CN104280497A - Method for detecting quality of two flavones in fructus aurantii - Google Patents
Method for detecting quality of two flavones in fructus aurantii Download PDFInfo
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Abstract
The invention provides a method for detecting the quality of 5,6,7,3',4'-pentamethoxyl flavones and 5,7,8, 3',4'-pentamethoxyl flavones in traditional Chinese medicine fructus aurantii and belongs to the field of biological medicine. The quality detecting method is a high performance liquid chromatography; chromatographic conditions and detection conditions are as follows: a mobile phase is formed by methyl alcohol and water according to a volume ratio of (45-60) to (40-55); the flow rate is 0.5-1.5mL/min; the column temperature is 25.0 DEG C; the sample feeding amount is 10-30 micro-liters; the detection wave length is 200-400nm. The main contribution of the method is characterized by establishing a method for detecting the HPLC content of sinensetin, namely 5,6,7,3',4'-pentamethoxyl flavones (E) and 5,7,8, 3',4'-pentamethoxyl flavones (I) in the fructus aurantii medicinal materials; a novel scientific basis is provided for completing the quality standard of the fructus aurantii medicinal materials and development and application of the fructus aurantii.
Description
Technical field
To the present invention relates in Fructus Aurantii 5,6,7,3 ', 4 '-pentamethoxyl flavones and 5,7,8, the quality determining method of 3 ', 4 '-pentamethoxyl flavones, belongs to biomedicine field.
Background technology
The dry immature fruit that Fructus Aurantii (Aurantii Fructus) is Rutaceae citrus plant bitter orange (Citrus aurantium L.) and variety thereof.Fructus Aurantii gas delicate fragrance, bitter, micro-acid, be used for the treatment of accumulation of food in the stomach and intes tine due to indigestion, the symptoms such as visceral organ ptosis, has the effect such as regulating the flow of QI to ease the stomach, the stagnant dissipate-swelling of row, be a kind of broad-spectrum Chinese crude drug, recorded by many editions Chinese Pharmacopoeias.
Chemical composition in Fructus Aurantii is primarily of flavones, alkaloid, cumarin and volatilization wet goods compounds composition.
Although Chinese scholars have studied chemical composition and the pharmacological action thereof of Fructus Aurantii widely, still there is the chemical composition be still not clear, the quality standard of Fructus Aurantii medicinal material need perfect, and the medical usage of Fructus Aurantii need further exploitation.
Summary of the invention
The technical problem to be solved in the present invention overcomes existing defect, and the basis furtherd investigate Fructus Aurantii chemical composition provides a kind of RP-HPLC method that can detect chemical composition content in Fructus Aurantii fast.
In order to solve the problems of the technologies described above, the invention provides following technical scheme:
The quality determining method of two kinds of flavones in Fructus Aurantii, described quality determining method is high performance liquid chromatography, chromatographic condition and testing conditions as follows:
Mobile phase: methanol-water, volume ratio is (45-60): (40-55)
Flow velocity: 0.5-1.5mLmin
-1
Column temperature: 25.0 DEG C
Sample size: 10-30 μ L
Determined wavelength: 200-400nm;
The preparation of need testing solution: it is appropriate that precision takes Fructus Aurantii medicinal powder (50-100 mesh sieve), using methyl alcohol as Extraction solvent, solvent load is 10 times (g/ml) of quality of medicinal material, refluxing extraction 2-4 time, and extraction time is 0.5-1.5h, extract is merged, filter, be concentrated into dry, dissolve with chromatogram methyl alcohol and be settled to designated volume, cross miillpore filter, get subsequent filtrate as need testing solution;
The preparation of reference substance solution: precision takes reference substance 5,6,7,3 ', 4 '-pentamethoxyl flavones and 5,7,8,3 ', 4 '-pentamethoxyl flavones is appropriate, is placed in volumetric flask, is mixed with concentration is 0.10-0.30mgmL by chromatogram methanol constant volume
-1mixing reference substance solution.
Further, above-mentioned quality determining method, chromatographic condition and testing conditions as follows:
Chromatograph: Agilent1200HPLC, diode array detector
Chromatographic column: Acchrom Unitary C18Column (4.6 × 250mm, 5 μm)
Mobile phase: methanol-water, volume ratio is 52:48
Flow velocity: 1.0mLmin
-1
Column temperature: 25.0 DEG C
Sample size: 20 μ L
Determined wavelength: 310nm.
Further, in above-mentioned quality determining method,
The preparation of need testing solution: it is appropriate that precision takes Fructus Aurantii medicinal powder (80 mesh sieve), using methyl alcohol as Extraction solvent, solvent load is 10 times (g/ml) of quality of medicinal material, refluxing extraction 3 times, and extraction time is respectively 1.5h, 1.0h, 0.5h, extract is merged, filter, be concentrated into dry, dissolve with chromatogram methyl alcohol and be settled to designated volume, cross 0.22 μm of miillpore filter, get subsequent filtrate as need testing solution.
Further, in above-mentioned quality determining method,
The preparation of reference substance solution: precision takes reference substance 5,6,7,3 ', 4 '-pentamethoxyl flavones 2.64mg and 5,7,8,3 ', 4 '-pentamethoxyl flavones 2.54mg, is placed in the volumetric flask of same 10mL, is mixed with concentration is respectively 0.26mgmL by chromatogram methanol constant volume
-1and 0.25mgmL
-1mixing reference substance solution.
Reference substance 5,6,7 used by the present invention, 3 ', 4 '-pentamethoxyl flavones and 5,7,8,3 ', 4 '-pentamethoxyl flavones is self-control, wherein, and 5,6,7,3 ', the purity of 4 '-pentamethoxyl flavones is 98.7%, 5,7,8, and the purity of 3 ', 4 '-pentamethoxyl flavones is 97.9%.5,6,7,3 ', 4 '-pentamethoxyl flavones and 5,7,8, the preparation method of 3 ', 4 '-pentamethoxyl flavones is as follows:
(1) extract
Get dry Fructus Aurantii medicinal material, pulverize, adopt the mode adding hot reflux to extract, Extraction solvent is ethanol, extracts 2-4 time, then merges extract, and filter, reduced pressure concentration, obtains crude extract;
(2) be separated
By crude extract, carry out silica gel column chromatography separation with mobile phase 1 as eluent, TCL inspection stream part, collect and merge component phase homogeneous turbulence part, recycling design, obtains 2 parts, wherein Part II is containing relatively large 5,6,7,3 ', 4 '-pentamethoxyl flavones and 5,7,8,3 ', 4 '-pentamethoxyl flavones;
(3) purifying
By step (2) gained 2 part respectively purifying repeatedly, obtain 10 compounds, be A be respectively limonin, B is cupreol, C is Nobiletin that is 5,6,7,8,3 ', 4 '-hexa methoxy flavones, D are Nomilin, E is senensetin that is 5,6,7,3 ', 4 '-pentamethoxyl flavones, F are naringenin that is 5,7,4 '-trihydroxy flavanone, G are 5,7,8,4 '-tetramethoxy flavones, H are 3,5,7,8,3 ', 4 '-hexa methoxy flavones, I are 5,7,8,3 ', 4 '-pentamethoxyl flavones, J are daucosterol.
