Analysis of myogenic cream quality standards

Study: Column Shishu Dong Guang Pei Ma Zhenlian

[Abstract] Objective: To study the development of myogenic cream quality standards. Methods: Preparation of select myogenic ointment, or active ingredients Smell Identification and determination of active ingredients of the three projects. Preparation focused on habitat, Angelica, extraction of raw turtle shell, change the temperature for the water to cook fried extraction method, a corresponding adjustment to other process areas; Smell Identification of the active ingredient or by thin layer chromatography, chemical method and the microscopic identification method ; active ingredient content was determined by high performance liquid chromatography. Results: The preparation process of success, made a series of important technical parameters of efficacy of new technology products increased by 36%; finished dosage prescriptions were implemented in the identification of all the testing; habitat, Angelica, Calamine shamisen successful determination of the main active ingredient. Conclusion: This study for the development of new quality standards laid a solid foundation.

[Keywords:] myogenic paste; quality standards; preparation process; prescription identification of content;

Myogenic cream recipe from the pearl, turtle shell, habitat, Angelica, gypsum, and calamine and other components, the effectiveness of Yin and detoxification, tissue regeneration convergence sores, and promote epithelial healing, treating unhealed sores, burns, burns and so on. Long-term clinical practice proved , similar preparations in the western, the myogenic unique cream treatment particularly significant. However, due to restrictions on the development of medical technology, the quality standard of work was stalled. In order to scientifically inherit and carry forward the essence of Chinese medicine, based on current scientific and technological development the new situation, our hospital pharmacy worker to the establishment of quality standards myogenic cream were studied. is from the preparation process, component identification and determination of three important aspects.

1 Preparation

1.1 Process

1.1.1 take pot heated sesame oil 2000g home heating to 260 ℃, adding more than Blood, to maintain oil temperature 260 ℃ ~ 300 ℃, 30min, to more than completely carbonized blood, remove blood over charcoal, and placing a small amount of milk bowl add oil Research into a fine paste, spare; oil also set aside.

1.1.2 take sesame oil 1000g, heated to 100 ℃, add fried Angelica mention 20min; oil temperature rose to 130 ℃, continued bombing 5min, to Angelica Pieces of carbonized micro-focus when the fish have not yet, spare oil filter.

1.1.3 The merger will be the oil residue Angelica habitat, decoction extracted 2 times 45min, filtered decoction and concentrated into a thick paste aside. Another raw turtle shell pieces and 0.5 × 0.5cm2 coarse, decoction extraction of 6 to 8 times, each time 60min, to hand twist for the degree of turtle shells that crisp, fried filtration, concentrated into a thick paste aside.

1.1.4 will be the new oil is blended with 5000g fried oil in the blood of more than carbon to maintain the oil temperature 220 ℃, put calamine powder raw gypsum powder and kneaded 30min; an alternative blended with filtered beeswax melted in the continued refining of oil 10min, cooling to maintain the oil temperature 80 ℃, stand.

1.1.5 to 1.1.2 1.1.4 Oil and oil mixture to a; to 1.1.3 two thick paste, add appropriate amount of anhydrous lanolin blending Research and even for the b; by the same amount with a sliding scale method b Research and uniform to maintain the temperature of 50 ℃, the c; take appropriate c cream and pearl powder, blood, Research and even more than carbon paste, the remaining sub-sub-equal c cream against the Research, a final d to paste.

1.2 Preparation

1.2.1 fried fried blood blood I meant more than charcoal. In an atmospheric pressure and 25 ℃ + -5 at room temperature, oil temperature below 260 ℃, charring reaction does not occur more than blood; oil temperature reached 260 ℃ start carbonization reaction occurs, but the reaction is not complete; only after a period of heat accumulation and conversion, I can gradually charring of Blood, will be showing up to 300 ℃, charring more complete response, when blood over charcoal honeycomb solid, with metal ointment knife hit the sound, that just right. oil 超过 300 ℃, blood over charcoal heated liquid, has the effect of flameless combustion and oil, carbon flaming. oil temperature exceeds 300 ℃ 30min, no significant changes in the blood over charcoal, but no preparation significance.

