CN109991213A - A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis - Google Patents
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis Download PDFInfo
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- CN109991213A CN109991213A CN201910026348.0A CN201910026348A CN109991213A CN 109991213 A CN109991213 A CN 109991213A CN 201910026348 A CN201910026348 A CN 201910026348A CN 109991213 A CN109991213 A CN 109991213A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis that the invention discloses a kind of, alkaloid dyeing is carried out with sharpleaf gambirplant root, stem free-hand section, each coloring site is removed according to coloration result and measures its total alkaloid, alkaloid memory structure position and content are specified, Uncaria is identified with this and determines the Strategies on Comprehensive of medicinal material.When conventional method harvests Uncaria, root, stem, leaf are unutilized, cause resource significant wastage.The present invention is using sharpleaf gambirplant root, stem as material, it carries out alkaloid dyeing observation and total alkaloid is extracted, so that it is determined that accumulation position and content of the alkaloid in uncaria, and as the effective ways for determining Uncaria quality, it is intended to improve traditional Land use systems of its medicinal material, root, stem, leaf are comprehensively utilized, to realize the maximization of its economic benefit.
Description
Technical field
The present invention relates to plant tissue slice, alkaloid dyeing and assay technologies, and in particular to uncaria free-hand section,
Alkaloid dyeing and Determination of Total Alkaloid method.
Background technique
Uncaria [Uncaria rhynchophylla (Miq.) Miq.ex Havil.] is Rubiaceae (Rubiaceae) hook
Calamus (Uncaria Schreber nom.cons.) perennial bejuco, is used as medicine with drying buckle stem branch.Its is sweet in flavor,
Property cool, returns liver, pericardium channel；There are dispelling wind and relieving convulsion, The flat liver of heat-clearing and other effects；For liver wind agitation, twitch, hyperpyretic convulsion, sense
It emits and presss from both sides the diseases such as frightened, children's crying with fear, pre-eclampsia and headache and dizziness.Main pharmacodynamics ingredient is indoles alkaloid in uncaria, including
Rhynchophyllin, isorhynchophylline, corynoxeine, ocrynoxine, Conan are because of over one hundred kind of alkali etc., wherein being contained with rhynchophyllin and isorhynchophylline
It measures higher.In uncaria acquisition, acquisition process, pharmacist is to avoid Uncaria adulterated, and only purchase buckle branch, rest part
It is dropped, though due to sharpleaf gambirplant root, stem and leaf not long hook, alkaloid is higher, is dropped the significant wastage for causing resource.
Due to that can not detect the alkaloid of sharpleaf gambirplant root, stem and leaf in existing acquisition process, pharmacist is to avoid uncaria
Medicinal material is adulterated, and only purchases buckle branch, and rest part is dropped, to cause the significant wastage of resource.
Summary of the invention
It is examined the object of the present invention is to provide a kind of based on the uncaria alkaloid of Histochemical localization and chemical composition analysis
Survey method.The invention is that Uncaria identifies and the comprehensive development and utilization of herb resource opened up a new way, and is avoided that uncaria medicine
Material is adulterated, while the advantages that reduce the waste of uncaria resource.
