CN102768254A - Method for establishing Anoectochilus roxburghii fingerprint, and Anoectochilus roxburghii fingerprint and Anoectochilus formosanus Hay fingerprint - Google Patents
Method for establishing Anoectochilus roxburghii fingerprint, and Anoectochilus roxburghii fingerprint and Anoectochilus formosanus Hay fingerprint Download PDFInfo
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- CN102768254A CN102768254A CN2012102037834A CN201210203783A CN102768254A CN 102768254 A CN102768254 A CN 102768254A CN 2012102037834 A CN2012102037834 A CN 2012102037834A CN 201210203783 A CN201210203783 A CN 201210203783A CN 102768254 A CN102768254 A CN 102768254A
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention discloses a method for establishing an Anoectochilus roxburghii fingerprint, and an Anoectochilus roxburghii fingerprint and an Anoectochilus formosanus Hay fingerprint. The method provided by the present invention comprises the following factors: (1) preparation of a test solution; (2) chromatographic condition: using an octadecylsilane bonded silica gel as a column filler; elution mode: using a gradient elution solution consisting of phosphoric acid and methanol as a mobile phase to conduct gradient elution; and ultraviolet detection with a detection wave length of 310-320nm; and (3) determination in accordance with a high performance liquid chromatography to obtain a fingerprint. The method provided by the invention employs high-performance liquid chromatography to establish the Anoectochilus roxburghii fingerprint and improve the level of quality control of Anoectochilus roxburghii; the invention provides an internal control peak fingerprint discrimination method to solve the problem of lack of reference substance for relevant components of Anoectochilus roxburghii; and the internal control peak is used as a reference to obtain stable relative retention time of each characteristic peak and relative peak area, and realize better precision of the method.
Description
Technical field
The invention belongs to traditional Chinese medicine quality control field, particularly relate to a kind of method for building up and Fujian roxburgh anoectochilus terminal bud and anoectochilus formosanus finger-print of roxburgh anoectochilus terminal bud finger-print.
Background technology
Roxburgh anoectochilus terminal bud is that the orchid family is opened lip epidendrum floral leaf and opened blue Anoectochilus roburghii (Wall.) Lindl of lip), anoectochilus formosanus Anoectochilus formosanus Hay, perennial rare Chinese herbal medicine.It is wider in usable range among the people, have " king of medicine ", " gold grass ", " refreshing medicine " and laudatory titles such as " bird gensengs " in Hong Kong and Taiwan and south east asia.The property flat, flavor is sweet, returns lung, liver, kidney, urinary bladder channel.Effect such as have clearing heat and cooling blood, removing toxicity for detumescence, expelling wind and removing dampness, relieving convulsion flat liver, kidney tonifying, diuresis, moisten the lung and relieve the cough.Be used to treat the high heat of acute infantile convulsion wind, rheumatic arthritis, hepatitis, ephritis, cystitis, chyluria, blood urine, bronchitis, spitting of blood, diabetes, hypertension etc.; Modern times are used for difficult and complicated cases such as antitumor, improve immunity, liver protecting, reducing blood lipid, auxiliary anti arteriosclerosis and cerebral thrombus, antiviral, control myasthenia gravis, dispel whelk and freckle etc.
Roxburgh anoectochilus terminal bud mainly is distributed in the states such as Japan, China, Sri Lanka, India and Nepal in Asia; All there is its herb resource that enriches in province such as Fujian, China south, Guangxi, Guangdong, Hainan, Guizhou, Sichuan, Yunnan, and Fujian and Taiwan two provinces are as medicinal mainly containing: three kinds of Fujian roxburgh anoectochilus terminal bud, anoectochilus formosanus and Kaohsiung roxburgh anoectochilus terminal buds.
The roxburgh anoectochilus terminal bud properties and characteristics: Fujian these article of roxburgh anoectochilus terminal bud are dry herb, slightly are shrunk, and stipes is obvious; Leaf alternate tool handle, the membranous sheath shape of phyllopodium is embraced stem, and the leaf yellow green is avette or oval, and the blade face is glossy, vein is golden yellow pinniform net arteries and veins, the blade back lilac red; Fragrance is special.
Anoectochilus formosanus and Fujian roxburgh anoectochilus terminal bud main difference point are blackish green for leaf, and vein is white pinniform net arteries and veins.
