CN108982504A - A kind of tomentose pummelo fruit quality of medicinal material detection method - Google Patents
A kind of tomentose pummelo fruit quality of medicinal material detection method Download PDFInfo
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
- G01B5/00—Measuring arrangements characterised by the use of mechanical techniques
- G01B5/08—Measuring arrangements characterised by the use of mechanical techniques for measuring diameters
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
- G01N5/04—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder
- G01N5/045—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder for determining moisture content
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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Abstract
The invention discloses the quality determining method of tomentose pummelo fruit medicinal material, this method includes appearance character identification, microscopical characters, thin layer identification, determination of moisture, total ash measurement, characteristic spectrum identifies and assay.The method all has stronger specificity and good reproducibility.Operability means are provided to establish unified feasible tomentose pummelo fruit quality of medicinal material standard.In favor of protecting high-quality medicinal material, avoids adulterating, preferably ensure clinical application safely, effectively.
Description
Technical field:
The invention belongs to traditional Chinese medicine quality detection fields, more particularly to tomentose pummelo fruit quality of medicinal material detection method.
Background technique:
Tomentose pummelo fruit is the immature fruit of prematurity of rutaceae Citrus grandis (Citrus grandis ' Tomentosa '),
Also known as tangerine pearl, tangerine tire, Exocarpium Citri Rubrum tire.Citrus grandis is grown on Guangdong Huazhou and its periphery region, and the kind that begins has about one in beam court so far
Thousand quincentenary cultivation histories.Citrus grandis is shaddock rather than tangerine, and why young fruit is known as " tomentose pummelo fruit ", is by means of Song, bright
Since Exocarpium Citri Rubrum reputation.Relevant literature record, first appeared in (Kangxu 14 years) " Guangdong leads to will " in 1675, in the category of Gaozhou county mansion medicine
Have under classification described below: " though claiming fertile soil in Guangdong, the right shallow tax matter of natural conditions and social customs of a place is soft crisp, and personage is with it, and different as clever rarity, object is also
Solely arrogate to oneself its beauty person, if ... the Exocarpium Citri Rubrum of Huazhou, the lichee of Zengcheng, the fragrant rhinoceros, Bulbus Fritillariae Thunbergii in fine jade south "." Guangdong Province Huazhou will " (1827)
Roll up 11 and draw " Exocarpium Citri Rubrum is distinguished ": " the produced Exocarpium Citri Rubrum in Huazhou, for doctor with its benefit gasification phlegm, function is high in other medicine.(fruit) has red white flesh two
Kind, obviously shaddock, no the slightest is different, and it is mixed exhale say tangerine, and adorn its color say it is red.Husband fruit is conducive to use, that is, deposits its name to write in fact
It can.Why must tangerine it is expensive and shaddock low-priced.Or the sincere shaddock of shaddock is said, how he produces and is unfavorable for using, and gives that say this just so-called genuine."
Record within letter out thread 15 years (1890) " Guangdong Province Gaozhou county mansion will ": " with regard to the non-tangerine of tangerine of Huazhou, shaddock, the people in the world is all
Tangerine it, I also have to tangerine it."
Citrus grandis early stage as it is medicinal when, concocting method also imitates Exocarpium Citri Rubrum " go white take skin ", is used as medicine using exocarp, i.e.,
Pummelo pee medicinal material.And due to Citrus grandis skin depth carnic acid, not middle food in actual use, is evolved as using prematurity young fruit
It is used as medicine.Most of in Citrus grandis young fruit is all middle pericarp, and wherein the pharmacological components content such as aurantiin, Rhoifolin is very high,
It is removed undoubtedly a kind of waste, and is spent human and material resources, the reasonable utilization of resource is unfavorable for.Tomentose pummelo fruit record earliest in
Second volume of " Guangxi Chinese medicinal herbal " (1963), for " quench the thirst, it is aid digestion, remove the stagnation of the circulation of vital energy in the heart.Control dyspepsia, abdominal mass "；It also records afterwards
In " dictionary of medicinal plant ", " China's book on Chinese herbal medicine ".By nearly market circulation in 50 years, tomentose pummelo fruit clinical efficacy confirmation has become at present
The mainstream of Citrus grandis processes specification.
Tomentose pummelo fruit medication is extensive, especially in Guangdong and Guangxi Provinces area；But current edition National Pharmacopeia and provincial standard are not yet received
Carry the kind.Lack unified quality standard, causes the confusion in circulation and use.
Summary of the invention
The invention discloses the quality determining method of tomentose pummelo fruit Chinese medicinal material, including it is appearance character identification, microscopical characters, thin
Layer identification, determination of moisture, total ash measurement, characteristic spectrum identifies and content assaying method.For foundation unification, feasible Exocarpium Citri Rubrum
Pearl quality of medicinal material standard provides operability means.
Tomentose pummelo fruit appearance character discrimination method of the present invention includes the following steps:
Discrimination method: the appearance character of test sample is inspected, and is photographed to record；Use vernier caliper measurement fruit trans D
With longitudinal diameter, using its average value as fruit diameter；
Test sample appearance should be in subsphaeroidal, hemispherical or cylinder, and surface is blackish green or celadon, densely covered fine hair have wrinkle
Line and small grease chamber, apex have the trace that style falls off, and base portion has the scar of rounded carpopodium；Matter is hard, is not easy to cut, section flat
Whole, yellow-white or light red brown have train of thought line.Outer rim has the irregular recessed grease chamber of a column.Gas fragrance, bitter, micro-pungent.
Selected fruit diameter as appearance character index, tomentose pummelo fruit medicinal material can be divided into it is first-class, second-class, third, four etc. 4
Grade, first-class: fruit diameter is no more than 4cm；Second-class: fruit diameter is no more than 5cm；It is third, four etc.: fruit diameter is no more than
6cm。
Tomentose pummelo fruit microscopical characters of the present invention include the following steps:
Test sample preparation: test sample crushes, and crosses No. two sieves, is uniformly mixed.It takes in right amount on glass slide, drips few drops of hydration chlorine
After aldehyde solution infiltrates powder, alcolhol burner heating, then appropriate dilute glycerite is added dropwise, it is sufficiently mixed, covered；
Discrimination method: the slide made is placed in microscopically observation, test sample powder yellowish-brown to sepia.Middle pericarp
Parenchyma cell is in irregular shape, and wall unevenly thickens, and some makees beaded or the special thickness at corner.Pericarp epidermal surface is seen
Polygonal, class are rectangular or rectangle, anticline thicken, stomata similar round, and 18~31 μm of diameter, accessory cell 5~7, side
By cuticula outside seeing, by the radial wall thickening of foreign side.It can be seen that cataclasm nonglandular hair, cataclasm cell up to ten is several, and the widest part is straight
About 33 μm of diameter, has wall wart or outer wall is smooth, inner wall coarse, cell includes faint yellow or granulated brown object.Calcium oxalate prismatic crystal at
Piece or embark on journey is present in middle pericarp parenchyma cell, is in Polyhedral, diamond shape, prismatic, rectangle or in irregular shape, diameter 1
It is~32 μm, 5~40 μm long.Conduit is spiral duct and reticulate vessel.Accidental lithocyte and fiber.
