CN104215491B - A kind of method extracted from plant and measure haemachrome - Google Patents
A kind of method extracted from plant and measure haemachrome Download PDFInfo
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Abstract
The present invention discloses a kind of method extracted from plant and measure haemachrome, comprise the following steps: step one, employing acetone and the mixed solution centrifugation of distilled water, remove chlorophyll, then with hydrochloric acid, acetone and the mixed solution centrifugation of distilled water, obtain mixed extract, extracting with ether, concentrate drying obtains the plant heme being dried, standby the most again；Step 2, employing ultraviolet spectrophotometer measure the absorbance at 574.5nm；Step 3, mean light absorbency A step 2 obtained substitute into equation of linear regression: A=2.6814496C+0.07506221, are calculated extension rate C, further according to Ci=0.5C mg/ml, calculate concentration C i of haemachrome in plant leaf blade.The method extracting and measuring haemachrome from plant that the present invention provides can content of hemachrome in Accurate Determining plant, play a significant role in the research of chlorophyll metabolism regulatory mechanism.
Description
Technical field
The present invention relates to extraction and the assay method of a kind of pigment, relate generally to one and extract from plant and measure blood red
The method of element.
Background technology
Haemachrome is the tetrapyrrole porphyrins that a class is important, is distributed widely in the blood of animal, muscle and one
In a little plant tissues.In animal, haemachrome is primarily present in the erythrocyte in blood, combines jointly with globin
Constitute hemoglobin；In plant, haemachrome take part in electron transmission as the prothetic group of cytochrome enzyme, peroxidase.
At present, it is more that the haemachrome in animal extracts research, the most ripe, has had and has compared rounded system, it is possible to achieve in batches
Produce.The haemachrome extracted from animal blood is widely used in medicine and health food, is used for treating nutritional anemia, evil
Property tumor, hepatitis etc..In plant, structure and the chlorophyll of haemachrome are closely similar, are all cyclized by four pyrroles and are formed
Porphyrin compound, has chelated an iron ion in the porphyrin ring of haemachrome, as it is shown in figure 1, and chelate in chlorophyll porphyrin ring
One magnesium ion, as shown in Figure 2.At biosynthetic preliminary stage, from ALA be synthesized to Proto IX synthesis be all haemachrome and
Chlorophyll is common, as shown in Figure 3.Haemachrome molecule can also (leaf be green as signal factor regulation and control glutamic acid-tRNA reductase
First crucial rate-limiting enzyme in element building-up process, this enzyme can directly affect whole Chlorophyll synthesis process) and light cooperation
Expression by relevant karyogene.Therefore, the content of hemachrome in Accurate Determining plant is to research chlorophyll metabolism regulatory mechanism
Extremely important.Content of hemachrome in animal blood is the highest, and research is a lot, extracts the easiest, and technology is the most ripe；Plant group
Content of hemachrome in knitting is the lowest, studies less, extraction comparison difficulty, does not also have relatively cheap and believable assay method,
Become a bottleneck in chlorophyll metabolism research work.
Summary of the invention
It is an object of the invention to for the problem of haemachrome extraction comparison difficulty in plant, it is provided that a kind of extraction from plant
With the method measuring haemachrome, the method can content of hemachrome in Accurate Determining plant, at chlorophyll metabolism regulatory mechanism
Research in play a significant role.
