CN104502511B - The detection method of four kinds of gradient elutions in Allium wallichii - Google Patents
The detection method of four kinds of gradient elutions in Allium wallichii Download PDFInfo
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Abstract
The invention provides adenine in Allium wallichii or its congener, guanosine, thymidine or/and the UPLC detection method of ribosidoadenine.Above-mentioned detection method, simply, accurate, analysis time is short, good stability, may be used for Allium wallichii and the detection of congener (such as Folium Allii tuberosi) Nucleosides thereof.
Description
Technical field
The present invention relates to the detection method of Allium wallichii.
Background technology
Allium wallichii is Liliaceae (Liliaceae) Allium (Allium) plant Allium wallichii Allium wallichii Kunth
Dry herb.Bulb is cylindric, or slightly makees avette cylindric, has the thickest root；Bulb crust yellowish-brown or light yellow, in sheet
Shape ruptures or in threadiness, the most subreticulate, and endothelium film quality, only top rupture.Leaf slat shape, to wide bar shaped, is upwards grown tapering,
The middle arteries and veins of tool and parallel thready pulse, shorter or long than scape or the most isometric, wide 2.5～15.5 centimetres, long 20～60 centimetres.Flower
Roripa is extracted out from leafage, three prismatic cylindricalitys, has 3 vertical ribs, and rib is narrow aliform sometimes, and high 20～60 centimetres, bottom is by sheath；Always
Bud is unilateral to ftracture, or 2 split, film quality, caducous；Umbel fan-shaped, to hemispherical, has majority evacuation or intensive flower；Pedicel is near
Isometric, than perianth length of a film 2～4 times, base portion is without squamella；Premium color, aubergine, purple are to black purple, and asterism shape is carried out；Perianth
Sheet square is circular oval to narrow square round shape, spends rear opisthotonos, and tip is blunt or notch, isometric, long 5～9 millimeters, wide 1.5～2 millimeters；
Filigree is isometric, taper, shorter or the most isometric than tapel, base portion symphysis and with tapel adhesion；The ovary shape of falling ovum is spherical, has 3
Circle rib, base portion does not have the sweet cave of depression；Style is longer than ovary.Flowering fruit bearing stage 7～JIUYUE.Produce Sichuan (west and south), (southeast, Tibet
Portion), Yunnan, Guizhou, In Northern Guangxi (Mao'er Shan Mountain) and Southern Hunan (big mountain).It is born in the moistening grass of height above sea level 2300～4800 meters
Under slope, border, shrubbery or limes marginis.Also there are distribution in north India, Nepal, Sillim and Bhutan[1-2]。
Allium wallichii is one of Yi nationality's medicine sieve nest source not, and all herbal medicine has promoting blood circulation to remove blood stasis, effect of dispelling wind for relieving itching, uses
In multiple diseases such as treatment ascarid pain, stomachache, pertussis, flu, traumatic injury wound, psoriasiss[3-4].Among the people, Allium wallichii is also a kind of
Medicinal and edible plant, " the southern regions of the Yunnan Province book on Chinese herbal medicine " is recorded: do vegetable edible, spleen invigorating of nourishing blood, bone and muscle strengthening, reinforcement gas[5].Allium wallichii is in border, Guizhou
Interior aboundresources, is mainly distributed on little Folium Allii tuberosi level ground and big Folium Allii tuberosi level ground, and locality is mainly used in tourism development.
Summary of the invention
Inventor finds under study for action, and Allium wallichii has good active anticancer (see test example 1), and by ultra high efficiency liquid
Phase chromatograph detects in Allium wallichii containing gradient elution.It is currently known gradient elution and participates in the metabolism of biological cell DNA
Journey, is that biological cell sustains life the basic component of activity, including ribosidoadenine, adenine, guanine, guanosint
Glycosides, thymus pyrimidine, thymidine, cytosine etc., have antiviral, antitumor, immunomodulating, antiinflammatory, resist myocardial ischemia,
Improve the multiple biological activitys such as cardiovascular and cerebrovascular circulation[6].Therefore, on the basis of finding that Allium wallichii has active anticancer, it is studied
The detection method of each gradient elution in medicinal and edible plant, can carry the quality-monitoring of such medicinal and edible plant or control
For possible.
