CN107167527A - A kind of lotus root polysaccharide binary finger-print and its construction method - Google Patents
A kind of lotus root polysaccharide binary finger-print and its construction method Download PDFInfo
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Abstract
The present invention relates to a kind of lotus root polysaccharide binary finger-print and its construction method, this method comprises the following steps：Lotus root skin, Leaf-feeding insects and rhizoma nelumbinis three are crushed respectively for examination position；Polyose extraction processing is carried out respectively for examination position by three of crushing, obtains lotus root polysaccharide sample；Carry out FTIR detections and HPLC detections respectively to lotus root polysaccharide sample, obtain polysaccharide infared spectrum and polysaccharide high-efficient liquid phase chromatogram；The lotus root skin for obtaining different cultivars lotus rhizome, the three polysaccharide infared spectrums and polysaccharide high-efficient liquid phase chromatogram for examination position of Leaf-feeding insects and rhizoma nelumbinis is repeated several times, then lotus root polysaccharide infrared standard finger-print and high performance liquid chromatography standard finger-print are built to obtain with average vector.It the advantage is that, multidimensional data based on polysaccharide in Nelumbo, its infrared standard finger-print and high performance liquid chromatography standard finger-print are constructed, the discriminating and quality control of lotus rhizome derived polysaccharide can be carried out with this binary finger-print, the degree of accuracy than single finger-print is higher.
Description
Technical field
The invention belongs to agricultural product and its quality analysis field of functional component, and in particular to a kind of lotus root polysaccharide binary refers to
Line collection of illustrative plates and its construction method.
Background technology
Lotus rhizome is China's cultivation most wide, sales volume and the maximum aquatic vegetable of sales range.According to described in dictionary of medicinal plant, lotus
Lotus root life with can heat-clearing, cool blood, dissipate it is tired, control pyreticosis polydipsia, it is ripe with can invigorating the spleen, appetizing, benefit blood, myogenic, antidiarrheal.Above-mentioned effect may
Part it is relevant with polysaccharide, lotus root polysaccharide through research confirm with antifatigue (Zhou Taoying, 2011), hypoglycemic (Luo Denghong, 2011),
Anti-oxidant (Luo Denghong, 2011；Yan Lang, 2008；Jiang, 2011) and immunological regulation (Jiang, 2011) isoreactivity.Fresh lotus rhizome
Middle Polyose extraction yield is up to 5% (Sun Jie, 2016；Wang Yu, 2007), and the traditional processing accessory substance such as lotus root skin, rhizoma nelumbinis and lotus root slag
In also containing abundant polysaccharide (Li Zhengyi, 2016；Yan Lang, 2007).At present, lotus rhizome processing scientific and technological level is weak, product " homogeneity
Change " phenomenon is serious, and economic value added is not high, and the accessory substances such as substantial amounts of lotus root skin, rhizoma nelumbinis and lotus root slag are produced in process not
Obtain effective exploitation utilization.Correspondingly, the high level of lotus root polysaccharide, which is developed, helps to improve product structure and expands industrial chain,
It is good in functional food and biomedicine field application prospect, and the quality control of polysaccharide is extremely crucial.
The fingerprint pattern technology of polysaccharide is widely used in terms of polysaccharide and its raw material quality control.LBP-X is (high
Eastwards, 2014), hericium erinaceum polysaccharide (Zhou Chunhui, 2016), Dendrobium officinale polysaccharide (Luo Jianping, 2009), cassia seeds polysaccharides are (high-new
Open, 2014), the fingerprint pattern technology of aloe polysaccharide (Dong Yinmao, 2015) can not only break through polysaccharide quality and the true and false and differentiate, also
The difference in raw material variety and the place of production can be realized.However, there is not been reported for the fingerprint pattern technology of lotus root polysaccharide.
