CN112213431A

CN112213431A - Detection method and quality control method of rhodiola rosea wall-broken decoction pieces

Info

Publication number: CN112213431A
Application number: CN201910619904.5A
Authority: CN
Inventors: 黄勇; 徐浩坤; 谢宇
Original assignee: Guizhou Guangjitang Pharmaceutical Co ltd
Current assignee: Guizhou Guangjitang Pharmaceutical Co ltd
Priority date: 2019-07-10
Filing date: 2019-07-10
Publication date: 2021-01-12

Abstract

The invention discloses a detection method and a quality control method of rhodiola rosea wall-broken decoction pieces. The detection method comprises the steps of (1) obtaining an HPLC (high performance liquid chromatography) spectrum of rhodiola rosea decoction pieces (wall breaking); (2) a step of confirming fingerprint information of the obtained HPLC chromatogram, wherein the fingerprint information includes the number of peaks, relative retention time, and relative peak area; (3) and detecting the quality of the rhodiola rosea decoction pieces (wall breaking) based on fingerprint information. The method can more effectively reflect the active ingredients and the content thereof which can be absorbed by the medicinal materials.

Description

Detection method and quality control method of rhodiola rosea wall-broken decoction pieces

Technical Field

The invention relates to the field of processing of traditional Chinese medicine decoction pieces, in particular to a rhodiola rosea medicinal material, and particularly relates to a detection method and a quality control method of wall-broken powder.

Background

The rhodiola rosea traditional Chinese medicine wall-breaking decoction pieces are prepared from rhodiola rosea serving as a raw material by a modern superfine grinding wall-breaking technology. The radix Rhodiolae is dry root and rhizome of rhodiola crenulata Rhodiolacrenulata (hook.f. et Thoms.) H.Ohba. Has effects of invigorating qi, promoting blood circulation, dredging collaterals and relieving asthma, and can be used for treating qi deficiency, blood stasis, thoracic obstruction, cardiodynia, apoplexy, hemiplegia, listlessness and asthma. Modern pharmacological research shows that rhodiola has the function of improving anoxia tolerance, and rhodiola can weaken the damage degree of anoxia heap in vivo to organism, and mainly contains alcohol and glycosides thereof, flavonoids, terpenoids, phenol and glycosides thereof, polysaccharides, coumarins, volatile oils and other components.

The rhodiola rosea traditional Chinese medicine wall-broken decoction pieces are prepared by processing rhodiola rosea medicinal materials meeting the legal standard requirements to powder with the D90 being less than or equal to 45 mu m (more than 300 meshes) by a modern superfine grinding wall-breaking technology, and preparing the powder into 30-200-mesh granular decoction pieces with no additive in all components by a micro powder inclusion technology. Compared with the traditional decoction pieces, the traditional Chinese medicine wall-broken decoction pieces have the advantages of uniform quality, high medicine utilization rate, good stability, convenience and quickness in application and the like.

The traditional Chinese medicine wall-broken decoction pieces are developed into a fourth generation novel traditional Chinese medicine decoction piece which is formed after the traditional decoction pieces, the formula granules and the extract granules. So far, nearly hundreds of plant medicinal materials are successfully developed into the traditional Chinese medicine wall-breaking decoction pieces and are sold on the market. But the research on the detection and quality control method of the rhodiola wall-broken decoction pieces is less at present.

CN 104055826A discloses the technical field of a Chinese medicinal preparation. In particular to a rhodiola wall-breaking preparation. The rhodiola wall-breaking preparation has high bioavailability, strong preparation stability and good disintegration by crushing rhodiola into wall-breaking powder, further granulating by an ethanol-water wet method, carrying out extrusion forming at a certain rotating speed and drying. The patent does not mention the quality control method of the wall-broken powder.

For another example, CN108152431A discloses a method for constructing HPLC fingerprint of rhodiola rosea wall-broken decoction pieces and a method for detecting quality, which only researches the method for constructing HPLC fingerprint of rhodiola rosea wall-broken decoction pieces and detecting quality. However, the quality detection method constructed in the patent still cannot reflect the quality of the decoction pieces well.

Disclosure of Invention

In order to solve at least part of technical problems in the prior art, the inventor conducts intensive research and finds that the quality of the rhodiola wall-broken powder can be effectively detected by selecting specific information of an HPLC (high performance liquid chromatography) spectrum as an index. The present invention has been accomplished, at least in part, based on this. Specifically, the present invention includes the following.

The invention provides a detection method of rhodiola rosea decoction pieces (wall breaking), which comprises the following steps:

(1) obtaining an HPLC (high performance liquid chromatography) spectrum of rhodiola rosea decoction pieces (wall breaking);

(2) a step of confirming fingerprint information of the obtained HPLC chromatogram, wherein the fingerprint information includes the number of peaks, relative retention time, and relative peak area;

(3) and detecting the quality of the rhodiola rosea decoction pieces (wall breaking) based on the fingerprint information.

Preferably, in the step (1) of the present invention, the condition for obtaining the HPLC chromatogram of the rhodiola rosea decoction pieces (wall-broken) comprises: the chromatographic column is an Agilent C18 column; mobile phase: acetonitrile (a) -0.02% phosphoric acid water (B), gradient elution: 6% A-6% A (0-18min), 6% A-20% A (18-45min), 20% A-20% A (45-55min), 20% A-22% A (55-65min), 22% A-22% A (65-90min), DAD detection wavelength of 278nm, flow rate of 0.8 mL/min^-1The column temperature was 30 ℃.

Preferably, the method further comprises a processing step of rhodiola rosea decoction pieces (wall breaking) before the step (1): weighing radix Rhodiolae decoction pieces (wall-broken) 1g, adding 50% methanol, ultrasonic treating for 20-60min, cooling, adding 50% methanol to make up the weight loss, and filtering with microporous membrane.

