CN102429966B - A kind of concocting method without gallbladder Radix Aconiti Lateralis Preparata - Google Patents

Abstract

The invention provides a kind of concocting method without gallbladder Radix Aconiti Lateralis Preparata or Radix Aconiti Lateralis Preparata, it comprises the following steps: a, get fresh mud Radix Aconiti Lateralis Preparata, cleans; Moderate heat keeps boiling to boil 10-20min; B, section, leaching drift 20-30min, 105 DEG C of-110 DEG C of temperature, normal pressure steams 2-10h; C, oven dry, to obtain final product.The present invention also provide this concocting method to prepare without gallbladder Radix Aconiti Lateralis Preparata or Radix Aconiti Lateralis Preparata, do not add in concocting process edible gallbladder bar.Present invention substantially reduces the content of calcium ion in processed product, magnesium ion, chloride ion and other metal ions, while ensure that clinical efficacy, reduce toxic and side effects, improve drug safety.

Description

A method for processing gallbladder-free tablets

technical field

The invention relates to Wudanheishun tablets or Baifu tablets and a processing method thereof, belonging to the field of medicines.

Background technique

Aconite is a processed product of the root of Aconitum carmichaelii Debx., a plant of the Ranunculaceae family. It has the effects of restoring yang and rescuing the adversity, replenishing fire and supporting yang, dispelling cold and relieving pain, and is used for yang collapse, cold limbs, weak pulse, impotence and cold uterus, It is an important Chinese medicinal material for cold pain in the abdomen, vomiting and diarrhea due to deficiency and cold, edema due to yin and cold, exogenous pathogenesis due to yang deficiency, and arthralgia due to cold and dampness. Due to the severe toxicity of aconite, doctors in ancient and modern times paid special attention to its processing methods in order to reduce its toxicity and improve its clinical efficacy. Aconite is a commonly used Chinese herbal medicine, and Heishun Pian or Baifu Pian is one of the most common specifications for its decoction pieces in clinical application.

The traditional processing method of Heishun slices is: take the aconite, wash it, immerse it in the aqueous solution of edible gallbladder for several days, boil it together with the immersion liquid until it is thoroughly cooked, remove it, float it in water, and cut it into slices about 0.5cm thick. Then soak in water and bleach, use toner to dye the attached piece into a strong brown color, take it out, steam until the oily surface and luster appear, bake until half dry, and then dry in the sun or continue to dry. After the aconite is bleached in water, the toxic components of diester-type alkaloids are hydrolyzed into less toxic mono-ester-type alkaloids through the heat treatment of cooking, which not only destroys the toxic components, but also does not affect its cardiotonic components. The attenuation effect ensures the clinical curative effect at the same time. [Lu Nan. Analysis of the processing method of aconite. "Journal of Practical Internal Medicine of Traditional Chinese Medicine". 2010, 24: 98-100]

The traditional processing method of Baifupian is: select aconites of uniform size, wash them, immerse them in the aqueous solution of edible gallbladder for several days, boil them together with the soaking liquid until they are thoroughly cooked, remove them, peel off the outer skin, and cut them longitudinally to a thickness of about 0.3 The slices of cm are soaked in water, taken out, steamed thoroughly, and dried in the sun. They are commonly called "Baifu slices".

However, modern studies have proved that the gallbladder used in the processing of Heishun Tablets or Baifu Tablets contains a large amount of calcium ions, magnesium ions, chloride ions and other metal ions. The contents of calcium, magnesium and chloride ions in the current traditional processing methods are about : 1.7%, 0.3%, 3.8%. Calcium and magnesium ions have a strong corrosive effect on the stomach, coagulating the proteins in human organs, and magnesium ions can inhibit the cardiovascular and nervous systems after being absorbed, which is highly toxic to humans. Although the aconite should be washed with running water for 5 to 6 times after being soaked in the gallbladder, it is impossible to completely remove the gallbladder, and there are more criminals who pour a large amount of gall into the aconite and increase the weight of the aconite. Therefore, Heishun tablets or Baifu tablets prepared by traditional processing methods have relatively large toxic and side effects, and there are certain hidden dangers in safety.

In recent years, although many scholars have carried out continuous exploration, using new technologies to improve the processing technology of aconite. For example, Liu Huiru et al. adopted the uniform experimental design method to optimize the best processing technology as fresh aconite plus 0.1% gallbladder, decocted for 3 hours, bleached for 2 days, steamed for 2 hours, and the processing time can be shortened by 8 days; Hu Sumei et al. used saturated salt Water replaces bile liquid to process aconite. The author believes that aconite must be soaked for a long time to reduce its toxicity; the preparation process of the above method is complicated and requires high equipment, and these processing techniques have not been verified by large-scale production and cannot meet large-scale production. need.

