CN113295789B

CN113295789B - Method for establishing UPLC fingerprint spectrum of Wanglaoji cold tea concentrated juice and detection method thereof

Info

Publication number: CN113295789B
Application number: CN202110529640.1A
Authority: CN
Inventors: 张丰源; 叶文才; 徐文流; 翁少全; 王英; 郑荣波; 范春林; 李词周; 熊煜; 杨方明; 王艳芳
Original assignee: Wang Lao Ji Great Health Industry Meizhou Co ltd; Guangzhou Wanglaoji Major Health Industry Co ltd
Current assignee: Wang Lao Ji Great Health Industry Meizhou Co ltd; Guangzhou Wanglaoji Major Health Industry Co ltd
Priority date: 2021-05-14
Filing date: 2021-05-14
Publication date: 2022-11-08
Anticipated expiration: 2041-05-14
Also published as: CN113295789A

Abstract

The invention belongs to the technical field of quality control of herbal tea plant beverages, and particularly relates to a method for establishing UPLC fingerprint spectrum of Wanglaoji cold tea concentrated juice and a detection method thereof. The establishing method provided by the invention comprises the following processes: (1) preparing a reference substance solution; (2) preparing a test solution; (3) Through establishment and measurement of measurement conditions of the ultra-high performance liquid chromatography, 14 characteristic common peaks can be detected. The establishment method provided by the invention is simple and convenient to operate, short in analysis time, strong in specificity, good in precision, stability and reproducibility, capable of rapidly and comprehensively detecting main chemical components in the WangLoKAT cold tea concentrated juice, reflecting the quality information of the WangLoKAT cold tea concentrated juice and the like, and providing scientific reference for production and quality control of WangLoKAT cold tea.

Description

Establishment method and detection method of UPLC fingerprint spectrum of Wanglaoji cold tea concentrated juice

Technical Field

The invention belongs to the technical field of herbal tea plant beverage quality control, and particularly relates to a method for establishing UPLC fingerprint spectrum of Wanglaoji herbal tea concentrated juice and a detection method thereof.

Background

Herbal tea is a Chinese herbal medicine which is guided by the theory of traditional Chinese medicine and flexibly applies cold and cool property or has the effect of dispelling internal heat by combining local climatic conditions and water and soil characteristics in the exploration process of long-term disease prevention and health preservation of people in the south of Ling, is summarized to have the effects of clearing heat and detoxicating, promoting the production of body fluid to quench thirst, clearing internal heat and removing dampness and the like, can be used as tea beverage in daily life, and the herbal tea is used as a culture in the south of Ling, has been recognized by the nation, and is listed as a first national level 'non-material culture heritage' in 2006. The existing prepared and sold herbal tea pavements still appear in the major street and the minor street of the Guangdong, but the canned and paper-box-packed herbal tea developed in recent years successfully pushes the herbal tea culture to the whole country and the world.

The Wanglaoji cold tea is based on the traditional formula of three flowers, three grass and one leaf (chrysanthemum, honeysuckle, frangipani, chinese mesona herb, liquorice, selfheal and microcos paniculata) and is continuously improved in the aspects of extraction of medicinal materials, concentration proportion of liquid medicine, dosage of white granulated sugar and sterilization and canning processes, and the flavor of the Wanglaoji cold tea is optimized, so that the Wanglaoji cold tea becomes a plant cold tea beverage which is popular with consumers and has high popularity. The Wanglaoji cold tea concentrated juice is an intermediate product of Wanglaoji cold tea and is obtained by extracting and concentrating seven medicinal materials in a formula.

The Wanglaoji herb tea belongs to plant beverage drinks, has more formula medicinal materials, has complex chemical components of extract concentrated juice, has only 17 reported chemical components at present, and comprises 7 phenolic acid compounds, 6 flavonoid compounds, 4 triterpenes and saponin compounds (CongGeing and other Chinese medicinal materials, 2018,41 (4): 889-893), and the research on the chemical components is not complete. At present, the quality control of WangLoKAI herbal tea is only based on the sensory detection of plant beverage products and the determination of the total dietary fiber content specified in national Standard of people's republic of China (plant beverages). The published patent (Guangzhou WangLooji herb tea intermediate volatile component fingerprint construction and detection method [ P ]. CN 106324143A, 2017.01.11) constructs the WangLooji herb tea intermediate volatile component fingerprint and detection method, the method only aims at volatile components, and the WangLooji concentrated juice is obtained by water extraction and mainly contains water-soluble components, wherein the volatile component content is very low, so that the method is used for controlling the quality of the WangLooji concentrated juice and has strong limitation.

Disclosure of Invention

The invention separates and purifies the main components of the WangLoKAT herbal tea condensed juice and identifies the structure by a plurality of separation means, and adopts UPLC technology to establish a fingerprint spectrum and a detection method which can quickly and comprehensively reflect the internal quality (chemical components) of the WangLoKAT herbal tea condensed juice, thereby providing an effective method for the production and quality control of the WangLoKAT herbal tea.

In order to achieve the purpose, the invention adopts the following specific scheme:

a method for establishing UPLC fingerprint of concentrated juice of Wanglaoji cold tea comprises the following steps:

s1, preparing a reference solution: precisely weighing reference substances including danshensu, protocatechuic acid, neochlorogenic acid, 15-demethyl frangipanin, chlorogenic acid, secologenin, caffeic acid, cerbernic acid B, mesonolic acid D, isorosmarinic acid, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid and isochlorogenic acid A, dissolving in 30-50% methanol to obtain 0.03-0.06 mg/mL solution, filtering with 0.22 μm microporous membrane, and collecting filtrate;

s2, preparing a test solution: centrifuging 10-20mL of WangLogji cold tea concentrated juice at 5000r/min for 15min, taking 5-10mL of centrifuged supernatant, adding 15-30 mL of methanol for dilution and precipitation, centrifuging again at 5000r/min, filtering the supernatant through a 0.22-micron microporous filter membrane, and taking subsequent filtrate to obtain the product;

s3, high performance liquid chromatography determination: and (3) precisely absorbing the reference substance solution prepared in the step (S1) and the test substance solution prepared in the step (S2) respectively, and injecting the reference substance solution and the test substance solution into an ultra-high performance liquid chromatograph for determination to obtain the fingerprint of the WangLoKAI cold tea concentrated juice with the common characteristic peak.

Preferably, the ultra high performance liquid chromatograph measuring conditions of step S3 include: a chromatographic column: octadecylsilane chemically bonded silica; column temperature: the column temperature is 25-35 ℃; mobile phase: using aqueous solution of formic acid with volume concentration of 0.08-0.12% as a mobile phase A, and using acetonitrile as a mobile phase B; flow rate: 0.2-0.5 mL/min; sample introduction amount: 1-5 mu L; detection wavelength: 210-280 nm.

Preferably, the measurement conditions of the ultra high performance liquid chromatograph in step S3 further include: elution was performed according to the following elution procedure: at 0-6 min, the volume percentage of the mobile phase B is increased from 5% to 7%; at 6-7 minutes, the volume percent of the mobile phase B is reduced from 7 percent to 5 percent; at 7-12 minutes, the volume percent of the mobile phase B is increased from 5 percent to 8 percent; at 12-15 minutes, the volume percent of the mobile phase B is increased from 8% to 13%; at 15-32 minutes, the volume percentage of mobile phase B rose from 13% to 18%.

Preferably, the number of the common characteristic peaks in step S3 is 14, which are: no. 1-danshensu, no. 2-protocatechuic acid, no. 3-neochlorogenic acid, no. 4-15-demethylfrangipanin, no. 5-chlorogenic acid, no. 6-seco-majorin, no. 7-caffeic acid, no. 8-pimaric acid B, no. 9-mesonolic acid D, no. 10-isorosmarinic acid glycoside, no. 11-isochlorogenic acid B, no. 12-isochlorogenic acid A, no. 13-rosmarinic acid and No. 14-isochlorogenic acid C.