1.2.2 fried fried Angelica Angelica purpose of eluates volatile oil. 100 ℃ provided by Angelica Ma frying, boiling point 100 ℃ lower than most of the volatile oil can be dissolved, but the Pieces of the surface does not change color, indicating the oil temperature of 100 ℃ Angelica Pieces of dehydration is not yet clear. Oil temperature than 100 ℃, the higher the boiling point of the volatile oil began stripping, when Angelica cross section from the yellow-white becoming dark yellow to prove Angelica began dehydration. But if the oil temperature 超过 130 ℃ or 130 ℃ more than 5min, the Angelica quickly charring. Angelica Once carbonized, it loses its value after the water extraction. Therefore, special emphasis must be fried Angelica control the furnace, both eluates after volatile oil and aqueous soluble components.

1.2.3 Health and landlords Angelica aqueous habitat containing a large number of catalpol and polysaccharides, angelica master containing addition to the above essential oil, still containing ferulic acid and polysaccharides. Two active ingredients are sparse, Department of hydrophilic substances in oil. The old process oil by high temperature 260 ℃ Fried, medicated oil and torment by 60min to make more than loss of almost 100% composition, identify the ingredients in the finished preparations were negative. the new standard by water extraction, fundamentally changed this situation, the ingredients in the finished dosage identification were positive reaction, the content of the ingredients and Chinese Herbal Medicine dose parallel relationship, so that finished in the determination of the ingredients possible.

1.2.4 Health and water extraction plates of turtle shell turtle is the main component of amino acids, the oil substance is hydrophilic sparse. The old process has been frying temperature 260 ℃, 60min by medicated oil and torment, causing 100% loss. A new standard based on the similarity phase dissolution laws, changed the reference to fried method for water, so that the finished product identification and determination of amino acids possible. aqueous turtle board, must pay attention to the students of shells fried, not fried sand into the hot products. sand hot products While easy to fry the active ingredients, but the concept of traditional Chinese medicine theory, students are good at nourishing Yin and Yang of turtle shell, turtle shell and the sand hot yin yang effect reduced. In order to maximize the amino acid in the turtle board proposed, in addition to appropriate increase in the number of extraction and solvent addition, students should be broken into small pieces or coarse turtle shell, increasing the contact area with the water solvent. By comparing the calculation shows, a 12 × 18 × 0.2cm3 raw turtle shell, if broken into 0.5 × 0.5 × 0.2 cm3 pieces, the surface area will increase 863-fold, corresponding extraction efficiency greatly improved. Turtle plate to make amino acids mentioned the "degree" is the turtle plate cooking "crisp" hand twist turtle shell, a twist that is broken.

1.2.5 when the temperature is the temperature reduction control large components of the matrix will refine oil temperature; reduction is appropriate to reduce the boil when the oil time. This standard, in addition to blood over charcoal frying process, so that 25% of the matrix sesame oil to 300 ℃, the remaining 75% of the matrix are controlled by sesame oil temperature within 220 ℃, a large fraction of time will refine oil from the past 60min reduced to 40min. so when the temperature reduction can significantly reduce the length of sesame oil at high temperature time under a lot of decomposition, oxidation, polymerization, a significant reduction of low molecular weight aldehydes and ketones generated, thereby maximizing the production of the drug to avoid the fire. myogenic ointment cream is for external use traditional Chinese medicine is not necessary as the system of black plaster, as sesame oil and a high degree of decomposition of the high degree of polymerization, on the contrary, sesame oil liquid boil the "tender" a little, but can significantly improve the efficacy of myogenic cream for breakfast, three surgical sore after all evidence, are appropriate.

