CN117263790A - Longan essence, preparation method thereof and identification method of longan honey - Google Patents
Longan essence, preparation method thereof and identification method of longan honey Download PDFInfo
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- CN117263790A CN117263790A CN202310972864.9A CN202310972864A CN117263790A CN 117263790 A CN117263790 A CN 117263790A CN 202310972864 A CN202310972864 A CN 202310972864A CN 117263790 A CN117263790 A CN 117263790A
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- 235000012907 honey Nutrition 0.000 title claims abstract description 143
- 240000001008 Dimocarpus longan Species 0.000 title claims abstract description 136
- 235000000235 Euphoria longan Nutrition 0.000 title claims abstract description 136
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title abstract description 9
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 claims abstract description 42
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 34
- 235000007586 terpenes Nutrition 0.000 claims description 22
- 230000014759 maintenance of location Effects 0.000 claims description 16
- 150000003505 terpenes Chemical class 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- JLIDBLDQVAYHNE-WEYXYWBQSA-N 2-trans-abscisic acid Chemical compound OC(=O)\C=C(/C)\C=C\C1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-WEYXYWBQSA-N 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- ZCHPKWUIAASXPV-UHFFFAOYSA-N acetic acid;methanol Chemical group OC.CC(O)=O ZCHPKWUIAASXPV-UHFFFAOYSA-N 0.000 claims description 4
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- 238000004519 manufacturing process Methods 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 38
- 238000001514 detection method Methods 0.000 abstract description 9
- 239000012071 phase Substances 0.000 description 51
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- 241000196324 Embryophyta Species 0.000 description 9
- 150000001793 charged compounds Chemical class 0.000 description 9
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- 238000001819 mass spectrum Methods 0.000 description 8
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- -1 terpene compounds Chemical class 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 244000020518 Carthamus tinctorius Species 0.000 description 3
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 3
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- 238000004949 mass spectrometry Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 235000017784 Mespilus germanica Nutrition 0.000 description 2
- 244000182216 Mimusops elengi Species 0.000 description 2
- 235000000560 Mimusops elengi Nutrition 0.000 description 2
- 240000003152 Rhus chinensis Species 0.000 description 2
- 235000014220 Rhus chinensis Nutrition 0.000 description 2
- 241000793823 Tilia amurensis Species 0.000 description 2
- 235000007837 Vangueria infausta Nutrition 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
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- 235000019253 formic acid Nutrition 0.000 description 2
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- 230000007017 scission Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 210000000051 wattle Anatomy 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000345998 Calamus manan Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 235000000604 Chrysanthemum parthenium Nutrition 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 235000000802 Leonurus cardiaca ssp. villosus Nutrition 0.000 description 1
- 244000241838 Lycium barbarum Species 0.000 description 1
- 235000015459 Lycium barbarum Nutrition 0.000 description 1
- 241000219071 Malvaceae Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241000218682 Pseudolarix amabilis Species 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
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- 238000004440 column chromatography Methods 0.000 description 1
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- 238000001914 filtration Methods 0.000 description 1
- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical compound OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
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- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
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- 235000012950 rattan cane Nutrition 0.000 description 1
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- 238000001196 time-of-flight mass spectrum Methods 0.000 description 1
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- 238000005303 weighing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
- C07C49/647—Unsaturated compounds containing a keto groups being part of a ring having unsaturation outside the ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/79—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- 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
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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
- 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
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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
- 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
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
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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
- 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
- G01N2030/3007—Control of physical parameters of the fluid carrier of temperature same temperature for whole column
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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
- 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
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- General Health & Medical Sciences (AREA)
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Abstract
The invention relates to the technical field of honey detection, in particular to a longan extract and a preparation method thereof as well as a longan honey identification method. The longan extract has a structure shown in formula 1:the invention discovers a novel compound longan essence in longan honey through researching longan honey. The invention also discovers that when the honey to be detected contains the anti-trans abscisic acid, the longan extract, the cis-trans abscisic acid, the honey to be detected can be judged to be the longan honey. The invention also provides a new method for distinguishing the longan honey producing areas.
