Papaya decoction piece standard decoction and characteristic spectrum construction method thereof

Abstract

The invention provides a standard decoction of pawpaw decoction pieces, further provides a characteristic spectrum construction method of the standard decoction of pawpaw decoction pieces and an identification method of the standard decoction of pawpaw decoction pieces, and further provides a method for identifying production places of pawpaw medicinal materials in the standard decoction. According to the construction method of the specific chromatogram, the special requirements of a chromatographic column are reduced, the analysis time is shortened, the detection efficiency is improved, the preparation of a test solution and the selection of characteristic peaks are optimized, the precision is high, the stability is good, the repeatability is good, and a new choice is provided for the quality detection of papaya medicinal materials, decoction pieces, standard decoction and formula granules.

Description

Technical Field

[0001] The invention relates to a papaya slice standard decoction and a characteristic spectrum construction method thereof. Background Art

[0002] The Chinese medicinal papaya (Chaenomelis Fructus) is the dried nearly mature fruit of the Rosaceae plant Chaenomelesspeciosa (Sweet) Nakai. The Chinese medicinal papaya has the effects of relaxing muscles and tendons, activating collaterals, and harmonizing the stomach and eliminating dampness. It is used for dampness, stiffness, soreness and pain in the waist and knee joints, vomiting and diarrhea due to summer heat, cramps, beriberi and edema, etc. The Chinese medicinal papaya is a traditional Chinese medicine that can be used as both medicine and food. It is grown in most parts of China. It is mainly produced in Anhui, Hubei, Sichuan and other places, and is commonly known as "wrinkled papaya". The Chinese medicinal papaya contains a variety of chemical components such as triterpenes, phenylpropanoids, flavonoids, organic acids, amino acids, oils and fats, steroids, etc., and has pharmacological activities such as analgesia, anti-inflammatory, immune enhancement, liver protection, anti-gastric ulcer and intestinal damage, and anti-tumor (Zou Yan, et al., Research Progress on Chemical Composition and Pharmacological Activity of Chinese Medicinal Papaya, International Journal of Pharmaceutical Research, July 2019).

[0003] Chinese medicine formula granules are made from single Chinese medicine slices through water extraction, concentration, drying and granulation. They are a supplement to traditional Chinese medicine slices and are used for clinical prescriptions of traditional Chinese medicine. Due to its advantages of convenient administration, definite efficacy and easy quality control, it has a very broad development prospect in recent years, but there is still no unified quality standard for formula granules. The "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules (Draft for Comments)" compiled and issued by the State Pharmacopoeia Committee in 2016 proposed that in the research of Chinese medicine formula granule standards, a comparative study of raw materials, intermediates, finished products and "standard decoctions" should be conducted, and pointed out that the characteristics or fingerprints of Chinese medicinal materials, Chinese medicine slices, intermediates and Chinese medicine formula granules should be correlated.

[0004] "Papaya Formula Granules" have been included in the Guangdong Provincial Quality Standard for Traditional Chinese Medicine Formula Granules (Guangdong PFKL20210211). This standard discloses the preparation method, properties, identification method and characteristic spectrum of papaya formula granules. It has been verified that the standard characteristic spectrum method of Guangdong Provincial Papaya Formula Granules that has been promulgated has the following defects: ① The use of a special hydrophilic chromatographic column has low method adaptability; ② The analysis time is long; ③ The preparation method of the test solution is complicated, and the elution strength of the solvent and the initial mobile phase is quite different, and solvent effects such as bifurcation may occur in the earlier eluting chromatographic peaks; ④ Characteristic peak 1 is easily interfered by the solvent peak (retention time is about 3 minutes), and the relative retention time is not specified.