In step (1), the volume fraction as the ethanol of Extraction solvent is 60%-90%, and the Extraction solvent volumetric usage extracting three times is the 20-30 of quality of medicinal material doubly (g/ml), and extraction time is 0.5-2h; Wherein, preferably, the volume fraction as the ethanol of Extraction solvent is 80%, and the Extraction solvent volumetric usage extracting three times is respectively 10 times, 8 times, 8 times (g/ml) of quality of medicinal material, and extraction time is respectively 1.5h, 1.0h, 1.0h.
Purification process in step (3) is selected from one or more in following method: carry out silica gel column chromatography separation with mobile phase 1, ODS column chromatography for separation is carried out as eluent with mobile phase 2, ODS column chromatography for separation is carried out with mobile phase 3, ODS column chromatography for separation is carried out with mobile phase 4, ODS column chromatography for separation is carried out as eluent with mobile phase 5, silica gel column chromatography separation is carried out as eluent with mobile phase 6, be separated by preparative chromatograph as eluent with mobile phase 8, undertaken being separated or using recrystallizing methanol by preparative high performance liquid chromatography instrument for eluent with mobile phase 9.
The methanol-water that the methanol-water that in above-mentioned steps (2) and (3), the boiling of the water-methanol of the boiling of the water-methanol of the ethyl acetate-light petrol of mobile phase 1 to be volume ratio be 1:15, mobile phase 2 to be volume ratios be 1:3, mobile phase 3 to be volume ratios be 1.5:1, mobile phase 4 to be volume ratios be 1.5:1, mobile phase 5 to be volume ratios be 2:1, mobile phase 6 are volume ratios is 1.5:1 methylene chloride-sherwood oil, mobile phase 8 is 82%, mobile phase 9 are 62%.
The present invention utilizes Wave Spectrum means, consulting pertinent literature, being divided into from identifying 10 monomeric compounds in conjunction with physicochemical property, wherein Nomilin i.e. (D), 3,5,7,8,3 ', 4 '-hexa methoxy flavones (H) and 5,7,8,3 ', 4 '-pentamethoxyl flavones (I) is separated first to obtain from Fructus Aurantii; The main contribution of the present invention to establish in Fructus Aurantii medicinal material senensetin that is 5,6,7,3 ', 4 '-pentamethoxyl flavones (E) and 5,7,8, the HPLC content assaying method of 3 ', 4 '-pentamethoxyl flavones (I), for the exploitation of the quality standard and Fructus Aurantii of improving Fructus Aurantii medicinal material provide new scientific basis.By detection method of the present invention, record compound 5,6,7 in Fructus Aurantii, the average content of 3 ', 4 '-pentamethoxyl flavones (E) is 0.16mgg
-1, compound 5,7,8, the average content of 3 ', 4 '-pentamethoxyl flavones (I) is 0.024mgg
-1.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for instructions, together with embodiments of the present invention for explaining the present invention, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is HPLC figure (I) of Fructus Aurantii medicinal material and HPLC figure (II) of reference substance compd E and I;
Fig. 2 is compound 5,6,7, the uv atlas of 3 ', 4 '-pentamethoxyl flavones (E);
Fig. 3 is compound 5,7,8, the uv atlas of 3 ', 4 '-pentamethoxyl flavones (I);
Fig. 4 is the peak values of two kinds of compounds in different solvents;
The peak value of two kinds of compounds when Fig. 5 is Different Extraction Method;
The peak value of two kinds of compounds when Fig. 6 is different solvents volume;
Fig. 7 is the linear relationship curve of two kinds of reference substance compounds.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein is only for instruction and explanation of the present invention, is not intended to limit the present invention.
One, the extracting and developing of chemical composition and purifying in Fructus Aurantii
1, medicinal material and reagent
Medicinal material
Extracting and developing and the purifying medicinal material of chemical composition are purchased from Hebei Anguo.
Reagent
Ethyl acetate, sherwood oil, ethanol, chloroform, methylene chloride, methyl alcohol, acetone, absolute ethyl alcohol, above reagent is all purchased from Beijing Chemical Plant, is analysis pure; Chromatogram methyl alcohol is purchased from Sigma-Aldrich company.
Thin-layer chromatography developping agent
The developping agent of positive thin-layer chromatography is generally: chloroform-methanol (15:1), petroleum ether-ethyl acetate (5:1);
The developping agent of inverse thin layer chromatography is generally: acetone-water (3:1), methanol-water (5:1).
Developer
5% sulfuric acid-ethanolic solution (105 DEG C of heating colour developings)
2, the Extraction and separation of chemical composition
(1) extract
Get the Fructus Aurantii medicinal material of 20.0kg drying, pulverize, adopt the mode adding hot reflux to extract, Extraction solvent is 80% ethanol, the consumption of solvent is respectively 10 times of quality of medicinal material, 8 times, 8 times, extract three times, extraction time is respectively 1.5h, 1.0h, 1.0h, merges extract, filters, reduced pressure concentration, obtains crude extract 3.0kg.
(2) separation, purifying
By 3.0kg crude extract, carry out silica gel column chromatography separation with mobile phase 1 as eluent.TCL inspection stream part, collect and merge component phase homogeneous turbulence part, recycling design, obtains Part I and Part II, and wherein Part II detects containing relatively large 5,6,7 by TCL, 3 ', 4 '-pentamethoxyl flavones and 5,7,8,3 ', 4 '-pentamethoxyl flavones.
(1) separation of Part I
Part I mobile phase 1 is carried out silica gel column chromatography separation as eluent, by the portion collection containing a large amount of same stream part obtained at first, then carry out ODS column chromatography for separation with mobile phase 2 as eluent, obtain compd B (900mg).The portion collection of a large amount of two same stream parts will be contained while finally obtaining, then silica gel column chromatography separation is carried out with mobile phase 1, obtain two parts, and then use mobile phase 3 respectively, mobile phase 4 carries out ODS column chromatography for separation, obtain compd A (298mg) respectively, D (30mg).
(2) separation of Part II
Part II mobile phase 1 is carried out silica gel column chromatography separation as eluent, according to the sequencing flowed out from chromatographic column, collects and obtain L
1-L
7the part of 7, these 7 parts are respectively containing a large amount of phase homogeneous turbulence part.By L
1carry out ODS column chromatography for separation with mobile phase 3 as eluent, obtain Compound C (85mg).By L
3carry out ODS column chromatography for separation with mobile phase 5 as eluent, obtain compd E (370mg).By L
4carry out silica gel column chromatography separation with mobile phase 6 as eluent, and then carry out ODS column chromatography for separation with mobile phase 7 as eluent and obtain compound F 17-hydroxy-corticosterone (1.0g).By L
5with mobile phase 8 as eluent, be separated with preparative chromatograph, obtain G (151mg) and H (18mg) two compounds.By L
6with mobile phase 9 as eluent, be separated with preparative high performance liquid chromatography instrument, obtain Compound I (83mg).By L
7with reagent recrystallizations such as methyl alcohol, obtain compound J (1.0g).