Preferably, the preparation method of the rhodiola rosea decoction pieces (wall breaking) comprises the following steps:

(1') drying the pretreated rhodiola root raw material until the moisture content is below 7 wt% to obtain dried rhodiola root;

(2') crushing the dried rhodiola rosea to 800 meshes of 300 meshes to obtain rhodiola rosea wall-broken powder;

(3') carrying out micro-powder inclusion granulation on the rhodiola wall-broken powder without adding any auxiliary material or additive to obtain the rhodiola wall-broken decoction piece granules with 30-200 meshes.

Preferably, step (2') of the present invention comprises:

(2' -1) a step of subjecting the dried rhodiola rosea to first pulverization to obtain a first pulverized material of 60-100 mesh;

(2' -2) a step of subjecting the first pulverized material to second pulverization to obtain a second pulverized material of 100-300 mesh; and

(2' -3) a step of subjecting the second pulverized material to third pulverization to obtain a third pulverized material of more than 300 to 800 meshes; wherein the first pulverization, the second pulverization, and the third pulverization are different types of pulverization.

Preferably, the first pulverization is universal pulverization, and the feed is dried rhodiola rosea with 5-7 wt% of water and 3.2-8mm of particle size; the second crushing is ball milling crushing; and the third crushing is airflow crushing, wherein the conditions of the airflow crushing comprise feeding pressure of 0.5-0.9MPa, frequency of 35-45Hz, induced air pressure difference of 0.2-0.8MPa, feeding mesh number of 100-300 meshes and crushing to be more than 300-800 meshes, thereby obtaining the wall-broken powder.

Preferably, in the step (3), if the relative retention time of the No. 1 peak is between 0.3000 and 0.3065, the relative retention time of the No. 4 peak is between 1.1000 and 1.1200, and the relative retention time of the No. 14 peak is between 2.1500 and 2.2300, the rhodiola rosea decoction pieces (wall breaking) are detected to be in accordance with the requirement; wherein the relative retention time is the relative retention time obtained with the retention time of peak No. 2 as a reference.

Preferably, if the relative peak area of the No. 1 peak is 8.7000-9.0000, the relative peak area of the No. 4 peak is 1.3000-1.5000 and the relative peak area of the No. 14 peak is 1.500-1.8000, the rhodiola rosea decoction pieces (wall breaking) are detected to meet the requirement; the relative peak area is a relative peak area obtained by taking the peak area of the No. 2 peak as a reference.

In a second aspect of the present invention, a quality control method of rhodiola rosea decoction pieces (wall breaking) is provided, which takes the method of the first aspect of the present invention as a step.

The quality control method of the rhodiola rosea decoction pieces (wall breaking) can more effectively reflect the active ingredients and the content thereof, and can be used for evaluating the influence of different processing methods on the rhodiola rosea medicinal materials and the like. In addition, the method can also be used for judging the purity of the rhodiola rosea decoction pieces (wall breaking) in the sample.

Drawings

FIG. 1 is an overlay of rhodiola rosea decoction pieces (wall-broken) by fingerprint.

FIG. 2 is a superposition graph of fingerprint spectra of traditional decoction pieces of radix Rhodiolae.

FIG. 3 is a fingerprint of rhodiola rosea.

Fig. 4 is a comparison graph of fingerprint spectra of radix Rhodiolae decoction pieces (wall-broken) and traditional decoction pieces.

FIG. 5 shows the effect of different dose of radix Rhodiolae decoction pieces (wall-broken) and traditional decoction pieces on the survival rate of myocardial cells induced by ischemia and anoxia of H9c 2.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

The detection method of rhodiola rosea decoction pieces (wall breaking) provided by the invention (sometimes referred to as the detection method of the invention) is not only used for identifying whether a sample is the rhodiola rosea decoction pieces (wall breaking), but also more importantly used for judging or evaluating/evaluating whether the rhodiola rosea decoction pieces (wall breaking) meet or meet the specified requirements. The 'specified requirements' in the invention include that the traditional decoction pieces have more absorbable active ingredients and higher content, or the traditional wall-broken decoction pieces have higher active ingredients and higher content, so that the traditional decoction pieces have more excellent effects, such as the survival rate of ischemic and anoxic myocardial cells. Preferably, the "specified requirements" include at least a relative retention time of peak No. 1 between 0.3000 and 0.3065, a relative retention time of peak No. 4 between 1.1000 and 1.1200, and a relative retention time of peak No. 14 between 2.1500 and 2.2300 in the HPLC fingerprint of the sample; the relative peak area of No. 1 peak is 8.7000-9.0000, the relative peak area of No. 4 peak is 1.3000-1.5000, and the relative peak area of No. 14 peak is 1.500-1.8000.

The detection method of the rhodiola rosea decoction pieces (wall breaking) is a method based on an HPLC (high performance liquid chromatography) spectrum, and specifically comprises the following steps of:

(2) a step of confirming fingerprint information in the obtained HPLC profile, wherein the fingerprint information includes a relative retention time and a relative peak area;

The respective steps are explained in detail below.

[ procedure for obtaining HPLC chromatogram of rhodiola rosea decoction pieces (wall breaking) ]

The step (1) is a step of obtaining the HPLC (high performance liquid chromatography) spectrum of the rhodiola rosea decoction pieces (wall breaking), and in order to establish a unified standard, the HPLC spectrum of the rhodiola rosea decoction pieces (wall breaking) is preferably obtained under the same condition. More preferably, the conditions for obtaining the HPLC profile of the rhodiola rosea decoction pieces (wall-broken) include:

the chromatographic column is an Agilent C18 column; mobile phase: acetonitrile (a) -0.02% phosphoric acid water (B), gradient elution: 6% A-6% A (0-18min), 6% A-20% A (18-45min), 20% A-20% A (45-55min), 20% A-22% A (55-65min), 22% A-22% A (A: (1))65-90min), the DAD detection wavelength is 278nm, and the flow rate is 0.8 mL/min^-1The column temperature is 30 ℃ and a predetermined amount of sample is taken, for example, 10. mu.L. The Agilent C18 column is preferably a uniform size column, for example, 4.6mm by 250mm, 5 μm.

