CN101078713A - Fingerprint quality control method of gynostemma pentaphylla medicine added with internal standard - Google Patents
Fingerprint quality control method of gynostemma pentaphylla medicine added with internal standard Download PDFInfo
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a quality control method for fingerprint of herba gynostemae medicinal materials with internal standard in it, comprising (1) measuring method for fingerprint of herba gynostemae medicinal material, (2) building standard fingerprint, (3) comparing HPLC fingerprint of sample with reference fingerprint according to technology demand of quality control of fingerprint that similarity of six common peaks calculated is not smaller than 0.9. The invention is provided with high sensitivity, good reproducibility, strong specialization and reliable method. It can evaluate quality of herba gynostemae medical material scientifically and effectively. It can be applied in quality control in practical production and can satisfy the demand of identification and quality control for enterprise medicinal material.
Description
Technical field
The invention belongs to gynostemma pentaphylla raw material quality control method field, particularly relate to the gynostemma pentaphylla medicinal materials fingerprint method of quality control that adds internal standard compound in a kind of body resistance-strengthening stasis-eliminating plant drug.
Background technology
Gynostemma pentaphylla (Gynostemma pentaphfllum (Thunb) Makino.) is the perennial draft of overgrowing, and is Curcurbitaceae gynostemma pentaphyllum genus type species plants.Beginning is stated from herbal for Relief of Famines.Traditional Chinese medical science pharmacology is thought the gynostemma pentaphylla bitter, and is cold in nature, nontoxic, have clearing heat and detoxicating, relieving cough and reducing sputum, tonifying QI to produce body fluid, the merit of spleen-benefiting mind-tranquilizing.Modern pharmacology proof gynostemma pentaphylla has the inhibition tumour; prevent aging; reduce blood fat; enhance immunity; prevent glucocorticoid side effect, protection heart and liver, blood sugar lowering; pharmacological action such as sedation-analgesia and antiulcer, thus make gynostemma pentaphylla be widely used in clinical at aspects such as treatment tracheitis, catarrhal jaundice, internal lesion caused by overexertion are deficient.
Gynostemma pentaphylla is distributed in India, Nepal, Sillim, Bangladesh, Sri Lanka, Burma, Vietnam, Laos, Malaysia, Indonesia, New Guinea, Korea, Japan and Chinese.Wherein, China is distribution and the differentiation center of this plant.It mainly is distributed in subtropics and torrid areas on the south the line of the Qinling Mountains to Huaihe River, and all there is distribution in Jiangsu, Zhejiang, Anhui, Fujian, Guangdong, Hainan, Guangxi, Henan, Hubei, Hunan, Sichuan, Chongqing, Guizhou, Yunnan, Tibet, Shaanxi, Gansu etc.Gynostemma pentaphylla is a kind of vertical distribution amplitude maximum in the gynostemma pentaphyllum genus, and it is widely distributed in the hillside, mountain valley thick forest of height above sea level 300-3200m, under the sparse woods of hillside, in the shrubbery, the dark and damp meadow in cheuch, riverbank, roadside; Be fragmentary distribution more; Gynostemma pentaphylla mainly is grown in the lower floor of evergreen broad-leaved, mixed deciduous forest, and general ubac sees that tailo is rare more, in the strongly fragrant pine forest of covering, growth and the distribution of gynostemma pentaphylla is arranged seldom all in other coniferous forests, theropencedrymion and the mao bamboo woods.In Japanese documentation, usually the medicine source plant resource of gynosaponin is defined as kindred plant that " gynostemma pentaphylla and kindred plant thereof " clearly mention and has only three kinds of Burma gynostemma pentaphylla (Gynostemma burmanicum King ex Chakr.), light leaf gynostemma pentaphyllum (Gynostemmalaxum) and full edge gynostemma pentaphylla (Gynoiemma integrifoliola Cogn.).