Present invention aim at providing the detection method of a kind of Allium wallichii Nucleosides.Another object of the present invention exists
In the detection method of offer Allium wallichii medicinal and edible plant, it is not limited to the detection to gradient elution.
Ultra Performance Liquid Chromatography (Ultra Performance Liquid Chromatography UPLC) (has another name called superelevation
Pressure liquid chromatography, ultrahigh speed liquid chromatograph) it is a brand-new classification in separation science, UPLC is by means of HPLC (efficient liquid phase
Color method) theory and principle, cover little granular filler, the lowest system bulk and the quick brand new technical such as detection means, increase
Add flux, sensitivity and the chromatographic peak capacity analyzed.The present invention uses UPLC to carry out detecting just.
Specifically, the invention provides adenine, guanosine, thymus pyrimidine core in Allium wallichii or its congener
The UPLC detection method of one or more compositions in glycosides, ribosidoadenine, it includes following operating procedure:
(1) measuring samples is taken, extracting in water, collect extracting solution, prepare need testing solution；
(2) one or more mixed in adenine, guanosine, thymidine, ribosidoadenine is taken
Compound, prepares reference substance solution；
(3) need testing solution, reference substance solution are injected separately in Ultra Performance Liquid Chromatography instrument, use external standard method detection i.e.
Can；Wherein, chromatographic condition is as follows:
Chromatographic column: octadecylsilane chemically bonded silica is filler
Detection wavelength: 255～265nm
Flowing phase: water-methanol system, its Gradient program is as follows:
Further, in step (1), the mode of extracting in water is ultrasonic.
Further, in step (2), the solvent of reference substance solution is selected from water, methanol or methanol aqueous solution.
Further, in step (3), described detection wavelength is 260nm.
Further, in step (3), the particle diameter of described chromatographic column filler is 1.7～3.5 μm, is 1.7～1.8 μm, more excellent
Elect 1.8 μm as.
Further, described column size is: 2.1 × 100mm.
Preferably, the model of described chromatographic column is: ACQUITY UPLC HSS T3,1.8 μm, 2.1 × 100mm.
Further, described flow rate of mobile phase is 0.2ml/min；Chromatographic column column temperature is 30 DEG C.
Further, described measuring samples be Liliaceae allium Allium wallichii Allium wallichii Kunth or its
The root of congener and stem, leaf, flower, seed or herb.Closer, described congener is Folium Allii tuberosi A.tuberosum
Rottl.ex Spreng.。
Above-mentioned detection method, simply, accurate, analysis time is short, good stability, may be used for Allium wallichii and belongs to medicine food together
The detection of homologous plant (such as Folium Allii tuberosi) Nucleosides.
Present invention also offers the quality determining method of Allium wallichii, it uses UPLC method to detect, including as follows
Operating procedure:
(1) measuring samples is taken, extracting in water, collect extracting solution, prepare need testing solution；
(2) need testing solution is injected in Ultra Performance Liquid Chromatography instrument, detect；Wherein, chromatographic condition is as follows:
Chromatographic column: octadecylsilane chemically bonded silica is filler
Detection wavelength: 255～265nm
Flowing phase: water-methanol system, its Gradient program is as follows:
Further, in step (1), the mode of extracting in water is ultrasonic.
Further, in step (2), described detection wavelength is 260nm.
Further, in step (2), the particle diameter of described chromatographic column filler is 1.7～3.5 μm, is 1.7～1.8 μm, more excellent
Elect 1.8 μm as.
Further, described column size is: 2.1 × 100mm.
Preferably, the model of described chromatographic column is: ACQUITY UPLC HSS T3,1.8 μm, 2.1 × 100mm.
Further, described flow rate of mobile phase is 0.2ml/min；Chromatographic column column temperature is 30 DEG C.