[0005] There are also other literature reports on the quality research of papaya formula granules, such as: Wu Beili, Research on the quality standard of papaya formula granules, Anhui University of Traditional Chinese Medicine, 2016. This paper discloses the quality standard of papaya formula granules, including the determination of total organic acids: malic acid (C4 H 6 O 5 ) per 1g of total organic acid shall not be less than 192.0mg; water-soluble organic acid determination: per 1g of quinic acid, malic acid, shikimic acid, protocatechuic acid and chlorogenic acid, the total amount shall not be less than 35.22mg; triterpene acid determination: per 1g of oleanolic acid and ursolic acid, the total amount shall not be less than 5.0mg; papaya formula granules HPLC characteristic spectrum requires that the sample spectrum should present 5 characteristic peaks, with the protocatechuic acid peak as the S peak, and by calculating the relative retention time of each characteristic peak and the peak, the ratio is specified to be within ±5% of the specified value. The specified values ​​are: 0.23 (peak 1), 0.25 (peak 2), 0.85 (peak 3), 1.00 (peak S), 1.63 (peak 4). The papaya formula granule samples used in this document were prepared by water extraction of the drug residue after alcohol extraction, which is inconsistent with the traditional extraction method (water extraction) of drugs; the established HPLC characteristic spectrum method has a relatively complicated pre-treatment of the sample, which is first treated with 50% ethanol for 1 hour and then concentrated, re-dissolved in water, acidified with HCl, and then extracted twice with n-butanol, and the solvent was recovered under reduced pressure. The residue was dissolved and fixed to volume, and the processing time was long; the mobile phase aqueous phase used in the HPLC characteristic spectrum is diammonium phosphate solution (adjusted to pH 2.2 with phosphoric acid) buffer salt, which is complicated to prepare; the chromatographic analysis time is relatively long, requiring 55 minutes; the method identified 5 characteristic peaks, 3 of which are contained within a retention time of 6 minutes, and the solvent peaks will interfere with the identification of characteristic peaks. Xue Liangyan, et al., Study on HPLC fingerprint spectrum of papaya formula granules, Strait Pharmacy 2019, Vol. 31, No. 8, Establishment of HPLC fingerprint analysis method for papaya formula granules. Methods: High performance liquid chromatography was used with Megres5-C18 (Hanbon, 250×4.6 mm, 5 μm) as the chromatographic column; acetonitrile-0.05% phosphoric acid solution as the mobile phase for gradient elution; the detection wavelength was 280 nm; the flow rate was 1.0 mL·min -1 ; Column temperature 30℃. The chromatographic analysis time of this method is relatively long, requiring 60 minutes; this method determined 5 characteristic peaks, but only identified 1 characteristic peak (chlorogenic acid). The quality detection methods of papaya formula granules or decoction pieces reported so far cannot meet the requirements. Summary of the invention

[0006] The technical solution of the present invention is to provide a standard decoction of papaya slices, and another technical solution of the present invention is to provide a method for constructing a characteristic spectrum of the standard decoction of papaya slices, and a method for identifying the papaya decoction or formula granules.

[0007] The invention provides a papaya slice standard decoction, which is an extract prepared by decocting papaya with water, filtering and concentrating the filtrate, and its HPLC characteristic spectrum contains 5 characteristic peaks, and the relative retention times are respectively: peak 1: 0.6, peak 2: 0.72, peak 3: 1.000, peak 4: 1.553, peak 5: 3.113, and the relative retention time fluctuates within the range of ±10%;

[0008] The chromatographic conditions are as follows: chromatographic column: octadecylsilane bonded silica gel as filler; methanol as mobile phase A, 0.3% acetic acid solution as mobile phase B, gradient elution as specified in the following table; column temperature 30°C; detection wavelength 290nm, theoretical plate number calculated based on protocatechuic acid peak should be not less than 5000;

[0009]

[0010] Among them, the preparation process of the standard decoction of papaya slices is as follows: take 100g of papaya slices, add water and decoct twice, add 10 times of water for the first decoction, soak for 30 minutes, boil, keep boiling for 30 minutes, filter with a 200-mesh sieve, and immediately cool to room temperature; add 8 times of water for the second decoction, boil, keep boiling for 20 minutes, filter with a 200-mesh sieve, combine the decoctions, immediately cool to room temperature, concentrate, freeze-dry, and package to obtain.

[0011] Wherein, the HPLC characteristic spectrum is as follows Figure 1 shown.

[0012] The present invention also provides a method for constructing a characteristic spectrum of the standard decoction of papaya slices, which is characterized in that it comprises the following steps:

[0013] a. Preparation of reference solution: Take papaya reference medicinal material, add water and ultrasonically extract for 15-30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution;

[0014] Separately, take protocatechuic acid reference substance, chlorogenic acid reference substance, and cinnamic acid reference substance, add water to prepare a mixed solution as the reference substance solution;

[0015] b. Preparation of test solution: Take the freeze-dried powder of papaya slices standard decoction, add water, ultrasonically extract for 15-30 minutes, cool, shake well, filter, and take the filtrate;

[0016] c. Determination: Accurately pipette 10 μl of reference solution and test solution, inject into liquid chromatograph, and determine;

[0017] The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler; methanol is used as mobile phase A, 0.3% acetic acid solution is used as mobile phase B, and gradient elution is performed as specified in the following table; column temperature is 25-35°C; detection wavelength is 290nm, and the number of theoretical plates calculated based on the protocatechuic acid peak should be no less than 5000;

[0018]

[0019] Preferably, the ultrasonic extraction conditions in step a and step b are: power 240W, frequency 40kHz, and time 30 minutes.