The content of the invention
In order to solve lotus root polysaccharide quality control present in prior art and the problems such as true and false authentication technique lacks, this hair
It is bright that a kind of lotus root polysaccharide binary finger-print and its construction method are provided.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：A kind of structure side of lotus root polysaccharide binary finger-print
Method, it comprises the following steps：
1) it is lotus root section and rhizoma nelumbinis to take lotus rhizome to clean rear cutting, and lotus root section digs skin zone, and rhizoma nelumbinis excision fibrous root obtains lotus root skin, eaten
With position and rhizoma nelumbinis three for examination position, 5~20 mesh are crushed to respectively stand-by；
2) by step 1) in lotus root skin, Leaf-feeding insects and the rhizoma nelumbinis three crushed carried out respectively at Polyose extraction for examination positions
Reason, respectively obtains corresponding three parts of lotus root polysaccharide samples；
3) using step 2) obtained three parts of lotus root polysaccharide samples carry out FTIR detections and HPLC inspections as test object, respectively
Survey, obtain lotus root skin, Leaf-feeding insects and rhizoma nelumbinis three for the examination corresponding polysaccharide infared spectrum in position and polysaccharide high-efficient liquid phase chromatogram；
4) step 1 is repeated several times), respectively obtain 2) and 3) lotus root skin, Leaf-feeding insects and the rhizoma nelumbinis of multiple different cultivars lotus rhizomes
The three polysaccharide infared spectrums and polysaccharide high-efficient liquid phase chromatogram for examination position, with the average vector structure of all polysaccharide infared spectrums
Lotus root polysaccharide infrared standard finger-print is built to obtain, lotus root polysaccharide is built to obtain with the average vector of all polysaccharide high-efficient liquid phase chromatograms
High performance liquid chromatography standard finger-print, then construct described lotus root polysaccharide binary finger-print.
Above-mentioned technical proposal can have following further specifically chosen.
Specifically, step 2) in Polyose extraction processing particular content be：Solid-liquid ratio 1g will be pressed for examination position:15mL adds
Enter distilled water, be transferred to 90 DEG C of constant temperature water bath extraction 2h after homogenization 5min under 12000r/min rotating speed；After extraction terminates,
10min is centrifuged using 4500r/min, polysaccharide extraction liquid is separated by filtration；Polysaccharide extraction liquid pH to 6.5 is adjusted, alpha-amylase is added
Digest to iodine solution reaction and be negative in 70 DEG C of water-baths, be cooled to after room temperature and adjust to pH 4.5, add carbohydrase in 55 DEG C of water
Digest to content of reducing sugar and be not further added by bath, be warming up to 95 DEG C and keep 10min to go out enzyme；Enzyme-extracting solution vacuum at 70 DEG C
Original volume 1/4 is concentrated into, is taken off using Sevage methods and absolute ethyl alcohol is added after albumen to 80% ethanol volumetric concentration, in 4 DEG C of standings
Overnight；10min precipitation and separation polysaccharide is centrifuged using 4500r/min；Polysaccharide volatilizees ethanol after being washed through 80% ethanol, with a small amount of water
Freeze-drying obtains lotus root polysaccharide sample after redissolution.
Specifically, step 3) in FTIR detect particular content be：By step 2) obtained lotus root polysaccharide sample and KBr powder
End in mass ratio 1:100 mix grinding in mortar, are placed in mould and are pressed into transparent sheet, in 4000~400cm-1Wave-number range
Inside it is scanned acquisition polysaccharide infared spectrum.
Specifically, step 3) in HPLC detect particular content be：By step 2) obtained lotus root polysaccharide sample adds distillation
Water is configured to 5mg/mL polysaccharide solution, takes 100 μ L polysaccharide solutions in ampoule bottle, adds 100 μ L trifluoroacetic acid aqueous solution,
Inflated with nitrogen tube sealing, is placed in hydrolyzing 8h in 110 DEG C of baking ovens；After hydrolysis terminates, the hydrolyzate of cooling is transferred to centrifuge tube, is divided to two
It is secondary to wash ampoule bottle with 150 μ L methanol, and centrifuge tube is moved into the lump；Hydrolyzate N2Drying, is repeated plus methanol is dried up 2 times, is gone
Except trifluoroacetic acid, hydrolyzate sample is obtained after adding the dissolving of 100-150 μ L ultra-pure waters；Take 50 μ L hydrolyzates samples and 50 μ L
NaOH solution is mixed, and the PMP methanol solutions for adding 100 μ L react 100min after lucifuge in 70 DEG C of baking ovens；Reaction terminates rear chamber
Warm lucifuge cools down 10min, and the HCl solution for adding 100 μ L is neutralized, moisturizing to 2mL；Isometric chloroform is added, whirlpool is mixed, and is stood
Chloroform phase is discarded after layering, is repeated 3 times；Fetch water phase, after 0.45 μm of filtering with microporous membrane for HPLC it is qualitative, quantitative point
Analysis, obtains polysaccharide high-efficient liquid phase chromatogram.
