CN110658282B - Construction method of UPLC characteristic spectrum of fresh herba Houttuyniae standard decoction and detection method of fresh herba Houttuyniae preparation - Google Patents
Construction method of UPLC characteristic spectrum of fresh herba Houttuyniae standard decoction and detection method of fresh herba Houttuyniae preparation Download PDFInfo
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Abstract
The invention relates to a construction method of a UPLC characteristic spectrum of a standard decoction of fresh houttuynia cordata and a detection method of a fresh houttuynia cordata preparation. The construction method of the UPLC characteristic map comprises the following steps: preparing a reference substance solution; preparing freeze-dried powder of standard decoction of houttuynia cordata; preparation of a test solution: adding 65-75% ethanol water solution into the freeze-dried powder of the houttuynia cordata standard decoction for extraction, filtering the extracting solution, and taking the subsequent filtrate to obtain a test solution; and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, a mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution. The characteristic spectrum constructed by the construction method can comprehensively reflect the characteristics of the standard houttuynia cordata decoction, and has comprehensive and reliable quality detection and short time.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine quality detection, in particular to a construction method of a UPLC characteristic spectrum of a standard decoction of fresh houttuynia cordata and a detection method of a fresh houttuynia cordata preparation.
Background
Fresh Houttuynia cordata is a fresh whole plant of Houttuynia cordata (Houttuynia cordiata Thunb.) of Saururaceae, is widely distributed in northwest, North China, China and south of Yangtze river, and is one of famous plant resources with homology of medicine and food. The fresh cordate houttuynia is used as a medicine by the whole herb, has pungent taste and slightly cold property, and has the effects of clearing heat and removing toxicity, eliminating carbuncle and expelling pus, and inducing diuresis and treating stranguria.
Because of the limitation of the factors of difficult preservation and storage, single supply channel, deficient variety of fresh medicinal preparations, inconvenient clinical dispensing and use and the like, the traditional medicinal fresh houttuynia cordata is gradually replaced by the dry houttuynia cordata. However, research shows that the bacteriostatic, anti-inflammatory and cough-relieving pharmacological activities of the fresh houttuynia cordata are superior to those of dry houttuynia cordata, so that if the fresh houttuynia cordata is clinically used in a formula with dry houttuynia cordata replacing fresh houttuynia cordata, the special pharmacological effects of the fresh houttuynia cordata cannot be exerted, and the traditional Chinese medicine theory is contradictory.
The traditional Chinese medicine formula particle is prepared by taking single traditional Chinese medicine decoction pieces as raw materials, extracting, concentrating and granulating by adopting the modern pharmaceutical technology, is used for the traditional Chinese medicine clinical formula, and has the advantages of no need of decoction, direct taking, small dosage, sanitation, safety, convenient carrying and storage and the like. If the fresh houttuynia cordata is prepared into the formula particles by using the modern process, the problems of difficult fresh-keeping and storage, high transportation cost, inconvenient clinical dispensing and the like of the fresh houttuynia cordata can be solved, and the characteristics of fresh medicine of the fresh houttuynia cordata are exerted.
The standard decoction of Chinese medicinal decoction pieces is prepared by standard process based on Chinese medicinal theory and clinical application, and referring to modern extraction method, and can be used for marking clinical medication and ensuring accuracy and dosage consistency. At present, because the traditional fresh medicines have various usages such as decoction, juice smashing, external application and the like, the application of a standard decoction on the fresh medicines is not common, but the usage of the fresh houttuynia cordata specified by pharmacopoeia is decoction with water or juice smashing, so the decoction of the fresh houttuynia cordata standard decoction can be used as a standard substance of a fresh houttuynia cordata formula granule or a novel preparation thereof. The freeze-dried powder is prepared by carrying out low-temperature reduced pressure concentration and vacuum freeze drying on a decoction, and can fully keep the chemical stability and the biological activity of the material components in the standard decoction, so the prepared fresh houttuynia cordata standard decoction freeze-dried powder is used for standardizing the quality of fresh houttuynia cordata formula granules or a novel preparation thereof, and the accuracy of clinical medication and the consistency of curative effect can be ensured.
The characteristic map of the traditional Chinese medicine is used for obtaining comprehensive chemical component characteristic information of the traditional Chinese medicine and is widely applied to quality control of the traditional Chinese medicine, the traditional Chinese medicine characteristic map is used for expressing and reflecting the integral chemical characteristics of the traditional Chinese medicine, and the consistency of the characteristic map is also one of important indexes of the consistency of formula granules and standard decoction. Therefore, it is very necessary to establish a characteristic map of the standard decoction of fresh houttuynia cordata.
Disclosure of Invention
Therefore, a method for constructing a UPLC characteristic spectrum of a standard decoction of fresh houttuynia cordata is needed. The characteristic spectrum constructed by the construction method can comprehensively reflect the characteristics of the standard decoction of the fresh houttuynia cordata, has comprehensive and reliable quality detection and short time, can well control the quality of the fresh houttuynia cordata formula granules or the novel preparation thereof, and ensures the consistency of the curative effect of the fresh houttuynia cordata and the traditional decoction.
A method for constructing a UPLC characteristic spectrum of a standard decoction of fresh houttuynia cordata comprises the following steps:
preparation of control solutions: dissolving chlorogenic acid, cryptochlorogenic acid, hyperoside and quercetin reference substance with solvent to obtain reference substance solution;
freeze-dried powder preparation of fresh houttuynia cordata standard decoction: taking fresh houttuynia cordata decoction pieces, adding water for extraction, carrying out solid-liquid separation, concentrating the obtained extracting solution, and carrying out freeze drying to obtain freeze-dried powder of the fresh houttuynia cordata standard decoction;
preparation of a test solution: adding 65-75% ethanol water solution into the freeze-dried powder of the fresh houttuynia cordata standard decoction for extraction, filtering the extracting solution, and taking the subsequent filtrate to obtain a test solution;
and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, a mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution.
