CN111208224B - Liquid chromatography detection method of herpetospermum pedunculosum seed shell extract - Google Patents

Liquid chromatography detection method of herpetospermum pedunculosum seed shell extract Download PDF

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CN111208224B
CN111208224B CN202010036029.0A CN202010036029A CN111208224B CN 111208224 B CN111208224 B CN 111208224B CN 202010036029 A CN202010036029 A CN 202010036029A CN 111208224 B CN111208224 B CN 111208224B
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methanol
herpetospermum
extract
shells
herpetospermum pedunculosum
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CN111208224A (en
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汪俊松
陈剑锋
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Nanjing University of Science and Technology
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Nanjing University of Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate

Abstract

The invention discloses a liquid chromatography detection method of a pedunculate herpetospermum seed shell extract. According to the method, herpetospermum pedunculosum shells are used as raw materials, methanol and water are used as mobile phases, 0.1% of formic acid is added into the methanol, and the optimal elution condition is obtained by adjusting the ratio of the methanol to the water under different elution times, so that a liquid phase map is obtained. The method is simple, convenient and stable, and has high precision and good reproducibility. By the detection method, the chemical component information in the herpetospermum pedunculosum seed shells is fully reflected. The liquid phase analysis spectrogram of the herpetospermum pedunculosum fruit shell extract established by the invention has important significance for the production and development of herpetospermum pedunculosum fruit shells.

