CN111122804A - A quality control method of caulis Bauhihiae Championii - Google Patents
A quality control method of caulis Bauhihiae Championii Download PDFInfo
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Abstract
The invention discloses a quality control method of a bauhinia championii medicinal material, and relates to the technical field of traditional Chinese medicinal materials. Comprises (1) microscopic identification of medicinal powder; (2) the ranges of water content, total ash content and extract content of the medicinal materials are as follows: the water content is not more than 15.0%, the total ash content is not more than 9.0%, and the extract is measured by hot dipping method under alcohol-soluble extract measuring method (2201 in the four-part general rule of the Chinese pharmacopoeia 2015 edition), and diluted ethanol is used as solvent, and is not less than 15.0%; (3) identifying by thin-layer chromatography; the test solution should have fluorescence spots with the same color at the corresponding positions on the chromatogram of the gallic acid control solution. The invention establishes a scientific, complete, reliable and effective quality control method of the bauhinia championii medicinal material, and the method has strong specificity and good reproducibility; meanwhile, the quality standard of the bauhinia championii medicinal material is established by adopting the method, and the internal quality and the medication quality of the medicinal material can be effectively evaluated and controlled.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicinal materials, and in particular relates to a quality control method of a bauhinia championii medicinal material.
Background
Caulis Bauhiniae Championii is fresh or dried rattan of Bauhinia championii (Benth.) of Leguminosae. Collected all the year round, removed impurities, and used fresh or dried in the sun. It is first recorded in the Bing Cao Yao Bian (treatise on herb Property), and Yun: "leaves like dovetail, roots red, as flower center. Also named as caulis Bagguo (the Res. of raw herbal medicine), glaucescent fissistigma root (the Classification of Chinese trees), glaucescent fissistigma root, yangtao (the plant of Chinese veterinary medicine in Guangxi), swallow tail (the record of medicine in Nanning City), pig foot fork, sheep hoof (the Chinese herbal medicine in Guangxi), Dioscorea bulbifera, spatholobus suberectus, Wuli mushroom, Shuangmu crab (the herbal medicine commonly used in Zhejiang folk), and herba Polygoni Multiflori, dried hammer, nine-dried beef cattle, and caulis Sargentodoxae; marsdenia tenacissima (J. am. Han) and Feiyang rattan and Rumex japonicus (J. am. Yam. ex Pak. et al., records of famous records of Guangxi medicine), and folk medicine is also known as Jiulongteng, and is used by minority nationalities in our province. Collected in the book of 1983 edition "Chinese medicinal materials and national medicinal materials quality Standard" in Guizhou province, the Bauhinia championii is a fresh or dried rattan of the Leguminosae Bauhinia championii (Benth.) Benth.
The bauhinia championii is a woody vine of bauhinia championii: there are tendrils. Small soft hair with closely attached tender branches and inflorescences. Leaf intergrowth; the petiole is 1-2.5cm long, fine and slightly hairy; the leaf is paper, oval or heart-shaped, 3-10cm long, 2.5-6.5cm wide, sharp and tapered at the tip, slightly concave or 2-split to no-split, and slightly concave or heart-shaped at the base; the upper surface is hairless, the lower surface is closely attached with short and soft hair, the hair gradually becomes hairless or nearly hairless, and the powder is brownish when being dried; 5-7 pulses are appeared. The flower is amphoteric, the inflorescence is long and narrow, the axilla is grown, sometimes the inflorescence is grown opposite to the leaf or a plurality of inflorescences are gathered at the top of the branch to form a compound inflorescence, and the length is 7-20 cm; the bracts and the small bracts are small, conical tips and early fall; the flower stalks are fine and 1-1.5cm long; the receptacle is funnel-shaped and about 2mm long; calyx, lobe 5, needle-shaped, about 3mm long; the petals 5 are colorful, have petal handles and petal spoon shapes, and have the middle part outside for dredging the silk hairs; fertile stamens 3, hairless, degenerated stamens 2; the ovary has a short handle, is only hairy along two suture lines, has short style and small stigma. The pod has inverted egg-shaped oblong or belt shape, flat shape, length of 7-12cm, width of 2.5-3cm, and no pellucida. The seeds are 2-5, round, flat and about 1.2cm in diameter. The flowering period is 6-10 months, and the fruit period is 7-12 months.
At present, the bauhinia championii has the functions of expelling wind, relieving pain and removing blood stasis, and the specific drug effect of the bauhinia championii is widely accepted by folks. At present, medicines containing the bauhinia championii as an active ingredient are developed in the market, but the quality of the bauhinia championii medicinal material is difficult to control due to the lack of a good quality control method, so that the normal production and operation are difficult, and the quality control method needs to be standardized to control the quality of the bauhinia championii medicinal material. At present, the medicinal material of the bauhinia championii has not established relevant standards. The prior art only relates to the examination of the characters of the bauhinia championii medicinal material (quality standard of Chinese medicinal materials and national medicinal materials in Guizhou province), has low pertinence, and cannot achieve the aim of really controlling the quality of the medicinal materials by using the methods.