Specifically, the concentration of the trifluoroacetic acid aqueous solution is 4mol/L, the concentration of the NaOH solution is 0.6mol/L,
The concentration of the PMP methanol solutions is 0.5mol/L, and the concentration of the HCL solution is 0.3mol/L.
Specifically, the lotus root polysaccharide infrared standard finger-print includes 5 common characteristic peaks, it is followed successively by：1076cm-1、
1420cm-1、1630cm-1、2930cm-1、3410cm-1。
Specifically, the lotus root polysaccharide high performance liquid chromatography standard finger-print includes 7 common characteristic peaks, when it retains
Between be followed successively by：12.94min、18.39min、20.35min、23.03min、27.90min、31.49min、33.67min.
Specifically, step 4) in multiple different cultivars lotus rhizomes quantity be more than 13.
In addition, the lotus root polysaccharide binary finger-print obtained by above method is also claimed in the present invention.
Beneficial effects of the present invention are：
1) lotus root polysaccharide is extracted respectively as confession examination position using lotus root skin, rhizoma nelumbinis and the Leaf-feeding insects of different cultivars lotus rhizome and obtain
Corresponding lotus root polysaccharide infrared finger print atlas is obtained, and lotus rhizome is constructed with the average vector of multiple lotus root polysaccharide infrared finger print atlas
Polysaccharide infrared standard finger-print, as with reference to collection of illustrative plates, using coefficient correlation (R) and the different product of included angle cosine (cos θ) analysis
Plant and different parts polysaccharide FTIR finger-print similitudes, find the infrared fingerprint figure of different cultivars lotus rhizome and different parts polysaccharide
Spectrum it is very much like, can as discriminating and the quality control of lotus rhizome derived polysaccharide foundation, it can also be used to lotus rhizome based food it is true
Property differentiate；
2) likewise, the present invention also constructs lotus with the average vector of multiple lotus root polysaccharide efficient liquid-phase chromatograph finger print atlas
Lotus root polysaccharide high performance liquid chromatography standard finger-print, using it as reference, different cultivars different parts are found through similarity analysis
The high performance liquid chromatography fingerprint characteristic of lotus root polysaccharide is more similar, therefore lotus root polysaccharide high performance liquid chromatography standard finger-print also may be used
Differentiate as the foundation of discriminating and the quality control of lotus rhizome derived polysaccharide, and applied to the authenticity of lotus rhizome based food；
3) present invention is whole using the polysaccharide of lotus root skin, rhizoma nelumbinis and the Leaf-feeding insects extraction from multiple kind lotus rhizomes as research object
The multidimensional data system of polysaccharide in Nelumbo is closed, the comprehensive and reliability of finger-print information is added；
4) lotus root polysaccharide infrared standard finger-print and high performance liquid chromatography standard finger-print are constructed, is referred to this binary
Line collection of illustrative plates carries out the discriminating and quality control of lotus rhizome derived polysaccharide, and compared to single finger-print, the degree of accuracy is higher.
Brief description of the drawings
Fig. 1 is the FTIR finger-prints of lotus root polysaccharide, and wherein a is that lotus rhizome Leaf-feeding insects polysaccharide FTIR finger-prints, b are lotus root
Skin polysaccharide FTIR finger-prints, c are that rhizoma nelumbinis polysaccharide FTIR finger-prints, d are lotus root polysaccharide FTIR standard finger-prints, figure acceptance of the bid
Number 1 to 13 corresponding site for representing 13 kinds extracts the corresponding collection of illustrative plates that polysaccharide carries out testing to obtain, and curve is from up in figure
Under be corresponding in turn to the collection of illustrative plates of 1 to 13 kind；
Fig. 2 is the HPLC chromatogram of monose hybrid standard product；
Fig. 3 is monosaccharide composition analysis HPLC chromatogram in lotus root polysaccharide, and it is that lotus rhizome Leaf-feeding insects are more that wherein a and b, which are respectively,
Sugared HPLC finger-prints and its standard diagram, c and d are respectively lotus root skin polysaccharide HPLC finger-prints and its standard diagram, e and f points
Wei not rhizoma nelumbinis polysaccharide HPLC finger-prints and its standard diagram；G is the HPLC standard finger-prints of 39 lotus root polysaccharide samples.