Preferably, the relative retention time of each characteristic peak is within ± 10% of a specified value by taking the mesolic acid D No. 9 as a reference peak, and the specified values of each peak are respectively: 0.13, 0.16, 0.21, 0.24, 0.40, 0.47, 0.53, 0.60, 1.00, 1.16, 1.20, 1.25, 1.40 and 1.43.

Preferably, the reference peak is No. 9 mesolic acid D, the relative peak area of each characteristic peak is within +/-20% of the specified value, and the specified values of each peak are respectively: 0.53, 0.13, 0.40, 0.19, 0.46, 0.11, 0.64, 0.32, 1.00, 0.11, 0.24, 0.21, 0.42, and 0.19; and respectively constructing UPLC fingerprint spectrums consisting of the common characteristic peaks.

The invention also provides a UPLC fingerprint detection method for the concentrated juice of Wanglaoji cold tea, which comprises the following steps:

(1) Preparation of a reference solution: accurately weighing reference substances including danshensu, protocatechuic acid, neochlorogenic acid, 15-demethyl frangipanin, chlorogenic acid, secologenin, caffeic acid, cerberinic acid B, mesonolic acid D, isorosmarinic acid glycoside, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid and isochlorogenic acid A, dissolving in 30-50% methanol to obtain 0.03-0.06 mg/mL solution, filtering with 0.22 μm microporous membrane, and collecting filtrate;

(2) Preparing a test solution: centrifuging 10-20mL of Wanglaoji cold tea concentrated juice at 5000r/min for 15min, taking 5-10mL of centrifuged supernatant, adding 15-30 mL of methanol to dilute and precipitate, centrifuging again at 5000r/min, filtering the supernatant by a 0.22-micron microporous filter membrane, and taking subsequent filtrate to obtain the product;

(3) And (3) high performance liquid chromatography determination: precisely sucking the reference solution prepared in the step S1 and the test solution prepared in the step S2 respectively, detecting according to the chromatographic conditions of claims 2-3, comparing the chromatogram of the test solution obtained by detection with the fingerprint obtained by the establishing method of claim 1, calculating the similarity, and evaluating the quality of UPLC.

Compared with the prior art, the establishing method provided by the invention has the technical advantages that: on the basis of carrying out systematic research on chemical components of the WangLo concentrated juice, the invention adopts a reference substance and an HPLC-MS method to carry out structure confirmation on 14 specific peaks in a fingerprint spectrum, and simultaneously adopts a characteristic fingerprint spectrum constructed by ultra-high performance liquid chromatography (UPLC) to carry out more comprehensive quality evaluation on the WangLo concentrated tea juice, thereby improving the quality control level of intermediate products in the production process of the herbal tea, making up the blank of UPLC control on the intermediate quality of the herbal tea from the perspective of the chemical components, simultaneously adopting the LC technology to establish the fingerprint spectrum, having the advantages of simple and convenient operation, high analysis speed, high accuracy and good reproducibility, and providing scientific basis for the quality evaluation and control of the WangLo concentrated tea juice.

Description of the drawings:

FIG. 1 is the HR-ESI-MS diagram of the compound salvianic acid A in example 1;

FIG. 2 is the NMR spectrum of danshensu compound in example 1;

FIG. 3 is the NMR carbon spectrum of the compound danshensu of example 1;

FIG. 4 is a HR-ESI-MS plot of protocatechuic acid of the compound of example 1;

FIG. 5 is a NMR spectrum of protocatechuic acid of the compound of example 1;

FIG. 6 is a NMR carbon spectrum of protocatechuic acid of the compound of example 1;

FIG. 7 is a HR-ESI-MS graph of neochlorogenic acid of the compound of example 1;

FIG. 8 is the NMR spectrum of neochlorogenic acid of example 1;

FIG. 9 is the NMR carbon spectrum of neochlorogenic acid as the compound in example 1;

FIG. 10 is a HR-ESI-MS diagram of compound 15-demethylfrangipanin of example 1;

FIG. 11 is the NMR chart of compound 15-demethyl plumericin of example 1;

FIG. 12 is the NMR spectrum of compound 15-demethylfrangipanin of example 1;

FIG. 13 is a HR-ESI-MS diagram of chlorogenic acid of the compound of example 1;

FIG. 14 is a NMR spectrum of chlorogenic acid of example 1;

FIG. 15 is the NMR carbon spectrum of chlorogenic acid of example 1;

FIG. 16 is a HR-ESI-MS plot of the compound secologanin of example 1;

FIG. 17 is a NMR chart of the compound secologanin of example 1;

FIG. 18 is a NMR C-spectrum of the compound secologanin of example 1;

FIG. 19 is a HR-ESI-MS graph of caffeic acid, the compound of example 1;

FIG. 20 is a NMR spectrum of caffeic acid, the compound of example 1;

FIG. 21 is a NMR carbon spectrum of caffeic acid, the compound of example 1;

FIG. 22 is a HR-ESI-MS plot of cerbera Mangifera indica B in example 1;

FIG. 23 is a NMR spectrum of the compound cerbera mango acid B in example 1;

FIG. 24 is a NMR carbon spectrum of the compound cerbera mango acid B in example 1;

FIG. 25 is a HR-ESI-MS diagram of the compound mesolic acid D in example 1;

FIG. 26 is a nuclear magnetic resonance hydrogen spectrum of the compound mesonolic acid D in example 1;

FIG. 27 is a nuclear magnetic resonance carbon spectrum of the compound mesonolic acid D in example 1;

FIG. 28 is the HR-ESI-MS graph of isorosmarinic acid glycoside, a compound of example 1;

FIG. 29 is a NMR chart of isorosmarinic acid glycoside compound of example 1;

FIG. 30 is the NMR spectrum of isorosmarinic acid glycoside compound of example 1;

FIG. 31 is a HR-ESI-MS plot of isochlorogenic acid B, the compound of example 1;

FIG. 32 is the NMR spectrum of isochlorogenic acid B of example 1

FIG. 33 is the NMR spectrum of isochlorogenic acid B, the compound of example 1;

FIG. 34 is a HR-ESI-MS plot of isochlorogenic acid C, the compound of example 1;

FIG. 35 is a NMR spectrum of isochlorogenic acid C, the compound of example 1;

FIG. 36 is a NMR carbon spectrum of isochlorogenic acid C, the compound of example 1;

FIG. 37 is a HR-ESI-MS plot of rosmarinic acid, the compound of example 1;

FIG. 38 is a NMR spectrum of rosmarinic acid, the compound of example 1;

FIG. 39 is a NMR carbon spectrum of rosmarinic acid, the compound of example 1;

FIG. 40 is a HR-ESI-MS plot of isochlorogenic acid A, the compound of example 1;

FIG. 41 is a NMR spectrum of isochlorogenic acid A, the compound of example 1;

FIG. 42 is the NMR C-spectrum of isochlorogenic acid A as the compound in example 1;

FIG. 43 is a UPLC chromatogram superposition chart of the concentrated solution of WangLoKAT herbal tea in example 2 as a sample and as a reference;

FIG. 44 is a UPLC fingerprint of the WangLoKAT herbal tea concentrate of example 2;

FIG. 45 is a UPLC chromatogram overlay of 16 lots of WangLo herbal tea concentrate of example 2;

FIG. 46 is a comparative fingerprint spectrum of UPLC of the Wanglaoji cool tea concentrate of example 2;

FIG. 47 is a UPLC detection spectrum of the Wanglaoji cool tea concentrated juice sample to be detected in example 3.

Detailed Description

The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. The method is carried out according to the conventional technical method and the content of the instrument instruction in the field, wherein the specific technology or condition is not indicated in the embodiment; the reagents and instruments used are not indicated by manufacturers, and are all conventional products commercially available.