Disclosure of Invention
In order to solve the problems in the background art, the invention provides a quality control method of a bauhinia championii medicinal material, which can effectively evaluate and control the internal quality and the medication quality of the medicinal material.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a quality control method of bauhinia championii medicinal material comprises (1) microscopically identifying whether the medicinal material powder is light brown or brown, conduit with fringe holes, most broken, with diameter of about 10-300 μm, numerous calcium oxalate cubic crystals, diameter of 2-30 μm, slender fiber, diameter of 10-25 μm, and often bundled or singly scattered, wherein some peripheral cells contain the calcium oxalate cubic crystals to form crystal fiber; (2) the ranges of water content, total ash content and extract content of the medicinal materials are as follows: the water content is not more than 15.0%, the total ash content is not more than 9.0%, and the extract is measured by hot dipping method under alcohol-soluble extract measuring method (2201 in the four-part general rule of the Chinese pharmacopoeia 2015 edition), and diluted ethanol is used as solvent, and is not less than 15.0%; (3) identifying by thin-layer chromatography; the test solution should have fluorescence spots with the same color at the corresponding positions on the chromatogram of the gallic acid control solution.
Wherein, the thin-layer chromatography adopts a silica gel G thin-layer plate, toluene-ethyl acetate-formic acid is used as a developing agent, and 10% phosphomolybdic acid ethanol solution is used as a color developing agent.
In the invention, the thin-layer chromatography is carried out according to the following operations: respectively dropping 5-10 μ l of sample solution and 5-10 μ l of reference solution on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-formic acid solution at volume ratio of 4-6:3-5:1 as developing agent, taking out, and air drying; heating 10% phosphomolybdic acid ethanol solution at 105 deg.C until spots are clear, and spots with the same color appear on the chromatogram of the test solution at the position corresponding to the chromatogram of the control solution.
Preferably, the volume ratio of the developing solvent toluene-ethyl acetate-formic acid is 5:4: 1.
In the invention, the test sample is prepared by the following method: the test sample is prepared by the following method: putting the powder of the test sample into a conical flask with a plug, adding an organic solvent, carrying out ultrasonic treatment, filtering, and concentrating to obtain a test sample solution.
Preferably, the test article is prepared by the following method: taking about 2g of the coarse powder of the product, adding 20ml of chloroform-methanol (8:2), performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue with 1ml of methanol to obtain a test solution.
In the invention, the reference substance is prepared by the following method: taking gallic acid control, adding methanol to make 1ml solution containing 1mg as control solution.
The quality control method of the bauhinia championii medicinal material further comprises content measurement, wherein a mobile phase adopted by the content measurement is a methanol-0.1% phosphoric acid solution, the column temperature is 25 ℃, and the detection wavelength is 275 nm.
Furthermore, octadecylsilane chemically bonded silica is used as a filler; the volume ratio of the components is 4: 96% methanol-0.1% phosphoric acid solution as mobile phase; the column temperature is 25 ℃, and the flow rate is 1.0 ml/min; the detection wavelength is 272 nm; the number of theoretical plates is not less than 2000 calculated according to gallic acid peak, respectively and precisely sucking 10 μ l of reference solution and sample solution, respectively, injecting into liquid chromatograph, and measuring.
Preferably, taking a proper amount of gallic acid reference substance, precisely weighing, and adding 80% methanol to obtain 45ug solution per 1ml to obtain reference substance solution; taking 0.5g of medicinal powder (passing through a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 4mol/L hydrochloric acid solution, sealing the plug, weighing, heating and refluxing for 3 hours, cooling, weighing again, complementing the weight loss by 4mol/L hydrochloric acid solution, shaking up, filtering, and taking the subsequent filtrate to obtain the sample solution.
Compared with the prior art, the invention has the following beneficial effects: the invention establishes a scientific, complete, reliable and effective quality control method of the bauhinia championii medicinal material by the microscopic identification of the medicinal material and combining the water content, the total ash content, the extract and the thin-layer chromatography, and the method has strong specificity and good reproducibility; meanwhile, the quality control method also comprises a content detection method which adopts gallic acid as a reference substance. The method establishes quality standard of the bauhinia championii medicinal material, and can effectively evaluate and control the internal quality and the medication quality of the medicinal material.