A kind of technical solution of the present invention: uncaria alkaloid detection side based on Histochemical localization and chemical composition analysis
Method, comprising the following steps:
A, using sharpleaf gambirplant root, uncaria stem as material, distilled water clean after by free-hand section, select it is uniform in size, with a thickness of
The thin slice of 0.1~0.5mm is placed on glass slide, respectively with improvement bismuth potassium iodide coloring agent, bromocresol green reagent and concentrated nitric acid pair
Sharpleaf gambirplant root, uncaria stem slice are dyed；
It b,, will be in sharpleaf gambirplant root, uncaria stem according to alkaloid coloration result in step 1 after separately taking sharpleaf gambirplant root, uncaria stem to clean
It being placed in insulating box after coloring structure and the removing of uncolored structure, drying temperature is 35~75 DEG C, and drying time is 3~8 days,
Be milled sieving after taking-up, collects spare；
C, 0.5~2.0g of Gambir Plant in step 2 is taken to be placed in conical flask, addition 2~10ml ammonia solution wetting 10~
90min, adds methanol, and solid-liquid ratio is 1:30~1:70g/mL, 20~60min of ultrasonic extraction, 55 DEG C of water bath methods after filtering
Methanol is added Potassium Hydrogen Phthalate-hydrochloride buffer 30mL of pH3.6, shakes up, filters, and buffer constant volume is to 50mL
Test liquid；Test liquid 4mL is taken, the color condition of acid-dye colorimetry is Potassium Hydrogen Phthalate-hydrochloride buffer of pH3.6
10mL, bromocresol green dye liquor 10mL, extractant extract three times, and 4~8mL, is settled to 25mL every time, in ultraviolet specrophotometer
Total alkaloid OD value is measured by sampling under 413nm wavelength, makes standard solution by reference substance of rhynchophyllin, makes standard curve, most
After calculate total alkaloid content in uncaria sample.
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis above-mentioned, the step
The dyeing processing time of each reagent is 15min in a, and dyeing terminates, and distilled water cleaning is added dropwise in reagent after blotting, and is closed the lid
Slide is placed in microscopically observation alkaloid accumulation position, in this, as the foundation of next step institutional framework removing.
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis above-mentioned, the step
It will be placed in insulating box after coloring structure and the removing of uncolored structure in b according to sharpleaf gambirplant root, uncaria stem coloration result, drying temperature
Degree is 55 DEG C, is dried 5 days, be milled 40 meshes.
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis above-mentioned, the step
In c, 1g Gambir Plant is weighed in conical flask, and 5ml ammonia solution is added and soaks 30min, adds methanol, solid-liquid ratio 1:50g/
Potassium Hydrogen Phthalate-hydrochloride buffer of pH3.6 is added in mL, ultrasonic extraction 40min, 55 DEG C of water bath method methanol after filtering
30mL shakes up, filters, and buffer constant volume to 50mL is test liquid；Take test liquid 4mL, the colour developing item of acid-dye colorimetry
Part be pH3.6 Potassium Hydrogen Phthalate-hydrochloride buffer 10mL, bromocresol green dye liquor 10mL, extractant extract three times, often
Secondary 4mL is settled to 25mL, and total alkaloid OD value is measured by sampling under ultraviolet specrophotometer 413nm wavelength, is with rhynchophyllin
Reference substance makes standard curve, finally calculates total alkaloid content in uncaria sample.
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis above-mentioned, in step c
The Potassium Hydrogen Phthalate of the pH3.6-hydrochloride buffer 30mL by 0.2mol/L Potassium Hydrogen Phthalate solution 25ml with
0.1mol/L hydrochloric acid 6ml mixing adds distilled water to be diluted to 100ml and is made, PH 3.6.
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis above-mentioned, in step c
The extractant is chloroform.
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis above-mentioned, the improvement
Bismuth potassium iodide is that 0.85g bismuth subnitrate is dissolved in 10mL glacial acetic acid and 40mL distilled water, then is mixed with the potassium iodide of 20mL 40%
It closes.
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis above-mentioned, the ammonia are molten
Liquid is obtained by the ratio preparation of 9:30:16 by volume of ammonium hydroxide, distilled water, dehydrated alcohol.
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis above-mentioned, the bromine first
The green reagent of phenol is dissolved in 2.8ml by 0.1g bromocresol green, and in 0.05mol/L NaOH solution, distilled water is settled to 200ml and is made.
Beneficial effects of the present invention: compared with prior art, technology of the invention has the advantage that
When conventional method harvests Uncaria, sharpleaf gambirplant root, uncaria stem, uncaria leaf are unutilized, cause resource very big
Waste.The present invention carries out alkaloid dyeing observation and total alkaloid is extracted, so that it is determined that raw using sharpleaf gambirplant root, uncaria stem as material
Accumulation position and content of the alkaloids in uncaria, and as the effective ways for determining Uncaria quality, it is intended to improve its medicinal material
Traditional Land use systems, root, stem, leaf are comprehensively utilized, to realize the maximization of its economic benefit.
