CN111650312A - Quality detection method of wine for relaxing muscles and tendons and treating rheumatism - Google Patents
Quality detection method of wine for relaxing muscles and tendons and treating rheumatism Download PDFInfo
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Abstract
The application is a divisional application with the application number of 201410851229.6, the application date of 2014 of 12 and 31 and the invention name of 'a quality detection method for tendon relaxing rheumatic wine'. The invention discloses a quality detection method of a rheumatic wine capable of relaxing muscles and tendons, which comprises the following steps: preparing a test solution, preparing a reference solution and carrying out chromatographic conditions. The detection method provided by the invention simplifies the sample extraction step; the reflux time is shortened; the dosage of chloroform serving as an extraction reagent is reduced; the efficiency is improved, and the device is more economical and environment-friendly.
Description
The application is a divisional application with the application number of 201410851229.6, the application date of 2014 of 12 and 31 and the invention name of 'a quality detection method for tendon relaxing rheumatic wine'.
Technical Field
The invention belongs to the technical field of detection. More particularly, relates to a quality detection method of rheumatic wine capable of relaxing muscles and tendons.
Background
The prescription of the wine for relaxing muscles and tendons and treating rheumatism is 30g of chunguteng, 30g of Chinese starjasmine stem, 30g of suberect spatholobus stem, 20g of caulis kadsurae, 30g of Wunian, 10g of giant knotweed, 30g of Shuidong and 20g of Kadsura coccinea root. The preparation method comprises the following steps: pulverizing the above eight medicinal materials into fine powder, adding 1050ml Chinese liquor, soaking for 1 hr, heating under reflux for 5.5 hr, filtering, and collecting filtrate; preparing sucrose into simple syrup, mixing with the above filtrate, adding Chinese liquor to 1000ml, mixing, filtering, and packaging. The functions and indications are as follows: dispel wind-damp, relax tendons and activate collaterals. Can be used for treating rheumatic arthralgia, traumatic injury, arthralgia and myalgia, and soreness of waist and limbs.
The published quality detection method for wine for relaxing muscles and tendons and rheumatism is recorded in page 654-657 of meridian limb brain series booklet of 'national Chinese patent drug standardization compilation', and specifically comprises the following steps:
preparation of a test solution: precisely measuring 10ml of the product, evaporating to dryness, adding 30ml of 2.5mol/l sulfuric acid solution into residues for dissolving, transferring to a conical flask, placing in a boiling water bath, heating and refluxing for 1 hour, cooling, adding 30ml of trichloromethane, adding reflux for 1 hour, cooling, placing in a separating funnel, washing with a small amount of trichloromethane washing solution, adding into the separating funnel, separating trichloromethane solution, shaking and extracting acid liquor with trichloromethane for 5 times, 30nl each time, combining the trichloromethane solution, dehydrating with a proper amount of anhydrous sodium sulfate, evaporating to dryness, adding a proper amount of methanol into residues for dissolving, placing in a 25ml measuring flask, adding methanol for diluting to a scale, and shaking uniformly to obtain the product.
Preparation of control solutions: taking appropriate amount of emodin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
Chromatographic conditions and system adaptability: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid solution (82:18) is used as a mobile phase; the detection wavelength was 254 nm. The number of theoretical plates is not less than 1500 calculated according to emodin peak.
The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
The detection method has the defects that the sample processing method is complicated; long time consumption and low efficiency; and the extraction reagent dosage is large, the environment is polluted, the defects of being uneconomical and environment-friendly and the like are overcome, the applicant provides a new method for detecting the tendon-relaxing rheumatic wine, and the sample extraction step can be simplified; the reflux time is shortened; the dosage of chloroform as an extraction reagent is reduced; improving the efficiency and being economical and environment-friendly.
Under the test item of the original medicinal materials of giant knotweed in the first part of the 2010 edition 'Chinese pharmacopoeia', the content determination of emodin is disclosed, wherein the preparation and chromatographic test conditions of a test solution are as follows:
preparation of a test solution: taking about 0.1g of the product powder (passing through a third sieve), precisely weighing, precisely adding 25ml of trichloromethane and 20ml of 2.5mol/L sulfuric acid solution, weighing, heating and refluxing in a water bath at 80 ℃ for 2 hours, cooling to room temperature, weighing again, supplementing the reduced weight with trichloromethane, shaking up, collecting trichloromethane solution, precisely weighing 10ml, evaporating to dryness, adding methanol into residues for dissolving, transferring to a 10ml measuring flask, adding methanol for diluting to a scale, shaking up, filtering, and taking a subsequent filtrate to obtain the product.