Example 1 separation, purification and structural identification of the major chemical components of Wanglaoji cool tea concentrated juice

1. Test samples: concentrated juice of Wanglaoji cold tea provided by Guangzhou Wanglaoji big health industry limited company;

2. the test instrument: the optical rotation value is measured by a Jasco P-2000 polarimeter; ultraviolet (UV) spectroscopy using a Jasco V-550 UV/Vis spectrometer; infrared Spectrum (IR) was measured using a JASCO FT/IR-4600 Japan Plus Fourier Transform type infrared spectrometer (KBr pellet); nuclear magnetic resonance spectroscopy (NMR) was performed using a Bruker AV-300, bruker AV-400 or Bruker AV-600 nuclear magnetic resonance apparatus; high resolution mass spectrometry (HR-ESI-MS) was performed using an Agilent 6210LC/MSD TOF type mass spectrometer; the analytical High Performance Liquid Chromatography (HPLC) adopts Agilent 1260 high performance liquid chromatograph (G1311C quaternary pump system and G1315D diode array detector); the Preparative High Performance Liquid Chromatography (PHPLC) adopts Agilent 1260 high performance liquid chromatograph (G1310B unit pump system and G1365D MWD detector);

3. test materials and reagents: the macroporous adsorption resin HP-20 is purchased from Shanghai Kayin chemical Co., ltd; sephadex LH-20 column chromatography packing was purchased from Amersham Biosciences; analytical HPLC column Cosmosil 5C18-MS-II column (4.6 mm. Times.250mm, 5 μm); preparative HPLC column was a Cosmosil 5C18-MS-II column (10 mm. Times.250mm, 5 μm); the deuterated reagent is produced by CIL company in the United states; the chemical reagents are analytically pure or chromatographically pure and purchased from Merck company and Shandong Yuwang company;

4. separation and purification of main chemical components of the Wanglaoji cold tea concentrated juice: the concentrated juice of Wanglaoji tea is diluted to 0.05kg/L by adding purified water, and then subjected to column chromatography by a macroporous adsorption resin (HP-20), and gradient elution is performed by using ethanol-water (10 → 90 → 20 → 80 → 30 → 70 → 40 → 100, 0, V/V) as a mobile phase, wherein each gradient elution is performed for 3 to 4 column volumes (36 to 48L), so as to obtain a water fraction (220 g), a 10% ethanol fraction (218 g), a 20% ethanol fraction (59 g), a 30% ethanol fraction (40 g), a 40% ethanol fraction (33 g) and an anhydrous ethanol fraction (48 g).

Subjecting the 10% ethanol elution fraction to Sephadex LH-20 gel column chromatography, gradient elution with methanol-water (1); fr.5 by preparative HPLC separation purification to give compounds WLJ-2 (52 mg) and W LJ-7 (65 mg); fr.2 was separated and purified by 2 preparative HPLC to give compounds WLJ-4 (55 mg) and W LJ-6 (50 mg); fr.4 was isolated and purified by preparative HPLC to give compound WLJ-8 (46 mg).

Fr.7 is subjected to Sephadex LH-20 gel column chromatography and gradient elution by methanol and the like to obtain 4 fractions (fr.1-fr.4), and fr.2 is subjected to preparative HPLC separation and purification to obtain a compound WLJ-10 (51 mg); fr.9 is subjected to gradient elution by S ephadex LH-20 gel column chromatography, such as methanol and the like, to obtain 3 fractions (fr.1-fr.3), and fr.3 is subjected to separation and purification by preparative HPLC to obtain a compound WLJ-11 (40 mg).

Subjecting the 20% ethanol elution part to Sephadex LH-20 gel column chromatography, gradient elution with methanol-water (1; fr.3 by preparative HPLC separation purification to give compound WLJ-12 (64 mg); fr.5 by preparative HPLC separation purification gave compounds WLJ-13 (37 mg) and WLJ-14 (50 mg).

The 14 separated compounds are tested to have the purity of over 95 percent by HPLC-DAD scanning and an area normalization method. The structures of the compounds are identified by methods such as HR-ESI-MS, NMR and the like, and are respectively danshensu, protocatechuic acid, neochlorogenic acid, 15-demethylfrangipanin, chlorogenic acid, secologenin, caffeic acid, cerberanoic acid B, mesonolic acid D, isorosmarinic acid glycoside, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid and isochlorogenic acid A; the spectra and physicochemical constants of the 14 compounds are as follows:

danshensu: a yellow amorphous powder having the chemical formula:

spectral data:

HR-ESI-MS:m/z 221.0422[M+Na] ⁺ (C ₉ H ₁₀ O ₅ Na,calcd 221.0420)；UVλ _max :241,283nm；IR(KBr)ν _max :3395(OH), 1596,1404(Ar)cm ^-1 ； ¹ H-NMR(300MHz,CD ₃ OD)δ _H :6.71(1H,s,H-2),6.67(1H, d,J＝7.7Hz,H-5),6.57(1H,dd,J＝7.7,1.5Hz,H-6),4.25(1H,dd,J＝7.7,4.4Hz, H-8),2.95(1H,dd,J＝13.8,4.4Hz,Ha-7),2.74(1H,dd,J＝13.8,7.7Hz,Hb-7)； ¹³ C-NMR(75MHz,CD ₃ OD)δ _C 177.4 (C-9), 146.0 (C-3), 144.9 (C-4), 130.3 (C-1), 121.9 (C-6), 117.7 (C-2), 116.1 (C-5), 73.1 (C-8), 41.1 (C-7). The above data are consistent with the danshensu reported in the literature (Li Xue Shi et al, shenyang pharmaceutical university, 2012,29 (3): 194-198, 266.), so the compound is identified as danshensu (tanshinol).

Protocatechuic acid: an amorphous powder having the chemical formula:

spectral data: HR-ESI-MS:177.0155[ m ] +Na] ⁺ (C ₇ H ₆ O ₄ Na,calcd 177.0158)；UVλ _max : 254,295nm；IR(KBr)ν _max :3452(OH),1642(C＝O),1510,1381(Ar)cm ^-1 ； ¹ H-NMR (400MHz,CD ₃ OD)δ _H :7.43(2H,m,H-2,H-6),6.80(1H,d,J＝8.2Hz,H-5)； ¹³ C-NMR(100MHz,CD ₃ OD)δ _C 170.2 (C-7), 151.5 (C-4), 146.0 (C-3), 123.9 (C-6), 123.1 (C-1), 117.7 (C-2), 115.8 (C-5). The above data are consistent with those reported in the literature (Wang, 26256, et al, yunnan plant research, 2009,31 (3): 284-288.) and the compound was identified as protocatechuic acid (protocatechuic acid).

Chlorogenic acid: a pale yellow amorphous powder having the chemical formula:

spectral data:

HR-ESI-MS:m/z 377.0841[M+Na] ⁺ (C ₁₆ H ₁₈ O ₉ Na,calcd 377.0843)；UVλ _max :239,299,329nm；IR(KBr)ν _max :3366 (-OH),1690(C＝O),1610,1519,1436(Ar)cm ^-1 ； ¹ H-NMR(400MHz,CD ₃ OD)δ _H : 7.59(1H,d,J＝15.9Hz,H-7′),7.05(1H,d,J＝1.8Hz,H-2′),6.94(1H,dd,J＝8.2, 1.8Hz,H-6′),6.78(1H,d,J＝8.2Hz,H-5′),6.31(1H,d,J＝15.9Hz,H-8′),5.36(1H, m,H-3),4.14(1H,ddd,J＝12.6,9.3,4.2Hz,H-5),3.66(1H,dd,J＝8.3,3.2Hz, H-4),2.18(2H,m,H ₂ -6),2.11(1H,m,Hα-2),1.97(1H,m,Hβ-2)； ¹³ C-NMR(100 MHz,CD ₃ OD)δ _C 176.5 (C-7), 169.0 (C-9 '), 149.4 (C-4 '), 146.8 (C-7 '), 146.7 (C-3 '), 128.0 (C-1 '), 122.9 (C-6 '), 116.5 (C-5 '), 115.8 (C-8 '), 115.2 (C-2 '), 75.5 (C-1), 74.7 (C-4), 73.0 (C-3), 68.5 (C-5), 41.4 (C-6), 36.8 (C-2). The above data are consistent with the new chlorogenic acid reported in the literature (Chinesemedicines, 2010,41 (4): 542-544.), so the compound is identified as new chlorogenic acid.

