AU2020459328A1

AU2020459328A1 - Endothelium corneum gigeriae galli specific polypeptide and application thereof in identification of endothelium corneum gigeriae galli

Info

Publication number: AU2020459328A1
Application number: AU2020459328A
Authority: AU
Inventors: Xiangdong Chen; Xueren CHENG; Guangming He; Qiping HU; Guowei Li; Dongmei SUN; Mei Wei; Xiaolong Yang
Original assignee: Guangdong Yifang Pharmaceutical Co Ltd
Current assignee: Guangdong Yifang Pharmaceutical Co Ltd
Priority date: 2020-07-23
Filing date: 2020-12-28
Publication date: 2023-02-23
Also published as: CN115980247A; CN115718159A; CN113970611A; CN116087399A; WO2022016809A1; CN113970611B

Abstract

An endothelium corneum gigeriae galli specific polypeptide and an application thereof in the identification of endothelium corneum gigeriae galli. By using high-resolution liquid chromatography-mass spectrometry technology and combining proteomics theory, a characteristic polypeptide of the endothelium corneum gigeriae galli is screened out, and the use of the characteristic polypeptide can quickly and accurately identify an endothelium corneum gigeriae galli component in an endothelium corneum gigeriae galli sample (e.g. an endothelium corneum gigeriae galli standard decoction), and can quickly identify whether the endothelium corneum gigeriae galli standard decoction is mixed with the membrane of poultry gizzard such as the membrane of duck gizzard, the membrane of goose gizzard, and the membrane of pigeon gizzard.

Description

ENDOTHELIUM CORNEUM GIGERIAE GALLI SPECIFIC POLYPEPTIDE AND APPLICATION THEREOF IN IDENTIFICATION OF ENDOTHELIUM CORNEUM GIGERIAE GALLI ECHNICAL FIELD

)001] The present disclosure relates to the field of analytical chemistry, in particular to an endothelium orneum gigeriae galli specific polypeptide and application thereof in identification of endothelium corneum

igeriae galli.

ACKGROUND

)002] Endothelium comeum gigeriae galli (i.e., chicken gizzard endothelium) refers to dried gizzard

ndothelium of Gallus gallus domesticus of Phasianidae and is obtained by killing the Gallus gallus omesticus, collecting the gizzard, peeling off the endothelium of the gizzard immediately, and washing and

rying the endothelium. Endothelium corneum gigeriae galli is formerly called a yellow skin in chicken

izzard, chicken gizzard, a yellow skin within chicken gizzard, chicken's yellow skin, Jishipi, Jihezi, zhongjin, Huashidan, Huagudan or the like, which is originally from the description of "the yellow skin of

bicken gizzard is mainly used for promoting excretion" in "Shen Nong's herbal classic".

)003] Endothelium corneum gigeriae galli is sweet in taste and mild in nature, and selectively functions in pleen, stomach, small intestine and bladder. It plays a role in invigorating the stomach and promoting

igestion, arresting seminal emission, treating stranguria and eliminating calculus. It is commonly used in

eating food stagnation, vomiting and diarrhea, infantile malnutrition, enuresis, spermatorrhea, urolithiasis ud astringent pain, biliary distension and hypochondriac pain. Modern clinical studies have shown that the

ndothelium corneum gigeriae galli can effectively alleviate clinical symptoms of consumptive thirst and its

implications. )004] However, the study of quality standard for the endothelium comeum gigeriae galli is incomplete.

egarding the standard for the endothelium corneum gigeriae galli in the 2015 edition of the Chinese

harmacopoeia, the properties, moisture, total ash and extracts of the endothelium corneum gigeriae galli are managed without requirement on the identification method. As the dried endothelium comeum gigeriae galli

re brittle and easily broken, it is difficult to distinguish the endothelium corneum gigeriae galli from

izzard endothelium of other bird species such as duck gizzard endothelium, goose gizzard endothelium, igeon gizzard endothelium or the like in the appearance. Further, for the decocted solution obtained by

ecocting in water or the prepared formula granules, due to lack of trait of the decoction pieces, it is more

ifficult to distinguish the endothelium corneum gigeriae galli by using the existing managing means.

)005] The applicant has searched literature reports on methods of identifying endothelium corneum

igeriae galli from gizzard endothelium of other bird species. The traditional techniques are mainly based on

ait for identification, such as "Identification of chicken gizzard endothelium from duck gizzard ndothelium and goose gizzard endothelium" by Honglai Zou et al. and "How to identify chicken gizzard

ndothelium from duck gizzard endothelium" by Xiaozhuan Liu. Zhende Chen et al. identified chicken

izzard endothelium from duck gizzard endothelium by the high-performance capillary electrophoresis JPCE) for protein polypeptide. The difference of the HPCE profiles between the chicken gizzard

ndothelium and the duck gizzard endothelium indicates the difference between the chicken gizzard

ndothelium and the duck gizzard endothelium. Wuqing Lv et al. showed the differences among chicken izzard endothelium, duck gizzard endothelium and goose gizzard endothelium according to the differences

i amylase activity, protease activity, amino acid contents and trace element contents. However, the above

methods are complicated. Further, for the water-decocted solution of chicken gizzard endothelium, the

lentification methods described in the above literature reports are not applicable or not specific for

distinguishing the chicken gizzard endothelium from duck gizzard endothelium, goose gizzard endothelium

ud pigeon gizzard endothelium.