Contents of the invention

The technical solution of the present invention is to provide a method for processing gallbladder-free Hei Shun tablets or Baifu tablets, which does not add gallbladder in the process of processing. Another technical solution of the present invention is to provide the gallbladder-free black shun tablets or baifu tablets prepared by the processing method.

The present invention provides a kind of processing method of gallbladder-free Hei Shun tablet or Bai Fu tablet, it comprises the following steps:

a. Take fresh aconite, wash it; keep boiling on medium heat for 10-20 minutes;

b. Slices, dipping and bleaching for 20-30 minutes, bleaching until the surface of the slices has no stickiness, steaming at 105°C-110°C for 2-10h under normal pressure;

c, drying, that is to say.

Wherein, the steaming time described in step b is 8h.

Wherein, the drying method described in step c is drying at 40°C-70°C for 17h-73h.

Wherein, the drying method described in step c is drying at 55° C. for 24 hours.

Wherein, the processing method of the described Wudanbaitian is as follows: peel off the outer skin after boiling, and cut into longitudinal slices with a thickness of 0.3cm-0.5cm.

Wherein, the processing method of the non-dark black slices is as follows: do not peel after boiling, cut into longitudinal slices, and the thickness is 0.3cm-0.5cm.

The present invention also provides the gallbladder-free black shun tablets or baifu tablets prepared by the processing method.

It contains monoester alkaloids such as benzoyl neoaconine (C ₃₁ H ₄₃ NO ₁₀ ), benzoyl aconine (C ₃₂ H ₄₅ NO ₁₀ ) and benzoyl aconitine ( C ₃₁ H ₄₃ NO ₉ ), not less than 0.010%; containing diester-type alkaloids such as neoaconitine (C ₃₃ H ₄₃ NO ₁₀ ), hypoaconitine (C ₃₂ H ₄₅ NO ₁₀ ) Based on the total amount of aconitine (C ₃₃ H ₄₃ NO ₁₁ ), it should not exceed 0.020%. It contains not more than 0.5% w/w calcium ions, not more than 0.2% w/w magnesium ions and not more than 0.5% w/w chloride ions. It contains 0.01% to 0.5% w/w of calcium ions, 0.01% to 0.2% w/w of magnesium ions and 0.01% to 0.5% w/w of chloride ions.

The processing method of Heishun Tablet or Baifu Tablet of the present invention does not undergo soaking, and the alkaloid content of the aconite treated at normal temperature and pressure also complies with the provisions of the 2010 edition of "Chinese Pharmacopoeia". The gallbladder-free Heishun tablets or Baifu tablets prepared by the processing method provided by the present invention, because no edible gallbladder is added in the processing process, the processed products have greatly reduced calcium ions in them compared with traditional Heishun tablets and Baifu tablets. , magnesium ions, chloride ions and other metal ions, which reduces the toxic and side effects. At the same time, Wudanheishun Tablets and Baifu Tablets were tested according to the quality standards under the item of Aconite in the "Chinese Pharmacopoeia" 2010 Edition, and all the indicators can be tested. Meet the requirements of the Pharmacopoeia, indicating that the gallbladder-free Heishun Tablets and Baifu Tablets without edible gall can achieve the same effect as Heishun Tablets and Baifu Tablets in the processing process. In addition, the present invention also monitors and controls the toxic components of diester alkaloids, heavy metals and pesticide residues in Wudanheishun Tablets or Baifu Tablets, in order to comprehensively control the quality and quality of Wudanheishun Tablets or Baifu Tablets. Security is guaranteed.

Description of drawings

Fig. 1 Diagram of medicinal materials in Wudanfu Tablets (wherein, A: Wudanhei Shun Tablet; B: Wudanbai Tablet)

Figure 2 Micrograph of Wudanfu Tablet powder

Fig. 3 TLC of Wudan Fuzi (No. 1 is the Fuzi purchased in the traditional market, No. 2-11 are the self-made Wudan Fuzi and Baifu slices in Quxian Fuzi respectively, and Butuo Fuzi's self-made Wudan Black Shun and Baifu slices respectively. Fu Tablets, Jiangyou Aconitum Self-made No Gallbladder Black Shun Tablets, Baifu Tablets (2010), Jiangyou Aconite Homemade No Gallbladder Black Shun Tablets, Baifu Tablets (2011), Jiangyou Fuzi Homemade No Gallbladder Black Shun Tablets, Baifu Tablets (Middle test), No. 12-14 are respectively benzoyl neoaconitine, benzoyl aconitine, benzoyl hypoaconitine reference substance)