The chemical constitution and the content of roxburgh anoectochilus terminal bud are definite, have the active material of valuable pharmacological and are mainly: flavone compound (Quercetin, Isorhamnetin), steroid compound (24～isopropenyl cholesterol, open the blue sterol of lip, ergosterol, stigmasterol, campesterol, β～sitosterol etc.); Triterpene compound (suberone, succinic acid, coumaric acid, forulic acid, daucosterol, palmitic acid, oleanolic acid, ursolic acid); Polysaccharide (polysaccharide 13.326%, compound sugar 11.243%, reducing sugar 9.73%); Alkaloid (huperzine and aconitine); With the cardiac stimulant glucoside, ester class, taurine etc.
Aspect the quality assessment of roxburgh anoectochilus terminal bud medicinal material; In the document of delivering at present, generally adopt microscopical identification, thin-layer chromatography to identify both at home and abroad, or the single component assay; Like flavonoids, anoectochilus roxburghii glycosides etc.; And as the quality evaluation index of roxburgh anoectochilus terminal bud medicinal material, obviously, the roxburgh anoectochilus terminal bud quality of medicinal material can not accurately differentiated and estimate to this quality evaluating method.
Summary of the invention
The objective of the invention is to overcome the prior art defective, a kind of method for building up of differentiating the finger-print that the roxburgh anoectochilus terminal bud true and false is good and bad is provided.
Another object of the present invention provides the finger-print of Fujian roxburgh anoectochilus terminal bud.
A purpose more of the present invention provides the finger-print of anoectochilus formosanus.
Technical scheme of the present invention is following:
A kind of method for building up of roxburgh anoectochilus terminal bud finger-print adopts high performance liquid chromatography, specifically comprises the steps:
(1) preparation of need testing solution: precision takes by weighing the test sample fine powder and is dissolved in the methyl alcohol, carry out sonicated after, the centrifuging and taking supernatant with this supernatant liquid filtering, promptly gets need testing solution;
(2) chromatographic condition: chromatographic column is filler with the octadecylsilane chemically bonded silica; Type of elution: the gradient eluent of forming with phosphoric acid and methyl alcohol is a moving phase, carries out gradient elution; Ultraviolet detection: the detection wavelength is 310～320nm.
(3) measure: the accurate need testing solution of drawing, be injected into high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtain finger-print.
In a preferred embodiment of the invention, said step (1) is: precision takes by weighing test sample fine powder 0.1～5g and places tool plug Erlenmeyer flask, the accurate methyl alcohol 10～100mL that adds; Close plug, the accurate title, decide, and sonicated is after 35～55 minutes; Put and be chilled to room temperature, supply the weight that subtracts mistake, shake up with methyl alcohol; Get supernatant after centrifugal 7～15 minutes, this supernatant through membrane filtration, is promptly got need testing solution.
In a preferred embodiment of the invention, said step (1) is: precision takes by weighing test sample fine powder 0.2g and places tool plug Erlenmeyer flask, the accurate methyl alcohol 20mL that adds; Close plug, the accurate title, decide, and sonicated is after 45 minutes; Put and be chilled to room temperature, supply the weight that subtracts mistake, shake up with methyl alcohol; Get supernatant after centrifugal 10 minutes, this supernatant through 0.25 μ m membrane filtration, is promptly got need testing solution.
In a preferred embodiment of the invention, said step (2) is: chromatographic column is filler with the octadecylsilane chemically bonded silica; Type of elution: the gradient eluent of forming with 0.1～0.6% phosphoric acid and methyl alcohol is a moving phase, carries out gradient elution; Column temperature: 25～30 ℃; Ultraviolet detection: the detection wavelength is 313～318nm; Flow velocity: 0.8～1.2mL/min.
In a preferred embodiment of the invention, said step (2) is: chromatographic column is filler with the octadecylsilane chemically bonded silica; Type of elution: the gradient eluent of forming with 0.4% phosphoric acid and methyl alcohol is a moving phase, carries out gradient elution; Column temperature: 30 ℃; Ultraviolet detection: the detection wavelength is 316nm; Flow velocity: 1.0mL/min.
