CN105136949B - A kind of Kiwi berry stem triterpenes HPLC finger-prints are set up and its finger-print - Google Patents
A kind of Kiwi berry stem triterpenes HPLC finger-prints are set up and its finger-print Download PDFInfo
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Abstract
Set up the invention discloses a kind of Kiwi berry stem triterpenes HPLC finger-prints and its finger-print, including：Medicinal material is dried in the shade, Kiwi berry cauline leaf, bud, delicious root herb powder, plus methanol are taken respectively, accurately weighed, heating and refluxing extraction 60min is let cool, with methanol polishing weight, filtering, container and filter residue are respectively washed several times with methanol, filtrate and cleaning fluid are merged, it is evaporated with Rotary Evaporators, again with methanol dissolving and cleaning container, cross 0.45um apertures miillpore filter, take subsequent filtrate as need testing solution after merging；Efficient liquid phase chromatographic analysis, mobile phase is the aqueous formic acid of acetonitrile 0.2%, and gradient elution obtains Kiwi berry branch, the finger-print of leaf medicinal material.The test sample of the present invention is simple for production, and chromatographic condition is easily realized；The stability and reappearance of method are all relatively good；Suitable for the discriminating and control to Kiwi berry branch, leaf, the flower pesticide material true and false, the place of production and quality.
Description
Technical field
Set up the invention belongs to food and medicine management domain, more particularly to a kind of Kiwi berry stem triterpenes HPLC finger-prints
And its finger-print.
Background technology
Traditional Chinese medicine fingerprint refers in particular to the Chinese medicine each component group that Chinese medicine sample is obtained through the modern analytical technique such as spectrum or chromatogram
The collection of illustrative plates or image of body characteristicses.Traditional Chinese medicine fingerprint is as a kind of synthesis, quantifiable quality control method, at present, by
Numerous international medical institutions are recognized, it has also become one of Chinese patent drug, natural drug quality control most efficient method.Eat in the U.S.
What product drug administration formulated in 1996《Guides of the FDA on vegetable products》In, it is desirable in the middle of plant material, autonomic drug
Product and botanical drug product provide corresponding finger-print.British Herbal Pharmacopoeia, India herbal medicine allusion quotation and Canadian medicinal and fragrant plant
Thing association, German medicinal plant association also receive chromatographic fingerprinting.China National Drug supervisory and management department was sent out in 2000
Cloth《The technical requirements (provisional) of the finger-print research of traditional Chinese medicine》, be distinctly claimed to the traditional Chinese medicine newly declared and
The traditional Chinese medicine listed carries out finger-print standard.Traditional Chinese medicine fingerprint have passed through the research of many decades, and having used includes
Ultraviolet, infrared, gas phase, efficient liquid phase, thin layer, nuclear magnetic resonance, SEM, computer image analysis, electrophoretic techniques,
Many modern analysis and test means such as isoenzyme analysis method, Protocols in Molecular Biology, Clustering Analysis Technology, are the material mirror of Chinese medicine
Fixed and constituent analysis have accumulated substantial amounts of data.But from the point of view of Chinese medicine overall quality control, also reach far away effectively anti-
Reflect and control Chinese medicine total quality purpose.High performance liquid chromatography (HPLC) finger-print is readily available accreditation in the world,
Many countries including the U.S., Britain, France, Canada, Germany, Japan and India are more willing to receive HPLC fingerprint images
Spectrum.Therefore, it is that Chinese medicine sets up corresponding HPLC finger-prints, is the effective means for controlling Chinese medicine inherent quality at this stage.Four
River is one of maximum main producing region of Kiwi berry, and its root, Chinese actinidia root exists as detoxicating and fighting cancer medicinal material《Jiangxi herbal medicine》、《Hunan medicine
Will》、《Zhejiang conventional herbal medicine among the people》、《Fujian traditional herbal medicine》、《Fujian east book on Chinese herbal medicine》、《Shaanxi Chinese herbal medicine》In it is all on the books, report
The compounds such as carbohydrate, triterpene, flavones are successively isolated in root；Another tool investigation, because Root Resource is limited, current Chinese Medicinal Materials Markets purchase
The Chinese actinidia root 90% entered fills root with stem.In terms of Kiwifruit Culture, entering the winter every year has a large amount of prunings, and cauline leaf, flower thinning are discarded；Therefore build
Vertical branch, leaf, flower active ingredient finger-print, are that both to have solved quality evaluation mode single, are not enough to system, intactly show medicine
The total quality of material, needs more advanced, the more effective quality control method problem of exploration badly；Actinidia can be made fully sharp again
With.