Chromatographic conditions and system applicability test: octadecylsiloxane bonded silica gel is used as a filler; methanol-0.1 phosphoric acid solution (80: 20) is used as a mobile phase; the detection wavelength is 254nm, and the number of theoretical plates is not less than 3000 calculated according to emodin peak.
If the content of the muscle-relaxing rheumatism wine is directly detected by adopting the content detection method of the giant knotweed raw medicinal materials, the following problems can occur: 1. the sample of the muscle-relaxing rheumatism wine is a liquid sample and contains volatile solvent ethanol, and the volume ratio is measured by a pipette and the weighing is more convenient and accurate. 2. In the method for measuring the content of the giant knotweed rhizome, the heating reflux time of a test sample is longer, 2 hours are needed, and the content of the emodin is reduced when the reflux is more than 30min through actual examination. 3. If the giant knotweed rhizome method is adopted for extraction liquid separation, one extraction is less than that of the product method, and experiments prove that emodin is incompletely extracted by adopting one liquid separation. 4. The giant knotweed rhizome detection method adopts anhydrous sodium sulfate for dehydration treatment of the chloroform solution combined with the split solution, and a small amount of sulfuric acid solution enters the chloroform solution to blacken and carbonize the emodin in the process of evaporation to dryness, so that the content of the emodin is seriously influenced.
Therefore, a method which has the advantages of simple steps, less solvent consumption, economy, environmental protection, short time consumption and high content of extracted emodin is needed to be provided so as to detect the tendon relaxing rheumatism wine and improve the product quality.
Disclosure of Invention
The invention aims to provide a quality detection method of a rheumatic wine capable of relaxing muscles and tendons.
The invention provides a quality detection method of a tendon-relaxing rheumatic wine, which comprises the preparation of a test solution, wherein the test solution is prepared by extracting the tendon-relaxing rheumatic wine in a solvent, and the solvent comprises a sulfuric acid solution and trichloromethane.
Preferably, the sulfuric acid solution is 1-4mol/L sulfuric acid solution; the volume ratio of the tendon relaxing rheumatism wine to the sulfuric acid solution to the trichloromethane is 10 (15-30) to 15-30.
More preferably, the sulfuric acid solution is 1-2.5mol/L sulfuric acid solution; the volume ratio of the tendon relaxing rheumatism wine to the sulfuric acid solution to the trichloromethane is 10 (15-30) to 15-30.
Most preferably, the sulfuric acid solution is a 2.5mol/L sulfuric acid solution; the volume ratio of the tendon relaxing rheumatism wine to the sulfuric acid solution to the trichloromethane is 10:30: 30.
Preferably, the extraction comprises heating in a water bath at 80 ℃ under reflux for 15-90 minutes.
Further preferably, the preparation of the test solution comprises the steps of: precisely measuring 10ml of muscle-relaxing rheumatism wine, sequentially adding 15-30ml of sulfuric acid solution and chloroform each of which is 1-4mol/L, heating and refluxing for 15-90 minutes in a water bath at 80 ℃, cooling, and separating chloroform solution for later use.
More preferably, the preparation of the test solution further comprises: after the chloroform solution is separated, the acid solution is shaken and extracted for 1-2 times by 15-30ml of chloroform, dehydrated by anhydrous sodium sulfate and then combined with the separated chloroform solution after heating and refluxing.
More preferably, the preparation of the test solution comprises the steps of: precisely measuring 10ml of muscle-relaxing rheumatism wine, sequentially adding 15-30ml of each of 1-2.5mol/l sulfuric acid solution and chloroform, heating and refluxing in 80 ℃ water bath for 30-60 minutes, cooling, and separating chloroform solution; extracting the acid solution with 15-30ml chloroform under shaking for 1-2 times, dehydrating with anhydrous sodium sulfate, mixing with chloroform solution obtained after heating reflux, evaporating to dryness, dissolving residue with appropriate amount of methanol, diluting with methanol to 25ml, shaking, filtering, and collecting filtrate.
In addition, the quality detection method of the tendon relaxing rheumatism wine comprises the following steps: preparing a reference substance solution, preparing a test substance solution and carrying out chromatographic conditions.
The preparation of the test solution comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine, sequentially adding 15-30ml of each of 1-4mol/l of sulfuric acid solution and trichloromethane, heating and refluxing for 15-90 minutes in a water bath at 80 ℃, cooling, taking trichloromethane liquid, shaking and extracting acid liquor for 1-2 times by using 15-30ml of trichloromethane, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating to dryness, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering, and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine.
