A kind of method for building up of roxburgh anoectochilus terminal bud finger-print
Technical field
The invention belongs to traditional Chinese medicine quality control field, particularly relate to a kind of method for building up and Anoectochilus roxburghii 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 Anoectochilus Roxburghii Anoectochilus roburghii (Wall.) Lindl), anoectochilus formosanus Anoectochilus formosanus Hay, perennial Valuable Herbal Medicine.It is wider in usable range among the people, have the laudatory titles such as " king of medicine ", " gold grass ", " refreshing medicine " and " bird ginseng " in Hong Kong and Taiwan and south east asia.Property flat, taste is sweet, returns lung, liver, kidney, urinary bladder channel.The effect such as there is 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 for the treatment of the high heat of acute infantile convulsion wind, rheumatic arthritis, hepatitis, ephritis, cystitis, chyluria, blood urine, bronchitis, spitting of blood, diabetes, hypertension etc.; Modern for the difficult and complicated cases such as antitumor, improve immunity, liver protecting, reducing blood lipid, auxiliary anti arteriosclerosis and cerebral thrombus, antiviral, control myasthenia gravis, removing acne and freckle etc.
Roxburgh anoectochilus terminal bud is mainly distributed in the states such as Japan, China, Sri Lanka, India and the Nepal in Asia, there is its abundant herb resource in the provinces such as south China Fujian, Guangxi, Guangdong, Hainan, Guizhou, Sichuan, Yunnan, and Fujian and Taiwan two provinces are as medicinal mainly containing: three kinds of Anoectochilus roxburghii, anoectochilus formosanus and Kaohsiung roxburgh anoectochilus terminal buds.
Roxburgh anoectochilus terminal bud properties and characteristics: Anoectochilus roxburghii this product, for dry herb, is slightly shrunk, and stipes is obvious; Leaf alternate tool handle, the membranous sheath shape of phyllopodium is embraced stem, and leaf yellow green is avette or oval, and blade face is glossy, vein is golden yellow pinniform net arteries and veins, blade back lilac red; Fragrance is special.
Anoectochilus formosanus and Anoectochilus roxburghii main difference point are blackish green for leaf, and vein is white pinniform net arteries and veins.
Chemical composition and the content of roxburgh anoectochilus terminal bud are definite, the material with valuable pharmacological activity is 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), and Cardiac glycosides, ester class, taurine etc.
Aspect the quality assessment of roxburgh anoectochilus terminal bud medicinal material, in the document of delivering both at home and abroad at present, general Microscopic Identification, the thin-layer chromatography of adopting identified, or single component assay, as flavonoids, anoectochilus roxburghii glycosides etc., and as the quality evaluation index of roxburgh anoectochilus terminal bud medicinal material, obviously, roxburgh anoectochilus terminal bud quality of medicinal material can not accurately be differentiated and evaluate to this quality evaluating method.
Summary of the invention
The object of the invention is to overcome prior art defect, a kind of method for building up of the finger-print of differentiating roxburgh anoectochilus terminal bud true and false quality is provided.
Another object of the present invention, is to provide the finger-print of Anoectochilus roxburghii.
A further object of the present invention, is to provide the finger-print of anoectochilus formosanus.
Technical scheme of the present invention is as follows:
A method for building up for roxburgh anoectochilus terminal bud finger-print, adopts high performance liquid chromatography, specifically comprises the steps:
(1) preparation of need testing solution: precision takes test sample fine powder and is dissolved in methyl alcohol, carries out after ultrasonic processing, and centrifuging and taking supernatant, by this supernatant liquid filtering, obtains need testing solution;
(2) chromatographic condition: chromatographic column is take octadecylsilane chemically bonded silica as filler; Type of elution: take the gradient eluent of phosphoric acid and methyl alcohol composition as mobile phase, carry out gradient elution; Ultraviolet detects: 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, described step (1) is: precision takes test sample fine powder 0.1~5g and is placed in tool plug Erlenmeyer flask, and precision adds methyl alcohol 10~100mL, close plug, accurately weighed, ultrasonic processing, after 35~55 minutes, lets cool to room temperature, supplies the weight of less loss with methyl alcohol, shake up, after centrifugal 7~15 minutes, get supernatant, this supernatant, through membrane filtration, is obtained to need testing solution.