Current Kiwi berry branch, leaf, flower triterpenes finger-print have no report.
The content of the invention
Set up it is an object of the invention to provide a kind of Kiwi berry stem triterpenes HPLC finger-prints and its finger-print, purport
Due to the exhaustion of wild Fructus actinidiae chinensis resource, medicinal material market is pretended to be with other or substitutes Chinese actinidia root medicine with Kiwi berry branch stem
Can material turns into normality, and monitor the inherent quality evidence for lacking and judging to substitute, and can not judge different cultivars, position, the place of production
Kiwi berry rhizome makees the quality (i.e. active component content height) of medical material quanlity.
The present invention is achieved in that a kind of Kiwi berry stem triterpenes HPLC fingerprints, described macaque
Peach stem triterpenes HPLC fingerprints use high performance liquid chromatography, and specific step is as follows：
Step one, the preparation of reference substance solution, precision weighs oleanolic acid reference substance and ursolic acid reference substance, plus methanol system
Into each mixed solutions containing 0.1mg of every 1ml, shake up, produce；
Step 2, the preparation of need testing solution after pulverizing medicinal materials, takes 4.0g, is placed in round-bottomed flask, adds 40ml first
Alcohol, weighed weight, is heated to reflux 1h, lets cool, and with methanol polishing weight, shakes up, takes supernatant to centrifuge, produce；
Step 3, using octadecylsilane chemically bonded silica as filler；It is water-soluble with 0.2% formic acid using acetonitrile as mobile phase A
Liquid is that the regulation in Mobile phase B, according to the form below carries out gradient elution；
Step 4, it is accurate respectively to draw the μ l of the reference substance solution 10 and μ l of need testing solution 10, liquid chromatograph is injected, is surveyed
It is fixed, produce, total triterpene contentses are calculated with oleanolic acid and ursolic acid respectively, final result is in terms of average value.
Further, the gradient elution is configured with following volumetric concentration and carried out：
At 0 minute, mobile phase A is 30% acetonitrile solution, and Mobile phase B is 70% 0.2% aqueous formic acid；
At 16 minutes, mobile phase A is 45% acetonitrile solution, and Mobile phase B is 55% 0.2% aqueous formic acid；
At 22 minutes, mobile phase A is 46% acetonitrile solution, and Mobile phase B is 54% 0.2% aqueous formic acid；
At 30 minutes, mobile phase A is 54% acetonitrile solution, and Mobile phase B is 46% 0.2% aqueous formic acid；
At 34 minutes, mobile phase A is 66% acetonitrile solution, and Mobile phase B is 34% 0.2% aqueous formic acid；
At 38 minutes, mobile phase A is 70% acetonitrile solution, and Mobile phase B is 30% 0.2% aqueous formic acid；
At 46 minutes, mobile phase A is 90% acetonitrile solution, and Mobile phase B is 10% 0.2% aqueous formic acid；
At 56 minutes, mobile phase A is 90% acetonitrile solution, and Mobile phase B is 10% 0.2% aqueous formic acid；
At 57 minutes, mobile phase A is 30% acetonitrile solution, and Mobile phase B is 70% 0.2% aqueous formic acid；
At 69 minutes, mobile phase A is 30% acetonitrile solution, and Mobile phase B is 70% 0.2% aqueous formic acid.