15-demethyl frangipanin: a light yellow oil having the chemical formula:

spectral data:

HR-ESI-MS:m/z 479.1160[M+Na] ⁺ (C ₂₀ H ₂₄ O ₁₂ Na,calcd 479.1160)；UVλ _max :238nm；IR(KBr)ν _max :3435(OH),1720 (C＝O),1642(C＝C)； ¹ H-NMR(400MHz,CD ₃ OD)δ _H :7.50(1H,s,H-10),7.38(1H,s, H-3),6.49(1H,dd,J＝5.5,2.4Hz,H-6),5.51(1H,dd,J＝5.5,1.9Hz,H-7),5.26 (1H,d,J＝4.8Hz,H-1),4.69(1H,d,J＝7.9Hz,H-1′),4.55(1H,q,J＝6.3Hz,H-13), 3.89(2H,m,H-5,Ha-6′),3.69(1H,m,Hb-6′),3.37(3H,m,overlapped,H-3′,H-4′, H-5′),3.19(1H,t,J＝7.9Hz,H-2′),2.93(1H,dd,J＝7.6,4.9Hz,H-9),1.41(3H,d,J ＝6.3Hz,H-14)； ¹³ C-NMR(100MHz,CD ₃ OD)δ _C 172.8 (C-12), 169.7 (C-15), 152.5 (C-3), 150.4 (C-10), 141.7 (C-6), 138.5 (C-11), 129.7 (C-7), 114.1 (C-4), 100.0 (C-1 '), 98.0 (C-8), 94.1 (C-1), 78.4 (C-3'), 77.8 (C-5 '), 74.6 (C-2'), 71.3 (C-4 '), 63.5 (C-13), 62.5 (C-6'), 50.6 (C-9), 40.4 (C-5), 22.4 (C-14). The above data and textThe compound was identified as 15-demethylPlumeride because 15-demethylfranin reported in (ZHao M, etc. Separation Science and Technology,2015,50 (15): 2360-2366.) is identical.

Chlorogenic acid: a pale yellow amorphous powder having the chemical formula:

spectral data:

HR-ESI-MS:m/z 377.0840[M+Na] ⁺ (C ₁₆ H ₁₈ O ₉ Na,calcd 377.0843)；UVλ _max :241,299,330nm；IR(KBr)ν _max :34 26(OH),1691(C＝O),1616,1527,1453(Ar)cm ^-1 ； ¹ H-NMR(400MHz,CD ₃ OD)δ _H :7.56(1H,d,J＝15.9Hz,H-7′),7.05(1H,d,J＝1.8Hz,H-2′),6.9 5(1H,dd,J＝8.2,1.8Hz,H-6′),6.78(1H,d,J＝8.2Hz,H-5′),6.26(1H, d,J＝15.9Hz,H-8′),5.34(1H,td,J＝9.2,4.4Hz,H-5),4.18(1H,m H-3), 3.73(1H,dd,J＝8.6,2.8Hz,H-4),2.04～2.26(4H,m,H-2,H-6)； ¹³ C-N MR(100MHz,CD ₃ OD)δ _C 177.4 (C-7), 168.7 (C-9 '), 149.5 (C-4 '), 147.1 (C-7 '), 146.8 (C-3 '), 127.8 (C-1 '), 123.0 (C-6 '), 116.5 (C-5 '), 115.3 (C-8 '), 115.2 (C-2 '), 76.4 (C-1), 73.6 (C-4), 72.0 (C-5), 71.4 (C-3), 38.9 (C-6), 38.2 (C-2). The data are consistent with the chlorogenic acid reported in the literature (Chinesemedicinal, 2010,41 (4): 542-544), so that the compound is identified as chlorogenic acid (chlorogenic acid).

Secologanin: a light yellow oil having the chemical formula:

spectral data

HR-ESI-MS:m/z 413.1053[M+Na] ⁺ (C ₁₆ H ₂₂ O ₁₁ Na,calcd 413.1054)；UVλ _max :240nm；IR(KBr)ν _max :3414(OH),1642 (C＝C)cm ^-1 ； ¹ H-NMR(400MHz,CD ₃ OD)δ _H :7.50(1H,d,J＝1.8Hz,H-3),5.64(1H, dt,J＝17.2,10.0Hz,H-8),5.48(1H,d,J＝3.8Hz,H-1),5.26(1H,dd,J＝17.2,1.3 Hz,H-10a),5.23(1H,dd,J＝10.0,1.3Hz,H-10b),4.66(1H,d,J＝7.9Hz,H-1′), 3.89(1H,dd,J＝12.0,1.7Hz,Hb-6′),3.67(1H,dd,J＝12.0,5.5Hz,Ha-6′),3.37 (1H,t,J＝8.9Hz,H-3′),3.31(1H,m,H-5′),3.31(1H,m,overlapped,H-4′),3.29(1H, m,overlapped,H-5),3.22(1H,dd,J＝9.1,7.9Hz,H-2′),3.06(1H,dd,J＝16.7,4.0 Hz,Hb-6),2.82(1H,m,H-9),2.24(1H,dd,J＝16.7,9.6Hz,Ha-6)； ¹³ C-NMR(100 MHz,CD ₃ OD)δ _C 176.3 (C-7), 170.2 (C-11), 153.6 (C-3), 134.5 (C-8), 120.5 (C-10), 110.1 (C-4), 99.9 (C-1 '), 97.5 (C-1), 78.3 (C-5'), 77.9 (C-3 '), 74.6 (C-2'), 71.5 (C-4 '), 62.7 (C-6'), 45.2 (C-9), 34.8 (C-6), 28.4 (C-5). The data are consistent with those reported in the literature (summer-long. Chinese modern Chinese medicine, 2012,14 (4): 26-32.) and the compound is identified as secologanin.

Caffeic acid: a pale yellow amorphous powder having the chemical formula:

spectral data: ¹ H-NMR(500MHz,CD ₃ OD)δ _H: 7.53(1H,d,J＝15.9Hz,H-7), 7.04(1H,d,J＝2.0Hz,H-2),6.93(1H,dd,J＝8.2,2.0Hz,H-6),6.78(1H, d,J＝8.2Hz,H-5),6.22(1H,d,J＝15.9Hz,H-8)； ¹³ C-NMR(125MHz, CD ₃ OD)δ _C: 171.1 (C-9), 149.4 (C-4), 147.0 (C-7), 146.8 (C-3), 127.8 (C-1), 1.22 (C-6), 116.5 (C-8), 115.6 (C-2), 115.1 (C-5). The above data are consistent with caffeic acid reported in the literature (Ching. Chinese herbal medicine, 2010,41 (1): 29-32.) and the compound was identified as caffeic acid.