Description
Technical Field
The invention relates to the technical field of honey detection, in particular to longan essence, a preparation method thereof and a longan honey identification method.
Background
The honey source plant of the longan honey is longan, and the general flowering period is 3-6 months per year. The longan honey is amber, fragrant, sweet and not easy to crystallize. Longan is produced in China, has long cultivation history and is deeply favored by people. China and Thailand are the most dominant longan producing countries worldwide.
The longan honey has high market value, has adulteration phenomenon, seriously affects the benefits of consumers and bee farmers, and restricts the development of the longan honey industry.
Therefore, it is necessary and urgent to establish a method for distinguishing longan honey.
Disclosure of Invention
In order to solve the technical problems, the invention provides a longan extract, a preparation method thereof and a method for identifying longan honey. Can be used for judging authenticity of longan honey
In a first aspect, the present invention provides a longan extract having a structure represented by formula 1:
through research on longan honey, the invention discovers that a new compound longan essence is contained in the longan honey, and the compound can be used as a characteristic plant compound marker of the longan honey. In particular, the compound combines trans-abscisic acid and cis-trans-abscisic acid, and can be used for accurately judging the authenticity of longan honey.
In a second aspect, the invention also provides a preparation method of the longan extract, which is obtained by separating terpene components in honey by high performance liquid chromatography, wherein the honey comprises longan honey.
Preferably, in the high performance liquid chromatography separation, the mobile phase A is an aqueous acetic acid solution with a concentration of 0.18-0.22%, and the mobile phase B is a methanol acetic acid solution with a concentration of 0.18-0.22%; and/or, gradient elution is employed; and/or the chromatographic column adopts a Phenomenex Gemini C18 chromatographic column.
Further preferably, the gradient elution procedure is:
0-11 min, the mobile phase B rises from 9% to 14%;
11-14 min, the mobile phase B rises from 14% to 15%;
14-17 min, and the mobile phase B is kept at 15%;
17-24 min, the mobile phase B rises from 15% to 16%;
the mobile phase B rises from 16% to 17% after 24-28 min;
28-30 min, the mobile phase B rises from 17% to 22%;
30-38 min, the mobile phase B rises from 22% to 25%;
the mobile phase B rises from 25% to 30% after 38-41 min;
41-46 min, the mobile phase B rises from 30% to 33%;
46-55 min, and 33% of mobile phase B;
55-60 min, the mobile phase B rises from 33% to 34%;
60-70 min, and the mobile phase B rises from 34% to 35%;
70-80 min, and the mobile phase B rises from 35% to 36%;
80-90 min, the mobile phase B rises from 36% to 45%;
90-100 min, the mobile phase B rises from 45% to 52%;
100-110 min, the mobile phase B rises from 52% to 57%;
110-120 min, the mobile phase B rises from 57% to 65%;
120-130 min, the mobile phase B rises from 65% to 70%;
130-135 min, the mobile phase B rises from 70% to 80%;
the volume sum of the mobile phase A and the mobile phase B is 100%. In the invention, the mobile phase A changes along with the volume change of the mobile phase B, and the sum of the volumes of the mobile phase A and the mobile phase B is 100%. The longan honey separation system of the invention combines the specific elution program and conditions on the basis of adopting the fixity and the mobile phase, so that the separation and enrichment effect is greatly improved.
Preferably, a Phenomenex Gemini C18 column is used; the flow rate is 0.6-0.8 mL/min, the sample injection amount is 20+ -2 mu L, the column temperature is 34-36 ℃, and the detection wavelength is 280+ -10 nm.
Further preferably, mobile phase a is an aqueous acetic acid solution having a concentration of 0.2%, and mobile phase B is a methanol acetic acid solution having a concentration of 0.2%; the flow rate was 0.7mL/min and the column temperature was 35 ℃.