[0020] Wherein, the concentrations of the protocatechuic acid reference substance, chlorogenic acid reference substance, and cinnamic acid reference substance solutions prepared in step a are: 0.1 mg protocatechuic acid, 15 μg chlorogenic acid, and 0.1 mg cinnamic acid per 1 ml, respectively.

[0021] Wherein, the chromatographic column described in step c is: ZORBAX Eclipse Plus C18 chromatographic column (4.6×250mm, 5μm), Luna C18(2) chromatographic column (4.6×250mm, 5μm), Agilent Eclipse XDB-C18 chromatographic column (4.6×250mm, 5μm); the column temperature is 30°C; and the flow rate is 0.8-1.2ml / min.

[0022] The present invention also provides a method for identifying a papaya slice standard decoction or papaya formula granules, which uses the papaya slice standard decoction as a standard product and prepares a sample to be tested into a test product;

[0023] The standard solution and the test solution are respectively injected into a high performance liquid chromatograph, and the detection is performed according to the chromatographic conditions of claim 1 to obtain the corresponding characteristic chromatogram to be identified;

[0024] The characteristic chromatograms of the standard and test solutions are compared. If the characteristic chromatogram of the test sample is consistent with that of the standard, it is identified as qualified.

[0025] The present invention also provides a method for identifying the origin of papaya medicinal materials, which comprises adopting the characteristic spectrum construction method to detect the characteristic spectrum of the standard decoction of papaya medicinal pieces to be tested; then using multivariate statistical analysis software SIMCA 14.1 to perform partial least squares discriminant analysis (PLS-DA) on the relative peak area results of the characteristic peaks of the samples to be tested and the relative peak area results of the characteristic peaks of the standard decoctions of papaya medicinal pieces from different origins, and obtaining the origin of the papaya medicinal materials by PLS-DA scatter plot analysis of the samples to be tested and the standard papaya samples from different origins.

[0026] The method of the present invention has the following advantages:

[0027] (1) Reduced special requirements for chromatographic columns

[0028] The original Guangdong Provincial Standard Method Characteristic Spectrum Method used Luna Omega PS C18, a special hydrophilic chromatographic column, and the method had low applicability. After optimization, it can be detected using a common C18 column. The method investigated three conventional chromatographic columns: ZORBAX Eclipse Plus C18 column (4.6×250mm, 5μm), Luna C18 (2) column (4.6×250mm, 5μm), and Agilent Eclipse XDB-C18 column (4.6×250mm, 5μm). The durability of different types of chromatographic columns was good, which improved the applicability of the method.

[0029] (2) Shorten analysis time and improve detection efficiency

[0030] The original gradient elution conditions of the Guangdong Provincial Standard Method are shown below. The analysis time is 55 minutes. After optimization, the detection can be completed in only 35 minutes, which is 20 minutes shorter and improves the detection efficiency.

[0031]

[0032] (3) Optimize the preparation of test solution

[0033] The original Guangdong Provincial Standard Method used 75% methanol reflux for 30 minutes, and after optimization, water ultrasound was used for 30 minutes. The ultrasound time is short and the operation is simpler; and the solvent is water, which reduces the difference in elution intensity with the initial mobile phase and avoids solvent effects such as bifurcation of early elution chromatographic peaks. In addition, the method of the present invention uses 0.3% acetic acid as the aqueous phase, which is simpler to prepare and has less damage to the chromatographic column.

[0034] (4) Optimize characteristic peak selection

[0035] The original Guangdong Provincial Standard Method selected 5 characteristic peaks and identified 3 characteristic peaks, of which Peak 1 had a retention time of about 3 minutes, which was easily interfered by solvent peaks, and the relative retention time of Peak 1 was not specified. After optimization, 5 chromatographic peaks were determined, and 4 characteristic peaks (5-hydroxymethylfurfural, protocatechuic acid, chlorogenic acid, and cinnamic acid) were identified, with one more characteristic peak (5-hydroxymethylfurfural) than the original method; the chromatographic peak with a retention time of about 6 minutes was selected as Peak 1 to avoid interference from solvent peaks, and its relative retention time was specified to make the method more perfect.