Tomentose pummelo fruit thin-layer identification method of the present invention includes the following steps:
The preparation of test solution: taking test sample powder 0.5g, adds methanol 5ml, is ultrasonically treated 15 minutes, and centrifugation takes
Clear liquid is as test solution；
The preparation of reference substance solution: taking aurantiin reference substance, adds methanol that solution of every 1ml containing 1mg is made, as reference substance
Solution；
Solvent: ethyl acetate: acetone: glacial acetic acid: water=8: 4: 0.3: 1；
Discrimination method: each 2 μ l of test solution and control solution is drawn, is put respectively in same high-efficient silica gel G lamellae
On, it is unfolded, takes out, dry, spray 5% alchlor ethanol solution, is heated 1 minute at 105 DEG C, set wavelength 365nm ultraviolet lamp
Under inspect；In sample chromatogram, at the position corresponding to the chromatogram of the reference substance, the fluorescence spot of same color is shown.
Tomentose pummelo fruit determination of moisture method of the present invention includes the following steps:
Test sample preparation: test sample crushes, and crosses No. two sieves, takes 2~5g, is laid in the dry flat weighing bottle to constant weight
In；
Measuring method: the accurately weighed weighing bottle equipped with test sample, unlatching bottle cap is 5 hours dry at 100~105 DEG C, will
Bottle cap covers, and in dislocation drier, lets cool 30 minutes, accurately weighed, then 1 hour dry in above-mentioned temperature, lets cool, weighs, until
Until the difference weighed twice in succession is no more than 5mg.According to the weight of less loss, water content is calculated in test sample less than 12.0%.
Tomentose pummelo fruit total ash measuring method of the present invention includes the following steps:
Test sample preparation: test sample crushes, and crosses No. two sieves, after mixing, takes 2~3g, set the crucible of ignition to constant weight
In, weighed weight (it is slowly red-hot accurately to 0.01g), until completely charing when, gradually rise temperature to 500~600 DEG C, made
Full ashing and to constant weight；
Measuring method: according to residue weight, the content of total ash in test sample is calculated less than 4.5%.
Tomentose pummelo fruit characteristic spectrum method of the present invention includes the following steps:
The preparation of test solution: taking test sample powder, crosses No. two sieves about 0.5g, accurately weighed, sets in stuffed conical flask,
Precision plus methanol 10ml, weighed weight, ultrasonic treatment 30 minutes, let cool, then weighed weight, the weight of less loss are supplied with methanol,
It shakes up, filters, subsequent filtrate is taken, as test solution；
The preparation of reference solution: taking aurantiin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing aurantiin
The solution of 500 μ g, as reference solution.
Chromatographic condition: using octadecylsilane chemically bonded silica as filler, using methanol as mobile phase A, with 2% acetum
For Mobile phase B, gradient elution program are as follows: 0~25 minute, 30% mobile phase A, 70% Mobile phase B；25~75 minutes, 30% →
45% mobile phase A, 70% → 55% Mobile phase B；75~110 minutes, 45% → 100% mobile phase A, 55% → 0% mobile phase
B；110~120 minutes, 100% mobile phase A, 0% Mobile phase B；Flow velocity is 1ml per minute, Detection wavelength 320nm；
Measuring method: it is accurate respectively to draw reference solution and each 5 μ l of test solution, inject liquid chromatograph, record
120 minutes chromatograms；4 characteristic peaks should be presented in test sample characteristic spectrum, wherein characteristic peak 1 should be with corresponding object of reference peak
Retention time is identical；Peak corresponding with aurantiin object of reference peak is the peak S, calculates the relative retention time of characteristic peak 2~4, feature
The relative retention time at peak 2,3 should within ± the 5% of specified value, the relative retention time of characteristic peak 4 should specified value ±
, it is specified that value within 6% are as follows: 1.49 (peaks 2), 1.74 (peaks 3), 3.19 (peaks 4).
Tomentose pummelo fruit content assaying method of the present invention includes the following steps:
The preparation of test solution: taking test sample powder, crosses No. two sieves, takes about 50mg, accurately weighed, sets 10ml volumetric flask
In, methanol 5ml is added in precision, is ultrasonically treated 30 minutes (150W, 40kHz), lets cool, and adds 50% methanol to scale, shakes up, filter
It crosses, takes subsequent filtrate, as test solution；
The preparation of reference substance solution: taking aurantiin, Rhoifolin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made
Solution containing 400 μ g of aurantiin, 10 μ g of Rhoifolin respectively, as reference substance solution；
Chromatographic condition: using octadecylsilane chemically bonded silica as filler；With -0.3% acetum of methanol, (volume ratio is
35: 65) being mobile phase；Detection wavelength is 283nm, 338nm；
Measuring method: accurate absorption reference substance solution and each 5 μ l of test solution respectively, injection liquid chromatograph, measurement,
By external standard method with calculated by peak area content；Selected aurantiin and Rhoifolin content are as inherent index, by tomentose pummelo fruit medicinal material point
It is first-class: to be no less than 11.8% containing aurantiin, Rhoifolin is no less than 0.93% for first-class, second-class, third, 4 grades such as four；
It is second-class: to be no less than 8.5% containing aurantiin, Rhoifolin is no less than 0.77%；It is third: to be no less than 7.5% containing aurantiin, Rhus succedanea
Glycosides is no less than 0.50%；Four etc.: being no less than 6.5% containing aurantiin, Rhoifolin is no less than 0.37%.
The present invention is that the quality testing done for the first time to tomentose pummelo fruit Chinese medicine provides specificity, reproducibility strong, Quan Mianke
Capable detection method to establish tomentose pummelo fruit quality of medicinal material standard unify, feasible, and further carries out its credit rating
Division provides comprehensive feasible detection means and avoids adulterating, substantially increase tomentose pummelo fruit in favor of protecting high-quality medicinal material
The quality control level of medicinal material.
Detailed description of the invention:
Fig. 1 is the microscopical characters feature diagram of tomentose pummelo fruit；
Fig. 2 is the investigation result diagram of thin layer detection point sample volume；
Fig. 3 is 1~11 batch of tomentose pummelo fruit sample thin layer identification result figure；
Fig. 4 is tomentose pummelo fruit compare feature map；
Fig. 5 is the cluster analysis result figure of 28 batches of tomentose pummelo fruit samples.
Specific embodiment:
Claim of the invention is described in further detail combined with specific embodiments below.
The appearance character of 1 tomentose pummelo fruit medicinal material of embodiment identifies
28 batches, representative sample is had collected from tomentose pummelo fruit medicinal material Genuine producing area, as shown in table 1.Inspect each batch of sample
Appearance character.Tomentose pummelo fruit sample is in subsphaeroidal, hemispherical or cylinder, 3~6cm of diameter.Surface is blackish green or celadon, gathers
Fine hair has wrinkle and small grease chamber, and apex has the trace that style falls off, and base portion has the scar of rounded carpopodium；Matter is hard, is not easy to cut
It opens, section is smooth, and yellow-white or light red brown have train of thought line.Outer rim has the irregular recessed grease chamber of a column.Gas fragrance, taste
Bitter, micro-pungent.