Embodiment
Below in conjunction with drawings and the specific embodiments, the present invention is described in further detail, and example is served only for explaining
The present invention, is not intended to limit the scope of the present invention.
The present invention provides a kind of lotus root polysaccharide binary fingerprint map construction method, and this method includes following particular content：
1. the preparation of lotus rhizome test sample
Lotus root, bar river lotus root, Bai Pao are floated in Hubei Province lotus No. 5, E Lian 6, E Lian 7, E Lian 8, Yingcheng City's white lotus, sheep of galloping along on horseback, Guixi
Son, Bobai lotus root, military plant 2,8143, Changzhou drift river, 13 kind lotus rhizomes are by national germplasm Wuhan aquatic vegetable resource garden altogether
There is provided, sample time is in October, 2015.
It is lotus root section and rhizoma nelumbinis to take lotus rhizome to clean rear cutting；Lotus root section digs skin zone, and rhizoma nelumbinis excision fibrous root obtains lotus root skin, eaten
Position and rhizoma nelumbinis three are for examination position；It is stand-by that 5~20 mesh are crushed to respectively for examination position.2. the preparation of lotus root polysaccharide
Weigh step 1) in lotus rhizome test sample 100g, by solid-liquid ratio (g/mL) 1:15 add distilled water, in 12000r/
90 DEG C of constant temperature water bath extraction 2h are transferred under min rotating speed after homogenization 5min；After extraction terminates, centrifuged using 4500r/min
10min, is separated by filtration polysaccharide extraction liquid；Adjust polysaccharide extraction liquid pH to 6.5, add alpha-amylase digested in 70 DEG C of water-baths to
Iodine solution reaction is negative, and is cooled to after room temperature and adjusts to pH 4.5, and addition carbohydrase is digested to reduced sugar in 55 DEG C of water-baths to be contained
Amount is not further added by, and is warming up to 95 DEG C and is kept 10min to go out enzyme；Enzyme-extracting solution is concentrated in vacuo to original volume 1/4 at 70 DEG C, adopts
Taken off with Sevage methods and absolute ethyl alcohol is added after albumen to 80% ethanol volumetric concentration, stood overnight in 4 DEG C；Using 4500r/min
Centrifuge 10min precipitation and separation polysaccharide；Polysaccharide volatilizees ethanol after being washed through 80% ethanol, is freeze-dried and obtains after being redissolved with a small amount of water
Lotus root polysaccharide.
3. structure and the analysis of lotus root polysaccharide binary standard finger-print
The FTIR fingerprint map analyzings of 3-1. lotus root polysaccharides
Take polysaccharide sample and KBr powder in mass ratio 1:100 mix grinding in mortar, are placed in mould and are pressed into thin transparent
Piece, in 4000~400cm-1Acquisition FTIR collection of illustrative plates is scanned in wave-number range.
The Leaf-feeding insects of 13 kind lotus rhizomes, skin zone, section portion prepare the FTIR finger-prints of gained polysaccharide and with more than 39
The average vector of sugar-like product FTIR finger-prints builds standard FTIR finger-prints, as shown in Figure 1.Referred to the standard FTIR of structure
Line collection of illustrative plates is, with reference to collection of illustrative plates, different cultivars and different parts polysaccharide to be analyzed using coefficient correlation (R) and included angle cosine (cos θ)
FTIR finger-print similitudes, related data is as shown in table 1.The correlation coefficient value of 39 polysaccharide samples is 0.9856 ± 0.2049
(n=39), and included angle cosine value be 0.9991 ± 0.0014 (n=39), thus illustrate that different cultivars lotus rhizome and different parts are more
Sugar FTIR finger-prints it is very much like, can as lotus root source property polysaccharide discriminating foundation.
The similarity analysis of the lotus root polysaccharide FTIR finger-prints of table 1.
The HPLC fingerprint map analyzings of 3-2. lotus root polysaccharides
(1) structure of the PCD-HPLC methods of monosaccharide composition analysis
Compound concentration is 5mmol/L mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, Portugal respectively
Grape sugar, galactolipin, arabinose standard solution；Compound concentration is respectively 1,2,3,4,5mmol/L monose hybrid standard liquid.