Another object of the present invention is to provide a kind of Kiwi berry stem triterpenes HPLC finger-prints, the Kiwi berry stem three
The foundation of terpene HPLC finger-prints, specifically includes following steps：
Step one, the preparation of reference substance solution, precision weighs oleanolic acid reference substance and ursolic acid reference substance, plus methanol system
Into each mixed solutions containing 0.1mg of every 1ml, shake up, produce；
Step 2, the preparation of need testing solution after pulverizing medicinal materials, takes 4.0g, is placed in round-bottomed flask, adds 40ml first
Alcohol, weighed weight, is heated to reflux 1h, lets cool, and with methanol polishing weight, shakes up, takes supernatant to centrifuge, produce；
Step 3, using octadecylsilane chemically bonded silica as filler；It is water-soluble with 0.2% formic acid using acetonitrile as mobile phase A
Liquid is that the regulation in Mobile phase B, according to the form below carries out gradient elution；
Step 4, it is accurate respectively to draw the μ l of the reference substance solution 10 and μ l of need testing solution 10, liquid chromatograph is injected, is surveyed
It is fixed, produce, total triterpene contentses are calculated with oleanolic acid and ursolic acid respectively, final result is in terms of average value；
There is shared 15, peak in the finger-print；
No. 1 peak, Average residence time RT is 6.56min, and peak area is that 18213, RSD is 6.10%；
No. 2 peaks, Average residence time RT is 11.21min, and peak area is that 23654, RSD is 7.93%；
No. 3 peaks, Average residence time RT is 12.68min, and peak area is that 2630, RSD is 0.88%；
No. 4 peaks, Average residence time RT is 13.46min, and peak area is that 5384, RSD is 1.80%；
No. 5 peaks, Average residence time RT is 15.71min, and peak area is that 2009, RSD is 0.67%；
No. 6 peaks, Average residence time RT is 19.29min, and peak area is that 2286, RSD is 0.76%；
No. 7 peaks, Average residence time RT is 25.39min, and peak area is that 2758, RSD is 0.92%；
No. 8 peaks, Average residence time RT is 28.89min, and peak area is that 3299, RSD is 1.10%；
No. 9 peaks, Average residence time RT is 29.29min, and peak area is that 6543, RSD is 2.19%；
No. 10 peaks, Average residence time RT is 35.31min, and peak area is that 2784, RSD is 0.93%；
No. 11 peaks, Average residence time RT is 40.86min, and peak area is that 73140, RSD is 24.51%；
No. 12 peaks, Average residence time RT is 41.74min, and peak area is that 6092, RSD is 2.04%；
No. 13 peaks, Average residence time RT is 46.16min, and peak area is that 1662, RSD is 0.55%；
No. 14 peaks, Average residence time RT is 50.54min, and peak area is that 4755, RSD is 1.59%；
No. 15 peaks, Average residence time RT is-min, and peak area is smaller, RSD for-.
The invention has the advantages that：
Chinese actinidia root (Chinese actinidia root, Actinidia arguta) is used as internal heat anticance, clearing heat for detumescence medicinal material, existing thousands of in China
Year history, but as medicinal material tomography occurs in the exhaustion of wild resource, Chinese Medicinal Materials Markets often pretend to be root with Kiwi berry branch, and fish mesh is mixed
Miscellaneous, quality is difficult to ensure that.Finger-print research of the present invention, can be by different cultivars, different parts (branch, flower, root) and date-plum persimmon Mi
Monkey peach root is compared, by comparing its shared peak, it was demonstrated that branch is the extending part of root, with homologous homogeneity attribute, and is spent
Nuance can also be highlighted between branch, root；Set up, can be distinguished for substituting Chinese gooseberry by Kiwi berry triterpenes finger-print
Root makees the Kiwi berry branch of medicinal material and the difference of other plant roots, is the system that Kiwi berry branch substitutes Chinese actinidia root quality of medicinal material standard
Surely lay the foundation.
In addition, the present invention treats Kiwi berry branch, leaf, flower pesticide material as an entirety, can by comparing its shared peak
To find out the nuance between different medicinal materials；Test sample is simple for production, and chromatographic condition is easily realized；The stability of method and again
Existing property is all relatively good；Suitable for the discriminating and control to Kiwi berry branch, leaf, the flower pesticide material true and false, the place of production and quality.
Brief description of the drawings
Fig. 1 is Kiwi berry stem triterpenes HPLC fingerprint flow charts provided in an embodiment of the present invention；
Fig. 2 is that branch provided in an embodiment of the present invention is compared 20 shared peaks of triterpenes with root, and bud is compared triterpenes with root
15 shared peak schematic diagrames；
Fig. 3 is that 8 kinds Kiwi berry branch provided in an embodiment of the present invention is compared triterpenes fingerprint similarity height with root,
Bud is compared the low schematic diagram of triterpenes fingerprint similarity with root.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Below in conjunction with the accompanying drawings and specific embodiment to the present invention application principle be further described.