In one embodiment, the gradient elution comprises the following procedure:
the volume percentage of the mobile phase A is increased from 5% to 11% in 0-7 min;
the volume percentage of the mobile phase A is increased from 11% to 11.5% within 7-10 min;
the volume percentage of the mobile phase A is increased from 11.5% to 20% in 10-13 min;
and (3) 13-20 min, wherein the volume percentage of the mobile phase A is increased from 20% to 25%.
In one embodiment, the conditions of the ultra high performance liquid chromatograph include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 200-400 nm; flow rate: 0.25-0.35 mL/min; column temperature: 25-35 ℃.
In one embodiment, the detection wavelength is a program wavelength: 326nm for 0-13 min and 254nm for 13-20 min.
In one embodiment, the flow rate is 0.3 mL/min; the column temperature was 30 ℃.
In one embodiment, in the preparation of the test solution, the extraction method is ultrasonic extraction, the ultrasonic power is 200-300W, and the frequency is 30-50 kHZ; the extraction time is 25-35 min.
In one embodiment, in the preparation of the freeze-dried powder of the standard decoction of fresh houttuynia cordata, the water extraction method comprises two times of decoction: adding 8-14 times of water into the first decoction, heating the mixture to boil with strong fire, and decocting the mixture with slow fire for 20-30 min; adding 6-12 times of water into the second decoction, heating the second decoction with strong fire until the second decoction is boiled, and decocting the second decoction with slow fire for 15-25 min; and/or the mesh number of the screen mesh adopted for solid-liquid separation is 200-400 meshes; and/or the concentration temperature is 40-70 ℃, preferably 50-60 ℃.
In one embodiment, the freeze-drying procedure comprises: the pre-freezing temperature is-50 to-30 ℃, and the pre-freezing time is 1 to 2 hours; the sublimation drying temperature is-45-0 ℃, the sublimation drying time is 40-80 hours, and the vacuum degree is 0.1-0.3 mbar; the analysis drying temperature is 10-30 ℃, the analysis drying time is 3-10 hours, and the vacuum degree is 0 mbar.
In one embodiment, the dissolution is performed using a solvent identical to the extracted solvent used in the preparation of the test solution.
In one embodiment, the characteristic profile comprises 7 common peaks, the 7 common peaks comprising characteristic peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, hyperin and quercetin.
The invention also provides a detection method of the fresh houttuynia cordata preparation, which comprises the following steps:
preparing a sample solution to be tested: taking a to-be-detected fresh houttuynia cordata preparation, grinding, adding 65-75% ethanol water solution by volume fraction, extracting, filtering the extracting solution, and taking a subsequent filtrate to obtain a to-be-detected sample solution;
and injecting the reference substance solution and the sample solution to be detected into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, a mobile phase B is a phosphoric acid water solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution.
In one embodiment, the gradient elution comprises the following procedure:
the volume percentage of the mobile phase A is increased from 5% to 11% in 0-7 min;
the volume percentage of the mobile phase A is increased from 11% to 11.5% within 7-10 min;
the volume percentage of the mobile phase A is increased from 11.5% to 20% in 10-13 min;
and (3) 13-20 min, wherein the volume percentage of the mobile phase A is increased from 20% to 25%.
In one embodiment, the conditions of the ultra high performance liquid chromatograph include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 200-400 nm; flow rate: 0.25-0.35 mL/min; column temperature: 25-35 ℃.
In one embodiment, the detection wavelength is a program wavelength: 326nm for 0-13 min and 254nm for 13-20 min.
In one embodiment, the flow rate is 0.3 mL/min; the column temperature was 30 ℃.
In one embodiment, in the preparation of the sample solution to be detected, the extraction method is ultrasonic extraction, the ultrasonic power is 200-300W, and the frequency is 30-50 kHZ; the extraction time is 25-35 min.
In one embodiment, the fresh houttuynia cordata preparation is a fresh houttuynia cordata granule prepared by taking fresh houttuynia cordata decoction pieces as raw materials, taking water as a solvent for extraction, concentrating, drying and adding auxiliary materials.
In one embodiment, the solvent used for dissolving the control solution is the same as the extracted solvent used for preparing the sample solution to be tested.
Compared with the prior art, the invention has the following beneficial effects:
the UPLC characteristic spectrum establishing method of the fresh houttuynia cordata standard decoction can establish the characteristic spectrum of the obtained fresh houttuynia cordata standard decoction, comprehensively reflects the main chemical component information of the houttuynia cordata standard decoction, is beneficial to quality control and evaluation of fresh houttuynia cordata preparations (such as formula granules), enables the main index components of the fresh houttuynia cordata preparation to be consistent with those of the traditional decoction, and provides reference for quality research and standard establishment of the fresh houttuynia cordata preparation.
Furthermore, the freeze-dried powder of the standard decoction of fresh houttuynia cordata prepared by the invention has the advantages of smooth surface, uniform color and small hygroscopicity, can be used as a standard reference substance to accurately reflect the component information of the standard decoction of fresh houttuynia cordata, and also provides a quality standard reference substance for a fresh houttuynia cordata preparation.
The detection method of the fresh houttuynia cordata preparation can comprehensively reflect the main chemical component information of the fresh houttuynia cordata preparation, and is convenient for comprehensively monitoring the quality of the fresh houttuynia cordata preparation. The detection method is simple and convenient to operate, short in time consumption, high in precision, high in accuracy, good in stability and good in reproducibility, and is convenient for large-scale popularization and application.
Drawings
FIG. 1 is a characteristic map of lyophilized powder of standard decoction of fresh houttuynia cordata according to an embodiment of the present invention (Peak 1: neochlorogenic acid; Peak 2 (S1): chlorogenic acid; Peak 3: cryptochlorogenic acid; Peak 5: hyperin; Peak 7: quercitrin (S2));
FIG. 2 is a chromatogram for absorption wavelength investigation in the above-mentioned characteristic map creation process;
FIG. 3 is a chromatogram for mobile phase investigation in the above-mentioned feature map creation process;
FIG. 4 is a chromatogram for column temperature investigation in the above-mentioned characteristic map establishing process;
FIG. 5 is a chromatogram for flow rate investigation in the above-mentioned feature map creation process;
FIG. 6 is a chromatogram for the overall examination in the above-mentioned feature map creation process;
FIG. 7 is a chromatogram of chromatogram peak (characteristic peak) of lyophilized powder of standard decoction of fresh herba Houttuyniae during the establishment of the above characteristic chromatogram (S1: mixed reference substance; S2: lyophilized powder of standard decoction of fresh herba Houttuyniae; 1: neochlorogenic acid; 2: chlorogenic acid; 3: cryptochlorogenic acid; 4: hyperoside; 5: quercetin);
FIG. 8 is a characteristic spectrum of 17 batches of lyophilized powder of standard decoction of fresh herba Houttuyniae;
FIG. 9 is a chromatogram for quality detection of three batches of fresh herba Houttuyniae granule.