Further preferably, the high performance liquid chromatography further comprises the step of extracting terpene components in the honey by a solid phase extraction column before the high performance liquid chromatography separation; specifically, the Strata-X-A solid phase extraction column is adopted for extraction, methanol is used as an activating agent, water is used as a balancing agent and a eluting agent, a methanol solution of formic acid with the concentration of 9-11% (preferably 10%) is used as an eluting agent, and finally, the obtained eluting solution is dried, re-dissolved by methanol and filtered to obtain the product.
Further preferably, the pore size of the filter membrane at the time of filtration is 0.22. Mu.m.
Further preferably, the eluent is a methanol solution of formic acid with a concentration of 10%.
According to the invention, by adopting the extraction method, the interference of moisture and sugar in the longan honey can be effectively removed, and the effective enrichment of the target component longan extract is realized.
Preferably, the method comprises the steps of preprocessing honey before separating terpene components in the honey by high performance liquid chromatography, and preferably comprises the following steps: mixing honey with water, regulating pH value to 6.5-7, centrifuging at 8800-9200 rpm for 18-22 min, and collecting supernatant. Preferably, the pH is adjusted to 6.8 and centrifuged at 9000rpm for 20min.
The pretreatment of the invention is preferably carried out with the addition of water in an amount sufficient to dissolve the honey and facilitate the subsequent operation.
Preferably, after the terpene component in the honey is separated by high performance liquid chromatography, the target peak eluent is collected, wherein the retention time of the target peak is 67.45min.
In a third aspect, the invention also provides the application of the longan extract or the longan extract prepared by the preparation method of the longan extract in longan honey identification.
In a fourth aspect, the invention also provides a method for identifying longan honey, which comprises the steps of detecting terpene components in the honey to be detected through a high performance liquid chromatography, and judging that the honey to be detected is the longan honey when the terpene components contain trans-abscisic acid, longan extract and cis-and trans-abscisic acid; the longan extract is described above.
In the identification method, the method for detecting the honey to be detected by the high performance liquid chromatography is as described above.
The high performance liquid chromatography can effectively separate the trans-abscisic acid, the longan extract, the cis-abscisic acid and the trans-abscisic acid in the honey to be detected, thereby realizing the accurate judgment of authenticity.
Preferably, when the content of the trans-abscisic acid in the honey to be detected is greater than 4.08mg/kg, the content of the longan extract is greater than 3.39mg/kg, and the content of the cis-trans-abscisic acid is greater than 9.67mg/kg, the honey to be detected is judged to be the longan honey.
Further preferably, when the ratio of the content of the longan element to the content of the cis-trans abscisic acid in the honey to be detected is greater than 1.1, the honey to be detected is determined to be Thailand longan honey.
In a fifth aspect, the present invention provides a finger print of longan honey, which is obtained by processing and constructing longan honey by high performance liquid chromatography, wherein in the high performance liquid chromatography finger print of longan honey, the retention time of a characteristic peak of longan extract is 67.45min, the retention time of a characteristic peak of trans-abscisic acid is 60.32min, and the retention time of a characteristic peak of cis-abscisic acid is 68.84min. The high performance liquid chromatography is described above. The method specifically comprises the steps of pretreatment of the honey, extraction of terpene components in the honey by a solid phase extraction column and high performance liquid chromatography detection.
