Electron beam irradiation sterilization and quality detection method for Platycodon grandiflorum

Abstract

The invention discloses a platycodon electron beam irradiation sterilization and quality detection method, which belongs to the field of Chinese medicine sterilization and quality detection of Chinese medicine, and comprises the following steps: (1) establishing a fingerprint of platycodon; (2) sealing and packaging the platycodon in a packaging bag, and irradiating the packaged platycodon using a high-energy electron accelerator; (3) storing the irradiated platycodon at room temperature; (4) performing quality detection on the irradiated platycodon. The application can significantly reduce the number of microorganisms in platycodon; the test results show that after 360 days of storage, the number of microorganisms in the irradiated platycodon meets the requirements of the pharmacopoeia, and the sensory properties and physical and chemical indicators thereof are not significantly changed. At the same time, under the same chromatographic conditions, the application not only establishes the fingerprint of platycodon, but also simultaneously determines the content of platycodon saponin D in platycodon. The whole method is simple to operate and has both qualitative and quantitative functions. The application overcomes the singleness of the original quality detection method and has a high application value.

Description

Technical Field

[0001] The present application relates to the field of traditional Chinese medicine, in particular to the field of traditional Chinese medicine sterilization and traditional Chinese medicine quality inspection, and specifically to a method for sterilizing and quality inspection of Platycodon grandiflorum by electron beam irradiation. More specifically, the present application relates to a method for sterilizing Platycodon grandiflorum by electron beam irradiation, and a method for quality inspection of Platycodon grandiflorum after irradiation. Background Art

[0002] Platycodon grandiflorum Platycodon grandiflorus ) As a commonly used Chinese medicine, it has the effects of clearing the lungs, relieving sore throat, removing phlegm, and draining pus. In addition, the pharmacological activities of Platycodon grandiflorum in immune regulation, anti-inflammatory, anti-tumor, liver protection, and hypolipidemic have also attracted widespread attention. In addition to its medicinal uses, as a traditional "medicine and food" medicinal material, Platycodon grandiflorum is also a very good functional food. In some areas, it is very common to use Platycodon grandiflorum as an edible vegetable, and beverages, candied fruits, preserved fruits and other products made from Platycodon grandiflorum have a large market sales volume. At the same time, cosmetics made from Platycodon grandiflorum extracts also have the effect of beauty and skin care, and are favored by the cosmetics industry.

[0003] Chinese medicinal materials are easily contaminated with microorganisms and insect eggs during the harvesting, drying, processing and storage stages, causing them to deteriorate, rot, mildew, and be infested by insects, greatly affecting their quality and efficacy.

[0004] The storage and maintenance technologies of Chinese medicinal materials are mainly divided into two categories: chemical storage and maintenance technologies and physical storage and maintenance technologies. Among them, chemical maintenance technologies include sulfur fumigation, aluminum phosphide fumigation, ethylene oxide fumigation, gas conditioning maintenance, low temperature and microwave treatment storage and maintenance technologies, etc. Due to its significant insect and mildew prevention effects, low cost, and easy operation, sulfur fumigation has been widely used in medicinal material processing and storage, but excessive use of sulfur fumigation will cause dangerous components such as sulfur dioxide and arsenic trioxide (arsenic component) to remain. Aluminum phosphide fumigation can effectively kill pests and microorganisms in Chinese medicinal materials, but aluminum phosphide is toxic to both humans and animals. It mainly acts on the nervous system, respiratory system, cardiovascular system and liver, causing convulsions, coma, and even death. Ethylene oxide fumigation uses ethylene oxide as a drug, and ethylene oxide itself has a huge carcinogenic risk; therefore, this method cannot be used on a large scale. The controlled atmosphere storage required by the controlled atmosphere curing method is expensive, requires frequent leak checks, temperature and humidity measurements, and is not suitable for long-term storage. Low temperature and microwave treatment storage and curing technology is only suitable for a small amount of precious medicinal materials and has a narrow application range.

[0005] Therefore, a new device and / or method is urgently needed to solve the above problems. Summary of the invention

[0006] Electron beam irradiation sterilization has the characteristics of no pollution, no residue, significant insecticidal and sterilization effect, and high safety, and is expected to be used in the sterilization of traditional Chinese medicines.60 Compared with Co-γ, the penetration ability of electron beam is weaker, and the appropriate irradiation dose and processing technology vary greatly for different irradiation objects.

[0007] Furthermore, ensuring the stability of the quality of Chinese medicine after irradiation is a prerequisite for the application of irradiation sterilization in the field of Chinese medicine preservation. Therefore, establishing corresponding quality inspection standards for Chinese medicine after irradiation is of great significance for ensuring the efficacy of Chinese medicine. However, in the prior art, there is no comprehensive quality inspection method for Platycodon grandiflorum after irradiation.