Cerbera mangifera acid B: a yellow oil having the chemical formula:

spectral data:

HR-ESI-MS:m/z 233.0423[M+Na] ⁺ (C ₁₀ H ₁₀ O ₅ Na,calcd 233.0420)；UVλ _max :240,283nm；IR(KBr)ν _max :3426(O H),1639,1398(Ar)cm ^-1 ； ¹ H-NMR(400MHz,CD ₃ OD)δ _H :7.96(1H,s,H-2), 7.88(1H,d,J＝7.5Hz,H-4),7.52(1H,d,J＝7.2Hz,H-6),7.39(1H,t, J＝7.6Hz,H-5),4.37(1H,m,H-8),3.17(1H,dd,J＝13.3,3.8Hz,Ha-7), 2.99(1H,dd,J＝13.3,7.6Hz,Hb-7)； ¹³ C-NMR(100MHz,CD ₃ OD)δ _C 177.3 (C-9), 170.0 (C-10), 139.4 (C-1), 135.4 (C-6), 131.9 (C-3), 131.8 (C-2), 129.3 (C-5), 129.0 (C-4), 72.7 (C-8), 41.3 (C-7). The above data are consistent with those reported in the literature (Zhao M, etc. Se corporation Science and Technology,2015,50 (15): 2360-2366.) and this compound was identified as cerberic acid B.

mesonolic acid D: novel rosmarinic acid derivatives, yellow oil, having the chemical formula:

spectral data:

HR-ESI-MS:m/z 561.0995[M+Na] ⁺ (C ₂₇ H ₂₂ O ₁₂ Na,calcd 561.1003)；UVλ _max :241,287,335nm；IR(KBr)ν _max :3435 (OH),1688(C＝O),1619,1513,1435(Ar)cm ^-1 ； ¹ H-NMR(400MHz,CD ₃ OD)δ _H : 7.71(1H,s,H-4),6.83(1H,d,J＝8.0Hz,H-5),6.75(1H,d,J＝8.0Hz,H-6),6.71 (1H,d,J＝2.0Hz,H-2′),6.64(1H,d,J＝7.9Hz,H-5′),6.58(1H,d,J＝8.2Hz, H-15),6.55(1H,d,J＝2.0Hz,H-12),6.48(1H,dd,J＝7.9,2.0Hz,H-6′),6.43(1H, dd,J＝8.2,2.0Hz,H-16),5.21(1H,overlapped,H-8′),4.52(1H,s,H-2),4.25(1H,d, J＝1.1Hz,H-1),2.99(2H,m,Ha-7′,Hb-7′)； ¹³ C-NMR(100MHz,CD ₃ OD)δ _C 176.3 (C-18), 173.4 (C-9 '), 168.1 (C-17), 149.3 (C-7), 146.0 (C-3 '), 145.8 (C-13), 145.1 (C-8, C-4 '), 145.0 (C-14), 139.8 (C-4), 134.5 (C-11), 129.1 (C-1 '), 126.5 (C-3), 126.0 (C-10), 122.5 (C-5), 122.0 (C-6 '), 120.6 (C-9), 119.8 (C-16), 117.4 (C-2 '). 116.4 (C-5 '), 116.3 (C-15), 115.3 (C-12), 114.7 (C-6), 74.8 (C-8 '), 47.4 (C-1), 41.8 (C-2), 37.8 (C-7 '). The above data are consistent with the reports in the literature (Lu Y, etc. tetrahedron Letters,2001, 42.

Isorosmarinic acid glycoside: a yellow oil having the chemical formula:

spectral data:

HR-ESI-MS:m/z 545.1261[M+Na] ⁺ (C ₂₄ H ₂₆ O ₁₃ Na,calcd 545.1266)；UVλ _max :242,292,318nm；IR(KBr)ν _max :3449 (OH),1688(C＝O),1613,1519,1453(Ar)cm ^-1 ； ¹ H-NMR(400MHz,CD ₃ OD)δ _H : 7.60(1H,d,J＝15.9Hz,H-7),7.53(1H,s,H-2),7.19(1H,d,J＝8.3Hz,H-6),6.87 (1H,d,J＝8.3Hz,H-5),6.76(1H,d,J＝2.0Hz,H-2′),6.70(1H,d,J＝8.0Hz,H-5′), 6.61(1H,dd,J＝8.0,2.0Hz,H-6′),6.37(1H,d,J＝15.9Hz,H-8),5.18(1H,dd,J＝ 8.3,4.3Hz,H-8′),4.83(1H,d,J＝7.4Hz,H-1″),3.96(1H,dd,J＝12.1,2.1Hz, Hb-6″),3.73(1H,dd,J＝12.1,6.1Hz,Ha-6″),3.51(3H,m,H-2″,H-3″,H-5″),3.39 (1H,m,H-4″),3.09(1H,dd,J＝14.3,4.3Hz,Hb-7′),3.01(1H,dd,J＝14.3,8.3Hz, Ha-7′)； ¹³ C-NMR(100MHz,CD ₃ OD)δ _C 173.3 (C-9 '), 168.3 (C-9), 151.2 (C-4), 147.1 (C-3), 147.0 (C-7), 146.1 (C-3 '), 145.2 (C-4 '), 129.3 (C-1 '), 127.9 (C-1), 126.3 (C-6), 121.9 (C-6 '), 118.2 (C-2), 117.7 (C-2 '), 117.4 (C-5), 116.3 (C-5 '), 115.4 (C-8), 104.2 (C-1 '), 78.5 (C-3 '), 77.6 (C-5 '), 74.9 (C-2 '), 74.6 (C-8 '), 71.5 (C-4 '), 62.5 (C-6 '), 37.9 (C-7 '). The above data are consistent with the isorosmarinic acid glycoside reported in the literature (Ha TJ, etc. food Chem,2012,135 (3): 1397-403), so the compound was identified as isorosmarinic acid glycoside (salviaflaside).

Isochlorogenic acid B: a white amorphous powder having the chemical formula:

spectral data: ¹ H-NMR(300MHz,CD ₃ OD)δ _H :7.57(1H,d,J＝15.9Hz,H-7″),7.54 (1H,d,J＝15.9Hz,H-7′),7.05(1H,d,J＝2.0Hz,H-2″),7.03(1H,d,J＝2.0Hz, H-2′),6.93(1H,dd,J＝8.2,2.0Hz,H-6″),6.86(1H,dd,J＝8.2,2.0Hz,H-6′),6.78 (1H,d,J＝8.2Hz,H-5″),6.73(1H,d,J＝8.2Hz,H-5′),6.30(1H,d,J＝15.9Hz, H-8″),6.25(1H,d,J＝15.9Hz,H-8′),5.64(1H,m,H-3),4.99(1H,overlapped,H-4), 4.40(1H,td,J＝9.9,4.4Hz,H-5),2.06～2.40(4H,m,H ₂ -2，H ₂ -6)； ¹³ C-NMR(75 MHz,CD ₃ OD)δ _C 178.0 (C-7), 168.7 (C-9 '), 168.6 (C-9'), 149.5 (C-4 '), 147.4 (C-7'), 147.3 (C-7 '), 146.7 (C-3'), 146.6 (C-3 '), 127.7 (C-1'), 127.6 (C-1 '), 123.3 (C-6'), 123.2 (C-6 '), 116.5 (C-5'), 116.4 (C-5 '), 115.1 (C-2' ), 115.0 (C-8 '), 114.8 (C-8'), 76.6 (C-1), 75.2 (C-4), 70.1 (C-3), 65.7 (C-5), 41.9 (C-6), 36.9 (C-2). The above data are consistent with isochlorogenic acid B reported in the literature (Zhaoshan et al Chinese herbal medicine, 2017,48 (8): 1513-1518), so the compound is identified as isochlorogenic acid B.

Isochlorogenic acid C: a white amorphous powder having the chemical structure:

spectral data:

HR-ESI-MS:m/z 539.1159[M+Na] ⁺ (C ₂₅ H ₂₄ O ₁₂ Na,calcd 539.1160)；UVλ _max :241,301,329nm；IR(KBr)ν _max :3292 (OH),1683(C＝O),1602,1522,1453(Ar)cm ^-1 ； ¹ H-NMR(400MHz,CD ₃ OD) δ _H :7.60(1H,d,J＝15.9Hz,H-7″),7.52(1H,d,J＝15.9Hz,H-7′),7.02 (1H,d,J＝2.4Hz,H-2″),7.01(1H,d,J＝2.4Hz,H-2′),6.92(1H,dd,J＝ 8.2,2.2Hz,H-6″),6.90(1H,dd,J＝8.2,2.2Hz,H-6′),6.76(1H,d,J＝8. 2Hz,H-5″),6.74(1H,d,J＝8.2Hz,H-5′),6.29(1H,d,J＝15.9Hz,H-8″), 6.19(1H,d,J＝15.9Hz,H-8′),5.63(1H,m,H-3),5.12(1H,dd,J＝9.0, 3.0Hz,H-4),4.38(1H,m,H-5),2.10～2.32(4H,m,H ₂ -2,H ₂ -6)； ¹³ C-NMR (100MHz,CD ₃ OD)δ _C 176.8 (C-7), 168.5 (C-9 '), 168.2 (C-9 '), 149.6 (C-4 ' ), 147.7 (C-7 '), 147.6 (C-7 '), 146.7 (C-3 ' ), 127.71 (C-1 '), 127.65 (C-1 '), 123.1 (C-6 ' ), 116.5 (C-5 ' ), 115.2 (C-2 ' ), 114.8 (C-8 '), 11.7 (C-8 '), 76.1 (C-1), 75.8 (C-4), 69.4 (C-3), 69.0 (C-5), 39.4 (C-6), 38.4 (C-2). The data are consistent with isochlorogenic acid C reported in the literature (Zhaoshan et al, chinese herbal medicine, 2017,48 (8): 1513-1518), so the compound is identified as isochlorogenic acid C.