The invention has the advantages that: the invention discovers a novel compound namely longan extract which can be used as a characteristic plant compound marker of longan honey. When the honey to be detected contains the trans-abscisic acid, the longan extract and the cis-trans-abscisic acid, the honey to be detected can be judged to be the longan honey. The invention establishes a solid phase extraction and high performance liquid chromatography separation detection method of terpene compounds in longan honey, and identifies 4 representative plant compounds contained in the longan honey by an HPLC-QTOF method, wherein the representative plant compounds are respectively trans, trans abscisic acid, longan extract, cis, trans abscisic acid and 3-carbonyl-ionone with lower content. And introducing the obtained longan honey terpene compound HPLC chromatogram into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and performing preliminary simulation to generate a longan honey standard fingerprint with representative significance. In addition, the invention can not detect the trans, trans abscisic acid, longan essence, cis-trans abscisic acid in other single nectar except longan nectar by comparing with the HPLC (high performance liquid chromatography) maps of the three single nectar types including the wattle nectar, the locust nectar and the tilia amurensis nectar and analyzing and comparing the plant compounds of other collected mass honey such as the safflower nectar, the gallnut nectar, the medlar nectar, the leonurus nectar, the nine-Dragon nectar and the like. The three components can be used as characteristic plant compound markers of the longan honey, and are applied to the authenticity and quality evaluation of the longan honey, and have important practical significance for protecting the legal rights and interests of honey consumers and maintaining the healthy development of the honey consumption industry.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the prior art, reference will be made to the accompanying drawings which are required to be used in the description of the embodiments or the prior art, wherein like elements or parts are generally identified by like reference numerals throughout. In the drawings, elements or portions thereof are not necessarily drawn to scale. It is evident that the drawings in the following description are only some embodiments of the present invention and that other drawings may be obtained from these drawings by those of ordinary skill in the art without inventive effort.
FIG. 1 is a liquid-mass spectrum of longan extract in example 1 of the present invention;
FIG. 2 is a liquid-mass spectrum of longan extract in example 1 of the present invention;
FIG. 3 is a chart showing nuclear magnetic resonance spectrum of longan extract in example 1 of the present invention;
FIG. 4 is a chromatogram of terpene components in longan honey in example 1 of the present invention;
FIG. 5 is an HPLC superposition spectrum of twenty different earthworm eye raw material honeys produced in example 2 of the present invention; wherein R represents a standard fingerprint generated based on twenty different types of earthworm eye raw material honey, and S1-S20 represent twenty different types of earthworm eye raw material honey.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The equipment used is not pointed out by manufacturers, and is a conventional product which can be purchased by a regular channel manufacturer. The methods are conventional methods unless otherwise specified, and the starting materials are commercially available from the public sources unless otherwise specified.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1
The embodiment provides a longan extract and a preparation method thereof and establishment of a finger print of characteristic components of longan honey, wherein the method comprises the following steps:
1. preparation of longan honey solution: accurately weighing 20.0g of longan honey in a beaker, adding 80mL of deionized water, stirring with a glass rod until the honey is fully dissolved, adding 5% ammonia water solution, adjusting the pH value to about 6.8, centrifuging for 20min at 9000rmp, and taking supernatant for later use.
2. Extracting and enriching longan honey terpene compounds: extracting terpene components in honey by using a Strata-X-A solid phase extraction column, firstly activating the extraction column by using 3mL of methanol and balancing the extraction column by using 3mL of deionized water, then adding the supernatant, leaching by using deionized water, finally eluting by using 10% formic acid methanol solution, and collecting eluent. The eluent was dried with nitrogen, redissolved in 2.0mL of methanol and filtered through a 0.22 μm filter membrane for further use.
3. Separation and detection of longan honey terpene compounds: separating and detecting terpene components in the honey by using high performance liquid chromatography, wherein the chromatographic column is Phenomenex Gemini C18 chromatographic column, the mobile phase A is 0.2% acetic acid aqueous solution, the mobile phase B is 0.2% acetic acid methanol solution, and the gradient elution procedure is as follows: 0-11 min, the mobile phase B rises from 9% to 14%; 11-14 min, the mobile phase B rises from 14% to 15%; 14-17 min, and the mobile phase is kept at 15%; 17-24 min, the mobile phase B rises from 15% to 16%; the mobile phase B rises from 16% to 17% after 24-28 min; 28-30 min, the mobile phase B rises from 17% to 22%; 30-38 min, the mobile phase B rises from 22% to 25%; the mobile phase B rises from 25% to 30% after 38-41 min; 41-46 min, the mobile phase B rises from 30% to 33%; 46-55 min, and 33% of mobile phase B; 55-60 min, the mobile phase B rises from 33% to 34%; 60-70 min, and the mobile phase B rises from 34% to 35%; 70-80 min, and the mobile phase B rises from 35% to 36%; 80-90 min, the mobile phase B rises from 36% to 45%; 90-100 min, the mobile phase B rises from 45% to 52%; 100-110 min, the mobile phase B rises from 52% to 57%; 110-120 min, the mobile phase B rises from 57% to 65%; 120-130 min, the mobile phase B rises from 65% to 70%; 130-135 min, the mobile phase B rises from 70% to 80%. The sample injection amount is 20 mu L, the column temperature is 35 ℃, and the detection wavelength is 280nm. Detection, component separation were performed according to the above conditions, and a chromatogram was recorded, see fig. 4. The eluate at each peak time was collected and identified.