In a preferred embodiment of the invention, the program of the gradient elution in the said step (2) is undertaken by following volumetric concentration configuration:
In the time of 0 minute, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution;
In the time of 30 minutes, moving phase is 50% methyl alcohol and 50% 0.4% phosphoric acid solution;
In the time of 45 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid solution;
In the time of 55 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid solution;
In the time of 60 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution;
In the time of 65 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution.
In a preferred embodiment of the invention, said step (3) is: the accurate need testing solution 5 μ L that draw, be injected into high performance liquid chromatograph, and according to high effective liquid chromatography for measuring, obtain finger-print.
The finger-print of the Fujian roxburgh anoectochilus terminal bud of the method for building up gained of the above-mentioned finger-print of a kind of usefulness; Said finger-print length is 65 minutes; Have 10 characteristic peaks; Wherein No. 5 characteristic peaks be in the forulic acid with reference to the peak, relative retention time and the relative peak area of calculating each characteristic peak respectively with No. 5 characteristic peaks get said finger-print, and be specific as follows:
No. 1 peak, relative retention time 0.16, relative peak area 0.27～2.08;
No. 2 peaks, relative retention time 0.59～0.60, relative peak area 0.44～0.69;
No. 3 peaks, relative retention time 0.91～0.92, relative peak area 0.60～2.21;
No. 4 peaks, relative retention time 0.96, relative peak area 0.27～0.88;
No. 5 peaks, relative retention time 1.00, relative peak area 1.00;
No. 6 peaks, relative retention time 1.12, relative peak area 0.52～10.18;
No. 7 peaks, relative retention time 1.20, relative peak area 0.74～2.22;
No. 8 peaks, relative retention time 1.35, relative peak area 0.36～2.13;
No. 9 peaks, relative retention time 1.74, relative peak area 2.56～5.53;
No. 10 peaks, relative retention time 2.13～2.14, relative peak area 0.47～1.00;
The retention time reappearance RSD of above-mentioned characteristic peak is less than 2%, and RSD is less than 3% for the peak area reappearance.
The finger-print of the anoectochilus formosanus of the method for building up gained of the above-mentioned finger-print of a kind of usefulness; Said finger-print length is 65 minutes; Have 11 characteristic peaks; Wherein 4 ' number characteristic peak be in the forulic acid with reference to the peak, relative retention time and the relative peak area of calculating each characteristic peak respectively with 4 ' number characteristic peak get said finger-print, and be specific as follows:
1 ' number peak, relative retention time 0.16, relative peak area 0.48～1.34;
2 ' number peak, relative retention time 0.91～0.92, relative peak area 1.25～2.86;
3 ' number peak, relative retention time 0.96, relative peak area 0.24～0.33;
4 ' number peak, relative retention time 1.00, relative peak area 1.00;
5 ' number peak, relative retention time 1.12, relative peak area 0.61～1.94;
6 ' number peak, relative retention time 1.20, relative peak area 0.65～2.58;
7 ' number peak, relative retention time 1.35～1.36, relative peak area 0.19～0.39;
8 ' number peak, relative retention time 1.70～1.72, relative peak area 0.14～0.40;
9 ' number peak, relative retention time 2.48～2.49, relative peak area 0.61～1.15;
10 ' number peak, relative retention time 2.53～2.54, relative peak area 1.27～4.64;
11 ' number peak, relative retention time 2.55～2.57, relative peak area 0.25～0.62;
The retention time reappearance RSD of above-mentioned characteristic peak is less than 2%, and RSD is less than 3% for the peak area reappearance.
The invention has the beneficial effects as follows:
1 method utilization high performance liquid chromatography of the present invention is set up the finger-print of roxburgh anoectochilus terminal bud, improves the level of roxburgh anoectochilus terminal bud quality control.
2 methods of the present invention rise to a new stage with the index that the single index components or the active component of routine is quality control, with the fingerprint characteristic of chromatogram with through the quantization parameter that finger-print obtains, more effectively discern the false from the genuine control of quality.
3 the present invention propose a kind of interior fingerprint discrimination method with reference to the peak, have solved the difficult problem of the relevant composition reference substance of present shortage roxburgh anoectochilus terminal bud.In adopting with reference to the peak as reference, the relative retention time and the relative peak area of each characteristic peak that obtains are more stable, method precision is better.
The good stability of 4 need testing solutions of the present invention, at room temperature, within 48 hours, it is stable that need testing solution all can keep.