The present invention realizes above-mentioned purpose and the technical scheme is that a kind of side extracted from plant and measure haemachrome
Method, comprises the following steps:
Step one, the extraction of plant heme
The most accurately weigh the fresh blade 1g treating measuring plants, be placed in the mortar of pre-cooling, add liquid nitrogen and be ground to powder, so
After be transferred in centrifuge tube, add the mixed solution I of acetone and distilled water, centrifugal after concussion uniformly, abandoning supernatant, repeat to
Centrifugal product adds mixed solution I, concussion and the centrifugally operated of acetone and distilled water, until the centrifugal product obtained is nothing
Color, obtains Primary product, standby；Adding the mixed solution II of acetone and distilled water again in the Primary product obtained, concussion is all
Even rear centrifugal, abandoning supernatant, repeats to add acetone and the mixed solution II of distilled water in centrifugal product, shake and be centrifuged behaviour
Make, until the centrifugal product obtained is colourless, obtain secondary species, standby；
In described mixed solution I, the ratio shared by acetone is more than the ratio in mixed solution II；
B. in the secondary species that step a obtains, add the mixed solution of hydrochloric acid, acetone and distilled water, after concussion uniformly from
The heart, collects supernatant, standby；Then in the centrifugal precipitation obtained, again add the mixed solution of hydrochloric acid, acetone and distilled water,
It is centrifuged after concussion uniformly, collects supernatant, merge twice supernatant, obtain mixed extract, standby；
C. in the mixed extract that step b obtains, add ether, join after mix homogeneously in separatory funnel, by separatory
Funnel be repeatedly inverted after stratification, collect upper solution, standby；And in lower floor's solution, again add ether, mix homogeneously
After join in separatory funnel, after stratification collect upper solution, merge the upper solution obtained for twice, be placed in separatory funnel
In, it is subsequently adding distilled water isopyknic with the upper solution merged and washs, take ether layer after stratification, join rotation
Turn in evaporimeter and concentrate, then enriched product is poured in centrifuge tube, dry up with nitrogen, i.e. obtain the plant heme being dried, standby
With；
Step 2, plant heme assay
7.44ml distilled water and 0.48ml 5N NaOH, stirring is added in the plant heme being dried that step one obtains
Add 12 ml distilled water and 4.08 ml pyridine, again mix homogeneously after dissolving to plant heme, use ultraviolet spectrometry light
Degree meter measures the absorbance at 574.5nm, adds the iron cyanide of several dithionates and 50mmol/L during measurement in the sample,
The blade of the plant taking more than three independent strains or three strains is measured, and repeatedly measures and averages, obtains mean light absorbency
A, standby；
Step 3, the calculating of plant heme concentration
Mean light absorbency A step 2 obtained substitutes into equation of linear regression: A=2.6814496C+0.07506221, meter
Calculation obtains extension rate C, further according to Ci=0.5C mg/ml, calculates concentration C i of haemachrome in plant leaf blade.
Concussion described in step a uniformly required time is 10-25min, centrifugal condition be centrifugal force 4000g, centrifugal time
Between 8-20min.
Acetone described in step a and acetone and volume ratio 99:1 of distilled water, mixed solution in the mixed solution I of distilled water
I each addition is 10ml.
Acetone described in step a and acetone and volume ratio 80:20 of distilled water in the mixed solution II of distilled water, mix molten
The each addition of liquid II is 5ml.
Hydrochloric acid, acetone described in step b are 5 with the volume ratio of hydrochloric acid, acetone and distilled water in the mixed solution of distilled water:
80:15, each addition is 5ml.
The concussion time described in step b is 10-25min, and centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min.
The each addition of ether in step c is 10ml.
Beneficial effect
The method extracting and measuring haemachrome from plant of one, present invention offer can successfully carry from plant tissue
Taking haemachrome, extraction effect is the most stable, can play a significant role in the research work of Chlorophyll synthesis regulatory mechanism, solves
The extraction of current animal blood red pigment is studied the most deeply, it is already possible to realize scale, industrialization, and blood red in plant
Cellulose content is the lowest, extracts difficulty, studies less problem.
The present invention is only with some common agents and ultraviolet spectrophotometer, it is possible to measure the haemachrome in plant sample
Content, and results contrast is reliable, can meet the research needs in laboratory completely, not only low cost, suitable popularization and application, also
Substantially overcome the haemachrome in current plant and measure the technical problem not having cheap and reliable method.
Accompanying drawing explanation
Fig. 1 is the schematic arrangement of haemachrome；
Fig. 2 is chlorophyllous schematic arrangement；
Fig. 3 is haemachrome and chlcrophyll biosynthesis approach schematic diagram；
Fig. 4 is that the haemachrome standard test solution ultraviolet spectrophotometer of variable concentrations is swept in the range of 350-600nm
Situation about retouching；
Fig. 5 is the canonical plotting of the haemachrome of SPSS software analysis.