Gynostemma pentaphylla is the herbaceous species plant, has the property of climbing up by holding on to.Root system is the system of fibrous root of being made up of adventitious root.Primary structure of root is made of epidermis, cortex and center pillar, xylem 2～4 prototypes, and the tool casparian strip, phelloderm is thicker in the secondary structure.Stem has the branch of the aerial stem of plant and subterranean stem.The aerial stem of plant is fine and soft, tool groove line, and the rich toughness of five prismatics does not have hair or by pubescence.The tool tendril is born in axil, the many minutes y-bends in top.Be made of epidermis, cortex, vascular cylinder and marrow, bicollateral vascular bundle is lined up two circles, 5 of outer rings, and 4～5 of inner rings, fiber is linked to be a ring on every side.Underground old stem is cylindrical, and fiber is discontinuous ring-type on every side, and vascular bundle tool secondary xylem and secondary phloem are lined up a circle, and external phloem is obvious, the xylem prosperity, and conduit diameter 20～155gm, starch-containing in the medullary ray broad, medullary ray and marrow parenchyma cell.Stem contacts to earth and locates to bear adventitious root.5～7 on bird foot shape compound leaf, back of the body abdomen type structure does not wait the type stomatal apparatus, and petiole tool 5 bundle vascular bundles are divided into 7～9 bundles when entering leaflet.The gynostemma pentaphylla monoid in the different places of production, the shape and the size of its little number of sheets, leaflet exist variation.Along with the ecological condition difference, the differentiation degree of mesophyll palisade tissue and spongy tissue is different, the leaf epidermis form is also different, be grown in than the population in the dry environment, blade gets fairly perfect at the structural development aspect the control moisture loss, and in air humidity than the population in the overall situation since control moisture loss ability, can only be distributed in the extremely limited zone.
Applied histochemistry's location technology finds that gypenoside mainly is distributed in the assimilating tissue and phloem parenchyma cells of nutrition organs, and the phelloderm of collenchyma, epidermis and perithelium also has a small amount of distribution, and its relative content is leaf＞stem＞root; In the year growth cycle, the highest with the saponin content in florescence.
Since the seventies in 20th century, Chinese scholars adopts modern separation technology and analytical technology that strand oil blackeite composition is carried out extensive studies.Find to contain multiple composition in the gynostemma pentaphylla medicinal material, comprise saponin(e, flavones, amino acid, polysaccharide, vitamin and trace element.Wherein gypenoside is main effective constituent.The basic chemical structure of gynosaponin is the glucoside unit of 18 kinds of dammarane types.Now from this plant, isolate the dammarane type gynosaponin of kind more than 100 and the similar skeleton of panaxoside, wherein gynosaponin-3 and Ginsenoside Rb
1Identical, gynosaponin-4 ,-8 ,-7 chemical constitution then respectively with the Ginsenoside Rb
3, Rd, F
2Consistent.
Aspect the quality assessment of gynostemma pentaphylla medicinal material, in the document of delivering both at home and abroad at present, general colourimetry or the thin layer chromatography scanning of adopting measured gypenosides, and as the quality evaluation index of gynostemma pentaphylla medicinal material, obviously, the gynostemma pentaphylla medicinal material can not accurately be differentiated and estimate to this quality evaluating method.The present invention will remedy the blank of gynostemma pentaphylla medicinal materials fingerprint research.
Summary of the invention
Technical matters to be solved by this invention provides the gynostemma pentaphylla medicinal materials fingerprint method of quality control that adds internal standard compound in a kind of body resistance-strengthening stasis-eliminating plant drug, and this method can be controlled the quality of raw medicinal material comprehensively, guarantees the steady quality of product.
A kind of gynostemma pentaphylla medicinal materials fingerprint method of quality control that adds internal standard compound of the present invention comprises the following steps:
1. the detection method of gynostemma pentaphylla medicinal materials fingerprint
(1) chromatographic column: Eclipse XDB-C18 (250 * 4.6nm, 0.5 μ m); Moving phase: water A-acetonitrile B.
(2) preparation method of test sample: the gynostemma pentaphylla pulverizing medicinal materials is crossed 40 mesh sieves, adopt sample quarterlies, take by weighing 2.0-4.0g gynostemma pentaphylla medicinal material.Extracted 12-24 hour with the sherwood oil Soxhlet, solution is colourless to the extraction apparatus.Take out the dregs of a decoction, volatilize solvent, add 60-100ml methyl alcohol ultrasonic Extraction 30-60min.With the extract filtration under diminished pressure, filtrate is concentrated into dried, with dissolve with methanol and be settled to 10ml, solution A.With solution A behind sample on the HZ-818 macroporous absorbent resin, with 95% ethanol elution of 5 times of bed volume.The solvent of evaporated under reduced pressure eluent, the dissolving of residue water is settled to 10ml, gets solution B.With solution B sample on the AB-8 macroporous absorbent resin, water, 50% ethanolic solution and 95% ethanolic solution wash-out are collected 95% ethanol eluate successively, evaporated under reduced pressure solvent, residue with 60% methanol constant volume to 10ml.Solution pipettes filtrate 300 μ l with 0.45 μ m filtering with microporous membrane, and adding concentration is the ginsenoside Rb of 1.00mg/ml
2 Reference substance solution 50 μ l mix, and carry out the HPLC fingerprint map analyzing.
(3) column temperature: 20 ℃-40 ℃
(4) detect wavelength: 203nm
(5) flow velocity: 0.6-1.0ml/min
(6) sample size: 10-20 μ l
(7) the isocratic elution condition is: moving phase is water (A)-acetonitrile (B), 0-60min, 34%B isocratic elution.