Detailed Description
The method for constructing UPLC characteristic spectrum of standard decoction of fresh houttuynia cordata and the method for detecting fresh houttuynia cordata preparation of the present invention are further described in detail with reference to the following specific examples.
The instruments and reagents used in the examples of the invention were as follows:
1. instrument for measuring the position of a moving object
Ultra-high performance liquid chromatograph: waters H-Class UPLC, Thermo Vanqish UPLC, Agilent Infinity 1290 UPLC;
an electronic balance: ME203E, ME204E, XP26(METTLER TOLEDO Co.)
An ultra-pure water machine: MiliQ Direct 8 (Merck Millipop Co., Ltd.)
A chromatographic column: waters ACQUITY HSS T3(2.1 mm. times.100 mm, 1.8 μm), Agilent SB C18(2.1 mm. times.100 mm, 1.8 μm), Agilent Eclipes Plus C18(2.1 mm. times.100 mm, 1.8 μm)
2. Reagent
Acetonitrile and phosphoric acid are chromatographically pure, and water is ultrapure water; the rest are actually analytically pure.
Neochlorogenic acid (Vickqi Biotech Co., Ltd., Sichuan province, lot number: wkq 18030107); chlorogenic acid (China institute for testing food and drug, batch No. 110753-201817); cryptochlorogenic acid (Vickqi Biotech Co., Ltd., Sichuan province, lot number: wkq 16081903); hyperin (China institute for testing and drug testing, lot number: 111521-201708); quercetin (Chinese food and drug testing institute, batch No.: 111538-201606)
Standard decoction of fresh houttuynia cordata: lot numbers BT1, BT2, BT3, BT4, BT5, BT6, BT7, BT8, BT9, BT10, BT11, BT12, BT13, BT14, BT15, BT16, BT 17.
Fresh houttuynia cordata formula granules: the lot numbers of KL1, KL2 and KL3 are fresh herba houttuyniae granules which are prepared by taking fresh herba houttuyniae decoction pieces as raw materials, taking water as a solvent for extraction, concentrating, drying and adding auxiliary materials.
Unless otherwise specified, the percentages in the embodiments of the invention are volume percentages, wherein the strong fire refers to 180-200 ℃, and the slow fire refers to 120-150 ℃.
Example 1 method for establishing characteristic spectrum of standard decoction of fresh houttuynia cordata
The establishing method comprises the following steps:
1) preparation of standard decoction: taking a fresh houttuynia cordata medicinal material, removing impurities, and cutting into sections to obtain fresh houttuynia cordata decoction pieces; weighing 200g of fresh houttuynia cordata decoction pieces, placing the decoction pieces in an electric ceramic pot, adding water for decocting twice, adding 12 times of water for the first time of decoction, soaking for 30 minutes, boiling with strong fire, keeping slightly boiling for 20 minutes with slow fire, filtering the decoction while the decoction is hot through a 350-mesh screen, and quickly cooling the filtrate with cold water; adding 10 times of water for the second time, heating with strong fire to boil, keeping boiling with slow fire for 15 minutes, filtering the decoction with 350 mesh sieve while it is hot, rapidly cooling the filtrate with cold water, and mixing the two decoctions; and (3) carrying out reduced pressure and low temperature concentration by adopting a rotary evaporator until the material feeding amount is: the mass of the clear paste is 1:0.5 of extract; subpackaging into 10ml brown penicillin bottles under magnetic stirring, transferring into a vacuum freeze dryer for freeze-drying after subpackaging.
2) Control reference solution preparation: precisely weighing appropriate amount of neochlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, hyperoside reference substance, and quercetin reference substance, and respectively adding 70% ethanol water solution to obtain reference substance solution containing 10 μ g of reference substance per 1 mL.
3) Preparing a test solution: taking 0.1g of fresh houttuynia cordata standard decoction powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 70% ethanol aqueous solution, sealing the plug, weighing, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% ethanol aqueous solution, shaking up, filtering, and taking the subsequent filtrate.
4) And (3) detection: respectively sucking 1 μ L of reference substance solution and sample solution of reference substance, respectively, and injecting into an ultra high performance liquid chromatograph under the following chromatographic conditions:
octadecylsilane chemically bonded silica was used as a filler (Waters ACQUITY HSS T3, column length 100mm, inner diameter 2.1mm, particle diameter 1.8 μm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid water solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table 1; the flow rate is 0.3mL per minute; the column temperature is 30 ℃; the detection wavelength is 326nm in 0-13 min and 254nm in 13-25 min. The number of theoretical plates should not be less than 30000 calculated by chlorogenic acid peak.
TABLE 1
5) As a result: the test sample characteristic map should present 7 characteristic peaks, the peak corresponding to the chlorogenic acid reference peak is the S1 peak, the peak corresponding to the quercitrin reference peak is the S2 peak, the relative retention time of the peak 1, the peak 3 and the S1 peak, the relative retention time of the peak 4, the peak 5, the peak 6 and the S2 peak should be respectively calculated, the relative retention time should be within the range of +/-10% of the specified value, the specified value is: 0.66 (neochlorogenic acid), 1.09 (cryptochlorogenic acid), 1.80 (peak 4), 1.82 (hyperin), 1.85 (peak 6). (as shown in figure 1) preparation method of lyophilized powder of standard decoction of fresh herba Houttuyniae
1. Investigation of extraction Process
Selection of a decocting utensil: according to the regulations of a decoction container (the decoction container should be made of ceramic, stainless steel, copper and other materials, and the like, which are easy to corrode are forbidden) in the national traditional Chinese medicine issue (2009) No. 3 text of the medical institution traditional Chinese medicine decoction room management Specification and the habit of decocting decoction at home, a 3L automatic decoction ceramic pot is selected as the standard decoction decocting equipment for fresh houttuynia cordata.