Description of drawings
Fig. 1 is the HPLC finger-print of Fujian of the present invention roxburgh anoectochilus terminal bud (sharp leaf) group training article;
Fig. 2 is the HPLC finger-print of Fujian of the present invention roxburgh anoectochilus terminal bud (roundleaf) group training article;
Fig. 3 is the HPLC finger-print of the wild article of Fujian of the present invention roxburgh anoectochilus terminal bud (Da Ye);
Fig. 4 is the HPLC finger-print of the wild article of Fujian of the present invention roxburgh anoectochilus terminal bud (leaflet);
Fig. 5 is the HPLC finger-print of anoectochilus formosanus of the present invention (first-class) group training article;
Fig. 6 is the HPLC finger-print of anoectochilus formosanus of the present invention (second-class) group training article;
Fig. 7 is the HPLC finger-print of anoectochilus formosanus of the present invention (third-class) group training article;
Fig. 8 is the HPLC finger-print of anoectochilus formosanus of the present invention (pure scented tea) group training article;
Fig. 9 is the HPLC finger-print of the pseudo-article 1 of commercially available roxburgh anoectochilus terminal bud;
Figure 10 is the HPLC finger-print of the pseudo-article 2 of commercially available roxburgh anoectochilus terminal bud;
Figure 11 is the HPLC finger-print of the pseudo-article 3 of commercially available roxburgh anoectochilus terminal bud;
Figure 12 is the HPLC finger-print of the pseudo-article 4 of commercially available roxburgh anoectochilus terminal bud;
Figure 13 is the blue HPLC finger-print of commercially available variegated leaf;
Embodiment
Below technical scheme of the present invention further explained and described through embodiment.
Set up Fujian roxburgh anoectochilus terminal bud finger-print:
(1) get 4 batches of preparations of carrying out need testing solution of certified products Fujian roxburgh anoectochilus terminal bud group training article respectively, be specially: the accurate respectively fine powder 0.2g that takes by weighing above-mentioned test sample places tool plug Erlenmeyer flask, the accurate methyl alcohol 20mL that adds; Close plug, the accurate title, decide, and sonicated is after 45 minutes; Put and be chilled to room temperature, supply the weight that subtracts mistake, shake up with methyl alcohol; Get supernatant after centrifugal 10 minutes, this supernatant through 0.25 μ m membrane filtration, is promptly got need testing solution;
(2) chromatographic condition: chromatographic column is Aglient Extend C18 (4.6 * 250mm, 5 μ m); Type of elution: the gradient eluent of forming with 0.4% phosphoric acid and methyl alcohol is a moving phase, carries out gradient elution; Column temperature: 30 ℃; Ultraviolet detection: the detection wavelength is 316nm; Flow velocity: 1.0mL/min; Wherein the program of gradient elution is undertaken by following volumetric concentration configuration:
In the time of 0 minute, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution;
In the time of 30 minutes, moving phase is 50% methyl alcohol and 50% 0.4% phosphoric acid solution;
In the time of 45 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid solution;
In the time of 55 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid solution;
In the time of 60 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution;
In the time of 65 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution.
(3) measure: the accurate respectively above-mentioned need testing solution 5 μ L of absorption are injected into high performance liquid chromatograph, according to high effective liquid chromatography for measuring; And carry out analysis-by-synthesis, set up following Fujian roxburgh anoectochilus terminal bud finger-print: said finger-print length is 65 minutes, has 10 characteristic peaks; No. 5 characteristic peaks are forulic acid; Get this peak as interior with reference to the peak, relative retention time and the relative peak area of calculating each characteristic peak respectively with No. 5 characteristic peaks get said finger-print, and be specific as follows:
No. 1 peak, relative retention time 0.16, relative peak area 0.27～2.08;
No. 2 peaks, relative retention time 0.59～0.60, relative peak area 0.44～0.69;
No. 3 peaks, relative retention time 0.91～0.92, relative peak area 0.60～2.21;
No. 4 peaks, relative retention time 0.96, relative peak area 0.27～0.88;
No. 5 peaks, relative retention time 1.00, relative peak area 1.00;
No. 6 peaks, relative retention time 1.12, relative peak area 0.52～10.18;
No. 7 peaks, relative retention time 1.20, relative peak area 0.74～2.22;
No. 8 peaks, relative retention time 1.35, relative peak area 0.36～2.13;
No. 9 peaks, relative retention time 1.74, relative peak area 2.56～5.53;
No. 10 peaks, relative retention time 2.13～2.14, relative peak area 0.47～1.00;
The retention time reappearance RSD of above-mentioned characteristic peak is less than 2%, and RSD is less than 3% for the peak area reappearance.