Two, the Structural Identification of chemical composition
The structure analysis of compd A
Compd A is white powder, is soluble in the organic solvents such as chloroform, and fusing point is: 297.5 ~ 298.5 DEG C.Molish reaction presents feminine gender, shows in this compound molecule not sugary.
ESI-MS:m/z471.2 [M+H]+, point out the molecular weight of this compound to be 470.2.
13C NMR (CDCl3, the 150MHz) spectrum of compd A provides 26 carbon signals altogether, known in conjunction with DEPT spectrum, containing 4 methyl signals (being δ C30.15 respectively, 21.37,20.68,17.61) in molecule; 5 methylene signals (being δ C65.34 respectively, 36.38,35.65,30.81,18.90); 8 methine signals (being δ C143.22 respectively, 141.11,109.66,79.14,77.79,60.53,53.85,48.10); All the other 9 is quaternary carbon signal (being δ C206.09 respectively, 169.08,166.60,119.98,80.29,65.69,51.32,45.93,37.94).
δ H1.296 (3H, s) in 1H NMR (CDCl3,600MHz) spectrum, (1.181 3H, s), 1.181 (3H, s), the existence of 1.077 (3H, s) 4 groups of proton signals can confirm in molecule further containing 4 methyl.Methylene signals δ C65.34 may be connected with oxygen.In methine signals, δ C79.14,77.79,53.85 may be the carbon signal be connected with oxygen or carbonyl respectively.In quaternary carbon signal, δ C206.09 is carbonyl carbon signals, δ C169.08, and 166.60 may be the carbonyl carbon signals on ester group, δ C80.29, and 65.69 may be the carbon signal be connected with oxygen, and δ C45.93 may be the carbon signal be connected with carbonyl.
Low field end δ C143.22,141.11,119.98,109.66 may be double key carbon signal, and known in conjunction with DEPT spectrum, δ C119.98 is quaternary carbon signal, and all the other are methine signals, points out in molecule thus and may contain furan nucleus.
1δ during H NMR composes
h7.417 (1H, s), 7.402 (1H, s), the appearance of 6.343 (1H, s) 3 groups of proton signals, confirms in molecule further containing furan nucleus.
According to more than
13c NMR and
1the analysis of H NMR signal, preliminary deduction compd A is limonoid, through contrasting with document, confirms that this compound is limonin (Limonin, C
26h
30o
8), structural formula is as follows:
The structure analysis of compd B
Compd B is white crystal, is soluble in chloroform, methyl alcohol, acetone and other organic solvent, and fusing point is: 141.5 ~ 142.5 DEG C.
Use different developping agent system to carry out TLC to the standard items of compd B and cupreol, result shows that both Rf values are all consistent with the color of spot.Fusing point test is carried out in both mixing, found that the fusing point of potpourri does not decline, therefore determine that B is cupreol (β-sitosterol, C
29h
50o).Structure is as follows:
The structure analysis of Compound C
Compound C is white crystal, is soluble in the organic solvent such as chloroform, methyl alcohol, and fusing point is: 136.5 ~ 137.0 DEG C.The positive is presented with the reaction of hydrochloric acid-magnesium powder, but negative to ferric chloride reaction display, show that Compound C may for the Flavonoid substances without phenolic hydroxyl group.Molish reaction presents feminine gender, shows in this compound molecule not sugary.
ESI-MS:m/z403.2 [M+H]
+, point out the molecular weight of this compound to be 402.2.
Compound C
13c NMR (CDCl
3, 125MHz) spectrum provide 21 carbon signals altogether.Known in conjunction with DEPT spectrum, having 6 methyl signals in molecule (is δ respectively
c62.28,61.98,61.83,61.69,56.11,55.99), 4 methine signals (are δ respectively
c119.69,111.26,108.59,106.80), all the other 11 is quaternary carbon signal (is δ respectively
c177.33,161.15,152.00,151.49,149.32,148.43,147.73,144.14,138.02,123.99,114.78), wherein δ
c177.33 are speculated as carbonyl carbon signals, and all the other each carbon signals appearing at low field may be the carbon signal on flavones parent nucleus.In addition, above methyl signals all appears at low field, illustrates that the atom large with electronegativity or group are connected, and infers probably containing 6 methoxyls in molecule,
1h NMR (CDCl
3, 500MHz) spectrum in δ
h4.112 (3H, s), 4.034 (3H, s), 3.983 (3H, s), 3.969 (3H, s), 3.959 (6H, s) appearance of 6 groups of proton signals, can confirm to have 6 methoxyls in this compound molecule further, tentatively can confirm that this compound is the flavone compound containing six methoxyls.
1during H NMR composes, δ
h6.657 (1H, s) may be the proton signals on flavones C ring, δ
h7.589 (1H, dd, J=8.5,2.0Hz), 7.423 (1H, d, J=2.0Hz), 6.992 (1H, d, J=8.5Hz) may be proton signal on flavones B ring, can infer that on flavones parent nucleus C ring, No. 3 positions are without replacement thus, A ring and B ring are connected with 4 and 2 methoxyls respectively.
According to more than
13c NMR and
1the analysis of H NMR signal, through contrasting with document, confirms that this compound is 5,6,7,8,3 ', 4 '-hexa methoxy flavones, i.e. Nobiletin (Nobiletin, C
21h
22o
8), structural formula is shown in:
*62.28 61.98,61.83,61.69,55.99 and 56.11 is interchangeable;
*4.112 4.034,3.983,3.969,3.959 and 3.959 is interchangeable.
The structure analysis of Compound D
Compound D is white crystal, is soluble in the organic solvent such as chloroform, methyl alcohol, and fusing point is: 271.0 ~ 272.0 DEG C.Molish reaction presents feminine gender, shows in this compound molecule not sugary.
ESI-MS:m/z515.2 [M+H]
+, point out the molecular weight of this compound to be 514.2.
Compound D
13c NMR (CDCl
3, 125MHz) spectrum provide 28 carbon signals altogether.Known in conjunction with DEPT spectrum, have 6 methyl signals in molecule and (be respectively δ
c33.47,23.39,20.83,20.77,17.08,16.52); 4 methylene signals (are respectively δ
c38.80,35.32,32.28,17.19); Methine signals 8 (is respectively δ
c143.27,141.01,109.64,78.01,70.71,53.40,51.01,44.37), all the other are that 10 quaternary carbon signals (are respectively δ
c206.76,169.24,169.15,166.75,120.06,84.38,65.46,52.87,44.17,37.48).
