CN108548889B - Quality standard of Mongolian medicinal material Jinyazi - Google Patents
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Abstract
The invention discloses a quality standard of a Mongolian medicinal material Jinyazi, which comprises a microscopic characteristic standard of whole herb powder, a moisture content, ash content and extract content standard, a thin-layer chromatography standard and a content standard of 2.42-3.20% of 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-O- β -D-glucoside, wherein fluorescent spots with the same color are displayed at positions corresponding to a chromatogram of a test sample, and the content standard of the 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-O- β -D-glucoside serving as the Jinyazi is simple in operation and strong in specificity.
Description
Technical Field
The invention relates to the field of traditional Chinese medicinal materials, and in particular relates to a quality standard of a Mongolian medicinal material Jinyazi.
Background
Mongolian medicinal material Chrysosplenium nudum Bunge is dry whole plant of Gymnospermum nudum of Chrysosplenium of Saxifragaceae, Mongolian name Alatan-Bo-Ri, perennial herb, and whole plant is used as medicine. Grown in the forest with elevation 3900-. Distributed in Gansu, Qinghai, Xinjiang, Sichuan, Yunnan and Tibet. Bitter taste and cold nature. When the medicine is used in coordination with the above-mentioned herbs, it can clear heat and alleviate stabbing pain. Can be used for treating pyretic and solar diseases, liver heat, yellow stain of eye skin, headache due to blood circulation, and subarachnoid headache. According to literature reports, the 90% ethanol extract of the nude stem chrysosporium has good anti-tumor activity on 5 human tumor cells (breast cancer, lung cancer, rectal cancer, oral epithelial cancer, prostate cancer and the like), and the inhibition rate on the tumor cells reaches 75%.
At present, a large amount of literature data show that systematic research on a plurality of Mongolian medicine quality standards is gradually carried out, qualitative identification combined with modern technological means and content measurement contents are continuously carried out, firstly, only identification items are specified for the chrysosporium in the Mongolian medicine standard, people in 2004 to 2006 carry out preliminary research on chemical components and pharmacological actions of the chrysosporium nudum waist, and self-made 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside is used as a reference substance to measure the content of the chrysosporium waist, but the standards for examination items such as water, ash, extracts and the like, microscopic identification, thin-layer chromatography identification with special components as indexes and the like are still lack, the research and application of the chrysosporium are rapidly developed, and the existing quality control and quality of the chrysosporium cannot be met.
Disclosure of Invention
In order to solve the problems, the invention provides a quality standard of a Mongolian medicinal material Jinyao, which can effectively evaluate and control the inherent quality and the medication quality of the medicinal material.
In order to achieve the purpose, the invention adopts the technical scheme that:
the quality standard of Mongolian medicinal material Jinyao is characterized in that: the method comprises the following steps:
(1) microscopic characteristics of whole grass powder: the powder is yellow-green to green; the pollen grains are round or triangular, the diameter is 17-22 mu m, fine granular sculptures are formed on the surface, and 3 germination grooves are formed; the epidermal cells on the leaves are polygonal, the vertical walls of the leaves are thickened like pearls, and pores are rare; the cell wall of the lower epidermal cell is wavy and curved, and pores are more inequality and less inequality;
(2) the content ranges of water, ash and extract of the medicinal materials are as follows: the water content is between 7.52 and 10.28 percent, and the average value is 8.78 percent; the ash content is 18.24-14.51%, and the average value is 16.91%; the extract content is between 34.06 percent and 27.74 percent, and the average value is 30.43 percent;
(3) the thin-layer chromatography result shows that the positions of the chromatogram of the test sample, which correspond to the chromatogram of the compound 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside, show fluorescence spots with the same color;
(4) the content of 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside as the cashew nut is 2.42-3.20%.
Further, the thin layer chromatography is operated by:
s1, precisely weighing 5.1mg of compound 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside, transferring into a volumetric flask, adding methanol to a constant volume of 5ml, and making into 1.0 mg/ml-1The control solution of (4).