(1) foundation of test sample finger-print comprises the steps:
The preparation of a need testing solution: get Fujian roxburgh anoectochilus terminal bud (sharp leaf) group training article, Fujian roxburgh anoectochilus terminal bud (roundleaf) group training article, the wild article of Fujian roxburgh anoectochilus terminal bud (Da Ye), the wild article of Fujian roxburgh anoectochilus terminal bud (leaflet), the pseudo-article 1 of commercially available roxburgh anoectochilus terminal bud, the pseudo-article 2 of commercially available roxburgh anoectochilus terminal bud, the pseudo-article 3 of commercially available roxburgh anoectochilus terminal bud, the pseudo-article 4 of commercially available roxburgh anoectochilus terminal bud and commercially available variegated leaf orchid respectively and carry out the preparation of need testing solution, be specially: the accurate respectively fine powder 0.2g that takes by weighing above-mentioned test sample places tool plug Erlenmeyer flask, the accurate methyl alcohol 20mL that adds; Close plug; The accurate title, decide, and sonicated was put and is chilled to room temperature after 45 minutes; Supply the weight that subtracts mistake with methyl alcohol; Shake up, get supernatant after centrifugal 10 minutes, with this supernatant warp 0.25 μ m membrane filtration; Promptly get need testing solution, respectively number consecutively good fortune 1, good fortune 2, good fortune 3, good fortune 4, puppet 1, puppet 2, puppet 3, puppet 4 and variegated leaf;
The b chromatographic condition: chromatographic column is Aglient Extend C18 (4.6 * 250mm, 5 μ m); Type of elution: the gradient eluent of forming with 0.4% phosphoric acid and methyl alcohol is a moving phase, carries out gradient elution; Column temperature: 30 ℃; Ultraviolet detection: the detection wavelength is 316nm; Flow velocity: 1.0mL/min; Wherein the program of gradient elution is undertaken by following volumetric concentration configuration:
In the time of 0 minute, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid; In the time of 30 minutes, moving phase is 50% methyl alcohol and 50% 0.4% phosphoric acid; In the time of 45 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid; In the time of 55 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid; In the time of 60 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid; In the time of 65 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid.
C measures: the above-mentioned need testing solution 5 μ L of accurate respectively absorption, be injected into high performance liquid chromatograph, and according to high effective liquid chromatography for measuring, obtain the finger-print of each test sample, like Fig. 1 to Fig. 4, Fig. 9 is to shown in Figure 13.
(2) be interior with reference to the peak with No. 5 forulic acids all with above-mentioned each test sample finger-print, the relative retention time and the relative peak area of each test sample characteristic peak are as shown in the table:
The analyzing comparison and can know that method of the present invention can be differentiated Fujian roxburgh anoectochilus terminal bud genuine piece and the pseudo-article of commercially available roxburgh anoectochilus terminal bud of the Fujian roxburgh anoectochilus terminal bud finger-print that data in the last table and embodiment 1 are set up, and the quality of Fujian roxburgh anoectochilus terminal bud is estimated.
Embodiment 3
Set up the anoectochilus formosanus finger-print:
(1) get 4 batches of preparations of carrying out need testing solution of certified products anoectochilus formosanus respectively, be specially: the accurate respectively fine powder 0.2g that takes by weighing above-mentioned test sample places tool plug Erlenmeyer flask, the accurate methyl alcohol 20mL that adds; Close plug, the accurate title, decide, and sonicated is after 45 minutes; Put and be chilled to room temperature, supply the weight that subtracts mistake, shake up with methyl alcohol; Get supernatant after centrifugal 10 minutes, this supernatant through 0.25 μ m membrane filtration, is promptly got need testing solution;
(2) chromatographic condition: chromatographic column is Aglient Extend C18 (4.6 * 250mm, 5 μ m); Type of elution: the gradient eluent of forming with 0.4% phosphoric acid and methyl alcohol is a moving phase, carries out gradient elution; Column temperature: 30 ℃; Ultraviolet detection: the detection wavelength is 316nm; Flow velocity: 1.0mL/min; Wherein the program of gradient elution is undertaken by following volumetric concentration configuration:
In the time of 0 minute, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution;
In the time of 30 minutes, moving phase is 50% methyl alcohol and 50% 0.4% phosphoric acid solution;
In the time of 45 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid solution;
In the time of 55 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid solution;
In the time of 60 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution;
In the time of 65 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution.