Water addition amount investigation: because the quality and the water absorption rate of the traditional Chinese medicine decoction pieces are greatly different, the water adding amount is determined according to different decoction pieces. According to technical requirements for quality control and standard formulation of traditional Chinese medicine formula granules, standard decoction is prepared, and the water is preferably added to be 2-5 cm above the medicine surface.
The water content of the fresh houttuynia cordata is high, and compared with the traditional dry medicinal materials, the water absorption capacity of the fresh houttuynia cordata has larger difference, so 5 water adding quantities with different levels are drawn up, the influence on the standard decoction of the fresh houttuynia cordata is inspected, and the optimal water adding quantity range is preferably selected so as to meet the requirement that the water adding quantity is 2-5 cm higher than the liquid level in the technical requirement of quality control and standard formulation of Chinese medicinal formula granules.
Taking 100g of fresh houttuynia cordata decoction pieces, putting the fresh houttuynia cordata decoction pieces into a 3L ceramic pot, measuring 6-12 times of water for the first decoction and 4-10 times of water for the second decoction, respectively, soaking the height of the medicine surface with the water, and measuring the cream rate and the content of quercetin, wherein the measurement is carried out for 2 times in parallel. The results are shown in Table 2.
TABLE 2
Experimental results show that except for adding 8 times of water for the first decoction and 6 times of water for the second decoction, the water adding amount of the rest of the water for each level meets the requirement that the solvent leaching liquid level is 2-5 cm, and the difference of the paste yield is small. When 16 times of water is added in the first decoction, the liquid level of the solvent is nearly 5cm, the upper limit of the water addition amount is easily exceeded, and the concentration time is prolonged due to too much water addition amount, which is not beneficial to the retention of the material components. Therefore, the final amount of water is preferably 8-14 times of water in the first decoction, and 6-12 times of water in the second decoction.
Investigating the dosage of the decoction pieces: according to the suggestion under the preparation item of 'standard decoction' in the technical requirements for quality control and standard formulation of traditional Chinese medicine formula granules, the amount of decoction pieces used per decoction is generally 100-200 g. According to the preparation method of the standard soup, 100g of decoction pieces and 200g of decoction pieces are respectively weighed, and the dosage and cream yield of different decoction pieces and the difference of the content of the target component quercetin are inspected. The results are shown in Table 3.
TABLE 3
Water addition amount investigation and decoction piece dosage investigation experimental results show that the cream yield and the quercetin content of different decoction pieces have no obvious difference. However, the amount of freeze-dried powder produced by preparing standard decoction from 100g of fresh houttuynia cordata is about 4.5g, which cannot meet the dosage of UPLC characteristic spectrum study of the subsequent standard decoction freeze-dried powder, so the dosage of decoction pieces is 200 g.
Investigation of the decoction time: according to the suggestion under the item of 'preparation of standard decoction' in the technical requirements for quality control and standard formulation of traditional Chinese medicine formula granules, each dose of medicine is generally decocted twice. Therefore, according to the preparation method of the standard decoction, different decoction times are set, and the influence of the different decoction times on the quality of the standard decoction is inspected. The results are shown in Table 4.
TABLE 4
The observation result of the decoction time shows that the cream yield is gradually increased along with the extension of the decoction time, but the content of the quercetin is gradually reduced. When the decoction time is 10min for one decoction and 5min for two decoction, the dissolution of the substances in the fresh houttuynia decoction pieces is insufficient, and the extraction rate and the content of quercetin are both low; when the first decoction exceeds 30min and the second decoction exceeds 25min, the content of the lyophilized powder quercetin is obviously reduced. Considering that the fresh houttuynia cordata is a heat-clearing medicine, the decoction time is not suitable to be too long, so the standard decoction of the fresh houttuynia cordata is determined to be decocted twice, the decoction time is preferably that the decoction is carried out for 20-30 minutes after the decoction is boiled in the first decoction, and the decoction is carried out for 15-25 minutes after the decoction is boiled in the second decoction.
And (3) investigating solid-liquid separation conditions: the mesh number of the solid-liquid separation filter material is recommended to be more than 100 meshes under the item of 'standard decoction preparation' in the technical requirements of quality control and standard formulation of traditional Chinese medicine formula granules. In order to obtain decoction consistent with the traditional decoction by a modern solid-liquid separation method, the filtering effects of 100-mesh, 200-mesh, 350-mesh and 400-mesh sieves on the fresh houttuynia cordata decoction are mainly considered.
Taking 4 parts of fresh houttuynia cordata decoction pieces, 200g of each fresh houttuynia cordata decoction piece, putting the fresh houttuynia cordata decoction pieces into a 3L ceramic pot, adding 12 times of water into the decoction pieces for the first time, soaking the decoction pieces for 30 minutes, boiling the decoction pieces with strong fire, keeping the decoction pieces slightly boiling for 20 minutes with slow fire, filtering the decoction pieces while the decoction pieces are hot by using screens of 100 meshes, 200 meshes, 350 meshes and 400 meshes respectively, rapidly cooling the filtrate by using cold water, and rapidly cooling the filtrate by using the cold water. Adding 10 times of water for the second time, heating with strong fire to boil, keeping boiling with slow fire for 15min, filtering the decoction with 100 mesh, 200 mesh, 350 mesh and 400 mesh screens, rapidly cooling the filtrate with cold water, and mixing the filtrates. Observing the difficulty degree of the fresh houttuynia cordata decoction when the decoction is filtered by screens with different apertures, the clarity of the filtrate and the sediment amount after standing to determine proper solid-liquid separation conditions. The results are shown in Table 5.