Detailed description of the invention
A kind of method extracted from plant and measure haemachrome, comprises the following steps:
Step one, the extraction of plant heme
The most accurately weigh the fresh blade 1g treating measuring plants, be placed in the mortar of pre-cooling, add liquid nitrogen and be ground to powder, so
After be transferred in centrifuge tube, add the mixed solution I of acetone and distilled water, centrifugal after concussion uniformly, abandoning supernatant, repeat to
Centrifugal product adds mixed solution I, concussion and the centrifugally operated of acetone and distilled water, until the centrifugal product obtained is nothing
Color, obtains Primary product, standby；Adding the mixed solution II of acetone and distilled water again in the Primary product obtained, concussion is all
Even rear centrifugal, abandoning supernatant, repeats to add acetone and the mixed solution II of distilled water in centrifugal product, shake and be centrifuged behaviour
Make, until the centrifugal product obtained is colourless, obtain secondary species, standby；
In described mixed solution I, the ratio shared by acetone is more than the ratio in mixed solution II；
B. in the secondary species that step a obtains, add the mixed solution of hydrochloric acid, acetone and distilled water, after concussion uniformly from
The heart, collects supernatant, standby；Then in the centrifugal precipitation obtained, again add the mixed solution of hydrochloric acid, acetone and distilled water,
It is centrifuged after concussion uniformly, collects supernatant, merge twice supernatant, obtain mixed extract, standby；
C. in the mixed extract that step b obtains, add ether, join after mix homogeneously in separatory funnel, by separatory
Funnel be repeatedly inverted after stratification, collect upper solution, standby；And in lower floor's solution, again add ether, mix homogeneously
After join in separatory funnel, after stratification collect upper solution, merge the upper solution obtained for twice, be placed in separatory funnel
In, it is subsequently adding distilled water isopyknic with the upper solution merged and washs, take ether layer after stratification, join rotation
Turn in evaporimeter and concentrate, then enriched product is poured in centrifuge tube, dry up with nitrogen, i.e. obtain the plant heme being dried, standby
With；
Step 2, plant heme assay
7.44ml distilled water and 0.48ml 5N NaOH, stirring is added in the plant heme being dried that step one obtains
Add 12 ml distilled water and 4.08 ml pyridine, again mix homogeneously after dissolving to plant heme, use ultraviolet spectrometry light
Degree meter measures the absorbance at 574.5nm, adds the iron cyanide of several dithionates and 50mmol/L during measurement in the sample,
The blade of the plant taking more than three independent strains or three strains is measured, and repeatedly measures and averages, obtains mean light absorbency
A, standby；
Step 3, the calculating of plant heme concentration
Mean light absorbency A step 2 obtained substitutes into equation of linear regression: A=2.6814496C+0.07506221, meter
Calculation obtains extension rate C, further according to Ci=0.5C mg/ml, calculates concentration C i of haemachrome in plant leaf blade.
Concussion described in step a uniformly required time is 10-25min, centrifugal condition be centrifugal force 4000g, centrifugal time
Between 8-20min.
Acetone described in step a and acetone and volume ratio 99:1 of distilled water, mixed solution in the mixed solution I of distilled water
I each addition is 10ml.
Acetone described in step a and acetone and volume ratio 80:20 of distilled water in the mixed solution II of distilled water, mix molten
The each addition of liquid II is 5ml.
Hydrochloric acid, acetone described in step b are 5 with the volume ratio of hydrochloric acid, acetone and distilled water in the mixed solution of distilled water:
80:15, each addition is 5ml.
The concussion time described in step b is 10-25min, and centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min.
The each addition of ether in step c is 10ml.
Measuring method reliability detects
(1) precision weighs 0.012 gram of haemachrome standard substance and is placed in small beaker, adds 7.44 ml distilled water, 0.48 ml
The NaOH of 5N, stirs and adds 12 ml distilled water, 4.08 ml pyridines after dissolving to haemachrome, be made into after being again stirring for uniformly
Standard test solution；
(2) the haemachrome standard test solution configured is diluted to 4 × 10 respectively-2, 5 × 10-2, 1 × 10-1, 1.5 ×
10-1, 2 × 10-1, 3 × 10-1, 4 × 10-1And 5 × 10-1Times, it is then respectively adding the ferrum of several dithionates and 50mmol/L
Cyanide, scans and records maximum absorbance in the range of 350-600nm, and result is as shown in table 1:
Table 1: the absorbance of variable concentrations haemachrome standard solution
* concentration C represents the extension rate of haemachrome standard solution.