The chromatographic conditions mentioned above are preferred conditions after optimization and are not the only conditions for achieving the object of the present invention, and indeed, on the basis of the above conditions, those skilled in the art can make modifications or adjustments and easily obtain other equivalent conditions or similar conditions, which are also feasible for the object of the present invention.

In order to establish a standardized detection method, the rhodiola rosea decoction pieces (wall breaking) are preferably processed, namely the rhodiola rosea decoction pieces (wall breaking) are prepared into samples which can be used for HPLC sample loading. The treatment step comprises weighing a prescribed amount (e.g., 1g) of radix Rhodiolae decoction pieces (wall-broken), adding a prescribed amount of 50% methanol (e.g., 50mL), sonicating for 20-60min, preferably 30-50min, more preferably 40-48min, cooling, supplementing the lost weight with 50% methanol, and filtering with a microporous membrane (e.g., 0.45 μm).

The rhodiola rosea decoction pieces (wall-broken) are preferably rhodiola rosea powder, and more preferably rhodiola rosea raw materials are processed into powder with the granularity of more than 300 meshes by a grinding technology and then are prepared into granular decoction pieces with the granularity of 30-200 meshes.

In certain embodiments, the rhodiola rosea decoction pieces (wall-broken) of the present invention are decoction pieces obtained by a specific method. The specific method combines the advantages of the jet milling technology and the micro powder inclusion technology, and the two technologies generate a synergistic effect by optimizing the characteristics of the technologies, so that the loss of active ingredients of the rhodiola rosea in the processing process is avoided, and the detection method is more objective and accurate. It is known in the art that different processing or treatment methods of rhodiola root herbs have different effects on the active ingredients or the efficacy of the herbs. Some processing or treatment methods may greatly reduce the content of active ingredients and even destroy their structure. The existing wall breaking method has great influence on active ingredients, and decoction pieces can be obtained, but the medicinal effect of the decoction pieces needs to be further improved. The detection method can well distinguish the decoction pieces obtained by the existing wall breaking method from the decoction pieces with higher drug effect obtained by the method.

In an exemplary embodiment, the preparation method of the rhodiola rosea decoction pieces (wall breaking) at least comprises the following steps:

The step (1') of the present invention is a step of obtaining a dried rhodiola rosea. Drying may be carried out using any known method. In an exemplary embodiment, the drying of the present invention is freeze-drying. The rhodiola rosea is rich in polysaccharide, volatile oil and other substances, the freeze drying technology is suitable for retaining the components in the rhodiola rosea, and the components are prevented from being damaged and lost by the traditional drying method. Specifically, the freeze-drying step may include the following three substeps.

(1' -1) freezing step:

pre-cooling the treated rhodiola rosea raw material to a first temperature under normal pressure and keeping the temperature for 1 to 2 hours, then cooling to a second temperature under the first pressure and keeping the temperature for 0.5 to 3 hours, preferably 0.8 to 2.5 hours, more preferably 1 to 2 hours, and then further cooling to a third temperature and keeping the temperature for 0.5 to 2 hours, preferably 0.8 to 1.5 hours, more preferably 0.9 to 1.0 hour. Such a freezing step, unlike the traditional linear rate cooling, is more conducive to the formation of ice crystals of a particular size, reduces the resistance to sublimation of water vapor, and makes the resulting dried product more brittle, which is more conducive to the subsequent comminution steps. Preferably, the first temperature is 1.5-2.5 ℃, the second temperature is-45 ℃ to-35 ℃, and the third temperature is-60 ℃ or lower. Preferably, the first pressure is from 10 to 15KPa and the second pressure is from 1 to 1.5 KPa.

(1' -2) sublimation step:

and reducing the pressure from the first pressure to the second pressure for sublimation under the condition of keeping freezing, and gradually increasing the temperature back during sublimation. Most of the water can be removed in the process. For example, thermal energy is provided by way of shelf heating. During sublimation, which is carried out under a high vacuum, the frozen state of the contents of the box must be maintained during the pressure reduction to prevent spillage out of the container. When the pressure in the box is reduced to 1.33KPa and the temperature is below-60 ℃, the ice begins to sublimate, and the sublimed water vapor is frozen into ice crystals in the condenser. To ensure sublimation of the ice, the heating system is preferably turned on and the shelf is heated to a temperature of-28 ℃ to-10 ℃ at the eutectic point. Generally, lower gas pressures favor sublimation and water vapor evolution, but gas pressures below 1.00KPa have been found to be detrimental to sublimation. Therefore, the sublimation pressure of the present invention is generally 1.00KPa to 1.33 KPa. During sublimation, step (1-2) is terminated when the temperature reaches-28 ℃ to-10 ℃.

(1' -3) redrying step: the temperature is raised to 30-35 ℃ and re-drying is carried out in the process until the moisture content is 5-9 wt%. The bound water is removed by further drying, and at this time, the water vapor pressure on the surface of the solid is reduced to a different extent, and the drying rate is significantly reduced. Preferably, the shelf temperature should be increased appropriately during this stage to facilitate evaporation of the water, typically by heating the shelf to 30-35 ℃ until the sample temperature coincides with the shelf temperature to achieve dryness. The water content of the lyophilized sample is controlled to be 5 wt% or less, preferably 3 wt% or less, and more preferably 1 wt%.

It should be noted that, in the step (1') of the present invention, the parameters of each step are set not only to obtain the desired dried rhodiola root, but also to improve the brittleness of the rhodiola root and to facilitate the subsequent pulverization.

Step (2') of the present invention is a three-stage pulverization step comprising:

In the three-stage pulverization of the present invention, the first pulverization, the second pulverization, and the third pulverization are different types of pulverization. The invention finds that through three different types of crushing, preferably, the raw material medicine is subjected to universal crushing → ball milling crushing → airflow crushing, so that the yield of wall breaking crushing can be improved after three times of crushing, for example, rhodiola root raw material medicine is directly subjected to airflow crushing without universal crushing and ball milling crushing, so that a plurality of fibers cannot meet the requirements of wall breaking crushing, the waste of the raw material medicine can be caused, and the yield of the wall breaking powder obtained by performing airflow crushing on the Chinese material medicine powder subjected to universal crushing and ball milling crushing is more than 95% (if the previous two times of crushing are not performed, the yield can only be about 84% by only airflow crushing). The jet milling uses compressed air or superheated steam to pass through a nozzle to generate high-speed airflow, a high-speed gradient is formed near the nozzle, supersonic high-turbulence generated by the nozzle is used as a particle carrier to mill unstable substances such as thermosensitive components and the like, and the retention of components such as alcohol and glycosides thereof, flavonoids, terpenoids, phenols and glycosides thereof, polysaccharides, coumarins, volatile oils and the like which are rich in the traditional Chinese medicinal materials is ensured.