Figure 4 standard curve diagram of six alkaloids

Figure 5 reference substance spectrum (6 peaks are successively benzoyl neoaconitine, benzoyl aconitine, benzoyl hypoaconitine, neoaconitine, aconitine, hypoconitine reference substance peak )

Figure 6 Atlas of No Gallbladder Attachment Sheet

Detailed ways

Embodiment 1 The gallbladder-free process investigation of the present invention

Take about 10kg of fresh aconite, wash it, and boil it on medium heat (keep boiling) for about 15 minutes (until the heart is through).

Divide the boiled aconite evenly into two parts, cut one part into longitudinal slices (about 0.3-0.5cm thick), soak and bleach the cut medicinal materials (20-30min), and then evenly divide into 5 equal parts (for processing black shun slices) ); the other part was peeled and cut into longitudinal slices (about 0.3-0.5cm thick), soaked and bleached the cut medicinal materials (20-30min), and then evenly divided into 5 equal parts (for processing Baifu tablets).

1.1 Inspection of steaming time and drying method

Five equal parts of Heishun slices and Baifu slices were steamed for about 2h, 4h, 6h, 8h, and 10h (normal pressure) respectively. The steamed medicinal materials are evenly divided into four equal parts, and dried by four methods of drying, microwave drying, far-infrared drying, and reduced-pressure drying. Count the drying time, and number the dried medicinal materials as follows:

Table 1 Comparison of four different drying methods: (except for microwave drying, the drying temperature is 55°C)

In summary, the appearance of samples obtained by drying by four methods, microwave and far-infrared drying is not good, and does not meet the pharmacopoeia standards. The appearance of samples obtained by drying and vacuum drying basically meets the pharmacopoeia standards, but vacuum drying is time-consuming and It is a waste of energy, so the final selection of drying is the drying method of the non-biliary black smooth sheet and the non-biliary ratio attached sheet.

The contents of alkaloids in the slices obtained by different steaming times are different, as shown in Table 2:

Table 2 Determination results of alkaloid content in Wudanheishun tablets and Baifu tablets at different steaming times

According to the provisions of the first part of the "Chinese Pharmacopoeia" 2010 edition, Heishun tablets and Baifu tablets contain diester-type alkaloids such as neoaconitine (C ₃₃ H ₄₃ NO ₁₀ ), hypoaconitine (C ₃₂ H ₄₅ NO ₁₀ ) and aconitine (C ₃₃ H ₄₃ NO ₁₁ ), the total amount shall not exceed 0.020%. , containing benzoyl neoaconitine (C ₃₁ H ₄₃ NO ₁₀ ), benzoyl aconitine (C ₃₂ H ₄₅ NO ₁₀ ) and benzoyl hypoaconitine (C ₃₁ H ₄₃ NO ₉ ) shall not be less than 0.010%. It can be seen that the samples dried after steaming for 8 hours are in compliance with the provisions of the Pharmacopoeia.

1.2 Examination of drying temperature

Select 4kg of fresh aconite, wash it, boil it on medium heat for about 15 minutes (keep boiling), then divide it into two equal parts, cut one part into longitudinal slices (about 0.3-0.5cm thick), soak and bleach the cut herbs (20 -30min), steam for about 8 hours, and then divide the steamed medicinal materials into four equal parts; peel the other part, slice (about 0.3-0.5cm thick), soak and bleach the cut medicinal materials (20-30min), steam After about 8 hours, it is evenly divided into four equal parts; it is dried at different temperatures respectively. The test results are shown in the table below:

Table 3 Comparison table of appearance and alkaloid content of Wudanfu Tablets at different drying temperatures

Generally, the higher the content of monoester alkaloids and the lower the content of diester alkaloids in the prepared sputum, the better the quality of sputum. From the experimental data in Table 3, it can be known that the drying temperature is 40-70°C, the content of the prepared monoester alkaloid is high, and the content of the diester type alkaloid is low; wherein, when the optimum temperature for drying is 55°C, The quality is better, and the drying time is significantly shorter than that at 40°C, and the drying time is 24 hours.

To sum up, the processing technology of Wudanhei Shun slices is as follows: take fresh aconite, wash it, boil it on medium heat for about 15 minutes (keep boiling), cut into longitudinal slices (0.3-0.5cm), soak and bleach the cut medicinal materials ( 20-30min), steam for about 8h (normal pressure), take out, and dry (55°C, 24h).