UMMARY

)006] A purpose of the present disclosure is to provide an endothelium corneum gigeriae galli specific olypeptide and application thereof in identification of endothelium comeum gigeriae galli. The

ndothelium comeum gigeriae galli specific polypeptide has high specificity and thus can distinguish the

ndothelium corneum gigeriae galli from goose gizzard endothelium, duck gizzard endothelium and pigeon izzard endothelium.

)007] The present disclosure relates to an endothelium corneum gigeriae galli specific polypeptide

-lected from one or more of polypeptides shown in SEQ ID NOs: 1 to 4. )008] According to a further aspect of the present disclosure, the present disclosure also relates to use of

ie endothelium corneum gigeriae galli specific polypeptide as described above in identification of

udothelium corneum gigeriae galli. )009] Compared to the existing technology, the present disclosure has the following advantages.

ccording to the present disclosure, characteristic polypeptides of endothelium comeum gigeriae galli were

:reened out by using a liquid chromatography-mass spectrometry combined method with a high resolution i combination with proteomics theory. With the characteristic polypeptides described above, the

ndothelium corneum gigeriae galli component in an endothelium comeum gigeriae galli sample, such as a

andard decoction of endothelium comeum gigeriae galli, can be identified quickly and accurately, and hether the standard decoction of endothelium corneum gigeriae galli contains the gizzard endothelium of other bird species, such as duck gizzard endothelium, goose gizzard endothelium, pigeon gizzard ndothelium or the like, can be determined. The present method has a strong specificity and can provide a :ientific experimental basis for the quality evaluation study of the sample of endothelium comeum gigeriae alli, thereby achieving the specific identification of the quality of the endothelium corneum gigeriae galli simple, which can remedy the defect of inaccurate identification of the standard decoction due to lack of the appearance trait of the decoction pieces, reduce the adverse effects of adulterants in the Chinese medicine market, and ensure that the high-quality and authentic endothelium corneum gigeriae galli preparations are available to the public. Further, the present disclosure provides a new means to improve the quality standard f the decoction pieces of endothelium corneum gigeriae galli.

RIEF DESCRIPTION OF THE DRAWINGS )010] In order to more clearly describe the technical solutions in the specific embodiments of the present disclosure or in the prior art, brief description will be made below to the drawings that used in the specific mbodiments or in the prior art. Obviously, the drawings as described below are some embodiments of the resent disclosure. For those of ordinary skill in the art, other drawings can be derived from these drawings ithout creative work. )011] Figure 1 shows the mass spectra of endothelium corneum gigeriae galli, duck gizzard endothelium, oose gizzard endothelium and pigeon gizzard endothelium extracted at 379.21 of mass charge ratio (m/z) recording to an example of the present disclosure; )012] Figure 2 shows the mass spectra of endothelium corneum gigeriae galli, duck gizzard endothelium, oose gizzard endothelium and pigeon gizzard endothelium extracted at 785.41 of mass charge ratio (m/z) recording to an example of the present disclosure; )013] Figure 3 shows the mass spectra of endothelium corneum gigeriae galli, duck gizzard endothelium, oose gizzard endothelium and pigeon gizzard endothelium extracted at 487.24 of mass charge ratio (m/z) recording to an example of the present disclosure; )014] Figure 4 shows the mass spectra of endothelium corneum gigeriae galli, duck gizzard endothelium, oose gizzard endothelium and pigeon gizzard endothelium extracted at 607.98 of mass charge ratio (m/z) recording to an example of the present disclosure; )015] Figure 5 shows the mass spectra of endothelium corneum gigeriae galli, duck gizzard endothelium, oose gizzard endothelium and pigeon gizzard endothelium extracted at 683.32 of mass charge ratio (m/z) recording to an example of the present disclosure; )016] Figure 6 shows the mass spectra of endothelium corneum gigeriae galli, duck gizzard endothelium, oose gizzard endothelium and pigeon gizzard endothelium extracted at 362.72 of mass charge ratio (m/z) recording to an example of the present disclosure; )017] Figure 7 shows the mass spectra of endothelium corneum gigeriae galli, duck gizzard endothelium, oose gizzard endothelium and pigeon gizzard endothelium extracted at 426.70 of mass charge ratio (m/z) recording to an example of the present disclosure;

)018] Figure 8 shows the mass spectra of endothelium corneum gigeriae galli, duck gizzard endothelium, oose gizzard endothelium and pigeon gizzard endothelium extracted at 400.22 of mass charge ratio (m/z)

recording to an example of the present disclosure;

)019] Figure 9 shows the mass spectra of endothelium corneum gigeriae galli, duck gizzard endothelium, oose gizzard endothelium and pigeon gizzard endothelium extracted at 415.74 of mass charge ratio (m/z)

recording to an example of the present disclosure;

)020] Figure 10 shows the mass spectra of endothelium corneum gigeriae galli, duck gizzard endothelium, oose gizzard endothelium and pigeon gizzard endothelium extracted at 421.76 of mass charge ratio (m/z)

recording to an example of the present disclosure;

)021] Figure 11 is a diagram showing the comparison of secondary ion mass spectra of the polypeptide of udothelium corneum gigeriae galli at 379.21 of mass charge ratio (m/z) and an artificially synthesized

olypeptide thereof according to an example of the present disclosure;

