阿胶

English text reference: Chinese Pharmacopoeia (2020 Edition)

Overview

Donkey-hide Gelatin is the solid gelatin prepared by decocting and concentrating the dried skin or fresh skin of the donkey, an animal of the Equidae family.

Preparation

The donkey skin is soaked to remove the hair, cut into pieces and washed. It is decocted in water in several portions, filtered, and the filtrate is combined. The combined filtrate is concentrated (with the addition of appropriate amounts of yellow rice wine, rock sugar, and soybean oil) to obtain a thick paste. The paste is cooled, cut into pieces, and dried to obtain the final product.

Description

Donkey-hide Gelatin is in the form of rectangular blocks, square blocks, or pieces. It is brown to dark brown in color and has a glossy appearance. The texture is hard and brittle, and the fractured surface is shiny. When the fragments are held up to the light, they appear brown and translucent. It has a slight odor and a slightly sweet taste.

Identification

Take 0.1g of the powder, add 50ml of 1% ammonium bicarbonate solution, and sonicate for 30 minutes. Filter the solution through a microporous membrane, take 100μl of the filtrate, and place it in a microsampling bottle. Add 10μl of trypsin solution (prepare a solution containing 1mg of trypsin per ml of 1% ammonium bicarbonate solution for sequence analysis, and prepare it before use). Shake well and incubate at 37°C for 12 hours to obtain the test solution. Take 0.1g of the reference drug Ejiao, and prepare a solution using the same method as the test solution. Perform the test according to the chromatographic and mass spectrometric conditions under the characteristic peptide item in the determination of content. Select the mass-to-charge ratios (m/z) 539.8 (double charge)→612.4 and m/z 539.8 (double charge)→923.8 as the detection ion pairs. Take 5μl of the Ejiao reference drug solution and inject it into the high-performance liquid chromatography-mass spectrometry instrument for analysis. In the ion flo w chromatogram extracted with the ion pairs of m/z 539.8 (double charge)→612.4 and m/z 539.8 (double charge)→923.8, a chromatographic peak with the same retention time as the reference drug should be present.

Examination

Water

Take 1g of the sample, accurately weigh, add 2ml of water, dissolve by heating, evaporate on a water bath until the thickness does not exceed 2mm, and determine according to the method for water determination <0832, method 2>, not more than 15.0 per cent.

Heavy metals and harmful elements

Determine according to the method for determination of lead, cadmium, arsenic, mercury, and copper <2321, atomic absorption spectrophotometry or inductively coupled plasma mass spectrometry>. The lead content should not exceed 5mg/kg; the cadmium content should not exceed 0.3mg/kg; the arsenic content should not exceed 2mg/kg; the mercury content should not exceed 0.2mg/kg; and the copper content should not exceed 20mg/kg.

Insoluble matter

Take 1.0g of the sample, accurately weigh, add 5ml of water, heat to dissolve, transfer to a centrifuge tube with a constant weight of 10ml, wash with 5ml of warm water in 3 portions, combine the washings into the centrifuge tube, shake well. Keep warm in a 40°C water bath for 15 minutes, centrifuge (at a speed of 2000 rpm) for 10 minutes, remove the floating oil on the tube wall, pour off the supernatant along the tube wall, add warm water to the mark along the tube wall, centrifuge, repeat the washing process 3 times, pour off the supernatant, heat the centrifuge tube at 105°C for 2 hours, take out, cool in a desiccator for 30 minutes, accurately weigh, calculate, and obtain the result.

The water insoluble substance of this product shall not exceed 2.0%.

IOther

The relevant provisions under Adhesives <0184> shall be complied with.

Assay

Amino acids

Carry out the method for high performance liquid chromatography <0512>.

Chromatographic conditions and system suitability test

Octadecylsilane-bonded silica gel was used as filler; acetonitrile-0.1 mol/L sodium acetate solution (pH adjusted to 6.5 with acetic acid) (7:93) was used as the mobile phase A, and acetonitrile-water (4:1) was used as the mobile phase B. The gradient elution was performed according to the following table; the detection wavelength was 254 nm; and the column temperature was 43 ℃. The theoretical plate count should be not less than 4000 based on the L-hydroxyproline peak.

Preparation of reference solution

Take the appropriate amount of L-hydroxyproline control, glycine control, alanine control, L-proline control, weigh it precisely, add 0.1 mol/L hydrochloric acid solution to make a mixed solution containing 80 μg of L-hydroxyproline, 0.16 mg of glycine, 70 μg of alanine, 0.12 mg of L-proline per 1 ml, that is to say.

Preparation of test solution

Take about 0.25g of crude powder of this product, weigh it precisely, put it in 25ml measuring flask, add 0.1mol/L hydrochloric acid solution 20ml, ultrasonic treatment (power 500W, frequency 40kHz) for 30 minutes, let it cool down, add 0.1mol/L hydrochloric acid solution to the scale, shake well. Precision measure 2ml, put in 5ml ampoule, add 2ml hydrochloric acid, 150 ℃ hydrolysis for 1 hour, cooled, transferred to the evaporating dish, washed with 10ml of water in batches, the washings into the evaporating dish, evaporation, residue add 0.1mol/L hydrochloric acid solution dissolved, transferred to a 25ml measuring flask, add 0.1mol/L hydrochloric acid solution to the scale, shaking, that is, obtained.