[0036] The characteristic spectrum construction method of the present invention has high precision, good stability and good repeatability, and provides a new option for quality detection of papaya slices, standard decoctions and formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1Reference characteristic spectrum (wherein, peak 2: 5-hydroxymethylfurfural; peak 3 (S): protocatechuic acid; peak 4: chlorogenic acid; peak 5: cinnamic acid; chromatographic column: Agilent Eclipse plus C18, 4.6mm×250mm, 5μm)

[0038] Figure 2 Influence of solvent effect (A. 75% methanol, B. methanol)

[0039] Figure 3 Overlay of characteristic spectra of 15 batches of standard decoctions of papaya slices

[0040] Figure 4 Comparative characteristic spectrum of standard decoction of papaya slices (peak 2: 5-hydroxymethylfurfural; peak 3 (S): protocatechuic acid; peak 4: chlorogenic acid; peak 5: cinnamic acid; chromatograph: Thermo UPLC; column: Agilent Eclipse plus C18 (4.6mm×250mm, 5μm))

[0041] Figure 5 PLS-DA score scatter plot of standard decoctions from different origins DETAILED DESCRIPTION

[0042] Example 1 Method for constructing characteristic spectrum of standard decoction of papaya slices of the present invention

[0043] [Characteristic spectrum] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0044] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel is used as filler; methanol is used as mobile phase A, 0.3% acetic acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; column temperature is 30°C; detection wavelength is 290nm. The theoretical plate number calculated based on the protocatechuic acid peak should not be less than 5000.

[0045]

[0046] Preparation of reference solution: Take 1 g of papaya reference medicinal material, place it in a stoppered conical flask, add 20 ml of water, and treat it with ultrasound (power 240 W, frequency 40 kHz) for 30 minutes. Cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution.

[0047] Take appropriate amount of protocatechuic acid reference substance, chlorogenic acid reference substance and cinnamic acid reference substance, add water to make a mixed solution containing 0.1 mg of protocatechuic acid, 15 μg of chlorogenic acid and 0.1 mg of cinnamic acid per 1 ml, as the reference substance solution.

[0048] Preparation of test solution: Take 0.5 g of the product, place it in a stoppered conical flask, add 20 ml of water, and treat it with ultrasound (power 240 W, frequency 40 kHz) for 30 minutes. Cool, shake well, filter, and take the filtrate to obtain the solution.

[0049] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0050] The test sample chromatogram should show 5 characteristic peaks, and the retention time should correspond to the 5 characteristic peaks in the reference drug chromatogram. Peak 3, peak 4, and peak 5 should correspond to the retention time of the corresponding reference substance peak. The peak corresponding to the protocatechuic acid reference peak is the S peak. The relative retention time of peak 1, peak 2 and S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified value is: 0.60 (peak 1), 0.72 (peak 2) (see Figure 1 ).

[0051] Example 2 Screening test of the characteristic spectrum construction method of the present invention

[0052] The present invention is based on the provincial characteristic spectrum standard of papaya formula granules, quotes the chromatographic conditions of the characteristic spectrum of papaya formula granules, studies the preparation method of the test solution of the characteristic spectrum of the standard decoction of papaya slices, establishes the characteristic spectrum method of the standard decoction of papaya slices, and conducts methodological verification.

[0053] 1. Instruments, reagents and test drugs

[0054] Instruments: Waters high performance liquid chromatograph (Acquity, Waters Corporation), Thermo ultra high performance liquid chromatograph (UltiMate 3000, Thermo Fisher Scientific); one ten-thousandth balance (BNT204, Nanjing Bonita Scientific Instrument Co., Ltd.), one millionth balance (XPE105, Mettler, Switzerland), ultrasonic cleaning instrument (1990QTD, Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Milli-QDirect, Merck & Co., Ltd.).

[0055] Reagents: methanol (analytical grade, Xilong Scientific Co., Ltd.), ethanol (analytical grade, Xilong Scientific Co., Ltd.), glacial acetic acid (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.), methanol (chromatographic grade, RCI Labscan), acetonitrile (chromatographic grade, RCI Labscan), and ultrapure water (self-made in the laboratory).

[0056] Test drugs: standard decoction of papaya slices; 5-hydroxymethylfurfural protocatechuic acid (batch number: 111626-202316, 98.4%, China Food and Drug Inspection Institutes), (batch number: 110809-202207, 97.5%, China Food and Drug Inspection Institutes), chlorogenic acid (batch number: 110753-202119, 96.3%, China Food and Drug Inspection Institutes), cinnamic acid (batch number: 110786-202305, 99.8%, China Food and Drug Inspection Institutes), papaya control medicinal materials (batch number: 121003-201206, China Food and Drug Inspection Institutes).

[0057] 2 Chromatographic conditions

[0058] Chromatographic column: ZORBAX Eclipse Plus C18 (4.6×250mm, 5μm); methanol as mobile phase A, 0.3% acetic acid solution as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 290nm. Injection volume 10μl.