)022] Figure 12 is a diagram showing the comparison of secondary ion mass spectra of the polypeptide of udothelium corneum gigeriae galli at 785.41 of mass charge ratio (m/z) and an artificially synthesized

olypeptide thereof according to an example of the present disclosure;

)023] Figure 13 is a diagram showing the comparison of secondary ion mass spectra of the polypeptide of udothelium corneum gigeriae galli at 487.24 of mass charge ratio (m/z) and an artificially synthesized

olypeptide thereof according to an example of the present disclosure;

)024] Figure 14 is a diagram showing the comparison of secondary ion mass spectra of the polypeptide of udothelium corneum gigeriae galli at 607.98 of mass charge ratio (m/z) and an artificially synthesized

olypeptide thereof according to an example of the present disclosure;

)025] Figure 15 is a diagram showing the comparison of secondary ion mass spectra of the polypeptide of uck gizzard endothelium and goose gizzard endothelium at 683.32 of mass charge ratio (m/z) and an

artificially synthesized polypeptide thereof according to an example of the present disclosure;

)026] Figure 16 is a diagram showing the comparison of secondary ion mass spectra of the polypeptide of uck gizzard endothelium and goose gizzard endothelium at 362.72 of mass charge ratio (m/z) and an

)027] Figure 17 is a diagram showing the comparison of secondary ion mass spectra of the polypeptide of uck gizzard endothelium and goose gizzard endothelium at 426.70 of mass charge ratio (m/z) and an artificially synthesized polypeptide thereof according to an example of the present disclosure;

)028] Figure 18 is a diagram showing the comparison of secondary ion mass spectra of the polypeptide of igeon gizzard endothelium at 400.22 of mass charge ratio (m/z) and an artificially synthesized polypeptide

1ereof according to an example of the present disclosure;

)029] Figure 19 is a diagram showing the comparison of secondary ion mass spectra of the polypeptide of igeon gizzard endothelium at 415.74 of mass charge ratio (m/z) and an artificially synthesized polypeptide

1ereof according to an example of the present disclosure;

)030] Figure 20 is a diagram showing the comparison of secondary ion mass spectra of the polypeptide of igeon gizzard endothelium at 421.76 of mass charge ratio (m/z) and an artificially synthesized polypeptide

1ereof according to an example of the present disclosure;

)031] Figure 21 shows the mass spectrum of detected endothelium comeum gigeriae galli component in a ecoction piece mixture including endothelium comeum gigeriae galli according to an example of the

resent disclosure;

)032] Figure 22 shows the mass spectrum of detected endothelium comeum gigeriae galli component in a andard decoction mixture including endothelium corneum gigeriae galli according to an example of the

resent disclosure;

)033] Figure 23 shows the mass spectrum of detected duck gizzard endothelium and goose gizzard ndothelium at a ratio of 5% in a decoction piece mixture including endothelium comeum gigeriae galli

recording to an example of the present disclosure;

)034] Figure 24 shows the mass spectrum of detected pigeon gizzard endothelium at a ratio of 5% in a ecoction piece mixture including endothelium corneum gigeriae galli according to an example of the

resent disclosure;

)035] Figure 25 shows the mass spectrum of detected duck gizzard endothelium and goose gizzard ndothelium at a ratio of 5% in a standard decoction mixture including endothelium corneum gigeriae galli

recording to an example of the present disclosure; and

)036] Figure 26 shows the mass spectrum of detected pigeon gizzard endothelium at a ratio of 5% in a andard decoction mixture including endothelium corneum gigeriae galli according to an example of the

resent disclosure.

ETAILED DESCRIPTION OF THE EMBODIMENTS

)037] Reference to embodiments of the present disclosure will be provided in detail and one or more

examples of which are described below. Each example is provided for explanation but not for limiting the resent disclosure. In practice, it is obvious to those skilled in the art to make a variety of modifications and variations of the present disclosure without departing from the scope or spirit of the present disclosure. For example, features illustrated or described as a part of an embodiment may be used in another embodiment to generate a further embodiment. )038] Accordingly, it is intended that the present disclosure covers such modifications and variations that ill within the scope of the appended claims and their equivalents. Other objects, features and aspects of the resent disclosure are disclosed in the following detailed description or are obvious therefrom. Those of rdinary skill in the art should understand that this discussion is merely a description of exemplary mbodiments and is not intended to limit the broader aspects of the present disclosure. )039] The present disclosure relates to an endothelium corneum gigeriae galli specific polypeptide -lected from one or more of polypeptides shown in SEQ ID NOs: 1 to 4. )040] According to a further aspect of the present disclosure, the present disclosure also relates to use of ie endothelium corneum gigeriae galli specific polypeptide as described above in identification of udothelium corneum gigeriae galli. )041] In some embodiments, the use is for distinguishing the endothelium comeum gigeriae galli from izzard endothelium of other bird species, in which the gizzard endothelium of other bird species is one or iore selected from goose gizzard endothelium, duck gizzard endothelium and pigeon gizzard endothelium.