Preferably, the preparation of the test solution: precisely measuring 10ml of muscle-relaxing rheumatism wine, sequentially adding 15-30ml of each of 1-2.5mol/l sulfuric acid solution and trichloromethane, heating and refluxing for 30-60 minutes in a water bath at 80 ℃, cooling, taking trichloromethane liquid, shaking and extracting acid liquor for 1-2 times by using 15-30ml of trichloromethane, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating to dryness, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering, and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine.
Further preferably, the preparation of the test solution: precisely measuring 10ml of muscle-relaxing rheumatism wine, sequentially adding 30ml of 2.5mol/l sulfuric acid solution and 30ml of trichloromethane respectively, heating and refluxing in a water bath at the temperature of 80 ℃ for 30-60 minutes, cooling, separating trichloromethane liquid, shaking and extracting acid liquor for 1-2 times by using 30ml of trichloromethane, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating to dryness, adding a proper amount of methanol into residues to dissolve the residues, adding methanol to dilute the residues to 25ml, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine.
The chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid solution (82:18) as mobile phase; the detection wavelength was 254 nm.
Specifically, the quality detection method provided by the invention comprises the following steps:
preparation of a test solution: precisely measuring 10ml of muscle-relaxing rheumatism wine, sequentially adding 15-30ml of each of 1-2.5mol/l sulfuric acid solution and trichloromethane, heating and refluxing for 30-60 minutes in a water bath at 80 ℃, cooling, taking trichloromethane liquid, shaking and extracting acid liquor for 1-2 times by using 15-30ml of trichloromethane, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating the trichloromethane liquid in a water bath at 70 ℃, adding a proper amount of methanol into residues for dissolving, diluting to 25ml by adding methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
preparation of control solutions: accurately weighing appropriate amount of emodin reference substance, and adding methanol to obtain solution containing 50 μ g per 1 ml;
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid solution (82:18) as mobile phase; the detection wavelength is 254 nm;
the detection method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Preferably, the method comprises the steps of:
preparation of a test solution: precisely measuring 10ml of muscle-relaxing rheumatism wine, sequentially adding 30ml of 2.5mol/l sulfuric acid solution and 30ml of trichloromethane, heating and refluxing in a water bath at 80 ℃ for 30 minutes, cooling, separating trichloromethane liquid, shaking and extracting acid liquor by using 30ml of trichloromethane for 1 time, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating the trichloromethane liquid in a water bath at 70 ℃ to dryness, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute the residues to 25ml, shaking up, filtering, and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
preparation of control solutions: accurately weighing appropriate amount of emodin reference substance, and adding methanol to obtain solution containing 50 μ g per 1 ml;
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid solution (82:18) as mobile phase; the detection wavelength is 254 nm;
the detection method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
In the detection method provided by the invention, emodin is not less than 90 mug in every 1ml of the wine for relaxing muscles and tendons and rheumatism.
The method provided by the invention has the following advantages:
1. compared with the original method, the method has the following differences:
2. the invention has the advantages that:
1) compared with the original detection method: improved sample processing methods include: the sample extraction step is simplified; the reflux time is shortened; the dosage of chloroform serving as an extraction reagent is reduced; the efficiency is improved, and the device is more economical and environment-friendly.
2) The method for processing the test solution can ensure that the content of the emodin is higher and the detection result is more stable in the content detection of the tendon relaxing rheumatism wine, and can also be used for detecting the content of the emodin in other medicinal materials, thereby having wide application range.
3) The content determination method of the invention has good stability and high sensitivity, and the product quality detected by the method is stable.
Drawings
FIG. 1-A, FIG. 1-B, and FIG. 1-C are HPLC chromatograms, wherein A is a reference solution, B is a test solution, and C is a negative sample solution;
FIG. 2: emodin linear relation curve chart.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
1. Experimental Material
1.1, sample source:
the tendon-relaxing rheumatism wine is provided by Qianjin pharmaceutical industry, Inc. of Tazhou province.
1.2 Instrument and reagents:
the instrument comprises the following steps: high performance liquid chromatography instrument: agilent 1200; a VWD detector;
ultra pure water machine (ABW-1001-U model Eicopu).