1 tomentose pummelo fruit sample list of table
The microscopical characters of 2 tomentose pummelo fruit medicinal material of embodiment
Test sample crushes, and crosses No. two sieves, is uniformly mixed.It takes in right amount on glass slide, few drops of chloraldurate solution of drop are by powder
After the infiltration of end, alcolhol burner heating, then appropriate dilute glycerite is added dropwise, it is sufficiently mixed, covered, is placed under microscope and sees
It examines.Representative microscopic features are shown in Fig. 1.Sample powder yellowish-brown is to sepia, and middle pericarp parenchyma cell is in irregular shape, and wall is uneven
Even to thicken, some makees beaded or the special thickness at corner.Pericarp epidermal surface sees that polygonal, class be rectangular or rectangle, hangs down
All wall thickenings, stomata similar round, 18~31 μm of diameter, accessory cell 5~7.It can be seen that cataclasm nonglandular hair, broken section of cell is up to
Ten is several, and about 33 μm of the widest part diameter, cell includes faint yellow or granulated brown object.Calcium oxalate prismatic crystal in flakes or presence of embarking on journey
It is in Polyhedral, diamond shape, prismatic, rectangle or in irregular shape in middle pericarp parenchyma cell, 1~32 μm of diameter, long by 5~
40 μm of conduits are spiral duct and reticulate vessel.Accidental lithocyte and fiber.
The thin layer of 3 tomentose pummelo fruit medicinal material of embodiment identifies
1 instrument
Card Ma thin-layer chromatography auto sample applicator (ATS 4), Automatic-expanding instrument (ADC 2), ultraviolet imagery system (CAMAG,
Muttenz,Switzerland)。
2 reagents
Ethyl acetate, acetone, glacial acetic acid, methanol, ethyl alcohol are that analysis is pure.
Aurantiin reference substance (110722-200610, National Institute for Food and Drugs Control), the control of meranzin hydrate
Product (BBP00486, Yunnan ethylmercuric cloride object technical concern Co., Ltd), Rhoifolin reference substance (12110232, the same Tian Sheng in Shanghai
Object technical concern Co., Ltd).
3 preparation method of test article are investigated
(1) investigation of Extraction solvent
Prepare the 0.98mg/mL of reference substance containing aurantiin, the mixed solution of meranzin hydrate reference substance 0.19mg/mL.
Different solvents are investigated: methanol, methanol aqueous solution (7:3, v/v), ethyl alcohol, ethanol water (7:3,
v/v).Test sample preparation step is as follows:
No. 5 samples are taken, No. two sieves is crushed, takes 0.5g powder in conical flask, it is molten to be separately added into the above-mentioned different extractions of 5mL
Agent, ultrasound 15 minutes, centrifugation take supernatant as test solution.2 μ L test solutions are respectively taken, point sample is in High Performance Thin silicon
Glue G plate.Solvent selects ethyl acetate, acetone, acetic acid and water (8:4:0.3:1), puts excellent thin layer according to preliminary result
Plate is expanded to 8cm in saturation expansion cylinder.Lamellae is taken out, it is dry, after spraying appropriate 5% alchlor ethanol solution color developing agent,
It heats one minute in 105 DEG C, and is observed under 365nm ultraviolet wavelength.The result shows that Extraction solvent is to test solution thin layer exhibition
It opens effect and has no significant effect, final choice methanol is as Extraction solvent.
(2) investigation of Extraction solvent volume
It taking 0.5g sample powder in conical flask, is separately added into 5mL, 10mL, 15mL, 20mL methanol is 15 minutes ultrasonic, from
The heart takes supernatant as test solution.Respectively take 2 μ L test solution point samples, with method expansion, colour developing the result shows that, with mentioning
The reduction of volume is taken, aurantiin and meranzin hydrate band clarity increase.Therefore 5mL is selected to extract volume progress subsequent
Experiment.
(3) investigation of extraction time
It takes 0.5g sample powder in conical flask, 5mL methanol is added, respectively ultrasound 5min, 15min, 25min and 35min,
Centrifugation, takes supernatant as test solution.2 μ L test solution point samples are respectively taken, with method expansion, colour developing.The result shows that ultrasound
Time has no significant effect extraction effect.Select 15 minutes as extraction time.
The investigation of 4 spotting methods
(1) investigation of point sample volume
0.5 μ L is taken respectively, and 1 μ L, 2 μ L, 5 μ L, 8 μ L test solution points are unfolded in High Performance Thin plate, dry, spray colour developing
Agent is placed under 365nm ultraviolet wavelength and observes after 105 ° are heated 1 minute.As a result see Fig. 2.The result shows that with point sample volume
Increase, aurantiin and meranzin hydrate band clarity increase.But when point sample volume is 8 μ L, aurantiin band is under
Square band separation is poor.Therefore 2 μ L of final choice is as point sample volume.
(2) investigation of strip width
Take 2 μ L test solution points on High Performance Thin plate, strip width is respectively 2mm, 4mm, 6mm, 8mm
And10mm is unfolded in saturation expansion cylinder, dries, and dries 1 minute after spraying appropriate color developing agent in 105 °, and in 365nm ultraviolet waves
Long lower observation.The result shows that band seems narrower thin with the increase of point sample width, separating degree is more preferable.Therefore select 8mm's
Strip width carries out follow-up test.
5 methodological studies
(1) specificity
According to the condition determined, test solution is prepared, (0.10g/mL, methanol are molten for Exocarpium Citri Grandis control medicinal material solution
Solution), aurantiin reference substance solution (0.98mg/mL, methanol dissolution), (0.46mg/mL, methanol are molten for Rhoifolin reference substance solution
Solution), meranzin hydrate reference substance solution (0.36mg/mL, methanol dissolution).
The above solution respectively takes 2 μ L points to be unfolded in High Performance Thin plate, dry, sprays appropriate color developing agent, heats 1 minute in 105 °
Afterwards, it is placed under 365nm length ultraviolet light and observes.365nm length ultraviolet light result: in test solution, blue bands RF
0.52, two green stripes Rs corresponding with meranzin hydrate reference substanceFRespectively 0.21,0.18, respectively correspond aurantiin
Reference substance and Rhoifolin reference substance.Specificity meets test request as the result is shown.
(2) stability
For test solution after being placed at room temperature for different time (0,1,2,3 day), point sample, expansion, colour developing are placed in 365nm wave
It is observed under long ultraviolet light.The result shows that test solution is good in 3 days internal stabilities.
(3) durability
Different-grain diameter silica gel thin-layer plate
Sample is put respectively in (TLC and HPTLC plate) on different-grain diameter lamellae.Expansion, colour developing, set 365nm length ultraviolet
It is observed under light.The result shows that High Performance Thin lath band is relatively sharp, but common lamellae also can reach identification and require.
The investigation of fluorescent plate
Sample is put respectively in GF254 plate and silica gel g thin-layer plate.Expansion, colour developing, set and observe under 365nm length ultraviolet light.Knot
Fruit shows that the band of silica gel g thin-layer plate is more concentrated, is narrow, and GF254 plate is not applicable for this experiment.
Different brands lamellae
Sample is put respectively on different brands lamellae, and expansion, colour developing are set and observed under 365nm length ultraviolet light.As a result table
Bright, different brands lamellae has no significant effect test solution band.
The influence of humidity is unfolded
After point sample, it is unfolded under the conditions of different humidity (46%, 65%, 75%, 84%) respectively, after colour developing, is placed in 365nm
It is observed under length ultraviolet light.The result shows that band clarity is without significant difference under different humidity.