TABLE 5
The experimental results show that: when the fresh houttuynia cordata is decocted and then subjected to solid-liquid separation, the 100-mesh, 200-mesh, 350-mesh and 400-mesh sieves are easy to filter, but the clarity of filtrate obtained by filtering through the 100-mesh sieve is poor, the amount of precipitate obtained after standing is large, and the clarity of filtrate obtained by filtering through the 200-mesh, 350-mesh and 400-mesh sieves is good, and the amount of precipitate obtained after standing is small. Therefore, the mesh number of the screen for solid-liquid separation and filtration of the standard decoction of fresh houttuynia cordata is preferably 200-400 meshes.
In conclusion, the extraction process of the standard decoction of the fresh houttuynia cordata is determined as follows: taking about 200g of fresh houttuynia cordata decoction pieces, placing the fresh houttuynia cordata decoction pieces in an electric ceramic pot, adding water for decoction twice, adding 8-14 times of water for the first decoction, soaking for 30 minutes, boiling with strong fire, keeping the slight boiling for 20-30 minutes with slow fire, filtering the decoction while the decoction is hot through a 200-400-mesh screen, and quickly cooling the filtrate with cold water. Adding 6-12 times of water for the second time, heating with strong fire to boil, keeping slightly boiling with slow fire for 15-25 minutes, filtering the decoction while hot by using a 200-400-mesh screen, quickly cooling the filtrate with cold water, and combining the two decoctions.
2. Investigation of concentration Process
According to the proposal of 'preparation of standard decoction' in the 'technical requirements for quality control and standard formulation of traditional Chinese medicine formula granules', a reduced pressure concentration method is adopted for low-temperature concentration to obtain an extract with a specified amount. Now, the concentration state was observed by comparing the different concentration temperatures to determine the appropriate concentration temperature and temporarily determining the concentration ratio to be 1:0.5 (feed-to-liquid ratio, g/ml).
Taking 200g of fresh houttuynia cordata decoction pieces, paralleling 2 parts, placing the fresh houttuynia cordata decoction pieces in a 3L ceramic kettle, adding 12 times of water into the decoction pieces for the first time, soaking the decoction pieces for 30 minutes, boiling the decoction pieces with strong fire, keeping the decoction pieces slightly boiling for 20 minutes with slow fire, filtering the decoction while the decoction is hot by using a 350-mesh screen, quickly cooling the filtrate by using cold water, and quickly cooling the filtrate by using the cold water. Adding 10 times of water for the second time, heating with strong fire to boil, keeping slightly boiling with slow fire for 15 minutes, filtering the decoction while hot with 350 mesh sieve, rapidly cooling the filtrate with cold water, and mixing the two filtrates. Transferring the decoction into a round-bottom flask, and concentrating under reduced pressure and low temperature (temperature: 50 deg.C, 65 deg.C; vacuum degree: -0.10MPa) to 100ml by using a rotary evaporator. Comparing and measuring the density and the paste yield of the standard decoction of the fresh houttuynia cordata at different concentration temperatures and observing the state of the concentrated solution. The results are shown in Table 6.
TABLE 6
The experimental results show that: the state, the density and the content of the quercetin of the concentrated solution are basically not different when the concentration temperature is 40-70 ℃. When the concentration temperature is 40 ℃, the concentration time is as long as 4 hours, and the concentration efficiency is low; when the concentration temperature is 70 ℃, the content of the quercetin is slightly reduced. Therefore, the concentration temperature is preferably 50 to 60 ℃.
3. Investigation of freeze drying Process
According to the recommended standard decoction drying under the item of 'standard decoction preparation' in the technical requirements for quality control and standard formulation of traditional Chinese medicine formula granules, the freeze drying is preferably adopted, so that the quality stability and easy dissolution of the granules can be ensured, and auxiliary materials are not added. Therefore, the drying method of freeze drying to the standard decoction of fresh houttuynia cordata is selected firstly.
Taking 200g of fresh houttuynia cordata decoction pieces, placing the fresh houttuynia cordata decoction pieces in a 3L ceramic pot, adding 12 times of water into the decoction pieces for the first time, soaking the decoction pieces for 30 minutes, boiling the decoction pieces with strong fire, keeping the decoction slightly boiling for 20 minutes with slow fire, filtering the decoction while the decoction is hot by using a 350-mesh screen, quickly cooling the filtrate by using cold water, and quickly cooling the filtrate by using the cold water. Adding 10 times of water for the second time, heating with strong fire to boil, keeping slightly boiling with slow fire for 15 minutes, filtering the decoction while hot with 350 mesh sieve, rapidly cooling the filtrate with cold water, and mixing the two filtrates. Transferring the decoction to a round-bottom flask, concentrating under reduced pressure and low temperature by using a rotary evaporator to obtain 100ml of extract, and freeze-drying the extract in a freeze dryer. And (5) investigating the influence of different freeze-drying parameters on the quality of the freeze-dried powder. The results are shown in Table 7.
TABLE 7
The experimental results show that: the freeze drying process of the concentrated solution of fresh houttuynia cordata is smooth under the different parameter levels set as above, and light purplish red flocculent solid with loose texture can be obtained.
In summary, the freeze-drying process of fresh houttuynia cordata is determined as follows: the prefreezing temperature of the concentrated solution is-30 to-50 ℃, the prefreezing time is 1 to 2 hours, the sublimation drying temperature is-45 to 0 ℃, the sublimation drying time is 36 to 80 hours, the vacuum degree is 0.1 to 0.3mbar, the desorption drying temperature is 0 to 30 ℃, the desorption drying time is 3 to 10 hours, and the vacuum degree is 0 mbar.
Second, chromatographic condition and system adaptability test
Investigation of the optimum absorption wavelength: and (3) scanning the sample solution at full wavelength by using a PDA (personal digital assistant) detector, collecting chromatograms of the sample solution at 254nm, 280nm, 326nm and program wavelength (326 nm in 0-13 min and 254nm in 13-25 min), comparing the collected chromatograms, and determining the optimal wavelength. The results are shown in Table 8 and FIG. 2.
TABLE 8
The result shows that the program wavelength detection mode is adopted, the chromatographic peak has good peak type, and the absorption intensity of the main chromatographic peak is high, so the program wavelength detection mode is selected.