50 μ L monose titer and the mixed standard solution of various concentrations gradient are drawn respectively, respectively add 50 μ L NaOH solution
(0.6mol/L) is placed in 5mL tool plug test tubes, and whirlpool is mixed；Plus 100 μ L PMP (0.5mol/L) methanol solution, whirlpool mix,
Lucifuge reacts 100min in 70 DEG C of baking ovens, takes out lucifuge and cools down 10min to room temperature, adds 100 μ L HCl solution (0.3mol/
L) neutralize, moisturizing to 2mL；Add isometric chloroform whirlpool to mix, chloroform phase is discarded after stratification, so extraction 3 times；By water
Mutually with being analyzed after 0.45 μm of filtering with microporous membrane for HPLC, the foundation of qualitative, quantitative is used as.Monose is detected using HPLC methods
Constitute and content, chromatographiccondition is：UV detector wavelengths are 250nm, and chromatographic column is AgilentExtend-C18 chromatographic columns
(4.6 × 250mm, 5 μm), column temperature is 30 DEG C, mobile phase A (0.1mol/L sodium phosphate buffer, pH 6.7) and Mobile phase B
(acetonitrile) (0~25min, mobile phase A and B volume ratio maintain 84 using gradient elution:16；25~36min, mobile phase A and B
Volume ratio be down to 80:20；36~40min, mobile phase A and B volume ratio rise to 84:16), elution flow rate is 1.0mL/min,
Sampling volume is 20 μ L.The HPLC chromatogram of obtained monose standard items is as shown in Figure 2.
From Figure 2 it can be seen that 8 kinds of monose hybrid standard product are preferably separated in chromatogram, each monose (1 → 8) correspondence
Retention time be followed successively by：Mannose (Man) 12.87min, ribose (Rib) 16.99min, rhamnose (Rha) 18.32min, Portugal
Grape alditol (GlcUA) 20.13min, galacturonic acid (GalUA) 22.85min, glucose (Glc) 27.62min, galactolipin
(Gal) 31.19min, arabinose (Ara) 33.45min.The concentration of 8 kinds of monose and the linearly dependent coefficient of peak area between
0.9925~0.9951.
(2) monosaccharide composition analysis and evaluation of methodology of lotus root polysaccharide
100 μ L polysaccharide solutions (5mg/mL) are taken in ampoule bottle, the trifluoroacetic acid (TFA, 4mol/L) for adding 100 μ L is molten
Liquid, inflated with nitrogen tube sealing is placed in hydrolyzing 8h in 110 DEG C of baking ovens；After hydrolysis terminates, the hydrolyzate of cooling is transferred to centrifuge tube,
Ampoule bottle is washed with 150 μ L methanol in two times, and moves into centrifuge tube in the lump；Hydrolyzate is dried up with N2, is repeated plus methanol drying 2
It is secondary, TFA is removed, hydrolyzate sample is obtained after adding ultra-pure water dissolving；Take 50 μ L hydrolyzates and 50 μ L NaOH solution
(0.6mol/L) is mixed, and the PMP methanol solutions (0.5mol/L) for adding 100 μ L react 100min after lucifuge in 70 DEG C of baking ovens；
Reaction terminates rear room temperature lucifuge cooling 10min, and the HCl solution (0.3mol/L) for adding 100 μ L is neutralized, moisturizing to 2mL；Add etc.
Volume of chloroform, whirlpool is mixed, and is discarded chloroform phase after stratification, is repeated 3 times；Water intaking phase, through 0.45 μm of miillpore filter mistake
HPLC qualitative and quantitative analysis is supplied after filter.
The PMP derivatives of mixing monose standard items are taken, sample introduction are repeated 5 times, the RSD of 8 kinds of monose peak areas and retention time
Value is respectively less than 5%, illustrates that detection method precision is better.
Hydrolysis, derivatization and the lotus root polysaccharide sample neutralized after extraction are placed into measure monose group after 0,4,8 and 12h respectively
Into and content, as a result show the peak area standard deviation RSD of each chromatographic peak of sample between 2.11%~2.98%, retention time
RSD values are between 1.32%~2.03%, and detection method has good stability in 12h.
To 5 parts of lotus root polysaccharide sample solutions, be hydrolyzed respectively, derivatization, neutralize extracting operation prepare it is parallel
5 parts of lotus root polysaccharide HPLC test liquids.Determine monose composition and content finds that the peak area RSD values of each chromatographic peak are between 1.35%
~3.56%, retention time RSD values show that the repeatability of detection method is good between 1.13%~3.31%.