Reagent: methanol (chromatographically pure, Miro chemical reagents Ltd, Tianjin);
absolute ethanol (analytical grade, hunan hui hong reagent limited);
cyclohexane (analytical grade, hunan hui hong reagent limited);
ethyl acetate (analytical grade, west longa chemical ltd);
petroleum ether (60-90 deg.C) (Mi European chemical reagent Co., analytically pure Tianjin);
glacial acetic acid (analytical pure lake south hui rainbow reagents ltd);
trichloromethane (analytically pure, fujin, fuyu fine chemical industries, ltd);
toluene (analytical pure lake south hui rainbow reagents ltd);
acetone (analytically pure, riches quartz glass ltd);
water (redistilled, prepared just before use);
phosphoric acid (analytical purity, hunan hui hong reagent limited).
1.3 control
Emodin reference (China institute for testing food and drug, for content determination, batch No. 110756-and 200110).
2. Chromatographic conditions
A chromatographic column: agilent TC-C18 (4.6X 250mm, 5 μm); mobile phase: methanol-0.1% phosphoric acid solution (82: 18);
flow rate: 1.0 ml/min; detection wavelength: 254 nm; column temperature: 35 ℃; sample introduction amount: 10 mu L of the solution; the number of theoretical plates is not less than 1500 calculated according to emodin peak.
3. Preparation of a test solution:
the method comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch No. 20130103), sequentially adding 30ml of each of 2.5mol/l sulfuric acid solution and chloroform, heating and refluxing for 15 minutes in a water bath at 80 ℃, cooling, separating and taking chloroform solution, shaking and extracting acid solution for 1 time by using 30ml of chloroform, dehydrating by using anhydrous sodium sulfate, combining the chloroform solutions, evaporating the chloroform solution in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
the second method comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch No. 20130103), sequentially adding 30ml of 2.5mol/l sulfuric acid solution and 30ml of trichloromethane, heating and refluxing in a water bath at 80 ℃ for 30 minutes, cooling, separating trichloromethane liquid, extracting acid solution for 1 time by shaking with 30ml of trichloromethane, dehydrating with anhydrous sodium sulfate, combining the trichloromethane liquids, evaporating the trichloromethane liquid in a water bath at 70 ℃ to dryness, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering with a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the medicinal liquor;
the third method comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch No. 20130103), sequentially adding 30ml of each of 2.5mol/l sulfuric acid solution and chloroform, heating and refluxing for 60 minutes in a water bath at 80 ℃, cooling, separating and taking chloroform solution, shaking and extracting acid solution for 1 time by using 30ml of chloroform, dehydrating by using anhydrous sodium sulfate, combining the chloroform solutions, evaporating the chloroform solution in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
the method four comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch No. 20130103), sequentially adding 30ml of each of 2.5mol/l sulfuric acid solution and chloroform, heating and refluxing for 90 minutes in a water bath at 80 ℃, cooling, taking chloroform solution, shaking and extracting acid solution for 1 time by using 30ml of chloroform, dehydrating by using anhydrous sodium sulfate, combining the chloroform solutions, evaporating the chloroform solution in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the wine;
the method five comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch number 20130103), sequentially adding 30ml of 2.5mol/l sulfuric acid solution and 30ml of trichloromethane, placing in a water bath at 80 ℃, heating and refluxing for 30 minutes, cooling, separating and taking trichloromethane liquid, shaking and extracting the acid solution for 30ml and trichloromethane for 2 times, 30ml each time, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating the trichloromethane liquid in the water bath at 70 ℃, adding a proper amount of methanol into residues for dissolving, diluting the residues to 25ml by adding methanol, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the wine;
comparing different extraction times by using a first method, a second method, a third method and a fourth method; the second method and the fifth method compare different extraction times.