Durability investigate the result shows that, different-grain diameter (High Performance Thin plate or common lamellae), different brands and humidity pair
Sample and reference substance solution thin-layer developing result have no significant effect.
The thin layer of 6 tomentose pummelo fruit samples identifies
Using the thin-layer identification method of foundation, the tomentose pummelo fruit sample being collected into is identified.Representative result such as Fig. 3 institute
Show.In test solution, on position corresponding with aurantiin reference substance, identical green fluorescence spot, R are shownFAbout
0.28。
The characteristic spectrum of 4 tomentose pummelo fruit medicinal material of embodiment
1 material
1.1 instrument
Electronic analytical balance (BP211D, Sartorius company of Switzerland)；A ten thousandth electronic analytical balance (ME204, it is auspicious
Scholar Mettler toledo company, instrument number: TP604-2)；3000 high performance liquid chromatograph of Ultimate (U.S. Dionex
Company, the bis- ternary pumps of DGP-3600SD, SRD-3600 degasser, UPS-3000SL autosampler, TCC3000-RS column oven,
DAD detector, 7.2 data processing software of Chromeleon；Instrument number: 418-3)；Chromatographic column: Welch XB-C18(4.6×
250mm, 5 μm, S.N.211604929), Yi Lite Hypersil BDS C18(4.6mm × 250mm, 5 μm) wears peace Acclaim
120C18(4.6mm × 250mm, 5 μm)；Numerical control ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instruments Co., Ltd.；Instrument
Number: CS605-1)；Superpure water machine (Simplicity 185personal, Millipore company of the U.S.)；10ml pipette.
1.2 reagent
Aurantiin, Isomperatorin reference substance are purchased from Nat'l Pharmaceutical & Biological Products Control Institute；Rhoifolin reference substance, purchase
From Shanghai Tongtian Biotechnology Co., Ltd.；Meranzin hydrate reference substance is laboratory self-control, and HPLC detection purity is higher than
98%.Methanol (Tianjin great Mao chemical reagent factory is analyzed pure)；Methanol (Honeywell, lot number: R11G4H, chromatographically pure)；Ice
Acetic acid (aladdin, lot number: E1519047, chromatographically pure).
1.3 sample
Condition is groped and methodological study sample used is No. 18 tomentose pummelo fruit samples, and the sample used of establishing of characteristic spectrum is
1~No. 15.
2 experimental methods are groped
2.1 chromatographic condition
Using methanol as mobile phase A, using 6% acetum as Mobile phase B, gradient elution is carried out by the regulation in table 2；Flow velocity
For 1ml/min, Detection wavelength 320nm.
2 mobile phase gradient of table
The investigation of 2.2 sample solution preparation methods
It is respectively compared Different Extraction Method (water-bath reflux, ultrasound) and different extraction time (ultrasonic 20min, ultrasound
30min) to the influence of testing result.
No. 18 sample powders (crossing No. two sieves) 0.5g is taken, it is accurately weighed, it sets in stuffed conical flask, methanol is added in precision
50ml, weighed weight, water-bath refluxing extraction 1h are let cool, then weighed weight, and the weight of less loss is supplied with methanol, is shaken up, 25ml is taken
Subsequent filtrate is concentrated into and closely does, methanol is added quantitatively to be transferred in 5ml measuring bottle, and adds methanol to scale, as test solution 1；
No. 18 sample powders (crossing No. two sieves) 0.5g is separately taken, it is accurately weighed, it sets in stuffed conical flask, methanol is added in precision
10ml, weighed weight, ultrasonic 20min or 30min, let cool, then weighed weight, and the weight of less loss is supplied with methanol, is shaken up, it is taken
Subsequent filtrate, as test solution 2, test solution 3.
It is resulting with ultrasonic 30min and water-bath reflux two kinds of extracting methods of 1h by 2.1 " chromatographic conditions " sample introduction measurement respectively
Calculated by peak area recovery rate.The result shows that (being shown in Table 3): ultrasound and water-bath reflux two methods extraction efficiency do not have notable difference,
It can be used as the preparation method of test solution, ultrasound procedure is more convenient time saving；But sonication treatment time is not preferably less than
30min。
The peak area of 3 Different Extraction Method of table compares
The selection of 2.3 Detection wavelengths
Detection wavelength should ensure that composing main chromatographic peak entirely has fine response, investigate using diode array detector different
Chromatogram situation under wavelength, preferably Detection wavelength are 320nm.
The selection of 2.4 chromatographic columns
Yi Lite Hypersil BDS C is investigated respectively18(4.6mm × 250mm, 5 μm), peace Acclaim 120C is worn18
(4.6mm × 250mm, 5 μm), Welth XB-C18(4.6mm × 250mm, 5 μm) three root chromatogram column separating degrees, retention time and reason
By the number of plates, the results showed that the C of different brands18It is used equally for tomentose pummelo fruit characteristic spectrum to identify, the results are shown in Table 4 final determine and use
Welch XB-C18(4.6mm × 250mm, 5 μm) chromatographic column carries out subsequent experimental.
The comparison of 4 chromatographic column of table
3 methodological studies
The preparation of 3.1 solution
The preparation of reference substance solution: precision weighs aurantiin reference substance 10.16mg, Rhoifolin reference substance 10.25mg, orange
Intradermal ester hydrate reference substance 10.45mg, Isomperatorin 5.48mg, add methanol to dissolve, shake up, and every 1ml is respectively prepared containing shaddock
The reference substance solution of 508 μ g of skin glycosides, 16.4 μ g of Rhoifolin, 8.36 μ g of meranzin hydrate, 54.8 μ g of Isomperatorin.
The preparation of test solution: taking test sample powder (crossing No. two sieves) about 0.5g, accurately weighed, sets stuffed conical flask
In, precision plus methanol 10ml, weighed weight are ultrasonically treated (power 120W, frequency 40kHz) 30min, let cool, then weighed weight,
The weight that less loss is supplied with methanol, shakes up, and filtration takes subsequent filtrate, as test solution.
3.2 chromatographic condition
With 2.1 " chromatographic conditions ".
3.3 precision test
No. 18 samples are taken, are made test solution, METHOD FOR CONTINUOUS DETERMINATION 6 times, and commented using chromatographic fingerprints of Chinese materia medica similarity
Valence system (2004A editions) software carries out similarity evaluation, the results are shown in Table 5.The result shows that instrument precision is good.
5 precision similarity evaluation result of table
3.4 repetitive test
No. 18 samples are taken, prepare 6 parts of test solutions, sample introduction measurement in parallel, and use chromatographic fingerprints of Chinese materia medica similar
It spends evaluation system (2004A editions) software and carries out similarity evaluation, the results are shown in Table 6.The result shows that repeatability is good.
The repeated similarity evaluation result of table 6
3.5 stability test
No. 18 samples are taken, test solution is made, place 0,2,4,6,12, rear sample introduction measurement for 24 hours at room temperature respectively, and
Similarity evaluation is carried out using similarity evaluation (2004A editions) software, the results are shown in Table 7.As a result
Show that test solution is good in internal stability for 24 hours.