In the present invention, the first-stage pulverization is preferably conducted by pulverizing the dried raw material to 60 to 100 mesh, preferably 70 to 100 mesh, by universal pulverization. The second stage pulverization includes pulverizing the first stage pulverized material to about 100-. The third stage of crushing comprises crushing the second stage crushed material to more than 300 meshes, preferably 400-1200 meshes, more preferably 400-800 meshes by air flow crushing, thereby obtaining the traditional Chinese medicine wall-broken powder. If it is less than 400 mesh, the inclusion effect in the final inclusion granulation is poor, and particles of 30 to 200 mesh, preferably 30 to 100 mesh, more preferably 35 to 80 mesh cannot be formed efficiently. On the other hand, if it is larger than 800 mesh, the resulting particles become poor in dispersibility.

The first stage comminution of the present invention is preferably universal comminution. In an exemplary embodiment, the universal comminution of the present invention is as follows: the production capacity is 300-1000kg/h, the feed granularity is 8-32mm, the feed moisture is less than 7 wt%, preferably 5-7 wt%, the spindle rotation speed is 3600rpm, and the yield is more than 98%. The above-mentioned pulverization conditions can give the desired 60-100 mesh particles.

The second-stage pulverization of the present invention is preferably ball-milling pulverization, which includes a process of ball-milling the first-stage pulverized material into a crude powder of the Chinese medicinal material. The yield of ball milling and crushing is 97-98%.

The air flow crushing of the invention comprises the steps of placing the Chinese medicinal material coarse powder in an air flow crusher for air flow crushing, and crushing to 300-1500 meshes to obtain the Chinese medicinal material wall-broken powder. Specifically, the pulverizing conditions include material pressure of 0.5-0.9MPa, preferably 0.6-0.8MPa, frequency of 35-45Hz, induced air pressure difference of 0.2-0.8MPa, and material water content of less than 7 wt%, preferably 5-7 wt%.

In the invention, compressed air after being filtered and dried is injected into a crushing cavity at a high speed, materials are repeatedly collided, rubbed and sheared at the intersection point of a plurality of high-pressure air flows to be crushed, the crushed materials move to a grading area along with ascending air flow under the suction action of a fan, under the action of strong centrifugal force generated by a grading turbine rotating at a high speed, the thick and thin materials are separated, fine particles meeting the particle size requirement enter a cyclone separator and a dust remover to be collected through a grading wheel, and the coarse particles descend to the crushing area to be continuously crushed. The freeze drying condition of the invention is the condition for obtaining the brittle material, and the freeze drying obtains the traditional Chinese medicine raw material with higher brittleness, thereby ensuring that the traditional Chinese medicine can be more easily crushed by air current crushing, and obtaining the excellent wall-broken decoction pieces.

In addition, it should be noted that the ball milling and pulverizing equipment needs to be cleaned for the next time of pulverizing, and the operation is complex, time-consuming and labor-consuming. The jet milling of the invention uses compressed air or superheated steam to pass through the nozzle to generate high-speed airflow, forms high speed gradient near the nozzle, and uses supersonic high turbulence generated by the nozzle as particle carrier to mill, especially for milling unstable substances containing heat-sensitive components.

Step (3') of inclusion and granulation of micropowder:

the step (3') of the invention is a micro powder inclusion and granulation step, which comprises the step of performing micro powder inclusion and granulation on the rhodiola rosea ultra-micro powder under the condition of not adding any auxiliary material or additive to obtain the rhodiola rosea wall-broken decoction piece granules with 30-200 meshes.

After the rhodiola rosea is subjected to wall breaking and crushing, the powder surface area is increased, the flowability is reduced, the adsorbability is increased, water in air is easily adsorbed, and an adsorption machine is difficult to clean in the filling process, so that the bottleneck technologies such as difficult subpackage and the like are caused. In order to solve the problem, the invention researches the micro powder inclusion granulation of the wall-broken decoction pieces obtained by wall-breaking and crushing the rhodiola root traditional Chinese medicinal materials. The invention takes the angle of repose as an evaluation index, and carries out micro powder granulation on the premise of not adding any glidant. Namely, the rhodiola rosea powder with good wall breaking is aggregated by a physical method. In the step (3'), the wall-broken and pulverized powder is subjected to micro-powder inclusion. The physical method is that one wall-broken powder molecule wraps another or more wall-broken powder molecules to form a unique molecular complex, and the packing material and the core material of the complex are the wall-broken powder of the same medicinal material. Preferably, the angle of repose of the molecular complex of the invention is between 30 ° and 40 °, and the stability and flowability are significantly improved.

The preparation method has the advantages that the obtained decoction pieces have the characteristics of strong liquidity, good stability, high safety, easy dispersion, convenient administration and the like.

The step (1) of the present invention preferably further comprises a process of processing the obtained HPLC data. Preferably, the obtained chromatogram data parameters are introduced into software of 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system' 2012.1 edition, and the information of each chromatogram peak is inspected.

[ procedure for confirming fingerprint information in the obtained HPLC chromatogram ]

Step (2) of the present invention is a step of confirming fingerprint information in the obtained HPLC profile. The HPLC fingerprint of the rhodiola rosea decoction pieces (wall-broken) obtained by the steps is provided with 22 peaks which are numbered as 1, 2, 3, 4, 5, 6, 7, … … and 22 from left to right. The fingerprint information of the present invention includes not only information on the number of peaks in the HPLC chromatogram, but also the retention time of the peaks, the peak area, particularly the relative retention time and the relative peak area. The calculation of relative retention time and relative retention peak area is known in the art.