The method six: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch number 20130103), sequentially adding 15ml of 1mol/l sulfuric acid solution and chloroform, heating and refluxing for 30 minutes in a water bath at 80 ℃, cooling, separating chloroform solution, shaking and extracting acid solution for 1 time by using 15ml of chloroform, dehydrating by using anhydrous sodium sulfate, combining the chloroform solutions, evaporating the chloroform solution in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
the method comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch No. 20130103), sequentially adding 30ml of each of 2.5mol/l sulfuric acid solution and chloroform, heating and refluxing for 60 minutes in a water bath at 80 ℃, cooling, separating and taking chloroform solution, shaking and extracting acid solution for 2 times by using 30ml of chloroform, dehydrating by using anhydrous sodium sulfate, combining the chloroform solutions, evaporating the chloroform solution in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
the method eight: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch number 20130103), sequentially adding 15ml of 4mol/l sulfuric acid solution and chloroform, heating and refluxing for 90 minutes in a water bath at 80 ℃, cooling, separating chloroform solution, shaking and extracting acid solution for 1 time by using 15ml of chloroform, dehydrating by using anhydrous sodium sulfate, combining the chloroform solutions, evaporating the chloroform solution in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
the method comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch number 20130103), sequentially adding 20ml of 4mol/l sulfuric acid solution and 20ml of chloroform, heating and refluxing for 15 minutes in a water bath at 80 ℃, cooling, separating chloroform solution, shaking and extracting acid solution for 2 times by using 20ml of chloroform, dehydrating by using anhydrous sodium sulfate, combining the chloroform solutions, evaporating the chloroform solution in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute the residues to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
the method comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch No. 20130103), sequentially adding 25ml of 3mol/l sulfuric acid solution and 25ml of trichloromethane, heating and refluxing for 80 minutes in a water bath at 80 ℃, cooling, separating trichloromethane liquid, shaking and extracting acid solution for 1 time by using 25ml of trichloromethane, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating the trichloromethane liquid in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, diluting to 25ml by adding methanol, shaking uniformly, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
the method eleven comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch No. 20130103), sequentially adding 20ml of 2mol/l sulfuric acid solution and 20ml of trichloromethane, heating and refluxing for 20 minutes in a water bath at 80 ℃, cooling, separating trichloromethane liquid, shaking and extracting acid solution for 2 times by using 20ml of trichloromethane, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating the trichloromethane liquid in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
the method twelve: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch No. 20130103), sequentially adding 30ml of 1.5mol/l sulfuric acid solution and 30ml of trichloromethane, placing in a water bath at 80 ℃, heating and refluxing for 50 minutes, cooling, separating and taking trichloromethane liquid, shaking and extracting acid solution for 2 times by using 15ml of trichloromethane, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating the trichloromethane liquid by using a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding the methanol to dilute to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
the method thirteen comprises the following steps: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch No. 20130103), sequentially adding 20ml of 1.5mol/l sulfuric acid solution and 20ml of trichloromethane, heating and refluxing in a water bath at 80 ℃ for 40 minutes, cooling, separating trichloromethane liquid, shaking and extracting acid solution for 1 time by using 20ml of trichloromethane, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating the trichloromethane liquid in a water bath at 70 ℃ to dryness, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute the residues to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
the method is fourteen: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch number 20130103), sequentially adding 25ml of 2mol/l sulfuric acid solution and 25ml of trichloromethane, heating and refluxing for 30 minutes in a water bath at 80 ℃, cooling, separating trichloromethane liquid, shaking and extracting acid solution for 1 time by using 30ml of trichloromethane, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating the trichloromethane liquid in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering by using a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
fifteen methods: precisely measuring 10ml of muscle-relaxing rheumatism wine (batch No. 20130103), sequentially adding 30ml of 1mol/l sulfuric acid solution and 30ml of trichloromethane, heating and refluxing for 90 minutes in a water bath at 80 ℃, cooling, separating trichloromethane solution, extracting acid solution for 1 time by shaking 15ml of trichloromethane, dehydrating by anhydrous sodium sulfate, combining the trichloromethane solutions, evaporating the trichloromethane solution in a water bath at 70 ℃, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering by a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine.
4. Preparation of negative sample solution
Taking other medicinal materials except the giant knotweed rhizome according to the formula proportion, preparing a giant knotweed rhizome-lacking negative sample according to the process, and preparing a giant knotweed rhizome-lacking negative sample solution according to the preparation method of the test sample solution.
5. Preparation of control solutions
Taking appropriate amount of emodin reference substance, precisely weighing, adding methanol to obtain solution containing 50 μ g per 1ml, shaking, and filtering with microporous membrane (0.45 μm).
6. Chromatographic conditions and specificity tests
Octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid solution (82:18) as mobile phase; the detection wavelength was 254 nm. The number of theoretical plates is not less than 1500 calculated according to emodin peak. See FIGS. 1-A, 1-B and 1-C.
The results of FIGS. 1-A, 1-B and 1-C show: according to the chromatographic conditions, the emodin is well separated, the area of the negative sample emodin peak is within 4 percent of the sample, and the specificity is strong.