(3) measure: the accurate respectively above-mentioned need testing solution 5 μ L of absorption are injected into high performance liquid chromatograph, according to high effective liquid chromatography for measuring; And carry out analysis-by-synthesis, set up following anoectochilus formosanus finger-print: said finger-print length is 65 minutes, has 10 characteristic peaks; No. 5 characteristic peaks are forulic acid; Get this peak as interior with reference to the peak, relative retention time and the relative peak area of calculating each characteristic peak respectively with No. 5 characteristic peaks get said finger-print, and be specific as follows:
1 ' number peak, relative retention time 0.16, relative peak area 0.48～1.34;
2 ' number peak, relative retention time 0.91～0.92, relative peak area 1.25～2.86;
3 ' number peak, relative retention time 0.96, relative peak area 0.24～0.33;
4 ' number peak, relative retention time 1.00, relative peak area 1.00;
5 ' number peak, relative retention time 1.12, relative peak area 0.61～1.94;
6 ' number peak, relative retention time 1.20, relative peak area 0.65～2.58;
7 ' number peak, relative retention time 1.35～1.36, relative peak area 0.19～0.39;
8 ' number peak, relative retention time 1.70～1.72, relative peak area 0.14～0.40;
9 ' number peak, relative retention time 2.48～2.49, relative peak area 0.61～1.15;
10 ' number peak, relative retention time 2.53～2.54, relative peak area 1.27～4.64;
11 ' number peak, relative retention time 2.55～2.57, relative peak area 0.25～0.62;
The retention time reappearance RSD of above-mentioned characteristic peak is less than 2%, and RSD is less than 3% for the peak area reappearance.
(1) foundation of test sample finger-print comprises the steps:
The preparation of a need testing solution: get anoectochilus formosanus (first-class) group training article, anoectochilus formosanus (second-class) group training article, anoectochilus formosanus (third-class) group training article, anoectochilus formosanus (pure scented tea) group training article, the pseudo-article 1 of commercially available roxburgh anoectochilus terminal bud, the pseudo-article 2 of commercially available roxburgh anoectochilus terminal bud, the pseudo-article 3 of commercially available roxburgh anoectochilus terminal bud, the pseudo-article 4 of commercially available roxburgh anoectochilus terminal bud and commercially available variegated leaf orchid respectively and carry out the preparation of need testing solution, be specially: the accurate respectively fine powder 0.2g that takes by weighing above-mentioned test sample places tool plug Erlenmeyer flask, the accurate methyl alcohol 20mL that adds; Close plug; The accurate title, decide, and sonicated was put and is chilled to room temperature after 45 minutes; Supply the weight that subtracts mistake with methyl alcohol; Shake up, get supernatant after centrifugal 10 minutes, with this supernatant warp 0.25 μ m membrane filtration; Promptly get need testing solution, number platform 1, platform 2, platform 3, platform pure, pseudo-1, pseudo-2, pseudo-3, pseudo-4 and variegated leaf respectively;
The b chromatographic condition: chromatographic column is Aglient Extend C18 (4.6 * 250mm, 5 μ m); Type of elution: the gradient eluent of forming with 0.4% phosphoric acid and methyl alcohol is a moving phase, carries out gradient elution; Column temperature: 30 ℃; Ultraviolet detection: the detection wavelength is 316nm; Flow velocity: 1.0mL/min; Wherein the program of gradient elution is undertaken by following volumetric concentration configuration:
In the time of 0 minute, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid; In the time of 30 minutes, moving phase is 50% methyl alcohol and 50% 0.4% phosphoric acid; In the time of 45 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid; In the time of 55 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid; In the time of 60 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid; In the time of 65 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid.
