic buffer is the Tris-glycine buffer.
Wherein, in the step 1), described sample buffer is 0.1 times of supernatant; In boiling water, heat 3min behind the mixing; Described centrifugal condition is the centrifugal 30s of 4000r/min.
Wherein, in the step 1), described sample buffer also is added with 20%(v/v) glycerine and 10%(v/v) beta-mercaptoethanol.
Wherein, step 2) in, the immobile liquid of described fixedly employing is 10% trichloroacetic acid solution; The dyeing liquor that described dyeing is adopted is for examining Ma Shi light blue dyeing liquor; The eluent that described wash-out adopts is for examining Ma Shi light blue eluent.
Preferably, described dyeing is to soak gel with examining Ma Shi light blue dyeing liquor, and 50 ℃ ~ 60 ℃ heating water bath 15min, and constantly vibration let cool hypsokinesis and remove dyeing liquor.
The inventive method can accurately detect the certified products tuber of pinellia, and accuracy is high, and detection method is simple, quick, can substitute traditional detection method, has a good application prospect.
Obviously, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area, not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.
The embodiment of form is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
Fig. 1 sample treatment testing result, wherein, swimming lane 1～9: method 1～9, M: standard protein;
Fig. 2 colouring method testing result;
Fig. 3 is fond of the sample detection result;
Fig. 4 is fond of the sample detection result;
Fig. 5 is fond of the sample detection result.
Embodiment
Reagent
A liquid: 1.5MTris-HCL solution (separation gel storage liquid)
Precision takes by weighing Tris alkali (analyzing pure) 18.15g, adds an amount of ultrapure water dissolving, transfers pH value to 8.8 with concentrated hydrochloric acid (analyzing pure), adds ultrapure water and is settled to 100mL.Stick labeling, 4 ℃ of preservations.This solution effective service life, be limited to three months.
B liquid: 1MTris-HCL solution (concentrated glue storage liquid)
Precision takes by weighing Tris alkali (analyzing pure) 12.1g, adds an amount of ultrapure water dissolving, with concentrated hydrochloric acid (analyzing pure) adjust pH to 6.8, adds ultrapure water and is settled to 100mL.Stick labeling, 4 ℃ of preservations.This solution effective service life, be limited to three months.
C liquid: 30% acrylamide storage liquid
Precision takes by weighing acrylamide (Acr analyzes pure) 29.2g, N, and N '-methylene diacrylamide (analyzing pure) 0.8g adds an amount of ultrapure water dissolving, is settled to 100mL again, uses Filter paper filtering, sticks labeling, 4 ℃ of preservations of lucifuge.This solution effective service life, be limited to three months.
D liquid: 10%SDS solution
Precision takes by weighing SDS (analyzing pure) 10g, is dissolved to 100mL with ultrapure water, sticks labeling, room temperature storage.This solution effective service life, be limited to three months.
E liquid: 10% Ammonium Persulfate 98.5 (AP) solution
Precision takes by weighing Ammonium Persulfate 98.5 (analyzing pure) 10g, is dissolved to 100mL with ultrapure water, sticks labeling ,-20 ℃ of preservations.This solution is better than before use configuration most.
F liquid: 1%TEMED solution
The accurate T liquid 1mL that draws adds ultrapure water and is settled to 100mL, places brown bottle in 4 ℃ of preservations.
Get above-mentioned raw materials, compound concentration is that 5% concentrated glue and concentration are 12% separation gel.
Electrophoretic buffer: precision takes by weighing Tris alkali (analyzing pure) 6g, glycocoll (analyzing pure) 28.8g, SDS (analyzing pure) 10g, adds ultrapure water and is settled to 1000mL, adds before use 10 times of ultrapure water dilutions, sticks labeling, room temperature storage.
Sample buffer: precision takes by weighing SDS (analyzing pure) 4g, bromophenol blue (analyzing pure) 0.2g, add 20mL glycerine (analyzing pure), 10mL1MTris-HCl(pH6.8) solution, 10mL beta-mercaptoethanol (analyzing pure), add ultrapure water and be dissolved to 100mL, stick labeling, room temperature storage.
Examine Ma Shi light blue dyeing liquor: precision takes by weighing examines Ma Shi light blue R-250 (analyzing pure) 1g, adds methyl alcohol (analyzing pure) 200mL, and glacial acetic acid (analyzing pure) 50mL is settled to 500mL with ultrapure water, sticks labeling, room temperature storage.
Examine Ma Shi light blue eluent: measure ethanol (analyzing pure) 250mL, glacial acetic acid (analyzing pure) 80mL adds ultrapure water and is settled to 1000mL, sticks labeling, room temperature storage.
Dyeing immobile liquid: 10% trichloroacetic acid solution.