[0008] The invention purpose of the present application is to provide a method for electron beam irradiation sterilization and quality detection of Platycodon grandiflorum in view of the problem that there is no mature electron beam irradiation sterilization and preservation method for Platycodon grandiflorum. More specifically, the present application provides a method for electron beam irradiation sterilization of Platycodon grandiflorum and quality detection after irradiation. The present application can significantly reduce the number of microorganisms in Platycodon grandiflorum. The test results show that after the present application is used to treat Platycodon grandiflorum, the number of microorganisms in the irradiated Platycodon grandiflorum after storage for 360 days meets the requirements of the pharmacopoeia, and its sensory properties and physical and chemical indicators do not change significantly. At the same time, the fingerprint established by the present application can more comprehensively reflect the chemical components in Platycodon grandiflorum, and can accurately detect the content of platycodon saponin D in Platycodon grandiflorum. The control fingerprint obtained can objectively evaluate the quality of Platycodon grandiflorum after irradiation, overcome the singleness of the original quality detection method, and has a high application value.

[0009] In order to achieve the above purpose, this application adopts the following technical solutions:

[0010] A method for electron beam irradiation sterilization and quality detection of Platycodon grandiflorum comprises the following steps:

[0011] S1. Establishing the fingerprint of Platycodon grandiflorum;

[0012] S2, after sealing and packaging the Platycodon grandiflorum in a packaging bag, irradiating the packaged Platycodon grandiflorum using a high-energy electron accelerator;

[0013] S3, storing the Platycodon grandiflorum irradiated in step S2 at room temperature;

[0014] S4. Conducting quality inspection on the irradiated Platycodon grandiflorum;

[0015] The operation steps of step S1 are as follows:

[0016] S11. Preparation of reference melt

[0017] The platycoside D reference substance, the platycoside D2 reference substance, and the platycoside D3 reference substance are respectively mixed in a set ratio, and methanol is added to the mixture to obtain a first intermediate; after filtering the first intermediate, a reference substance melt containing the platycoside D reference substance, the platycoside D2 reference substance, and the platycoside D3 reference substance is prepared;

[0018] S12, preparing a test solution;

[0019] S13. Graph establishment

[0020] The reference solution prepared in step S11 and the test solution prepared in step S12 are respectively injected into a liquid chromatograph for measurement to obtain a fingerprint of Platycodon grandiflorum;

[0021] In the step S4, the irradiated Platycodon grandiflorum is prepared into an irradiated Platycodon grandiflorum solution, and the solution is injected into a liquid chromatograph and measured by high performance liquid chromatography to obtain a fingerprint of the irradiated Platycodon grandiflorum.

[0022] In the step S11, a reference substance melt is obtained by filtering with a microporous filter membrane having a pore size of 0.22 μm.

[0023] The operation of step S12, preparing the test solution is as follows:

[0024] Grind the Platycodon grandiflorum, sieve it, and accurately weigh 2.0g of Platycodon grandiflorum powder;

[0025] The weighed Platycodon grandiflorum powder was placed in a stoppered conical flask, 50 ml of methanol was accurately added, and the weight was weighed to obtain the second intermediate;

[0026] The second intermediate is subjected to ultrasonic treatment for 20-200 minutes to obtain a third intermediate;

[0027] After the ultrasonic treatment is completed, the third intermediate is placed at room temperature, weighed, and the lost weight is supplemented with methanol, and then shaken and filtered to obtain a fourth filtrate;

[0028] Accurately measure 25 mL of the fourth filtrate, evaporate to dryness in a water bath at 90°C, dissolve the obtained residue in water, and extract with a water-saturated n-butanol solution; combine the extracts, wash with 50 mL of ammonia test solution, discard the ammonia solution, continue washing with 50 mL of n-butanol saturated aqueous solution, discard the water solution, and obtain a fifth solution;

[0029] Finally, the fifth solution was evaporated to dryness, and an appropriate amount of methanol was added to the residue to dissolve the residue, and then the volume was fixed to 5 mL. After shaking and filtering, the filtrate was collected to obtain the test solution.

[0030] In the step S12, the platycodon grandiflorum is crushed, passed through a 100-mesh sieve, and 2.0 g of platycodon grandiflorum powder is accurately weighed; the weighed 2.0 g of platycodon grandiflorum powder is placed in a stoppered conical flask, 50 ml of methanol with a volume fraction of 50% is accurately added, and the weight is weighed to obtain a second intermediate; the second intermediate is placed in an ultrasonic instrument with a power of 200 W and a frequency of 40 kHz, and ultrasonically treated for 40 minutes to obtain a third intermediate; after the ultrasonic treatment is completed, the third intermediate is brought to room temperature, weighed, and the lost weight is supplemented with methanol with a volume fraction of 50%, and then shaken and filtered to obtain a fourth filtrate;

[0031] Accurately measure 25 mL of the fourth filtrate, evaporate it to dryness in a water bath at 90°C, add 20 mL of water to the residue, and shake and extract it with a water-saturated n-butanol solution for 3 times, each time using 20 mL of a water-saturated n-butanol solution; combine the water-saturated n-butanol extracts, wash with 50 mL of ammonia test solution, discard the ammonia solution, continue to wash with 50 mL of water saturated with n-butanol, discard the water solution, and obtain the fifth solution; finally, evaporate the fifth solution at 65-75°C, add an appropriate amount of methanol to the obtained residue to dissolve the residue, and make the volume to 5 mL, shake well, filter, collect the filtrate, and obtain the test solution.