(3) standard curve result: as shown in Figure 4, the haemachrome standard solution of all concentration is all about for result
Having the absworption peak of a feature at 574.5nm, and as shown in Figure 4, hemoglobin concentration is the highest, absworption peak is the most obvious.With blood red
Element relative concentration is abscissa, and maximum absorbance 574.5nm at is that vertical coordinate is mapped, and obtains Fig. 5, and carries out statistics and divide
Analysis.Standard curve data SPSS software is analyzed,
(4) as seen from Figure 5, absorbance A and relative concentration C i.e. extension rate is linear, and the phase of the two
Closing property is the highest, R2=0.989.These results suggest that the haemachrome assay method used in the present invention is the most reliable.With
After detection in, all use the absorbance at 574.5nm to weigh the content of hemachrome in plant.
Linear regression is carried out, then with method of least square:
∑Ci=0.04+0.05+0.1+0.15+0.2+0.3+0.4+0.5=1.74
∑Ai=5.26622
∑ Ci2=0.5991, ∑ Ai2=5.25538733, ∑ CiAi=1.7370647, (i=1,2,3,4,5,6,7,8)
Set up regression equation: A=bC+a,
∑Ci2∑Ai-∑Ci∑CiAi
Wherein, a=---------------------------=0.07506221
n∑Ci2-(∑Ci)2
n∑CiAi-∑Ci∑Ai
b=------------------------------=2.6814496
n∑Ci2-(∑Ci)2
Therefore, regression equation is: A=2.6814496C+0.07506221.
A is absorbance, and C is standard solution extension rate.Concentration of standard solution is 0.0005 g/ml, therefore testing sample
In haemachrome actual content be: Ci=0.0005C g/ml, i.e. 0.5C mg/ml.Measuring samples absorbance at 574.5nm
Value A, can be extrapolated C by regression equation, further converse the haemachrome actual content Ci in sample.
Below in conjunction with specific embodiment, the present invention will be further described:
Embodiment 1:
A kind of method extracted from Brassica Oleracea Var.Acephala pigeon and measure haemachrome, comprises the following steps:
Step one, the extraction of Brassica Oleracea Var.Acephala pigeon haemachrome
The most accurately weigh the fresh blade 1g of Brassica Oleracea Var.Acephala pigeon, be placed in the mortar of pre-cooling, add liquid nitrogen and be ground to powder
Shape, is then transferred in centrifuge tube, adds the acetone of 10ml volume ratio 99:1 and the mixed solution I of distilled water, after concussion uniformly
Centrifugal, centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min, abandoning supernatant, repeats to add in centrifugal product third
Ketone and mixed solution I, concussion and the centrifugally operated of distilled water, until the centrifugal product obtained is colourless, obtain Primary product, standby
With；Adding the acetone of 5ml volume ratio 80:20 and the mixed solution II of distilled water again in the Primary product obtained, concussion is uniformly
Rear centrifugal, centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min, abandoning supernatant, repeats to add in centrifugal product
Acetone and mixed solution II, concussion and the centrifugally operated of distilled water, until the centrifugal product obtained is colourless, obtain secondary product
Thing, standby；
B. adding 5ml volume ratio in the secondary species that step a obtains is the mixed of 5:80:15 hydrochloric acid, acetone and distilled water
Closing solution, centrifugal after concussion uniformly, the concussion time is 10-25min, and centrifugal condition is centrifugal force 4000g, centrifugation time 8-
20min, collects supernatant, standby；Then in the centrifugal precipitation obtained, hydrochloric acid, acetone are again added molten with the mixing of distilled water
Liquid, centrifugal after concussion uniformly, the concussion time is 10-25min, and centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min, receives
Collection supernatant, merges twice supernatant, obtains mixed extract, standby；
C. in the mixed extract that step b obtains, add 10ml ether, join after mix homogeneously in separatory funnel, will
Separatory funnel be repeatedly inverted after stratification, collect upper solution, standby；And in lower floor's solution, again add 10ml ether,
Join after mix homogeneously in separatory funnel, collect upper solution after stratification, merge the upper solution obtained for twice, be placed in
In separatory funnel, it is subsequently adding distilled water isopyknic with the upper solution merged and washs, after stratification, take ether layer,
Joining in Rotary Evaporators and concentrate, then poured into by enriched product in centrifuge tube, dry up with nitrogen, the garment or robe made of feathers i.e. obtaining being dried is sweet
Blue pigeon haemachrome, standby；
Step 2, Brassica Oleracea Var.Acephala pigeon content of hemachrome measure
7.44ml distilled water and 0.48ml 5N is added in the Brassica Oleracea Var.Acephala pigeon haemachrome being dried that step one obtains