In a preferred embodiment of the invention, described step (1) is: precision takes test sample fine powder 0.2g and is placed in tool plug Erlenmeyer flask, and precision adds methyl alcohol 20mL, close plug, accurately weighed, ultrasonic processing, after 45 minutes, lets cool to room temperature, supplies the weight of less loss with methyl alcohol, shake up, after centrifugal 10 minutes, get supernatant, this supernatant, through 0.25 μ m membrane filtration, is obtained to need testing solution.
In a preferred embodiment of the invention, described step (2) is: chromatographic column is take octadecylsilane chemically bonded silica as filler; Type of elution: take the gradient eluent of 0.1~0.6% phosphoric acid and methyl alcohol composition as mobile phase, carry out gradient elution; Column temperature: 25~30 ℃; Ultraviolet detects: detection wavelength is 313~318nm; Flow velocity: 0.8~1.2mL/min.
In a preferred embodiment of the invention, described step (2) is: chromatographic column is take octadecylsilane chemically bonded silica as filler; Type of elution: take the gradient eluent of 0.4% phosphoric acid and methyl alcohol composition as mobile phase, carry out gradient elution; Column temperature: 30 ℃; Ultraviolet detects: detection wavelength is 316nm; Flow velocity: 1.0mL/min.
In a preferred embodiment of the invention, the program of the gradient elution in described step (2) is undertaken by following volumetric concentration configuration:
0 minute time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid solution;
30 minutes time, the methyl alcohol that mobile phase is 50% and 50% 0.4% phosphoric acid solution;
45 minutes time, the methyl alcohol that mobile phase is 75% and 25% 0.4% phosphoric acid solution;
55 minutes time, the methyl alcohol that mobile phase is 75% and 25% 0.4% phosphoric acid solution;
60 minutes time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid solution;
65 minutes time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid solution.
In a preferred embodiment of the invention, described 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 Anoectochilus roxburghii of the method for building up gained of the above-mentioned finger-print of a kind of use, described finger-print length is 65 minutes, have 10 characteristic peaks, wherein No. 5 characteristic peaks are forulic acid internal reference peak, relative retention time and the relative peak area of calculating respectively each characteristic peak with No. 5 characteristic peaks obtain described finger-print, 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 reproducibility RSD of above-mentioned characteristic peak is less than 2%, and peak area reappearance RSD is less than 3%.
The finger-print of the anoectochilus formosanus of the method for building up gained of the above-mentioned finger-print of a kind of use, described finger-print length is 65 minutes, have 11 characteristic peaks, wherein 4 ' number characteristic peak is forulic acid internal reference peak, relative retention time and the relative peak area of calculating respectively each characteristic peak with 4 ' number characteristic peak obtain described finger-print, 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 reproducibility RSD of above-mentioned characteristic peak is less than 2%, and peak area reappearance RSD is less than 3%.
The invention has the beneficial effects as follows:
1 method of the present invention uses high performance liquid chromatography to set up the finger-print of roxburgh anoectochilus terminal bud, improves the level of roxburgh anoectochilus terminal bud quality control.
The index that 2 methods of the present invention are quality control by the single index components of routine or active component rises to a new stage, with the fingerprint characteristic of chromatogram, and the quantization parameter obtaining by finger-print, more effectively discern the false from the genuine, control quality.
3 the present invention propose the fingerprint discrimination method at a kind of internal reference peak, have solved the difficult problem of current shortage roxburgh anoectochilus terminal bud about composition reference substance.Adopt internal reference peak as reference, relative retention time and the relative peak area of the each characteristic peak obtaining are more stable, and 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.