Examination of mobile phase: the separation effect of 3 different mobile phases was examined, and acetonitrile was used as the mobile phase a, and 0.1% phosphoric acid water, 0.1% formic acid water, and 0.2% acetic acid water were used as the mobile phase B, respectively, to perform gradient elution. The results are shown in FIG. 3 and Table 9.
TABLE 9
The results showed that when 3 different mobile phases were compared, it was found that the gradient elution effect of acetonitrile-0.1% phosphoric acid and acetonitrile-0.1% formic acid was not very different from each other as a whole, and the separation effect of each chromatographic peak was comparable to the peak pattern, but that when acetonitrile-0.2% acetic acid was used as the mobile phase, peak 1 (neochlorogenic acid) appeared to split. Considering that acetonitrile-0.1% phosphoric acid water is adopted as a mobile phase, the chromatographic column has good tolerance and the base line of the chromatogram is stable, and finally the acetonitrile-0.1% phosphoric acid water is determined as the mobile phase.
Column temperature investigation: based on the experimental conditions set forth above, the column temperatures were examined at 28 ℃, 30 ℃ and 32 ℃. The results are shown in FIG. 4. The result shows that the chromatogram has better peak shape and moderate separation degree when the column temperature is 30 ℃. The column temperature was determined to be 30 ℃.
And (3) flow rate investigation: based on the experimental conditions set forth above, the flow rates were 0.28mL/min, 0.3mL/min, and 0.32mL/min, respectively. The results are shown in FIG. 5. The result shows that when the flow rate is 0.3mL/min, the chromatogram has better peak shape and moderate separation degree. Therefore, the flow rate was determined to be 0.3 mL/min.
And (4) overall inspection: on the basis of the experimental conditions set forth above, the chromatographic peak acquisition time was extended to 50 min. The results are shown in FIG. 6. The results show that after 20 minutes, the column still elutes isocratically to 50 minutes in 25% acetonitrile and no chromatographic peak is observed after 20 minutes. Therefore, when the chromatogram is collected for 20 minutes, the chromatographic information is completely collected. The chromatogram acquisition time was determined to be 20 minutes.
In conclusion, the characteristic spectrum chromatographic conditions and the system adaptability test of the standard decoction of the fresh houttuynia cordata are determined as follows: octadecylsilane chemically bonded silica is used as filler (Waters ACQUITY HSS T3 chromatographic column with column length of 100mm, inner diameter of 2.1mm, and particle diameter of 1.8 μm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid water solution is taken as a mobile phase B, and gradient elution is carried out according to the specification of the table 1; the flow rate is 0.3mL per minute; the column temperature is 30 ℃; the detection wavelength is 326nm in 0-13 min and 254nm in 13-25 min. The number of theoretical plates should not be less than 30000 calculated by chlorogenic acid peak.
Third, investigation of preparation of test solution
Investigation of extraction solvent: taking 0.1g of fresh houttuynia cordata standard decoction powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50% methanol aqueous solution, 90% methanol aqueous solution, methanol, 70% ethanol aqueous solution and 25mL of ethanol, sealing the plug, weighing, ultrasonically treating for 30 minutes (power 250W, frequency 40kHz), cooling, weighing again, respectively supplementing the weight loss with extraction solvent, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the final product. The results are shown in Table 10.
The result shows that the extraction effect of the 70% ethanol aqueous solution is better, so the extraction solvent is determined to be the 70% ethanol aqueous solution.
And (3) researching an extraction mode: taking 0.1g of fresh houttuynia cordata standard decoction powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 70% ethanol aqueous solution, sealing the plug, weighing, respectively extracting by reflux and ultrasound (power 250W, frequency 40kHz) for 30min, cooling, weighing again, complementing the lost weight with 70% ethanol aqueous solution, shaking up, filtering, and taking the subsequent filtrate to obtain the final product. The results are shown in Table 11.
TABLE 11
The result shows that the extraction effect of the ultrasonic and the backflow is not very different, and the extraction mode is determined to be ultrasonic (power 250W, frequency 40kHz) extraction based on the consideration of convenient operation.
Examining the amount of the extraction solvent: precisely weighing 0.1g of fresh herba Houttuyniae standard decoction powder, placing into a conical flask with a plug, precisely adding 70% ethanol water solution 25mL, 50mL and 75mL respectively, sealing, weighing, ultrasonically treating for 30min, cooling, weighing again, respectively supplementing with extraction solvent, shaking, filtering, and collecting the filtrate. The results are shown in Table 12.
TABLE 12
The results show that when the amount of the extraction solvent is 25mL, 50mL and 75mL, the difference of the characteristic maps of the three is not large, and the amount of the extraction solvent is determined to be 25mL based on the consideration of saving the solvent.
And (3) extracting time investigation: taking 0.1g of fresh herba Houttuyniae standard decoction powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 70% ethanol water solution, sealing the plug, weighing, performing ultrasonic treatment for 15min, 30min, 45min and 60min respectively, cooling, weighing again, supplementing with 70% ethanol water solution, shaking, filtering, and collecting the filtrate. The results are shown in Table 13.
The results showed that the extraction time was 30 minutes, since the extraction effect was the best when the extraction time was 30 minutes.
In conclusion, the preparation method of the test solution of the characteristic spectrum of the standard decoction of the fresh houttuynia cordata is determined as follows: taking 0.1g of fresh houttuynia cordata standard decoction powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 70% ethanol aqueous solution, sealing the plug, weighing, performing ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with 70% ethanol aqueous solution, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.
Fourth, chromatographic peak identification
Preparation of a test solution: the test solution of the standard decoction of fresh herba Houttuyniae is prepared by the above-mentioned experimental method.
Preparation of negative control solution: the negative control solution of the standard decoction of the fresh-lacking houttuynia cordata is prepared according to the experimental method drawn up as above.
Preparation of control reference solutions: taking appropriate amount of chlorogenic acid, cryptochlorogenic acid, hyperoside, and quercitrin as reference substances, and adding 70% ethanol water solution to obtain reference substance solution containing 100 μ g of reference substance per 1 mL. The peak of the characteristic diagram of the standard decoction of fresh herba Houttuyniae is located, and is shown in FIG. 7.