Description

Liquid chromatography detection method of herpetospermum pedunculosum seed shell extract
Technical Field
The invention belongs to the technical field of natural medicinal chemistry, and relates to a liquid chromatography detection method of a pedunculate herpetospermum seed shell extract.
Background
Herpetospermum pedunculosum (Herpetospermum pedunculosum) is a plant species of the genus Herpetospermum of the family Cucurbitaceae. The pedunculate herpetospermum fruit is a climbing herbaceous plant, the stem and the branch are slender, and the outer surface of the pedunculate herpetospermum fruit is provided with a ditch-shaped structure. When the pedunculate herpetospermum fruit grows out, sparse velveteen grows on the surface, and finally the surface becomes very smooth. The pedunculate herpetospermum is an annual plant, the flowers are unisexual and are hermaphroditic, widely distributed in himalayas in china, nipaler and india, and are often found in roadside and shrub clusters at an altitude of 2000-3500 m.
The herpetospermum pedunculosum seeds are dry and mature seeds of herpetospermum pedunculosum of Cucurbitaceae, are traditional Tibetan medicine of China, and have the Tibetan language of Se-Ji-Mei-Duo, which is recorded in the drug Standard of the Ministry of health of the people's republic of China. The herpetospermum pedunculosum seeds have the efficacy of clearing heat and removing toxicity, and are widely used for treating inflammation of 'red bar' (one of three diseases of Tibetan medicines). The history of the use of the pedunculate herpetospermum fruit is quite long, and the use of the pedunculate herpetospermum fruit as a medicament is mainly Tibetan medicine and Mongolian medicine. The records of Di Qing Tibetan medicine and Zhong Hua Ben Cao are: seeds are bitter in taste, cold in nature and acute in effect; can clear heat in the lower limbs and gallbladder; it can be used for treating visceral heat, liver heat, gallbladder heat (hepatitis, cholecystitis), and dyspepsia. The method comprises the steps of separating the carissonin (herpetin) from an ethanol extract of Herpetospermum pedunculosum seeds, such as Yuan-Hailong, lixian, a herpetin compound, a preparation method thereof, a pharmaceutical composition thereof and a use thereof, CN 1305468C [ P ].2007, and separating the carissonin, dehydroconiferyl alcohol (dehydroconiferyl alcohol), herpetospermum I (Herpesolide A), herpetospermum II (Herpesolide B) and other active compounds with medicinal value, such as ZHANG M, DENG Y, ZHANG H B, et al.
According to records of Chinese Tibetan medicine and Tibetan common Chinese herbal medicine, the main part of the herpetospermum pedunculosum seeds used as the Tibetan medicine is herpetospermum pedunculosum seeds, but parts such as seed coats, flowers and the like can also be utilized, and the effect is similar to that of the seeds. According to research, no explanation about the use of the herpetospermum pedunculosum seed shells as a medicament is found at present, and the research on chemical components, pharmacological toxicity and the like is not involved. In the actual production process, the ripe pedunculate herpetospermum seeds are conveyed to the next step of processing after being dried and shelled, and the shells of the pedunculate herpetospermum seeds are discarded, so that huge waste is caused.
Disclosure of Invention
The invention aims to provide a liquid chromatography detection method of a pedunculate herpetospermum seed shell extract. The method takes the herpetospermum pedunculosum fruit shells as raw materials, utilizes the chemical means of natural products, adds a certain proportion of formic acid into an organic phase, and establishes a liquid phase map of the herpetospermum pedunculosum fruit shells by using high performance liquid chromatography, thereby establishing quality standards for the industrialized utilization process of the herpetospermum pedunculosum fruit shells.
The technical scheme for realizing the purpose of the invention is as follows:
the liquid chromatography detection method of the herpetospermum pedunculosum seed shell extract comprises the following steps:
step 1, crushing the herpetospermum pedunculosum shells; heating and refluxing the crushed herpetospermum pedunculosum shells by 95% ethanol to extract to obtain an ethanol extract, extracting the herpetospermum pedunculosum ethanol extract by using ethyl acetate, and carrying out rotary steaming on the extract liquid under reduced pressure to obtain an ethyl acetate extract;
step 2, adding the aequorea peduncularis shell ethyl acetate extract into chromatographic grade methanol for mixing, performing ultrasonic treatment to fully dissolve the aequoria peduncularis shell ethyl acetate extract, and then performing sample injection through a microporous filter membrane;
and 3, performing gradient elution by using a high performance liquid chromatograph and a C18 chromatographic column as analytical columns, using methanol added with 0.1% formic acid as an A phase and water as a B phase, and setting an elution program for elution for 60min to obtain the fingerprint of the pedunculate herpetospermum fruit shell extract.
Preferably, in step 1, the reflux extraction is performed 3 times or more, and each reflux extraction time is 1 to 3 hours, preferably 3 hours for the first time, 2 hours for the second time, and 1 hour for the third time.
Preferably, in step 2, the pore size of the microporous filter membrane is 0.5 μm.
Preferably, in step 3, the elution procedure is as follows: 0-10 min, 10-25% methanol; 10-25min, 25-36% methanol; 25-29min, 36-58% methanol; 29-40min, 58-60% methanol; 40-45 min;60 to 100 percent of methanol; 45-60min, 100% methanol.
Preferably, in step 3, the column temperature is 30 ℃.
Preferably, in step 3, the fluorescence detection wavelength is 230nm,254nm and 260nm.
Preferably, in step 3, the flow rate of the mobile phase is 0.8mL/min.
Compared with the prior art, the invention has the following advantages:
according to the invention, the liquid chromatography analysis is carried out on the seed shells of the pedunculate herpetospermum seeds for the first time, the separation degree of the extract is increased by adding 0.1% formic acid into the mobile phase, and the obtained liquid chromatogram compound has good separation degree and a large amount of compounds. The method is simple, convenient, stable, high in precision and good in reproducibility. The detection method can more fully reflect the chemical component information in the herpetospermum pedunculosum shells, and the selected fluorescence detection wavelength provides more abundant compound information. The liquid phase analysis spectrogram of the raw material in industrial production is an important reference for quality control, and the liquid phase analysis spectrogram of the herpetospermum pedunculosum seed shell extract established by the invention has important significance for the production and development of the herpetospermum pedunculosum seed shell.