Accompanying drawing explanation
Fig. 1 is the HPLC finger-print of Anoectochilus roxburghii of the present invention (sharp leaf) group training product;
Fig. 2 is the HPLC finger-print of Anoectochilus roxburghii of the present invention (roundleaf) group training product;
Fig. 3 is the HPLC finger-print of the wild product of Anoectochilus roxburghii of the present invention (great Ye);
Fig. 4 is the HPLC finger-print of the wild product of Anoectochilus roxburghii of the present invention (leaflet);
Fig. 5 is the HPLC finger-print of anoectochilus formosanus of the present invention (first-class) group training product;
Fig. 6 is the HPLC finger-print of anoectochilus formosanus of the present invention (second-class) group training product;
Fig. 7 is the HPLC finger-print of anoectochilus formosanus of the present invention (third-class) group training product;
Fig. 8 is the HPLC finger-print of anoectochilus formosanus of the present invention (pure scented tea) group training product;
Fig. 9 is the HPLC finger-print of commercially available roxburgh anoectochilus terminal bud adulterant 1;
Figure 10 is the HPLC finger-print of commercially available roxburgh anoectochilus terminal bud adulterant 2;
Figure 11 is the HPLC finger-print of commercially available roxburgh anoectochilus terminal bud adulterant 3;
Figure 12 is the HPLC finger-print of commercially available roxburgh anoectochilus terminal bud adulterant 4;
Figure 13 is the HPLC finger-print of commercially available variegated leaf orchid;
Embodiment
By embodiment, technical scheme of the present invention is further detailed and is described below.
Embodiment 1
Set up Anoectochilus roxburghii finger-print:
(1) get respectively 4 batches of certified products Anoectochilus roxburghii group training product and carry out the preparation of need testing solution, be specially: the accurate fine powder 0.2g that takes above-mentioned test sample is placed in tool plug Erlenmeyer flask respectively, and precision adds methyl alcohol 20mL, close plug, accurately weighed, ultrasonic processing, after 45 minutes, lets cool to room temperature, supplies the weight of less loss with methyl alcohol, shake up, after centrifugal 10 minutes, get supernatant, this supernatant, through 0.25 μ m membrane filtration, is obtained to need testing solution;
(2) chromatographic condition: chromatographic column is Aglient Extend C18(4.6 × 250mm, and 5 μ m); Type of elution: take the gradient eluent of 0.4% phosphoric acid and methyl alcohol composition as mobile phase, carry out gradient elution; Column temperature: 30 ℃; Ultraviolet detects: detection wavelength is 316nm; Flow velocity: 1.0mL/min; Wherein the program of gradient elution is undertaken by following volumetric concentration configuration:
0 minute time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid solution;
30 minutes time, the methyl alcohol that mobile phase is 50% and 50% 0.4% phosphoric acid solution;
45 minutes time, the methyl alcohol that mobile phase is 75% and 25% 0.4% phosphoric acid solution;
55 minutes time, the methyl alcohol that mobile phase is 75% and 25% 0.4% phosphoric acid solution;
60 minutes time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid solution;
65 minutes time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid solution.
(3) measure: the accurate above-mentioned need testing solution 5 μ L of absorption respectively, be injected into high performance liquid chromatograph, according to high effective liquid chromatography for measuring, and carry out total score and analyse, set up following Anoectochilus roxburghii finger-print: described finger-print length is 65 minutes, have 10 characteristic peaks, No. 5 characteristic peaks are forulic acid, get this peak as internal reference peak, relative retention time and the relative peak area of calculating respectively each characteristic peak with No. 5 characteristic peaks obtain described finger-print, 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 reproducibility RSD of above-mentioned characteristic peak is less than 2%, and peak area reappearance RSD is less than 3%.