Detailed description of the invention
Fig. 1 is sharpleaf gambirplant root, alkaloid figure in each structure of uncaria rhizome；
Fig. 2 is that alkaloid accumulates station diagram in uncaria；
Fig. 3 is uncaria material structure removing figure.
Label in attached drawing:
It is dense that bismuth potassium iodide stained slice × 100,2- root bromocresol green stained slice × 100,3- root is improved in the root 1-
Nitric acid treatment slice × 100,4- stem improvement bismuth potassium iodide stained slice × 100,5- stem bromocresol green stained slice ×
100,6- stem's concentrated nitric acids processing slice × 100；A- biology alkali precipitation, A1- root xylem, B1- root bast, C1- stem marrow
Portion, D1- stem xylem, E1- stem bast.
Specific embodiment
The embodiment of the present invention 1
The present invention is further illustrated with reference to the accompanying drawings and examples, but be not intended as to the present invention limit according to
According to.
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis is constituted such as Fig. 1-3 institute
Show, comprising the following steps:
A, be material by sharpleaf gambirplant root, uncaria stem, distilled water clean after by free-hand section, select it is uniform in size, with a thickness of
The thin slice of 0.2~0.3mm is placed on glass slide, respectively with improvement bismuth potassium iodide coloring agent, bromocresol green reagent and concentrated nitric acid pair
Sharpleaf gambirplant root, uncaria stem slice are dyed, and the dyeing processing time of each reagent is 15min；
It b,, will be in sharpleaf gambirplant root, uncaria stem according to alkaloid coloration result in step 1 after separately taking sharpleaf gambirplant root, uncaria stem to clean
Coloring structure and uncolored structure are removed, and are placed in insulating box, and drying temperature is 55 DEG C, and drying time is 5 days, are ground after taking-up
Powder sieving, is collected spare；
C, it takes Gambir Plant 1g in step 2 to be placed in conical flask, 5ml ammonia solution is added and soaks 30min, adds methanol,
Solid-liquid ratio 1:50g/mL, ultrasonic extraction 40min, 55 DEG C of water bath method methanol after filtering, are added the hydrogen phthalate of pH3.6
Potassium-hydrochloride buffer 30mL, shakes up, filters, and buffer constant volume to 50mL is test liquid；Take test liquid 4mL, acid dyes ratio
The color condition of color method is Potassium Hydrogen Phthalate-hydrochloride buffer 10mL of pH3.6, bromocresol green dye liquor 10mL, three chloromethanes
Alkane extracts three times, and each 4mL is settled to 25mL, and total alkaloid OD is measured by sampling under ultraviolet specrophotometer 413nm wavelength
Value makes standard solution by reference substance of rhynchophyllin, calculates total alkaloid content in uncaria sample according to standard curve.Test
As a result as shown in fig. 1, total alkaloid content is respectively 1.80mg/g and 2.55mg/g in root bast and stem bast.