1h NMR (CDCl
3, 500MHz) spectrum in δ
h1.094 (3H, s), 1.179 (3H, s), 1.333 (3H, s), 1.471 (3H, s), 1.555 (3H, s), the appearance of 2.011 (3H, s) 6 groups of proton signals confirms in molecule further containing 6 methyl.In methine signals, δ
c53.40 may be connected with oxygen or carbonyl, δ
c78.01,70.71 is the carbon signal be connected with oxygen.In quaternary carbon signal, δ
c206.76 is carbon signals of carbonyl, δ
c169.24 169.15,166.75 is the carbonyl carbon signals of ester group respectively, δ
c84.38 65.46 may be the carbon signal be connected with oxygen, δ
c52.87 may be the carbon signal be connected with carbonyl.
Low field end δ
c143.27,141.01,120.06,109.64 may be double key carbon signal, known in conjunction with DEPT spectrum, δ
c120.06 is quaternary carbon signals, and all the other are methine signals, points out in molecule thus and may contain furan nucleus.In conjunction with
1δ during H NMR composes
h6.328 (1H, s), 7.400 (1H, s), the appearance of 7.400 (1H, s) 3 groups of proton signals, confirms in molecule further containing furan nucleus.
According to more than
13c NMR and
1the analysis of H NMR signal, preliminary deduction Compound D is limonoid, through contrasting with document, confirms that this compound is Nomilin (Nomilin, C
28h
34o
9), known by literature search, this compound is separated first and obtains from Fructus Aurantii, and its structural formula is as follows:
The structure analysis of compd E
Compd E is yellow crystals, is soluble in the organic solvent such as chloroform, methyl alcohol, and fusing point is: 178.5 ~ 179.5 DEG C.The positive is presented with the reaction of hydrochloric acid-magnesium powder, but negative to ferric chloride reaction display, show that compd E may for the Flavonoid substances without phenolic hydroxyl group.Molish reaction presents feminine gender, shows in this compound molecule not sugary.
ESI-MS:m/z373.1 [M+H]
+, point out the molecular weight of this compound to be 372.1.
Compd E
13c NMR (CDCl
3, 125MHz) spectrum provide 20 carbon signals altogether.Known in conjunction with DEPT spectrum, may (be δ respectively containing 5 methyl signals in molecule
c62.21,61.55,56.35,56.14,56.09); 5 methine signals (are δ respectively
c119.67,111.16,108.68,107.23,96.28); All the other are 10 quaternary carbon signals (is δ respectively
c177.18,161.29,157.77,154.52,152.57,151.89,149.29,140.42,124.04,112.75), wherein δ
c177.18 are speculated as carbonyl carbon signals, and all the other each carbon signals appearing at low field may be the carbon signal on flavones parent nucleus.In addition, above methyl signals all appears at low field, illustrates that the atom large with electronegativity or group are connected, and infers probably containing 5 methoxyls in molecule,
1h NMR (CDCl
3, 500MHz) spectrum in δ
h3.997 (6H, s), 3.981 (3H, s), 3.962 (3H, s), 3.925 (3H, s) appearance of 5 groups of proton signals, confirms that this compound contains 5 methoxyls further, tentatively can confirm that this compound is the flavone compound containing five methoxyls.
1during H NMR composes, δ
h6.642 (1H, s) may be the proton signals on flavones C ring, δ
h6.809 (1H, s) may be the proton signals on flavone A ring, δ
h7.329 (1H, d, J=2.0Hz), 6.962 (1H, d, J=8.5Hz), 7.519 (1H, dd, J=8.5,2.0Hz) may be proton signal on flavones B ring, can infer that on flavones parent nucleus C ring, No. 3 positions are without replacement thus, A ring and B ring are connected with 3 and 2 methoxyls respectively.
According to more than
13c NMR and
1the analysis of H NMR signal, through contrasting with document, confirms that this compound is 5,6,7,3 ', 4 '-pentamethoxyl flavones, i.e. senensetin (Sinensetin, C
20h
20o
7), structural formula is as follows:
*62.21 61.55,56.35,56.14 and 56.09 is interchangeable;
*3.981 3.997,3.997,3.962 and 3.925 is interchangeable.
The structure analysis of compound F 17-hydroxy-corticosterone
Compound F 17-hydroxy-corticosterone is off-white powder, is soluble in the organic solvents such as methyl alcohol, and fusing point is: 250.0 ~ 251.0 DEG C.The positive is presented with the reaction of hydrochloric acid-magnesium powder, and positive to ferric chloride reaction display, show that compound F 17-hydroxy-corticosterone may for the Flavonoid substances containing phenolic hydroxyl group.Molish reaction presents feminine gender, shows in this compound molecule not sugary.
ESI-MS:m/z273.1 [M+H]
+, point out the molecular weight of this compound to be 272.1.
Compound F 17-hydroxy-corticosterone
13c NMR (CD
3oD, 125MHz) spectrum provide 13 carbon signals altogether.Wherein δ
c116.32, δ
c129.03 signal intensities are comparatively large, are about 2 times of other signal intensities, infer it is to be superposed by the carbon signal that two chemical environments are identical and to produce thus, illustrate in molecule probably have symmetrical structure.
1h NMR (CDCl
3, 500MHz) spectrum in δ
h6.842 (2H, d, J=9.0Hz), the appearance of 7.322 (2H, d, J=8.5Hz) two groups of proton signals, proves further to contain symmetrical structure in molecule, infers that this symmetrical structure should be phenyl ring from the chemical shift of proton.Therefore, this compound is altogether containing 15 carbon signals, consistent with the parent nucleus carbon number of flavone compound.
Known in conjunction with DEPT spectrum, containing 1 methylene signals (δ in compound
c44.04); 7 methine signals (are δ respectively
c129.03,129.03,116.32,116.32,97.04,96.15,80.48); 7 quaternary carbon signals (are δ respectively
c197.79,168.35,165.48,164.89,159.03,131.09,103.35), wherein δ
c197.79 be carbonyl carbon signals, remaining quaternary carbon signal should be the carbon signal on flavones parent nucleus.
1proton signal δ in H NMR
h3.123 (1H, dd, J=17.0,13.0Hz) and 2.692 (1H, dd, J=17.0,3.0Hz) should be methylene (δ
c44.04) proton signal, therefore infer that further F may be flavanone kind composition.
Due to
13c NMR and
1h NMR spectrum does not provide the methoxyl signal of feature, has three hydrogen to be substituted simultaneously, and this compound is positive to ferric chloride reaction display, and is positioned at the δ of low field
h11.264 (1H, s), 9.305 (1H, s), 8.812 (1H, s) are speculated as the proton signal on phenyl ring hydroxyl, therefore confirm that this compound is the flavanone kind composition that hydroxyl replaces further.