Fifth, methodology verification
And (3) precision test: taking about 0.1g of fresh herba Houttuyniae standard decoction powder, precisely weighing, preparing and measuring the test solution according to the above-mentioned formulated experimental method, and continuously injecting sample for 6 times, the results are shown in tables 14 and 15.
TABLE 14
The result shows that the sample introduction is carried out for 6 times, the relative retention time RSD of 7 characteristic peaks is in the range of 0.00-0.08%, and the RSD of the relative peak area is in the range of 0.00-0.07%, which shows that the precision of the characteristic spectrum is better.
And (3) repeatability test: 6 parts of fresh houttuynia cordata standard decoction are precisely weighed, and the test solution is prepared and measured according to the experimental method drawn up as above, and the results are shown in tables 16 and 17.
TABLE 16
TABLE 17
The results show that the relative retention time RSD of 7 characteristic peaks of 6 repeatability experiment samples is in the range of 0.00-0.06%, and the RSD of the relative peak area is in the range of 0.00-0.68%, which shows that the repeatability of the characteristic spectrum is good.
And (3) stability test: based on the experimental conditions as defined above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h and 24h, respectively, and the results are shown in tables 18 and 19.
The results show that the RSD of the relative retention time and the relative peak area of each chromatographic peak in 24 hours is less than 0.71%, and the test solution is stable in 24 hours.
Intermediate precision test: different persons operate on different chromatographic instruments on different dates, the same batch of fresh houttuynia cordata standard decoction is taken, and the test method is formulated to prepare and measure the test solution, and the results are shown in tables 20 and 21.
TABLE 21
The results show that different people operate under different dates and different chromatographs, the relative retention time RSD of 7 characteristic peaks is in the range of 0.00-0.25%, the relative peak area RSD of 7 characteristic peaks is in the range of 0.09-1.26%, and the relative retention time RSD of each chromatographic peak and the intermediate precision of the relative peak area are good.
Durability investigation: based on the experimental conditions thus prepared, the results of examining the columns of Waters ACQUITY HSS T3(2.1 mm. times.100 mm, 1.8 μm), Agilent SB C18(2.1 mm. times.100 mm, 1.8 μm), and Agilent Eclipes Plus C18(2.1 mm. times.100 mm, 1.8 μm) are shown in tables 22 and 23.
TABLE 22
TABLE 23
The results show that the RSD of each characteristic peak relative to the retention time is in the range of 0.04-2.26% and the RSD of the relative peak area is in the range of 0.74-4.77% when the sample is detected by using the 3 chromatographic columns, and that the durability of the chromatographic columns is good.
Sixthly, establishing the characteristic map of the standard decoction of the fresh houttuynia cordata
Using the finally determined analysis method, characteristic spectrum determination was performed on 17 batches of fresh houttuynia cordata standard decoction (batches BT1, BT2, BT3, BT4, BT5, BT6, BT7, BT8, BT9, BT10, BT11, BT12, BT13, BT14, BT15, BT16, BT17), and similarity and relative retention time were calculated.
According to the principle that the relative retention time is stable, samples of each batch can be detected, and the peaks are relatively high, 7 peaks with good repeatability are selected as characteristic peaks. The result shows that when the peak 2 (chlorogenic acid) is taken as the S peak, the relative retention time RSD of the characteristic peak of the standard decoction of 17 batches of fresh houttuynia cordata is less than 2.0 percent. Finally, the following steps are provided: the characteristic spectrum of the fresh houttuynia cordata standard decoction sample should present 7 characteristic peaks, wherein the retention time of peak 2 and peak 7 should be consistent with that of the corresponding reference peak respectively. The peak corresponding to the chlorogenic acid reference peak is the S1 peak, the peak corresponding to the quercitrin reference peak is the S2 peak, the relative retention time of the peaks 1, 3 and S1, and the relative retention time of the peaks 4, 5, 6 and S2 are respectively calculated, wherein the relative retention time is within +/-10% of a specified value, and the specified value is as follows: 0.66 (neochlorogenic acid), 1.09 (cryptochlorogenic acid), 1.89 (peak 4), 1.91 (hyperin), 1.93 (peak 6).
A traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) is adopted to automatically match the characteristic spectrums of the standard decoction of 17 batches of fresh houttuynia cordata (figure 8), and the similarity is shown in a table 24, so that the comparison spectrum of the characteristic spectrums of the standard decoction of the fresh houttuynia cordata (namely figure 1) is established.
Example 2 quality detection method of fresh houttuynia cordata granules
The quality detection method comprises the following steps:
1) preparing a sample solution to be detected: grinding appropriate amount of fresh herba Houttuyniae granule (lot numbers are KL1, KL2, KL3), collecting about 0.1g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 70% ethanol water solution, sealing, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing lost weight with 70% ethanol water solution, shaking, filtering, and collecting filtrate.
2) And (3) detection: respectively sucking 1 mu L of reference substance solution of the reference substance and 1 mu L of sample solution to be detected, and injecting the reference substance solution and the sample solution into an ultra-high performance liquid chromatograph under the following chromatographic conditions:
octadecylsilane chemically bonded silica was used as a filler (Waters ACQUITY HSS T3, column length 100mm, inner diameter 2.1mm, particle diameter 1.8 μm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid water solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table 25; the flow rate is 0.3mL per minute; the column temperature is 30 ℃; the detection wavelength is 326nm in 0-13 min and 254nm in 13-25 min. The number of theoretical plates should not be less than 30000 calculated by chlorogenic acid peak.