[0032] In step S12, the third intermediate is placed at room temperature, weighed, and supplemented with 50% by volume methanol to make up the lost weight; the calculation formula for making up the lost weight is as follows:

[0033] Lost weight = weight of the second intermediate - weight of the third intermediate.

[0034] In the step S12, the fifth solution is evaporated to dryness at 65-75° C. using a rotary evaporator, and an appropriate amount of methanol is added to the residue to dissolve the residue, and the volume is fixed to 5 mL, shaken well, filtered through a 0.22 μm microporous filter membrane, and the filtrate is collected to obtain a test solution.

[0035] In the step S1 and step S4, the chromatographic conditions are as follows: the selected chromatographic column is a Thermo Scientific Syncronis C18 chromatographic column, 5 μm, 4.6 mm×250 mm;

[0036] Mobile phase: water as phase A and acetonitrile as phase B; gradient elution was used, and the elution time and mobile phase ratio were: 0-65 min, phase B: 15%-28%; 65-91 min, phase B: 28%-93%; 91-95 min, phase B: 93%-15% B; 95-102 min, phase B: 15%-15% B; the flow rate was 0.8 mL / min;

[0037] The detection wavelength was 210 nm, the column temperature was 30 °C, and the injection volume was 30 μL.

[0038] In the step S1, the contents of the platycoside D reference substance, the platycoside D2 reference substance, and the platycoside D3 reference substance in step S11 are adjusted to obtain N groups of reference substance melts; N is a natural number, and N≥2;

[0039] In step S13, the N groups of reference solution prepared in step S11 and the test solution prepared in step S12 are respectively injected into a liquid chromatograph for measurement to obtain a fingerprint of Platycodon grandiflorum;

[0040] A regression equation was established based on the relationship between the concentrations of different reference substances and the chromatographic peak areas in the N groups of reference substance melts.

[0041] In the step S4, the peak area of ​​the fingerprint of Platycodon grandiflorum after irradiation is recorded and brought into the regression equation in the step S13 to calculate the content of the corresponding component.

[0042] In the step S1, a common characteristic reference spectrum peak map of the high performance liquid chromatography fingerprint of the test solution is obtained, and 19 peaks in the common peaks are selected from the common characteristic reference spectrum peak map according to the separation degree and the reproducibility of the sample as characteristic peaks; the sum of the peak areas of the 19 characteristic peaks accounts for more than 80% of the total peak area;

[0043] In step S4, the computer-aided similarity evaluation software for traditional Chinese medicine fingerprint is used to compare the common characteristic reference spectrum peaks of irradiated Platycodon grandiflorum with the characteristic reference spectrum peaks of the test solution, and the similarity calculated based on 19 common peaks is not less than 0.9.

[0044] Among the 19 common characteristic peaks, peak No. 8 is the platycoside D3 peak, peak No. 12 is the platycoside D2 peak, and peak No. 13 is the platycoside D peak.

[0045] In step S2, the total number of initial aerobic bacteria in Platycodon grandiflorum is less than 5.17×10 5 CFU / g, the total number of initial molds and yeasts was <1.67×10 5 CFU / g, moisture <12%.

[0046] In the step S2, the electron beam irradiation dose used is 6-8 kGy, the electron beam irradiation treatment is single-sided irradiation, and the stacking thickness of the single-sided irradiation is ≤6.3 cm.

[0047] In the step S4, the irradiated Platycodon grandiflorum is prepared into an irradiated Platycodon grandiflorum solution according to the method of step S12, and injected into a liquid chromatograph for measurement to obtain a fingerprint of the irradiated Platycodon grandiflorum.

[0048] The method of the present application can significantly reduce the number of microorganisms in Platycodon; the test results show that after 360 days of storage, the number of microorganisms in the irradiated Platycodon meets the requirements of the pharmacopoeia, and its sensory properties and physicochemical indicators have not changed significantly. At the same time, under the same chromatographic conditions, the present application not only established the fingerprint of Platycodon, but also simultaneously determined the content of Platycodon saponin D in Platycodon. The whole method is easy to operate and has both qualitative and quantitative effects. The fingerprint established by the present application can more comprehensively reflect the chemical components in Platycodon, and the chromatographic peaks are well separated, with good peak shape and good repeatability. The control fingerprint obtained can objectively evaluate the quality of Platycodon after irradiation. In summary, the present application overcomes the singleness of the original quality detection method and has a high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The following are actual pictures of Platycodon grandiflorum after irradiation in Examples 1 to 4 and without irradiation in Comparative Example 1.

[0050] Figure 2 Schematic diagram of the placement of the dosimeter in the embodiment.

[0051] Figure 3 It is the HPLC chromatogram of the reference substances of platycoside D, platycoside D2 and platycoside D3 in the examples.

[0052] Figure 4 This is a comparison diagram of the fingerprints of Platycodon grandiflorum in Examples 1 to 4 and Comparative Example 1.

[0053] Figure 5 This is a comparison diagram of the fingerprints of Platycodon grandiflorum in Examples 8 to 12 and Comparative Example 1. DETAILED DESCRIPTION

[0054] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0055] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Any feature disclosed in this specification, unless otherwise stated, can be replaced by other alternative features that are equivalent or have similar purposes. That is, unless otherwise stated, each feature is only an example of a series of equivalent or similar features. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments.