)042] In some embodiments, the use is for identifying the presence or absence of gizzard endothelium of other bird species in the endothelium comeum gigeriae galli, in which the gizzard endothelium of other bird species is one or more selected from goose gizzard endothelium, duck gizzard endothelium and pigeon izzard endothelium; in which, the presence or absence of goose gizzard endothelium and/or duck gizzard endothelium in a abstance to be tested is determined based on the presence or absence of at least one of polypeptides shown i SEQ ID NOs: 5 to 7; and the presence or absence of pigeon gizzard endothelium in a substance to be tested is determined based n the presence or absence of at least one of polypeptides shown in SEQ ID NOs: 8 to 10. )043] The identification method of the specific polypeptide can be selected from the methods well known > those skilled in the art, for example by using antibodies and antibody fragments, lectins, aptamers or the ke capable of specifically binding to the specific polypeptide. The specific binding means that the affinity f the binding agent to its corresponding target molecule (i.e., the specific polypeptide) can be at least 107 /mol, or 108 L/mol, or more preferably 10' L/mol. The identification of the specific polypeptide can also be conducted by the biomass spectrometry or the like. In view that the subject to be identified in the present isclosure often has complicated ingredients, the identification is preferably conducted based on the hromatography-mass spectrometry combined method.

)044] In some embodiments, in the use, a substance to be tested is identified based on a hromatography-mass spectrometry combined method, and mass charge ratios (m/z) of parent ions and daughter ions of the endothelium comeum gigeriae galli specific polypeptide by using multiple reaction

monitoring (MRM) under a positive ion mode of electron spray ionization (ESI) are shown below: polypeptide m/z of doubly charged parent ions m/z of daughter ions SEQ ID NO:1 379.21 571.36 274.10 SEQ ID NO:2 785.41 229.12 941.50 SEQ ID NO:3 487.24 278.11 349.15 polypeptide m/z of triply charged parent ions m/z of daughter ions SEQ ID NO:4 607.98 473.28 1075.46

)045] In some embodiments, mass charge ratios (m/z) of parent ions and daughter ions of polypeptides specific to the goose gizzard endothelium, the duck gizzard endothelium or the pigeon gizzard endothelium

y using multiple reaction monitoring (MRM) under a positive ion mode of electron spray ionization (ESI) re shown below: polypeptide m/z of doubly charged parent ions m/z of daughter ions SEQ ID NO:5 683.32 159.08 260.12 SEQ ID NO:6 362.72 253.06 334.11 SEQ ID NO:7 426.70 203.10 130.09 SEQ ID NO:8 400.22 186.09 299.17 SEQ ID NO:9 415.74 303.17 632.34 SEQ ID NO:10 421.76 199.14 312.23

)046] In some embodiments, the identification of the substance to be tested by chromatography is performed with a gradient elution using mobile phase A and mobile phase B;

the mobile phase A is acetonitrile; and the mobile phase B is an aqueous formic acid solution of 0.07 v/v% to 0.13 v/v%.

)047] In some embodiments, conditions for the gradient elution include: Time (min) mobile phase A (%) mobile phase B (%) Oto3 3 to8 97to92 3 to45 8 to22 92 to78 45 to 54 22 to 32 78 to 68 54 to 54.1 32 to 100 68 to 0 54.1 to 58 100 0 58 to 58.1 100 to 3 0 to 97 58.1 to 62 3 97

)048] In some embodiments, a flow rate of the gradient elution is 0.3 to 0.5 mL/min, optionally 0.4 iL/min.

)049] In some embodiments, a stationary phase for a chromatography column used in the chromatography as functional groups mainly being C18 functional groups.

)050] In some embodiments, the chromatography column is Waters ACQUITY UPLC BEH C18 bromatography column.

)051] In some embodiments, the chromatography column has a length of 100 mm to 200 mm, an inner diameter of 1.0 mm to 3.0 mm and a packing particle size of 1.4 m to 2.0 [m.

)052] In some embodiments, the chromatography column has a length of 150 mm, an inner diameter of .1 mm and a packing particle size of 1.7 [m. )053] In some embodiments, the chromatography column is of a temperature of 25°C to 35°C. Optionally, 1e temperature can be selected from 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C or 34°C.

)054] In some embodiments, the substance to be tested is filtered by passing through a microporous filter membrane of 0.20 m to 0.24 [m, preferably a microporous filter membrane of 0.22 [m.

)055] In some embodiments, conditions for the mass spectrometry include: a positive ion mode of electron spray ionization; a flow rate of sheath gas of 30 L/min;

a flow rate of auxiliary gas of 10 L/min;

spray voltage of 3.30 kV; capillary temperature of 320°C; and

auxiliary gas temperature of 320°C,

in which each numerical parameter in the conditions has a floating value ranging from 90% to 110% of value of the numerical parameter.

)056] In some embodiments, the substance to be tested is a decoction or decoction pieces of endothelium orneum gigeriae galli. )057] The decoction pieces can include decoction pieces of original medicinal materials and decoction pieces of medicinal materials subjected to chopping and/or processing via fire. Methods for processing via

re include, but are not limited to, steaming, boiling, frying, baking, roasting, concocting, calcining, dipping, ranching, or the like.

)058] The decoction of endothelium comeum gigeriae galli can be obtained by decocting the original >rm or the decoction pieces of the endothelium comeum gigeriae galli, and generally further including collecting the filtrate and discarding the residue, for example, through filtering with a 320 to 380 mesh sieve.

)059] In some embodiments, when the substance to be tested is decoction pieces, the substance to be sted is subjected to trypsin digestion. Further, the trypsin digestion is conducted for 8 to 16 hours. ltematively, the trypsin digestion is conducted for 12 hours.