The method for detecting the tandem diode array detector (HPLC-PDA) by the liquid chromatography and detecting the tandem quadrupole/time-of-flight mass spectrum (HPLC-QTOF) by the liquid chromatography is specifically adopted to carry out category identification on terpene compounds obtained by effective separation and enrichment.
The specific identification method is described in detail as follows:
1. carrying out structural identification on terpene components in the longan honey after detection under the liquid chromatography conditions:
liquid phase mass spectrometry conditions: the ion source is ESI source (electrospray), the ion source spray voltage is 4kV, the outlet voltage is 130V, the heating temperature is 350 ℃, and nitrogen (N) 2 ) The flow rate is 11L/min, the collision gas is helium, the flow rate of the atomization gas is 80kPa, the pressure of the atomizer is 40psi, and the mass scanning range m/z=100-900 Da.
2. Structural identification analysis process and result of terpene compounds in longan honey:
(1) The structure of the compound in the eluent with retention time of 60.32min was resolved as follows:
the molecular weight of the compound is determined to be 264 by a molecular ion peak of 265.1214M/z [ M+H ] + and a molecular ion peak of 287.1238M/z [ M+Na ] + under the first-order mass spectrum. And judging the compound as the trans-abscisic acid by combining the retention time and the maximum ultraviolet absorption wavelength of the high performance liquid chromatography of the compound with the retention time and the maximum ultraviolet absorption wavelength of the standard high performance liquid chromatography and the fracture rule of the secondary mass spectrum.
(2) The structure of the compound in the eluent with retention time of 67.45min was resolved as follows:
a primary mass spectrum and a secondary mass spectrum in a positive ion mode. The molecular weight of the compound was found to be 204 by having a molecular ion peak of 205.1206[ M+H ] + and a molecular ion peak of 227.1032M/z [ M+Na ] +. Collecting the compound fraction by high performance liquid chromatography, and performing nuclear magnetic resonance analysis to obtain nuclear magnetic resonance spectrum of the compound, as shown in figure 3, wherein the upper graph is hydrogen spectrum, and the lower graph is carbon spectrum; the chemical shifts are shown in table 1.
Chemical shifts of the compounds of Table 1
| δH | δC | |
| 1 | - | 196.93 |
| 1’ | - | 213.92 |
| 2 | 6 | 126.40 |
| 2’ | 6.33 | 101.93 |
| 3 | - | 148.44 |
| 3’ | - | 196.82 |
| 4 | - | 116.11 |
| 4’ | 2.00 | 20.81 |
| 5 | - | 36.49 |
| 6 | 2.43 | 50.09 |
| 7 | 1.21 | 28.19 |
| 8 | 1.18 | 27.34 |
| 9 | 2.23 | 27.97 |
The chemical formula of the compound is judged to be 4- (polydiene-3' -methyl ketone) -3, 5-trimethyl-2-cyclohexenone, and the compound is named as longan essence. The structure of the longan extract is shown as the following formula:
the cleavage pathway under mass spectrometry of the compound is as follows:
(3) The structure of the compound in the eluent with retention time of 68.84min was resolved as follows:
a first-order mass spectrum in positive ion mode, having 265.1256M/z [ M+H ]] + Molecular ion peak of 287.1056M/z [ M+Na ]] + Molecular ion peak of 529.2435m/z [2M+H ]] + Molecular ion peak of 551.2239m/z [2M+Na ]] + The molecular weight of the compound was determined to be 264. The maximum ultraviolet absorption of the compound is 264, and the maximum ultraviolet absorption is identical with the retention time of the standard product of cis-abscisic acid and trans-abscisic acid through the verification of the standard product. Thus, the compound was judged to be cis, trans abscisic acid.