Rosmarinic acid: a yellow oil having the chemical formula:

spectral data:

HR-ESI-MS:m/z 383.0734[M+Na] ⁺ (C ₁₈ H ₁₆ O ₈ Na,calcd 383.0737)；UVλ _max :242,292,318nm；IR(KBr)ν _max :3469(OH), 1702(C＝O),1605,1524,1447cm ^-1 (Ar)； ¹ H-NMR(400MHz,CD ₃ OD)δ _H :7.55(1H, d,J＝15.9Hz,H-7),7.05(1H,d,J＝1.8Hz,H-2),6.95(1H,dd,J＝8.0,1.8Hz,H-6), 6.78(1H,d,J＝8.2Hz,H-5′),6.76(1H,d,J＝1.8Hz,H-2′),6.70(1H,dd,J＝8.2, 1.8Hz,H-6′),6.62(1H,dd,J＝8.0,1.8Hz,H-5),6.27(1H,d,J＝15.9Hz,H-8),5.18 (1H,dd,J＝8.0,4.3Hz,H-8′),3.10(1H,dd,J＝14.3,4.3Hz,Ha-7′),3.01(1H,dd,J ＝14.3,8.0Hz,Hb-7′)； ¹³ C-NMR(100MHz,CD ₃ OD)δ _C 173.8 (C-9 '), 168.5 (C-9), 149.7 (C-4 '), 147.7 (C-7), 146.8 (C-3 '), 146.1 (C-4), 145.2 (C-3), 129.3 (C-1 '), 127.6 (C-1), 123.1 (C-6), 121.8 (C-6 '), 117.6 (C-5), 116.5 (C-2 '), 116.3 (C-8), 115.2 (C-5 '), 114.4 (C-5), 74.7 (C-8 '), 37.9 (C-7 '). The above data are consistent with those reported in literature (Kang NS, etc. food Chemistry, 2011,124 (2): 556-562), so the compound was identified as rosmarinic acid.

Isochlorogenic acid A: a white amorphous powder having the chemical formula:

spectral data: ¹ H-NMR(300MHz,CD ₃ OD)δ _H :7.62(1H,d,J＝16.0Hz,H-7″), 7.56(1H,d,J＝16.0Hz,H-7′),7.07(2H,s,H-2″,H-2′),6.98(1H,dd,J＝8.0,2.0Hz, H-6″),6.95(1H,dd,J＝8.0,2.0Hz,H-6′),6.80(1H,d,J＝8.0Hz,H-5″),6.78(1H,d, J＝8.0Hz,H-5′),6.35(1H,d,J＝16.0Hz,H-8″),6.27(1H,d,J＝16.0Hz,H-8′), 5.38～5.46(2H,m,H-3,H-5),3.99(1H,dd,J＝7.6,3.3Hz,H-4),2.16～2.35(4H,m, H ₂ -2,H ₂ -6)； ¹³ C-NMR(75MHz,CD ₃ OD)δ _C :177.3(C-7),168.9(C-9″),168.4(C-9′), 149.5(C-4″),149.4(C-4′),147.3(C-7″),147.0(C-7′),146.7(C-3′,C-3″),127.9 (C-1″),127.8(C-1′),123.1(C-6″),123.0(C-6′),116.5(C-5′,C-5″),115.5(C-2″), 115.3(C-2′),115.2(C-8 '), 115.1 (C-8'), 74.7 (C-1), 72.5 (C-4), 72.0 (C-3), 70.7 (C-5), 37.6 (C-6), 36.0 (C-2). The above data are consistent with isochlorogenic acid a reported in the literature (Chen J, etc. fitoteeraria, 2014, 99.

Example 2 UPLC fingerprint spectrum establishment method of Wanglaoji cold tea concentrated juice

The method for establishing the fingerprint comprises the following steps:

s1, preparation of a reference solution: preparing a reference substance of danshensu, protocatechuic acid, neochlorogenic acid, 15-demethyl plumericin, chlorogenic acid, secologanin, caffeic acid, cerberanoic acid B, mesonolic acid D, isorosemary acid glycoside, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid and isochlorogenic acid A into a standard solution of 0.05 mg/mL, and filtering the standard solution through a microporous filter membrane to obtain a reference substance solution;

s2, preparing a test solution: taking 10mL of Wanglaoji cold tea concentrated juice, centrifuging at 5000r/min for 15min, taking 5mL of supernatant, adding 15mL of methanol solution to dilute and precipitate, centrifuging the diluent again at 5000r/min for 15min, taking supernatant, filtering by using a microporous filter membrane, and taking subsequent filtrate to obtain the sample solution. Preparing test solution of concentrated juice of Wanglaoji cold tea in each batch by the same method;

the Wanglaoji herb tea concentrated juice used in this example was provided by Guangzhou Wanglaoji herb tea health industry Co., ltd, and the batch number of 16 batches of concentrated juice samples is shown in Table 1.

TABLE 1 batch number for 16 batches of Wanglaoji cold tea concentrated juice

Number of	Production lot number	Numbering	Production batch number
				S1	B120 17030266	S9	B120 17030267
S2	A30 17030250DM	S10	B120 17030260DM
				S3	A30 17030265	S11	B120 17030254DM
S4	B120 17030259DM	S12	A30 17030256
				S5	A150 17030257	S13	A150 17030252DM
S6	A150 17030251DM	S14	A150 17030253DM
				S7	A30 17030264	S15	A150 17030255DM
S8	B120 17030261DM	S16	B120 17030262DM

S3, ultra-high performance liquid chromatography determination: precisely absorbing the reference substance solution prepared in the step S1 and the test substance solution prepared in the step S2 respectively, and injecting the reference substance solution and the test substance solution into an ultra-high performance liquid chromatograph for determination to obtain the concentrated juice fingerprint of the Wanglaoji cold tea with the common characteristic peak; the chromatographic conditions are as follows: and (3) chromatographic column: an Agilent Eclipse Plus C18 RRHD column with the specification of 2.1 multiplied by 100mm and 1.8 mu m; mobile phase: acetonitrile (B) -0.1% strength by volume formic acid water (a); column temperature: 30 ℃; detection wavelength: 225nm; flow rate: 0.3mL/min; sample injection amount: 2 mu L of the solution; collecting time: 32min, according to the gradient elution procedure described in Table 2 below.

TABLE 1 UPLC gradient elution schedule

Time (min)	Mobile phase A (v/v%)	Mobile phase B (v/v%)
			0	95	5
6	93	7
			7	95	5
12	92	8
			15	87	13
32	82	18

The method is adopted to detect each sample and the reference sample of the WangLoKAT concentrated juice to obtain a chromatogram. Comparing the chromatographic peak of the test solution with the retention time of the chromatographic peak of each reference, simultaneously adopting a PDA detector to carry out online detection at the wavelength of 200-400 nm, and comparing the chromatographic peak of the test solution with the ultraviolet absorption of the chromatographic peak of each reference. The UPLC chromatogram superposition chart of the sample solution and the reference solution of the WangLoKAT herbal tea concentrated juice is shown in figure 43, and the UPLC fingerprint chromatogram which can be identified by the WangLoKAT herbal tea concentrated juice is shown in figure 44.