[0059]

[0060] 3. Preparation of reference solution

[0061] Take 1g of papaya reference medicinal material, place it in a stoppered conical bottle, add 20ml of water, ultrasonicate for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amounts of protocatechuic acid reference substance, chlorogenic acid reference substance, and cinnamic acid reference substance, add methanol to make a mixed solution containing 0.1mg of protocatechuic acid, 15μg of chlorogenic acid, and 0.1mg of cinnamic acid per 1ml, as the reference solution of the reference substance.

[0062] 4 Investigation of the preparation method of test solution

[0063] 4.1 Investigation of extraction methods

[0064] This experiment investigated the effects of different extraction methods on the characteristic spectrum of the standard decoction of papaya slices, mainly investigating the two extraction methods of ultrasound and reflux, observing the peak shape and separation of the five characteristic peaks, and calculating the "total peak area / sample amount" of the five characteristic peaks, and comparing the effects of different extraction methods on the characteristic spectrum of the standard decoction of papaya slices.

[0065] The results showed that there was no significant difference in the peak shape, separation effect, and total peak area / sample weight of each characteristic peak using different extraction methods. Considering the ease of operation, ultrasonic extraction was selected.

[0066] 4.2 Investigation of extraction solvent

[0067] This experiment investigated the effects of different extraction solvents on the characteristic spectrum of the standard decoction of papaya slices. 75% methanol, 50% methanol, 25% methanol, 25% ethanol and water were used as extraction solvents. The peak shape and separation of the five characteristic peaks were observed and the "total peak area / sample weight" of the five characteristic peaks was calculated to compare the effects of different extraction solvents on the characteristic spectrum of the standard decoction of papaya slices, and the optimal extraction solvent was selected.

[0068] The results showed that when 75% methanol was used for extraction, the peak shape of the characteristic peaks was slightly worse due to the solvent effect; there was no obvious difference in the peak shape and separation effect of the characteristic peaks of other different extraction solvents. Among them, when water was used as the extraction solvent, the "total peak area / sample amount" of the five characteristic peaks was the largest, so water was used as the extraction solvent.

[0069] 4.3 Extraction time investigation

[0070] This experiment investigated the effect of ultrasound time (15 minutes, 30 minutes, and 45 minutes) on the characteristic spectrum of the standard decoction of papaya slices. The effect of different ultrasound times on the characteristic spectrum of the standard decoction of papaya slices was compared by calculating the "total peak area / sample weight" of the five characteristic peaks.

[0071] The results showed that there was no significant difference in the "total peak area / sample weight" of the five characteristic peaks at different ultrasonic times, indicating that complete extraction was achieved by ultrasonic extraction for 15 minutes. Considering the influence of the experimental environment, the ultrasonic extraction time was selected as 30 minutes to ensure the durability of the method.

[0072] 4.4 Determination of the preparation method of the test solution

[0073] According to the above experimental results, the sample pretreatment method of the characteristic spectrum of the standard decoction of papaya slices was determined as follows: take about 0.5 g of the powder of the standard decoction of papaya slices, put it in a stoppered conical flask, add 20 ml of water, ultrasonically treat (power 240 W, frequency 40 kHz) for 30 minutes, take it out, cool, shake well, filter, and take the filtrate.

[0074] The experiment found that when the organic phase ratio of the test solution solvent is high, the chromatographic peak shape will deteriorate or even bifurcate due to the large difference in elution strength with the initial mobile phase. Figure 2 .

[0075] 5 Methodological Validation

[0076] 5.1 Specificity Investigation

[0077] Take the test sample solution, negative sample solution and reference solution of the characteristic spectrum of the standard decoction of papaya slices, inject and analyze according to the chromatographic conditions under "1.2", and record the chromatogram. The results show that the test sample chromatogram has the same chromatographic peak at the corresponding retention time of the reference sample chromatogram and the reference medicinal material chromatogram, and there is no interference with the negative, indicating that the method has good specificity.

[0078] 5.2 Precision investigation

[0079] Take 0.5g of the powder of this product, prepare the test solution according to the test sample preparation method determined under "1.4", and analyze the sample according to the chromatographic conditions under "1.2". The same sample was sampled 6 times continuously for determination. The protocatechuic acid peak was used as the reference peak S, and the relative retention time and relative peak area of ​​peak 1, peak 2, peak 4, peak 5 and S peak were calculated, and the RSD was calculated. The results showed that the relative retention time and relative peak area RSD of each characteristic peak and S peak were less than 3.0% when the same sample solution was sampled 6 times continuously, indicating that the instrument precision was good.