(4) The structure analysis process of the compound in the eluent with the retention time of 76.15min is as follows:
first-order mass spectrum (207.1363M/z [ M+H)] + Molecular ion peak of 229.1181M/z [ M+Na ]] + 435.2482m/z [2M+Na ]] + Molecular ion peaks of (2). The molecular weight of this compound was 206. And judging the compound to be 3-carbonyl-ionone according to the maximum ultraviolet absorption wavelength, the retention time and comparison between the standard substance and the standard substance after verification. The structure is shown as the following formula:
the cleavage pathway under mass spectrometry of the compound is as follows:
example 2
In this example, the high performance liquid chromatography of example 1 was used to detect longan honey in ten fields, and the detected longan honey components were quantitatively analyzed by an external standard method.
Specific honey source information is as follows in table 2:
table 2 longan raw honey sample
The obtained chromatogram overlapping map of the longan honey of twenty different producing areas is shown in figure 5.
In twenty longan honey samples, the average content of trans-abscisic acid is 14.29mg/kg; the average content of longan extract is 11.13mg/kg; the average content of cis, trans abscisic acid is 34.41mg/kg; the average content of 3-carbonyl-ionone was 7.39mg/kg. The results are shown in Table 3:
TABLE 3 Compound content in longan Honey (mg/kg)
Note that: "-" is undetected.
Example 3
In this example, the high performance liquid chromatography of example 1 was used to perform component analysis (specific raw material honey source information is shown in table 4) on the golden larch honey, locust honey, tilia amurensis honey, safflower honey, chinese gall honey, matrimony vine honey, leonurus honey and nine-Dragon vine honey by using the high performance liquid chromatography, and the HPLC profile analysis of the above honey and the longan honey of example 1 was compared. The results show that the compounds trans, trans-abscisic acid, longan extract and cis, trans-abscisic acid are not detected in other single nectar except the longan nectar. The specific detection results are shown in Table 5:
TABLE 4 information on other sources of raw honey
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TABLE 5 information about whether other honey contains a marker compound
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Example 4
The high performance liquid chromatography of example 1 was used in this example to detect and analyze 19 commercial longan nectar products (different brands from different sales platforms) including 6 taiwan longan nectar products, 6 continental longan nectar products, and 7 thailand longan nectar products using a high performance liquid chromatograph. By HPLC chromatogram analysis comparison, the nineteen brands of longan honey all detect trans, trans-abscisic acid, longan extract, cis, trans-abscisic acid and 3-carbonyl-ionone, and the specific average contents are 7.40mg/kg, 5.97mg/kg, 16.61mg/kg and 3.16mg/kg respectively. The ratio of the longan element to the cis-trans-abscisic acid in the sample of the Thailand longan honey is obviously higher than that of the Taiwan longan honey and the Daucian longan honey, so that the Thailand longan honey, the Daucian longan honey and the Taiwan longan honey can be distinguished through the ratio of the longan element to the cis-trans-abscisic acid, and the Thailand longan honey can be judged when the ratio of the longan element to the cis-trans-abscisic acid in the sample of the longan honey to be detected is higher than 1.1.