Embodiment 1 detection method of the present invention
1, detection method
(1) preparation of test sample:
Take by weighing Pinellia Ternate 2g, with frozen water 4mL, in mortar, grind fast, be transferred to immediately in the centrifuge tube centrifugal 2min under the 2000r/min condition;
Get supernatant 50 μ l, add sample damping fluid 5 μ l, in boiling water, heat 3min behind the mixing, centrifugal 30s under the 4000r/min condition, refrigerated storage is for subsequent use.
(2) electrophoresis
The concentrated glue of preparation and separation gel after glue to be separated and concentrated glue fully solidify, add the electrophoretic buffer of 10 times of dilutions in electrophoresis tank, addition was not there to be glass plate to be as the criterion.Then draw 20 μ l samples with liquid-transfering gun, inject well.Plugged, starting potential is 100V, after indicator entered separation gel, voltage transferred to 220V and carries out the constant voltage electrophoresis.To be instructed dose of stop electrophoresis when walking to 1cm place, electrophoresis tank lower end.
(3) fix, dye and decolouring
After electrophoresis stops, taking out glass plate, gel is slowly stripped down, place 10% trichloroacetic acid dyeing immobile liquid, remove immobile liquid behind the placement 30min, use purified rinse water;
Soak gel with examining Ma Shi light blue dyeing liquor, 50 ℃ ~ 60 ℃ heating water bath 15min, and constantly vibration let cool hypsokinesis and remove dyeing liquor, use first the distilled water rinsing several times, then repeatedly carry out wash-out with eluent, use at last distilled water flushing again.
(4) calculate the Rm value
The electrophoretic band that detects gel noly has bands of a spectrum in Rm value 0.208,0.232,0.312,0.335,0.383,0.405,0.429,0.491,0.615,0.85 and/or 0.897.
The choice of parameters experiment of embodiment 2 the inventive method
1, sample treatment is preferred
Sample treatment as follows 1 ~ 9, all the other methods are with embodiment 1.
Method 1: get supernatant 50 μ l, add sample damping fluid 50 μ l, in boiling water, heat 3min behind the mixing, centrifugal 30s under the 4000r/min condition.
Method 2: get supernatant 50 μ l, add sample damping fluid 50 μ l, heat treated not, centrifugal 30s under the 4000r/min condition.
Method 3: get supernatant 50 μ l, add sample damping fluid 50 μ l, add again loading buffer5 μ l, heat treated not, centrifugal 30s under the 4000r/min condition.
Method 4: get supernatant 50 μ l, add sample damping fluid 50 μ l, add again loading buffer5 μ l, in boiling water, heat 3min behind the mixing, centrifugal 30s under the 4000r/min condition.
Method 5: get supernatant 50 μ l, add beta-mercaptoethanol 100 μ l, add again loading buffer5 μ l, in boiling water, heat 3min behind the mixing, centrifugal 30s under the 4000r/min condition.
Method 6: get supernatant 50 μ l, add beta-mercaptoethanol 50 μ l, add again loading buffer5 μ l, in boiling water, heat 3min behind the mixing, centrifugal 30s under the 4000r/min condition.
Method 7: get supernatant 50 μ l, add beta-mercaptoethanol 50 μ l, add again loading buffer5 μ l, heat treated not, centrifugal 30s under the 4000r/min condition.
Method 8: get supernatant 50 μ l, add loading buffer5 μ l, in boiling water, heat 3min behind the mixing, centrifugal 30s under the 4000r/min condition.
Method 9: get supernatant 50 μ l, add loading buffer5 μ l, heat treated not, centrifugal 30s under the 4000r/min condition.
As shown in Figure 1, the band of the swimming lane (swimming lane 1 ~ 4 and 8 ~ 9) of the sample that the swimming lane of Marker and the inventive method are processed is all clear, especially the band of swimming lane 8 is the clearest, conditions of streaking then appears in the swimming lane (swimming lane 5 ~ 7) of the sample of other disposal route preparations, the sample treatment that said method 1 ~ 4 and 8 ~ 9 are described all is effective, wherein, optimum with the sample treatment of method 8.
2, colouring method
Colouring method as follows 1 ~ 9, all the other methods are with embodiment 1.
Method 1: soak gel 3h with examining Ma Shi light blue dyeing liquor, and constantly vibration, the dyeing liquor that inclines is used first the distilled water rinsing several times, then repeatedly carries out wash-out with eluent, uses at last distilled water flushing again.
Method 2: soak gel with examining Ma Shi light blue dyeing liquor, 50 ℃ ~ 60 ℃ heating water bath 15min, and constantly vibration let cool hypsokinesis and remove dyeing liquor, use first the distilled water rinsing several times, then repeatedly carry out wash-out with eluent, use at last distilled water flushing again.