7. Investigation of linear relationships
Taking a proper amount of emodin reference substance, precisely weighing, dissolving with methanol, and fixing volume. Prepare a control solution with the mass concentration of 330 mug/ml. The solutions were diluted one time and four times in sequence to obtain four concentrations of control solutions: 165.00 μ g/ml, 82.66 μ g/ml, 41.45 μ g/ml, 20.79 μ g/ml, 10.41 μ g/ml. And (3) carrying out sample injection analysis on the five concentrations of the reference substances, and determining the peak area of the emodin according to the chromatographic conditions under the item 2. The peak area is used as the ordinate (Y) and the sample concentration (μ g/ml) is used as the abscissa (X), so that the emodin regression equation Y is 36.91926X +26.44238(r is 0.99997).
See table 1 and fig. 2.
TABLE 1 emodin Linear relationship examination results
| Sample concentration (mu g/ml) | 10.41 | 20.79 | 41.45 | 82.66 | 165.00 | 330.00 |
| Sample volume (ug) | 0.1041 | 0.2079 | 0.4145 | 0.8266 | 1.6500 | 3.3000 |
| Peak area value | 386.72 | 773.64 | 1571.93 | 3094.43 | 6151.95 | 12188.95 |
The results in table 1 and figure 2 show that: emodin is linear well at 10.41-330.00 μ g/ml.
8. Precision test
Respectively and precisely sucking 10 μ l of the same test solution (lot No. 20130103), preparing the test solution by the second method in the preparation method 3 of the test solution, continuously measuring for 6 times, and measuring the peak area of emodin according to the chromatographic conditions under item 2, as shown in Table 2.
TABLE 2 emodin precision test results
Table 2 the results show: its RSD was 0.23%.
The results show that: the precision of the instrument is good,
9 stability test
Precisely sucking 10 μ l of the same sample solution (lot No. 20130103), preparing the sample solution by the second method in the sample solution preparation method 3, injecting samples at 0, 2, 4, 6, 8, 16, and 24 hr, and determining emodin content according to the chromatographic conditions under item 2. The results are shown in Table 3.
TABLE 3 emodin stability test investigation results
Table 3 the results show: the RSD was 0.31%.
The results show that: the test solution is stable within 24 h.
10 repeatability test
Accurately measuring 6 parts of 10ml of this product (lot No. 20130103), preparing test solution according to item 3 (preparing test solution by method II of test solution preparation method 3), and measuring emodin peak area according to chromatographic conditions of item 2, the results are shown in Table 4.
TABLE 4 emodin repeatability test investigation results
Table 4 the results show: the RSD was 1.8%.
The results show that the method has good repeatability.
11 sample application recovery test
Precisely sucking 1ml and 6 parts of control solution containing emodin 0.5944mg/ml, placing in a conical flask, volatilizing methanol in water bath, precisely sucking 5ml of the product (lot number 20130103) and Chinese liquor respectively, placing in the conical flask, performing preparation operation of test solution under item 3 (preparing test solution by method II in test solution preparation method 3), measuring content under chromatographic conditions under item 2, and calculating recovery rate, wherein the results are shown in Table 5.
Table 5 emodin loading recovery test (n ═ 6)
12, a detection method:
precisely sucking 10 μ l each of the test solution prepared by the test solution preparation method under item 3 and the reference solution prepared under item 5, injecting into a liquid chromatograph, measuring according to the chromatographic conditions under item 2, and recording chromatographic peak.
As a result: see Table 6
TABLE 6 test results of different test solution preparation methods
| Method for preparing test solution | Emodin content (μ g/ml) |
| Method 1 | 101 |
| Method two | 114 |
| Method III | 111 |
| Method IV | 108 |
| Method five | 114 |
| Method VI | 103 |
| Method seven | 111 |
| Method eight | 94 |
| Method nine | 97 |
| Method ten | 96 |
| Method eleven | 106 |
| Method twelve | 105 |
| Method thirteen | 109 |
| Method fourteen | 107 |
| Method fifteen | 99 |
Example 2: comparison of the existing method with the detection method of example 1
1. For samples with different batches, the fifteen test samples in the embodiment 1 are detected by adopting the method, and meanwhile, the method in the comparative example 1 and the comparative example 2 is adopted for detection, and the method specifically comprises the following steps:
comparative example 1: taking 10ml of the tendon-relaxing rheumatism wine (batch number 20130106) in the invention, preparing a test solution according to a preparation method of the test solution under the item of the original medicinal material of polygonum cuspidatum in the first part of 2010 edition of Chinese pharmacopoeia, and determining the content of emodin according to the disclosed chromatographic conditions under the item of the original medicinal material of polygonum cuspidatum.
