CN103852552B

CN103852552B - Method for simultaneously detecting seven flavones substances in hops

Info

Publication number: CN103852552B
Application number: CN201410089061.XA
Authority: CN
Inventors: 刘春凤; 李佳; 李崎; 王金晶; 李永仙; 郑飞云
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2014-03-12
Filing date: 2014-03-12
Publication date: 2015-05-13
Anticipated expiration: 2034-03-12
Also published as: CN103852552A

Abstract

The invention discloses a method for simultaneously detecting seven flavones substances in hops and belongs to the field of chemical analysis and detection. Through the method, seven substances comprising xanthohumol, isoxanthohumol, 6-isoprene flavonoids, 8-isoprene flavonoids, hexamethoxy flavone, kaempferol3-O-rutinoside and quercetin are simultaneously detected by using a reversed-phase high-performance liquid chromatography. The method is especially suitable for detecting change relationships of contents of flavone substances in the hops, detection change of the flavone substances in production process of beer and inspecting antioxidant functions and nutritive values of the beer. The method is excellent in stability and reproducibility and simple, accurate and reliable to operate.

Description

A kind of method of seven kinds of Flavonoid substances in Simultaneously test hops

Technical field

The present invention relates to a kind of method simultaneously detecting seven kinds of Flavonoid substances in hops, especially a kind of method adopting reversed-phase high-performance liquid chromatography simultaneously to detect seven kinds of Flavonoid substances such as xanthohumol, Isoxanthohumol in hops, belongs to chemical analysis detection field.

Background technology

The same brewing water of hops, Fructus Hordei Germinatus, yeast be called four of brewing large raw materials.By contrast, although the addition of hops in beer is little, it has very large impact for the quality that beer is final, is mainly reflected in non-biostability, raising nutritive value three aspects of improving beer flavor, improving beer.

Correlative study shows, and compared with adding the beer of hops, does not add the beer of hops with obvious unhappy Fructus Hordei Germinatus local flavor, and simultaneously sweet taste, ethanol taste are too dense, and associative perception working style of government organs taste is second-rate.More it is worth noting, hops are important sources of a lot of natural products, and flavones is wherein important one.Flavones is under the jurisdiction of plant polyphenol material, has important inoxidizability and metal-chelating character, and pharmacology and biological characteristics significantly, have very large Research Significance and value to the non-biostability and nutritive value aspect improving beer.Except hops, fruit, vegetables, tealeaves, cocoa power etc. are also the important sources of Flavonoid substances, and so far, existing 4000 kinds of Flavonoid substances are found and identify, but research shows, hops are the exclusive source of the natural isoprene containing base flavones found at present.In the diet of the mankind, absorb this flavonoid substances mainly through drinks beer.

For studying the Flavonoid substances in hops further, first detection method of content accurately to be set up.Critical function composition in hops, such as picric acid, single phenol etc. have established comparatively ripe liquid phase detection method, and for detecting while multiple Flavonoid substances, do not have efficient and accurate method.Therefore for ease of monitoring the sweat of beer, being investigated anti-oxidation efficacy and the nutritive value of beer by the content of this flavonoid substances, being badly in need of setting up efficient accurate liquid phase detection method.

Summary of the invention

The object of this invention is to provide a kind of method simultaneously detecting seven kinds of Flavonoid substances in hops, especially a kind of method adopting reversed-phase high-performance liquid chromatography simultaneously to detect seven kinds of Flavonoid substances such as xanthohumol, Isoxanthohumol in hops.

Described seven kinds of Flavonoid substances are xanthohumol, Isoxanthohumol, 6-prenyl flavones, 8-prenyl flavones, hexa methoxy flavones, Kaempferol 3-O-rutinoside and Quercetin.