Drawings
FIG. 1: microscopic characteristic diagram of bauhinia championii medicinal material powder, wherein: 1. a flanged-hole conduit; 2. calcium oxalate cubic crystal; 3. crystal fibers; 4. fibers;
FIG. 2: reflux-extracting caulis Bauhihiae Championii with 10% hydrochloric acid and 50% methanol solution, and performing thin layer chromatography with toluene (saturated with water) -ethyl acetate-formic acid (6:3:1) as developing agent; in the figure: 1. gallic acid control, 2, sample (producing area: Guangdong);
FIG. 3: caulis piperis sargentodoxae at temperature: 25 ℃, humidity: when the concentration is 54%, using toluene-ethyl acetate-formic acid (5:4:1) as developing agent to use thin layer identification method chromatogram; in the figure: 1. sample (producing area: Baicai), 2, 3 gallic acid reference, 4, sample (producing area: Guangdong), 5, sample (producing area: Sichuan);
FIG. 4: caulis piperis sargentodoxae at temperature: 5 ℃, humidity: when the concentration is 63 percent, toluene-ethyl acetate-formic acid (5:4:1) is used as a developing agent for a thin layer identification method chromatogram; in the figure: 1. gallic acid control, 2, sample (producing area: Sichuan), 3, sample (producing area: Baicai), 4, sample (producing area: Guangdong);
FIG. 5: caulis piperis sargentodoxae at temperature: 40 ℃, humidity: when the concentration is 40%, using toluene-ethyl acetate-formic acid (5:4:1) as developing agent for thin layer identification chromatogram; in the figure: 1. gallic acid control, 2, sample (producing area: Sichuan), 3, sample (producing area: Baicai), 4, sample (producing area: Guangdong);
FIG. 6: caulis piperis sargentodoxae at temperature: 25 ℃, humidity: when the concentration is 72 percent, toluene-ethyl acetate-formic acid (5:4:1) is used as a developing agent for a thin layer identification method chromatogram; in the figure: 1. gallic acid control, 2, sample (producing area: Sichuan), 3, sample (producing area: Baicai), 4, sample (producing area: Guangdong);
FIG. 7: caulis piperis sargentodoxae at temperature: 25 ℃, humidity: when 32 percent of the total content is needed, toluene-ethyl acetate-formic acid (5:4:1) is used as a developing agent for a thin layer identification method chromatogram; in the figure: 1. gallic acid control, 2, sample (producing area: Sichuan), 3, sample (producing area: Baicai), 4, sample (producing area: Guangdong);
FIG. 8: caulis piperis sargentodoxae at temperature: 25 ℃, humidity: when 54 percent of the total content is in the range, a Qingdao ocean silica gel G plate is used for a thin layer identification method chromatogram; in the figure: in the figure: 1. gallic acid control, 2, sample (producing area: Sichuan), 3, sample (producing area: Baicai), 4, sample (producing area: Guangdong);
FIG. 9: caulis piperis sargentodoxae at temperature: 25 ℃, humidity: when 54 percent of the total content is in the range, a silica gel G plate is laid by hands to be used for a chromatogram in a thin layer identification method; in the figure: 1. gallic acid control, 2, sample (producing area: Sichuan), 3, sample (producing area: Baicai), 4, sample (producing area: Guangdong);
FIG. 10: a specific chromatogram for an HPLC method;
FIG. 11: gallic acid linear graph in HPLC;
FIG. 12: gallic acid was fit to a linear plot in HPLC.
Detailed Description
In order to make the objects and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Examples
1. Experimental apparatus and medicinal materials
The medicinal materials are as follows:
bauhinia championii is provided by Guizhou Shengshilongfang pharmaceutical Co. The origin of the sample: (1) guangdong, (2) Baicai, (3) Sichuan. And identified as dry vine stem of the leguminous plant Bauhinia championii (Benth). The certificate specimen is stored in Guizhou Shengshi Longfang pharmaceutical products GmbH.
Instruments and reagents:
electronic balance type AUW-220D (Shimadzu, Japan); a numerical control ultrasonic cleaner KQ-500D model (ultrasonic instruments Co., Ltd., Kunshan city, Jiangsu province); digital display constant temperature water bath HH-4 (national electric appliance Co., Ltd.); type 101-1 constant temperature drying oven (Shanghai Puhong instrument factory), dryer; LC-2010AHT full-automatic high performance liquid chromatograph (Shimadzu, Japan); box type resistance furnace SX2-2.5-10 type (Lunan electric furnace oven factory); a WFH-201B ultraviolet transmission reflectometer (shanghai seminars industries, ltd.) olympson model MD-50 biomicroscope and a matched imaging system, and a blade, a slide cover, tweezers, chloral hydrate, diluted glycerin, distilled water, chloroform (analytically pure, chongqing chuan dong chemicals, ltd.), toluene (analytically pure, chongqing chudong chemicals, ltd.), formic acid (analytically pure, chongqing chudong chemicals, ltd.), methanol (analytically pure, chongqing chudong chemicals, ltd.), ethanol (analytically pure, chongqing chudong chemicals, ltd.), ethyl acetate (analytically pure, chengdong chemicals, reagent factory), methanol (chromatographically pure, seimer science and technology, ltd.); water (Wahaha purified water); phosphoric acid (analytically pure, Tianjin, Daloco chemical reagent works); hydrochloric acid (analytically pure, Tianjin, Dalochi chemical laboratories, Inc.), phosphomolybdic acid, and the like.