Comparative example 2: taking 10ml of the tendon-relaxing rheumatic wine (batch No. 20130106), preparing a test solution according to a preparation method of the test solution in the content determination method of the original tendon-relaxing rheumatic wine disclosed in the tendon-relaxing rheumatic wine standard, and determining the content of emodin according to the chromatographic conditions of the original method.
2. Results of the experiment
2.1 test result for sample batch 20130106: see Table 7
TABLE 7 comparison of emodin content measurement results
Table 7 the results show: the contents of comparative examples 1 and 2 were not higher than 85. mu.g/ml, and of the fifteen methods of example 1 of the present invention, 95. mu.g/ml was the lowest and 115. mu.g/ml was the highest.
2.2 test result for sample batch 20130108: see Table 8
TABLE 8 comparison of emodin content measurement results
Table 8 the results show: the results of comparative examples 1 and 2 were 83. mu.g/ml and 79. mu.g/ml, respectively, whereas the method of the present invention provided a minimum of 93. mu.g/ml and up to 112. mu.g/ml.
The above results show that: the test solution prepared by the preparation method of the test solution has higher emodin content than that of the comparative example 1 and that of the comparative example 2 in the content determination process.
Example 3: three-batch tendon-relaxing rheumatism wine for detecting polygonum cuspidatum standard
The sample solution used was the same as in the second method in item 3 of example 1.
The detection method adopts a content detection method under the original medicinal material items of polygonum cuspidatum in the version 2010 of Chinese pharmacopoeia, and the detection results are shown in table 9:
TABLE 9 comparison of the results of the content measurements of the three batches of products
By investigating the content of emodin in the three batches of products, the results in table 9 show that: the emodin content of the three batches of products adopting the detection method is far higher than that of the emodin content detected by adopting the giant knotweed raw medicinal material content detection method, which shows that the detection method is more effective.
Compared with a sample processing method, the sample processing method provided by the invention has the advantages of shorter reflux time and higher efficiency.
Example 4:
1. instruments and reagents: the same as in example 1.
2. Experimental samples: the same as in example 1.
Chromatographic conditions are as follows: agilent TC-C18 (4.6X 250mm, 5 μm) column; methanol-0.1% phosphoric acid solution (65:35) as mobile phase; the detection wavelength was 254 nm. The theoretical plate number is not less than 1500 calculated according to emodin peak.
3. Preparation of test solution
The test solution is prepared by the method for preparing the test solution under the test item of the giant knotweed raw medicinal material, and the parameters are the same as those in the example 1.
4. Preparation of control solutions
Taking appropriate amount of emodin reference substance, precisely weighing, adding methanol to obtain solution containing 50 μ g per 1ml, shaking, and filtering with microporous membrane (0.45 μm).
5. Chromatographic conditions and specificity tests
Octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid solution (82:18) as mobile phase; the detection wavelength was 254 nm. The number of theoretical plates is not less than 1500 calculated according to emodin peak.
6. Determination of sample content
Ten batches of the product were prepared according to the method for preparing the test solution in example 3, 10 μ l each of the test solutions was precisely aspirated, and the content of emodin in the sample was calculated according to the chromatographic conditions in example 5.
7. And (3) test results: see table 10.
TABLE 10 results of content determination of ten samples
By examining the contents of the above ten batches of product, the results in table 10 show: the ten batches of products adopt the detection method under the detection item of the giant knotweed raw medicinal material, and the average content is 82.4 mu g/ml which is far lower than the average content 105 in the embodiment 2 of the invention.
The results show that: the emodin is detected by using a detection method under the item of the giant knotweed raw medicinal material, and the emodin is greatly lost in the extraction process.
Example 5:
1. instruments and reagents: the same as in example 1.
2. Experimental samples: the same as in example 1.
Chromatographic conditions are as follows: agilent TC-C18 (4.6X 250mm, 5 μm) column; methanol-0.1% phosphoric acid solution (65:35) as mobile phase; the detection wavelength was 254 nm. The theoretical plate number is not less than 1500 calculated according to emodin peak.
3. Preparation of test solution
The preparation method of the test solution in the original detection method of the rheumatic wine for relaxing muscles and tendons is adopted to prepare the test solution, and the parameters are the same as those in the example 1.
4. Preparation of control solutions
Taking appropriate amount of emodin reference substance, precisely weighing, adding methanol to obtain solution containing 50 μ g per 1ml, shaking, and filtering with microporous membrane (0.45 μm).
5. Chromatographic conditions and specificity tests
Octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid solution (85:15) as mobile phase; the detection wavelength was 254 nm. The number of theoretical plates is not less than 1500 calculated according to emodin peak.