7 stability similarity evaluation of table
The detection of 4 samples and the establishment of characteristic spectrum
1-15 batches of tomentose pummelo fruit samples are taken, test solution is respectively prepared, are measured.Use chromatographic fingerprints of Chinese materia medica similarity
Evaluation system (2004A editions) software, matches main chromatographic peak, generates control map, sees Fig. 4.The result shows that tomentose pummelo fruit
4 principal character peaks, respectively aurantiin, Rhoifolin, meranzin hydrate, Isomperatorin are presented in sample.In figure,
Peak 1 (S): aurantiin peak 2: Rhoifolin peak 3: meranzin hydrate peak 4: Isomperatorin.
The determination of 5 relative retention time specified values
Using different chromatographic columns (Yi Lite Hypersil BDS C18, wear peace Acclaim 120C18、Welch XB-C18)
It is investigated.Using aurantiin reference substance, Isomperatorin reference substance as reference, with aurantiin peak (peak 1) for the peak S, wild paint is calculated
The relative retention time for setting glycosides, meranzin hydrate, is shown in Table 8.According to the flat of different chromatographic column characteristic peak relative retention times
Mean value is, it is specified that the relative retention time of characteristic peak 2,3,4 is 1.49 (peaks 2), 1.74 (peaks 3), 3.19 (peaks 4), the measurement at peak 2,3
It as a result should be within ± the 5% of specified value；For 4 Isomperatorin of peak because appearance is slower, the RSD of relative retention time is larger, will limit
Degree is relaxed within ± the 6% of specified value.
The calculated result of 8 relative retention time specified value of table
The relative retention time of institute's test sample is as shown in table 9, and the relative retention time at each peak meets above-mentioned regulation.
9 tomentose pummelo fruit sample characteristic map relative retention time result of table
It * is specified value range in bracket
The foundation of 5 tomentose pummelo fruit medicinal material content assaying method of embodiment
The main active of tomentose pummelo fruit is Flavonoid substances, with aurantiin (C27H32O14) and Rhoifolin (C27H30O14)
To represent.Wherein aurantiin accounts for 80% of general flavone or more；The Pharmacological experiment result shows that with good relieving cough and reducing sputum work
With, while there is preferable anti-airways inflammatory effect: lipopolysaccharide-induced chmice acute lung inflammation can not only be significantly inhibited, and
And also there is good protective effect for chronic airway inflammation；Also there is certain antioxidation.Rhoifolin has certain
Anti-inflammatory and antioxidation, have significant protective effect to the genotoxicity of NF induction, while also having a hepatoprotective effect.Therefore, originally
Invention selects aurantiin and Rhoifolin as the index components of tomentose pummelo fruit assay.
1 material
1.1 instrument
A ten thousandth electronic analytical balance (ME204, Mettler toledo company of Switzerland)；Ultimate 3000 is efficient
Liquid chromatograph (Dionex company of the U.S., the bis- ternary pumps of DGP-3600SD, SRD-3600 degasser, UPS-3000SL automatically into
Sample device, TCC3000-RS column oven, DAD detector, 7.2 data processing software of Chromeleon)；Chromatographic column: Dionex
Acclaim 120C18(4.6 × 150mm, 3 μm), Welch XB-C18(4.6 × 250mm, 5 μm)；Numerical control ultrasonic cleaner
(KQ500DE, Kunshan Ultrasonic Instruments Co., Ltd.)；Superpure water machine (Simplicity 185personal, the U.S.
Millipore company)；Pipette.
1.2 reagent
Aurantiin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110722-201111, purity: 93.2%),
Rhoifolin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 111919-201102, purity: 95.0%).Methanol (day
The big luxuriant chemical reagent factory in Jinshi City, is analyzed pure)；Methanol (Honeywell, lot number: R11G4H, chromatographically pure)；Glacial acetic acid (aladdin,
Lot number: E1519047, chromatographically pure).
1.3 samples: condition is groped and methodological study sample used is No. 18 samples.
The investigation of 2 preparation method of test article
2.1 chromatographic condition
Chromatographic column: Dionex Acclaim 120C18(4.6 × 150mm, 3 μm)；Mobile phase: -0.3% acetic acid of methanol (A)
Solution (B)=35: 65；Flow velocity: 1.0ml/min；Detection wavelength: 320nm；Sample volume: 10 μ l；Column temperature: 35 DEG C.
The preparation of 2.2 reference substance solutions
Precision weighs appropriate aurantiin reference substance, Rhoifolin reference substance and meranzin hydrate reference substance, adds methanol
Every 1ml solution containing 496.5 μ g of aurantiin, 20.23 μ g of Rhoifolin, 9.12 μ g of meranzin hydrate respectively is made.
The preparation of 2.3 test solutions
Sample is extracted using water-bath reflux and ultrasonic two methods, wherein ultrasonic time investigate respectively 20 minutes,
30 minutes, compare the aurantiin, Rhoifolin and meranzin hydrate extraction effect of three of the above method.
Test solution A (water-bath reflux): taking test sample powder (crossing No. two sieves) 0.5g, accurately weighed, sets 150mL round bottom
In flask, methanol 50mL is added in precision, and weighed weight water-bath refluxing extraction 60 minutes, is let cool, then weighed weight, is mended with methanol
The weight of sufficient less loss, shakes up, and takes 25mL subsequent filtrate, and methanol is added quantitatively to be transferred in 5mL measuring bottle, and adds methanol to scale, shakes up,
It crosses 0.45 μm of filter membrane, takes subsequent filtrate to obtain the final product.
Test solution B (ultrasonic 20min): taking test sample powder (crossing No. two sieves) 0.5g, accurately weighed, sets 100mL tool
It filling in conical flask, methanol 50mL, weighed weight is added in precision, and 20 minutes (150W, 40kHz) of ultrasound is let cool, then weighed weight,
The weight that less loss is supplied with methanol, shakes up, and crosses 0.45 μm of filter membrane, takes subsequent filtrate to obtain the final product.
Test solution C (ultrasonic 30min): taking test sample powder (crossing No. two sieves) 0.5g, accurately weighed, sets 100mL tool
It filling in conical flask, methanol 50mL, weighed weight is added in precision, and 30 minutes (150W, 40kHz) of ultrasound is let cool, then weighed weight,
The weight that less loss is supplied with methanol, shakes up, and crosses 0.45 μm of filter membrane, takes subsequent filtrate to obtain the final product.
More than sample introduction 3 kinds of 10 μ L of test solution respectively, the peak area of ingredient needed for recording.The result shows that being shown in Table 10, surpass
Sound 30 minutes close with the extraction effect that water-bath flows back 60 minutes, and the peak area of two kinds of ingredients is slightly above water-bath reflux, says
This bright method extraction effect is preferable and more time saving, convenient.Therefore the extracting method for selecting ultrasound 30 minutes as sample.
The comparison of 10 Different Extraction Method of table
Finally determining sample solution preparation method are as follows: sample powder (crossing No. two sieves) about 0.5g is taken, it is accurately weighed, it sets
In 100ml stuffed conical flask, methanol 50ml is added in precision, and weighed weight is ultrasonically treated 30 minutes (150W, 40kHz), lets cool,
Weighed weight again is supplied the weight of less loss with methanol, is shaken up, and filtration, precision measures subsequent filtrate 5ml, sets in 10ml volumetric flask, adds
50% methanol shakes up to scale, filters, take subsequent filtrate to get.