[0056] 1. Sample preparation

[0057] Example 1

[0058] This embodiment provides a method for sterilizing Platycodon grandiflorum by electron beam irradiation, and a method for detecting the quality of Platycodon grandiflorum after irradiation sterilization. The operation steps are as follows:

[0059] S1. Establishing the fingerprint of Platycodon grandiflorum;

[0060] S2, after sealing and packaging the Platycodon grandiflorum in a packaging bag, irradiating the packaged Platycodon grandiflorum using a high-energy electron accelerator;

[0061] S3, storing the Platycodon grandiflorum irradiated in step S2 at room temperature;

[0062] S4. Conduct quality inspection on the irradiated Platycodon grandiflorum.

[0063] The operation steps of step S1 are as follows.

[0064] S11. Preparation of reference melt

[0065] Separately take Platycodon saponin D reference substance, Platycodon saponin D2 reference substance, and Platycodon saponin D3 reference substance and mix them, then add methanol to the mixture to obtain a first intermediate; the first intermediate is filtered through a 0.22 μm microporous filter membrane to prepare a reference substance melt containing Platycodon saponin D reference substance, Platycodon saponin D2 reference substance, and Platycodon saponin D3 reference substance.

[0066] S12. Prepare the test solution

[0067] Take Platycodon grandiflorum and grind it, pass it through a 100-mesh sieve, and accurately weigh 2.0g of Platycodon grandiflorum powder; place the weighed 2.0g Platycodon grandiflorum powder in a stoppered conical flask, accurately add 50ml of methanol with a volume fraction of 50%, weigh it, and obtain a second intermediate. Then, place the second intermediate in an ultrasonic instrument with a power of 200W and a frequency of 40kHZ, and ultrasonically treat it for 40 minutes to obtain a third intermediate. After the ultrasonic treatment is completed, put the third intermediate to room temperature, weigh it, and make up the lost weight with methanol with a volume fraction of 50%, then shake it well and filter it to obtain a fourth filtrate. Accurately measure 25 mL of the fourth filtrate, evaporate it to dryness in a water bath at 90°C, add 20 mL of water to the residue, and shake and extract it with water-saturated n-butanol for 3 times, each time with 20 mL of water-saturated n-butanol; combine the n-butanol extracts, wash with 50 mL of ammonia test solution, discard the ammonia solution, continue to wash with 50 mL of water saturated with n-butanol, discard the water solution, and obtain the fifth solution; finally, use a rotary evaporator to evaporate the fifth solution at 65-75°C, add an appropriate amount of methanol to the residue to dissolve it, and make it up to 5 mL, shake well, filter through a 0.22 μm microporous filter membrane, collect the filtrate, and obtain the test solution.

[0068] S13. Graph establishment

[0069] Finally, the mixed reference solution and the test solution were injected into a liquid chromatograph for determination to obtain the fingerprint of Platycodon grandiflorum.

[0070] The liquid chromatography conditions were as follows:

[0071] The selected chromatographic column was a Thermo Scientific Syncronis C18 column (column length, 25 cm, inner diameter, 4.6 mm, particle size, 5 μm); water-acetonitrile was used as the mobile phase for gradient elution, and the elution conditions were: 0~65min (15%~28% B), 65~91min (28%~93% B), 91~95min (93%~15% B), 95~102min (15%~15% B), with a flow rate of 0.8mL / min; the detection wavelength was 210nm, the column temperature was 30℃, and the injection volume was 30μL.

[0072] In the step S1, the contents of the platycodon saponin D reference substance, the platycodon saponin D2 reference substance, and the platycodon saponin D3 reference substance in the step S11 are adjusted to obtain N groups of reference substance melts. N is a natural number, and N≥2. In the step S13, the N groups of reference substance melts prepared in the step S11 and the test solution prepared in the step S12 are respectively injected into a liquid chromatograph for measurement to obtain a fingerprint of Platycodon. According to the relationship between the concentration of different reference substances and the chromatographic peak area in the N groups of reference substance melts, a regression equation is established.

[0073] Specifically, when establishing a regression equation, a regression equation for a single reference substance can be established (e.g., only a regression equation for a platycoside D reference substance, a platycoside D2 reference substance, or a platycoside D3 reference substance can be established; in this case, only the concentration of the N group of reference substance melt corresponding to a single reference substance can be adjusted); or a regression equation for multiple reference substances can be established (e.g., only a regression equation for a platycoside D reference substance, a platycoside D2 reference substance can be established, or a regression equation for a platycoside D reference substance, a platycoside D3 reference substance can be established, or a regression equation for a platycoside D2 reference substance, a platycoside D3 reference substance can be established, or a regression equation for a platycoside D reference substance, a platycoside D2, a platycoside D3 reference substance can be established, or a regression equation for a combination of other reference substances in Platycodon grandiflorum; in this case, only the concentration of the N group of reference substance melt corresponding to multiple reference substances can be adjusted). In the fingerprint, the concentration of the reference substance is correlated with the area of ​​the peak, so a regression equation can be established based on the relationship between the concentration of different reference substances and the area of ​​the chromatographic peak in the N group of reference substance melts.