The embodiment of the present invention 2
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis is constituted such as Fig. 1-3 institute
Show, comprising the following steps:
A, be material by sharpleaf gambirplant root, uncaria stem, distilled water clean after by free-hand section, select it is uniform in size, with a thickness of
The thin slice of 0.1~0.2mm is placed on glass slide, respectively with improvement bismuth potassium iodide coloring agent, bromocresol green reagent and concentrated nitric acid pair
Sharpleaf gambirplant root, uncaria stem slice are dyed, and the dyeing processing time of each reagent is 15min；
It b,, will be in sharpleaf gambirplant root, uncaria stem according to alkaloid coloration result in step 1 after separately taking sharpleaf gambirplant root, uncaria stem to clean
Coloring structure and uncolored structure are removed, and are placed in insulating box, and drying temperature is 35 DEG C, and drying time is 8 days, are ground after taking-up
Powder sieving, is collected spare；
C, it takes Gambir Plant 0.5g in step 2 to be placed in conical flask, 2ml ammonia solution is added and soaks 10min, adds first
The phthalic acid of pH3.6 is added in alcohol, solid-liquid ratio 1:30g/mL, ultrasonic extraction 60min, 55 DEG C of water bath method methanol after filtering
Hydrogen potassium-hydrochloride buffer 30mL, shakes up, filters, and buffer constant volume to 50mL is test liquid；Take test liquid 4mL, acid dyes
The color condition of colorimetric method is Potassium Hydrogen Phthalate-hydrochloride buffer 10mL of pH3.6, bromocresol green dye liquor 10mL, trichlorine
Methane extracts three times, and each 6mL is settled to 25mL, and total alkaloid is measured by sampling under ultraviolet specrophotometer 413nm wavelength
OD value makes standard solution by reference substance of rhynchophyllin, calculates total alkaloid content in uncaria sample according to standard curve.
The embodiment of the present invention 3
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis, comprising the following steps:
A, be material by sharpleaf gambirplant root, uncaria stem, distilled water clean after by free-hand section, select it is uniform in size, with a thickness of
The thin slice of 0.3~0.5mm is placed on glass slide, respectively with improvement bismuth potassium iodide coloring agent, bromocresol green reagent and concentrated nitric acid pair
Sharpleaf gambirplant root, uncaria stem slice are dyed, and the dyeing processing time of each reagent is 15min；
It b,, will be in sharpleaf gambirplant root, uncaria stem according to alkaloid coloration result in step 1 after separately taking sharpleaf gambirplant root, uncaria stem to clean
Coloring structure and uncolored structure are removed, and are placed in insulating box, and drying temperature is 75 DEG C, and drying time is 3 days, are ground after taking-up
Powder sieving, is collected spare；
C, it takes Gambir Plant 2.0g in step 2 to be placed in conical flask, 10ml ammonia solution is added and soaks 90min, adds first
Alcohol, by solid-liquid ratio 1:75g/mL, ultrasonic extraction 40min, the O-phthalic of pH3.6 is added in 55 DEG C of water bath method methanol after filtering
Potassium hydrogen phthalate-hydrochloride buffer 30mL, shakes up, filters, and buffer constant volume to 50mL is test liquid；Take test liquid 4mL, acidity dye
The color condition for expecting colorimetric method is Potassium Hydrogen Phthalate-hydrochloride buffer 10mL of pH3.6, bromocresol green dye liquor 10mL, three
Chloromethanes extracts three times, and each 8mL is settled to 25mL, and total biology is measured by sampling under ultraviolet specrophotometer 413nm wavelength
Alkali OD value makes standard solution by reference substance of rhynchophyllin, calculates total alkaloid content in uncaria sample according to standard curve.