To save solvent, preparation method of test article is adjusted in this standard draft are as follows: take this product powder (crossing No. two sieves) about
50mg, it is accurately weighed, it sets in 10ml volumetric flask, methanol 5ml is added in precision, and it is ultrasonically treated 30 minutes (150W, 40kHz), is let cool,
Add 50% methanol to shake up to scale, filter, take subsequent filtrate to get.
3 chromatographic systems are investigated
The preparation of 3.1 solution
(1) preparation of reference substance solution
Precision weighs appropriate aurantiin reference substance, Rhoifolin reference substance, adds methanol that every 1ml is made and contains aurantiin respectively
The solution of 420.2 μ g, 12.42 μ g of Rhoifolin.
(2) preparation of test solution
Test sample powder (crossing No. two sieves) about 0.5g is taken, it is accurately weighed, it sets in 100mL stuffed conical flask, first is added in precision
Alcohol 50mL, weighed weight are ultrasonically treated 30min (150W, 40kHz), let cool, then weighed weight, the weight of less loss is supplied with methanol
Amount, shakes up, and filters, and precision measures subsequent filtrate 5ml, sets in 10ml volumetric flask, adds 50% methanol to scale, shake up.Cross 0.45 μm
Filter membrane, take subsequent filtrate to get.
3.2 chromatographic condition
Mobile phase and ratio select -0.3% acetum of methanol (35:65) isocratic elution.
The selection of 3.3 Detection wavelengths
According to aurantiin, the ultraviolet wavelength scanning result of Rhoifolin reference substance solution, maximum absorption wavelength λ is determinedmax。
Select 283nm and 338nm as the Detection wavelength of aurantiin and Rhoifolin respectively.
The selection of 3.4 chromatographic columns
(1) investigation of different brands chromatographic column
Three kinds of different brands chromatographic columns: Dionex Acclaim C are investigated altogether18(5 μm, 4.6 × 250mm), Agilent
eclipse plus C18(5 μm, 2.1 × 250mm), Welch XB C18(5 μm, 4.6 × 250mm).It is molten using same reference substance
Liquid compares aurantiin, the retention time of Rhoifolin, theoretical cam curve and tailing factor.The result shows that table 11, for above three
Kind brand chromatographic column, aurantiin, Rhoifolin can obtain preferable separating effect.
The selection of 11 chromatographic column of table
(2) investigation of different column lengths
Using same test solution, the separating effect of different column length chromatographic columns: Dionex Acclaim 120C is investigated18
(3 μm, 4.6 × 150mm), Dionex Acclaim 120C18(5 μm, 4.6 × 250mm).The result shows that 12 are shown in Table, two kinds of length
Chromatographic column all have preferable separating degree for aurantiin and Rhoifolin.In view of shortening retention time, 150mm column length is selected
Chromatographic column carry out follow-up test.
The investigation of 12 column length of table
The selection of 3.5 mobile phases
(1) pH value
Using same test solution, separating effect when flowing phase pH value is respectively 2.6,2.8,3.0,3.2 is investigated.Knot
Fruit shows to be shown in Table 13, and with the reduction of pH value, retention time is elongated.But when pH is 2.8,3.0,3.2, retention time is distinguished very
It is small.In view of obtaining better column effect and separating degree, selective flow phase pH value is 3.0.
The selection of 13 flowing phase pH value of table
(2) mobile phase ratio
Using same test solution, different methanol ratios (30%, 33%, 35%, 37%, 40%) in mobile phase are investigated
To isolated influence.The result shows that being shown in Table 14, different proportion methanol has a significant impact the retention time of two kinds of ingredients to be measured.First
Alcohol ratio is higher, and retention time is shorter.In view of separating degree and shorten analysis time, final choice methanol ratio is 35%.
The selection of 14 mobile phase methanol ratio of table
(3) flow velocity
Using same test solution, the influence of (0.8,1.0,1.2ml/min) different in flow rate to separating effect is investigated.Knot
Fruit is shown in Table 15.The result shows that: in addition to retention time, flow velocity has no significant effect each parameter.After final choice 1.0ml/min is carried out
Continuous experiment.
The selection of 15 flow velocity of table
The selection of 3.6 column temperatures
Using same test solution, influence of the different column temperatures (25,30,35,40 DEG C) to separating effect is investigated.As a result table
Bright to be shown in Table 16, as column temperature rises, the separating degree of aurantiin and Rhoifolin reduces.In view of suitable separation therebetween
Degree, 35 DEG C of progress subsequent experimentals of final choice.
The selection of 16 column temperature of table
Finally determining chromatographic condition are as follows: chromatographic column: Dionex Acclaim 120C18(4.6 × 150mm, 3 μm)；Flowing
Phase: -0.3% acetum (B)=35: 65 of methanol (A)；Flow velocity: 1.0ml/min；Detection wavelength: aurantiin 283nm, Rhus succedanea
Glycosides 338nm；Sample volume: 10 μ l；Column temperature: 35 DEG C.
4 methodological studies
By mixed reference substance solution and test solution, aurantiin and Rhoifolin are calculated in sample using external standard method
In content.Its formula is as follows:
(ru/rs)×(Cs/Cu)×100
ruFor the peak area that in test solution, aurantiin is under 283nm wavelength, Rhoifolin is under 338nm wavelength；
rsFor the peak area that in hybrid standard product solution, aurantiin is under 283nm wavelength, Rhoifolin is under 338nm wavelength.CuFor
The concentration of test solution, unit mg/ml；CsThe respectively concentration of aurantiin, Rhoifolin in hybrid standard product solution,
Unit mg/ml.
4.1 the range of linearity
The preparation of reference substance mother liquor: precision weighs 16.34mg Rhoifolin reference substance in 10ml volumetric flask, and methanol is fixed
Hold, Rhoifolin reference substance mother liquor is made.According to purity, the concentration for calculating Rhoifolin mother liquor is 1.552mg/ml.
Precision weighs 42.02mg aurantiin reference substance in 10ml volumetric flask, adds a small amount of methanol to dissolve, adds above-mentioned open country
Lacquer tree glycosides reference substance mother solution 0.8ml, methanol constant volume shake up to get reference substance mother liquor is mixed, wherein aurantiin, Rhoifolin
Concentration is respectively 4.202,0.1242mg/ml.
The preparation of linearity control product solution: quantitatively pipette respectively different volumes 0.1,0.2,0.5,1.0,1.5,2.0,
The above-mentioned mixing reference substance mother liquor of 2.5ml adds 50% methanol constant volume in 10ml volumetric flask, mixes.
The linearity control product solution of each concentration of sample introduction respectively records aurantiin peak area, 338nm wavelength under 283nm wavelength
Lower Rhoifolin peak area.According to the peak area measured under each concentration, obtains following calibration curve and is shown in Table 17, the results showed that, shaddock
Skin glycosides and Rhoifolin all have good linear.
The standard curve of 17 aurantiin of table, Rhoifolin and meranzin hydrate
4.2 repetitive test
Same batch of sample is taken, prepares six parts of test solutions, sample introduction measurement in parallel.The result shows that being shown in Table 19), two kinds to be measured
The RSD of component content is respectively less than 2%.
19 repetitive test result of table
The test of 4.3 Intermediate precisions
Not same date: taking same batch of sample, 3 not same date prepare test solution, parallel 3 parts every time, sample introduction is surveyed
It is fixed, it the results are shown in Table 20, RSD less than 2%.