From chromatogram it can be seen that the chromatogram that draws under above-mentioned chromatographic condition of Allium wallichii not only contains the present invention
The above-mentioned four kinds of gradient elutions found, other are unknown or do not verify composition more (see Fig. 1), and therefore, research worker also can be adopted
With above-mentioned chromatographic condition, other compositions of Allium wallichii are detected, or, the chromatogram that also above-mentioned chromatographic condition can be drawn
Carry out finger printing research comparison, all can realize the comprehensive study to Allium wallichii quality.
Accompanying drawing explanation
Fig. 1 mixing reference substance (A) and the UPLC chromatogram of need testing solution (B), wherein, 1-adenine 2-guanosint
Glycosides 3-thymidine 4-ribosidoadenine
Fig. 2 water bath reflux method chromatogram
Fig. 3 ultrasonic extraction chromatogram
Fig. 4 water is solvent extraction chromatogram
Fig. 5 10% ethanol solution extraction chromatography figure
Fig. 6 20% ethanol solution extraction chromatography figure
Fig. 7 30% ethanol solution extraction chromatography figure
Fig. 8 40% ethanol solution extraction chromatography figure
Fig. 9 acetonitrile-0.1% glacial acetic acid aqueous systems chromatogram
Figure 10 methanol-0.1% glacial acetic acid aqueous systems chromatogram
Figure 11 methanol-0.2% glacial acetic acid aqueous systems chromatogram
Figure 12 methanol-water solution chromatogram
Figure 13 gradient 1 chromatogram
Figure 14 gradient 2 chromatogram
Figure 15 gradient 3 chromatogram
Figure 16 gradient 4 chromatogram
Detailed description of the invention
The detection of embodiment 1 gradient elution of the present invention
1 instrument and reagent
Waters Acquity H-Class UPLC Ultra Performance Liquid Chromatography instrument, including quaternary gradient pump, auto injection
Device, column oven, diode array detector and Empower2 work station (Waters, US)；DHG-9246 type electric heating is permanent
Temperature air dry oven (above Nereid's grand experimental facilities company limited), (city of Kunshan's ultrasonic instrument is limited for KQ-250B ultrasonic cleaner
Company), AE240S prunus mume (sieb.) sieb.et zucc. Teller electronic analytical balance [prunus mume (sieb.) sieb.et zucc. Teller-torr benefit (Shanghai) Co., Ltd.], IKA RV8V type rotates and steams
Send out instrument [Chinese mugwort blocks (Guangzhou) instrument and equipment company limited]；HX-200 type high speed Chinese medicine grinder (Yongkang, Zhejiang small stream bank five metals medical instrument
Factory), TGL-16 type table-type high-speed refrigerated centrifuge (Sichuan Shu Ke Instrument Ltd.).
Reference substance adenine (lot number: X-060-140801), guanosine (lot number: N-021-140730, lower abbreviation bird
Glycosides), thymidine (lot number: X-074-131208, lower abbreviation thymidine), ribosidoadenine (lot number: X-022-140108,
Lower abbreviation adenosine) it is purchased from Rui Fensi bio tech ltd, Chengdu, all reference substances calculate through HPLC areas of peak normalization method
Mass fraction is all higher than 98%.
Allium wallichii medicinal and edible plant is collected in Hezhang County, Bijie City, Guizhou Province respectively in JIUYUE, 2012 and in July, 2014
Big Folium Allii tuberosi level ground, is accredited as the herb of Liliaceae allium Allium wallichii through Southwest University for Nationalities professor Liu Yuan.Family plants Folium Allii tuberosi and gathers
In big roadside, yellow mud village, area just outside a city gate town, Hezhang County, Bijie City, Guizhou Province, leek seed medical material (decoction pieces) be purchased from Chengdu pharmacy of Tongrentang and
Company of Xing Lin chain drug store.Methanol (chromatographically pure, Fischer company), water is ultra-pure water, and glacial acetic acid is chromatographically pure.