The embodiment of the present invention 4
Vegetable material in the present embodiment is derived from 3 years raw uncarias of Guizhou Province Qiandongnan Prefecture of Guizhou Province uncaria planting base
[Uncaria rhynchophylla(Miq.)Miq.ex Havil.]。
A kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis, comprising the following steps:
A, be material by sharpleaf gambirplant root, uncaria stem, distilled water clean after by free-hand section, select it is uniform in size, with a thickness of
The thin slice of 0.1~0.5mm is placed on glass slide, respectively with improvement bismuth potassium iodide coloring agent, bromocresol green reagent and concentrated nitric acid pair
Sharpleaf gambirplant root, uncaria stem slice are dyed, and the dyeing processing time of each reagent is 15min；
It b,, will be in sharpleaf gambirplant root, uncaria stem according to alkaloid coloration result in step 1 after separately taking sharpleaf gambirplant root, uncaria stem to clean
It is placed in insulating box after coloring structure and the removing of uncolored structure, drying temperature is 55 DEG C, and drying time is 5 days, after taking-up
Be milled 40 meshes, collects spare；
C, it takes Gambir Plant 1.0g in step 2 to be placed in conical flask, ammonia solution wetting is added by solid-liquid ratio 1:50g/mL
30min, adds methanol, ultrasonic extraction 40min, and the hydrogen phthalate of pH3.6 is added in 55 DEG C of water bath method methanol after filtering
Potassium-hydrochloride buffer 30mL, shakes up, filters, and buffer constant volume to 50mL is test liquid；Using rhynchophyllin as reference substance, first is used
The reference substance solution of 43.2 μ g/mL is made in alcohol, in a linear relationship, the regression equation in 43.2~259.2 μ g ranges are as follows: C=
0.0431A+0.0199, R2=0.998；Test liquid 4mL is taken, the color condition of acid-dye colorimetry is the adjacent benzene two of pH3.6
First potassium hydrogen phthalate-hydrochloride buffer 10mL, bromocresol green dye liquor 10mL, extractant extract three times, and each 4mL is settled to 25mL, and
Total alkaloid OD value is measured by sampling under ultraviolet specrophotometer 413nm wavelength, finally calculates total alkaloid in uncaria sample
Content, data analysis and utilization Excel and SPSS 22.0.
Inventor carries out biology to sharpleaf gambirplant root, each structure of uncaria stem using the method for the embodiment described 4 using the present invention
Alkali content detection, as a result, it has been found that, in Fig. 1, in root bast and stem bast total alkaloid content be respectively 1.80mg/g and
Alkaloid is not detected in 2.55mg/g, root xylem, stem xylem and stem marrow；In Fig. 2, alkaloid accumulation knot in sharpleaf gambirplant root
Structure is bast, alkaloid accumulation structure is bast in uncaria stem；In Fig. 3, A1- root xylem, B1- root bast, C1- stem
Marrow, D1- stem xylem, E1- stem bast.
The traditional picking methods of Uncaria have abandoned root, stem and leaf, but many results of study show that its alkaloid contains
Amount is higher, therefore causes the waste of a large amount of herb resources.The present invention is using histotomy technology, alkaloid dyeing and extracts skill
Art specifies uncaria alkaloid accumulation structure, while removing the structure and carrying out alkaloid content determination, identifies Uncaria with this,
To improve the purchase method of medicinal material, root, stem, leaf are used, and realize maximized economic benefit.
1 uncaria alkaloid of table extracts optimal conditions
Note: different lowercases indicate that the two has significant difference (P < 0.05) in table.
Claims (9)
1. a kind of uncaria alkaloid detection method based on Histochemical localization and chemical composition analysis, which is characterized in that including
Following steps:
A, using sharpleaf gambirplant root, uncaria stem as material, distilled water clean after by free-hand section, select it is uniform in size, with a thickness of 0.1~
The thin slice of 0.5mm is placed on glass slide, respectively with improvement bismuth potassium iodide coloring agent, bromocresol green reagent and concentrated nitric acid to uncaria
Root, uncaria stem slice are dyed；
B, it after separately taking sharpleaf gambirplant root, uncaria stem to clean, according to alkaloid coloration result in step 1, will be coloured in sharpleaf gambirplant root, uncaria stem
It is placed in insulating box after structure and the removing of uncolored structure, drying temperature is 35~75 DEG C, and drying time is 3~8 days, is taken out
Milling sieving afterwards is collected spare；
C, it takes 0.5~2.0g of Gambir Plant in step 2 to be placed in conical flask, 2~10ml ammonia solution is added and soaks 10~90min,
Methanol is added, solid-liquid ratio is 1:30~1:70g/mL, and 20~60min of ultrasonic extraction, 55 DEG C of water bath method methanol, adds after filtering
Potassium Hydrogen Phthalate-hydrochloride buffer the 30mL for entering pH3.6, shakes up, filters, buffer constant volume to 50mL is test liquid；
Test liquid 4mL is taken, the color condition of acid-dye colorimetry is Potassium Hydrogen Phthalate-hydrochloride buffer 10mL of pH3.6, bromine
The green dye liquor 10mL of cresols, extractant extract three times, and 4~8mL, is settled to 25mL every time, in ultraviolet specrophotometer 413nm wavelength
Lower sampling and measuring total alkaloid OD value makes standard solution by reference substance of rhynchophyllin, makes standard curve, finally calculate hook
Total alkaloid content in rattan sample.