Different analysis personnel: taking same batch of sample, prepares 3 parts of test solutions in parallel respectively by three analysis personnel, into
Sample detection, the results are shown in Table 21, RSD less than 2%.
20 Intermediate precision test result of table-not same date
21 Intermediate precision test result of table-difference analysis personnel
4.4 accuracy testing
By the rate of recovery of two kinds of ingredients to be measured of additional amount known to detection come the accuracy of judgment method.It prepares respectively
The test solution of basic, normal, high three concentration, each concentration prepare three parts in parallel, the method is as follows:
Low concentration: precision pipettes methanol 50ml, sets in 100ml stuffed conical flask, then accurate removal 1.0ml.Take aurantiin
Reference substance 10mg, sample powder 250mg, it is accurately weighed, it moves in above-mentioned conical flask, and 1.0ml Rhoifolin reference substance is added
Solution (concentration 0.3105mg/ml, solvent are methanol).Weighed weight is ultrasonically treated 30min (150W, 40kHz), lets cool, then
Weighed weight is supplied the weight of less loss with methanol, is shaken up, and filtration, precision measures subsequent filtrate 5ml, sets in 10ml measuring bottle, adds 50%
Methanol shakes up to scale.It crosses 0.45 μm of filter membrane, takes subsequent filtrate to obtain the final product.
Middle concentration: precision pipettes methanol 50ml, sets in 100ml stuffed conical flask, then accurate removal 2.0ml.Take aurantiin
Reference substance 20mg, sample powder 250mg, it is accurately weighed, it moves in above-mentioned conical flask, and 2.0ml Rhoifolin reference substance is added
Solution (concentration 0.3105mg/ml, solvent are methanol), remaining step is same as above.
High concentration: precision pipettes methanol 50ml, sets in 100ml stuffed conical flask, then accurate removal 3.0ml.Take aurantiin
Reference substance 30mg, sample powder 250mg, it is accurately weighed, it moves in above-mentioned conical flask, and 3.0ml Rhoifolin reference substance is added
Solution (concentration 0.3105mg/ml, solvent are methanol), remaining step is same as above.
According to the measured amount of sample, original amount and additional amount, its rate of recovery is calculated.The result shows that (table 22, table 23), recycling
Rate range is between 95%-105%, and RSD < 3%.
22 aurantiin recovery test result of table
The rate of recovery (%)=100 × (the original amount of measured amount -)/additional amount, similarly hereinafter
23 Rhoifolin recovery test result of table
4.5 stability test
Using same test solution, the sample detection after room temperature stores different time compares the peak of three kinds of ingredients to be measured
Area, and calculate RSD.(table 24) as the result is shown, three kinds of ingredients are good in 24 hours internal stabilities in test solution.
24 test solution stability test result of table
4.6 serviceability test
Using the chromatographic column (150mm, 250mm) of two kinds of length, aurantiin, Rhoifolin content are detected respectively, passes through phase
The assay error of two kinds of column lengths is assessed average deviation (RAD).The result shows that (table 25), two kinds of component content calculated results
RAD be respectively less than 3%, illustrate that influence of the column length to testing result is small.
25 serviceability test result --- column length of table
* chromatographic column brand is Dionex acclaim
Each index determining result of 6 tomentose pummelo fruit medicinal material sample of embodiment and comprehensive analysis
Determination of moisture: test sample crushes, and crosses No. two sieves, takes 2~5g, is laid in drying into the flat weighing bottle of constant weight,
Accurately weighed, unlatching bottle cap is 5 hours dry at 100~105 DEG C, and bottle cap is covered, in dislocation drier, is let cool 30 minutes, essence
It is close weighed then 1 hour dry in above-mentioned temperature, it lets cool, weighs, until the difference weighed twice in succession is no more than 5mg.According to
The weight of less loss calculates water content (%) in test sample.Determination of moisture result is as shown in table 26.
Total ash measurement: test sample crushes, and crosses No. two sieves, after mixing, takes 2~3g, set the crucible of ignition to constant weight
In, weighed weight (it is slowly red-hot accurately to 0.01g), until completely charing when, gradually rise temperature to 500~600 DEG C, made
Full ashing simultaneously calculates the content (%) of total ash in test sample according to residue weight to constant weight.Total ash measurement result such as table 26
It is shown.
Aurantiin, Rhoifolin assay: according to established content assaying method, 28 batches of tomentose pummelo fruit samples are carried out
Aurantiin, Rhoifolin assay, as a result as shown in table 26.
Each index determining result of 26 28 batches of tomentose pummelo fruit samples of table
The correlation analysis of each index of 27 tomentose pummelo fruit of table
Data analysis: 19.0 software data processing of SPSS is utilized.Statistical is carried out to 5 quantifiable indicators detected
Tomentose pummelo fruit medicinal material grade classification index and limit are screened in analysis.
As shown in Table 26, the coefficient of variation of moisture and total ash is smaller, illustrates that dispersion degree is small, and difference is unobvious, prompts
It is not suitable as the index of sample grade classification.Therefore, the unified limit using average value floating 20% as moisture and total ash,
Regulation must not cross 12.0% and 4.5% respectively.
As shown in Table 27, aurantiin and Rhoifolin and fruit diameter are negatively correlated, and wherein fruit diameter is aesthetic appearance
Shape index, aurantiin, Rhoifolin content are inherent quality index, therefore combine these three indexs, to tomentose pummelo fruit quality of medicinal material
Superiority and inferiority is evaluated.
According to evaluation index detection data, 28 batches of tomentose pummelo fruit samples are subjected to hierarchial-cluster analysis, as a result as figure 5 illustrates.Spectrum
Be that distance in figure represents similarity, the remoter difference of distance is bigger, wherein No. 20 samples when distance is 25 just can individually and its
His sample is classified as one kind, therefore not as mean cluster numerical value.K- mean cluster analysis further is made to 3 evaluation indexes, as a result
It is shown in Table 28.
The final cluster centre value of 28 tomentose pummelo fruit sample of table
Tomentose pummelo fruit is finally divided into first-class, second-class, third and four etc. by binding analysis result and actual conditions:
First-class: fruit diameter is no more than 4cm, is no less than 11.8%, Rhoifolin containing aurantiin and is no less than 0.93%；Two
Deng: fruit diameter is no more than 5cm, is no less than 8.5%, Rhoifolin containing aurantiin and is no less than 0.77%；Third: fruit diameter is not
More than 6cm, 7.5%, Rhoifolin is no less than containing aurantiin and is no less than 0.50%；Four etc.: fruit diameter is no more than 6cm, contains shaddock
Skin glycosides is no less than 6.5%, Rhoifolin and is no less than 0.37%.The grade classification of 28 batches of samples the results are shown in Table 26, be divided into first-class
Sample have 4 batches, it is 6 batches second-class, 11 batches third, four etc. 4 batches, separately have 3 batches of failed test samples.Illustrate the tomentose pummelo fruit that the present invention establishes
Medicinal material detection method and by this method establish Quality Grade standard it is scientific, effective, feasible.