NaOH, stirs and adds 12 ml distilled water and 4.08 ml pyridines after dissolving to Brassica Oleracea Var.Acephala pigeon haemachrome, and mixing is all again
Even, use ultraviolet spectrophotometer measure 574.5nm place absorbance, add in the sample during measurement several dithionates with
The iron cyanide of 50mmol/L.The blade taking three independent strain Brassica Oleracea Var.Acephala pigeons is measured, and repeatedly measures and averages,
Being 0.188833 to mean light absorbency A, result is as shown in table 2, standby；
Table 2: the absorbance A of Brassica Oleracea Var.Acephala pigeon sample
Step 3, the calculating of Brassica Oleracea Var.Acephala pigeon hemoglobin concentration
Mean light absorbency A step 2 obtained substitutes into equation of linear regression: A=2.6814496C+0.07506221, meter
It is 0.04243 that calculation obtains extension rate C, further according to Ci=0.5C mg/ml, calculates haemachrome in Brassica Oleracea Var.Acephala pigeon blade
Concentration C i be 0.021215 mg/ml.
Embodiment 2:
A kind of method extracted from Plantula Brassicae chinensis and measure haemachrome, comprises the following steps:
Step one, the extraction of Plantula Brassicae chinensis haemachrome
The most accurately weigh the fresh blade 1g of Plantula Brassicae chinensis, be placed in the mortar of pre-cooling, add liquid nitrogen and be ground to powder, then
It is transferred in centrifuge tube, adds the acetone of 10ml volume ratio 99:1 and the mixed solution I of distilled water, centrifugal after concussion uniformly, from
Heart condition is centrifugal force 4000g, centrifugation time 8-20min, abandoning supernatant, repeats to add acetone and distillation in centrifugal product
The mixed solution I of water, concussion and centrifugally operated, until the centrifugal product obtained is colourless, obtain Primary product, standby；Again to
The Primary product obtained adds the acetone of 5ml volume ratio 80:20 and the mixed solution II of distilled water, centrifugal after concussion uniformly,
Centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min, abandoning supernatant, repeats to add acetone and steaming in centrifugal product
The mixed solution II of distilled water, concussion and centrifugally operated, until the centrifugal product obtained is colourless, obtain secondary species, standby；
B. adding 5ml volume ratio in the secondary species that step a obtains is the mixed of 5:80:15 hydrochloric acid, acetone and distilled water
Closing solution, centrifugal after concussion uniformly, the concussion time is 10-25min, and centrifugal condition is centrifugal force 4000g, centrifugation time 8-
20min, collects supernatant, standby；Then in the centrifugal precipitation obtained, hydrochloric acid, acetone are again added molten with the mixing of distilled water
Liquid, centrifugal after concussion uniformly, the concussion time is 10-25min, and centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min, receives
Collection supernatant, merges twice supernatant, obtains mixed extract, standby；
C. in the mixed extract that step b obtains, add 10ml ether, join after mix homogeneously in separatory funnel, will
Separatory funnel be repeatedly inverted after stratification, collect upper solution, standby；And in lower floor's solution, again add 10ml ether,
Join after mix homogeneously in separatory funnel, collect upper solution after stratification, merge the upper solution obtained for twice, be placed in
In separatory funnel, it is subsequently adding distilled water isopyknic with the upper solution merged and washs, after stratification, take ether layer,
Join in Rotary Evaporators and concentrate, then enriched product is poured in centrifuge tube, dry up with nitrogen, i.e. obtain the Plantula Brassicae chinensis being dried
Haemachrome, standby；
Step 2, Plantula Brassicae chinensis content of hemachrome measure
In the Plantula Brassicae chinensis haemachrome being dried that step one obtains, add 7.44ml distilled water and 0.48ml 5N NaOH, stir
Mix and add 12 ml distilled water and 4.08 ml pyridine, again mix homogeneously after dissolving to dry Plantula Brassicae chinensis haemachrome, use
Ultraviolet spectrophotometer measures the absorbance at 574.5nm, adds several dithionates and 50mmol/L during measurement in the sample
The iron cyanide.The blade taking three independent strain Plantula Brassicae chinensis is measured, and repeatedly measures and averages, obtains mean light absorbency A
Being 0.102403, result is as shown in table 3, standby；
Table 3: the absorbance A of Plantula Brassicae chinensis sample
Step 3, the calculating of Plantula Brassicae chinensis hemoglobin concentration
Mean light absorbency A step 2 obtained substitutes into equation of linear regression: A=2.6814496C+0.07506221, meter
It is 0.010196 that calculation obtains extension rate C, further according to Ci=0.5C mg/ml, calculates the dense of haemachrome in Plantula Brassicae chinensis blade
Degree Ci is 0.005098mg/ml.