[ quality of rhodiola rosea decoction pieces (wall breaking) detected based on fingerprint information ]

The step (3) of the invention is a step of detecting the quality of rhodiola rosea decoction pieces (wall breaking) based on fingerprint information. In the present invention, first, specific information needs to be selected from the fingerprint information as a detection index. The detection index of the present invention may be a part of the fingerprint information or may be the entire fingerprint information. Preferably, the detection index of the present invention is only a part of the fingerprint information. For example fingerprint information of a part of the peaks.

In the invention, researches show that when the relative retention time of the No. 1 peak is between 0.3000 and 0.3065, preferably between 0.3005 and 0.3060, the relative retention time of the No. 4 peak is between 1.1000 and 1.1200, preferably between 1.1050 and 1.1180, and the relative retention time of the No. 14 peak is between 2.1500 and 2.2300, preferably between 2.1700 and 2.2250, the obtained rhodiola rosea decoction pieces (wall breaking) have more excellent effect. Therefore, the invention can detect or judge the quality of the rhodiola root decoction pieces (wall breaking) by taking the information of the relative retention time and the like of the range including the No. 1, the No. 4 and the No. 14 peaks as the detection index of the performance of the rhodiola root. Further preferably, the relative peak area of peak No. 1 is between 8.7000 and 9.0000, preferably between 8.7000 and 8.9500; the relative peak area of the No. 4 peak is between 1.3000 and 1.5000, preferably between 1.3500 and 1.4500; the relative peak area of peak No. 14 is 1.500-1.8000, preferably 1.6000-1.7500. The relative peak areas further show that the compound has better activity or drug effect. In particular, has higher efficacy in treating ischemic and hypoxic conditions. The ischemic and hypoxic conditions of the invention comprise qi deficiency and blood stasis, chest stuffiness and pains, stroke hemiplegia, listlessness and asthma, etc.

It should be noted that the relative retention time is the relative retention time obtained by taking the retention time of peak No. 2 as a reference; the relative peak area is a relative peak area obtained by taking the peak area of peak No. 2 as a reference. In addition, as long as the information of peaks No. 1, 2, 4, and 14 is used as an index for judgment, the index of the present invention is within the protection scope of the detection method of the present invention, and in other embodiments, the index of the present invention further includes any one of the information of other peaks. For example, the index may include information on peak No. 3, but is not limited thereto. Further studies found that peak 1 corresponds to gallic acid, peak 2 corresponds to salidroside, peak 4 corresponds to tyrosol, and peak 14 corresponds to rutin.

Example 1

The preparation process of rhodiola rosea decoction pieces (wall breaking) mainly comprises the following steps:

1. sorting and removing non-medicinal parts of rhodiola root raw medicinal materials;

2. leaching and crushing: washing and sorting the rhodiola rosea and crushing the rhodiola rosea into small sections of 10-15 cm;

3. and (3) drying: drying the rhodiola rosea until the water content of the raw medicinal materials is 5 to 9 percent, and taking out;

4. coarse crushing: the rhodiola root is crushed to 60 to 100 meshes by universal crushing, and then ball-milled and crushed to 100 meshes and 500 meshes to obtain raw material coarse powder;

5. breaking the wall and crushing: placing the rhodiola rosea coarse powder into a jet mill, carrying out jet milling under the conditions that the feeding pressure is 0.6-0.8MPa, the frequency is 35-45Hz, the induced air pressure difference is 0.2-0.7MPa, the water content of the material is below 7 percent, and the mesh number is 60-100 meshes, and crushing to 800 meshes of 300 meshes to obtain the rhodiola rosea wall-broken powder;

6. coating with micropowder: micronizing radix Rhodiolae micropowder without adjuvant or additive, and granulating to obtain 30-200 mesh radix Rhodiolae wall-broken decoction pieces granule;

7. total mixing: the rhodiola granules are put into a three-dimensional motion mixer to be mixed evenly, and the angle of repose is ensured to be between 30 and 40 degrees.

8. Filling: the medicine is packaged in small bags with the weight of less than 5g, and is convenient to take and carry.

Second, the choice of the superfine grinding mode of rhodiola

Pulverizing radix Rhodiolae into 60-100 mesh powder, and ball milling into 500 mesh powder to obtain medicinal powder.

Ball-milling crushing can produce heat, causes the serious loss of components such as polysaccharide, volatile oil class, etc., and ball-milling crushing equipment smashes once and need to clean it just can carry out next operation, complex operation, waste time and energy. The airflow crushing adopts compressed air or superheated steam to pass through a nozzle to generate high-speed airflow, a high-speed gradient is formed near the nozzle, supersonic high-turbulence generated by the nozzle is used as a particle carrier for crushing, and the airflow crushing method is particularly suitable for crushing unstable substances containing heat-sensitive components and the like, and is particularly suitable for crushing rhodiola rosea medicinal materials rich in alcohol and glycosides, flavonoids, terpenes, phenols and glycosides, polysaccharides, coumarins, volatile oils and other components through series research on rhodiola rosea wall-breaking decoction pieces by using an airflow ultramicro crushing mode, so that the optimal parameters of the airflow crushing rhodiola rosea are finally determined as follows: feeding pressure of 0.6-0.8MPa, frequency of 35-45Hz, induced air pressure difference of 0.2-0.7MPa, material water content of below 7%, and jet milling with mesh number of 80-100 mesh.

Third, the selection of the mesh number of the wall breaking powder for the best granulation

In the production and preparation process of the granules, the particle size of the granulated powder is theoretically considered to be large, and the compressibility is poor when dry-pressing granulation is adopted, so that the granules are easy to loosen and uneven in the production process; the particle size of the granulated powder is too fine, the powder adsorbability is greatly enhanced, the granulation is difficult in production due to the fact that moisture in equipment and air is adsorbed in the production process, and the dissolubility of the granules is poor. To prove the above theory, the optimum selection of the mesh number of the granulated and wall-broken powder is tested by taking the measurement of fluidity, moldability, and dispersibility as indexes. 1. Detection instrument

Model JA2003X electronic analytical balance (manufacturer: Shanghai precision scientific instruments Co., Ltd.), bulk density tester (self-made), measuring cylinder, and funnel.