Method 3: soak gel with examining Ma Shi light blue dyeing liquor, place micro-wave oven (moderate heat) heating 3min, take out, let cool hypsokinesis and remove dyeing liquor, use first the distilled water rinsing several times, then repeatedly carry out wash-out with eluent, use again at last distilled water flushing.
The result as shown in Figure 2, the present invention can effectively dye with the method for Coomassie brilliant blue, wherein, 1 time of colouring method is longer, and dyeing after be difficult for wash-out, band is clear not; Colouring method 3 must be processed through micro-wave oven, though easy wash-out after the dyeing, band is also more clear, and band is many because microwave heating deforms, the in addition many shrinkages in gel edge in the heating process; Colouring method 2 dyeing are very fast, easier wash-out, and band is clear, is optimum colouring method.
Below in the mode of experimental example beneficial effect of the present invention is described:
Experimental example 1 usefulness the inventive method detects sample to be checked
Experiment material: 4 batches of medicinal materials buying from market, 9 parts of the 1st batch of medicinal materials (B1～B9), 9 parts of the 2nd batch of medicinal materials (S1～S9), and 16 parts of the 3rd batch of medicinal materials (N1～N16), 11 parts of (F1～F11), detect with the inventive method and traditional detection method respectively of the 4th batch of medicinal material.
1, detection method
(1) the present invention detects
Take by weighing sample 2g, with frozen water 4mL, in mortar, grind fast, be transferred to immediately in the centrifuge tube centrifugal 2min under the 2000r/min condition, get supernatant 50 μ l, add sample damping fluid 50 μ l, add again loading buffer5 μ l, in boiling water, heat 3min behind the mixing, centrifugal 30s under the 4000r/min condition, refrigerated storage is for subsequent use.
After glue to be separated and concentrated glue fully solidify, add the electrophoretic buffer of 10 times of dilutions in electrophoresis tank, addition was not there to be glass plate to be as the criterion.Then draw 20 μ l samples with liquid-transfering gun, inject well.Plugged, starting potential is 100V, after indicator entered separation gel, voltage transferred to 220V and carries out the constant voltage electrophoresis.To be instructed dose of stop electrophoresis when walking to 1cm place, electrophoresis tank lower end.
After electrophoresis stops, taking out glass plate, gel is slowly stripped down, place 10% trichloroacetic acid dyeing immobile liquid, remove immobile liquid behind the placement 30min, use purified rinse water.Then soak gel with examining Ma Shi light blue dyeing liquor, heating water bath 15min, and constantly vibration let cool hypsokinesis and remove dyeing liquor, use first the distilled water rinsing several times, then repeatedly carry out wash-out with eluent, use at last distilled water flushing again.
The electrophoretic band that detects gel is 0.208,0.232,0.312,0.335,0.383,0.405,0.429,0.491,0.615,0.85 and/or 0.897 no bands of a spectrum to be arranged at Rm.
(2) traditional detection method
Form and physicochemical property are identified.
2 experimental results
(1) the inventive method testing result
Testing result is as shown in table 1:
The testing result of table 1 the inventive method
As can be seen from Table 1, B1 ~ B9 sample has bands of a spectrum in Rm value 0.208,0.232,0.312,0.335,0.383,0.405,0.429,0.491,0.615,0.85 and 0.897, and all the other samples do not have bands of a spectrum in Rm value 0.208,0.232,0.312,0.335,0.383,0.405,0.429,0.491,0.615,0.85 and 0.897, illustrate that B1 ~ B9 sample is the certified products tuber of pinellia, all the other samples are the adulterant of the tuber of pinellia.
(2) traditional detection method testing result
B1～B9: be the class sphere, the slightly deflection minority that has is long-range sphere, diameter 1 ~ 1.5cm; Surface white or light yellow, there is the stem trace of depression on the top, and pit shape root trace on every side gathers; Below blunt circle, more smooth; Matter is solid, and section is pure white, rich mealiness, the nose of choking that powder is smelt; Gas is little, distinguishes the flavor of pungent, numb tongue and stings larynx.
S1～S9: be subcircular, ellipse, taper shape or obovate, diameter 0.5 ~ 1.5cm, high 0.8 ~ 3cm.Surface off-white color or faint yellow, unsmooth, all over the body shape root trace that mays be seen indistinctly; The upper end similar round, commonly used have deflection and leaf scar or the bud trace of projection, yellowish-brown, the lower end is point a bit slightly.Matter is solid, section white, mealiness.Gas is little, and it is pungent to distinguish the flavor of, numb tongue and sting larynx.