Drawings
FIG. 1 is a graph comparing the number of peaks in example 1, comparative example 1 and comparative example 2.
FIG. 2 is a liquid phase spectrum at a detection wavelength of 260nm in example 1.
FIG. 3 is a liquid phase spectrum at a detection wavelength of 254nm for example 1.
FIG. 4 is a liquid phase spectrum at a detection wavelength of 230nm in example 1.
FIG. 5 is a liquid phase spectrum of comparative example 1 at a detection wavelength of 260nm.
FIG. 6 is a liquid phase spectrum of comparative example 1 at a detection wavelength of 254 nm.
FIG. 7 is a liquid phase spectrum of comparative example 1 at a detection wavelength of 230 nm.
FIG. 8 is a liquid phase spectrum of comparative example 2 at a detection wavelength of 260nm.
FIG. 9 is a liquid phase spectrum of comparative example 2 at a detection wavelength of 254 nm.
FIG. 10 is a liquid phase spectrum of comparative example 2 at a detection wavelength of 230 nm.
Detailed Description
The present invention will be described in further detail with reference to the following examples and the accompanying drawings.
Example 1
(1) Crushing the hull of the herpetospermum pedunculosum; carrying out heating reflux extraction on the crushed herpetospermum pedunculosum shells by using 95% ethanol for three times, wherein the first time is 3 hours, the second time is 2 hours, and the third time is 1 hour to obtain an ethanol extracting solution; performing rotary evaporation and evaporation on the ethanol extract under reduced pressure to obtain an ethanol extract, extracting the ethanol extract by using ethyl acetate, and performing rotary evaporation and evaporation on the extract under reduced pressure to obtain an ethyl acetate extract;
(2) Adding the ethyl acetate extract of the herpetospermum pedunculosum into chromatographic grade methanol for mixing, filtering by using an organic filter membrane of 0.5um after fully mixing by using ultrasonic, transferring to a special sample injection bottle of a chromatograph, and waiting for sample injection;
(3) Opening the liquid chromatograph, setting an elution program after each component is ready, wherein the elution program is set as follows: 0-10 min,10% -25% methanol; 10-25min, 25-36% of methanol; 25-29min, 36-58% methanol; 29-40min, 58-60% methanol; 40-45 min;60 to 100 percent of methanol; 45-60min and 100% methanol. Setting the column temperature at 30 ℃, setting the flow rate at 0.8mL/min, setting the fluorescence detection wavelengths at 230nm,254nm and 260nm respectively, selecting a C18 chromatographic column as a detection column with specifications of 4.6mm multiplied by 200mm and 5 mu m, and adding 0.1% formic acid into A-phase methanol;
(4) Starting the high performance liquid chromatograph, executing a set program, and obtaining a liquid phase spectrogram by using the obtained data through a printing command.
In this example, the three detection wavelengths obtained chromatographic peaks were 51, 89, and 97, respectively.
Comparative example 1
(1) Crushing the hull of the herpetospermum pedunculosum; carrying out heating reflux extraction on the crushed pedunculate herpetospermum seed shells by using 95% ethanol for three times, wherein the first time is 3 hours, the second time is 2 hours, and the third time is 1 hour to obtain an ethanol extracting solution; performing rotary evaporation and evaporation on the ethanol extract under reduced pressure to obtain an ethanol extract, extracting the ethanol extract by using ethyl acetate, and performing rotary evaporation and evaporation on the extract under reduced pressure to obtain an ethyl acetate extract;
(2) Adding the ethyl acetate extract of the herpetospermum pedunculosum into chromatographic grade methanol for mixing, filtering by using an organic filter membrane of 0.5um after fully mixing by using ultrasonic, transferring to a special sample injection bottle of a chromatograph, and waiting for sample injection;
(3) Opening the liquid chromatograph, and setting an elution program after each component is ready, wherein the elution program is set as follows: 0-10 min,10% -25% methanol; 10-25min, 25-36% methanol; 25-29min, 36-58% methanol; 29-40min, 58-60% methanol; 40-45 min;60 to 100 percent of methanol; 45-60min, 100% methanol. The column temperature is set to be 30 ℃, the flow rate is set to be 0.8mL/min, the fluorescence detection wavelengths are respectively set to be 230nm,254nm and 260nm, a C18 chromatographic column is selected as a detection column, the specification is 4.6mm multiplied by 200mm and 5 mu m, and the A-phase methanol of the comparative example is the conventional chromatographic grade methanol without adding formic acid;
(4) Starting the high performance liquid chromatograph, executing a set program, and obtaining data to obtain a liquid phase spectrogram by using a printing command.
In this comparative example, the chromatographic peaks obtained at the three detection wavelengths were 24, 84, and 77, respectively.
Comparative example 2
(1) Crushing the herpetospermum pedunculosum shells; carrying out heating reflux extraction on the crushed pedunculate herpetospermum seed shells by using 95% ethanol for three times, wherein the first time is 3 hours, the second time is 2 hours, and the third time is 1 hour to obtain an ethanol extracting solution; performing rotary evaporation and evaporation on the ethanol extract under reduced pressure to obtain an ethanol extract, extracting the ethanol extract by using ethyl acetate, and performing rotary evaporation and evaporation on the extract under reduced pressure to obtain an ethyl acetate extract;
(2) Adding the ethyl acetate extract of the herpetospermum pedunculosum into chromatographic grade methanol for mixing, filtering by using an organic filter membrane of 0.5um after fully mixing by using ultrasonic, transferring to a special sample injection bottle of a chromatograph, and waiting for sample injection;
(3) The liquid chromatograph was turned on, and after the components were ready, the elution procedure for this comparative example was set according to the conventional method, with the elution procedure set to: 0-60min and 10-100% of methanol. The column temperature is set to be 30 ℃, the flow rate is set to be 0.8mL/min, the fluorescence detection wavelengths are respectively set to be 230nm,254nm and 260nm, a C18 chromatographic column is selected as a detection column, the specification is 4.6mm multiplied by 200mm and 5 mu m, and 0.1% formic acid is added into the phase A methanol of the comparative example;
(4) Starting the high performance liquid chromatograph, executing a set program, and obtaining data to obtain a liquid phase spectrogram by using a printing command.
In this comparative example, the chromatographic peaks obtained at three detection wavelengths were 36, 85, and 86, respectively.