Precisely measure 5ml of each of the above control solution and test solution, respectively, placed in a 25ml measuring flask, add 2.5ml of acetonitrile solution of 0.1mol/L phenyl isothiocyanate (PITC), 2.5ml of acetonitrile solution of lmol/L triethylamine, shaking well, leave for 1 hr at room temperature, add 50% acetonitrile to the scale, shaking well. Take 10ml, add n-hexane 10ml, shaking, placed for 10 minutes, take the lower layer of the solution, filtered, take the renewed filtrate, that is obtained.

Assay method

Precisely aspirate 5μl each of the derivatised control solution and test solution respectively, inject into the liquid chromatograph and determine, it is obtained.

This product contains not less than 8.0% of L-hydroxyproline, not less than 18.0% of glycine, not less than 7.0% of alanine, and not less than 10.0% of L-proline when calculated on a dry basis.

Signature peptide

Determined by high performance liquid chromatography-mass spectrometry < 0512,0431>.

Chromatography, mass spectrometry conditions and system suitability tests

Octadecylsilane-bonded silica gel was used as the filler (column inner diameter 2.1 mm); acetonitrile was used as the mobile phase A, and 0.1% formic acid solution as the mobile phase B. Gradient elution was carried out according to the following table at a flow rate of 0.3 ml per minute.

A triple quadrupole mass spectrometry detector was used with electrospray ionisation (ESI) multiple reaction monitoring (MRM) in positive ion mode, and the monitored ion pairs are shown in the table below:

Theoretical plate counts should be no less than 4000 based on donkey-derived peptide A1 peaks.

Preparation of reference solution

Take the appropriate amount of donkey-derived polypeptide A1 control, donkey-derived polypeptide A2 control, precision weighing, add 1% ammonium bicarbonate solution were made into a mixture of 2.5 μg per 1 ml, i.e., obtained.

Preparation of test solution

Take 0.1g of this product powder, precision weighing, placed in a 50ml measuring flask, add 1% ammonium bicarbonate solution 40ml, ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, add 1% ammonium bicarbonate solution to dilute to the scale, shake well. Precision measure lml to 5ml measuring flask, add trypsin solution (take serial analytical grade trypsin, add 1% ammonium bicarbonate solution to make a solution containing 1mg per 1ml, fresh before use) lml, add 1% ammonium bicarbonate solution diluted to the scale, shaking, 37 ℃ constant temperature enzyme solution for 12 hours, filtration, and take the renewed filtrate, that is, obtained.

Assay method

Precisely measure 1ml, 2ml, 5ml, 10ml, 20ml and 25ml of the control solution, respectively, in a 50ml measuring flask, add 1% ammonium bicarbonate solution to dilute to the scale, make a standard curve solution. The standard curve solution and test solution of different concentrations were sucked up 5μl each, injected into the high performance liquid chromatography-mass spectrometer, and the standard curve was prepared with the peak area of the control product as the vertical coordinate and the concentration of the control product as the horizontal coordinate. The amount of donkey-derived peptide A1 and donkey-derived peptide A2 in the test solution was read out from the standard curve and calculated.

This product shall contain not less than 0.15% of the total amount of donkey-derived polypeptide A1 (C41H68N12O13) and donkey-derived polypeptide A2 (C51H82N18O18), calculated as a dry product.

Prepared slices

Donkey-hide gelatin

Processing

Crush into small pieces.

Appearance

The product is irregular in shape and varies in size. The rest is the same as the medicinal material.

Examination

Water

Same as the medicinal material.

Insoluble matter

Same as the medicinal material.

Donkey-hide gelatin pill

Processing

Take donkey-hide gelatin, heat and soften it, cut it into pieces about 1cm in size, use the frying method <0213> to cook with clam powder until it forms a pill, remove it when there is no uncooked part inside, sieve off the clam powder, and let it cool.

Appearance

The product is spherical. The surface is brownish-yellow or grayish-white with white powder attached. It is light in weight, crisp in texture, and fragile. The cross-section is hollow or porous, pale yellow to brown. It has a slight odor and a slightly sweet taste.

Examination

Water

Same as the medicinal material, not more than 10.0 per cent.

Total ash

Not more than 4.0 per cent <2302>.

Identification.

Same as the medicinal material.

Content determination

Amino acids

Same as the medicinal material.

Property

Neutral.

Flavor

Sweet.

Meridian tropism

归肺、肝、肾经。

Actions

Nourishes blood and nourishes yin, moisturizes dryness, and stops bleeding.

Indications

Used for blood deficiency and withered yellow, dizziness and palpitations, muscular atrophy and weakness, restlessness and insomnia, deficiency wind stirring internally, lung dryness cough, cough with blood due to exertion, vomiting blood and hematuria, bleeding and collapse, and threatened abortion during pregnancy.

Dosage

3-9g.

Administration

Closed and blended.

Storage

Store in a sealed container.