2.1 microscopic identification:
now, by observing and measuring the powder of 3 medicinal material samples ((1) Guangdong, (2) Baicai, (3) Sichuan), the microscopic identification characteristics are summarized as follows:
the powder is light brown or dark brown. The conduit with the edge-grain holes is mostly broken and has a diameter of about 10 to 300 μm. The calcium oxalate has a plurality of cubic crystals with a diameter of about 2 to 30 μm. The fiber is slender, has the diameter of about 10-25 mu m, is often bundled or singly scattered, and some peripheral cells contain calcium oxalate square crystals to form crystal fiber. As shown in fig. 1.
2.2 thin layer identification
2.2.1 identification by thin-layer chromatography with gallic acid as reference
(1) Collecting about 2g of the powder, adding 50ml of 10% hydrochloric acid 50% methanol solution, heating and refluxing for 2 hr, cooling, filtering, extracting the filtrate with hydrochloric acid saturated diethyl ether for 2 times, each time 25ml, mixing the diethyl ether solutions, evaporating to dryness, and dissolving the residue with 1ml of methanol to obtain a sample solution. Taking gallic acid control, adding methanol to obtain solution containing 0.5mg per 1ml, and making into control solution. Performing thin layer chromatography (0502 of the four ministry of the national pharmacopoeia 2015), collecting 5-10 μ l of the sample solution and 5 μ l of the reference solution, respectively dropping on the same silica gel G thin layer plate, developing with toluene (saturated with water) -ethyl acetate-formic acid (6:3:1) as developing agent, taking out, air drying, spraying with 1% ferric trichloride ethanol solution, heating at 105 deg.C for several minutes, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test sample, spots of the sample are relatively fuzzy and unclear at the position corresponding to the chromatogram of the reference substance, and the thin-layer identification test is not ideal and needs further research. So the method is not included in the text of the new revised quality standards. See figure 2:
(2) taking about 2g of the coarse powder of the product, adding 20ml of chloroform-methanol (8:2), performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue with 1ml of methanol to obtain a test solution. And performing thin-layer chromatography (0502 of the four ministry of the national pharmacopoeia 2015), sucking 5-10 μ l of the test solution and 5 μ l of the reference solution, respectively dropping on the same silica gel G thin-layer plate, developing with toluene-ethyl acetate-formic acid (5:4:1) as developing agent, taking out, air drying, spraying 10% phosphomolybdic acid ethanol solution, heating at 105 deg.C until the spots are clear, and displaying spots of the same color in the chromatogram of the test solution at the position corresponding to the chromatogram of the reference solution.
2.2.2 thin layer chromatography methodology durability examination
The following methodology durability examination was conducted according to the requirements of the State Committee for pharmacopoeia, the "guidelines for the implementation of the analytical methods of Chinese medicine" guidelines.
① temperature examination of the temperature at room temperature (25 ℃), low temperature (5 ℃) and high temperature (40 ℃) were carried out, and the results were good (see FIGS. 3, 4 and 5)
② examination of humidity the results were good when both high humidity (72%) and low humidity (32%) were examined (see FIGS. 6 and 7)
③ investigation of different thin silica gel G plates two thin silica gel G plates were tested in total, Qingdao ocean chemical Co., Ltd. and hand-laid silica gel G plate (see FIGS. 8 and 9)
And (4) conclusion: under the test condition, spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control solution. The results show that the separation is good and the spots are clearly developed, so the selection is put into the text of quality standard.
2.3 inspection of moisture
Taking medicinal powder of caulis Bauhihiae Championii of different origins (sieved by No. 2 sieve) about 2.0g, and measuring according to the second method of water content measurement method (0832 in the fourth Productivity of the version 2015 in Chinese pharmacopoeia). The measurement result of the sample is 11.7-12.5%, the limit of 120% of the highest measurement value is 15.0% according to the measurement result of the moisture, and the moisture is determined to be not more than 15.0% by combining the factors such as the characteristics of medicinal materials, the harvesting and processing process, the storage process and the like, so the income quality standard text is recommended. The samples from different origins were determined as shown in Table 1.
2.4 inspection of Total Ash
Taking the medicinal powder of caulis Bauhihiae Championii of different producing areas (sieved by No. 2 sieve) about 4.0g, and measuring according to ash content determination method (the four-part general rule 2302 of the version in 2015 of Chinese pharmacopoeia). The measurement result of the sample is 5.4-7.5%, the limit of 120% of the highest measurement value is 9.0% according to the measurement result of the total ash content, and the total ash content is determined to be not more than 9.0% by combining the characteristics of medicinal materials, the harvesting, processing, storage processes and other factors, so that the income quality standard text is recommended. The samples from different origins were determined as shown in Table 1.