TABLE 6 Compound content in longan commercial Honey (mg/kg)
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In conclusion, according to the invention, the components of the longan honey and the plant compounds of the wattle honey, the locust honey, the basswood honey, the safflower honey, the Chinese gall honey, the medlar honey, the motherwort honey and the nine-dragon rattan honey are analyzed and compared, and the compounds of the trans, trans-abscisic acid, the longan essence, cis-trans abscisic acid are not detected in other single flower honey except the longan honey. Therefore, the trans-abscisic acid, the longan extract and the cis-trans-abscisic acid can be used as characteristic plant compound markers of the longan honey and can be applied to authenticity and identification of the longan honey.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The longan extract is characterized by having a structure shown in a formula 1:
2. the method for preparing longan extract according to claim 1, wherein the terpene component in the honey is separated by high performance liquid chromatography, and the honey comprises longan honey.
3. The method for preparing longan pulp according to claim 2, wherein in the high performance liquid chromatography separation, mobile phase a is an aqueous acetic acid solution with a concentration of 0.18-0.22%, and mobile phase B is a methanol acetic acid solution with a concentration of 0.18-0.22%; and/or, gradient elution is employed; and/or the chromatographic column adopts a Phenomenex Gemini C18 chromatographic column; and/or, the gradient elution procedure is:
0-11 min, the mobile phase B rises from 9% to 14%;
11-14 min, the mobile phase B rises from 14% to 15%;
14-17 min, and the mobile phase B is kept at 15%;
17-24 min, the mobile phase B rises from 15% to 16%;
the mobile phase B rises from 16% to 17% after 24-28 min;
28-30 min, the mobile phase B rises from 17% to 22%;
30-38 min, the mobile phase B rises from 22% to 25%;
the mobile phase B rises from 25% to 30% after 38-41 min;
41-46 min, the mobile phase B rises from 30% to 33%;
46-55 min, and 33% of mobile phase B;
55-60 min, the mobile phase B rises from 33% to 34%;
60-70 min, and the mobile phase B rises from 34% to 35%;
70-80 min, and the mobile phase B rises from 35% to 36%;
80-90 min, the mobile phase B rises from 36% to 45%;
90-100 min, the mobile phase B rises from 45% to 52%;
100-110 min, the mobile phase B rises from 52% to 57%;
110-120 min, the mobile phase B rises from 57% to 65%;
120-130 min, the mobile phase B rises from 65% to 70%;
130-135 min, the mobile phase B rises from 70% to 80%;
the volume sum of the mobile phase A and the mobile phase B is 100%.
4. A method of preparing longan extract according to claim 2 or 3, characterized in that the separation of terpene components in honey by high performance liquid chromatography comprises a step of pretreatment of the honey, preferably comprising the steps of: mixing honey with water, regulating pH value to 6.5-7, centrifuging at 8800-9200 rpm for 18-22 min, and collecting supernatant.
5. The method for producing longan pulp according to any one of claims 2-4, characterized in that after separating terpene components in honey by high performance liquid chromatography, collecting target peak eluent, wherein the retention time of the target peak is 67.45min.
6. Use of the longan extract of claim 1 or the longan extract prepared by the method of any of claims 2-5 in the identification of longan honey.
7. The identification method of the longan honey is characterized by detecting terpene components in the honey to be detected through a high performance liquid chromatography, and judging that the honey to be detected is the longan honey when the terpene components contain trans-abscisic acid, longan extract and cis-abscisic acid; the longan extract is as claimed in claim 1.
8. The method according to claim 7, wherein the honey to be tested is determined to be longan honey when the content of trans-abscisic acid is greater than 4.08mg/kg, the content of longan extract is greater than 3.39mg/kg, and the content of cis-trans-abscisic acid is greater than 9.67 mg/kg.
9. The method for identifying longan honey according to claim 7 or 8, wherein when the ratio of the content of the longan extract to the content of the cis-trans abscisic acid in the honey to be detected is greater than 1.1, the honey to be detected is determined to be thailand longan honey.
10. The finger print of the longan honey is characterized in that the finger print is obtained by processing and constructing the longan honey through a high performance liquid chromatography, wherein the retention time of a characteristic peak of longan extract is 67.45min, the retention time of a characteristic peak of trans-abscisic acid is 60.32min, and the retention time of a characteristic peak of cis-abscisic acid is 68.84min.
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