2. Reagent

Under the condition that no auxiliary material or additive is added, the traditional Chinese medicine wall breaking powder (I level wall breaking powder) with the particle size less than 400 meshes, the traditional Chinese medicine wall breaking powder (II level wall breaking powder) uniformly mixed with the particle size of 400 plus 800 meshes and the traditional Chinese medicine wall breaking powder (III level wall breaking powder) with the particle size greater than 800 meshes are subjected to dry-pressure granulation by micro powder inclusion, wherein the forming pressure of the micro powder inclusion is 100kg, the screw rotating speed is 100rpm, the pressing shaft rotating speed is 18rpm and the whole grain rotating speed is 180rpm, so that I level particles, II level particles and III level particles are obtained respectively.

3. Test method

3.1 angle of repose: the method comprises the steps of adopting a fixed-hopper method, connecting three funnels in series and fixing the funnels at a height of 1cm on horizontally placed coordinate paper, carefully pouring particles made of different grades of powder into the uppermost funnel along the funnels until the cone rest tip of medicinal powder formed on the coordinate paper contacts the lower end of the funnel, measuring the diameter of the bottom of the cone by the coordinate paper (repeatedly measuring for 3 times), calculating the angle of repose (Ig ∞ H/R), wherein the smaller the angle of repose is, the better the flowability is and the friction force between particles is small. See table 1.

3.2 moldability: the wall-broken powder of 3 different mesh sizes was granulated under the same conditions by dry pressing to form the product, and the results are shown in Table 1.

3.3 solubility: respectively taking 10g of granules prepared from the traditional Chinese medicine wall-broken powder with different grades, adding 200ml of hot water, and stirring for 5 minutes until the soluble granules are completely dissolved or slightly turbid. The results are shown in Table 1.

4. Results

TABLE 1 selection of the mesh number of the wall-broken powder for the best granulation

5. And (4) conclusion:

(1) fluidity: the angle of repose theta is an important parameter of powder flowability, and the smaller the angle of repose is, the better the flowability is and the smaller the inter-particle friction force is. The fluidity of theta is generally considered to be better than 30 degrees and less than 40 degrees, and the fluidity of theta is basically capable of meeting the production requirement, and the fluidity of theta is poor when the theta is more than 45 degrees. As can be seen from the above table, the difference between the angle of repose of the grade iii particles and that of the grade ii particles is small, which indicates that the flowability of the particles prepared from the two-stage wall-breaking powder is similar, the angle of repose measured by the grade i particles is greater than that of the grade ii particles and that the flowability can only substantially meet the process production requirements, and indicates that: the fluidity of the granules prepared from the II-grade and I-grade wall-breaking powder is better than that of the granules prepared from the I-grade wall-breaking powder;

(2) formability: because the I-grade particles are dry-pressed and molded by adopting the powder with the particle size less than 400 meshes, compared with the powder with the particle size of II-grade particles (evenly mixed with 800 meshes of 400) and III-grade particles (larger than 800 meshes), the powder has uneven appearance and cracks during molding, mainly because the powder contains larger particles, the large particles are difficult to fully absorb moisture and heat in water vapor during tempering, are not easy to soften as fine wall-broken powder, and lead to the difference of shrinkage due to different softening degrees during cooling, so that the problems that the particles are easy to loosen during dry-pressing and molding and the prepared particles are uneven are solved. In general, a material having a small particle size is easily compressible, a material having a large particle size is hardly compressible, and the smaller the particle size of the material is, the larger the elongation and deformation of the particles are under the same pressure and conditions. That is, the smaller the particle size, the easier the molding, so that the dry pressing of the powder for producing the grade II particles and the grade III particles is easier. However, the powder has a limited particle size, and when the particle size is too small, the surface area of the powder is enlarged, the adsorbability is enhanced, so that the production equipment is difficult to clean, and the production efficiency is influenced. The above experimental results show that the molding results are from good to bad: the granules prepared from the II-grade wall-breaking powder are more than the granules prepared from the III-grade wall-breaking powder are more than the granules prepared from the I-grade wall-breaking powder;

(3) and (3) dissolubility: the larger the particle size of the powder is, the larger the surface tension is, the less aggregation is likely to occur, the larger the contact area with water is, the more easily the powder is dissolved in water, the smaller the particle size of the powder is, the smaller the mutual adsorption between the powder is, the agglomeration is, and the more difficult the powder is to be dissolved in water. As can be seen from the experimental results in Table 1, the particles prepared from the grade I and II powders were completely dissolved within 5 min; the granules prepared from the grade III powder are not completely dissolved within 5 min. The particle prepared by the grade I and II powder has better dissolvability than the particle prepared by the grade III powder, and the particle prepared by the grade I and II powder has similar compatibility.

In conclusion, the results of investigation from three aspects of angle of repose, molding results and solubility show that the powder with the powder mesh number between 400-800 meshes (class II) is the best mesh number for granulation.

Example 2

The present embodiment is a comparison study of rhodiola rosea decoction pieces (wall breaking) and traditional decoction piece fingerprint.

10 samples of the original samples were obtained from the production areas of Tibet, Sichuan and inner Mongolia and identified as rhodiola crenulata (hook.f. et Thoms.) H.Ohba, a Crassulaceae plant. Pulverizing traditional decoction pieces, sieving with No. 3 sieve, breaking cell wall, pulverizing, and granulating.

The chromatographic conditions are as follows: the chromatographic column is an Agilent C18 column (4.6 mm. times.250 mm, 5 μm); mobile phase: acetonitrile (a) -0.02% phosphoric acid water (B), gradient elution: 6% A-6% A (0-18min), 6% A-20% A (18-45min), 20% A-20% A (45-55min), 20% A-22% A (55-65min), 22% A-22% A (65-90min), DAD detection wavelength of 278nm, flow rate of 0.8 mL/min^-1The column temperature was 30 ℃ and the amount of sample was 10. mu.L.