The processing technology of Wudanbaitian is as follows: take fresh aconite, wash, cook on medium heat for about 15 minutes (keep boiling), peel, cut into longitudinal slices (0.3-0.5cm), soak and bleach the cut medicinal materials (20- 30min), steam for about 8h (normal pressure), take it out, and dry (55°C, 24h).

Quality Standards Research

1.1 Character identification

No gallbladder black smooth product: sliced longitudinally, wide at the top and narrow at the bottom, 1.7-5cm long, 0.9-3cm wide, and 0.2-0.5cm thick. The outer skin is dark brown, the cut surface is dark yellow, oily and shiny, translucent, and has longitudinal duct bundles. Hard and brittle, horny-like section. Slight gas, light taste.

Wubiliba Fupian: without skin, yellowish white, translucent, hard and brittle.

The properties of Heishun Tablets and Baifu Tablets processed by the traditional method are consistent (see attached picture 1: medicinal material map of Wudanfu Tablets)

2.2 Microscopic identification

The powder of Wudanhei Shun Tablet and Wudanbai Fu Tablet is grayish-yellow and white, and the powder has the following structure:

① There are many starch granules, single granules are spherical or round polygonal, a few are oblong, diameter 2-20μm, umbilical points are dot-shaped, cross-shaped, herringbone-shaped; compound granules are composed of 2-7 or more granules become.

②Fragments of epigenetic cortex are rare. They are polygonal in surface view. The anticlinal wall is unevenly thickened, and some protrude into the cell lumen in the form of tumors, and the cell lumen contains brown substances.

③ Stone cells are rare and scattered, with a diameter of about 53-125 μm and obvious pits.

④ Bordered pits and reticulate vessels with a diameter of 20-48 μm. The prepared tablets are mainly parenchyma fragments containing gelatinized starch granules. The microscopic characteristics of the appendages processed by the traditional method are consistent. (Attachment 2)

2.3 TLC identification

Traditionally process 1 batch of attached tablets, 5 batches of Wudanhei Shunpian, and 5 batches of Wudanbaifianpian were crushed separately, and passed through a No. 4 sieve:

Take 5g of this product powder, wet it with 5ml of ammonia test solution, add 50ml of ether, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 0.5ml of dichloromethane to dissolve the residue, and use it as the test solution. Take another benzoyl neoaconitine reference substance, benzoyl aconitine reference substance, benzoyl hypoaconitine reference substance, add isopropanol-dichloromethane (1:1) mixed solution to prepare A mixed solution containing 1 mg per 1 ml was used as the reference solution (monoester alkaloid). Then take neoaconitine reference substance, hypoconitine reference crystal, and aconitine reference substance, add isopropanol-dichloromethane (1:1) mixed solution to make a mixed solution containing 1 mg per 1 ml, as a control product solution (diester alkaloid). According to the thin-layer chromatography (Appendix VIB) test, absorb 5-10 μl of the test solution and the reference solution, respectively spot on the same silica gel G thin-layer plate, and mix with n-hexane-ethyl acetate-methanol (6.4:3.6: 1) As a developing agent, put it in a developing cylinder saturated with ammonia vapor for 20 minutes, develop it, take it out, dry it in the air, and spray it with dilute potassium bismuth iodide test solution. In the chromatogram of the test product, Heishun Tablet or Baifu Tablet is at the corresponding position in the chromatogram of the benzoyl neoaconitine reference substance, benzoyl aconitine reference substance, and benzoyl aconitine reference substance On, spots of the same color appear. It is consistent with the traditional method of processing TLC to identify spots. (Attachment 3)

2.4 Determination of the content of alkali metal and alkaline earth metal

Test samples: 3 batches of Jiangyou aconite processed without gallbladder tablets, 1 batch of Jiangyou aconite processed with gallbladder tablets, 2 batches of gallbladder soaked (7 days, 30 days) tablets, 1 batch of Hebei Anguo purchased tablets, a total of 7 batches of medicinal materials Carry out calcium, magnesium, chloride ion content determination and comparison.

2.4.1 Determination of Calcium and Magnesium Ions

The Chengdu Branch of the Chinese Academy of Sciences was entrusted with the determination of calcium and magnesium ions.

Detection method: inductively coupled plasma emission spectrometry (ICP-AES).

Test results: see Table 4.