阿胶

Text reference: Chinese Pharmacopoeia (2015 Edition)

Overview

Donkey-hide Glue is the solid glue prepared from the dried or fresh skin of Equus asinus Linnaeus (Fam. Equidae) by decoction and concentration.

Procedure

Soak the donkey-hide, remove the hairs, cut into small pieces, rinse again, decoct with water several times, filter and combine the filtrates. Concentrate the glutinous filtrate ( or add a quantity of yellow rice wine, crystal sugar, soya-bean oil, respectively) to thick glue, allow to cool and congeal, cut into pieces, and dry in the air.

Description

Rectangular, square piece or small piece, brown to dark-brown, lustrous. Texture hard and fragile, fracture lustrous, fragment brown and translucent when looking against the light. Odour, slight； taste, slightly sweet.

Identification

To 0.1 g of the powder, add 50 ml of 1% solution of ammonium bicarbonate, ultrasonicate for 30 minutes, filter with millipore filter. Transfer 100 μl of the successive filtrate into a trace sample bottle, add 10 μl of trypsin solution ( dissolve trypsin for sequence analysis in 1% solution of ammonium bicarbonate to produce a solution containing 1 mg per ml, prepare when use) , mix well, hydrolyze by enzyme at 37℃ for 12 hours, use the hydrolysate as the test solution. Prepare a solution of 0.1 g of Asini Corii Colla reference drug in the same manner as the reference drug solution. Carry out the method for high performance liquid chromatography-mass spectrometry 〈 0512 and 0431〉, use octadecylsilane bonded silica gel as the stationary phase (2.1 mm in inner diameter) , and use acetonitrile as the mobile phase A, 0.1% solution of formic acid as mobile phase B, elute in gradient at 0.3 ml per minute as the following:

As detector a mass spectrometer set in positive electrospray ionization ( ESI＋ ) interface, using multiple reactions monitoring (MRM) mode. Monitor the ion pairs at m/z 539.8 (double charged) →612.4 and m/z 539. 8 (double charged) →923.8. Inject 5 μl of each of the reference drug solution and the test solution respectively to the column and determine. The signal to noise ratio (SNR) of the peaks in the chromatogram obtained with the reference solution should be greater than 3 : 1. The peaks in the chromatogram obtained with the test solution correspond in retention time to the peaks in the chromatogram obtained with the reference drug solution.

Examination

Water

Heat to dissolve 1 g, accurately weighed, in 2 ml of water, and evaporate to dryness on a water bath. Keep the thickness not over than 2 mm. Carry out the method for the determination of water 〈 0832 , method 2 〉, not more than 15.0 per cent.

Heavy metals and harmful elements

Carry out the method for determination of lead (Pb), cadmium (Cd), arsenic (As), mercury (Hg) and copper (Cu)〈2321, atomic absorption spectrophotometry or inductively-coupled plasma mass spectrometry), not more than 5 mg/kg of Pb; not more than 0.3 mg/kg of Cd； not more than 2 mg/kg of As； not more than 0.2 mg/kg of Hg； not more than 20 mg/ kg of Cu.

Insoluble matter in water

Weigh accurately 1.0 g of the powder in a flask, add 5 ml of water and heat to dissolve. Transfer to a 10 ml stoppered centrifuge tube previously dried to constant weight. Wash the flask 3 times with 5 ml of warm water, combine the washings to the centrifuge tube and mix well. Keep warm on a water bath at 40℃ for 15 minutes, centrifuge ( 2000 rotations per minute) for 10 minutes, discard the oil slick on tube wall and supernatant. Add warm water to volume along the tube wall, centrifuge, wash 3 times in the same manner and discard the supernatant. Heat the centrifuge tube at 105℃ for 2 hours, take out, cool in a desiccator for 30 minutes, weigh accurately and calculate, not more than 2.0 per cent.

Other requirements

Complies with the general requirements for glues 〈 0184 〉.

Assay

Carry out the method for high performance liquid chromatography 〈0512).

Prepared slices

Asini Corii Colla

Processing

Pound into pieces.

Asini Corii Colla (beaded)

Soften by heating, cut into small squares (about 1 cm long), then stir-bake to beads as described under the method for stir-baking with powdered clam-shell〈 0213〉until the inner not runny, take out, remove the powdered clam-shell by sifting, and allow to cool.

Examination

Water

Same as the crude drug, not more than 10.0%.

Total ash

Same as the crude drug, not more than 4.0%.

Identification

Same as the crude drug.

Assay

Same as the crude drug.

Property and Flavor

Neutral ； sweet. Meridian tropism Lung, liver and kidney meridians.

Actions

To nourish blood, replenish yin, moisten dryness, and stanch bleeding. Indicated for blood deficiency with sallow complexion, dizziness, palpitations, weak muscle, lack of strength, insomnia caused by vexation, internal stirring of defiency wind, cough caused by lung dryness, cough in consumptive disease, hemoptysis, hematemesis and hematuria, bloody stool, memstrnal flooding and spotting, and vaginal bleeding during pregnancy.

Administration and dosage

3-9 g, taken after dissolving in boiling decoction.

Storage

Preserve in tightly closed container.