N1～N16: be subsphaeroidal, diameter 3 ~ 4cm, crust is coarse, brown, peelling off crust is off-white color or light brown yellow, and there is the stem trace that gets deeply stuck on the top, and the periphery that has has several oblate spheroid shape stem tubers, and shape such as tiger slap, and gas is little, and it is pungent to distinguish the flavor of.
F1～F11: ovalize or oval, diameter 1 ~ 2cm, high 2 ~ 5cm.Surface white or yellow-white, slightly coarse, ring grain and root trace are arranged, top tool depression stem trace, the blunt circle in lower end.Matter is hard, and difficulty fractures, section off-white color, mealiness.Gas is little, and is lightly seasoned, the spicy thorn tongue of chewing.
The testing result of table 2 traditional detection method
| Sample number into spectrum | Qualification result |
| B1～B9 | The tuber of pinellia |
| S1～S9 | RHIZOMA TYPHONII FLAGELLIFORMIS |
| N1～N16 | RHIZOMA ARISAEMATIS |
| F1～F11 | Rhizoma typhonii |
Can find out that B1 ~ B9 sample is the certified products tuber of pinellia, all the other samples are the adulterant of the tuber of pinellia, and this result is consistent with the testing result of the inventive method.
To sum up, detection method of the present invention is consistent with the result of traditional authentication method, and accuracy is high, and is easy and simple to handle, with low cost, has a good application prospect and economic benefit.
Claims (8)
1. the detection method of a certified products tuber of pinellia is characterized in that: comprise the steps:
(1) get sample to be checked, add the water of 1 ~ 3 times of weight, 0 ℃ ~ 5 ℃ of water temperatures, homogenate, centrifugal, get supernatant;
(2) supernatant of usefulness SDS-PAGE electrophoresis method detecting step (1), it has bands of a spectrum in Rm value 0.208,0.232,0.312,0.335,0.383,0.405,0.429,0.491,0.615,0.85 and/or 0.897, the concentrated gum concentration of described electrophoresis is 5%, and resolving gel concentration is 12%.
2. detection method according to claim 1, it is characterized in that: in the step (1), described water is 2 times of example weight to be checked; Described centrifugal be the centrifugal 2min of 2000r/min.
3. detection method according to claim 1, it is characterized in that: in the step (2), described supernatant has bands of a spectrum in Rm value 0.208,0.232,0.312,0.335,0.383,0.405,0.429,0.491,0.615,0.85 and 0.897.
4. detection method according to claim 1, it is characterized in that: described step (2) comprises following steps:
1) supernatant is added 0.1 ~ 1 times of volume sample damping fluid, mixing, centrifugal, get sample solution, described sample buffer is 1mol/l Tris-HCl damping fluid, and is added with 0.03mol/l bromophenol blue and 0.15mol/l SDS;
2) get the sample solution loading of step 1), electrophoresis, fixing, dyeing, wash-out gets final product, and described electrophoretic buffer is the Tris-glycine buffer.
5. detection method according to claim 4, it is characterized in that: in the step 1), described sample buffer is 0.1 times of supernatant; After described supernatant and the sample buffer mixing, in boiling water, heat 3min first, centrifugal again; Described centrifugal be the centrifugal 30s of 4000r/min.
6. detection method according to claim 4, it is characterized in that: in the step 1), described sample buffer also is added with 20%(v/v) glycerine and 10%(v/v) beta-mercaptoethanol.
7. detection method according to claim 4 is characterized in that: step 2) in, the immobile liquid of described fixedly employing is 10%(w/w) trichloroacetic acid solution; The dyeing liquor that described dyeing is adopted is for examining Ma Shi light blue dyeing liquor; The eluent that described wash-out adopts is for examining Ma Shi light blue eluent.
8. detection method according to claim 7, it is characterized in that: described dyeing is: soak gel with examining Ma Shi light blue dyeing liquor, 50 ~ 60 ℃ of heating water bath 15min, and constantly vibration, dyeing liquor is removed in the cooling hypsokinesis.
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| CN104359964A (en) * | 2014-11-03 | 2015-02-18 | 宁波市鄞州华谦电子有限公司 | Gradient glue and reparation method thereof |
| CN106404961A (en) * | 2016-11-25 | 2017-02-15 | 成都中医药大学 | Method for discriminating adulteration of Pinellia pedatisecta in pinellia tuber |
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| CN104359964A (en) * | 2014-11-03 | 2015-02-18 | 宁波市鄞州华谦电子有限公司 | Gradient glue and reparation method thereof |
| CN106404961A (en) * | 2016-11-25 | 2017-02-15 | 成都中医药大学 | Method for discriminating adulteration of Pinellia pedatisecta in pinellia tuber |
| CN106404961B (en) * | 2016-11-25 | 2020-09-04 | 成都中医药大学 | Method for identifying adulterated radix polygoni multiflori in pinellia ternata |
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