[0080] 5.3 Stability investigation

[0081] Take 0.5g of the powder of this product, prepare the test solution according to the test sample preparation method determined under "1.4.4", and analyze the samples at 0, 4, 8, 12, 16, and 24 hours according to the chromatographic conditions under "1.2". Take the protocatechuic acid peak as the reference peak S, calculate the relative retention time and relative peak area of ​​peak 1, peak 2, peak 4, peak 5 and S, and calculate RSD. The results show that the same test solution was analyzed at 0, 4, 8, 12, 16, and 24 hours, and the relative retention time and relative peak area RSD of each characteristic peak and S peak were less than 3.0%, indicating that the test solution was relatively stable within 24 hours.

[0082] 5.4 Repeatability Study

[0083] Take 0.5g of the powder of this product, and make 6 parallel samples. According to the sample preparation method determined under "1.4.4", prepare 6 sample solutions. According to the chromatographic conditions under "1.2", the sample is injected and analyzed. The protocatechuic acid peak is used as the reference peak S. The relative retention time and relative peak area of ​​peak 1, peak 2, peak 4, peak 5 and S are calculated, and the RSD is calculated. The results show that the same batch of samples was measured 6 times, and the relative retention time and relative peak area RSD of each characteristic peak and S peak were less than 3.0%, indicating that the method has good repeatability.

[0084] 5.5 Intermediate precision study

[0085] Different analysts operated on different instruments at different times, took about 0.5g of papaya slice standard decoction, and paralleled 6 portions. According to the test sample preparation method determined under "1.4.4", 6 test sample solutions were prepared, and the samples were injected and analyzed according to the chromatographic conditions under "1.2". The protocatechuic acid peak was used as the reference peak S, and the relative retention time and relative peak area of ​​peak 1, peak 2, peak 4, peak 5 and S were calculated, and the RSD was calculated. The results showed that different analysts operated on different instruments at different times, and the same batch of samples were repeated 6 times. The relative retention time and relative peak area RSD of each characteristic peak and S peak were less than 3.0%. The RSD of relative retention time, relative peak area and 6 data of repeatability test were in the range of 0.20% to 0.43%, and the RSD of 6 data of repeatability test were less than 3.0%, indicating that the relative retention time of each characteristic peak had good intermediate precision.

[0086] 5.6 Durability test

[0087] ① Investigation of different chromatographic columns

[0088] To ensure that the characteristic spectrum method can be reproduced on different types of chromatographic columns, this experiment compared the effects of ZORBAX Eclipse Plus C18 column (4.6×250mm, 5μm), Luna C18(2) column (4.6×250mm, 5μm), and Agilent Eclipse XDB-C18 column (4.6×250mm, 5μm) on the durability of the characteristic spectrum of papaya slice standard decoction.

[0089] Take about 0.5g of the standard decoction of papaya slices, prepare the test solution according to the test solution preparation method determined under "4.4". Except for the different chromatographic columns, other chromatographic conditions are the same as those specified under "1.2". Sample analysis, using the protocatechuic acid peak as the reference peak S, calculate the relative retention time and relative peak area of ​​peak 1, peak 2, peak 4 and peak 5 with S, and calculate RSD. The results show that the relative retention time RSD of each characteristic peak except characteristic peak 5 with S peak is less than 3%, and characteristic peak 5 is cinnamic acid, so the cinnamic acid reference substance is used as a reference, and the relative retention time of peak 5 is not calculated. The relative peak area RSD of each characteristic peak with S peak is less than 3.0%, indicating that the durability of different types of chromatographic columns is good.

[0090] ② Investigation of different column temperatures

[0091] The effects of different column temperatures, namely 25℃, 30℃ and 35℃, on the durability of the characteristic spectrum of the standard decoction of papaya slices were compared.

[0092] Take about 0.5g of the standard decoction of papaya slices, prepare the test solution according to the test solution preparation method determined under "1.4.4", except that the column temperature is 25℃, 30℃ and 35℃ respectively, the other chromatographic conditions are the same as those specified under "1.2", sample analysis, take the protocatechuic acid peak as the reference peak S, calculate the relative retention time and relative peak area of ​​peak 1, peak 2, peak 4 and peak 5 with S, and calculate RSD. The results show that at different column temperatures, the relative retention time RSD of each characteristic peak with the S peak is in the range of 0.90% to 6.11%, and the relative peak area RSD is less than 3%, indicating that when the column temperature is ±5℃, the change of column temperature has a greater impact on the relative retention time of each characteristic peak, so the fixed column temperature is 30℃.