According to more than
13c NMR and
1the analysis of H NMR signal, and contrast with document, confirm that compound F 17-hydroxy-corticosterone is 5,7,4 '-trihydroxy flavanone, i.e. naringenin (naringenin, C
15h
12o
5), structural formula is as follows:
The structure analysis of compound G
Compound G is yellow crystals, is soluble in the organic solvent such as chloroform, methyl alcohol, and fusing point is: 215.5 ~ 216.5 DEG C.The positive is presented with the reaction of hydrochloric acid-magnesium powder, but negative to ferric chloride reaction display, show that compound G may for the Flavonoid substances without phenolic hydroxyl group.Molish reaction presents feminine gender, shows in this compound molecule not sugary.
ESI-MS:m/z343.3 [M+H]
+, point out the molecular weight of this compound to be 342.3.
Compound G's
13c NMR (CDCl
3, 125MHz) spectrum provide 17 carbon signals altogether.Wherein δ
c127.69, δ
c114.45 signal intensities are about 2 times of other carbon signal intensity, and infer it is to be superposed by the carbon signal that two chemical environments are identical and to produce thus, illustrating in molecule probably has symmetrical structure, so may have 19 carbon signals in supposition molecule.
Composed from DEPT, containing 4 methyl in molecule (is δ respectively
c61.58,56.58,56.29,55.48); 6 methine signals (are δ respectively
c127.69,127.69,114.45,114.45,106.95,92.55); All the other are 9 quaternary carbon signals (is δ respectively
c177.91,162.17,160.70,156.46,156.30,151.96,130.78,123.85,109.02), wherein δ
c177.91 are speculated as carbonyl carbon signals, and all the other each carbon signals appearing at low field may be the carbon signal on flavones parent nucleus.In addition, above methyl signals all appears at low field, illustrates that the atom large with electronegativity or group are connected, and illustrates probably containing 4 methoxyls in molecule,
1h NMR (CDCl
3, 500MHz) spectrum in δ
h4.001 (3H, s), 3.979 (3H, s), 3.949 (3H, s), 3.876 (3H, s) appearance of 4 groups of proton signals, confirms to contain 4 methoxyls in this compound further, tentatively can confirm that this compound is the flavone compound containing four methoxyls.
1δ during H NMR composes
hthe deducibility of 6.588 (1H, s) proton signal is the proton signal on flavones C ring, δ
h6.433 (1H, s) deduction is the proton signal on flavone A ring, δ
h7.887 (2H, d, J=8.5Hz), 7.021 (2H, d, J=8.5Hz) deducibility is the proton signal on flavones B ring, can infer that on flavones parent nucleus C ring, No. 3 positions are without replacement thus, A ring and B ring are connected with 3 and 1 methoxyl respectively.
According to more than
13c NMR and
1the analysis of H NMR signal, and contrast with document, confirm that compound G is 5,7,8, and 4 '-tetramethoxy flavones (5,7,8,4 '-tetramethoxyflavone, C
19h
18o
6), structural formula is as follows:
*56.58,56.29,61.58 and 55.48 is interchangeable
*4.001,3.979,3.949 and 3.876can be exchanged.
The structure analysis of compound H
Compound H is yellow powder, is soluble in the organic solvent such as chloroform, methyl alcohol, and fusing point is: 173.5 ~ 174.5 DEG C.The positive is presented with the reaction of hydrochloric acid-magnesium powder, but negative to ferric chloride reaction display, show that compound H may for the Flavonoid substances without phenolic hydroxyl group.Molish reaction presents feminine gender, shows in this compound molecule not sugary.
ESI-MS:m/z403.1 [M+H]
+, point out the molecular weight of this compound to be 402.1.
Compound H
13c NMR (CDCl
3, 125MHz) spectrum provide 21 carbon signals altogether.Composed from DEPT, containing 6 methyl in molecule (is δ respectively
c61.50,59.88,56.60,56.37,55.97,55.87); 4 methine signals (are δ respectively
c121.83,111.01,110.94,92.30); All the other are in 11 quaternary carbon signals (be δ respectively
c174.26,156.37,156.26,152.32,150.91,150.88,148.71,140.88,130.52,123.58,109.36), wherein δ
c174.26 are speculated as carbonyl carbon signals, and all the other each carbon signals appearing at low field may be the carbon signal on flavones parent nucleus.In addition, above methyl signals all appears at low field, illustrates that the atom large with electronegativity or group are connected, and infers probably containing 6 methoxyls in molecule,
1hNMR (CDCl
3, 500MHz) spectrum in δ
h(4.006 3H, s), 3.998 (3H, s), 3.900 (3H, s), 3.940 (3H, s), 3.970 (3H, s), 3.970 (3H, s) appearance of 6 groups of proton signals, confirms that this compound contains 6 methoxyls further, tentatively can confirm that this compound is the flavone compound containing six methoxyls.
1δ during H NMR composes
h6.419 (1H, s) supposition is the proton signal on flavone A ring, δ
h(7.863 1H, d, J=2.0Hz), 7.841 (1H, d, J=2.0Hz), 7.018 (1H, d, J=8.0Hz) be speculated as the proton signal on flavones B ring, can infer flavone A ring thus, B ring and C ring is all connected with methoxy group.
According to more than
13c NMR and
1the analysis of H NMR signal, and contrast with document, confirm that compound H is 3,5,7,8,3 ', 4 '-hexa methoxy flavones (3,5,7,8,3 ', 4 '-hexamethoxyflavone, C
21h
22o
8), known by literature search, this compound is separated first and obtains from Fructus Aurantii, and its structural formula is as follows:
*61.50 56.60,56.37,59.88,55.97 and 55.87 is interchangeable
*3.998 4.006,3.900,3.940,3.970 and 3.970 is interchangeable
The structure analysis of Compound I
Compound I is white powder, is soluble in the organic solvent such as chloroform, methyl alcohol, and fusing point is: 199.5 ~ 200.5 DEG C.The positive is presented with the reaction of hydrochloric acid-magnesium powder, but negative to ferric chloride reaction display, show that Compound I may for the Flavonoid substances without phenolic hydroxyl group.Molish reaction presents feminine gender, shows in this compound molecule not sugary.
From ESI-MS::m/z373.1 [M+H]
+, point out the molecular weight of this compound to be 372.1.
Compound I
13c NMR (CDCl
3, 125MHz) spectrum provide 20 carbon signals altogether.Composed from DEPT, containing 5 methyl in molecule (is δ respectively
c61.47,56.54,56.29,56.05,55.93), 5 methine signals (are δ respectively
c119.56,111.19,108.52,107.08,92.49), all the other are 10 quaternary carbon signals (is δ respectively
c177.88,160.52,156.51,156.29,151.92,151.79,149.21,130.67,123.98,108.91), wherein δ
c177.88 are speculated as carbonyl carbon signals, and all the other each carbon signals appearing at low field may be the carbon signal on flavones parent nucleus.In addition, above methyl signals all appears at low field, illustrates that the atom large with electronegativity or group are connected, and infers probably containing 5 methoxyls in molecule,
1h NMR (CDCl
3, 500MHz) spectrum in δ
h4.004 (3H, s), 3.983 (3H, s), 3.966 (3H, s), 3.953 (3H, s), 3.953 (3H, s) appearance of 5 groups of proton signals, confirms to contain 5 methoxyls in this compound further, tentatively can confirm that this compound is the flavone compound containing five methoxyls.