Claims (10)
1. a kind of tomentose pummelo fruit quality of medicinal material detection method, including appearance character identification, microscopical characters, thin layer identification, determination of moisture,
Total ash measurement, characteristic spectrum identifies and assay, it is characterised in that the thin layer identify the following steps are included:
The preparation of test solution: taking test sample powder to be checked to add methanol by the solid-liquid ratio of 1:5-10, and ultrasonic treatment takes supernatant
Liquid is as test solution；
The preparation of reference substance solution: taking aurantiin reference substance, adds methanol that solution of every 1ml containing 1mg is made, molten as reference substance
Liquid；
Solvent: ethyl acetate: acetone: glacial acetic acid: water=8: 4: 0.3: 1；
Discrimination method: taking test sample and each 2 μ l of reference substance solution, is put respectively on same silica gel g thin-layer plate, is unfolded, taken out, dry in the air
It is dry, 5% alchlor ethanol solution is sprayed, is heated 1 minute at 105 DEG C, is set and inspected under wavelength 365nm ultraviolet lamp；Test sample color
In spectrum, at the position corresponding to the chromatogram of the reference substance, the fluorescence spot of same color is shown.
2. detection method as claimed in claim 1, it is characterised in that the appearance character identify the following steps are included:
The appearance character for inspecting test sample to be detected, photographs to record；It is straight using vernier caliper measurement fruit trans D and longitudinal direction
Diameter, using its average value as fruit diameter；Test sample is in subsphaeroidal, hemispherical or cylinder, and surface is blackish green or celadon, close
Cloth fine hair has wrinkle and small grease chamber, and apex has the trace that style falls off, and base portion has the scar of rounded carpopodium；Matter is hard, is not easy
It cutting, section is smooth, yellow-white or light red brown, there is train of thought line, and outer rim has the irregular recessed grease chamber of a column, and gas is fragrant,
Bitter, micro-pungent, fruit diameter are less than 6cm.
3. detection method as claimed in claim 2, it is characterised in that the fruit diameter is less than 4cm.
4. detection method as claimed in claim 1, it is characterised in that the microscopical characters the following steps are included:
Test sample preparation: test sample crushes, and takes in right amount after on glass slide, dripping chloraldurate solution and infiltrating powder, alcolhol burner
Heating, then appropriate dilute glycerite is added dropwise, sample is made；
Discrimination method: sample is placed in microscopically observation；Test sample powder yellowish-brown is to sepia；Middle pericarp parenchyma cell shape
Shape is irregular, and wall unevenly thickens, and some makees beaded or the special thickness at corner；Pericarp epidermal surface sees polygonal, class
Rectangular or rectangle, anticline thicken, stomata similar round, and 18~31 μm of diameter, accessory cell 5~7, side is seen outer by cutin
Layer, by the radial wall thickening of foreign side；It can be seen that cataclasm nonglandular hair, cataclasm cell up to ten is several, about 33 μm of the widest part diameter, tool
Wall wart or outer wall is smooth, inner wall coarse, cell includes faint yellow or granulated brown object；Calcium oxalate prismatic crystal in flakes or presence of embarking on journey
It is in Polyhedral, diamond shape, prismatic, rectangle or in irregular shape in middle pericarp parenchyma cell, 1~32 μm of diameter, long by 5~
40μm；Conduit is spiral duct and reticulate vessel, accidental lithocyte and fiber.
5. detection method as claimed in claim 1, it is characterised in that the determination of moisture the following steps are included:
Test sample preparation: test sample crushes, and crosses No. two sieves, takes 2~5g, is laid in dry into the flat weighing bottle of constant weight；
Measuring method: the accurately weighed weighing bottle equipped with test sample, unlatching bottle cap is 5 hours dry at 100~105 DEG C, by bottle cap
It covers, in dislocation drier, lets cool 30 minutes, it is accurately weighed then 1 hour dry in above-mentioned temperature, it lets cool, weighs, until continuous
Until the difference weighed twice is no more than 5mg, according to the weight of less loss, water content is calculated in test sample less than 12.0%.
6. detection method as claimed in claim 1, it is characterised in that the total ash measurement includes the following steps:
Test sample crushes, and crosses No. two sieves, takes 2~3g, set in the crucible of ignition to constant weight, weighed weight is slowly red-hot, until complete
When charing, temperature is gradually risen to 500~600 DEG C, according to residue weight, the content for calculating total ash in test sample is less than
4.5%.
7. detection method as claimed in claim 1, it is characterised in that the characteristic spectrum identification includes the following steps:
The preparation of test solution: taking test sample powder 0.5g, adds methanol 10ml, and weighed weight is ultrasonically treated 30 minutes, puts
It is cold, then weighed weight, the weight of less loss is supplied with methanol, is shaken up, and is filtered, subsequent filtrate is taken, as test solution；
The preparation of reference solution: taking aurantiin reference substance appropriate, adds methanol that the solution of every 1ml 500 μ g containing aurantiin is made, and makees
For reference solution；
Chromatographic condition: being stream with 2% acetum using methanol as mobile phase A using octadecylsilane chemically bonded silica as filler
Dynamic phase B, gradient elution program are as follows: 0~25 minute, 30% mobile phase A, 70% Mobile phase B；25~75 minutes, 30% → 45%
Mobile phase A, 70% → 55% Mobile phase B；75~110 minutes, 45% → 100% mobile phase A, 55% → 0% Mobile phase B；110
~120 minutes, 100% mobile phase A, 0% Mobile phase B；Flow velocity is 1ml per minute, Detection wavelength 320nm；
Measuring method: it is accurate respectively to draw reference solution and each 5 μ l of test solution, liquid chromatograph is injected, records 120 points
The chromatogram of clock；4 characteristic peaks are presented in test sample characteristic spectrum, when wherein characteristic peak 1 should retain with corresponding object of reference peak
Between it is identical；Peak corresponding with aurantiin object of reference peak is the peak S, calculates the relative retention time of characteristic peak 2~4, the phase of characteristic peak 2
It is 1.49 ± 5% to retention time, the relative retention time of characteristic peak 3 is 1.74 ± 5%, the relative retention time of characteristic peak 4
It is 3.19 ± 6%.
8. detection method as claimed in claim 1, it is characterised in that the assay includes the following steps:
The preparation of test solution: taking test sample powder 50mg, accurately weighed, sets in 10ml volumetric flask, and methanol is added in precision
5ml is ultrasonically treated 30 minutes, lets cool, and adds 50% methanol to scale, shakes up, filter, subsequent filtrate is taken, as test solution；
The preparation of reference substance solution: taking aurantiin, Rhoifolin reference substance appropriate, accurately weighed, adds methanol that every 1ml difference is made
Solution containing 400 μ g of aurantiin, 10 μ g of Rhoifolin, as reference substance solution；
Chromatographic condition: using octadecylsilane chemically bonded silica as filler；It is molten with -0.3% acetic acid of methanol that volume ratio is 35: 65
Liquid is mobile phase；Detection wavelength is 283nm, 338nm；
Measuring method: it is accurate respectively to draw reference substance solution and each 5 μ l of test solution, inject liquid chromatograph, measurement, by outer
Mark method is with calculated by peak area content；The content of aurantiin is greater than 6.5%, and Rhoifolin content is greater than 0.37%.
9. detection method as claimed in claim 8, it is characterised in that the content of the aurantiin is greater than 8.5%, Rhus succedanea
Glycosides content is greater than 0.77%.
10. detection method as claimed in claim 9, it is characterised in that the content of the aurantiin is greater than 11.8%, open country paint
It sets glycosides content and is greater than 0.93%.
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