(3) the PCD-HPLC fingerprint map constructions of lotus root polysaccharide
The monosaccharide composition analysis of lotus root polysaccharide, the PCD-HPLC of obtained lotus rhizome different parts are carried out according to the method for (2)
Finger-print is as shown in figure 3, from Fig. 3 a to Fig. 3 e, the PCD-HPLC finger-prints more phase of different parts lotus root polysaccharide
Seemingly, but also slightly has difference.The fingerprint characteristic of its common pattern, the concrete analysis data such as institute of table 2 are analyzed with minimum shared peak value 1%
Show, the shared peak containing Man, GalUA, Glc, Gal and Ara, and Glc are by the similarity of the visible different parts of table 2
Peak area percent with Gal character pairs peak is more than 91%.And be in place of faint difference：Leaf-feeding insects polysaccharide also contains
There is Rha shared peak；Shared peak of skin zone's polysaccharide also containing Rib；And rhizoma nelumbinis polysaccharide being total to containing Rha, Rib and GlcUA simultaneously
There is peak.
Lotus root polysaccharide standard HPLC finger-prints (Fig. 3 f are built to the average vector of 39 polysaccharide sample PCD-HPLC collection of illustrative plates
It is shown), as with reference to collection of illustrative plates, different cultivars is analyzed using coefficient correlation (R) and included angle cosine (cos θ) and different parts are more
The PCD-HPLC finger-print similitudes of sugar, as shown in table 3, the Pearon between two kinds of Similarity values is related for concrete analysis data
Property extremely significantly (P<0.001).In addition to lotus root Leaf-feeding insects polysaccharide and Bobai lotus root skin zone polysaccharide being floated except Guixi, remaining 37 polysaccharide sample
Correlation coefficient value and included angle cosine value are above 0.9.Thus illustrate, the HPLC fingerprint characteristics of different cultivars and different parts polysaccharide
It is more similar, the important evidence that its standard finger-print can differentiate as the lotus root polysaccharide true and false, and lotus root Leaf-feeding insects are floated in Guixi
The HPLC finger-print specificity of polysaccharide can be used for Variety identification reference.
The shared peak information of the PCD-HPLC finger-prints of the lotus root polysaccharide of table 2.
Note：When " nd " represents that it is 1% to take minimum shared peak value, the monose is not detected in common pattern.
The similarity analysis of the lotus root polysaccharide PCD-HPLC finger-prints of table 3.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (9)
1. a kind of construction method of lotus root polysaccharide binary finger-print, it is characterised in that comprise the following steps：
1) cutting is lotus root section and rhizoma nelumbinis after taking lotus rhizome clean, and lotus root section digs skin zone and obtains lotus root skin and Leaf-feeding insects, and rhizoma nelumbinis cuts off fibrous root,
Lotus root skin, Leaf-feeding insects and rhizoma nelumbinis three are crushed to 5~20 mesh respectively for examination position, it is stand-by；
2) by step 1) in lotus root skin, Leaf-feeding insects and the rhizoma nelumbinis three crushed carry out Polyose extraction processing respectively for examination position, point
Corresponding three parts of lotus root polysaccharide samples are not obtained；
3) using step 2) obtained three parts of lotus root polysaccharide samples carry out FTIR detections and HPLC detections as test object, respectively, obtain
To lotus root skin, Leaf-feeding insects and rhizoma nelumbinis three for the examination corresponding polysaccharide infared spectrum in position and polysaccharide high-efficient liquid phase chromatogram；
4) step 1 is repeated several times), respectively obtain 2) and 3) the lotus root skin, Leaf-feeding insects and rhizoma nelumbinis three of multiple different cultivars lotus rhizomes
Polysaccharide infared spectrum and polysaccharide high-efficient liquid phase chromatogram for trying position, are built with the average vector of all polysaccharide infared spectrums
Lotus root polysaccharide infrared standard finger-print, with the average vector of all polysaccharide high-efficient liquid phase chromatograms build lotus root polysaccharide is efficient
Liquid chromatogram standard finger-print, then construct described lotus root polysaccharide binary finger-print.