)060] In some embodiments, when the substance to be tested is decoction pieces, the substance to be sted is subjected to grinding and/or ultrasonic treatment. Further, the power for the ultrasonic treatment is

W to 300 W, the frequency for the ultrasonic treatment is 40 kHz to 60 kHz, and the sonication time is

to 40 minutes.

)061] After the pretreatment of the substance to be tested, the impurities can be effectively removed and ie specific polypeptides can be released, thus increasing the specificity and sensitivity for detection.

)062] Embodiments of the present disclosure will be described in detail in combination with examples as elow. )063] Example 1 Screening, artificial synthesis and validation of specific polypeptides )064] 1.1 Preparation of a test solution )065] An appropriate amount of a sample was ground finely. About 0.1 g of the ground sample was eighed and 50 mL of a solution of 1% ammonium hydrogen carbonate was added, followed by the

trasonic treatment with a power of 250W and a frequency of 50 kHz for 30 minutes, and filtered through

.22tm of microporous filter membrane. 100 L of filtered liquid was precisely pipetted and transferred to a iicrosample bottle. 10tL of lmg/1mL trypsin solution (prepared by adding a solution of 1% ammonium

ydrogen carbonate to the sequencing grade trypsin and prepared for immediate use) was added to the

1icrosample bottle, shook, and subjected to enzymatic digestion at a constant temperature of 37°C for 12 ours, and thus the test solution was obtained.

)066] 1.2 Preparation of a control solution of medicinal material )067] 0.5g of powder of endothelium corneum gigeriae galli as a control was weighed and 50 mL of a )lution of 1% ammonium hydrogen carbonate was added, followed by heating under reflux for 30 minutes,

took, and filtered through 0.22tm of microporous filter membrane. 100tL of filtrate was pipetted and

ansferred to a microsample bottle. 10tL of lmg/1mL trypsin solution (prepared by dissolving the trypsin ith a solution of 1% ammonium hydrogen carbonate and prepared for immediate use) was added to the

1icrosample bottle, shook, and subjected to enzymatic digestion at a constant temperature of 37°C for 12

ours, and thus the control solution was obtained. )068] 1.3 Conditions for chromatography )069] The chromatography was carried out through Waters ACQUITY UPLC BEH C18 chromatography column (in 150 mm x 2.1mm, 1.7am), with the gradient elution using acetonitrile and 0.1% aqueous formic cid solution as the mobile phases. The temperature for chromatography column was 30°C and the flow rate

as 0.3 mL/min. 2tL of the sample was injected.

Table for gradient elution Time (min) acetonitrile (%) 0.1% aqueous formic acid solution (%) Oto3 3 to8 97to92 3 to45 8 to22 92to78 45 to54 22to32 78 to68 54 to 54.1 32 to 100 68 to 0

54.1 to 58 100 0 58 to58.1 100 to 3 0 to 97 58.1 to 62 3 97

)070] 1.4 Conditions for mass spectrometry )071] The conditions for mass spectrometry include a positive ion mode of electron spray ionization iESI+), a flow rate of sheath gas of 30 L/min, a flow rate of auxiliary gas of 10 L/min, spray voltage of .30 kV, capillary temperature of 320°C, and auxiliary gas temperature of 320°C; and the Full-MS-ddms2

iode scan was carried out.

)072] 1.5 Measurement )073] 2 L of the control solution of medicinal material and a test solution of endothelium corneum

igeriae galli, a test solution of duck gizzard endothelium, a test solution of goose gizzard endothelium, and test solution of pigeon gizzard endothelium were respectively precisely pipetted, injected into the liquid

hromatograph and measured according to the above conditions and the mass spectra were acquired.

)074] 1.6 Data processing )075] The acquired mass spectrometry data were imported into the Proteome Discoverer 2.4 analysis )ftware. The Uniprot-Aves database was used for searching to find the characteristic polypeptides specific

> the endothelium comeum gigeriae galli, the duck gizzard endothelium, the goose gizzard endothelium, or 1e pigeon gizzard endothelium were respectively screened out. The information about the sequence and the

1ass charge ratios (m/z) of the specific polypeptides of relative gizzard endothelium was as follows: Species No. sequence m/z SEQ ID NO:1 GESVLPR 379.21

SEQ ID NO:2 IDEASIPELQEIGR 785.41 -ndotheliumcomeumgigeriaegalli SEQ ID NO:3 NYADVHVR 487.24 SEQ ID NO:4 LLGAPVPVDENDEGLQR 607.98

SEQ ID NO:5 ASTATDGSSVGPTSK 683.32 gizzardendothelium SEQ ID NO:6 LIHDVK 362.72 luck gizzard endothelium uckgizzardendothelium SEQ ID NO:7 TTTESWK 426.70 SEQ ID NO:8 GQIQPTR 400.22 >igeon gizzard endothelium SEQ ID NO:9 VVTVEQR 415.74 SEQ ID NO:10 VVIVEQR 421.76

)076] The specificity of the selected mass charge ratios to the species was further determined by the comparison of the mass spectra. Figs. 1-4 show that the endothelium corneum gigeriae galli has four characteristic polypeptides which are uniquely presented in the endothelium corneum gigeriae galli and are

sent in any of the goose gizzard endothelium, the duck gizzard endothelium and the pigeon gizzard

ndothelium. Figs. 5-7 show that the goose gizzard endothelium and the duck gizzard endothelium both ave three identical characteristic polypeptides which are absent in any of the endothelium corneum gigeriae alli and the pigeon gizzard endothelium. Figs. 8-10 show that the pigeon gizzard endothelium has three characteristic polypeptides which are uniquely presented in the pigeon gizzard endothelium and are absent in ny of the endothelium corneum gigeriae galli, the duck gizzard endothelium and the goose gizzard ndothelium.