1.2.6 references mixed with water to collect against the cream of the habitat, Angelica and raw turtle shells thick paste, then faced with the thick paste of water and large components mentioned oily substrate mix. Practice has proved that the need to address three issues: first is to select the appropriate W / O emulsifier, the aqueous emulsion cutting thick paste, then mixed with a large component oily matrix; the second is to select the appropriate temperature 70 ℃ ~ 80 ℃, the emulsion cutting success; third grinding for success. three indispensable, but the first condition is most important. have done several experiments using the water emulsion Monostearin mention thick paste, the same conditions at 70 ℃, the amount of grinding for work, over with anhydrous lanolin emulsion thick paste ground water extraction capacity for work 100% or more. Therefore, anhydrous lanolin for selected W / O emulsifier.

2 Component Identification

Component identification methods, including physical and chemical methods, thin layer chromatography and powder microscopy. Reagents used in a variety of methods, instruments, materials, the test provided by the hospital pharmaceutical companies, the use of reference substance by the Shandong Provincial Institute for Drug available.

2.1 calamine

2.1.1 take myogenic cream carbonate 1.0g, add 10ml diethyl ether solution washed 2 times, discard the ether solution, collect residue, add 50% ethanol 10ml swing washed 2 times, discard the ethanol solution, the residue add 10ml diluted hydrochloric acid that the bubble boiling occurs, the occurrence of carbon dioxide gas, into the test solution of calcium hydroxide, which form a white precipitate.

2.1.2 to take more zinc dilute hydrochloric acid solution calamine, Gaja potassium ferricyanide test solution, that is, white precipitate; separation, precipitate insoluble in dilute hydrochloric acid.

2.2 Gypsum

2.2.1 take myogenic calcium cream 1.0g, add 10ml diethyl ether solution washed 2 times, discard the ether solution, the residue add 10ml 50% ethanol wash finishes 2nd, discard the ethanol solution, the residue add 10ml diluted hydrochloric acid solution, the filtrate as the test solution. get wet with hydrochloric acid after platinum wire dipped into the test, in the colorless flame combustion, the flame that was brick red.

2.2.2 take the test solution sulfate, barium chloride test solution dropping, the white precipitate; separation, precipitation in hydrochloric acid or nitric acid are not soluble; the other to take the test solution, lead acetate test solution dropping, that is generated white precipitate, separation, precipitation in ammonium acetate test solution or sodium hydroxide dissolved in the test solution.

2.3 Pearl (powder)

Take myogenic cream 1.0g, add 10ml diethyl ether solution washed 2 times, discard the ether solution, the residue add 10ml 50% ethanol wash finishes 2nd, discard the ethanol solution, collect residue, dry, powdered, and placing under the microscope : (scattered in the state) irregular pieces, translucent, with a rainbow-like sheen. the surface of particles from a few to ten the number of overlapping thin, tightly packed lamellar structure, visible as a dense layer of microwave lines or extreme close-like texture. Home UV lamp (365mm) was observed, which was light blue or bright yellow, to circumferential some of the more bright.

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2.4 turtle shell (amino acids)

Take myogenic cream 1.0g, add 10ml diethyl ether solution washed 2 times, still, abandoned to the ether solution, the residue add 10ml of ethanol wash finishes, static, abandoned to the ethanol solution, the residue dissolved 10ml water, aqueous solution filtered, concentrated to 5ml as the test solution. to take the test solution, add ninhydrin test solution 0.5ml, water bath 15min, the solution was blue-purple. an alternative for the test solution 1ml, plus 1% copper sulfate solution, the new system and 40% sodium hydroxide solution (1:1) mixture of a few drops, shake the solution was purple.

2.5 to yellow (Catalpol)

Take myogenic cream 1.0g, add 10ml diethyl ether solution washed 2 times, still, abandoned to the ether solution, the residue dissolved in methanol 10ml and concentrated to 5ml, let cool, filtered, as the test solution. Catalpol an alternative control products, made from methanol containing 0.5mg per 1ml of the solution. According to thin layer chromatography test, take these two kinds of solution of 5μl, respectively, points on the same silica gel G thin-layer plate with chloroform methanol  water (14:6 : 1) as the agent, started out, dried, sprayed with anisaldehyde test solution, heated at 105 ℃ to the spot color definition. for the test products for chromatography, chromatography with the reference substance in the corresponding position, was the same color spots.