And (3) importing the data of the sample to be tested into software of a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A edition for analysis, and generating a control UPLC fingerprint of WangLoKAI cold tea concentrated juice by using a median method and taking a No. 9 peak (mesonolic acid D) as a reference peak through multi-point correction and automatic matching. Selecting chromatographic peaks with higher response values and better separation degrees in the chromatogram as common characteristic peaks, calibrating 14 common peaks as a result, and identifying the chromatographic peaks according to chromatogram retention time of a reference substance and a test substance, a UV absorption spectrum, LC-MS fragment ions and the like. The peak name, relative retention time and relative peak area of each chromatographic peak are shown in Table 3, the UPLC chromatogram superposition chart of 16 batches of WangLoKAT concentrated tea juice is shown in figure 45, and the comparison fingerprint chromatogram of the UPLC is shown in figure 46.

Table 3 shows the names, relative retention times and relative peak areas of the common peaks in the fingerprint

Peak number	Name of Compound	Relative retention time	Relative peak area
				Peak
1	Salvianic acid A	0.13	0.53
				Peak 2	Protocatechuic acid	0.16	0.13
Peak 3	Novel chlorogenic acid	0.21	0.40
				Peak 4	15-demethyl frangipanin	0.24	0.19
Peak 5	Chlorogenic acid	0.40	0.46
				Peak 6	Cycloartanin	0.47	0.11
Peak 7	Caffeic acid	0.53	0.64
				Peak 8	Cerbera Manghas acid B	0.60	0.32
Peak 9 (S)	mesonolic acid D	1.00	1.00
				Peak 10	Isorosmarinic acid glycoside	1.16	0.11
Peak 11	Isochlorogenic acid B	1.20	0.24
				Peak 12	Isochlorogenic acid A	1.25	0.21
Peak 13	Rosmarinic acid	1.40	0.43
				Peak 14	Isochlorogenic acid C	1.43	0.19

The invention records the retention time and peak area of each common peak in 16 batches of WangLo cool tea concentrated juice fingerprint spectrums, and calculates the ratio of the retention time and peak area of each common peak to the S peak in the same batch of the fingerprint spectrums, thereby obtaining the relative retention time and relative peak area of each common peak. The results show that the relative retention time RSD of each common peak in 16 batches is less than 1%, and the relative peak area RSD value is large, which indicates that the relative contents of all components in the concentrated herbal tea juice of different batches have certain difference, the relative retention time detection result of UPLC characteristic fingerprint spectrum of WangLoKAI concentrated herbal tea juice is shown in table 4, and the relative peak area detection result is shown in table 5.

TABLE 4 relative retention time detection results of UPLC characteristic fingerprint of 16 Wanglaoji cold tea concentrated juice

TABLE 5 relative peak area detection results of UPLC characteristic fingerprint of 16 batches of Wanglaoji cold tea concentrated juice

Example 3 UPLC fingerprint spectrum determination method for Wanglaoji cool tea concentrated juice

The determination method comprises the following steps:

(1) Preparation of a test solution: centrifuging concentrated juice of Wanglaoji cold tea at 10 mL/5000r/min for 15min, collecting supernatant 5mL, diluting with 15mL of methanol solution, centrifuging the diluted solution at 5000r/min for 15min, collecting supernatant, filtering with microporous membrane, and collecting filtrate to obtain the sample solution;

(2) Chromatographic conditions and system applicability: a chromatographic column: an Agilent Eclipse Plus C18 RRHD column with the specification of 2.1 × 100mm,1.8 μm; detection wavelength: 210nm; flow rate: 0.2mL/min; column temperature: 25 ℃; sample introduction amount: 1 mu L of the solution; mobile phase: the mobile phase A is 0.08% formic acid-water solution, the mobile phase B is acetonitrile, and the gradient elution procedure is the same as example 2.

(3) And (3) determination: and (3) measuring the test solution according to the ultra-high performance liquid chromatography.

Example 4 UPLC fingerprint spectrum determination method for Wanglaoji cold tea concentrated juice

The determination method comprises the following processes:

(2) Chromatographic conditions and system applicability: a chromatographic column: an Agilent Eclipse Plus C18 RRHD column with the specification of 2.1 × 100mm,1.8 μm; detection wavelength: 225nm; flow rate: 0.3mL/min; column temperature: 30 ℃; sample introduction amount: 2 mu L of the solution; mobile phase: the mobile phase A is formic acid-water solution with the volume percentage of 0.1 percent, the mobile phase B is acetonitrile, and the gradient elution procedure is the same as that of the example 4;

(3) And (3) determination: and (3) measuring the test sample solution according to ultra-high performance liquid chromatography.

Example 5 method for measuring UPLC fingerprint of Wanglaoji cold tea concentrated juice

The determination method comprises the following processes:

(1) Preparation of a test solution: centrifuging concentrated juice of WangLoKAT herbal tea at 10 mL/5000r/min for 15min, collecting supernatant 5mL, diluting with 15mL of methanol solution, centrifuging the diluted solution at 5000r/min for 1 min, collecting supernatant, filtering with microporous membrane, and collecting filtrate to obtain sample solution;

(2) Chromatographic conditions and system applicability: and (3) chromatographic column: agilent Eclipse Plus C18 RRHD column; the specification is 2.1 multiplied by 100mm,1.8 mu m; detection wavelength: 254nm; flow rate: 0.4mL/min; column temperature: 35 ℃; sample injection amount: 4 mu L of the solution; mobile phase: the mobile phase A is formic acid-water solution with the volume percentage of 0.12 percent, the mobile phase B is acetonitrile, and the gradient elution procedure is the same as that of the example 2;

Example 6 verification of UpLC fingerprint establishment method for Wanglaoji cold tea concentrated juice

1. Instrument precision experiment

Taking the Wanggaiji cool tea concentrated juice, preparing a sample solution of the Wanggaiji cool tea concentrated juice according to the method in the embodiment 2, analyzing according to the chromatographic condition described in the embodiment 2, continuously feeding the same sample for 6 times, calculating the similarity value of the obtained chromatogram by using similarity evaluation software, taking a No. 9 peak (mesonolic acid D) as a reference peak (S), and investigating the relative retention time of all the shared peaks and the consistency of the relative peak areas, wherein the results (Table 6, table 7 and Table 8) show that the similarity value of the obtained chromatographic peaks is 1, the relative retention time R SD% of 14 chromatographic peaks is less than 3.0%, and the RSD% of the relative peak areas is less than 5.0%, which indicates that the instrument precision is good.

TABLE 6 results of similarity analysis of precision experiments (n = 6)

Numbering	1	2	3	4	5	6
							Degree of similarity	1.000	1.000	1.000	1.000	1.000	1.000

TABLE 7 calculation of relative Retention time for precision experiments (n = 6)

TABLE 8 calculation of relative peak area for precision experiments (n = 6)

2. And (3) repeatability test:

taking the Wanggaiji cold tea concentrated juice, preparing 6 parts of Wanggaiji cold tea concentrated juice sample solution according to the method in the step S2, analyzing according to the chromatographic conditions in the step S3, calculating the similarity value of the obtained chromatogram by adopting similarity evaluation software, observing the relative retention time and the consistency of relative peak areas of all the common peaks by taking the No. 9 peak as a reference peak, and showing that the similarity value of the obtained chromatographic peaks is 1, the relative retention time RSD% of 14 chromatographic peaks is less than 3.0%, and the RSD% of the relative peak area is less than 5.0%, so that the method has good repeatability.