C measures: the above-mentioned need testing solution 5 μ L of accurate respectively absorption, be injected into high performance liquid chromatograph, and according to high effective liquid chromatography for measuring, obtain the finger-print of each test sample, like Fig. 5 to Fig. 8, Fig. 9 is to shown in Figure 13.
(2) be interior with reference to the peak with 4 ' number forulic acid all with above-mentioned each test sample finger-print, the relative retention time and the relative peak area of each test sample characteristic peak are as shown in the table:
The analyzing comparison and can know that method of the present invention can be differentiated anoectochilus formosanus genuine piece and the pseudo-article of commercially available roxburgh anoectochilus terminal bud of the anoectochilus formosanus finger-print that data in the last table and embodiment 3 are set up, and the quality of Fujian roxburgh anoectochilus terminal bud is estimated.
The above is merely preferred embodiment of the present invention, so can not limit the scope that the present invention implements according to this, the equivalence of promptly doing according to claim of the present invention and description changes and modifies, and all should still belong in the scope that the present invention contains.
Claims (9)
1. the method for building up of a roxburgh anoectochilus terminal bud finger-print is characterized in that adopting high performance liquid chromatography, specifically comprises the steps:
(1) preparation of need testing solution: precision takes by weighing the test sample fine powder and is dissolved in the methyl alcohol, carry out sonicated after, the centrifuging and taking supernatant with this supernatant liquid filtering, promptly gets need testing solution;
(2) chromatographic condition: chromatographic column is filler with the octadecylsilane chemically bonded silica; Type of elution: the gradient eluent of forming with phosphoric acid and methyl alcohol is a moving phase, carries out gradient elution; Ultraviolet detection: the detection wavelength is 310～320nm;
(3) measure: the accurate need testing solution of drawing, be injected into high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtain finger-print.
2. the method for building up of a kind of roxburgh anoectochilus terminal bud finger-print as claimed in claim 1 is characterized in that: said step (1) is: precision takes by weighing test sample fine powder 0.1～5g and places tool plug Erlenmeyer flask, the accurate methyl alcohol 10～100mL that adds; Close plug, the accurate title, decide, and sonicated is after 35～55 minutes; Put and be chilled to room temperature, supply the weight that subtracts mistake, shake up with methyl alcohol; Get supernatant after centrifugal 7～15 minutes, this supernatant through membrane filtration, is promptly got need testing solution.
3. the method for building up of a kind of roxburgh anoectochilus terminal bud finger-print as claimed in claim 2 is characterized in that: said step (1) is: precision takes by weighing test sample fine powder 0.2g and places tool plug Erlenmeyer flask, the accurate methyl alcohol 20mL that adds; Close plug, the accurate title, decide, and sonicated is after 45 minutes; Put and be chilled to room temperature, supply the weight that subtracts mistake, shake up with methyl alcohol; Get supernatant after centrifugal 10 minutes, this supernatant through 0.25 μ m membrane filtration, is promptly got need testing solution.
4. the method for building up of a kind of roxburgh anoectochilus terminal bud finger-print as claimed in claim 1 is characterized in that: said step (2) is: chromatographic column is filler with the octadecylsilane chemically bonded silica; Type of elution: the gradient eluent of forming with 0.1～0.6% phosphoric acid and methyl alcohol is a moving phase, carries out gradient elution; Column temperature: 25～30 ℃; Ultraviolet detection: the detection wavelength is 313～318nm; Flow velocity: 0.8～1.2mL/min.
5. the method for building up of a kind of roxburgh anoectochilus terminal bud finger-print as claimed in claim 4 is characterized in that: said step (2) is: chromatographic column is filler with the octadecylsilane chemically bonded silica; Type of elution: the gradient eluent of forming with 0.4% phosphoric acid and methyl alcohol is a moving phase, carries out gradient elution; Column temperature: 30 ℃; Ultraviolet detection: the detection wavelength is 316nm; Flow velocity: 1.0mL/min.
6. the method for building up of a kind of roxburgh anoectochilus terminal bud finger-print as claimed in claim 5 is characterized in that: the program of the gradient elution in the said step (2) is undertaken by following volumetric concentration configuration:
In the time of 0 minute, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution;
In the time of 30 minutes, moving phase is 50% methyl alcohol and 50% 0.4% phosphoric acid solution;
In the time of 45 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid solution;
In the time of 55 minutes, moving phase is 75% methyl alcohol and 25% 0.4% phosphoric acid solution;
In the time of 60 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution;
In the time of 65 minutes, moving phase is 25% methyl alcohol and 75% 0.4% phosphoric acid solution.