2 methods and result
2.1 chromatographic condition
Chromatographic column is ACQUITY UPLC HSS T3 (2.1 × 100mm 1.8 μm), column temperature 30 DEG C, volume flow 0.2mL/
Min, sample size 2 μ L, flowing is water-methanol mutually, and by table 1 gradient elution, detection wavelength is 260nm；It is fast that theoretical cam curve presses gland
Purine calculates and is not less than 5000.
Table 1 gradient elution program
The preparation of 2.2 reference substance solution
Precision weighs reference substance adenine, guanosine, thymidine, adenosine in right amount respectively, in 10mL brown volumetric flask, adds water super
Sound dissolves, and constant volume, to scale, is made containing adenine 0.34mg/mL, guanosine 0.335mg/mL, thymidine 0.325mg/mL, adenosine
The list mark solution of 0.36mg/mL, each single mark solution of precision absorption is in right amount in 10mL brown volumetric flask respectively, and the constant volume that adds water is to carving
Degree, is mixing reference substance solution, and 4 DEG C of Refrigerator stores are standby.
The preparation of 2.3 need testing solutions
0.5g is in 50mL centrifuge tube accurately to weigh Allium wallichii sample powder (crossing pharmacopeia 5 sieve), and add water 30mL, close plug,
Weighed weight, ultrasonic (power: 250W) processes 1h, lets cool to room temperature, more weighed weight, supplies the weight of less loss with ultra-pure water,
Shaking up, 10000r/min is centrifuged 10min, takes supernatant and crosses 0.22 μm filter membrane, to obtain final product.
2.4 specificity tests
Under " 2.1 " under chromatographic condition, adenine, guanosine, thymidine, the mixing reference substance solution of adenosine and Allium wallichii flower
The UPLC spectrogram of flower bud need testing solution is shown in Fig. 1, and in mixing reference substance solution and need testing solution, 4 kinds of gradient elution separating degrees are good
Good, reach baseline separation.
The investigation of 2.5 linear relationships
Respectively precision measure four kinds of reference substance solution 0.2,0.4,0.8,1.6,2.0,2.8mL in 10mL volumetric flask, add
Water is settled to scale, measures peak area with chromatographic condition under " 2.1 ".With peak area as vertical coordinate (Y), reference substance mass concentration
Carrying out linear regression for abscissa (X) and obtain regression equation, using signal to noise ratio S/N=3 as detection limit (LOD), S/N=10 is as fixed
Amount limit (LOQ), is shown in Table 2.
The linear equation of 2 four kinds of gradient elutions of table and correlation coefficient
2.6 precision test
The mixing reference substance solution of preparation under accurate absorption " 2.2 ", with chromatographic condition continuous sample introduction 6 times under " 2.1 ",
Record peak area.Result adenine, guanosine, thymidine, the RSD of adenosine peak area be respectively 1.55%, 0.78%, 1.43%,
0.70%.Result shows that the precision of instrument is good.
2.7 stability test
Take Allium wallichii sample powder appropriate, according to " 2.3 " lower section legal system available test sample solution, with chromatograph under " 2.1 "
Condition respectively 0,1,2,4,8,10,12h measure, record peak area.Result adenine, guanosine, thymidine, adenosine peak area
RSD is respectively 0.94%, 1.43%, 0.85%, 0.72%.Result shows that need testing solution is stable in 12h.
2.6 replica test
Precision weighs with a collection of Allium wallichii sample powder 6 parts, according to " 2.3 " lower section legal system available test sample solution, with
Under " 2.1 ", chromatographic condition measures respectively, records peak area.Result adenine, guanosine, thymidine, the RSD difference of adenosine peak area
It is 1.92%, 1.26%, 1.05%, 1.71%.Result shows, the repeatability of the method is good.
2.7 average recovery tests
Taking 6 parts of the Allium wallichii alabastrum powder of known content, accurately weighed, precision adds a certain amount of reference substance respectively, presses
" 2.3 " lower section legal system available test sample solution, measures respectively with chromatographic condition under " 2.1 ", records peak area, calculate and reclaim
Rate, average recovery rate 98.25%～101.38%, RSD≤2.32%, be shown in Table 3.