[0074] In step S2, the Platycodon grandiflorum is sealed in a packaging bag. The Platycodon grandiflorum is a Platycodon grandiflorum medicinal material, and its initial aerobic bacterial count is less than 5.17×10 5 CFU / g, the total number of initial molds and yeasts was <1.67×10 5 CFU / g, moisture <12%; the packaging bag is made of PET+PE (in one example, its product name is: PET transparent flat-bottom ziplock bag); the bag size is 16 × 18cm, and the packaging density is 0.15-0.25 g / cm 3The thickness of the Platycodon grandiflorum in the bag is 2.0 to 2.5 cm, preferably 2.1 cm.

[0075] In step S2, the power of the high-energy electron accelerator used is 19-21 kw, and the electron beam energy is 9.5-10.5 MeV. In this embodiment, single-sided irradiation is adopted, the single layer thickness is 2.1 cm, the number of stacking layers is 1 layer (that is, the stacking thickness of the sample is 2.1 cm), and the electron beam irradiation dose used is 2 kGy.

[0076] In the step S4, the irradiated Platycodon grandiflorum is prepared into an irradiated Platycodon grandiflorum solution according to the method of step S12, and injected into a liquid chromatograph for measurement to obtain a fingerprint of the irradiated Platycodon grandiflorum.

[0077] In this embodiment, the concentration of the platycodon saponin D reference substance in the reference substance melt is adjusted to obtain regression equations of reference substances with different concentrations of platycodon saponin D. The peak area of ​​the fingerprint of Platycodon grandiflorum after irradiation is recorded, and the regression equation of the platycodon saponin D reference substance is brought into the regression equation to calculate the content of platycodon saponin D in Platycodon grandiflorum, and the content shall not be less than 0.1%.

[0078] In step S4, the HPLC detection spectrum of the irradiated Platycodon grandiflorum is compared with the reference spectrum of the test solution, and the similarity between the fingerprint spectrum of the irradiated Platycodon grandiflorum and the reference spectrum of the test solution is calculated according to the similarity evaluation system of the traditional Chinese medicine fingerprint spectrum. If the similarity between the fingerprint spectrum of the irradiated Platycodon grandiflorum and the reference spectrum of the test solution is not less than 0.90, the sample is qualified.

[0079] Comparative Example 1

[0080] The packaged Platycodon grandiflorum is not subjected to electron beam irradiation, and the remaining steps are the same as those in Example 1.

[0081] Example 2

[0082] An electron beam with a dose of 4 kGy was used for irradiation treatment, and the remaining steps were the same as those in Example 1.

[0083] Example 3

[0084] The irradiation treatment was performed using an electron beam with a dose of 6 kGy, and the remaining steps were the same as those in Example 1.

[0085] Example 4

[0086] An electron beam with a dose of 8 kGy was used for irradiation treatment, and the remaining steps were the same as those in Example 1.

[0087] Example 5

[0088] The number of bags of Platycodon grandiflorum stacked was three, with a thickness of 6.3 cm, and the remaining steps were the same as in Example 3.

[0089] Example 6

[0090] The number of bags of Platycodon grandiflorum stacked is four, the thickness is 8.4 cm, and the remaining steps are the same as those in Example 3.

[0091] Example 7

[0092] The irradiation method of Platycodon grandiflorum is double-sided irradiation, and the remaining steps are the same as those in Example 5.

[0093] Example 8

[0094] In the HPLC chromatography system, a hypersil gold aQ chromatographic column (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm, purchased from Thermo Fisher Scientific) was used. Gradient elution was performed with water (A)-acetonitrile (B) as the mobile phase, and the chromatographic conditions were as follows: 0-60 min (20%-62% B), 60-75 min (62%-92% B), 75-78 min (92%-92% B), 78-80 min (92%-20% B), 80-87 min (20%-20% B). The remaining steps were the same as those in Comparative Example 1.

[0095] Example 9

[0096] In the HPLC chromatography system, an SB-C18 chromatography column (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm, purchased from Agilent) was used, and the remaining steps were the same as those in Comparative Example 1.

[0097] Example 10

[0098] In the HPLC chromatography system, an SB-C18 chromatographic column (column length: 25 cm, inner diameter: 4.6 mm, particle size: 5 μm) was used, and water (A)-acetonitrile (B) was used as the mobile phase for gradient elution: 0-40 min (15%-25% B), 40-70 min (25%-31% B), 70-90 min (31%-91% B), 90-95 min (91%-91% B), 95-97 min (91%-15% B), 97-104 min (15%-15% B). The remaining steps were the same as those in Comparative Example 1.

[0099] Embodiment 11

[0100] In the HPLC chromatography system, a detection wavelength of 230 nm was used, and the remaining steps were the same as those in Comparative Example 1.

[0101] Example 12

[0102] In the HPLC chromatography system, a detection wavelength of 250 nm was used, and the remaining steps were the same as those in Comparative Example 1.

[0103] Embodiment 13

[0104] In the HPLC chromatography system, a detection wavelength of 280 nm was used, and the remaining steps were the same as those in Comparative Example 1.

[0105] 2. Experimental testing

[0106] 1. Microbiological testing

[0107] After 360 days of storage, the number of microorganisms in the Platycodon grandiflorum samples of Examples 1 to 4 and Comparative Example 1 was analyzed.