)077] 1.7 Artificial synthesis and validation of selected polypeptides )078] The sequences of the screened polypeptides were synthesized by Gill Biochemistry (Shanghai) Co., td., measured according to the same conditions for chromatography and mass spectrometry as those for the

st solution. The retention times and the secondary ion fragments of the synthesized polypeptides and those

f the screened polypeptides from the samples at the same mass charge ratio were compared. The results towed that the retention times and the secondary ion fragments of all the specific polypeptides in the

ndothelium corneum gigeriae galli, the duck gizzard endothelium, the goose gizzard endothelium, or the

igeon gizzard endothelium were respectively consistent with those of the artificially synthesized olypeptides, demonstrating that the sequences of the screened polypeptides are accurate with reference to

igure 11 to Figure 20.

)079] Example 2 Detection of decoction pieces and standard decoction of endothelium comeum gigeriae alli mixed with duck gizzard endothelium, goose gizzard endothelium and pigeon gizzard endothelium

)080] The polypeptide of endothelium comeum gigeriae galli shown in SEQ ID NO:2, the polypeptide of uck gizzard endothelium and goose gizzard endothelium shown in SEQ ID NO:6, and the polypeptide of igeon gizzard endothelium shown in SEQ ID NO:9 in Example 1 were selected. The decoction pieces and

ie standard decoction of endothelium corneum gigeriae galli mixed with duck gizzard endothelium, goose

izzard endothelium and pigeon gizzard endothelium as samples were detected by selecting appropriate -condary ions and using the parallel reaction monitoring (PRM).

)081] The detailed detection is as follows. )082] 2.1 Preparation of a decoction piece mixture including endothelium corneum gigeriae galli as a simple

)083] An appropriate amount of crude powder of endothelium corneum gigeriae galli was weighed and iixed with powders of duck gizzard endothelium, goose gizzard endothelium and pigeon gizzard

ndothelium in an amount of 10%, 5% or 2% by weight based on the endothelium corneum gigeriae galli,

:spectively. Thus, three gizzard endothelium mixtures were obtained.

)084] 2.2 Preparation of a mixed decocted solution (standard decoction) )085] An appropriate amount of endothelium corneum gigeriae galli was put in a ceramic pot, and the uck gizzard endothelium, the goose gizzard endothelium and the pigeon gizzard endothelium were blended

i an amount of 10%, 5% or 2% by weight based on the endothelium corneum gigeriae galli, thus obtaining iree gizzard endothelium mixtures. Water was added twice for decocting. For a first decocting, 10 times of oiling water were directly added, followed by heating with a strong power of 500W until it boils, and then ept boiling mildly with a gentle power of 200W for 30 minutes. The decocted solution was filtered through 350 mesh sieve and the filtrate was cooled down quickly with cold water. For a second decocting, 8 times f water were added, followed by heating with a strong power of 500W until it boils, and then kept boiling wildly with a gentle power of 200W for 10 minutes. The decocted solution was filtered through a 350 mesh eve and the filtrate was cooled down quickly with cold water. The filtrate obtained in both decocting was combined and thus the standard decoction was obtained. )086] 2.3 Preparation of a test solution )087] 0.1g of the decoction piece mixture including endothelium corneum gigeriae galli and an appropriate amount of the mixed decocted solution (corresponding to 0.Ig of the decoction pieces of udothelium corneum gigeriae galli) were respectively added to individual volumetric flasks of 50mL, and a )lution of 1% ammonium hydrogen carbonate was added until it reached the mark, followed by the ltrasonic treatment with a power of 250W and a frequency of 50kHz for 30 minutes and filtered through .22tm of microporous filter membrane. 100tL of the filtrate was precisely pipetted and transferred to a iicrosample bottle. 10tL of lmg/1mL trypsin solution (prepared by adding a solution of 1% ammonium ydrogen carbonate to the sequencing grade trypsin and prepared for immediate use) was added to the iicrosample bottle, shook, and subjected to enzymatic digestion at a constant temperature of 37°C for 12 ours, and thus the test solution of decoction piece mixture and the test solution of mixed decocted solution ere obtained. )088] 2.4 Preparation of a control solution )089] An appropriate amount of artificially synthesized polypeptides shown in SEQ ID NO:2, SEQ ID :6 or SEQ ID NO:9 as a control were precisely weighed respectively, and methanol was respectively added to generate a solution containing 10tg of each control polypeptide per 1mL, and thus the control )lution was obtained. )090] 2.5 Conditions for chromatography )091] The chromatography was carried out through Waters ACQUITY UPLC BEH C18 chromatography column (in 150 mm x 2.1mm, 1.7[tm). The gradient elution was conducted by using acetonitrile and 0.1% queous formic acid solution as the mobile phases according to the following table. The temperature for hromatography column was 30°C and the flow rate was 0.3 mL/min. Table for gradient elution Time (min) mobile phase A (%) mobile phase B (%) Oto3 3 to8 97to92 3 to45 8 to22 92to78

45 to54 22 to32 78 to68 54 to 54.1 32 to 100 68 to 0 54.1 to 58 100 0 58 to 58.1 100 to 3 0 to 97 58.1 to 62 3 97