2.4.2 Determination of chloride ion content

2.4.2.1 Preparation of the test sample Take about 5g of the samples of Heishun Tablets and Baifu Tablets (passed through No. 4 sieve), weigh them precisely, and bake them at 80°C to constant weight. Take another appropriate amount of edible gallbladder, and bake it to constant weight in the same way. Add 50ml of water, sonicate (250W, 50KHz) for 30min, let cool, make up the weight, shake well, and filter. Another appropriate amount of gallbladder is taken, baked to constant weight, and processed in the same way.

2.4.2.2 Experimental method

Accurately pipette 5ml of the test solution and 1ml of the bile solution into a 250ml Erlenmeyer flask, add 6ml of distilled water and 1ml of potassium chromate indicator, and titrate with 0.1mol/L silver nitrate standard solution until the brick red precipitate does not disappear Record the volume of silver nitrate solution consumed. The measurement results are shown in Table 4.

Table 47 Batches of Fupian Gallbladder Content Determination Results

The measurement results show that the contents of calcium, magnesium and chlorine in F ₁ ~ F ₃ (Wu Dan Hei Tablets) are several times or even ten times lower than those in Wu Dan Hei Shun Tablets soaked in gallbladder. times, greatly ensuring the safety of clinical medication. Tentative Wudanfu tablets contain calcium ions not higher than 0.5% w/w, magnesium ions not higher than 0.2% w/w, and chloride ions not higher than 0.5% w/w.

2.5 Heavy metals and harmful elements

Test samples: 3 batches of Jiangyou aconite processed Wudanfu tablets, 1 batch of Butuo Fuzi processed Wudanfu tablets, and 1 batch of Quxian processed Wudanfu tablets were randomly selected, totaling 5 batches.

Measure according to lead, cadmium, arsenic, mercury, copper assay (appendix IX B atomic absorption spectrophotometry or inductively coupled plasma mass spectrometry), and the assay results are shown in Table 5.

2.5.1 Instruments and reagents

American Agilent ICP-MS7500 inductively coupled plasma mass spectrometer, Shimadzu 1/100,000 electronic sky

Flat, programmable temperature-controlled microwave digestion instrument (MARS5 type, American CEM company), Millipore Milli-Q ultrapure water system. Nitric acid (imported from abroad).

2.5.2 Preparation of standard solution

Lead single element standard solution: MERCK, Lot: HC813220 standard value: 1000±2mg/L

Arsenic single element standard solution: MERCK, Lot: HC888556 standard value: 998±5mg/L

Mercury single element standard solution: MERCK, Lot: HC810358 standard value: 1000±2mg/L

Cadmium single element standard solution: MERCK, Lot: HC813220 standard value: 1000±2mg/L

Copper single element standard solution: MERCK, Lot: HC781459 standard value: 1001±2mg/L

Accurately measure 200 μl each of the above-mentioned lead, arsenic, mercury, and cadmium single-element standard solutions, put them in a 100mL volumetric flask, use 5% nitric acid to make the volume up to the mark, shake well, and use it as a reserve standard solution (2000 μg/L of arsenic, lead, and cadmium); Precisely measure 1 mL of the above-mentioned copper single-element standard solution and put it in a 100 mL volumetric flask, dilute to the mark with 5% nitric acid, shake well, and use it as a reserve standard solution (copper 10000 μg/L); measure an appropriate amount of the above-mentioned reserve standard solution respectively, and use 5 % nitric acid was diluted into various series concentrations, and prepared into mixed standard series solutions.

2.5.3 Instrument conditions

The RF power is 1350W, the sampling depth is 143step, the carrier gas flow rate is 0.91L/min, the auxiliary gas flow rate is 0.8L/min, the cooling gas flow rate is 13L/min, the rotation speed of the peristaltic pump is 30r/min when collecting samples, the integration time is 40 seconds, repeat 3 times .

2.5.4 Preparation of the test solution

Take about 0.5g of the powder attached to the tablet, weigh it accurately, put it in a polytetrafluoroethylene microwave digestion tank, add 7mL of nitric acid, let it stand for about 2 hours, set the microwave digestion program (power 1600W, heat up to 120°C at 20°C per minute , hold for 5 minutes, then raise the temperature at 8°C per minute to 160°C, hold for 10 minutes, then raise the temperature at 5°C per minute to 185°C, and hold for 25 minutes). Smoke, transfer the digestion solution to a 25mL volumetric flask with deionized water, and dilute to the mark with deionized water, shake well, and use it as the test solution. At the same time, prepare the blank solution of all reagent samples in the same way.