[0108] The total number of aerobic bacteria, molds and yeasts was carried out in accordance with the Microbial Limit Test for Non-sterile Drugs in the 2020 Edition of the Chinese Pharmacopoeia: Microbial Count Method (General Chapter 1105). The test results are shown in Table 1.

[0109] Table 1 Sterilization effect of Platycodon grandiflorum

[0110]

[0111] As shown in Table 1, the amounts of TAMC and TYMC detected in the non-irradiated Platycodon grandiflorum sample (Comparative Example 1) were 5.17×10 5 and 1.67×10 5 , does not meet the requirements of the pharmacopoeia (TAMC<10 3 TYMC<10 2 ); After irradiation with 2 kGy and 4 kGy of electron beam (Example 1 and Example 2), the TAMC and TYMC of Platycodon grandiflorum samples still do not meet the requirements of the Pharmacopoeia; TAMC in Platycodon grandiflorum treated with 6 kGy and 8 kGy of electron beam (Example 3 and Example 4) is 6.7×10 1 and 3.3×10 1 , TYMC is 6.7×10 1 and 3.3×10 1 , in line with the provisions of the pharmacopoeia.

[0112] 2. Color detection

[0113] Use NH300 colorimeter to analyze the color difference of Platycodon grandiflorum before and after irradiation, record its L*, a*, b* values, and calculate the total color difference ΔE of the sample. The calculation formula is as follows:

[0114] (1).

[0115] In formula (1), ΔL*, Δa*, and Δb* are the differences between the irradiated sample and the control.

[0116] After 360 days of storage, the colors of the Platycodon grandiflorum samples in Examples 1 to 4 and Comparative Example 1 were analyzed. The test results are shown in Table 2.

[0117] Table 2 Effects of different doses of electron beam irradiation on the color of Platycodon grandiflorum

[0118]

[0119] Note: L, a, b and △E represent the differences in brightness, redness, yellowness and total color of the samples respectively; - represents no biostatistical significance between the data in the same column, and different lowercase letters represent significant differences between the data in the same column (p<0.05).

[0120] As shown in Table 2, 2-8 kGy electron beam irradiation (Examples 1-4) had no significant effect on the L value and b value of Platycodon grandiflorum, but 2 kGy and 8 kGy electron beam irradiation increased the a value by 8.617% and 7.157% respectively; compared with the unirradiated (Comparative Example 1) sample, the total color difference of the electron beam irradiated (Examples 1-4) samples was small. In general, 2-8 kGy electron beam irradiation had no obvious effect on the color of Platycodon grandiflorum.

[0121] 3. Physical and chemical properties test

[0122] The moisture content is determined according to the moisture determination method-drying method (General Rule 0832 Method 2) of the 2020 edition of the "Chinese Pharmacopoeia"; the total ash is determined according to the ash determination method (General Rule 2302) of the 2020 edition of the "Chinese Pharmacopoeia"; the hot soaking method under the alcohol-soluble extract determination method (General Rule 2201 Determination) of the 2020 edition of the "Chinese Pharmacopoeia" is used as the solvent (ethanol in the Chinese Pharmacopoeia refers to ethanol with a concentration of 95%).

[0123] After 360 days of storage, the physical and chemical indices of the Platycodon grandiflorum samples in Examples 1 to 4 and Comparative Example 1 were analyzed. The test results are shown in Table 3.

[0124] Table 3 Effects of different doses of electron beam irradiation on the physical and chemical properties of Platycodon grandiflorum

[0125]

[0126] As shown in Table 3, the moisture content, ash content and extract of Platycodon grandiflorum after 2-8 kGy electron beam irradiation (Examples 1-4) all meet the requirements of the Pharmacopoeia (moisture ≤ 15.0%; ash ≤ 6.0%; extract ≥ 17.0%).

[0127] 4. Determination of Platycodon saponin D content

[0128] After 360 days of storage, the platycodon saponin D content of the platycodon samples in Examples 1 to 4 and Comparative Example 1 was analyzed. The test results are shown in Table 4.

[0129] Table 4 Effects of different electron beam irradiation doses on the content of active ingredients in Platycodon grandiflorum

[0130]

[0131] As shown in Table 4, the content of platycodon saponin D in Platycodon grandiflorum after 2-8 kGy electron beam irradiation (Examples 1-4) meets the requirements of the pharmacopoeia (≥0.1%).

[0132] 5. Fingerprint determination

[0133] 5.1 The fingerprints of the Platycodon grandiflorum samples in Examples 1 to 4 and Comparative Example 1 were detected after storage for 360 days.

[0134] 19 common characteristic peaks were selected and calibrated for Platycodon grandiflorum fingerprint. Figure 3 Fingerprint comparison confirmed that peak 8 (peak 8 is the 8th peak from left to right) is platycodon saponin D3, peak 12 (peak 12 is the 12th peak from left to right) is platycodon saponin D2, and peak 13 is platycodon saponin D. The fingerprints of Platycodon grandiflorum irradiated with different doses of electron beam were calibrated and matched automatically using the Chinese medicine chromatographic fingerprint similarity evaluation system 2.0 software, and the reference spectrum R was generated using the median method (0.1), as shown in Figure 2. Figure 4 As shown; and calculate the similarity, the similarity results are shown in Table 5.