2.5 examination of acid-insoluble Ash
The ash content obtained in the total ash content test was measured by an ash content measuring method (the general rule 2302 in the four departments of the edition of the Chinese pharmacopoeia 2015). The test result of the sample is 0.8-2.8%, and the experimental result shows that the ash content of acid incompatibility is low, so the method does not fall into the text of a standard draft. The samples from different origins were determined as shown in Table 1.
TABLE 1 determination results of water content, total ash content, acid insoluble ash content, and extract of bauhinia championii
2.6 measurement of extract
2.6.1 measurement of Water-soluble extracts: the test sample for determination is pulverized, passed through a second sieve, and mixed uniformly.
(1) Cold soaking method: taking about 2.0g of bauhinia championii medicinal material powder (sample production place: Guangdong), precisely weighing, placing in a 100ml conical flask, precisely adding 50ml of water, sealing, cold soaking, shaking all the time within the first 6 hours, standing for 18 hours, rapidly filtering by using a drying filter, precisely taking 20ml of subsequent filtrate, placing in an evaporation dish dried to constant weight, drying by evaporation on a water bath, drying at 105 ℃ for 3 hours, placing in a dryer for cooling for 30 minutes, and rapidly precisely weighing. The content (%) of the water-soluble extract in the test sample was calculated from the dried product. The results are shown in Table 2.
(2) Hot dipping method: taking about 2.0g of bauhinia championii medicinal material powder (sample production place: Guangdong), precisely weighing, placing in a 100ml conical flask, precisely adding 50ml of water, tightly plugging, weighing, standing for 1 hour, connecting with a reflux condenser tube, heating to boil, and keeping slightly boiling for 1 hour. After cooling, the flask was removed, stoppered, weighed again, the lost weight was made up with water, shaken well and filtered through a dry filter. Precisely measuring 25ml of filtrate, placing the filtrate in an evaporating dish which is dried to constant weight, drying the filtrate by evaporation on a water bath for 3 hours at 105 ℃, placing the filtrate in a dryer for cooling for 30 minutes, rapidly and precisely weighing the filtrate, and calculating the content (%) of the water-soluble extract in the test sample according to the dry product. The results are shown in Table 2.
TABLE 2 Water-soluble extract content (%)
| Producing area | Cold dipping method | Hot dipping method |
| Guangdong (Chinese character of Guangdong) | 5.2％ | 10.2％ |
And (3) test results: the hot dipping method is selected because the extract content measured by the hot dipping method in the bauhinia championii medicinal material is higher than that measured by the cold dipping method.
2.6.2. Alcohol-soluble extract determination: the measurement is carried out by hot dipping method under the alcohol-soluble extract measurement item. Ethanol (95% ethanol, the same below), diluted ethanol (49.5% -50.5% ethanol, the same below), and 80% ethanol are used as solvents. Taking about 2.0g of a test sample of bauhinia championii medicinal material powder (sample producing area: Guangdong), precisely weighing 2 parts, respectively placing into 100ml conical flasks, precisely adding 50ml of ethanol, diluted ethanol and 80% ethanol, sealing, and testing according to a method 1.2. The results are shown in Table 3.
TABLE 3 alcohol soluble extract content (%)
| Solvent(s) | Extract of plant |
| Ethanol | 12.4％ |
| 80% ethanol | 18.4％ |
| Dilute ethanol | 21.9％ |
And (3) test results: according to the comparison of the determination results in the table 3, the extract content of the bauhinia championii medicinal material in the diluted ethanol is higher than that of the extract content of other solvents, and referring to the relevant literature data, the main components contained in the bauhinia championii medicinal material are easily dissolved in methanol or ethanol, so the selection of the alcohol-soluble extract for determining the content of the bauhinia championii medicinal material is reasonable. Therefore, the extract is measured by hot dipping in the alcohol-soluble extract measuring method, and diluted ethanol is used as the solvent.
And (3) determining the extract of the medicinal material of the bauhinia championii in different producing areas: taking about 2.0g of the powder (sieved by a second sieve) of the medicinal material of the bauhinia championii (1: Bai Cao, 2: Guangdong, 3: Sichuan) from 3 different producing areas, precisely weighing, respectively placing 2 parts of the powder into a 100mL conical flask, precisely adding 50mL of diluted ethanol, tightly plugging, and testing according to a 2.6.1(2) method. The results are shown in Table 1.
And (4) conclusion: according to the results of hot dipping method, the minimum value is 21.3%, the limit is set to 80% of the minimum value, 17.0%, and the alcohol-soluble extract should not be less than 15.0% by combining with factors such as the production area, harvesting season and processing. A text of suggested income criteria.