The preparation method of the test sample comprises the following steps: precisely weighing about 1g of rhodiola rosea sample, placing the rhodiola rosea sample in a 100mL conical flask, precisely adding 50mL of 50% methanol, weighing, carrying out ultrasonic treatment (300W, 33kHz) for 45min, cooling, weighing again, supplementing the loss weight with 50% methanol, shaking up, filtering, and filtering with a microporous filter membrane (0.45 mu m) for later use.

Preparation of control

Precisely weighing appropriate amount of gallic acid, salidroside, tyrosol and rutin, placing into brown measuring flask, respectively adding 50% methanol to obtain gallic acid 0.1 mg. mL^-10.2 mg/mL salidroside^-1Tyrosol 0.2 mg/mL^-1Rutin 0.1 mg/mL^-1And (4) obtaining the reference substance solution.

1. Stability test of repeatability test

1g and 6 parts of rhodiola rosea decoction piece (wall-broken) sample and a traditional decoction piece sample are respectively taken to prepare a sample solution for chromatographic detection, the collected data is calculated, and the similarity analysis result carried out by using the 'similarity evaluation system of traditional Chinese medicine chromatographic fingerprint spectrum' version 2012.1 shows that the chromatographic detection method has good repeatability and the sample solution has good stability within 24 hours.

2. Calibration and confirmation of common fingerprint peaks of rhodiola rosea traditional decoction pieces and wall-broken decoction pieces and similarity analysis

Taking 10 batches of rhodiola rosea decoction piece (wall-broken) samples and traditional decoction piece samples, preparing a sample solution for chromatographic determination by 1g each, introducing obtained spectrum data parameters into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012.1 edition, analyzing by a median method, automatically matching to generate a fingerprint, and obtaining the fingerprint superposition graph results of 10 batches of rhodiola rosea decoction piece (wall-broken) and traditional decoction piece fingerprints shown in figures 1 and 2. Marking the peak areas with larger peak area and better stability and repeatability in chromatographic peaks as common peaks, marking 22 common peaks in a result experiment, comparing the data with a reference HPLC chromatogram to determine that the peak 1 is gallic acid, the peak 2 is salidroside, the peak 4 is tyrosol, the peak 14 is rutin, and the chromatogram result is shown in figure 3. Salidroside is the main index component of rhodiola, the peak emergence time is stable, the separation degree is good, salidroside is selected as a reference peak, the relative peak area and the relative retention time of the common peak of the fingerprint spectrums of all samples are calculated, and as a result, the relative retention time RSD% of the common peak of the rhodiola decoction pieces (wall breaking) and the common peak of the fingerprint spectrums of the traditional decoction pieces is less than 3%, and the relative peak area RSD% is large, so that the full appearance of the common peak of each batch of rhodiola fingerprint spectrums is basically consistent, the composition and quantity of chemical components are not greatly different, and the content difference of the chemical components is large.

Chromatogram data parameters obtained by 10 batches of rhodiola rosea traditional decoction pieces and wall-broken decoction pieces are introduced into software 2012.1 version of Chinese medicine chromatogram fingerprint similarity evaluation system to examine consistency of similarity of chromatographic peaks. The similarity results show that the similarity difference between 10 batches of rhodiola rosea decoction pieces (wall breaking) and the traditional decoction pieces is small, and the sample similarity is over 0.9, which shows that the quality of the fingerprint spectrum of the rhodiola rosea decoction piece (wall breaking) sample established in the experiment is stable.

3. Rhodiola rosea decoction piece (wall-broken) and traditional decoction piece fingerprint comparison research

Comparing HPLC fingerprint spectra before and after wall breaking of radix Rhodiolae medicinal material, the result shows that the general appearance of radix Rhodiolae decoction pieces (wall-broken) is basically consistent with that of each main chromatographic peak of traditional decoction pieces, and there are 22 peaks in total, and the number of chromatographic peaks of radix Rhodiolae is not increased or disappeared after wall breaking and crushing, which indicates that the kind and number of chemical components of radix Rhodiolae are not changed after wall breaking and crushing; by comparing the peak areas of the chromatographic peaks before and after wall breaking, the results show that the peak areas of salidroside (No. 2), tyrosol (No. 4) and rutin (No. 14) are increased except that the peak areas of the

chromatographic peaks

1, 10, 12, 15, 16 and 18 are slightly reduced, wherein the peak areas of the

chromatographic peaks

3, 5, 11, 21 and 22 are obviously increased, and the peak areas of the other chromatographic peaks are increased to different degrees. The results are shown in FIG. 4.

Example 3

This example is a comparative study of the protection effect of rhodiola rosea decoction pieces (wall-broken) obtained by the present invention and traditional decoction pieces on ischemia and hypoxia H9c2 myocardial cells.

Weighing radix Rhodiolae decoction pieces (wall-broken) and traditional decoction pieces each 1g, placing in conical flask with plug, adding 50mL 50% methanol solution, sealing, weighing, ultrasonic extracting at 300W and 33kHz for 45min, cooling, weighing again, supplementing with 50% methanol solution, shaking, filtering, collecting filtrate, filtering with 0.22 μm microporous membrane, and making into 20 mg/mL decoction pieces^-1The mother liquor is ready for use and is diluted to the required concentration before use.