The average recovery of table 3 four Nucleosides
2.9 sample determination
Weigh Different Harvesting Time difference Allium wallichii sample powder 0.5g respectively, according to " 2.3 " lower section legal system available test product
Solution, measures respectively with chromatographic condition under " 2.1 ", records peak area.According to containing of 4 kinds of gradient elutions of regression equation calculation
Amount, the results are shown in Table 4.
Content/mg the g of table 4 Different Harvesting Time four kinds of nucleoside of difference plant parts-1
Note: " " represents and do not adopt shop) collect * to this sample；" * " represents that this sample is decoction pieces
3 conclusions and discussion
(1) test is investigated extracting method, be respectively compared heat reflow method and ultrasonic assistant extracts four
The extraction ratio of Nucleosides, result ultrasonic method extraction effect is substantially better than heat reflow method, and this method is simple, easily operated
(result see Fig. 2,3).
(2) test also investigated the impact on four kinds of constituents extraction rates of the different extraction solvent, compare water, 10% ethanol,
20% ethanol, 30% ethanol, 40% ethanol are extraction ratio during extraction conditions, found that water extraction rate is the highest, along with ethanol
The increase of concentration, extraction ratio is on a declining curve, and each one-tenth swarming occurs in that serious conditions of streaking, finally determines that selection water is for carrying
Take solvent, supersound extraction (result is shown in Fig. 4～8).
(3) aqueous solution of ucleosides is alkalescence, and polarity is bigger, it is more difficult to separate.Test has been investigated acetonitrile-0.1% ice
Acetic acid water, methanol-0.1% glacial acetic acid water, methanol-0.2% glacial acetic acid water, four flow visualizing of methanol-water to four kinds of one-tenth
The eluting effect divided, the eluting effect of result methanol-water solution is the most preferable, and each composition has all reached baseline separation, and (result is shown in Fig. 9
～12)；In addition to solvent species, early stage of the present invention has also carried out a large amount of examination, at tens of kinds of streams to the Gradient program of flowing phase
A kind of particular flow phase gradient program the most only finding applicable Allium wallichii of the present invention to detect in dynamic phase condition is (such as gradient 4, figure
16), remaining flowing is the most all unable to reach good separating effect, the most only enumerates gradient 1～3 and is used as contrast, and result is shown in figure
13～15, but correlational study data are not limited only to this.
Gradient 1:
Table 5
Gradient 2:
Table 6
Gradient 3:
Table 7
Gradient 4:(preferred gradient)
Table 8
Being found by full wavelength scanner in test, when maximum absorption wavelength selects 260nm, four kinds of compounds all have stronger
Absorb, and good separation, the final detection wavelength selecting 260nm to be this experiment.
Gradient elution has antitumor, the multiple bioactive ingredients such as anticancer, by measurement result it can be seen that Allium wallichii
Four kinds of gradient elution rich contents of each medicinal part are especially the highest with alabastrum and leaf content, it is seen that it has potential research
And using value.Allium wallichii enriches in china natural resources, and the most domestic and international research about its chemical composition and pharmacologically active is still
For blank, to a certain degree have impact on reasonable, the safe medication of Yi nationality's medicine, the foundation of this method, can be used for this medicinal and edible plant and
The quality control of similar medicinal and edible plant.
The detection method that this test is set up is simple, accurately, section analysis time, good stability, may be used for Allium wallichii and
The detection of similar medicinal and edible plant Nucleosides.
Test example 1
One, the preparation method of extract:
(1) ethanol ethanol extract: the fresh Allium wallichii flowing clear water gathered is rinsed well, is divided into root and stem, leaf, spends three
Part, shreds each several part, adds 95% ethanol by solid-liquid ratio 1:20 (g:mL), soaks 24h, filters, and concentrating under reduced pressure filtrate is to leaching
Paste, collects extractum, reclaims ethanol.Filtering residue adds the recovery ethanol identical with 95% ethanol volume for the first time again, repeats above-mentioned behaviour
Make.Each position repeats to extract three times, merges the extractum of three times.