S2, precisely weighing 0.5g of naked stem and golden waist powder which passes through a 60-mesh sieve, transferring the powder into a conical flask with a plug, adding 25ml of methanol, carrying out ultrasonic treatment at the temperature of 45 ℃ for one hour under the power of 100W and the frequency of 40KHz, transferring the powder into a volumetric flask, carrying out suction filtration by using a Buchner funnel, transferring the powder into the volumetric flask, fixing the volume to 25ml by using the methanol, and filtering by using a 0.45-micron microporous membrane to obtain a test solution;
s3, sucking 5 μ L of sample solution and 3 μ L of reference solution, respectively dropping on the same silica gel G thin layer plate, sealing and pre-balancing with ethyl acetate-methanol-glacial acetic acid mixed solution as developing agent, wherein the volume ratio of ethyl acetate, methanol and glacial acetic acid is 7.3: 2.3: 0.4, for 30min, developing (covering and sealing), taking out, air drying, spraying 3% aluminum trichloride ethanol solution, blowing with a blower for 2min, and inspecting under 365nm ultraviolet lamp.
Furthermore, the water content, ash content and extract content of the medicinal materials are respectively identified by referring to a water determination method, an ash determination method and an extract determination method in 'Chinese pharmacopoeia' of 2015 edition.
The method for identifying the medicinal material properties, the microscopic property and the thin layer has the advantages of simple operation and strong specificity, the average content of water is 8.78%, the average content of total ash is 16.91%, the average content of extract is 30.43%, under the selected HPLC condition, the linear relation of the compound 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside is good in the range of 1.909 mu g-12.32 mu g, the linear regression equation is Y2282.7 x +1107.2, the correlation coefficient r 0.9991, the average sample recovery rate is 98.40%, the RSD is 1.8%, the content of the compound in seven batches of medicinal materials is 2.42% -3.20%, the quality standard of the Mongolian medicinal material cashew is established according to the data, and the inherent quality and the medication quality of the medicinal materials can be effectively evaluated and controlled.
Drawings
FIG. 1 is a microscopic image of the epidermis over the leaves of the waist of bare stem King.
FIG. 2 is a microscopic image of the epidermis under the leaves of the nude stem Aurea.
FIG. 3 is a microscopic feature of the naked stem cashew pollen grains.
FIG. 4 is a thin layer chromatogram.
FIG. 5 is an HPLC chromatogram;
wherein: (a) a control; (b) naked stem gill waist sample.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to 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.
Example 1
Medicinal materials
The Chrysosplenium medicinal material is purchased from Qinghai province, the detailed information is shown in Table 1, and the medicine is identified as dry whole plant of Gymnophilum nudum (Chrysosplenium nudicaule Bunge) of Saxifragaceae by professor assisting in the pharmacy of college students of inner Mongolia medical science.
TABLE 1 golden cashew medicinal material information table
Chemical reagent
The reference substance 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside (content: 98%, self-made), acetonitrile as chromatographic pure, water as Milli-Q ultrapure water, and other reagents as analytical pure
The product has short rhizome, mostly grows fine roots, and is brown to tan. The leaves are shriveled and broken, the intact ones have slender and long handles, leaf leathery texture and kidney shape, the length is about 0.9cm, the width is about 1.3cm, the edges have 11-15 shallow teeth, the bent parts between the teeth have brown soft hair or nipple bulges, the teeth are oblate, the length is about 0.3cm, the width is about 0.4cm, the tips are sunken and have 1 wart point. The parasol inflorescence is dense and hemispherical, is made of bract leathery material, is wide in egg shape to fan shape, and has 3-9 shallow teeth. Yellow and green, without petals; the calyx is oblate and blunt; a stamen 8. Black brown seed, egg-shaped. Light smell, sour and astringent taste.
1 authentication
Microscopic identification
The herb is mainly aerial part, with the largest leaf proportion. In the case of the harvest in the flowering phase, the calyx is also green, but the color of each part gradually becomes lighter as the storage time is prolonged, so the powder color is from yellow green to green.
The microscopic identification of the product mainly observes the types of the tablets including the leaf surface tablets and the whole grass powder tablets, and experiments prove that the microscopic characteristics observed in the leaf surface tablets are all seen in the medicinal material powder tablets, so the product only establishes the standard for the microscopic characteristics of the whole grass powder.
A small amount of the powder is taken to prepare a powder tablet, and the following microscopic characteristics can be observed under an optical microscope: the powder was yellow-green to green. The pollen grains are round or triangular, the diameter is 17-22 mu m, the surface is provided with fine granular sculptures, and 3 germination grooves are formed. The epidermal cells on the leaves are polygonal, the vertical walls of the leaves are thickened like pearls, and pores are rare; the cell wall of the lower epidermal cell is wavy and curved, and the pores are more inequality and less inequality.