[0135] Table 5 Similarity evaluation results of Platycodon grandiflorum treated with different doses of electron beam irradiation

[0136]

[0137] Note: In Table 5, S1-S3 represent three repetitions of Comparative Example 1; S4-S6 represent three repetitions of Example 1; S7-S9 represent three repetitions of Example 2; S10-S12 represent three repetitions of Example 3; S13-S15 represent three repetitions of Example 4; R represents the control fingerprint map generated by the system.

[0138] As shown in Table 5, the similarity between the fingerprints of Platycodon grandiflorum treated in Examples 1 to 4 and Comparative Example 1 and the control reached 0.9, indicating that the electron beam irradiation treatment in the dose range of 2 kGy to 8 kGy had no significant effect on the consistency of the chemical components of Platycodon grandiflorum.

[0139] 5.2 The fingerprints of Platycodon grandiflorum samples in Examples 8 to 13 and Comparative Example 1 were determined after storage for 360 days.

[0140] The fingerprints of Examples 8 to 13 and Comparative Example 1 are as follows Figure 5 The spectra of Examples 8 to 13 have problems such as poor peak shape, too small peak area, and poor separation effect.

[0141] 6. Actual absorption

[0142] On the 0th day of storage, the actual absorbed doses of the Platycodon grandiflorum samples in Examples 1 to 7 were measured. The test results are shown in Table 6.

[0143] Table 6 Actual absorbed dose

[0144]

[0145] From Tables 1 to 6 and Figure 4 It can be seen that the effective dose range of electron beam irradiation of Platycodon grandiflorum is 6.00~9.40kGy (corresponding to irradiation dose of 6~8kGy); after single-sided irradiation with an electron beam of 6kGy, the number of layers is 3, and the thickness is 6.3cm, Dmax=6.87 kGy, Dmin=6.16 kGy, U=1.115, which meets the processing standards; after single-sided irradiation with an electron beam of 6kGy, the number of layers is 4, and the thickness is 8.4 cm, Dmin=5.76 kGy, which is lower than the minimum effective dose of 6 kGy and does not meet the processing standards; after double-sided irradiation with an electron beam of 6kGy, the number of layers is 3, and the thickness is 6.3cm, Dmax=13.33 kGy, Dmin=11.94kGy, which is higher than the requirement of "Technical Guidelines for Radiation Sterilization of Traditional Chinese Medicine" issued by the State Food and Drug Administration that the maximum tolerated dose of traditional Chinese medicine irradiation shall not exceed 10 kGy, and does not meet the processing standards.

[0146] The test results show that the irradiation method of the present application can significantly reduce the number of microorganisms in Platycodon grandiflorum; after storage for 360 days, the number of microorganisms in the irradiated Platycodon grandiflorum meets the requirements of the pharmacopoeia, and its sensory properties and physical and chemical indicators have not changed significantly. At the same time, the fingerprint established by the present invention can more comprehensively reflect the chemical components in Platycodon grandiflorum, and can accurately detect the content of platycodon saponin D in Platycodon grandiflorum. The obtained control fingerprint can objectively evaluate the quality of Platycodon grandiflorum after irradiation, overcome the singleness of the original quality detection method, and has a high application value.

[0147] The present invention is not limited to the above-mentioned specific embodiments, but extends to any new features or any new combination disclosed in this specification, as well as any new method or process steps or any new combination disclosed.