2. the foundation of standard finger-print
Choose the Shaanxi sample of 10 batches of different lot numbers, carry out the efficient liquid-phase chromatograph finger print atlas analysis.All peaks in the HPLC finger-print of appropriate samples are compared, determined 6 total peaks, and be numbered (Fig. 1) by appearance time precedence as characteristic peak.Wherein, No. 1 peak is internal standard compound ginsenoside Rb
2
According to the identification result of LC-MS and HPLC-DAD, the efficient liquid-phase chromatograph finger print atlas of gynostemma pentaphylla has been expressed the distribution character of gypenoside class component.With internal standard compound ginsenoside Rb
2As the index components peak.
The finger-print of choosing a collection of Shaanxi gynostemma pentaphylla medicinal material according to following principle from the HPLC finger-print of 10 batch samples is finger-print (Fig. 1) in contrast, in conjunction with the effect experiment situation, pays the utmost attention to the finger-print of the medicinal material of determining curative effect.In conjunction with finger-print and quantitative result, the selected characteristic peak separates better, and the retention time at peak and peak height are suitable, the typical finger-print of peak type (shape at peak and peak height ratio).Preferentially choose the finger-print that baseline is steady, signal to noise ratio (S/N ratio) is good.
3, the technical requirement of the quality control of finger-print
3.1 the discriminating of the sample true and false
Calculate in ten batches of medicinal materials fingerprints each characteristic peak with respect to internal standard compound ginsenoside Rb according to formula
2Relative retention time RRT (retention time at the retention time of RRT=characteristic peak/interior mark peak (index peak)), determined the relative retention time and the scope (as shown in table 1) of each characteristic peak.
Table 1 Shaanxi gynostemma pentaphylla medicinal material HPLC finger-print is differentiated peak relative retention time and scope
| Peak number | 1 | 2 | 3 | 4 | 5 | 6 |
| Ownership RRT scope | Ginsenoside Rb 2 1.00 - | - 1.84 ±0.04 | - 2.05 ±0.04 | - 2.39 ±0.05 | - 2.83 ±0.06 | - 3.60 ±0.07 |
According to the fingerprint map analyzing condition testing sample is measured, drawn the sample HPLC chromatogram that adds behind the internal standard compound, calculate the relative retention time of each characteristic peak, and compare with listed reference fingerprint and relative retention time table.All exist as each characteristic peak, and relative retention time (RRT) is identical, then can be defined as this medicinal material.
3.2 sample quality evaluation
Adopt traditional Chinese medicine fingerprint area of computer aided similarity evaluation software (Chinese Pharmacopoeia Commission), the HPLC finger-print and the reference fingerprint (or reference substance common pattern finger-print) of sample compared.Should be with the similarity that 6 total peaks calculate less than 0.9.
Beneficial effect of the present invention is as follows:
1, finger-print provides such method, the index that the single index components or the active component of routine is quality control rises to a new stage, with the fingerprint characteristic of chromatogram and the quantization parameter that obtains by finger-print, more effectively discern the false from the genuine control of quality.And do not have bibliographical information at present about the research of gynostemma pentaphylla finger-print.Therefore the present invention is with regard to the problem for the such gynostemma pentaphylla quality of medicinal material control of solution, the level that improves the quality and control.
2. the present invention proposes a kind of fingerprint discrimination method that adds internal standard compound, has solved a difficult problem that lacks the gypenoside reference substance in the market.Adopt very close with character ginsenoside with the gypenoside structure as internal standard compound, as the index components peak, the relative retention time and the relative peak area of each characteristic peak that obtains are more stable with this internal standard compound, and the extensive degree of method is better.
3, investigated the stability of need testing solution, the result shows, at room temperature, within 48 hours, it is stable that the gynostemma pentaphylla need testing solution keeps.Precision and replica test show that all similarity is more than 0.9.