[0093] Table 1 Results of durability study at different column temperatures (relative retention time)

[0094]

[0095] ③ Investigation of different flow rates

[0096] The effects of different flow rates, namely 0.8ml / min, 1.0ml / min and 1.2ml / min, on the durability of the characteristic spectrum of the standard decoction of papaya slices were compared.

[0097] Take about 0.5g of the standard decoction of papaya slices, weigh it accurately, and prepare the test solution according to the test solution preparation method determined in "1.4.4". Except that the flow rates are 0.8ml / min, 1.0ml / min and 1.2ml / min respectively, other chromatographic conditions are the same as those specified in "1.2". Sample injection and analysis are performed. Taking the protocatechuic acid peak as the reference peak S, calculate the relative retention time and relative peak area of ​​peak 1, peak 3, peak 4 and peak 5 to S, and calculate RSD. The results showed that at different flow rates, except for the known peaks (peak 4 is chlorogenic acid and peak 5 is cinnamic acid), the RSD of the relative retention time of each characteristic peak and the S peak was less than 3%. Therefore, chlorogenic acid and cinnamic acid were added as references, and only the relative retention time of peak 1, peak 2 and S peak were calculated; the RSD of the relative peak area of ​​each characteristic peak and the S peak was less than 3.0%, indicating that when the flow rate is ±0.2ml / min, the change in flow rate has little effect on the relative retention time of peak 1 and peak 2 and the relative peak area of ​​each characteristic peak, and the flow rate durability is good.

[0098] 6. Sample determination

[0099] 6.1 Feature map construction

[0100] Take 15 batches of papaya slices standard decoction, prepare the test solution according to the method under "4.4", accurately pipette the above test solution and papaya reference medicinal material reference solution, respectively, inject and measure according to the chromatographic conditions of "1.2" and record the chromatogram, import it into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software, use the S7 sample chromatogram as the reference spectrum, use the median method, the time window width is 0.1min, generate a common mode, perform multi-point correction and peak matching on the sample to obtain the control characteristic spectrum of the papaya slices standard decoction, such as Figure 3 , Figure 4 .

[0101] 6.2 Common peak calibration

[0102] The protocatechuic acid peak with high separation, good peak shape and moderate retention time was used as the reference peak S. The relative retention time and relative peak area of ​​peak 1, peak 2, peak 4, peak 5 and S were calculated, and the RSD was calculated. The results are shown in Tables 2 and 3.

[0103] Table 2 Characteristic spectrum of papaya slices standard decoction sample determination (relative retention time)

[0104]

[0105] Table 3 Characteristic spectrum sample determination of standard decoction of papaya slices (relative peak area)

[0106]

[0107] As shown in Table 2, the relative retention time RSD of each characteristic peak of the characteristic spectrum of 15 batches of papaya slices standard decoction is less than 3%, indicating that samples from different origins and different batches have more common components, and the method is relatively stable and can be used for the quality control of papaya slices standard decoction. According to the results of the previous durability investigation, the characteristic spectrum standard of papaya slices standard decoction is determined as follows: 5 characteristic peaks should be presented in the chromatogram of the test sample, and the retention time should correspond to the 5 characteristic peaks in the chromatogram of the reference medicinal material reference, of which 3 peaks should correspond to the retention time of the corresponding reference peak. The peak corresponding to the protocatechuic acid reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.60 (peak 1), 0.72 (peak 2).

[0108] 6.3 Partial Least Squares Discriminant Analysis (PLS-DA)

[0109] As shown in Table 3, the relative peak areas of the characteristic spectra of the 15 batches of papaya slices standard decoctions vary greatly, indicating that the content of samples from different batches and different origins varies greatly. The partial least squares discriminant analysis (PLS-DA) was used to establish an analysis model for the relative peak area results of the characteristic peaks using multivariate statistical analysis software SIMCA 14.1 ( Figure 5 ). Standard decoctions from different origins are grouped into one category.

[0110] 6.4 Identification of chromatographic peak content determination

[0111] Four common peaks were identified by reference substances. Peaks 2, 3, 4, and 5 were 5-hydroxymethylfurfural, protocatechuic acid, chlorogenic acid, and cinnamic acid, respectively. The contents in standard decoctions of different batches of papaya slices were determined, and the results are shown in Table 4.