2.6 Angelica (ferulic acid)

Take myogenic cream 1.0g, add ethyl ether 10ml eluates 2, static collection ether solution, waving to the ether, the residue dissolved in methanol 10ml, filtered, concentrated to 1ml as the test solution. Ferulic acid, an alternative control products, made from methanol containing 1mg per 1ml of solution as the reference solution. Thin layer chromatography test, to learn the two solutions of the 10μl, respectively, points on the same silica gel G TLC plate to acid B vinegar acid (4:1:0.1) as the agent, started out, dried, Home UV light (365nm), under review. for the test products for chromatography, chromatography with the reference substance in the corresponding position, was the same color of the fluorescent spots.

3 Determination

Determination methods include physical and chemical methods and high performance liquid chromatography, the use of reagents, equipment, materials and the test provided by the hospital pharmaceutical companies. Reference substance by the Shandong Provincial Institute for Drug available.

3.1 calamine (zinc oxide)

Precision set myogenic cream 1.000g, add 10ml diethyl ether solution washed 3 times with ethyl ether solution was discarded; the residue washed 2 times 10ml of methanol, methanol liquid and discard; residue washed 2 times 10ml water, aqueous solution discarded. Collection of residual residue, set conical flask, add dilute hydrochloric acid 10ml, shake allows the dissolved, add a strong ammonia solution and ammonia chloride buffer (PH10.0) each 10ml, shake, add 10ml test solution, disodium hydrogen phosphate , shaking, filtration. Erlenmeyer flask with the residue of a chloride with ammonia buffer solution (PH10.0) 1 were a mixture of 4 parts water to wash 3 times 10ml, lotion combined with the filtrate, add 15ml 30% triethanolamine solution and a small amount of chrome black T indicator, titration with disodium ethylene diamine tetraacetate solution (0.05mol / L) titrated to the solution from purple into a pure blue. Each 1ml disodium ethylene diamine tetraacetate titration solution (0.05mol / L) is equivalent to 4.069mg of zinc oxide. Pharmacopoeia [1], calamine containing not less than 40% zinc oxide. Myogenic Ointment containing furnace Gan Shi 6.15%, 1g myogenic per calamine cream containing zinc oxide calculated at not less than 2.46mg. by more than 5 batches finished preparations were detected, the results shown in Table 1.

3.2 to yellow (Catalpol)

3.2.1 Chromatographic conditions and system suitability test to octadecyl silane bonded silica as a filler; with acetonitrile 0.1% phosphoric acid (1:99) as mobile phase; detection wavelength of 210nm. Theoretical plate number calculated by the peak catalpol not less than 5000 resolution is greater than 1.5.

3.2.2 Preparation of control solution and Linear Relation catalpol accurately weighed amount of reference substance, add the mobile phase is made with 5mg per 1ml of solution. Precise amount of reference substance solution, 0.1ml, 0.2ml, 0.4ml, 0.6 ml, 0.9ml, each with mobile phase to volume to 5ml, the sample 10μl, respectively 3221,6466,13005,19293,28892 peak area. Let the peak area for the Y, the sample volume (or sample concentration) for X the peak area of ​​the sample volume regression equation was Y = 3206.3139X +67.6846, r = 0.9999, shows the sample volume in the range of 1μg ~ 9μg peak area and sample size (or sample concentration) showed good between linear relationship.

3.2.3 Preparation of test solution for precision set myogenic cream 1.000g, add 10ml diethyl ether solution washed 3 times, discard the ether solution; residue dissolved in methanol 10ml, filtered, the filtrate was concentrated to dryness; residue add the mobile phase dissolved, transferred to 10ml volumetric flasks, diluted to the mark with the mobile phase, shake, filtration, that is, too. Pharmacopoeia [1] provides Rehmanniae calculated on dry goods, including catalpol (C15H22O10) not less than 0.20% myogenic Rehmanniae containing 3.07% cream, ointment containing myogenic per 1g catalpol not less than 61.0μg.