4,, find that the medicinal material similarity is between 0.9-1.0 through the finger-print similarity of 10 batches of gynostemma pentaphylla medicinal materials is calculated.Illustrate that the Shaanxi gynostemma pentaphylla finger-print quality control method of setting up is highly sensitive, good reproducibility, specificity is strong, method is reliable, can carry out scientific and effective evaluation to Shaanxi gynostemma pentaphylla quality of medicinal material, can apply to quality control in the actual production, satisfy the demand of enterprise's medicinal material discriminating and quality control.
Description of drawings
Fig. 1 adds internal standard compound ginsenoside Rb
2The standard HPLC finger-print of Shaanxi gynostemma pentaphylla medicinal material.
Fig. 2 adds internal standard compound ginsenoside Rb
2The HPLC finger-print of Shaanxi gynostemma pentaphylla medicinal material.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
1. experimental technique
1.1 reagent and medicinal material
| Reagent name | Rank | Production firm |
| HZ-818 macroporous absorbent resin AB-8 type macroporous absorbent resin methyl alcohol pure water acetonitrile 95% ethanol Gynostemma pentaphyllum | Analyze pure chromatographically pure technical grade | Nankai University of the Shanghai China scientific and technological trading company of shake Shanghai development chemical industry one Coca-Cola of factory Tedia Shaanxi |
1.2 instrument
| Title | Model | Production firm |
| The multiplex vavuum pump glass chromatography column of electronic balance High Performance Liquid Chromatography post insulating box medicinal herb grinder miillpore filter supercentrifuge screen cloth ultrasonic cleaner Rotary Evaporators circulating water type constant-flux pump | AL104 type HP1100 HT-130 type 0.45um |
Kunshan ultrasonic instrument Co., Ltd of centrifuge factory Gongyi City, domestic Shanghai, METTLER TOLEDO Agilent Tianjin Hengao Technology Development Co., Ltd. Shandong gives magnificent Instr Ltd. Gongyi City and gives west, magnificent Instr Ltd. Qingpu Shanghai Shanghai scientific instrument factory of Shanghai Branch of the Chinese Academy of Sciences of instrument plant |
1.3 the preparation of gynostemma pentaphylla sample
Take by weighing 2.0g gynostemma pentaphylla medicinal material, extracted 12 hours with the sherwood oil Soxhlet, solution is colourless to the extraction apparatus.Take out the dregs of a decoction, volatilize solvent, add 60ml methyl alcohol ultrasonic Extraction 30min.With the extract filtration under diminished pressure, filtrate is concentrated into dried, with dissolve with methanol and be settled to 10ml, solution A.
With solution A behind sample on the chromatographic column that 10ml HZ-818 macroporous absorbent resin is housed, with 95% ethanol elution of 5 times of bed volume.The solvent of evaporated under reduced pressure eluent, the dissolving of residue water is settled to 10ml, gets solution B.
With solution B sample on the chromatographic column that 15ml AB-8 macroporous absorbent resin is housed, water-ethanolic solution (100: 0,50: 50,5: 95) wash-out is collected 95% ethanol eluate successively, evaporated under reduced pressure solvent, residue with 60% methanol constant volume to 10ml.Solution is got the ginsenoside Rb of filtrate 300 μ l and concentration 1.00mg/ml with 0.45 μ m filtering with microporous membrane
2Standard solution 50 μ l mix, and carry out the HPLC fingerprint map analyzing.
1.4 the HPLC analysis condition of gypenoside
Chromatographic column: Eclipse XDB-C18 (250 * 4.6nm, 0.5 μ m); Moving phase: water A-acetonitrile B, 34%B isocratic elution 60min; 30 ℃ of column temperatures; Flow velocity 0.8ml/min; Detect wavelength 203nm; Sample size 10 μ l.
1.5 system suitability
After the determining fingerprint pattern condition is determined, by at least three batch samples are measured, calculate correlation parameter, and propose the suitable parameters condition of highly effective liquid phase chromatographic system on this basis.Be ginsenoside Rb
2The chromatographic peak area reappearance (RSD) of reference substance solution (142.86 μ g/ml) is not higher than 3%, and retention time reappearance (RSD) is not higher than 2%; The chromatographic column post is imitated with ginsenoside Rb
2Meter is no less than 15,000 theoretical cam curves.
1.6 experimental result
HPLC finger-print such as Fig. 2 of Shaanxi gynostemma pentaphylla medicinal material sample, the relative retention time of characteristic peak and relative peak area such as table 2.