[0112] Table 4 Content of index components in standard decoction of papaya slices (mg / g)

[0113]

Claims

1. A standard decoction of papaya slices, characterized in that: The extract is prepared by boiling papaya with water, filtering and concentrating the filtrate. Its HPLC characteristic spectrum contains 5 characteristic peaks, and the relative retention times are: peak 1: 0.6, peak 2: 0.72, peak 3: 1.000, peak 4: 1.553, peak 5: 3.113, and the relative retention time fluctuates within the range of ±10%. The chromatographic conditions are as follows: chromatographic column: octadecylsilane bonded silica gel as filler; methanol as mobile phase A, 0.3% acetic acid solution as mobile phase B, gradient elution as specified in the following table; column temperature 30°C; detection wavelength 290nm, theoretical plate number calculated based on protocatechuic acid peak should be not less than 5000; 2. The standard decoction of papaya slices according to claim 1, characterized in that: The preparation process of the standard decoction of papaya slices is as follows: 100g of papaya slices are taken, and water is added and decocted twice; for the first decoction, 10 times of water is added, and the decoction is soaked for 30 minutes, boiled, and decocted for 30 minutes while keeping a slight boil, filtered through a 200-mesh sieve, and immediately cooled to room temperature; for the second decoction, 8 times of water is added, boiled, and decocted for 20 minutes while keeping a slight boil, filtered through a 200-mesh sieve, the decoction is combined, and the decoction is immediately cooled to room temperature, concentrated, freeze-dried, and packaged to obtain the decoction.

3. The standard decoction of papaya slices according to claim 1 or 2, characterized in that: The HPLC characteristic spectrum is shown in Figure 1.

4. A method for constructing a characteristic spectrum of a standard decoction of papaya slices according to any one of claims 1 to 3, characterized in that: It includes the following steps: a. Preparation of reference solution: Take papaya reference medicinal material, add water and ultrasonically extract for 15-30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution; Separately, take protocatechuic acid reference substance, chlorogenic acid reference substance, and cinnamic acid reference substance, add water to prepare a mixed solution as the reference substance solution; b. Preparation of test solution: Take the freeze-dried powder of papaya slices standard decoction, add water, ultrasonically extract for 15-30 minutes, cool, shake well, filter, and take the filtrate; c. Determination: Accurately pipette 10 μl of reference solution and test solution, inject into liquid chromatograph, and determine; The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as filler; Use methanol as mobile phase A and 0.3% acetic acid solution as mobile phase B, and perform gradient elution according to the provisions in the following table; the column temperature is 25-35°C; the detection wavelength is 290nm, and the theoretical plate number calculated based on the protocatechuic acid peak should be no less than 5000; 5. The method for constructing a characteristic spectrum of a standard decoction of papaya slices according to claim 4, characterized in that: The ultrasonic extraction conditions described in step a and step b are: power 240W, frequency 40kHz, and time 30 minutes.

6. The method for constructing a characteristic spectrum of a standard decoction of papaya slices according to claim 4, characterized in that: The concentrations of the protocatechuic acid reference substance, chlorogenic acid reference substance, and cinnamic acid reference substance solutions prepared in step a are: 0.1 mg protocatechuic acid, 15 μg chlorogenic acid, and 0.1 mg cinnamic acid per 1 ml, respectively.

7. The method for constructing a characteristic spectrum of a standard decoction of papaya slices according to claim 4, characterized in that: The chromatographic column described in step c is: ZORBAX Eclipse Plus C18 chromatographic column (4.6×250mm, 5μm), Luna C18(2) chromatographic column (4.6×250mm, 5μm), Agilent Eclipse XDB-C18 chromatographic column (4.6×250mm, 5μm); the column temperature is 30°C; and the flow rate is 0.8-1.2ml / min.

8. A method for identifying a standard decoction of papaya slices, characterized in that: It uses the papaya slice standard decoction according to claim 1 or 2 as a standard product, and prepares the sample to be tested into a test product; The standard solution and the test solution are respectively injected into a high performance liquid chromatograph, and the detection is performed according to the chromatographic conditions of claim 1 to obtain the corresponding characteristic chromatogram to be identified; The characteristic chromatograms of the standard and test solutions are compared. If the characteristic chromatogram of the test sample is consistent with that of the standard, it is identified as qualified.

9. A method for identifying the origin of papaya medicinal material, characterized in that: It uses the characteristic spectrum construction method described in any one of claims 4 to 7 to detect the characteristic spectrum of the standard decoction of papaya slices; then uses multivariate statistical analysis software SIMCA14.1 to perform partial least squares discriminant analysis (PLS-DA) on the relative peak area results of the characteristic peaks of the samples to be tested and the relative peak area results of the characteristic peaks of the standard decoctions of papaya slices from different origins, and the PLS-DA scatter plot analysis of the samples to be tested and the standard papaya samples from different origins is used to obtain the origin of the papaya medicinal material.

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