Claims (1)

1. The liquid chromatography detection method of the herpetospermum pedunculosum seed shell extract is characterized by comprising the following steps:
step 1, crushing the herpetospermum pedunculosum shells; heating and refluxing crushed pedunculate herpetospermum fruit shells with 95% ethanol to obtain an ethanol extract, extracting the pedunculate herpetospermum fruit shell ethanol extract with ethyl acetate, decompressing and steaming the extract liquor to obtain an ethyl acetate extract, wherein the refluxing extraction time is 3 times, the first refluxing extraction time is 3 hours, the second refluxing extraction time is 2 hours, and the third refluxing extraction time is 1 hour;
step 2, adding the ethyl acetate extract of the herpetospermum pedunculosum seed shells into chromatographic grade methanol for mixing, performing ultrasonic treatment to fully dissolve the extract, and then injecting a sample through a microporous filter membrane, wherein the pore diameter of the microporous filter membrane is 0.5 mu m;
step 3, using a high performance liquid chromatograph, using a C18 chromatographic column as an analysis column, using methanol added with 0.1% formic acid as an A phase and water as a B phase, performing gradient elution, and setting an elution program to elute for 60min to obtain a fingerprint of the pedunculate herpetospermum seed shell extract; the elution procedure is as follows: 0 to 10min,10 to 25 percent of methanol; 10 to 25min,25 to 36 percent of methanol; 25 to 29min,36 to 58 percent of methanol; 29 to 40min,58 to 60 percent of methanol; 40-45 min;60% -100% methanol; 45-60 min,100% methanol, the flow rate is 0.8mL/min; the column temperature is 30 ℃; the fluorescence detection wavelength is 230nm,254nm and 260nm.
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