The data of table 2 Shaanxi gynostemma pentaphylla medicinal materials fingerprint
* the retention time at the retention time of RRT (relative retention time)=characteristic peak/interior mark peak (index peak)
* the peak area at the peak area of RPA (relative peak area)=characteristic peak/interior mark peak (index peak)
2,, find that the medicinal material similarity is greater than between the 0.9-1.0 by the similarity of ten batches of gynostemma pentaphylla medicinal materials is calculated.The gynostemma pentaphylla finger-print quality control method of setting up is described, method is reliable, and easy and simple to handle, and confirmation can apply to quality control in the actual production, satisfies the demand of enterprise's medicinal material discriminating and quality control.
Claims (2)
1. a gynostemma pentaphylla medicinal materials fingerprint method of quality control that adds internal standard compound comprises the following steps:
(1) detection method of gynostemma pentaphylla medicinal materials fingerprint
The gynostemma pentaphylla pulverizing medicinal materials is crossed 40 mesh sieves, take by weighing 2.0～4.0g gynostemma pentaphylla.Extracted 12-24 hour with the sherwood oil Soxhlet, add 60-100ml methyl alcohol ultrasonic Extraction 30-60min.Filtration under diminished pressure is concentrated into 10ml, gets solution A; Last HZ-818 macroporous absorbent resin is with 95% ethanol elution of 5 times of bed volume.Evaporated under reduced pressure eluent solvent, the dissolving of residue water is settled to 10ml, gets solution B; Last AB-8 macroporous absorbent resin, water, 50% ethanolic solution and 95% ethanolic solution wash-out are collected 95% ethanol eluate successively, evaporated under reduced pressure solvent, residue with 60% methanol constant volume to 10ml; Solution pipettes filtrate 300 μ l with 0.45 μ m filtering with microporous membrane, and adding concentration is the ginsenoside Rb of 1.00mg/ml
2Reference substance solution 50 μ l mix, and carry out the HPLC fingerprint map analyzing;
(2) foundation of standard finger-print
Choose the sample of 10 batches of different lot numbers, all peaks are compared, determine that 6 total peaks are characteristic peak, are numbered by appearance time precedence.Wherein, No. 1 peak is internal standard compound ginsenoside Rb
2
According to the identification result of LC-MS and HPLC-DAD, the efficient liquid-phase chromatograph finger print atlas of gynostemma pentaphylla has been expressed the distribution character of gypenoside class component.Therefore with internal standard compound ginsenoside Rb
2As the index components peak;
The finger-print of choosing a collection of gynostemma pentaphylla medicinal material according to following principle from the HPLC finger-print of 10 batch samples is finger-print in contrast, in conjunction with the effect experiment situation, pays the utmost attention to the finger-print of the medicinal material of determining curative effect.In conjunction with finger-print and quantitative result, the selected characteristic peak separates better, and the retention time at peak and peak height are suitable, the typical finger-print of peak type.Preferentially choose the finger-print that baseline is steady, signal to noise ratio (S/N ratio) is good;
(3) technical requirement of the quality control of finger-print
The discriminating of the sample true and false
Calculate in ten batches of medicinal materials fingerprints each characteristic peak with respect to internal standard compound ginsenoside Rb
2Relative retention time RRT (R has determined the relative retention time and the scope of each characteristic peak;
Gynostemma pentaphylla medicinal material HPLC finger-print is differentiated peak relative retention time and scope
Peak number 1 2 3 4 5 6
Ownership RRT scope Ginsenoside Rb
2 1.00 -
- 1.84 ±0.04 - 2.05 ±0.04 - 2.39 ±0.05 - 2.83 ±0.06 - 3.60 ±0.07
According to the fingerprint map analyzing condition testing sample is measured, drawn the sample HPLC chromatogram that adds behind the internal standard compound, calculate the relative retention time of each characteristic peak, and compare with listed reference fingerprint and relative retention time table.All exist as each characteristic peak, and relative retention time (RRT) is identical, then can be defined as this medicinal material;
Sample quality is estimated
The HPLC finger-print and the reference fingerprint of sample are compared, and the similarity that 6 total peaks calculate should be less than 0.9.
2. a kind of gynostemma pentaphylla medicinal materials fingerprint method of quality control that adds internal standard compound according to claim 1, it is characterized in that: chromatographic column: Eclipse XDB-C18, moving phase: water A-acetonitrile B, 20 ℃-40 ℃ of column temperatures detect wavelength 203nm, flow velocity 0.6-1.0ml/min, sample size 10-20 μ l, the isocratic elution condition is: moving phase is water (A)-acetonitrile (B), 0-60min, 34%B isocratic elution.
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