Embodiment 2
(1) foundation of test sample finger-print, comprises the steps:
The preparation of a need testing solution: get respectively Anoectochilus roxburghii (sharp leaf) group training product, Anoectochilus roxburghii (roundleaf) group training product, the wild product of Anoectochilus roxburghii (great Ye), the wild product of Anoectochilus roxburghii (leaflet), commercially available roxburgh anoectochilus terminal bud adulterant 1, commercially available roxburgh anoectochilus terminal bud adulterant 2, commercially available roxburgh anoectochilus terminal bud adulterant 3, commercially available roxburgh anoectochilus terminal bud adulterant 4 and commercially available variegated leaf orchid carry out the preparation of need testing solution, be specially: the accurate fine powder 0.2g that takes above-mentioned test sample is placed in tool plug Erlenmeyer flask respectively, precision adds methyl alcohol 20mL, close plug, accurately weighed, ultrasonic processing is after 45 minutes, let cool to room temperature, supply the weight of less loss with methyl alcohol, shake up, after centrifugal 10 minutes, get supernatant, by this supernatant through 0.25 μ m membrane filtration, obtain need testing solution, number consecutively good fortune 1 respectively, good fortune 2, good fortune 3, good fortune 4, pseudo-1, pseudo-2, pseudo-3, puppet 4 and variegated leaf,
B chromatographic condition: chromatographic column is Aglient Extend C18(4.6 × 250mm, and 5 μ m); Type of elution: take the gradient eluent of 0.4% phosphoric acid and methyl alcohol composition as mobile phase, carry out gradient elution; Column temperature: 30 ℃; Ultraviolet detects: detection wavelength is 316nm; Flow velocity: 1.0mL/min; Wherein the program of gradient elution is undertaken by following volumetric concentration configuration:
0 minute time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid; 30 minutes time, the methyl alcohol that mobile phase is 50% and 50% 0.4% phosphoric acid; 45 minutes time, the methyl alcohol that mobile phase is 75% and 25% 0.4% phosphoric acid; 55 minutes time, the methyl alcohol that mobile phase is 75% and 25% 0.4% phosphoric acid; 60 minutes time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid; 65 minutes time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid.
C measures: the above-mentioned need testing solution 5 μ L of accurate absorption respectively, be injected into high performance liquid chromatograph, and according to high effective liquid chromatography for measuring, obtain the finger-print of each test sample, as shown in Figure 1 to Figure 4, shown in Fig. 9 to Figure 13.
(2) by above-mentioned each test sample finger-print all take No. 5 forulic acids as internal reference peak, relative retention time and the relative peak area of each test sample characteristic peak are as shown in the table:
Analyzing of the Anoectochilus roxburghii finger-print that data in upper table and embodiment 1 are set up is more known, and method of the present invention can be differentiated Anoectochilus roxburghii genuine piece and commercially available roxburgh anoectochilus terminal bud adulterant, and the quality of Anoectochilus roxburghii is evaluated.
Embodiment 3
Set up anoectochilus formosanus finger-print:
(1) get respectively 4 batches of certified products anoectochilus formosanus and carry out the preparation of need testing solution, be specially: the accurate fine powder 0.2g that takes above-mentioned test sample is placed in tool plug Erlenmeyer flask respectively, and precision adds methyl alcohol 20mL, close plug, accurately weighed, ultrasonic processing, after 45 minutes, lets cool to room temperature, supplies the weight of less loss with methyl alcohol, shake up, after centrifugal 10 minutes, get supernatant, this supernatant, through 0.25 μ m membrane filtration, is obtained to need testing solution;
(2) chromatographic condition: chromatographic column is Aglient Extend C18(4.6 × 250mm, and 5 μ m); Type of elution: take the gradient eluent of 0.4% phosphoric acid and methyl alcohol composition as mobile phase, carry out gradient elution; Column temperature: 30 ℃; Ultraviolet detects: detection wavelength is 316nm; Flow velocity: 1.0mL/min; Wherein the program of gradient elution is undertaken by following volumetric concentration configuration:
0 minute time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid solution;
30 minutes time, the methyl alcohol that mobile phase is 50% and 50% 0.4% phosphoric acid solution;
45 minutes time, the methyl alcohol that mobile phase is 75% and 25% 0.4% phosphoric acid solution;
55 minutes time, the methyl alcohol that mobile phase is 75% and 25% 0.4% phosphoric acid solution;
60 minutes time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid solution;
65 minutes time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid solution.