2. the construction method of a kind of lotus root polysaccharide binary finger-print according to claim 1, it is characterised in that step 2)
In Polyose extraction processing particular content be：Solid-liquid ratio 1g will be pressed for examination position:15mL adds distilled water, in 12000r/min
Rotating speed under 90 DEG C of constant temperature water baths extraction 2h are transferred to after homogenization 5min；After extraction terminates, centrifuged using 4500r/min
10min, is separated by filtration polysaccharide extraction liquid；Adjust polysaccharide extraction liquid pH to 6.5, add alpha-amylase digested in 70 DEG C of water-baths to
Iodine solution reaction is negative, and is cooled to after room temperature and adjusts to pH 4.5, and addition carbohydrase is digested to reduced sugar in 55 DEG C of water-baths to be contained
Amount is not further added by, and is warming up to 95 DEG C and is kept 10min to go out enzyme；Enzyme-extracting solution is concentrated in vacuo to original volume 1/4 at 70 DEG C, adopts
Taken off with Sevage methods and absolute ethyl alcohol is added after albumen to 80% ethanol volumetric concentration, stood overnight in 4 DEG C；Using 4500r/min
Centrifuge 10min precipitation and separation polysaccharide；Polysaccharide volatilizees ethanol after being washed through 80% ethanol, is freeze-dried and obtains after being redissolved with a small amount of water
Lotus root polysaccharide sample.
3. the construction method of a kind of lotus root polysaccharide binary finger-print according to claim 1, it is characterised in that step 3)
The particular content of middle FTIR detection is：By step 2) obtained lotus root polysaccharide sample and KBr powder in mass ratio 1:100 in mortar
It is middle to mix grinding, it is placed in mould and is pressed into transparent sheet, in 4000~400cm-1Acquisition polysaccharide is scanned in wave-number range red
Outer collection of illustrative plates.
4. the construction method of a kind of lotus root polysaccharide binary finger-print according to claim 1, it is characterised in that step 3)
The particular content of middle HPLC detection is：By step 2) obtained lotus root polysaccharide sample plus distilled water be configured to 5mg/mL polysaccharide it is molten
Liquid, takes 100 μ L polysaccharide solutions in ampoule bottle, adds 100 μ L trifluoroacetic acid aqueous solution, and inflated with nitrogen tube sealing is placed in 110 DEG C
8h is hydrolyzed in baking oven；After hydrolysis terminates, the hydrolyzate of cooling is transferred to centrifuge tube, ampoule is washed with 150 μ L methanol in two times
Bottle, and centrifuge tube is moved into the lump；Hydrolyzate N2Drying, is repeated plus methanol is dried up 2 times, is removed trifluoroacetic acid, is added 100-150
Hydrolyzate sample is obtained after the dissolving of μ L ultra-pure waters；Take 50 μ L hydrolyzates samples and 50 μ L NaOH solution to mix, add 100 μ L's
PMP methanol solutions react 100min after lucifuge in 70 DEG C of baking ovens；Reaction terminates rear room temperature lucifuge cooling 10min, adds 100 μ L
HCl solution neutralize, moisturizing to 2mL；Isometric chloroform is added, whirlpool is mixed, and is discarded chloroform phase after stratification, is repeated
3 times；Water intaking phase, HPLC qualitative and quantitative analysis is supplied after 0.45 μm of filtering with microporous membrane, polysaccharide high-efficient liquid phase chromatogram is obtained.
5. the construction method of a kind of lotus root polysaccharide binary finger-print according to claim 4, it is characterised in that described three
The concentration of the fluoroacetic acid aqueous solution is 4mol/L, and the concentration of the NaOH solution is 0.6mol/L, the concentration of the PMP methanol solutions
For 0.5mol/L, the concentration of the HCL solution is 0.3mol/L.
6. a kind of construction method of lotus root polysaccharide binary finger-print according to claim 1, it is characterised in that the lotus
Lotus root polysaccharide infrared standard finger-print includes 5 common characteristic peaks, is followed successively by：1076cm-1、1420cm-1、1630cm-1、
2930cm-1、3410cm-1。
7. a kind of construction method of lotus root polysaccharide binary finger-print according to claim 1, it is characterised in that the lotus
Lotus root polysaccharide high performance liquid chromatography standard finger-print includes 7 common characteristic peaks, and its retention time is followed successively by：12.94min、
18.39min、20.35min、23.03min、27.90min、31.49min、33.67min。
8. a kind of construction method of lotus root polysaccharide binary finger-print according to any one of claim 1 to 7, its feature exists
In step 4) in multiple different cultivars lotus rhizomes quantity be more than 13.
9. a kind of lotus root polysaccharide binary finger-print, it is characterised in that obtained by the construction method described in claim 1 to 8.
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