2.7 assay
2.7.1 measurement method
2.7.1.1 selection of chromatographic conditions:
(1) chromatographic conditions chromatographic column: WELCHR0M C18(5um, 250X 4.6 mm); mobile phase: methanol-0.1% phosphoric acid (4: 96); flow rate: 1.0 ml/min; column temperature: 30 ℃; detection wavelength: 272 nm.
(2) Mobile phase: through experimental investigation, methanol-0.1% phosphoric acid solution (5: 95) and methanol-0.1% phosphoric acid solution (4: 96) are adopted as mobile phase for separation; wherein, the methanol-0.1 percent phosphoric acid solution (4: 96) can better separate the gallic acid to be detected from other impurities.
(3) Detection wavelength: the detection wavelength of gallic acid was selected to be 272 nm.
(4) Theoretical plate number: according to the content measurement of the measurement results of chromatographic columns of different brands, the theoretical plate number is not less than 2000 calculated according to the peak of gallic acid.
2.7.1.2 preparation of test solution:
taking about 0.5g of the product powder (passing through a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 4mol/L hydrochloric acid solution, sealing the plug, weighing, heating and refluxing for 2 hours, cooling, weighing again, complementing the weight loss by 4mol/L hydrochloric acid solution, shaking up, filtering, and taking the subsequent filtrate to obtain the product.
2.7.1.3 preparation of control solutions: taking appropriate amount of gallic acid as reference, precisely weighing, and adding 80% methanol to obtain solution containing 45ug per 1 ml.
2.7.1.4 selection of extraction methods
Taking about 0.5g and 2 parts of bauhinia championii (producing area: Guangdong) sample powder, precisely weighing, respectively placing into conical flasks with stoppers, precisely adding 50ml of 4mol/L hydrochloric acid solution, sealing the stoppers, weighing, heating and refluxing one part for 2 hours, carrying out ultrasonic treatment (250W, frequency 35kHz) for 60min on one part, cooling, weighing again, complementing the lost weight with 4mol/L hydrochloric acid solution, shaking up, filtering, and taking the subsequent filtrate.
Precisely sucking 10 μ l of each of the reference solution and the two test solutions, injecting into a liquid chromatograph, measuring according to the above chromatographic conditions, and recording chromatogram (see attached figure). The results are given in Table 4 below:
TABLE 4 extraction method selection
The result shows that the content of the reflux for 2 hours is higher than that of the ultrasonic for 1 hour, so the extraction method selects the reflux for 2 hours.
2.7.1.5 examination of sample size
Taking 0.3g, 0.5g and 1.0g of bauhinia championii (producing area: Guangdong) sample powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 4mol/L hydrochloric acid solution, sealing the plug, weighing, heating and refluxing for 2 hours, weighing again, complementing the lost weight with 4mol/L hydrochloric acid solution, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the bauhinia championii (producing area: Guangdong).
Precisely sucking 10 μ l of each of the reference solution and the three test solutions, injecting into a liquid chromatograph, measuring according to the above chromatographic conditions, and recording chromatogram (see attached figure). The results are given in Table 5 below:
TABLE 5 sample size investigation
As a result: the content of 1g is low, the content of 0.3g and 0.5g is not greatly different, and the sampling amount is 0.5g to take the uniformity of sampling into consideration.
2.7.1.6 selection of extraction time
Taking about 0.5g and 3 parts of bauhinia championii (producing area: Guangdong) sample powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 4mol/L hydrochloric acid solution, sealing the plug, weighing, heating and refluxing for 1 hour, 2 hours and 3 hours, cooling, weighing again, supplementing the lost weight with 4mol/L hydrochloric acid solution, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the bauhinia championii (producing area: Guangdong). Precisely sucking 10 μ l each of gallic acid reference solution and three test solutions, injecting into liquid chromatograph, measuring according to the above chromatographic conditions, and recording chromatogram (see attached figure). The results are given in Table 6 below:
TABLE 6 extraction timing
The result shows that the content of the reflux extraction is higher after 3 hours, so the extraction time is 3 hours.
2.7.1.7 selection of extraction solvent
Taking about 0.5g and 3 parts of bauhinia championii (producing area: Guangdong) sample, precisely weighing, placing in a conical flask with a plug, precisely adding 2mol/L hydrochloric acid solution, 3mol/L hydrochloric acid solution and 50ml of 4mol/L hydrochloric acid solution, sealing the plug, weighing, heating and refluxing for 2 hours, cooling, weighing again, complementing the weight loss, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the bauhinia championii (producing area: Guangdong). Precisely sucking 10 μ l each of gallic acid reference solution and three test solutions, injecting into liquid chromatograph, measuring according to the above chromatographic conditions, and recording chromatogram (see attached figure). The results are given in Table 7 below:
TABLE 7 selection of extraction solvents
The results show that the acid hydrolysis of 2mol/L hydrochloric acid solution and 3mol/L hydrochloric acid solution is incomplete, the content is low, the impurities are more, the acid hydrolysis of 4mol/L hydrochloric acid solution is complete, the content is higher, and the separation degree with the impurities is better. Therefore, 4mol/L hydrochloric acid solution is selected as the extraction solvent.