Described method comprises the steps:

A. hops sample preparation: hop pellets sample is after pulverising mill is powdered, accurate weighing 1g hops powder add 7mL absolute ether shake 15min after ultrasound wave 5min, the centrifugal 10min of 1500 × g, remove supernatant, add 20mL50%(v/v again) methanol solution shake 15min after ultrasonic 60min, the centrifugal 10min of 1500 × g, supernatant, through 0.45 μm of filtering with microporous membrane, is stored in sample bottle and prepares sample introduction.

B. standard specimen preparation: accurately take flavonoid standards 0.0010g hplc grade methanol and fully dissolve, constant volume, to 10mL, is diluted to variable concentrations as required, fully mixes, and with 0.45 μm of organic membrane filtration, freezen protective is for subsequent use, prepares sample introduction.

C. chromatographic condition is as follows:

Chromatographic column: ZorbaxEclipseXDB-C18(4.6 × 250mm, 5m);

Mobile phase: organic B phase is acetonitrile; Inorganic A phase is containing formic acid 0.1%(v/v) aqueous solution;

Sampling volume 10 μ L, flow velocity 0.8mL/min, column temperature 30 DEG C;

Determined wavelength: 280nm and 370nm;

Adopt gradient elution method, elution program is: 0-20min, 25%B-80%B; 20-25min, 80%B-100%B; 25-28min, 100%B-100%B; 28-30min, 100%B-25%B.

Detection method provided by the invention can by the xanthohumol in hops, Isoxanthohumol, 6-prenyl flavones, 8-prenyl flavones, hexa methoxy flavones, Kaempferol 3-O-rutinoside and Quercetin this 7 kinds of materials are disposable separates, simple to operate, accurately and reliably, save time.In addition, because Flavonoid substances is of a great variety, in hops, content is lower, this method compares other flavones detection methods, can obtain good separating effect to seven kinds of compounds.

Accompanying drawing explanation

Fig. 1 is flavones standard model chromatogram; 1, Kaempferol 3-O rutinoside; 2, Quercetin; 3, Isoxanthohumol; 4, hexa methoxy flavones; 5,8-prenyl flavones; 6,6-prenyl flavones; 7, xanthohumol.

Fig. 2 is hop pellets sample chromatogram figure; 1, Kaempferol 3-O rutinoside; 2, Quercetin; 3, Isoxanthohumol; 4, hexa methoxy flavones; 5,8-prenyl flavones; 6,6-prenyl flavones; 7, xanthohumol.

Embodiment

The standard specimen of embodiment 17 kinds of Flavonoid substances detects

Accurately take flavonoid standards 0.0010g hplc grade methanol fully to dissolve, constant volume, to 10mL, is diluted to variable concentrations as required, prepares sample introduction and is stored in sample bottle, sample introduction analysis.

Reversed-phase liquid chromatography analysis condition:

Chromatographic column: Zorbax Eclipse XDB-C18(4.6 × 250mm, 5m);

Sampling volume 10 μ L, flow velocity 0.8mL/min, column temperature 30 DEG C;

Determined wavelength: 280nm and 370nm;

Mobile phase: organic B phase is acetonitrile, inorganic A phase is for containing 0.1%(v/v) first aqueous acid; Adopt gradient elution method, elution program is: 0-20min, 25%B-80%B; 20-25min, 80%B-100%B; 25-28min, 100%B-100%B; 28-30min, 100%B-25%B.

As shown in Figure 1, peak 1 is Kaempferol 3-O rutinoside to gained standard specimen chromatogram, and peak 2 is Quercetin, and peak 3 is Isoxanthohumol, and peak 4 is hexa methoxy flavones, and peak 5 is 8-prenyl flavones, and peak 6 is 6-prenyl flavones, and peak 7 is xanthohumol.

Typical curve and the detection line scope of gained 7 kinds of standard models are as shown in table 1.