2.5.5 Sample determination

75As, 65Cu, 111Cd, 82Se use 115In (concentration about 10ppb) as the internal standard, 202Hg, 208Pb, use Bi209 (concentration about 10ppb) as the internal standard, and the above-mentioned standard series solutions are injected and analyzed by the peristaltic pump under the above instrument conditions. Take the element concentration (ppb) as the abscissa and the corresponding instrument response (ICPS) as the ordinate to draw a standard curve; the test solution is injected and analyzed under the same conditions, and the reagent blank of the sample is automatically deducted from the standard curve. The concentration (ppb) of each element in the test solution, and calculate the content of each element in the sample. The formula is as follows:

The content of each element in the sample (mg/kg)=the concentration of the test solution (mg/kg)*25/sample amount/1000, the measurement results are shown in Table 5.

2.6 Organochlorine pesticide residues

2.6.1 Chromatographic conditions

Chromatograph: Agilent 6890N gas chromatograph, 63Ni-ECD electron capture detector; column: DB-130m×0.32mm×1μm; column temperature 70°C (1min), 180°C, 205°C (10min), 280°C (10min ) INJ: 250°C; DET: 310°C; nitrogen flow rate: 1.7mL/min; makeup: 50mL/min.

2.6.2 Preparation of reference solution

666 (total BHC) were accurately measured 1mL α-BHC reference substance (GBW(E)060081 in the National Standard Material Research Center), 1mL β-BHC reference substance (GBW(E)060082 in the National Standard Material Research Center), 1mL γ-BHC reference substance (GBW(E)060083 of National Standard Material Research Center), 1mL of δ-BHC reference substance (GBW(E)060084 of National Standard Material Research Center) was placed in a 25mL measuring bottle, and petroleum ether (60～90℃ ) dissolved and diluted to the mark, shake well, that is.

DDT (total DDT) were measured precisely:

1mLPP'-DDE reference substance (GBW(E)060104, National Center for Standard Materials Research),

1mLPP'-DDD reference substance (GBW(E)060105, National Standard Material Research Center),

1mLOP'-DDT reference substance (GBW(E)060103, National Standard Materials Research Center),

Put 1mL of PP'-DDT reference substance (GBW(E)060102 of the National Standard Materials Research Center) in a 25mL measuring bottle, dissolve it with petroleum ether (60-90°C) and dilute to the mark, shake well to get ready.

Pentachloronitrobenzene (PCNB) Accurately measure 1mL of pentachloronitrobenzene reference substance (50μg/mL from the National Standard Materials Research Center), put it in a 25mL measuring bottle, dissolve it with petroleum ether (60-90°C) and dilute to the mark, Shake well and serve.

2.6.3 Preparation of mixed reference solution

Accurately measure the above standard stock solutions 5mL each of α-BHC, γ-BHC, δ-BHC, 10mL of β-BHC, 5mL of pentachloronitrobenzene (PCNB), 5mL of each of PP'-DDE, PP'-DDD, OP Put 10mL each of '-DDT and PP'-DDT in a 100mL measuring bottle, dilute to the mark with petroleum ether (60-90°C), shake well to get (α-BHC, γ-BHC, δ-BHC concentration 100ng/ mL, β-BHC concentration 200ng/mL, pentachloronitrobenzene (PCNB) concentration 100ng/mL, PP'-DDE, PP'-DDD concentration 100ng/mL, OP'-DDT, PP'-DDT concentration 200ng/mL ).

2.6.4 Preparation of the test solution

Take Wudanheishun Tablets or Baifu Tablets and dry them at 60°C for 4 hours, crush them into fine powder, take about 2g, weigh them accurately, put them in a 100mL conical flask with a stopper, add 20mL of water to soak overnight, add 40mL of acetone precisely, and weigh Determine the weight, sonicate for 30 minutes, let cool, then weigh again, make up the lost weight with acetone, add about 6g of sodium chloride, add 30mL of dichloromethane accurately, weigh, and sonicate for 15min, then weigh again Weight, use dichloromethane to make up the lost weight, let stand to separate layers, quickly move the organic phase into a 100mL conical flask with a stopper filled with an appropriate amount of anhydrous sodium sulfate, and let it stand for 4h. Precisely measure 35 mL, concentrate in a water bath at 40°C to near dryness under reduced pressure, add a small amount of petroleum ether (60-90°C) and repeat the operation as before until the dichloromethane and acetone are removed, dissolve and transfer with petroleum ether (60-90°C) To a 10mL graduated centrifuge tube with stopper, add petroleum ether (60-90°C) to 5mL. Carefully add 1mL of sulfuric acid, shake for 1min, centrifuge for 10min, and accurately measure 2mL of supernatant to obtain.