)092] 2.6 Conditions for mass spectrometry )093] The conditions for mass spectrometry include a positive ion mode of electron spray ionization HSI+), a flow rate of sheath gas of 30 L/min, a flow rate of auxiliary gas of 10 L/min, spray voltage of 3.30 V, capillary temperature of 320°C, and auxiliary gas temperature of 320°C, and the parallel reaction

monitoring (PRM) mode scan was carried out according to ion pairs in the following table. Name sequence m/z of m/z of daughter m/z of daughter parent ions ions No.1 ions No.2 SEQ ID NO:2 IDEASIPELQEIGR 785.41 229.12 941.50 SEQ ID NO:6 LIHDVK 362.72 253.06 334.11 SEQ ID NO:9 VVTVEQR 415.74 303.17 632.34

)094] 2.7 Measurement

)095] 2 L of the control solution and each test solution of mixed decocted solution were respectively

recisely pipetted, injected into the liquid chromatograph, and measured according to the above conditions, ud the mass spectra were acquired.

)096] 2.8 Results and analysis

)097] The experiment results showed that both the decoction piece mixture and the mixed decocted Caution can derive chromatographic peaks which have the retention times consistent with that of the

artificially synthesized polypeptide corresponding to SEQ ID NO: 2 as the control (see, Figs. 21 - 22),

demonstrating that the endothelium corneum gigeriae galli component can be accurately detected through 1e present method.

)098] The decoction piece mixture can derive chromatographic peaks which have the retention times

:spectively consistent with that of the artificially synthesized polypeptide corresponding to SEQ ID NO: 6 s the control and that of the artificially synthesized polypeptide corresponding to SEQ ID NO: 9 as the

control (see, Figs. 23-24). That is, when the decoction pieces of endothelium corneum gigeriae galli is mixed

ith 5% of duck gizzard endothelium, 5% of goose gizzard endothelium, and 5% of pigeon gizzard ndothelium, the components of the duck gizzard endothelium, the goose gizzard endothelium, and the

igeon gizzard endothelium can be detected.

)099] The mixed decocted solution can derive chromatographic peaks which have the retention times :spectively consistent with that of the artificially synthesized polypeptide corresponding to SEQ ID NO: 6

s the control and that of the artificially synthesized polypeptide corresponding to SEQ ID NO: 9 as the

control (see, Figs. 25 - 26). That is, when the standard decoction of endothelium comeum gigeriae galli as a simple is mixed with 5% of duck gizzard endothelium, 5% of goose gizzard endothelium, and 5% of pigeon izzard endothelium, the components of the duck gizzard endothelium, the goose gizzard endothelium, and ie pigeon gizzard endothelium can be detected.

)0100]The results showed that the present method has high sensitivity and thus can effectively control the

dulterants in the endothelium corneum gigeriae galli.

)0101]The technical features of the examples as described above can be combined in any ways. For brief description, not all possible combinations of the individual technical features in the above examples are

described. However, the combinations of these technical features should be considered to be within the

:ope of this specification given that such combinations of these technical features are not contradictory. )0102]The examples as described above merely indicate several embodiments of the present disclosure,

hich are described in a detailed manner, but they should not be construed as a limitation of the scope of the

resent disclosure. It should be noted that several variations and improvements can be made by those of rdinary skill in the art without departing from the conception of the present disclosure, and these belong to

1e scope of protection of the present disclosure. Therefore, the protection scope of the present disclosure

ould be subject to the appended claims.

SEQUENCE LISTING

<110> GUANGDONG YIFANG PHARMACEUTICAL CO., LTD

<120> ENDOTHELIUM CORNEUM GIGERIAE GALLI SPECIFIC POLYPEPTIDE AND APPLICATION THEREOF IN IDENTIFICATION OF ENDOTHELIUM CORNEUM GIGERIAE GALLI

<130> SP22729689AU

<150> CN 2020107159156 <151> 2020-07-23

<150> PCT/CN2020/140104 <151> 2020-12-28

<160> 10

<170> PatentIn version 3.5

<210> 1 <211> 7 <212> PRT <213> Artificial Sequence

<220> <223> polypeptide of endothelium corneum gigeriae galli

<400> 1

Gly Glu Ser Val Leu Pro Arg 1 5

<210> 2 <211> 14 <212> PRT <213> Artificial Sequence

<220> <223> polypeptide of endothelium corneum gigeriae galli

<400> 2

Ile Asp Glu Ala Ser Ile Pro Glu Leu Gln Glu Ile Gly Arg

1 5 10

<210> 3 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> polypeptide of endothelium corneum gigeriae galli

<400> 3

Asn Tyr Ala Asp Val His Val Arg 1 5

<210> 4 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> polypeptide of endothelium corneum gigeriae galli