Refering to Fig. 1：
The present invention is achieved in that a kind of Kiwi berry stem triterpenes HPLC fingerprints by Kiwi berry branch
Bar, leaf, flower pesticide material are treated as an entirety, obtain Kiwi berry branch, the finger-print of leaf medicinal material, by comparing its shared peak
Find out the nuance between different medicinal materials.
Further, described Kiwi berry stem triterpenes HPLC fingerprints are concretely comprised the following steps：
S101, need testing solution preparation：Medicinal material is dried in the shade, Kiwi berry cauline leaf, bud, delicious root herb powder are taken respectively
End, plus methanol, accurately weighed, heating and refluxing extraction 60min, are let cool, and with methanol polishing weight, container and filter residue are used in filtering
Methanol is respectively washed several times, and filtrate and cleaning fluid are merged, are evaporated with Rotary Evaporators, again with methanol dissolves and cleans appearance
Device, crosses 0.45um apertures miillpore filter, takes subsequent filtrate as need testing solution after merging；
S102, efficient liquid phase chromatographic analysis, mobile phase are the aqueous formic acid of acetonitrile -0.2%, and gradient elution obtains macaque
The finger-print of peach branch, leaf medicinal material.
Further, efficient liquid phase chromatographic analysis, chromatography column is Kromasil 100-5C18,250 × 4.6mm, 5 μm；
Mobile phase is the aqueous formic acid of acetonitrile -0.2%, gradient elution, and elution program is：0min→16min→22min→
30min → 34min → 38min → 46min → 56min → 57min → 69min, acetonitrile 30% → 45% → 46% → 54% →
66% → 70% → 90% → 90% → 30% → 30%；
Flow velocity is 1.0mL/min, and detector is ELSD, 95 DEG C, 3.0L/min；
Sample size is 10 μ L, and column temperature is 35 DEG C.
The Kiwi berry stem triterpenes HPLC fingerprints of the embodiment of the present invention comprise the following steps：
1st, the preparation of reference substance solution
Precision weighs oleanolic acid reference substance and ursolic acid reference substance, plus methanol is made every 1ml respectively the mixing containing 0.1mg is molten
Liquid, shakes up, and produces.
2nd, the preparation of need testing solution
After pulverizing medicinal materials, 4.0g is taken, is placed in round-bottomed flask, 40ml methanol is added, weighed weight is heated to reflux 1h,
Let cool, with methanol polishing weight, shake up, take supernatant to centrifuge, produce.
3rd, chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.2% aqueous formic acid as stream
Regulation in dynamic phase B, according to the form below carries out gradient elution；Evaporate photodetector (ELSD, 105 DEG C, 3.0L/min)
4th, determination method
It is accurate respectively to draw the μ l of the reference substance solution 10 and μ l of need testing solution 10, liquid chromatograph is injected, determines, produces.
Total triterpene contentses are calculated with oleanolic acid and ursolic acid respectively, final result is in terms of average value.
The method provided according to the present invention, HPLC finger-prints are established to 19 batches of Kiwi berry branches, leaf medicinal material, by dividing
Analysis is compared, 7 batches of different Kiwi berry cauline leafs of contrast, bud, the HPLC finger-prints of delicious root, is as a result shown：Kiwi berry cauline leaf,
The substantially shared peak of root has 20, as shown in Figure 2.As shown in figure 3, the similarity of cauline leaf, root is very high, but their phases with bud
Seemingly spend lower slightly, obvious shared 15, peak.Can as Kiwi berry cauline leaf medicinal material standard finger-print.
Table 1
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.