Embodiment 3:
A kind of method extracted from Wuta-tsai and measure haemachrome, comprises the following steps:
Step one, the extraction of Wuta-tsai haemachrome
The most accurately weigh the fresh blade 1g of Wuta-tsai, be placed in the mortar of pre-cooling, add liquid nitrogen and be ground to powder, then
It is transferred in centrifuge tube, adds the acetone of 10ml volume ratio 99:1 and the mixed solution I of distilled water, centrifugal after concussion uniformly, from
Heart condition is centrifugal force 4000g, centrifugation time 8-20min, abandoning supernatant, repeats to add acetone and distillation in centrifugal product
The mixed solution I of water, concussion and centrifugally operated, until the centrifugal product obtained is colourless, obtain Primary product, standby；Again to
The Primary product obtained adds the acetone of 5ml volume ratio 80:20 and the mixed solution II of distilled water, centrifugal after concussion uniformly,
Centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min, abandoning supernatant, repeats to add acetone and steaming in centrifugal product
The mixed solution II of distilled water, concussion and centrifugally operated, until the centrifugal product obtained is colourless, obtain secondary species, standby；
B. adding 5ml volume ratio in the secondary species that step a obtains is the mixed of 5:80:15 hydrochloric acid, acetone and distilled water
Closing solution, centrifugal after concussion uniformly, the concussion time is 10-25min, and centrifugal condition is centrifugal force 4000g, centrifugation time 8-
20min, collects supernatant, standby；Then in the centrifugal precipitation obtained, hydrochloric acid, acetone are again added molten with the mixing of distilled water
Liquid, centrifugal after concussion uniformly, the concussion time is 10-25min, and centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min, receives
Collection supernatant, merges twice supernatant, obtains mixed extract, standby；
C. in the mixed extract that step b obtains, add 10ml ether, join after mix homogeneously in separatory funnel, will
Separatory funnel be repeatedly inverted after stratification, collect upper solution, standby；And in lower floor's solution, again add 10ml ether,
Join after mix homogeneously in separatory funnel, collect upper solution after stratification, merge the upper solution obtained for twice, be placed in
In separatory funnel, it is subsequently adding distilled water isopyknic with the upper solution merged and washs, after stratification, take ether layer,
Join in Rotary Evaporators and concentrate, then enriched product is poured in centrifuge tube, dry up with nitrogen, i.e. obtain the Wuta-tsai being dried
Haemachrome, standby；
Step 2, Wuta-tsai content of hemachrome measure
In the Wuta-tsai haemachrome being dried that step one obtains, add 7.44ml distilled water and 0.48ml 5N NaOH, stir
Mix and add 12 ml distilled water and 4.08 ml pyridine, again mix homogeneously after dissolving to dry Wuta-tsai haemachrome, use
Ultraviolet spectrophotometer measures the absorbance at 574.5nm, adds several dithionates and 50mmol/L during measurement in the sample
The iron cyanide.The blade taking three independent strain Wuta-tsais is measured, and repeatedly measures and averages, obtains mean light absorbency A
Being 0.149113, result is as shown in table 4, standby；
Table 4: the absorbance A of Wuta-tsai sample
Step 3, the calculating of Wuta-tsai hemoglobin concentration
Mean light absorbency A step 2 obtained substitutes into equation of linear regression: A=2.6814496C+0.07506221, meter
It is 0.02762 that calculation obtains extension rate C, further according to Ci=0.5C mg/ml, calculates the concentration of haemachrome in Wuta-tsai blade
Ci is 0.01381mg/ml.