6. Determination of sample content
Taking ten batches of the product, respectively preparing according to the second method in the preparation method of the test solution under the item 3, respectively and precisely sucking 10 mu l of each test solution, measuring according to the chromatographic conditions of the items 5, and calculating the content of the emodin in the sample.
7. And (3) test results: see table 11.
TABLE 11 results of content measurement of ten samples
By examining the contents of the above ten batches, the results in table 11 show: the average content of ten batches of products measured by the method of example 5 was 75.1. mu.g/ml, which is less than the average 105.0 content of example 2.
The results show that: the emodin is detected by using a preparation method of a test sample of the original wine for relaxing muscles and tendons and treating rheumatism, and the emodin is greatly lost in the extraction process.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The quality detection method of the tendon-relaxing rheumatism wine is characterized by comprising the preparation of a test solution, wherein the test solution is prepared by extracting the tendon-relaxing rheumatism wine in a solvent, and the solvent comprises a sulfuric acid solution and trichloromethane.
2. The quality inspection method according to claim 1, wherein the sulfuric acid solution is 1 to 4mol/L sulfuric acid solution; the volume ratio of the tendon relaxing rheumatism wine to the sulfuric acid solution to the trichloromethane is 10 (15-30) to 15-30.
3. The quality inspection method according to claim 1 or 2, wherein the sulfuric acid solution is 1 to 2.5mol/L sulfuric acid solution; the volume ratio of the tendon relaxing rheumatism wine to the sulfuric acid solution to the trichloromethane is 10 (15-30) to 15-30.
4. The quality inspection method according to claim 3, wherein the sulfuric acid solution is a 2.5mol/L sulfuric acid solution; the volume ratio of the tendon relaxing rheumatism wine to the sulfuric acid solution to the trichloromethane is 10:30: 30.
5. The quality detection method according to any one of claims 1 to 4, wherein the extraction comprises heating in a water bath at 80 ℃ and refluxing for 15 to 90 minutes.
6. The quality inspection method according to claim 5, wherein the preparation of the test solution comprises the steps of: precisely measuring 10ml of muscle-relaxing rheumatism wine, sequentially adding 15-30ml of sulfuric acid solution and chloroform each of which is 1-4mol/L, heating and refluxing for 15-90 minutes in a water bath at 80 ℃, cooling, and separating chloroform solution for later use.
7. The quality inspection method according to claim 6, wherein the preparation of the test solution further comprises: after the chloroform solution is separated, the acid solution is shaken and extracted for 1-2 times by 15-30ml of chloroform, dehydrated by anhydrous sodium sulfate and then combined with the separated chloroform solution after heating and refluxing.
8. The quality inspection method according to any one of claims 1 to 7, wherein the preparation of the test solution comprises the steps of: precisely measuring 10ml of muscle-relaxing rheumatism wine, sequentially adding 15-30ml of each of 1-2.5mol/l sulfuric acid solution and chloroform, heating and refluxing in 80 ℃ water bath for 30-60 minutes, cooling, and separating chloroform solution; extracting the acid solution with 15-30ml chloroform under shaking for 1-2 times, dehydrating with anhydrous sodium sulfate, mixing with chloroform solution obtained after heating reflux, evaporating to dryness, dissolving residue with appropriate amount of methanol, diluting with methanol to 25ml, shaking, filtering, and collecting filtrate.
9. A quality detection method according to any one of claims 1 to 8, wherein the detection method comprises the steps of:
preparation of a test solution: precisely measuring 10ml of muscle-relaxing rheumatism wine, sequentially adding 15-30ml of each of 1-2.5mol/l sulfuric acid solution and trichloromethane, heating and refluxing for 30-60 minutes in a water bath at 80 ℃, cooling, taking trichloromethane liquid, shaking and extracting acid liquor for 1-2 times by using 15-30ml of trichloromethane, dehydrating by using anhydrous sodium sulfate, combining the trichloromethane liquid, evaporating to dryness, adding a proper amount of methanol into residues to dissolve, adding methanol to dilute to 25ml, shaking up, filtering, and taking a subsequent filtrate to obtain the muscle-relaxing rheumatism wine;
preparation of control solutions: accurately weighing appropriate amount of emodin reference substance, and adding methanol to obtain solution containing 50 μ g per 1 ml;
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid solution (82:18) as mobile phase; the detection wavelength is 254 nm;
the detection method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
10. A quality control method according to any one of claims 1-9, wherein emodin is not less than 90 μ g per 1ml of the wine for relieving rigidity of muscles and rheumatism.
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