2.7.2 methodological validation
2.7.2.1 specificity
Taking caulis Bauhihiae Championii (producing area: Guangdong) sample powder, preparing sample solution according to the preparation method of test solution, injecting reference solution and sample solution into liquid chromatograph according to the above chromatogram conditions, wherein the chromatogram of the sample solution has corresponding chromatogram peak at the corresponding position of the chromatogram of the reference solution, and is shown in special chromatogram, fig. 10.
2.7.2.2 examination of Linear relationships
Under the above chromatographic conditions, 2. mu.l, 4. mu.l, 6. mu.l, 8. mu.l, 10. mu.l and 12. mu.l of the gallic acid control solution (74.60128. mu.g/ml) were precisely pipetted and sequentially injected into a liquid chromatograph, and the peak area was measured. And drawing a standard curve by taking the peak area (Y) of the chromatographic peak of the gallic acid reference substance as a vertical coordinate and the sample injection amount (X) as a horizontal coordinate, wherein the gallic acid has a good linear relation in the range of 0.1355-0.8129 mu g. The results are given in Table 8 below:
TABLE 8 Gallic acid Linear relationship investigation
The equation fitting the linear equation to the through-origin is: Y4013265.2000X.
And substituting the lowest point sample amount (0.1355 mu g) and the highest point sample amount (0.8129 mu g) in the linear relation test into two equations respectively to calculate, wherein the relative deviation of the obtained peak areas is 0.99% (low) to 0.08% (high). Therefore, the intercept can be approximate to zero, which indicates that the product can be used for calculating the content measurement result by an external standard one-point method.
2.7.2.3 precision test
Precisely sucking 10 μ l of control solution, repeatedly introducing sample for 6 times, measuring according to the above chromatographic conditions, measuring peak area of control, recording chromatogram (see figure), and calculating relative standard deviation, wherein the result shows good precision, and the result is shown in Table 9 below.
TABLE 9 Gallic acid precision test results
2.7.2.4 repeatability test
Taking the sample powder of the bauhinia championii (producing area: Guangdong), preparing 6 parts of test solution according to the preparation method of the test solution in the quality standard draft, determining the content of gallic acid according to the chromatographic conditions, and obtaining the result shown in the following table 10 and a repeatability chromatogram map which shows that the repeatability is good.
TABLE 10 Gallic acid repeatability test results
2.7.2.5 stability test
Taking the powder of the sample of the bauhinia championii (producing area: Guangdong), and preparing the test solution according to the preparation method of the test solution in the quality standard draft. After 10 μ l of the sample was injected at room temperature at intervals, the peak area was measured according to the above-mentioned chromatographic conditions, the chromatogram was recorded (see the drawing), the content was calculated, and the relative standard deviation was calculated, whereby the sample was stable for 12 hours (RSD 0.23%), and the results are shown in table 11 below.
TABLE 11 gallic acid stability test results
2.7.2.6 recovery test
Taking 0.25g of sargentgloryvine stem (producing area: Guangdong, average content of gallic acid is 5.28mg/g) with determined content, precisely weighing, placing in a conical flask with a stopper, precisely adding 50ml of 4mol/L hydrochloric acid solution containing a gallic acid reference substance (0.025954272mg/ml), preparing a sample solution according to a preparation method of a test solution, determining the content of the gallic acid according to the chromatographic conditions, and calculating the recovery rate, wherein the result is shown in the following table 12 and an accuracy chromatogram map, which indicates that the recovery rate is better.
TABLE 12 Gallic acid recovery test data (n ═ 6)
2.7.2.7 durability
The content of gallic acid in the bauhinia championii (producing area: Guangdong) is respectively measured by adopting chromatographic columns with octadecylsilane chemically bonded silica gel of different brands as a filler, and the influence of the chromatographic columns of different brands is investigated. The chromatogram is a durable chromatogram, and the content measurement results are shown in Table 13 below. The result shows that chromatographic columns using octadecylsilane chemically bonded silica of different brands as filling agents have no influence on content measurement.