Experimental observation shows that the microscopic characteristics of the bare-stem and golden-waist powder in different batches are completely consistent. The structures of pollen grains, epidermal cells, stomatal axis and the like have identification significance and are easy to observe, so the income standard is met.
2. Identification by thin layer chromatography
Through literature investigation and preliminary experiments, the thin-layer identification is finally determined by taking 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside as a control, the test can be carried out according to the thin-layer identification method of Chinese pharmacopoeia (2015 edition I, appendix VIB), the spots are clearly separated, the identification effect is satisfactory, and samples of different batches are positive.
2.1 preparation of Standard solutions
Accurately weighing 5.1mg of reference substance, transferring into volumetric flask, adding methanol to constant volume of 5ml, and making into 1.0 mg. m1-1The control solution of (4).
2.2 preparation of test solutions
Weighing bare stem and golden waist powder (sieved by a 60-mesh sieve) 0.5g precisely, transferring into a conical flask with a plug, adding methanol 25ml, adding methanol with power of 100W, frequency of 40KHz, temperature of 45 ℃, performing ultrasonic treatment for one hour, performing suction filtration by using a Buchner funnel, transferring into a volumetric flask, performing constant volume treatment to 25ml by using methanol, and filtering by using a 0.45-micron microporous membrane to obtain the product.
2.3 thin layer chromatography conditions and results
Sucking 5 μ L of sample solution and 3 μ L of reference solution, respectively dropping on the same silica gel G thin layer plate, using chloroform, methanol, glacial acetic acid (7.8: 1.8: 0.4), dichloromethane, methanol, glacial acetic acid (8.3: 1.3: 0.4), ethyl acetate, methanol, glacial acetic acid (7.3: 2.3: 0.4) as developing agent, sealing and pre-balancing with vapor for 30min, developing (covering and sealing), taking out, air drying, spraying 3% aluminum trichloride ethanol solution (3G of aluminum trichloride in 100mL of ethanol), blowing with blower for 2min, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test sample, the fluorescence spots having the same color as those in the chromatogram of the control sample are observed, and it is considered that ethyl acetate, methanol and glacial acetic acid (7.3: 2.3: 0.4) are preferable as developing agents. See fig. 4.
3 examination item and extract measurement
3.1 determination of moisture
Since the product is susceptible to dry or humid environments, the text specifies a limit check of moisture to control the purity of the medicinal material. The seven batches of the medicinal materials in the table 1 are measured according to the moisture measurement method in Chinese pharmacopoeia (2015 edition, general rules of the four departments, 0832, second method). The results show that the moisture content of seven samples is between 7.52% and 10.28%, and the average value is 8.78%. The results are shown in Table 2.
3.2 determination of Ash
The ash content of seven batches of the medicinal materials in the table 1 is measured according to the ash content measurement method in Chinese pharmacopoeia (2015 edition four-part general rule 2302). The results show that the ash content of seven samples is 18.24-14.51%, and the average value is 16.91%. The results are shown in Table 2.
3.3 extract determination
The seven batches of the medicinal materials in the table 1 are measured according to the extract measuring method in Chinese pharmacopoeia (2015 edition of the four ministry of general regulations 2201 water soluble extract cold immersion method). The results show that the extract content of seven samples is between 34.06% and 27.74%, and the average value is 30.43%. The results are shown in Table 2.
TABLE 2 measurement results of medicinal material of naked-stem Jinyao
4 content determination
The Mongolian medicinal material golden cashew nut mainly contains flavonol compounds, and 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside in the golden cashew nut is selected as a content determination component of the golden cashew nut from the comprehensive consideration of the characteristics of the components, the source and the content of a reference substance and the like.
4.1 chromatographic Condition and specificity Studies
Chromatographic conditions are as follows: mobile phase acetonitrile-water (25: 75), flow rate: 1 mL. min-1The detection wavelength is 350nm, the column temperature is 30 ℃, the test time is 50min, under the selected chromatographic condition, the separation degree of a reference substance 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside chromatographic peak and an adjacent chromatographic peak is more than 1.5, the tailing factors are within the range of 0.95-1.05, and the theoretical plate number is more than 10000 calculated according to the reference substance 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside peak.