In conjunction with
1δ during H NMR composes
h6.603 (1H, s) supposition is the proton signal on flavones C ring, δ
h6.436 (1H, s) supposition is the proton signal on flavone A ring, δ
h(7.411 1H, s), 6.990 (1H, d, J=8.5Hz), 7.581 (1H, d, J=8.0Hz) supposition is the proton signal on flavones B ring, can infer that on flavones parent nucleus C ring, No. 3 positions are without replacement thus, A ring and B ring are connected with 3 and 2 methoxyls respectively.
According to more than
13c NMR and
1the analysis of H NMR signal, and contrast with document, confirm that Compound I is 5,7,8,3 ' 4 '-pentamethoxyl flavones (5,7,8,3 ', 4 '-pentamethoxyflavone, C
20h
20o
7), known by literature search, this compound is separated first and obtains from Fructus Aurantii, and its structural formula is as follows:
*56.54 56.29,61.47,56.05 and 55.93 is interchangeable
*3.953 3.953,3.966,3.983 and 4.004 is interchangeable
The structure analysis of compound J
Compound J: white powder, is soluble in the organic solvent such as chloroform, methyl alcohol, and fusing point is: 292.0 ~ 293.0 DEG C.
Different developping agent system is used to carry out thin-layer chromatography contrast to the standard items of compound J and daucosterol, show that both Rf value Rf are all consistent with the color of spot, fusing point test is carried out in both mixing, found that fusing point does not decline, therefore determine that J is daucosterol (daucosterol, C
35h
60o
6), structural formula is as follows.
Three, RP-HPLC method measures the selection of liquid phase chromatogram condition in the content of two kinds of polymethoxyflavones in Fructus Aurantii
1, the selection of mobile phase condition
This experiment is tested various different mobile phase, tests the methanol-water of different volumes ratio respectively, acetonitrile-water and methanol-acetonitrile-water.From the degree of separation of chromatographic peak, peak shape and retention time integrated survey, find that the effect of methanol-water is better.When adopting acetonitrile-water, I is difficult to reach baseline separation with other peaks and retention time is longer; Adopt gradient elution, separating effect, still not as methanol-water, is therefore tentatively determined to adopt methanol-water as mobile phase.Through Optimum Experiment, finally determine that the ratio adopting methanol-water is 52:48, now in chromatogram, E and I degree of separation reaches more than 1.5.The chromatogram of the hybrid standard liquid of sample solution and two kinds of reference substances is shown in Fig. 1.Under this chromatographic condition, the degree of separation of compd E and I is respectively 2.58 and 1.74.
2, the selection of determined wavelength
Dissolve a small amount of E and I respectively with chromatogram methyl alcohol, its concentration is respectively 0.13mgmL-1 and 0.13mgmL-1, then scans in 200 ~ 400nm wavelength coverage E and I respectively with ultraviolet spectrophotometer.From scanning result, E has absorption maximum at 328.1nm and 214.9nm place, and I has absorption maximum at 339.0nm and 206.0nm place.Use DAD multiwavelength detector to find the common best detection wavelength of this two kinds of compounds, peak areas both making are comparatively large and baseline is steady.Find through repetition test, 310nm is best detection wavelength.Therefore, this experiment adopts 310nm to be determined wavelength.The uv-spectrogram of E and I is shown in Fig. 2 and Fig. 3.
3, the selection of extraction conditions
In order to make these two kinds of compounds of E and I can extract from Fructus Aurantii medicinal material completely, reach maximum extracted rate, the factor that four affect extraction ratio has been investigated in this experiment, and these four factors are respectively: Extraction solvent, extracting method, Extraction solvent consumption, extraction time.Using the inspection target of the chromatographic peak area of compd E and I as extraction ratio influence factor.
3.1, the selection of Extraction solvent
By Fructus Aurantii pulverizing medicinal materials, cross 80 mesh sieves, precision takes 6 parts, every part of 2.5000g.Adopt methyl alcohol, ethyl acetate, ethanol, chloroform, sherwood oil, acetone is as Extraction solvent, and solvent load is 10 times of quality of medicinal material.Extracting mode is for adding hot reflux, and reflux three times, the time is respectively 1.5h, 1.0h, 0.5h.Phegma is merged, filters, be concentrated into dry, by chromatogram methanol constant volume in 10mL volumetric flask, as need testing solution.Cross 0.22 μm of miillpore filter, get subsequent filtrate 20 μ L, inject high performance liquid chromatograph, parallel sample introduction twice, measure E and I peak area respectively, obtain its mean value.Measurement result is shown in Fig. 4.As can be seen from measurement result, when using methyl alcohol to make Extraction solvent, E and I peak area is maximum, illustrates that extraction effect is best, therefore adopts methyl alcohol as Extraction solvent.
3.2, the selection of extracting method
The Fructus Aurantii medicinal material of (80 mesh sieve) after pulverizing and sieving, precision takes 4 parts, every part of 2.5000g.Use following four kinds of extracting modes: add hot reflux 1 hour, mechanical shaking extraction 1 hour, ultrasonic extraction 1 hour and cold soaking extract for 24 hours.Extraction solvent is methyl alcohol, and solvent load is 10 times of quality of medicinal material.Extract is merged, filters, be concentrated into dry, by chromatogram methanol constant volume in 10mL volumetric flask, as need testing solution.Cross 0.22 μm of miillpore filter, get subsequent filtrate 20 μ L, inject high performance liquid chromatograph, measure peak area, parallel sample introduction twice, measure E and I peak area, obtain its mean value.Measurement result is shown in Fig. 5.Result shows, use and add hot reflux when making extracting mode, E and I peak area is maximum, illustrates that extraction effect is best, therefore employing adds hot reflux as extracting mode.
3.3, the selection of Extraction solvent consumption
The Fructus Aurantii medicinal material of (80 mesh sieve) after pulverizing and sieving, precision takes 3 parts, every part of 2.5000g.Use methyl alcohol as Extraction solvent, solvent load is respectively 8 times, 10 times, 12 times (V/W) of quality of medicinal material, and heating return time is all 1 hour.Extract is merged, filters, be concentrated into dry, by chromatogram methanol constant volume in 10mL volumetric flask, as need testing solution.Cross 0.22 μm of miillpore filter, get subsequent filtrate 20 μ L, inject high performance liquid chromatograph, parallel sample introduction twice, measure E and I peak area, obtain its mean value.Measurement result is shown in Fig. 6.Measurement result shows, when solvent load is 10 times of quality of medicinal material, substantially can extract completely, therefore adopt the methyl alcohol of 10 times of volumes (V/W) as Extraction solvent.