TABLE 13 measurement results of columns of different brands
2.7.3 sample determination
Taking 3 batches of samples, and determining the content of gallic acid according to a quality standard draft method (the result is shown in a table 14). The content is calculated according to the following formula:
in the formula: a. theSample (A)-peak area integral value of gallic acid in test sample
ATo pair-gallic acid control peak area integral value
CTo pairGallic acid control concentration (mg/ml)
MSample (A)-sample size (g) of sample
50-test dilution volume (ml)
TABLE 14 determination of gallic acid content in samples from different production areas
And (4) conclusion: according to the above 3 medicinal materials determination results, the lowest content value is 0.52%, the limit is 80% of the lowest value, and the product is formulated to contain gallic acid (C) calculated according to dry product by combining with factors such as medicinal material production area, harvesting season, processing and storage process7H6O5) Not less than 0.30%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (10)
1. A quality control method of bauhinia championii medicinal materials is characterized by comprising the following steps: comprises (1) microscopic identification of medicinal powder; the powder is light brown or brown, the conduit with the edge grain holes is mostly broken, the diameter is about 10-300 mu m, the calcium oxalate has numerous cubic crystals, the diameter is 2-30 mu m, the fiber is slender, the diameter is 10-25 mu m, the fiber is usually bundled or singly scattered, and some peripheral cells contain the calcium oxalate cubic crystals to form crystal fibers; (2) the ranges of water content, total ash content and extract content of the medicinal materials are as follows: the water content is not more than 15.0%, the total ash content is not more than 9.0%, and the extract is measured by hot dipping method under alcohol-soluble extract measuring method (2201 in the four-part general rule of the Chinese pharmacopoeia 2015 edition), and diluted ethanol is used as solvent, and is not less than 15.0%; (3) identifying by thin-layer chromatography; the test solution should have fluorescence spots with the same color at the corresponding positions on the chromatogram of the gallic acid control solution.
2. The quality control method of the bauhinia championii medicinal material according to claim 1, which is characterized in that: the thin-layer chromatography adopts a silica gel G thin-layer plate, toluene-ethyl acetate-formic acid is used as a developing agent, and a 10% phosphomolybdic acid ethanol solution is used as a color developing agent.
3. The quality control method of the bauhinia championii medicinal material according to claim 2, which is characterized in that: the thin layer chromatography was performed as follows: respectively dropping 5-10 μ l of sample solution and 5-10 μ l of reference solution on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-formic acid solution at volume ratio of 4-6:3-5:1 as developing agent, taking out, and air drying; heating 10% phosphomolybdic acid ethanol solution at 105 deg.C until spots are clear, and spots with the same color appear on the chromatogram of the test solution at the position corresponding to the chromatogram of the control solution.
4. The quality control method of the bauhinia championii medicinal material according to claim 3, which is characterized in that: the volume ratio of the developing agent toluene to ethyl acetate to formic acid is 5:4: 1.
5. The quality control method of the bauhinia championii medicinal material according to claim 3, which is characterized in that: the test sample is prepared by the following method: putting the powder of the test sample into a conical flask with a plug, adding an organic solvent, carrying out ultrasonic treatment, filtering, and concentrating to obtain a test sample solution.
6. The quality control method of the bauhinia championii medicinal material according to claim 5, which is characterized in that: the test sample is prepared by the following method: taking about 2g of the coarse powder of the product, adding 20ml of chloroform-methanol (8:2), performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue with 1ml of methanol to obtain a test solution.
7. The quality control method of the bauhinia championii medicinal material according to claim 3, which is characterized in that: the reference substance is prepared by the following method: taking gallic acid control, adding methanol to make 1ml solution containing 1mg as control solution.
8. The quality control method of the bauhinia championii medicinal material according to claim 1, which is characterized in that: the method also comprises content measurement, wherein a mobile phase adopted by the content measurement is a methanol-0.1% phosphoric acid solution, the column temperature is 25 ℃, and the detection wavelength is 275 nm.
9. The quality control method of the bauhinia championii medicinal material according to claim 8, which is characterized in that: octadecylsilane chemically bonded silica is used as a filling agent; the volume ratio of the components is 4: 96% methanol-0.1% phosphoric acid solution as mobile phase; the column temperature is 25 ℃, and the flow rate is 1.0 ml/min; the detection wavelength is 272 nm; the number of theoretical plates is not less than 2000 calculated according to gallic acid peak, respectively and precisely sucking 10 μ l of reference solution and sample solution, respectively, injecting into liquid chromatograph, and measuring.
10. The quality control method of the bauhinia championii medicinal material according to claim 9, which is characterized in that: taking a proper amount of gallic acid reference substance, precisely weighing, and adding 80% methanol to obtain solution containing 45ug per 1ml to obtain reference substance solution; taking 0.5g of medicinal powder (passing through a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 4mol/L hydrochloric acid solution, sealing the plug, weighing, heating and refluxing for 3 hours, cooling, weighing again, complementing the weight loss by 4mol/L hydrochloric acid solution, shaking up, filtering, and taking the subsequent filtrate to obtain the sample solution.
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