(2) water extract: reclaim the filtering residue after the alcohol extraction of each position, is put in ventilation and dries to without ethanol taste, weigh each portion
The dried medical material in position is appropriate, adds distilled water by solid-liquid ratio 1:20 (g:mL), soaks 30min, little fire heating, keeps micro-boiling 30min,
Let cool to room temperature, filter, collect filtrate.Filtering residue continues to repeat above operation twice, merges three filtrates, is evaporated to extractum
Shape, collects extractum.
The Allium wallichii each position alcohol extraction obtained by said method and the water extracted immersing paste are dried respectively under the conditions of freezing, extremely
Powder, obtains each extractive part:
The each extractive part of table 9 Allium wallichii
Two, test cell line
Take the logarithm the cell of phase, the culture medium in culture bottle of inclining, rinse twice with aseptic PBS, add trypsinization thin
Born of the same parents, observation of cell matter contraction under inverted microscope, iuntercellular are away from becoming big, and incline pancreas enzyme-EDTA, and aseptic PBS rinses once.Add training
Support base piping and druming cell, notice that piping and druming action is soft.Counting cell, adjusting concentration of cell suspension is 1 × 105/mL, every hole 100 μ
L is inoculated in 96 orifice plates.Put in cell culture incubator, 37 DEG C, 5% carbon dioxide cellar culture 24 hours, treat that cell is paved with plate
The end, inhale and abandon supernatant, add fresh culture and add test medicine.Test arranges blank group, positive drug group, given the test agent group,
Often 5 concentration of group, the multiple hole of each concentration 3, every hole final volume is 200 μ L.Putting into incubator, after 48 hours, every hole adds 50 μ L
TCA fixative, puts into 4 DEG C of fixing 1h, with distilled water flushing 5 times, dries.Every hole adds 50 μ L SRB dyeing liquors, uses after 15min
1% glacial acetic acid rinses 5 times, dries.Every hole 200 μ L Tris dissolves, and shaking bed shaking 20min, in microplate reader, 490nm wavelength is surveyed
Value.Test in triplicate with method.Calculate medicine needed for cell proliferation rate (Percentage Growth, PG) and 50% growth inhibited
Substrate concentration (GI50).
Result of the test:
Allium wallichii root and stem water extract A1, ethanol extraction B4 and Allium wallichii flower ethanol extraction B6 are to hepatoma carcinoma cell
SMMC7721 has obvious inhibitory action, and GI50 value is respectively 12.6 μ g/mL, 9.6 μ g/mL and 12.0 μ g/mL；Allium wallichii root
And stem and flower ethanol extraction B4 and B6 have obvious inhibitory action to breast cancer cell T47D, GI50 value is respectively 14.7 μ
G/mL and 18.8 μ g/mL；Allium wallichii root and stem and flower ethanol extraction B4 and B6 have significantly suppression to lung carcinoma cell LU-04
Effect, GI50 value is respectively 14.7 μ g/mL and 18.8 μ g/mL.
List of references
[1] Chinese Academy of Sciences's Chinese Plants will editorial board. Chinese Plants will volume 14 [M]. Science Press,
1980:210.
[2] Plants From Guizhou will editorial board. Plants From Guizhou will volume 3 [M]. the Guizhou People's Press, 1982:362-363
[3] State Administration of Traditional Chinese Medicine's " China's book on Chinese herbal medicine " editorial board. China book on Chinese herbal medicine volume 22 [M]. Shanghai: Shanghai science
Technology publishing house, 1999:50.
[4] Li Gengdong, He Tingchao. Yi nationality doctor's plant amedica (sequel) [M]. Chengdu: Sichuan Nationalities Press, 1992:180.
[5] Lan Mao. the southern regions of the Yunnan Province book on Chinese herbal medicine [M]. Kunming: Yunnan Science Press, 2004:573.