3.4, the selection of extraction time
The Fructus Aurantii medicinal material 2.5000g of (80 mesh sieve) after precision takes and pulverizes and sieves, adds hot reflux 1.5h with 25mL methyl alcohol, extracts as first time, and by extracting liquid filtering, filtrate is retained; Residue 25mL methyl alcohol adds hot reflux 1.0h, extracts as second time, and by extracting liquid filtering, filtrate is retained; Residue 25mL methyl alcohol adds hot reflux 0.5h, extracts as third time, and by extracting liquid filtering, filtrate is retained; Residue 25mL methyl alcohol adds hot reflux 0.5h, extracts, by extracting liquid filtering as the 4th time.The filtrate of four parts of extracts is boiled off solvent respectively, by chromatogram methanol constant volume in 10mL volumetric flask, as need testing solution.Cross 0.22 μm of miillpore filter, get subsequent filtrate 20 μ L, inject high performance liquid chromatograph, E and I do not detected in No. the 4th extract as a result, illustrate to extract for three times and substantially extract completely, thus determine that extraction time is three times.
Embodiment 1
In Chinese medicine Fructus Aurantii 5,6,7,3 ', 4 '-pentamethoxyl flavones and 5,7,8, the quality determining method of 3 ', 4 '-pentamethoxyl flavones, described quality determining method is high performance liquid chromatography, and its concrete steps are as follows:
1, chromatographic condition and testing conditions:
Chromatograph: Agilent1200HPLC, diode array detector
Chromatographic column: Acchrom Unitary C18Column (4.6 × 250mm, 5 μm)
Mobile phase: methanol-water, volume ratio is 52:48
Flow velocity: 1.0mLmin
-1
Column temperature: 25.0 DEG C
Sample size: 20 μ L
Determined wavelength: 310nm.
2, the preparation of need testing solution
It is appropriate that precision takes Fructus Aurantii medicinal powder (80 mesh sieve), using methyl alcohol as Extraction solvent, solvent load is 10 times (V/W) of quality of medicinal material, refluxing extraction 3 times, and extraction time is 1.5h, 1.0h, 0.5h, extract is merged, filter, be concentrated into dry, dissolve with chromatogram methyl alcohol and be settled to designated volume, cross 0.22 μm of miillpore filter, get subsequent filtrate as need testing solution.
3, the preparation of reference substance solution
Precision takes reference substance compound 5,6,7,3 ', 4 '-pentamethoxyl flavones (E) and 5,7,8,3 ', 4 '-pentamethoxyl flavones (I) is placed in the volumetric flask of same 10mL, quality is respectively 2.64mg and 2.54mg, is mixed with by chromatogram methanol constant volume the mixing reference substance solution that concentration is respectively 0.26mgmL-1 and 0.25mgmL-1.
4, methodological study
4.1, linear relationship
Reference substance solution being diluted respectively is 2,5,10,20,50,100,200 and 500 times, and the concentration obtaining E in each mixed standard solution is respectively: 0.13,0.053,0.026,0.013,0.0053,0.0026,0.0013,0.00053mgmL-1; The concentration of I is respectively: 0.13,0.051,0.025,0.013,0.0051,0.0025,0.0013,0.00051mgmL-1.Get the above-mentioned each reference substance solution of 20 μ L respectively, in injection liquid chromatography, record chromatogram, measures the peak area of each reference substance.By peak area (ordinate) to sample size (horizontal ordinate) drawing standard curve, the equation of linear regression of two compounds and linearly dependent coefficient are in table one, and the measurement result of E and I peak area mean value is in table two, and typical curve is shown in Fig. 7.
The equation of linear regression of table one or two compound and linearly dependent coefficient
The peak area mean value of table two two compounds
Analysis design mothod result can draw: the sample size of compd E and I is good in the scope internal linear relation of 0.011 ~ 5.3 μ g and 0.010 ~ 5.1 μ g respectively.
4.2, detectability and quantitative limit
Accurately pipette reference substance solution 1.00mL, dissolve with chromatogram methyl alcohol and be settled in 10mL volumetric flask, in the mixing reference substance solution obtained, the concentration of E and I is respectively 0.026mgmL-1 and 0.025mgmL-1, is constantly diluted by this solution, accurately calculates its concentration after each dilution.Each solution after dilution is got 20 μ L, injection liquid chromatography respectively, and record chromatogram peak height, calculates signal to noise ratio (S/N ratio) (S/N).Show from experimental result, E and I dilute respectively be 4000 times and 1900 times time, now S/N is about 3.0, and sample size is now detectability; E and I dilute respectively be 1140 times and 450 times time, S/N is about 10.0, and sample size is now quantitative limit.Experimental result is in table three.
Table three detectability and quantitative limit
4.3, precision
Precision takes reference substance E and I, is mixed with the mixing reference substance solution that concentration is respectively 0.045 and 0.0076mgmL-1, gets 20 μ L and injects high performance liquid chromatograph.Parallel sample introduction 6 times in one day, measures the peak area of two compounds, calculates its withinday precision respectively.According to above method sample introduction, once a day, continuous six days, measure the peak area of two compounds, calculate its day to day precision respectively.The results are shown in Table four and table five.
Table four withinday precision
Note: peak area unit is mAUs
Table five day to day precision
Note: peak area unit is mAUs
As can be seen from above two tables, the RSD of two kinds of compounds is all less than 2%, illustrates that the precision of the method is good.
4.4, repeatability
6 parts of need testing solutions are prepared, single injected sampling amount 20 μ L, every part of parallel sample introduction twice, the content in medicinal material with external standard method compd E and I according to said method.Experimental result is in table six
Table sixfold renaturation experimental data
Result shows, the RSD of two kinds of compounds is all less than 2%, illustrates that repeatability is good.
4.5, stability
According to said method preparation need testing solution, by the solution after filtration in the 0-12h of a day, every 2 hours sample introductions, sample size 20 μ L, measures the peak area of compd E and I and records retention time.Experimental result is in table seven.
Table seven stability experiment data
As can be seen from the above table, the RSD of two kinds of compounds is all less than 2%, and explanation has good stability.