Claims (7)
1. the method extracted from plant and measure haemachrome, it is characterised in that: comprise the following steps:
Step one, the extraction of plant heme
The most accurately weigh the fresh blade 1g treating measuring plants, be placed in the mortar of pre-cooling, add liquid nitrogen and be ground to powder, then turn
Move in centrifuge tube, add the mixed solution I of acetone and distilled water, centrifugal after concussion uniformly, abandoning supernatant, repeats to centrifugal
Product adds mixed solution I, concussion and the centrifugally operated of acetone and distilled water, until the centrifugal product obtained is colourless,
To Primary product, standby；In the Primary product obtained, add the mixed solution II of acetone and distilled water again, after concussion uniformly from
The heart, abandoning supernatant, repeats mixed solution II, concussion and the centrifugally operated adding acetone in centrifugal product with distilled water, directly
It is colourless to the centrifugal product obtained, obtains secondary species, standby；
In described mixed solution I, the ratio shared by acetone is more than the ratio in mixed solution II；
B. in the secondary species that step a obtains, add hydrochloric acid, acetone and the mixed solution of distilled water, shake rear centrifugal,
Collect supernatant, standby；Then in the centrifugal precipitation obtained, again add hydrochloric acid, acetone and the mixed solution of distilled water, shake
It is centrifuged after swinging uniformly, collects supernatant, merge twice supernatant, obtain mixed extract, standby；
C. in the mixed extract that step b obtains, add ether, join after mix homogeneously in separatory funnel, by separatory funnel
Stratification after being repeatedly inverted, collects upper solution, standby；And in lower floor's solution, again add ether, add after mix homogeneously
Enter in separatory funnel, collect upper solution after stratification, merge the upper solution obtained for twice, be placed in separatory funnel,
The isopyknic distilled water of upper solution being subsequently adding and merge washs, and takes ether layer, join rotation after stratification
Evaporimeter concentrates, then enriched product is poured in centrifuge tube, dry up with nitrogen, i.e. obtain the plant heme being dried, standby；
Step 2, plant heme assay
Adding 7.44ml distilled water and 0.48ml 5N NaOH in the plant heme being dried that step one obtains, stirring is to planting
Thing haemachrome adds 12 ml distilled water and 4.08 ml pyridine, again mix homogeneously after dissolving, use ultraviolet spectrophotometer
Measure the absorbance at 574.5nm, during measurement, add the iron cyanide of several dithionates and 50mmol/L in the sample, take solely
The blade of vertical plant more than three strains is measured, and repeatedly measures and averages, obtains mean light absorbency A, standby；
Step 3, the calculating of plant heme concentration
Mean light absorbency A step 2 obtained substitutes into equation of linear regression: A=2.6814496C+0.07506221, calculates
To extension rate C, further according to Ci=0.5C mg/ml, calculate concentration C i of haemachrome in plant leaf blade.
A kind of method extracted from plant and measure haemachrome, it is characterised in that: step a institute
Time needed for the concussion uniformly stated is 10-25min, and centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min.
A kind of method extracted from plant and measure haemachrome, it is characterised in that: step a institute
The acetone stated and acetone and volume ratio 99:1 of distilled water in the mixed solution I of distilled water, each addition of mixed solution I is
10ml。
A kind of method extracted from plant and measure haemachrome, it is characterised in that: step a institute
The acetone stated and acetone and volume ratio 80:20 of distilled water in the mixed solution II of distilled water, each addition of mixed solution II
For 5ml.
A kind of method extracted from plant and measure haemachrome, it is characterised in that: step b institute
Hydrochloric acid, the acetone stated are 5:80:15 with the volume ratio of hydrochloric acid, acetone and distilled water in the mixed solution of distilled water, add every time
Amount is 5ml.
A kind of method extracted from plant and measure haemachrome, it is characterised in that: step b institute
The concussion time stated is 10-25min, and centrifugal condition is centrifugal force 4000g, centrifugation time 8-20min.
A kind of method extracted from plant and measure haemachrome, it is characterised in that: in step c
The each addition of ether be 10ml.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN1306002A (en) * | 2001-02-07 | 2001-08-01 | 尹兆明 | Process for extracting heme from animal blood |
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