This detection method of table 1. detects linear equation and the sensing range of seven kinds of Flavonoid substances

The detection of Flavonoid substances in embodiment 2 hops sample

Hop pellets is after pulverising mill is powdered, accurate weighing 1g hops powder add 7mL absolute ether shake 15min after ultrasound wave 5min, the centrifugal 10min of 1500 × g, remove supernatant, add again 20mL50% methanol solution shake 15min after the centrifugal 10min of ultrasonic 60min, 1500 × g, supernatant is through 0.45 μm of filtering with microporous membrane, be stored in sample bottle, sample introduction analysis.

Reversed-phase liquid chromatography analysis condition:

Chromatographic column: Zorbax Eclipse XDB-C18(4.6 × 250mm, 5m);

Sampling volume 10 μ L, flow velocity 0.8mL/min, column temperature 30 DEG C;

Determined wavelength: 280nm and 370nm;

Mobile phase: organic B phase is acetonitrile; Inorganic A phase is for containing 0.1%(v/v) first aqueous acid.Adopt gradient elution method, elution program is: 0-20min, 25%B-80%B; 20-25min, 80%B-100%B; 25-28min, 100%B-100%B; 28-30min, 100%B-25%B.

Gained sample chromatogram figure is as Fig. 2.

The stability of embodiment 3 detection method and reappearance assessment

Get the hops sample of known object content, add the standard model of the variable concentrations handled well according to the method for embodiment 1 respectively, it is as follows specifically to add standard specimen concentration: xanthohumol (80mg/L, 60mg/L, 40mg/L, 20mg/L, 10mg/L, 5mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L), Kaempferol 3-O rutinoside (80mg/L, 60mg/L, 40mg/L, 20mg/L, 10mg/L, 5mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L), Isoxanthohumol (10mg/L, 8mg/L, 6mg/L, 4mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L), 6-prenyl flavones (5mg/L, 4mg/L, 3mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L, 0.125mg/L), 8-prenyl flavones (5mg/L, 4mg/L, 3mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L, 0.125mg/L), hexa methoxy flavones (5mg/L, 4mg/L, 3mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L, 0.125mg/L), Quercetin (5mg/L, 4mg/L, 3mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L, 0.125mg/L).By the mixed liquor of variable concentrations be prepared into, respectively measure 6 times according to the method for embodiment 2 and condition, RSD value all about 5%, show the stability of the method and reappearance fine.The recovery gets its mean value, the recovery of xanthohumol, Isoxanthohumol, Kaempferol 3-O rutinoside, Quercetin, 6-prenyl flavones in the recovery of 90%-100%, 8-prenyl flavones, hexa methoxy flavones between 80%-90%.The method can seven kinds of Flavonoid substances in more reliable Simultaneously test hops sample as can be seen here.

Claims

1. the method for seven kinds of Flavonoid substances in Simultaneously test hops, it is characterized in that, described seven kinds of Flavonoid substances are xanthohumol, Isoxanthohumol, 6-prenyl flavones, 8-prenyl flavones, hexa methoxy flavones, Kaempferol 3-O-rutinoside and Quercetin; Said method comprising the steps of:

1) sample preparation: hop pellets sample is after pulverising mill is powdered, after weighing 1g hops powder adds 7mL absolute ether shake 15min, ultrasonic process 5min, the centrifugal 10min of 1500 × g, removes supernatant, then after adding 20mL50% methanol solution shake 15min, ultrasonic process 60min, the centrifugal 10min of 1500 × g, supernatant, through 0.45 μm of filtering with microporous membrane, is stored in sample bottle and prepares sample introduction;

2) sample rp-hplc analysis step 1) prepared, analysis condition is as follows:

Chromatographic column: ZorbaxEclipseXDB-C18;

Sampling volume 10 μ L, flow velocity 0.8mL/min, column temperature 30 DEG C;

Determined wavelength: 280nm and 370nm;

Mobile phase: organic B phase is acetonitrile; Inorganic A phase is contain the first aqueous acid that volume fraction is 0.1%;

Adopt gradient elution, elution program is: 0-20min, 25%B-80%B; 20-25min, 80%B-100%B; 25-28min, 100%B-100%B; 28-30min, 100%B-25%B.