4.6, average recovery
Precision takes 5.34mg reference substance E in the volumetric flask of 100mL, uses chromatogram methanol constant volume, obtains the list mark solution I that concentration is the E of 0.0534mgmL-1; Precision takes 1.25mg reference substance I in the volumetric flask of 100mL, uses chromatogram methanol constant volume, obtains the list mark solution II that concentration is the I of 0.0125mgmL-1.(80 mesh sieve) Fructus Aurantii medicinal material after pulverizing and sieving (compd E known to I content), precision takes 6 parts, and quality is 2.5000g.Be divided into three groups by 6 parts, pipette 3.6mL mono-mark solution I respectively and the mono-solution II of marking of 2.4mL is placed in two parts of medicinal materials of first group, the amount now adding E and I is about 80% of E and I content in medicinal material; Pipette 4.5mL mono-mark solution I respectively and the mono-solution II of marking of 3.0mL is placed in two parts of medicinal materials of second group, the amount now adding E and I is about 100% of E and I content in medicinal material; Pipette 5.4mL mono-mark solution I respectively and the mono-solution II of marking of 3.7mL is placed in two parts of medicinal materials of the 3rd group, the amount now adding E and I is about 120% of E and I content in medicinal material.The content of methyl alcohol in 6 parts of medicinal materials is all supplemented to 25mL, and then prepares need testing solution according to the method in " 3.3.5 " item, get each solution 20 μ L, injection liquid chromatography, measure peak area, calculated the content of compd E and I by external standard method, and calculate the recovery.Experimental data in table eight, table nine, table ten.
The volume that table eight adds and the quality added
Table nine compound 5,6,7, the recovery of 3 ', 4 '-pentamethoxyl flavones (E)
Table ten compound 5,7,8, the recovery of 3 ', 4 '-pentamethoxyl flavones (I)
5, in Fructus Aurantii 5,6,7,3 ', 4 '-pentamethoxyl flavones (E) and 5,7,8, the assay of 3 ', 4 '-pentamethoxyl flavones (I)
This measuring is purchased from the content of E and I in the Fructus Aurantii medicinal material of Hebei Anguo and Hui nationality.
5.1, the preparation of reference substance solution
Get reference substance 5,6,7,3 ', 4 '-pentamethoxyl flavones (E) and 5,7,8,3 ', 4 '-pentamethoxyl flavones (I), accurately weighed, dissolve and constant volume with methyl alcohol, be mixed with the mixing reference substance solution that concentration is respectively 0.045mgmL-1 and 0.0076mgmL-1, this solution is as External standards solutions.
5.2, the preparation of need testing solution
Precision takes Fructus Aurantii medicinal powder (80 mesh sieve) 5.0000g, using methyl alcohol as Extraction solvent, solvent load is 10 times (50mL) of quality of medicinal material, refluxing extraction 3 times, and extraction time is 1.5h, 1.0h, 0.5h, extract is merged, filter, be concentrated into dry, dissolve with chromatogram methyl alcohol and be settled in 10mL volumetric flask, cross 0.22 μm of miillpore filter, get subsequent filtrate as need testing solution.
5.3, the mensuration of sample size
Get reference substance solution and each 20 μ L of need testing solution, inject high performance liquid chromatograph respectively, carry out according to 1 conditional the peak area measuring E and I, replicate determination twice, utilize the content of two kinds of methoxy flavone compounds in each medicinal material of external standard method, the results are shown in Table 11, table ten two.
The content of table ten one compd E
The content of table ten two Compound I
From experimental result, in the medicinal material of Hebei Anguo and Hui nationality, the content of compd E is respectively 0.094mgg-1 and 0.22mgg-1, and average content is 0.16mgg-1; The content of Compound I is respectively 0.015mgg-1 and 0.032mgg-1, and average content is 0.024mgg-1.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. the quality determining method of two kinds of flavones in Fructus Aurantii, it is characterized in that, described quality determining method is high performance liquid chromatography, chromatographic condition and testing conditions as follows:
Mobile phase: methanol-water, volume ratio is (45-60): (40-55)
Flow velocity: 0.5-1.5mLmin
-1
Column temperature: 25.0 DEG C
Sample size: 10-30 μ L
Determined wavelength: 200-400nm;
The preparation of need testing solution: it is appropriate that precision takes 50-100 order Fructus Aurantii medicinal powder, using methyl alcohol as Extraction solvent, solvent load is 10 times (g/ml) of quality of medicinal material, refluxing extraction 2-4 time, and extraction time is 0.5-1.5h, extract is merged, filter, be concentrated into dry, dissolve with chromatogram methyl alcohol and be settled to designated volume, cross miillpore filter, get subsequent filtrate as need testing solution;
The preparation of reference substance solution: precision takes reference substance 5,6,7,3 ', 4 '-pentamethoxyl flavones and 5,7,8,3 ', 4 '-pentamethoxyl flavones is appropriate, is placed in volumetric flask, is mixed with concentration is 0.10-0.30mgmL by chromatogram methanol constant volume
-1mixing reference substance solution.
2. quality determining method as claimed in claim 1, is characterized in that, chromatographic condition and testing conditions as follows:
Chromatograph: Agilent1200HPLC, diode array detector
Chromatographic column: Acchrom Unitary C18Column (4.6 × 250mm, 5 μm)
Mobile phase: methanol-water, volume ratio is 52:48
Flow velocity: 1.0mLmin
-1
Column temperature: 25.0 DEG C
Sample size: 20 μ L
Determined wavelength: 310nm.
3. quality determining method as claimed in claim 1 or 2, is characterized in that:
The preparation of need testing solution: it is appropriate that precision takes 80 order Fructus Aurantii medicinal powders, using methyl alcohol as Extraction solvent, solvent load is 10 times (g/ml) of quality of medicinal material, refluxing extraction 3 times, and extraction time is respectively 1.5h, 1.0h, 0.5h, extract is merged, filter, be concentrated into dry, dissolve with chromatogram methyl alcohol and be settled to designated volume, cross 0.22 μm of miillpore filter, get subsequent filtrate as need testing solution.
4. quality determining method as claimed in claim 1 or 2, is characterized in that:
The preparation of reference substance solution: precision takes reference substance 5,6,7,3 ', 4 '-pentamethoxyl flavones 2.64mg and 5,7,8,3 ', 4 '-pentamethoxyl flavones 2.54mg, is placed in the volumetric flask of same 10mL, is mixed with concentration is respectively 0.26mgmL by chromatogram methanol constant volume
-1and 0.25mgmL
-1mixing reference substance solution.
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| CN106770768B (en) * | 2016-12-26 | 2019-12-17 | 上海微谱化工技术服务有限公司 | Separation and detection method of flavonoid glycoside compounds in fructus aurantii prepared in traditional Chinese medicine decoction pieces |
| CN109633033A (en) * | 2019-01-29 | 2019-04-16 | 江西省林业科学院 | A kind of characteristic pattern spectrometry identifying river Fructus Aurantii |
| CN109633033B (en) * | 2019-01-29 | 2022-01-28 | 江西省林业科学院 | Characteristic map method for identifying bitter orange in river |
| CN111198235A (en) * | 2020-01-13 | 2020-05-26 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Method for detecting content of isosinensetin in plasma |
| CN111198235B (en) * | 2020-01-13 | 2021-05-11 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Method for detecting content of isosinensetin in plasma |
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