Claims (2)
1. a kind of Kiwi berry stem triterpenes HPLC fingerprints, it is characterised in that described Kiwi berry stem triterpenes
HPLC fingerprints use high performance liquid chromatography, and specific step is as follows：
Step one, the preparation of reference substance solution, weighs oleanolic acid reference substance and ursolic acid reference substance, plus every 1ml is made in methanol
Respectively the mixed solution containing 0.1mg, shakes up, and produces；
Step 2, the preparation of need testing solution after pulverizing medicinal materials, takes 4.0g, is placed in round-bottomed flask, adds 40ml methanol,
Weighed weight, is heated to reflux 1h, lets cool, and with methanol polishing weight, shakes up, takes supernatant to centrifuge, produce；
Step 3, using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.2% aqueous formic acid as
Mobile phase B, carries out gradient elution；Detected using photodetector is evaporated, 105 DEG C of evaporation photodetector evaporating temperature, gas
Flow 3.0L/min；
Step 4, it is accurate respectively to draw the μ l of the reference substance solution 10 and μ l of need testing solution 10, liquid chromatograph is injected, is determined, i.e.,
, total triterpene contentses are calculated with oleanolic acid and ursolic acid respectively, final result is in terms of average value；The gradient elution is with such as
The configuration of lower volume concentration is carried out：
At 0 minute, mobile phase A is 30% acetonitrile solution, and Mobile phase B is 70% 0.2% aqueous formic acid；
At 16 minutes, mobile phase A is 45% acetonitrile solution, and Mobile phase B is 55% 0.2% aqueous formic acid；
At 22 minutes, mobile phase A is 46% acetonitrile solution, and Mobile phase B is 54% 0.2% aqueous formic acid；
At 30 minutes, mobile phase A is 54% acetonitrile solution, and Mobile phase B is 46% 0.2% aqueous formic acid；
At 34 minutes, mobile phase A is 66% acetonitrile solution, and Mobile phase B is 34% 0.2% aqueous formic acid；
At 38 minutes, mobile phase A is 70% acetonitrile solution, and Mobile phase B is 30% 0.2% aqueous formic acid；
At 46 minutes, mobile phase A is 90% acetonitrile solution, and Mobile phase B is 10% 0.2% aqueous formic acid；
At 56 minutes, mobile phase A is 90% acetonitrile solution, and Mobile phase B is 10% 0.2% aqueous formic acid；
At 57 minutes, mobile phase A is 30% acetonitrile solution, and Mobile phase B is 70% 0.2% aqueous formic acid；
At 69 minutes, mobile phase A is 30% acetonitrile solution, and Mobile phase B is 70% 0.2% aqueous formic acid.
2. the Kiwi berry stem that a kind of Kiwi berry stem triterpenes HPLC fingerprints as claimed in claim 1 are set up
Triterpenes HPLC finger-prints, it is characterised in that
There is shared 14, peak in the finger-print；
No. 1 peak, Average residence time RT is 6.56min, and peak area is that 18213, RSD is 6.10%；
No. 2 peaks, Average residence time RT is 11.21min, and peak area is that 23654, RSD is 7.93%；
No. 3 peaks, Average residence time RT is 12.68min, and peak area is that 2630, RSD is 0.88%；
No. 4 peaks, Average residence time RT is 13.46min, and peak area is that 5384, RSD is 1.80%；
No. 5 peaks, Average residence time RT is 15.71min, and peak area is that 2009, RSD is 0.67%；
No. 6 peaks, Average residence time RT is 19.29min, and peak area is that 2286, RSD is 0.76%；
No. 7 peaks, Average residence time RT is 25.39min, and peak area is that 2758, RSD is 0.92%；
No. 8 peaks, Average residence time RT is 28.89min, and peak area is that 3299, RSD is 1.10%；
No. 9 peaks, Average residence time RT is 29.29min, and peak area is that 6543, RSD is 2.19%；
No. 10 peaks, Average residence time RT is 35.31min, and peak area is that 2784, RSD is 0.93%；
No. 11 peaks, Average residence time RT is 40.86min, and peak area is that 73140, RSD is 24.51%；
No. 12 peaks, Average residence time RT is 41.74min, and peak area is that 6092, RSD is 2.04%；
No. 13 peaks, Average residence time RT is 46.16min, and peak area is that 1662, RSD is 0.55%；
No. 14 peaks, Average residence time RT is 50.54min, and peak area is that 4755, RSD is 1.59%.
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Effective date of registration: 20191217 Address after: 610000 No. 588, east route, Longquanyi District, Chengdu, Sichuan Province Patentee after: Sichuan Jinke pharmaceutical limited liability company Address before: 610066, B101, Vanke City Garden, Chengdu, Sichuan, Jinjiang District 77, China Patentee before: Song Yan |