(3) measure: the accurate above-mentioned need testing solution 5 μ L of absorption respectively, be injected into high performance liquid chromatograph, according to high effective liquid chromatography for measuring, and carry out total score and analyse, set up following anoectochilus formosanus finger-print: described finger-print length is 65 minutes, have 10 characteristic peaks, No. 5 characteristic peaks are forulic acid, get this peak as internal reference peak, relative retention time and the relative peak area of calculating respectively each characteristic peak with No. 5 characteristic peaks obtain described finger-print, 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 reproducibility RSD of above-mentioned characteristic peak is less than 2%, and peak area reappearance RSD is less than 3%.
Embodiment 4
(1) foundation of test sample finger-print, comprises the steps:
The preparation of a need testing solution: get respectively anoectochilus formosanus (first-class) group training product, anoectochilus formosanus (second-class) group training product, anoectochilus formosanus (third-class) group training product, anoectochilus formosanus (pure scented tea) group training product, commercially available roxburgh anoectochilus terminal bud adulterant 1, commercially available roxburgh anoectochilus terminal bud adulterant 2, commercially available roxburgh anoectochilus terminal bud adulterant 3, commercially available roxburgh anoectochilus terminal bud adulterant 4 and commercially available variegated leaf orchid carry out the preparation of need testing solution, be specially: the accurate fine powder 0.2g that takes above-mentioned test sample is placed in tool plug Erlenmeyer flask respectively, precision adds methyl alcohol 20mL, close plug, accurately weighed, ultrasonic processing is after 45 minutes, let cool to room temperature, supply the weight of less loss with methyl alcohol, shake up, after centrifugal 10 minutes, get supernatant, by this supernatant through 0.25 μ m membrane filtration, obtain need testing solution, number respectively platform 1, platform 2, platform 3, platform is pure, pseudo-1, pseudo-2, pseudo-3, puppet 4 and variegated leaf,
B chromatographic condition: chromatographic column is Aglient Extend C18(4.6 × 250mm, and 5 μ m); Type of elution: take the gradient eluent of 0.4% phosphoric acid and methyl alcohol composition as mobile phase, carry out gradient elution; Column temperature: 30 ℃; Ultraviolet detects: detection wavelength is 316nm; Flow velocity: 1.0mL/min; Wherein the program of gradient elution is undertaken by following volumetric concentration configuration:
0 minute time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid; 30 minutes time, the methyl alcohol that mobile phase is 50% and 50% 0.4% phosphoric acid; 45 minutes time, the methyl alcohol that mobile phase is 75% and 25% 0.4% phosphoric acid; 55 minutes time, the methyl alcohol that mobile phase is 75% and 25% 0.4% phosphoric acid; 60 minutes time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid; 65 minutes time, the methyl alcohol that mobile phase is 25% and 75% 0.4% phosphoric acid.
C measures: the above-mentioned need testing solution 5 μ L of accurate absorption respectively, be injected into high performance liquid chromatograph, and according to high effective liquid chromatography for measuring, obtain the finger-print of each test sample, as Fig. 5 to Fig. 8, shown in Fig. 9 to Figure 13.
(2) by above-mentioned each test sample finger-print all take 4 ' number forulic acid as internal reference peak, relative retention time and the relative peak area of each test sample characteristic peak are as shown in the table:
Analyzing of the anoectochilus formosanus finger-print that data in upper table and embodiment 3 are set up is more known, and method of the present invention can be differentiated anoectochilus formosanus genuine piece and commercially available roxburgh anoectochilus terminal bud adulterant, and the quality of Anoectochilus roxburghii is evaluated.
The above, be only preferred embodiment of the present invention, therefore can not limit according to this scope of the invention process, the equivalence done according to the scope of the claims of the present invention and description changes and modifies, and all should still belong in the scope that the present invention contains.