TABLE 25
The detection result is shown in fig. 9, the relative retention time of the common characteristic peak of 3 batches of formula granules is basically consistent with the standard decoction comparison characteristic spectrum, and the reproducibility is good, so that the established characteristic spectrum can be used for quality evaluation of the formula granules.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (14)
1. A method for constructing a UPLC characteristic spectrum of a standard decoction of fresh houttuynia cordata is characterized by comprising the following steps:
preparation of control solutions: dissolving chlorogenic acid, cryptochlorogenic acid, hyperoside and quercetin reference substance with solvent to obtain reference substance solution;
freeze-dried powder preparation of fresh houttuynia cordata standard decoction: taking fresh houttuynia cordata decoction pieces, adding water for extraction, carrying out solid-liquid separation, concentrating the obtained extracting solution, and carrying out freeze drying to obtain freeze-dried powder of the fresh houttuynia cordata standard decoction;
preparation of a test solution: adding 65-75% ethanol water solution into the freeze-dried powder of the fresh houttuynia cordata standard decoction for extraction, filtering the extracting solution, and taking the subsequent filtrate to obtain a test solution;
injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, a mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution;
the gradient elution included the following procedure:
the volume percentage of the mobile phase A is increased from 5% to 11% in 0-7 min;
the volume percentage of the mobile phase A is increased from 11% to 11.5% within 7-10 min;
the volume percentage of the mobile phase A is increased from 11.5% to 20% in 10-13 min;
13-20 min, increasing the volume percentage of the mobile phase A from 20% to 25%;
the chromatographic column adopted by the ultra-high performance liquid chromatograph takes octadecylsilane chemically bonded silica as a filler;
the detection wavelength adopted by the ultra-high performance liquid chromatograph is the program wavelength: 326nm for 0-13 min and 254nm for 13-20 min.
2. The method for constructing the UPLC characteristic spectrum of the fresh houttuynia cordata standard decoction according to claim 1, wherein the conditions of the ultra high performance liquid chromatograph comprise:
flow rate: 0.25-0.35 mL/min; column temperature: 25-35 ℃.
3. The method for constructing the UPLC feature map of the standard decoction of fresh houttuynia cordata as claimed in claim 2, wherein the flow rate is 0.3 mL/min; the column temperature was 30 ℃.
4. The method for constructing the UPLC feature map of the fresh houttuynia cordata standard decoction according to claim 1, wherein in the preparation of the test solution, the extraction method is ultrasonic extraction, the ultrasonic power is 200-300W, and the frequency is 30-50 kHZ; the extraction time is 25-35 min.
5. The method for constructing the UPLC feature map of the standard decoction of fresh houttuynia cordata as claimed in claim 1, wherein the water extraction method comprises two decoction steps in the preparation of the freeze-dried powder of the standard decoction of fresh houttuynia cordata: adding 8-14 times of water into the first decoction, heating the mixture to boil with strong fire, and decocting the mixture with slow fire for 20-30 min; adding 6-12 times of water into the second decoction, heating the second decoction with strong fire until the second decoction is boiled, and decocting the second decoction with slow fire for 15-25 min.
6. The method for constructing the UPLC characteristic spectrum of the standard decoction of fresh houttuynia cordata as claimed in claim 1, wherein the mesh number of the screen for solid-liquid separation is 200-400 meshes; and/or the concentration temperature is 40-70 ℃.
7. The method for constructing the UPLC profile of a standard decoction of fresh houttuynia cordata according to claim 1, wherein the freeze-drying process comprises the following steps: the pre-freezing temperature is-50 to-30 ℃, and the pre-freezing time is 1 to 2 hours; the sublimation drying temperature is-45 ℃ to 0 ℃, the sublimation drying time is 40 to 80 hours, and the vacuum degree is 0.1 to 0.3 mbar; the analytic drying temperature is 10-30 ℃, the analytic drying time is 3-10 hours, and the vacuum degree is 0 mbar.
8. The method for constructing the UPLC characteristic spectrum of the standard decoction of fresh houttuynia cordata as claimed in any one of claims 1 to 7, wherein the characteristic spectrum comprises 7 common peaks, and the 7 common peaks comprise characteristic peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, hyperoside and quercetin.
9. A UPLC characteristic spectrum detection method of a fresh houttuynia cordata preparation is characterized by comprising the following steps:
preparing a sample solution to be tested: taking a to-be-detected fresh houttuynia cordata preparation, grinding, adding 65-75% ethanol water solution by volume fraction, extracting, filtering the extracting solution, and taking a subsequent filtrate to obtain a to-be-detected sample solution;
injecting a sample solution to be detected into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, a mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution;
the gradient elution included the following procedure:
the volume percentage of the mobile phase A is increased from 5% to 11% in 0-7 min;
the volume percentage of the mobile phase A is increased from 11% to 11.5% within 7-10 min;
the volume percentage of the mobile phase A is increased from 11.5% to 20% in 10-13 min;
13-20 min, increasing the volume percentage of the mobile phase A from 20% to 25%;
the chromatographic column adopted by the ultra-high performance liquid chromatograph takes octadecylsilane chemically bonded silica as a filler;
the detection wavelength adopted by the ultra-high performance liquid chromatograph is the program wavelength: 326nm for 0-13 min and 254nm for 13-20 min.
10. The UPLC profile detection method of fresh houttuynia cordata preparation according to claim 9, wherein the conditions of the ultra high performance liquid chromatograph comprise:
flow rate: 0.25-0.35 mL/min; column temperature: 25-35 ℃.
11. The UPLC profile assay method of fresh houttuynia cordata formulation according to claim 10 wherein the flow rate is 0.3 mL/min; the column temperature was 30 ℃.
12. The UPLC feature spectrum detection method of fresh houttuynia cordata preparation according to claim 9, wherein in the preparation of the sample solution to be detected, the extraction method is ultrasonic extraction, the ultrasonic power is 200-300W, and the frequency is 30-50 kHZ; the extraction time is 25-35 min.
13. The method for detecting the UPLC profile of fresh houttuynia cordata preparation according to claim 12, wherein in the preparation of the sample solution to be detected, the extraction method is ultrasonic extraction, the ultrasonic power is 250W, and the frequency is 40 kHZ; the extraction time is 30 min.
14. The UPLC feature spectrum detection method of fresh houttuynia cordata preparation according to any one of claims 9 to 13, wherein the fresh houttuynia cordata preparation is fresh houttuynia cordata granules prepared by extracting fresh houttuynia cordata decoction pieces as a raw material with water as a solvent, concentrating, drying, and adding auxiliary materials.
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