TABLE 9 similarity analysis results of repeatability tests (n = 6)

Number of	1	2	3	4	5	6
							Degree of similarity	1.000	1.000	1.000	1.000	1.000	1.000

TABLE 10 relative retention time calculation of repeatability tests (n = 6)

TABLE 11 method relative peak area calculation of repeatability experiments (n = 6)

3. Stability test

Taking concentrated WangLoKAI cold tea juice, preparing a sample solution of the concentrated WangLoKAI cold tea juice according to the step S2 in the embodiment 2, analyzing the sample solution in the chromatographic conditions of 0, 2, 4, 8, 12 and 24 hours respectively according to the step S3, calculating the similarity value of the obtained chromatogram by adopting similarity evaluation software, and taking a No. 9 peak as a reference peak, and examining the consistency of the relative retention time and the relative peak area of each common peak, wherein the results (Table 12, table 13 and Table 14) show that the similarity value of the obtained chromatogram peaks is 1, the relative retention time RSD% of 14 chromatogram peaks is less than 3.0%, and the RSD% of the relative peak area is less than 5.0%, and the result shows that the sample solution has good stability in 24 hours.

Table 2 results of similarity analysis of stability experiments (n = 6)

Number of	1	2	3	4	5	6
							Similarity of the two	1.000	1.000	1.000	1.000	1.000	1.000

Table 13 calculation of relative retention time for stability experiments (n = 6)

TABLE 14 relative peak area calculation for stability experiments (n = 6)

Example 7 similarity evaluation of UPLC fingerprint of Wanglaoji herbal tea concentrate

The evaluation process specifically comprises the following steps:

(1) Determination of 16 batches of WangLoKAT herbal tea concentrated juice and acquisition of UPLC fingerprint spectrum

The source of 16 batches of Wanglaoji cold tea concentrate is shown in example 2. A sample solution was prepared according to step S2 in example 2, and 16 batches of the wanggaiji herb tea concentrate were each measured by the ultra high performance liquid chromatography in step S3, and the UPLC standard fingerprint of the wanggaiji herb tea concentrate obtained was as shown in fig. 46.

(2) Inspection of UPLC fingerprint similarity of Wanglaoji cool tea concentrated juice

The UPLC fingerprint spectrum of the concentrated juice of the WangLoKAT herbal tea is compared with the UPLC fingerprint spectrum of the concentrated juice of the WangLoKAT herbal tea in the embodiment 2 as a reference, and the similarity of the UPLC fingerprint spectrum of each batch of the concentrated juice of the WangLoKAT herbal tea is calculated by software of a Chinese medicine chromatogram fingerprint spectrum similarity evaluation system 2004A edition. The results are shown in table 15, the similarity of the 16 batches of Wanggoji cold tea concentrated juice is more than 0.98, which indicates that the quality of the cold tea concentrated juice of each batch is uniform and stable:

TABLE 13 UPLC characteristic fingerprint similarity evaluation results of Wanglaoji cool tea concentrated juice of batches

Example 8 detection method of Wanglaoji herb tea concentrate

1. Chromatographic conditions are as follows: chromatography was carried out according to the chromatographic conditions in example 2;

2. preparing a sample solution to be tested: taking 10mL of Wanglaoji cold tea concentrated juice, centrifuging for 15min at 5000r/min, taking 5mL of supernatant, adding 15mL of methanol solution for dilution, centrifuging the diluted solution again for 15min at 5000r/min, taking supernatant, filtering by using a microporous filter membrane, and taking subsequent filtrate to obtain the sample solution to be detected;

3. and (3) determination: precisely absorbing 2 mu L of the sample solution to be detected, injecting the sample solution into an ultra-high performance liquid chromatograph, and analyzing according to the chromatographic conditions under item 1 to obtain the UPLC spectrum of the sample solution to be detected.

As shown in fig. 47, as compared with the UPLC comparison fingerprint spectrum of the wanggaiji cool tea concentrated juice established in example 2, it can be seen that the similarity between the wanggaiji cool tea concentrated juice of the batch and the comparison fingerprint spectrum is high, and the quality of the cool tea concentrated juice of the batch can be considered to be qualified.

Finally, it should be noted that the above-described embodiments are described to facilitate understanding and use of the invention by those of ordinary skill in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make modifications and alterations without departing from the scope of the present invention.

Claims

1. A method for establishing UPLC fingerprint spectrum of Wanglaoji cool tea concentrated juice is characterized by comprising the following steps:

s1, preparation of a reference solution: precisely weighing reference substances including danshensu, protocatechuic acid, neochlorogenic acid, 15-demethyl frangipanin, chlorogenic acid, secologenin, caffeic acid, cerbernic acid B, mesonolic acid D, isorosmarinic acid, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid and isochlorogenic acid A, dissolving in 30-50% methanol to obtain 0.03-0.06 mg/mL solution, filtering with 0.22 μm microporous membrane, and collecting filtrate;

s3, high performance liquid chromatography determination: respectively and precisely sucking the reference substance solution prepared in the step S1 and the test solution prepared in the step S2, and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for determination to obtain fingerprint spectrums of the concentrated juice of the Wanglaoji cold tea, which have the common characteristic peak; the measuring conditions of the ultra-high performance liquid chromatograph comprise: and (3) chromatographic column: octadecylsilane chemically bonded silica; column temperature: the column temperature is 25-35 ℃; mobile phase: formic acid aqueous solution with volume concentration of 0.08-0.12% is used as a mobile phase A, and acetonitrile is used as a mobile phase B; flow rate: 0.2-0.5 mL/min; sample introduction amount: 1-5 microliter; detection wavelength: 210-280 nm; further comprising: elution was performed according to the following elution procedure: at 0-6 min, the volume percentage of the mobile phase B is increased from 5% to 7%; at 6-7 minutes, the volume percent of the mobile phase B is reduced from 7 percent to 5 percent; at 7-12 minutes, the volume percentage of the mobile phase B is increased from 5% to 8%; at 12-15 minutes, the volume percent of the mobile phase B is increased from 8% to 13%; at 15-32 minutes, the volume percent of mobile phase B increased from 13% to 18%.

2. The method according to claim 1, wherein the number of the common characteristic peaks in step S3 is 14, which are respectively: 1-danshensu, 2-protocatechuic acid, 3-neochlorogenic acid, 4-15-demethylfrangipanin, 5-chlorogenic acid, 6-secologanin, 7-caffeic acid, 8-cerbera mangifera acid B, 9-mesonolic acid D, 10-isorosmarinic acid glycoside, 11-isochlorogenic acid B, 12-isochlorogenic acid A, 13-rosmarinic acid and 14-isochlorogenic acid C.

3. The method of claim 2, wherein the reference peak is meso acid D No. 9, the relative retention time of each characteristic peak is within ± 10% of the specified value, and the specified values of each peak are: 0.13, 0.16, 0.21, 0.24, 0.40, 0.47, 0.53, 0.60, 1.00, 1.16, 1.20, 1.25, 1.40 and 1.43.

4. The method according to claim 2, wherein the reference peak is meso acid D No. 9, the relative peak area of each characteristic peak is within ± 20% of the specified value, and the specified values of each peak are: 0.53, 0.13, 0.40, 0.19, 0.46, 0.11, 0.64, 0.32, 1.00, 0.11, 0.24, 0.21, 0.42, and 0.19; and respectively constructing UPLC fingerprint spectra formed by the common characteristic peaks.

5. A detection method of UPLC fingerprint of concentrated juice of Wanglaoji cold tea is characterized by comprising the following steps:

(1) Preparation of a reference solution: precisely weighing reference substances including danshensu, protocatechuic acid, neochlorogenic acid, 15-demethyl frangipanin, chlorogenic acid, secologenin, caffeic acid, cerbernic acid B, mesonolic acid D, isorosmarinic acid, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid and isochlorogenic acid A, dissolving in 30-50% methanol to obtain 0.03-0.06 mg/mL solution, filtering with 0.22 μm microporous membrane, and collecting filtrate;

(3) And (3) high performance liquid chromatography determination: precisely sucking the reference solution prepared in the step S1 and the test solution prepared in the step S2 respectively, detecting according to the chromatographic conditions in the claim 1, comparing the chromatogram of the test solution obtained by detection with the fingerprint obtained by the establishing method in the claim 1, calculating the similarity, and evaluating the quality of the ULC of the concentrated juice of the WangLoKAI herbal tea.