2.6.5 Measurement methods and results

Accurately draw 1 μl each of the test solution and the corresponding concentration of the mixed reference solution, inject 3 consecutive samples respectively, take the average value of the 3 times, and calculate the organochlorine pesticide residues in the test product according to the external standard method. Results: Organochlorine pesticides (HCH, DDT, Pentachloronitrobenzene) residues were not detected in 5 batches of attached tablets.

Table 5 Determination of Heavy Metals and Pesticide Residues in Wudanfu Tablets of Fuzi Processing in Main Production Areas

It can be seen from Table 5 that the determination results of heavy metals and harmful elements are in line with the provisions of the first part of the "Chinese Pharmacopoeia" 2010 edition: lead shall not exceed 5 parts per million; cadmium shall not exceed 3 parts per million; arsenic shall not exceed 0 parts per million Second, mercury must not exceed 2 parts per million, and copper must not exceed 20 parts per million.

The amount of organochlorine pesticide residues determined according to the amount of pesticide residues is in line with the provisions of the first part of the "Chinese Pharmacopoeia" 2010 edition: 666 (total BHC) shall not exceed 2 parts per million; DDT (total DDT) shall not exceed 2 parts per million ; Pentachloronitrobenzene (PCNB) shall not exceed 1/10000000.

2.7 Determination of alkaloid content

Test samples: 3 batches each of Jiangyou aconite processed without gallbladder black slices and Baifu slices, Butuo aconite processed without gallbladder black slices and Baifu slices each, 1 batch of Quxian aconite processed without gallbladder black slices and Baifu slices each batches, a total of 10 batches.

2.7.1 Instruments and reagents

Agilent 1200 HPLC system: including 1200 pump, DAD detector, Millipore Milli-Q ultrapure water system, Shimadzu 1/100,000 electronic balance, Brasson ultrasonic cleaner, KQ-100 ultrasonic cleaner, aconitine (Lot No.: 110797-200404), Neoaconitine (Lot No.: 110799-200404), Hypoaconitine (Lot No.: 110798-200404) Reference Substance (provided by China Institute for the Control of Pharmaceutical and Biological Products); Benzoyl Neoaconitine , Benzoyl aconitine, Benzoyl aconitine (provided by Xinhehua Pieces Factory, no batch number yet). Methanol and acetonitrile used in HPLC are all imported chromatographic reagents, and the rest are domestic analytical reagents. The water used in HPLC is ultrapure water, and the rest of the water is purified water.

2.7.2 Chromatographic conditions and system suitability test

Octadecylsilane bonded silica gel is used as filler, column temperature is 20°C; mobile phase is two-phase gradient elution of A and B, A: acetonitrile, B: 40mmol/L ammonium acetate buffer (adjust pH=10.0 with ammonia water) , carry out gradient elution according to the table below, the flow rate is 1ml/min; the detection wavelength is 230nm. The number of theoretical plates should not be less than 3000 based on the peak of neoaconitine.

2.7.3 Preparation of the test solution: Accurately weigh about 2 g of the fine powder of the gallbladder powder passed through a 60-mesh sieve, weigh 3 parts in parallel, place them in 200 mL Erlenmeyer flasks, add 2 mL of 25% ammonia water each, and wet for 30 minutes. Accurately measure 100 mL of the extraction solvent isopropanol-ethyl acetate (1:1) mixture, add it to the Erlenmeyer flask containing the sample, and weigh it. After ultrasonication for 30 minutes, place the sample bottle at room temperature, weigh again, make up for the lost weight with extraction solvent, mix well and filter. Precisely measure 40 mL of the subsequent filtrate, concentrate to a small volume, and then completely transfer it to an evaporating dish with the extraction solvent and evaporate to dryness. The residue was dissolved with 0.01% hydrochloric acid-methanol and settled in a 2mL volumetric flask, shaken up, and filtered through a 0.45μm microporous membrane to obtain the test solution.

2.7.4 Preparation of reference substance solution: Accurately weigh 2.6, 1.3, and 5.19 mg of neoaconitine, hypoaconitine, and aconitine reference substance, put them in the same 10mL measuring bottle, dissolve with 0.01% methanolic hydrochloric acid solution and dilute to scale, and shake well to obtain a diester alkaloid reference solution. Precisely weigh 4.0256 mg, 2.56 mg, and 1.8 mg of benzoyl aconine, benzoyl hypoconine, and benzoyl neoaconine, respectively, and place them in a 1 mL measuring bottle, dissolve them with 0.01% methanolic hydrochloric acid solution and Dilute to the mark and shake well to obtain the reference solution of 3 monoester alkaloids.