[6] Lv Aijuan, Wu Hao. the progress [J] of Chinese medicine Nucleosides. China's Chinese medicine information magazine, 2006,
13(7):94-97.
Claims (9)
1. adenine, guanosine, thymidine, the UPLC detection side of four kinds of compositions of ribosidoadenine in Allium wallichii
Method, it is characterised in that: it includes following operating procedure:
(1) measuring samples is taken, extracting in water, collect extracting solution, prepare need testing solution, wherein, the mode of extracting in water is super
Sound；
(2) take adenine, guanosine, thymidine, the mixture of four kinds of compositions of ribosidoadenine, prepare reference substance
Solution；
(3) need testing solution, reference substance solution are injected separately in Ultra Performance Liquid Chromatography instrument, use external standard method to detect；
Wherein, chromatographic condition is as follows:
Chromatographic column: octadecylsilane chemically bonded silica is filler, the particle diameter of filler is 1.7～3.5 μm
Detection wavelength: 255～265nm
Flowing phase: water-methanol system, its Gradient program is as follows: 0 → 1min, water 95%, methanol 5%；1 → 3min, water 95% →
92%, methanol 5% → 8%；3 → 3.5min, water 92% → 91%, methanol 8% → 9%；3.5 → 4min, water 91%, methanol
9%；4 → 5min, water 91% → 90%, methanol 9% → 10%；5 → 6min, water 90% → 86%, methanol 10% → 14%；6
→ 6.5min, water 86%, methanol 14%；6.5 → 7.5min, water 86% → 84%, methanol 14% → 16%；7.5 → 11min,
Water 84%, methanol 16%.
Detection method the most according to claim 1, it is characterised in that: in step (2), the solvent of reference substance solution is selected from
Water, methanol or methanol aqueous solution.
Detection method the most according to claim 1, it is characterised in that: in step (3), described detection wavelength is 260nm.
Detection method the most according to claim 1, it is characterised in that: in step (3), the particle diameter of described chromatographic column filler is
1.7～1.8 μm.
Detection method the most according to claim 4, it is characterised in that: in step (3), the particle diameter of described chromatographic column filler is
1.8μm。
6. according to the detection method described in claim 1,4 or 5, it is characterised in that: described column size is: 2.1 ×
100mm。
Detection method the most according to claim 6, it is characterised in that: the model of described chromatographic column is: ACQUITY UPLC
HSS T3,1.8 μm, 2.1 × 100mm.
Detection method the most according to claim 1, it is characterised in that: described flow rate of mobile phase is 0.2ml/min；Chromatographic column
Column temperature is 30 DEG C.
Detection method the most according to claim 1, it is characterised in that: described measuring samples is the many stars of Liliaceae allium
The root of fragrant-flowered garlic Allium wallichii Kunth and stem, leaf, alabastrum, seed or herb.
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| KR100896700B1 (en) * | 2007-01-04 | 2009-05-14 | 고려대학교 산학협력단 | A.tuberosum Rottl. extract having an activity of activating choline acetyltransferase in brain nerve cells |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103926332A (en) * | 2013-01-15 | 2014-07-16 | 重庆医科大学 | Ultra performance liquid chromatography method for simultaneously determining contents of uridine, guanosine and adenosine in rhizoma pinelliae extract |
| CN103149301A (en) * | 2013-03-07 | 2013-06-12 | 通化华夏药业有限责任公司 | Method for simultaneously determining contents of 10 nucleoside components in sowthistle-leaf ixeris seedling injection by utilizing HPLC-DAD (High Performance Liquid Chromatography-Diode Array detector) method |
Non-Patent Citations (2)
| Title |
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| RP-HPLC定量测定冬虫夏草中5种核苷类成分;张占蓬等;《海峡药学》;20130331;第25卷(第3期);说明书第20段 * |
| 高效液相色谱法测定盐炙韭菜子中腺苷的含量;刘俊达等;《亚太传统医药》;20110930;第7卷(第9期);第26～30页 * |
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