2. a kind of uncaria alkaloid detection side based on Histochemical localization and chemical composition analysis according to claim 1
Method, it is characterised in that: the dyeing processing time of each reagent is 15min in the step a, and dyeing terminates, and reagent blots
Distilled water cleaning is added dropwise afterwards, covered is placed in microscopically observation alkaloid accumulation position, as next step knot of tissue
The foundation of structure removing.
3. a kind of uncaria alkaloid detection side based on Histochemical localization and chemical composition analysis according to claim 1
Method, it is characterised in that: removed coloring structure and uncolored structure according to sharpleaf gambirplant root, uncaria stem coloration result in the step b
After be placed in insulating box, drying temperature be 55 DEG C, dry 5 days, be milled 40 meshes.
4. a kind of uncaria alkaloid detection side based on Histochemical localization and chemical composition analysis according to claim 1
Method, it is characterised in that: in the step c, 1g Gambir Plant is weighed in conical flask, and 5ml ammonia solution is added and soaks 30min, then
Methanol, solid-liquid ratio 1:50g/mL, ultrasonic extraction 40min is added, the neighbour of pH3.6 is added in 55 DEG C of water bath method methanol after filtering
Potassium hydrogen phthalate-hydrochloride buffer 30mL, shakes up, filters, and buffer constant volume to 50mL is test liquid；Test liquid 4mL is taken,
The color condition of acid-dye colorimetry is Potassium Hydrogen Phthalate-hydrochloride buffer 10mL of pH3.6, bromocresol green dye liquor
10mL, extractant extract three times, and each 4mL is settled to 25mL, and is measured by sampling under ultraviolet specrophotometer 413nm wavelength
Total alkaloid OD value makes standard curve by reference substance of rhynchophyllin, finally calculates total alkaloid content in uncaria sample.
5. a kind of uncaria alkaloid detection side based on Histochemical localization and chemical composition analysis according to claim 1
Method, it is characterised in that: the Potassium Hydrogen Phthalate of pH3.6 described in step c-hydrochloride buffer 30mL is by 0.2mol/L neighbour's benzene
Dioctyl phthalate hydrogen potassium solution 25ml is mixed with 0.1mol/L hydrochloric acid 6ml, is added distilled water to be diluted to 100ml and is made, PH 3.6.
6. according to claim 1 or 4 a kind of based on the inspection of the uncaria alkaloid of Histochemical localization and chemical composition analysis
Survey method, it is characterised in that: extractant described in step c is chloroform.
7. a kind of uncaria alkaloid detection side based on Histochemical localization and chemical composition analysis according to claim 1
Method, it is characterised in that: the improvement bismuth potassium iodide coloring agent is that 0.85g bismuth subnitrate is dissolved in 10mL glacial acetic acid and 40mL distilled water
In, then mix with the potassium iodide of 20mL40%.
8. a kind of uncaria alkaloid detection side based on Histochemical localization and chemical composition analysis according to claim 1
Method, it is characterised in that: the ammonia solution is obtained by the ratio preparation of 9:30:16 by volume of ammonium hydroxide, distilled water, dehydrated alcohol.
9. a kind of uncaria alkaloid detection side based on Histochemical localization and chemical composition analysis according to claim 1
Method, it is characterised in that: the bromocresol green reagent is dissolved in 2.8ml by 0.1g bromocresol green, in 0.05mol/LNaOH solution, steams
Distilled water is settled to 200ml and is made.
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