3.2.4 Precision precision of drawing the reference solution 0.5ml, add the mobile phase to volume 5ml, injection 10μl, measured peak area, and even measuring 5, RSD = 2.16%.

3.2.5 Stability Test precision drawing from the same batch test solution 10μl, measured sample peak area, measured 5 times with an interval of time was 2h, 4h, 8h, 12h, 24h, RSD = 3.18%.

3.2.6 repeatability precision drawing five different batches of each test solution 10μl, parallel sampling, determination of peak area, RSD = 3.22%.

3.2.7 Test check recoveries of known habitat Catalpol content from the same batch myogenic cream 5 portions, each 1g, according to 3.2.3 deep into the test solution, reference substance, respectively, by adding appropriate amount of precision, The precision drawing 20μl, measured sample peak area, the average recovery was 98.22%, RSD = 1.8%.

3.2.8 Precision of determination results were drawing the reference solution and sample solution of the 10μl, the peak area of ​​sample measured by external standard content, results in Table 1.

3.3 Angelica (ferulic acid)

3.3.1 Chromatographic conditions and system suitability test to octadecyl silane bonded silica as a filler; with acetonitrile 0.085% phosphoric acid (17:83) as mobile phase; detection of flow length 316mm; column temperature was 35 ℃, theoretical plate calculated by the number of ferulic acid should not be less than 5000 resolution is greater than 1.5.

3.3.2 Preparation of control solution linear relationship between the study and ferulic acid reference substance, accurately weighed 25mg, 1ml each made of methanol containing 5mg of the control solution. Were precise amount of the reference solution 0.1ml, 0.2ml, 0.4ml , 0.6ml, 0.9ml, 1.0ml, 1.2ml, 5ml set brown volumetric flask, add methanol to volume, 10μl of each drawing sampling precision, peak area were 39186,78392,156789,235269,352978,392164,470598. Let the peak area A, the sample volume (or concentration) of C, the peak area of ​​the sample volume regression equation was A = 39221.3698C-50.4275, r = 0.9999, sample size in the range of 1.0μg ~ 12.0μg, peak area and sample volume (or concentration) showed a good linear relationship.

3.3.3 Preparation of the test solution, and determination of precision set myogenic cream 1.000g, add ethyl ether 10ml 3 times eluates collected ether solution, waving to the ether, the residue 10ml with 70% methanol eluates 2, combined methanol, filtered, concentrated to 0.8ml, as the test solution. are precision drawing with ferulic acid 10μg per 1ml of the reference substance solution and test solution 10μl, high performance liquid chromatography, determination, that is, too.

Pharmacopoeia [1] provides Angelica with ferulic acid (C10H10O4) not less than 0.050%, myogenic angelica extract containing 1.56%, 1g per kg of ferulic acid cream myogenic not be less than 7.8μg. According to the above detected 5 approved product formulations, the results shown in Table 1. Table 1 an important and effective component of myogenic determination cream results

4 Discussion

Preparation of myogenic paste standards, into certain elements of modern technology, high-temperature frying than did the traditional method, a lot of progress, significant clinical improvement of standards of the new process validation of scientific rationality. However, the overall group in terms of square and the overall process, pending further study. Meanwhile, the main active ingredient myogenic cream identification and determination also need further exploration. such as herbs pearl is sore myogenic convergence to the drug, determination of the project should be located . But the pearl of the principal components to be measured calcium carbonate, mixed in the case of three drugs, there are some interference. If measurement of calcium, gypsum, calcium also; If the measured carbonate, calamine also carbonate ion, so the test measurements of calcium carbonate can not solve the determination of calcium carbonate in pearls question. This idea of ​​forcing us to choose another to design a formula, the number of moles of calcium pearl calcium = total number of moles of the mixture - the number of moles of gypsum in sulfate, or carbonate in pearls mole = the total number of moles of a mixture of carbonate - the number of moles of zinc in calamine. This design when not, to be experimental answer.

[References]
1 Republic of China Pharmacopoeia (a), Beijing: Chemical Industry Press, 2005,82; 89; 157.

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