2.7.5 Preparation of standard curve: Precisely draw appropriate amount of monoester-type and diester-type reference substance mixture into the same 10ml volumetric flask, and add 0.01% hydrochloric acid methanol to dilute to the mark. Then draw an appropriate amount of intermediate solution to make 5 mixed reference substance solutions with different concentrations, analyze according to the above-mentioned chromatographic conditions, and perform regression calculation on the injection volume (X) with the peak area (Y) (based on the aconitine content and peak area as a standard curve), the regression equation is shown in Figure 4

2.7.6 Sample recovery test: Accurately weigh 1g of Aconitum officinalis fine powder, a total of 30 parts, and each 5 parts are used to determine the recovery rate of one alkaloid, 6 groups in total. Add the same amount of reference substance solution to each group of samples respectively, and carry out the preparation and HPLC analysis of the test solution according to the method under the preparation of the test solution. Calculate the recovery rate according to the measured amount and the added amount. The average sample recovery (RSD) of aconitine, hypoaconitine, neoaconitine, benzoyl aconitine, benzoyl hypoaconitine, and benzoyl neoaconitine were respectively 98.70%, 100.47%, 98.20%, 100.35%, 100.27%, and 98.47%.

2.7.7 Sample determination: Accurately weigh about 2g of each sample, weigh 3 copies of each sample in parallel, prepare according to the method under the preparation of the test solution, and analyze the three monoester alkaloids according to the above chromatographic conditions. The accompanying test solution was calculated on the same day, and the results are shown in Table 6. The HPLC spectra of the reference substance and Wudanfu Tablets are shown in Figure 5 and Figure 6 .

Table 6 Determination of self-made Wudanfu Tablets

According to the provisions of the first part of the "Chinese Pharmacopoeia" 2010 edition, the monoester alkaloids of Heishun Tablets and Baifu Tablets are benzoyl neoaconitine (C ₃₁ H ₄₃ NO ₁₀ ), benzoyl aconitine ( C ₃₂ H ₄₅ NO ₁₀ ) and benzoyl _aconitine (C ₃₁ H ₄₃ NO ₉ ), not less than 0.010%; ₄₃ NO ₁₀ ), hypoaconitine (C ₃₂ H ₄₅ NO ₁₀ ) and aconitine (C ₃₃ H ₄₃ NO ₁₁ ), the total amount shall not exceed 0.020%.

It can be seen that the alkaloid content of all batches of Wudanheishun Tablets and Wudanbaifu Tablets is in compliance with the provisions of Part I of the 2010 edition of the Chinese Pharmacopoeia.

In summary, the gallbladder-free black shun tablets or Baifu tablets prepared by the processing method provided by the present invention greatly reduce the concentration of calcium ions, magnesium ions, chloride ions and other metals in the process of processing because no edible gallbladder is added. The concentration of ions reduces the toxic and side effects. At the same time, Wudanheishun Tablets and Baifu Tablets were tested according to the quality standards under the item of Fuzi in the "Chinese Pharmacopoeia" 2010 Edition. The gallbladder-free Heishun Tablets and Baifu Tablets with edible gallbladder can achieve the same effect as Heishun Tablets and Baifu Tablets. In addition, the present invention also monitors and controls the toxic components of diester alkaloids, heavy metals and pesticide residues in Wudanhei Shun Tablets or Baifu Tablets, in order to comprehensively control the poisoning of Wudanhei Shun Tablets or Baifu Tablets. Quality and safety are guaranteed.

Claims (3)

1. A method for processing gallbladder-free tablets, characterized in that: it is processed according to the following steps: a. Take fresh aconite, wash it; keep boiling on medium heat for 10-20 minutes; b. Sliced, soaked and bleached for 20-30 minutes, steamed at 105°C-110°C for 8 hours under normal pressure; c. Drying to obtain the product; the drying method is drying at 55° C. for 24 hours. 2. The processing method according to claim 1, characterized in that: in step a of the processing method, peel off the outer skin after boiling, and cut into longitudinal slices with a thickness of 0.3cm-0.5cm. 3. The processing method according to claim 1, characterized in that: in step a of the processing method, the skin is not peeled after cooking, and the slices are cut into longitudinal slices with a thickness of 0.3cm-0.5cm.