Cell plating: collecting logarithmic growth phase H9c2 cells (purchased from Shanghai cell bank of Chinese academy of sciences), digesting with trypsin, resuspending complete cell culture solution to obtain cell suspension, and suspending at 1 × 10⁴The cell amount of each hole is inoculated in a 96-hole plate, 8 columns are multiplied by 3 rows in total and are respectively used for a normal group, a model group, a rhodiola rosea traditional decoction piece low, medium and high dose group and a rhodiola rosea wall-broken decoction piece low, medium and high dose group, and each group is provided with 3 parallel compound holes; at 37 ℃ with 5% CO₂Culturing for 24h in a constant-temperature incubator to synchronize cells and then performing a drug intervention experiment;

preparing different-concentration drug-containing culture media of the rhodiola wall-broken decoction pieces and the traditional decoction pieces: 0.2 mg/mL^-1、0.02mg·mL^-1、0.002mg·mL^-1；

After the cells were grouped, the normal group and the model group were completely cultured in the medium, and the other groups were cultured in the medium containing the corresponding drug at 37 ℃ and 5% CO₂After the cells are cultured in a constant temperature incubator for 1h, replacing the model group with a sugar-free and serum-free DMEM medium, replacing the cells of the other groups with a corresponding medicine-containing sugar-free and serum-free DMEM medium, placing the cells in an anoxic box, and carrying out closed anoxic culture at 37 ℃ for 8 h; normal replacement was done in complete medium with 5% CO at 37 deg.C₂Culturing for 8h in an incubator;

and (3) CCK-8 detection: the cell culture broth (stored separately for subsequent LDH detection) was removed, 30. mu.L of CCK-8 was added to the cells in the wells, incubated at 37 ℃ in a incubator for 4h, and 150. mu.L of ddH was added₂O, oscillating for 5min at low speed, and measuring the absorbance of each group at the position of 450nm wavelength of an enzyme-labeling instrumentValues, assay results, calculate cell viability.

The cell survival rate was (experimental OD value-blank well OD value)/(normal OD value-blank well OD value) × 100%.

As shown in fig. 5, compared with the normal group, the cell survival rate of the model group H9c2 was significantly reduced (P < 0.05), and the cell survival rate of the rhodiola wallichiana wall-broken decoction piece high-dose group (B3) and the cell survival rate of the traditional decoction piece high-dose group (A3) H9c2 were not significantly reduced.

Compared with the model group, the rhodiola rosea wall-broken decoction pieces low (B1), medium (B2), high (B3) dose group and the rhodiola rosea traditional decoction piece high dose group (A3) can obviously improve the survival rate of H9c2 cells (P is less than 0.05). Under the same concentration, the improvement of the H9c2 cell survival rate of the rhodiola wall-broken decoction pieces is better than that of the rhodiola traditional decoction pieces.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

Claims

1. A detection method of rhodiola wall-broken decoction pieces is characterized by comprising the following steps:

(1) obtaining an HPLC (high performance liquid chromatography) spectrum of the rhodiola wall-broken decoction pieces;

(3) and detecting the quality of the rhodiola wall-broken decoction pieces based on the fingerprint information.

2. The method for detecting the rhodiola rosea wall-broken decoction pieces as claimed in claim 1, wherein the conditions for obtaining the HPLC (high performance liquid chromatography) spectrum of the rhodiola rosea wall-broken decoction pieces in the step (1) comprise:

the chromatographic column is an Agilent C18 column; mobile phase: acetonitrile (a) -0.02% phosphoric acid water (B), gradient elution: 6% A-6% A (0-18min), 6% A-20% A (18-45min), 20% A-20% A (45 min)-55min), 20% A-22% A (55-65min), 22% A-22% A (65-90min), DAD detection wavelength of 278nm, flow rate of 0.8mL min^-1The column temperature was 30 ℃.

3. The method for detecting the rhodiola rosea wall-broken decoction pieces as claimed in claim 2, wherein the method further comprises the following processing steps of the rhodiola rosea wall-broken decoction pieces before the step (1): weighing 1g radix Rhodiolae wall-broken decoction pieces, adding 50% methanol, ultrasonic treating for 20-60min, cooling, adding 50% methanol to make up the weight loss, and filtering with microporous membrane.

4. The method for detecting the rhodiola rosea wall-broken decoction pieces as claimed in claim 1, wherein the rhodiola rosea wall-broken decoction pieces are prepared by pulverizing rhodiola rosea raw materials into powder of more than 300 meshes and then preparing the powder into granular decoction pieces of 30-200 meshes.

5. The method for detecting the rhodiola rosea wall-broken decoction pieces as claimed in claim 1, wherein the preparation method of the rhodiola rosea wall-broken decoction pieces comprises the following steps:

6. The method for detecting the rhodiola rosea wall-broken decoction pieces as claimed in claim 1, wherein the step (2') comprises:

(2' -2) a step of subjecting the first pulverized material to second pulverization to obtain a second pulverized material of 100-300 mesh;

7. The method for detecting the rhodiola rosea wall-broken decoction pieces as claimed in claim 6,

the first crushing is universal crushing, and the feed material is dry rhodiola rosea with the water content of 5-7 wt% and the granularity of 3.2-8 mm; the second crushing is ball milling crushing; and the third crushing is airflow crushing, wherein the conditions of the airflow crushing comprise feeding pressure of 0.5-0.9MPa, frequency of 35-45Hz, induced air pressure difference of 0.2-0.8MPa, feeding mesh number of 100-300 meshes and crushing to be more than 300-800 meshes, thereby obtaining the wall-broken powder.

8. The method for detecting the rhodiola rosea wall-broken decoction pieces as claimed in claim 1, wherein in the step (3), if the relative retention time of the No. 1 peak is between 0.3000 and 0.3065, the relative retention time of the No. 4 peak is between 1.1000 and 1.1200, and the relative retention time of the No. 14 peak is between 2.1500 and 2.2300, the rhodiola rosea wall-broken decoction pieces are detected to meet the requirements; wherein the relative retention time is the relative retention time obtained with the retention time of peak No. 2 as a reference.

9. The method for detecting the rhodiola rosea wall-broken decoction pieces as claimed in claim 1, wherein if the relative peak area of the No. 1 peak is 8.7000-9.0000, the relative peak area of the No. 4 peak is 1.3000-1.5000 and the relative peak area of the No. 14 peak is 1.500-1.8000, the rhodiola rosea wall-broken decoction pieces are detected to meet the requirements; the relative peak area is a relative peak area obtained by taking the peak area of the No. 2 peak as a reference.

10. A quality control method of rhodiola wall-broken decoction pieces, which takes the method according to any one of claims 1 to 9 as a step.