4.2 specialization examination
Taking the reference solution, the test solution and the mobile phase respectively, and determining under the chromatographic conditions. The result shows that the chromatogram of the test sample has a corresponding chromatographic peak (see figure 5) at the retention time corresponding to the reference substance, and the chromatogram of the mobile phase has no chromatographic peak at the corresponding retention time, which indicates that the method is negative and has no interference and good specificity.
4.3 methodological inspection
Investigation of linear relationships
The control solution under item 2.1 was sampled at 2. mu.L, 5. mu.L, 7.5. mu.L, 10. mu.L and 12.5. mu.L, respectively, with the amount of the sample taken as the abscissa X, and the peak area was recorded and taken as the ordinate Y to plot the standard curve of the control, and as a result, the linear regression equation was 2282.7X +1107.2 and the correlation coefficient r was 0.9991, indicating that 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside had good linearity in the range of 1.909. mu.g to 12.32. mu.g.
Precision test
And taking the prepared reference substance solution, continuously injecting samples for 6 times according to the chromatographic conditions of the experiment, recording the chromatographic peak area (A), and calculating the RSD value to be 0.9%. The result shows that the precision of the method is better and meets the requirement.
Reproducibility test
6 parts of the same batch of sample powder (sample 2) are precisely weighed, a sample solution is prepared according to the method, 20 mu L of the sample solution is precisely absorbed, the measurement is respectively carried out, and the chromatogram is recorded. The result shows that the RSD value is 1.2 percent, which indicates that the sampling reproducibility of the instrument is good.
Stability test
Precisely sucking 20 μ L of the same sample solution (sample 2), performing sample injection determination at 0, 1, 2, 4, 8, and 12h respectively according to the above chromatographic conditions, and recording chromatogram. The result RSD was 0.8%, indicating that the sample solution was stable over 12 h.
Sample application recovery test
A sample powder (sieved through a 60-mesh sieve) with a known content is precisely weighed, a reference substance is added in a ratio of 1: 0.5, 1: 1 and 1: 1.5, the sample powder is placed in a conical flask and plugged, the sample solution is prepared according to the method for preparing the test solution under item 2.2, the chromatographic condition is tested under item 4.1, and the recovery rate is calculated as shown in the following table 3. The results showed good recoveries of 95.40% -99.88%, with an average value of 98.40% and an RSD of 1.8%.
TABLE 3 sample recovery test results
Content determination 7 batches of samples are respectively taken 2 parts and precisely weighed, a sample solution is prepared according to the method under item 2.2, 20 microlitres is precisely absorbed, the content of 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside in 7 batches of samples is respectively determined by an external standard method, the content of 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-0- β -D-glucoside is 2.42-3.20 percent and the total average content is 2.69 percent, and the results are shown in Table 2.
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 (1)
1. The quality detection method of the Mongolian medicinal material Jinyao is characterized by comprising the following steps: the method comprises the following steps:
s1, precisely weighing 5.1mg of compound 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-O- β -D-glucoside, transferring into a volumetric flask, adding methanol to a constant volume of 5ml, and making into 1.0 mg/ml-1The control solution of (4);
s2, precisely weighing 0.5g of naked stem and golden waist powder which passes through a 60-mesh sieve, transferring the powder into a conical flask with a plug, adding 25ml of methanol, performing ultrasonic treatment for one hour at the power of 100W and the frequency of 40KHz and the temperature of 45 ℃, performing suction filtration by using a Buchner funnel, transferring the powder into a volumetric flask, performing constant volume to 25ml by using the methanol, and filtering by using a 0.45 mu m microporous membrane to obtain a test solution;
s3, sucking 5 muL of a test sample solution and 3 muL of a reference solution, respectively dropping the test sample solution and the reference solution on the same silica gel G thin-layer plate, taking a mixed solution of ethyl acetate, methanol and glacial acetic acid with the volume ratio of 7.3: 2.3: 0.4 as a developing agent, sealing and pre-balancing the developing agent by using steam for 30min, developing, taking out, airing, spraying a 3% aluminum trichloride ethanol solution, blowing for 2min by using a blower, and inspecting under an ultraviolet lamp with 365nm, wherein in a chromatogram of the test sample, fluorescent spots with the same color as that of a compound 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-O- β -D-glucoside show at positions corresponding to the chromatogram, and the content of the 5,4 '-dihydroxy-3, 6, 3' -trimethoxy-flavone-7-O- β -D-glucoside is 2.42% -3.20%.
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