[0148] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for electron beam irradiation sterilization and quality inspection of Platycodon grandiflorum, characterized in that: The steps include: S1. Establishing the fingerprint of Platycodon grandiflorum; S2, after sealing and packaging the Platycodon grandiflorum in a packaging bag, irradiating the packaged Platycodon grandiflorum using a high-energy electron accelerator; S3, storing the Platycodon grandiflorum irradiated in step S2 at room temperature; S4. Conducting quality inspection on the irradiated Platycodon grandiflorum; The operation steps of step S1 are as follows: S11. Preparation of reference solution Respectively taking platycoside D reference substance, platycoside D2 reference substance, and platycoside D3 reference substance and mixing them in a set ratio, and then adding methanol to the mixture to obtain a first intermediate; after filtering the first intermediate, a reference substance solution containing platycoside D reference substance, platycoside D2 reference substance, and platycoside D3 reference substance is prepared; S12, preparing a test solution; S13. Graph establishment The reference solution prepared in step S11 and the test solution prepared in step S12 are respectively injected into a liquid chromatograph for measurement to obtain a fingerprint of Platycodon grandiflorum; In the step S4, the irradiated Platycodon grandiflorum is made into an irradiated Platycodon grandiflorum solution, and the solution is injected into a liquid chromatograph and measured by high performance liquid chromatography to obtain a fingerprint of the irradiated Platycodon grandiflorum; The operation of step S12, preparing the test solution, is as follows: Grind the Platycodon grandiflorum, sieve it, and accurately weigh 2.0g of Platycodon grandiflorum powder; The weighed Platycodon grandiflorum powder was placed in a stoppered conical flask, 50 ml of methanol was accurately added, and the weight was weighed to obtain the second intermediate; The second intermediate is subjected to ultrasonic treatment for 20-200 minutes to obtain a third intermediate; After the ultrasonic treatment is completed, the third intermediate is placed at room temperature, weighed, and the lost weight is supplemented with methanol, and then shaken and filtered to obtain a fourth filtrate; Accurately measure 25 mL of the fourth filtrate, evaporate to dryness in a water bath at 90°C, dissolve the obtained residue in water, and extract with a water-saturated n-butanol solution; combine the extracts, wash with 50 mL of ammonia test solution, discard the ammonia solution, continue washing with 50 mL of n-butanol saturated aqueous solution, discard the water solution, and obtain a fifth solution; Finally, the fifth solution was evaporated to dryness, and an appropriate amount of methanol was added to the residue to dissolve the residue, and then the volume was fixed to 5 mL, shaken and filtered, and the filtrate was collected to obtain the test solution; In the step S12, the platycodon grandiflorum is crushed, passed through a 100-mesh sieve, and 2.0 g of platycodon grandiflorum powder is accurately weighed; the weighed 2.0 g of platycodon grandiflorum powder is placed in a stoppered conical flask, 50 ml of methanol with a volume fraction of 50% is accurately added, and the weight is weighed to obtain a second intermediate; the second intermediate is placed in an ultrasonic instrument with a power of 200 W and a frequency of 40 kHz, and ultrasonically treated for 40 minutes to obtain a third intermediate; after the ultrasonic treatment is completed, the third intermediate is brought to room temperature, weighed, and the lost weight is supplemented with methanol with a volume fraction of 50%, and then shaken and filtered to obtain a fourth filtrate; Accurately measure 25 mL of the fourth filtrate, evaporate to dryness in a water bath at 90°C, add 20 mL of water to the residue, and shake and extract with a water-saturated n-butanol solution for 3 times, using 20 mL of a water-saturated n-butanol solution each time; combine the water-saturated n-butanol extracts, wash with 50 mL of ammonia test solution, discard the ammonia solution, continue to wash with 50 mL of water saturated with n-butanol, discard the water solution, and obtain the fifth solution; finally, evaporate the fifth solution at 65-75°C, add an appropriate amount of methanol to the obtained residue to dissolve the residue, and make it dilute to 5 mL, shake well, filter, collect the filtrate, and obtain the test solution; The chromatographic conditions were as follows: the selected chromatographic column was a Thermo Scientific Syncronis C18 column, 5 μm, 4.6 mm × 250 mm; Mobile phase: water as phase A and acetonitrile as phase B; gradient elution was used, and the elution time and mobile phase ratio were: 0-65 min, phase B: 15%-28%; 65-91 min, phase B: 28%-93%; 91-95 min, phase B: 93%-15%; 95-102 min, phase B: 15%-15%; the flow rate was 0.8 mL / min; The detection wavelength was 210 nm, the column temperature was 30 °C, and the injection volume was 30 μL.

2. The method according to claim 1, characterized in that In the step S12, the fifth solution is evaporated to dryness at 65-75° C. using a rotary evaporator, and an appropriate amount of methanol is added to the residue to dissolve the residue, and the volume is fixed to 5 mL, shaken well, filtered through a 0.22 μm microporous filter membrane, and the filtrate is collected to obtain a test solution.

3. The method according to claim 1, characterized in that In the step S1, the contents of the platycoside D reference substance, the platycoside D2 reference substance, and the platycoside D3 reference substance in step S11 are adjusted to obtain N groups of reference substance solutions; N is a natural number, and N≥2; In step S13, the N groups of reference solution prepared in step S11 and the test solution prepared in step S12 are respectively injected into a liquid chromatograph for measurement to obtain a fingerprint of Platycodon grandiflorum; A regression equation was established based on the relationship between the concentrations of different reference substances and the chromatographic peak areas in the N groups of reference substance solutions.

4. The method according to claim 3, characterized in that In the step S4, the peak area of ​​the fingerprint of Platycodon grandiflorum after irradiation is recorded and brought into the regression equation in the step S13 to calculate the content of the corresponding component.

5. The method according to any one of claims 1 to 4, characterized in that: In the step S1, a common characteristic reference spectrum peak map of the high performance liquid chromatography fingerprint of the test solution is obtained, and 19 peaks in the common peaks are selected from the common characteristic reference spectrum peak map according to the separation degree and the reproducibility of the sample as characteristic peaks; the sum of the peak areas of the 19 characteristic peaks accounts for more than 80% of the total peak area; In step S4, the computer-aided similarity evaluation software for traditional Chinese medicine fingerprint is used to compare the common characteristic reference spectrum peaks of irradiated Platycodon grandiflorum with the characteristic reference spectrum peaks of the test solution, and the similarity calculated based on 19 common peaks is not less than 0.

9.

6. The method according to claim 5, characterized in that Among the 19 common characteristic peaks, peak No. 8 is the platycoside D3 peak, peak No. 12 is the platycoside D2 peak, and peak No. 13 is the platycoside D peak.

7. The method according to claim 1, characterized in that In the step S2, the electron beam irradiation dose used is 6-8 kGy, the electron beam irradiation treatment is single-sided irradiation, and the stacking thickness of the single-sided irradiation is ≤6.3 cm.

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