<400> 4

Leu Leu Gly Ala Pro Val Pro Val Asp Glu Asn Asp Glu Gly Leu Gln 1 5 10 15

Arg

<210> 5 <211> 15 <212> PRT <213> Artificial Sequence

<220> <223> polypeptide of goose/duck gizzard endothelium

<400> 5

Ala Ser Thr Ala Thr Asp Gly Ser Ser Val Gly Pro Thr Ser Lys 1 5 10 15

<210> 6 <211> 6 <212> PRT <213> Artificial Sequence

<220> <223> polypeptide of goose/duck gizzard endothelium

<400> 6

Leu Ile His Asp Val Lys 1 5

<210> 7 <211> 7 <212> PRT <213> Artificial Sequence

<220> <223> polypeptide of goose/duck gizzard endothelium

<400> 7

Thr Thr Thr Glu Ser Trp Lys 1 5

<210> 8 <211> 7 <212> PRT <213> Artificial Sequence

<220> <223> polypeptide of pigeon gizzard endothelium

<400> 8

Gly Gln Ile Gln Pro Thr Arg 1 5

<210> 9 <211> 7 <212> PRT <213> Artificial Sequence

<220> <223> polypeptide of pigeon gizzard endothelium

<400> 9

Val Val Thr Val Glu Gln Arg 1 5

<210> 10 <211> 7 <212> PRT <213> Artificial Sequence

<220> <223> polypeptide of pigeon gizzard endothelium

<400> 10

Val Val Ile Val Glu Gln Arg 1 5

Claims

Vhat is claimed is: 1. An endothelium corneum gigeriae galli specific polypeptide selected from one or more of

olypeptides shown in SEQ ID NOs: 1 to 4.
2. Use of the endothelium corneum gigeriae galli specific polypeptide according to claim 1 in

lentification of endothelium corneum gigeriae galli.
3. The use according to claim 2, which is for distinguishing the endothelium comeum gigeriae galli -om gizzard endothelium of other bird species, wherein the gizzard endothelium of other bird species is one

r more selected from goose gizzard endothelium, duck gizzard endothelium and pigeon gizzard

ndothelium.
4. The use according to claim 2, which is for identifying the presence or absence of gizzard

ndothelium of other bird species in the endothelium corneum gigeriae galli, wherein the gizzard

ndothelium of other bird species is one or more selected from goose gizzard endothelium, duck gizzard ndothelium and pigeon gizzard endothelium;

wherein the presence or absence of goose gizzard endothelium and/or duck gizzard endothelium in a

abstance to be tested is determined based on the presence or absence of at least one of polypeptides shown i SEQ ID NOs:5 to 7; and

the presence or absence of pigeon gizzard endothelium in a substance to be tested is determined based

n the presence or absence of at least one of polypeptides shown in SEQ ID NOs:8 to 10.
5. The use according to any one of claims 2 to 4, wherein a substance to be tested is identified based on

chromatography-mass spectrometry combined method, and mass charge ratios of parent ions and daughter

s of the endothelium comeum gigeriae galli specific polypeptide by using multiple reaction monitoring VIRM) under a positive ion mode of electron spray ionization (ESI) are shown below: polypeptide m/z of doubly charged parent ions m/z of daughter ions SEQ ID NO:1 379.21 571.36 274.10 SEQ ID NO:2 785.41 229.12 941.50 SEQ ID NO:3 487.24 278.11 349.15 polypeptide m/z of triply charged parent ions m/z of daughter ions SEQ ID NO:4 607.98 473.28 1075.46
6. The use according to claim 4, wherein mass charge ratios of parent ions and daughter ions of olypeptides specific to the goose gizzard endothelium, the duck gizzard endothelium or the pigeon gizzard ndothelium by using multiple reaction monitoring (MRM) under a positive ion mode of electron spray mization (ESI) are shown below: polypeptide m/z of doubly charged parent ions m/z of daughter ions SEQ ID NO:5 683.32 159.08 260.12 SEQ ID NO:6 362.72 253.06 334.11 SEQ ID NO:7 426.70 203.10 130.09

SEQ ID NO:8 400.22 186.09 299.17 SEQ ID NO:9 415.74 303.17 632.34 SEQ ID NO:10 421.76 199.14 312.23
7. The use according to claim 5 or 6, wherein the identification of the substance to be tested by hromatography is performed with a gradient elution of mobile phase A and mobile phase B;

the mobile phase A is acetonitrile; and

the mobile phase B is an aqueous formic acid solution of 0.07 v/v% to 0.13 v/v%.
8. The use according to claim 7, wherein conditions for the gradient elution comprise: Time (min) mobile phase A (%) mobile phase B (%) 0to3 3 to8 97to92 3 to 45 8 to 22 92 to 78 45 to54 22 to32 78 to68 54 to 54.1 32 to 100 68 to 0 54.1 to 58 100 0 58 to 58.1 100 to 3 0 to 97 58.1 to 62 3 97
9. The use according to claim 8, wherein a flow rate of the gradient elution is 0.3 to 0.5 mL/min;

a stationary phase for a chromatography column used in the chromatography has functional groups mainly being C18 functional groups; and

the chromatography column is of a temperature of 25°C to 35°C.
10. The use according to any one of claims 5, 6, 8 and 9, wherein conditions for the mass spectrometry comprise:

a positive ion mode of electron spray ionization;

a flow rate of sheath gas of 30 L/min; a flow rate of auxiliary gas of 10 L/min;

spray voltage of 3.30 kV;

capillary temperature of 320°C; and auxiliary gas temperature of 320°C,

wherein each numerical parameter in the conditions has a floating value ranging from 90% to 110% of

value of the numerical parameter.
11. The use according to any one of claims 5, 6, 8 and 9, wherein the substance to be tested is a

ecoction or decoction pieces of endothelium corneum gigeriae galli.
12. The use according to claim 11, wherein when the substance to be tested is a decoction pieces, the abstance to be tested is subjected to trypsin digestion.

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