7. the method for building up of a kind of roxburgh anoectochilus terminal bud finger-print as claimed in claim 1; It is characterized in that: said step (3) is: the accurate need testing solution 5 μ L that draw; Be injected into high performance liquid chromatograph,, obtain finger-print according to high effective liquid chromatography for measuring.
8. finger-print of the Fujian roxburgh anoectochilus terminal bud of the method for building up gained of finger-print according to claim 1; It is characterized in that: said finger-print length is 65 minutes; Have 10 characteristic peaks; Wherein No. 5 characteristic peaks be in the forulic acid with reference to the peak, relative retention time and the relative peak area of calculating each characteristic peak respectively with No. 5 characteristic peaks get said finger-print, and be specific as follows:
No. 1 peak, relative retention time 0.16, relative peak area 0.27～2.08;
No. 2 peaks, relative retention time 0.59～0.60, relative peak area 0.44～0.69;
No. 3 peaks, relative retention time 0.91～0.92, relative peak area 0.60～2.21;
No. 4 peaks, relative retention time 0.96, relative peak area 0.27～0.88;
No. 5 peaks, relative retention time 1.00, relative peak area 1.00;
No. 6 peaks, relative retention time 1.12, relative peak area 0.52～10.18;
No. 7 peaks, relative retention time 1.20, relative peak area 0.74～2.22;
No. 8 peaks, relative retention time 1.35, relative peak area 0.36～2.13;
No. 9 peaks, relative retention time 1.74, relative peak area 2.56～5.53;
No. 10 peaks, relative retention time 2.13～2.14, relative peak area 0.47～1.00;
The retention time reappearance RSD of above-mentioned characteristic peak is less than 2%, and RSD is less than 3% for the peak area reappearance.
9. finger-print of the anoectochilus formosanus of the method for building up gained of finger-print according to claim 1; It is characterized in that: said finger-print length is 65 minutes; Have 11 characteristic peaks; Wherein 4 ' number characteristic peak be in the forulic acid with reference to the peak, relative retention time and the relative peak area of calculating each characteristic peak respectively with 4 ' number characteristic peak get said finger-print, and be specific as follows:
1 ' number peak, relative retention time 0.16, relative peak area 0.48～1.34;
2 ' number peak, relative retention time 0.91～0.92, relative peak area 1.25～2.86;
3 ' number peak, relative retention time 0.96, relative peak area 0.24～0.33;
4 ' number peak, relative retention time 1.00, relative peak area 1.00;
5 ' number peak, relative retention time 1.12, relative peak area 0.61～1.94;
6 ' number peak, relative retention time 1.20, relative peak area 0.65～2.58;
7 ' number peak, relative retention time 1.35～1.36, relative peak area 0.19～0.39;
8 ' number peak, relative retention time 1.70～1.72, relative peak area 0.14～0.40;
9 ' number peak, relative retention time 2.48～2.49, relative peak area 0.61～1.15;
10 ' number peak, relative retention time 2.53～2.54, relative peak area 1.27～4.64;
11 ' number peak, relative retention time 2.55～2.57, relative peak area 0.25～0.62;
The retention time reappearance RSD of above-mentioned characteristic peak is less than 2%, and RSD is less than 3% for the peak area reappearance.
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| CN106546677A (en) * | 2016-10-27 | 2017-03-29 | 邵武市林业科学技术推广中心 | A kind of method based on finger print identification variety classes Herba Anoectochili roxburghii |
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| CN109022610A (en) * | 2018-09-13 | 2018-12-18 | 杭州师范大学 | A kind of molecular specificity labeled primers and its discrimination method of roxburgh anoectochilus terminal bud |
| CN109022610B (en) * | 2018-09-13 | 2021-08-10 | 杭州师范大学 | Molecular specificity marker primer of anoectochilus formosanus and identification method thereof |
| CN112240914A (en) * | 2019-07-18 | 2021-01-19 | 福建中医药大学 | Method for detecting flavone components in anoectochilus formosanus with different appearance phenotypes |
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