CN104013655B - Method for preparing radix isatidis extract - Google Patents
Method for preparing radix isatidis extract Download PDFInfo
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- CN104013655B CN104013655B CN201410250141.9A CN201410250141A CN104013655B CN 104013655 B CN104013655 B CN 104013655B CN 201410250141 A CN201410250141 A CN 201410250141A CN 104013655 B CN104013655 B CN 104013655B
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Abstract
The invention provides a method for preparing a radix isatidis extract. The extract contains DL-epigoitrin. The preparation method comprises the following steps: crushing, adding water, leaching at the temperature of 20-60 DEG C for 3-6 hours, filtering, concentrating and drying the filtrate. According to the method, the conventional radix isatidis extraction process is greatly simplified, and the extraction rate of the DL-epigoitrin is obviously improved.
Description
Technical field
The present invention relates to a kind of preparation method of Radix Isatidis extract, particularly a kind of easy and can improve dl- announcement according to
The Radix Isatidis extract preparation method of spring content.
Background technology
Radix Isatidis are the dry root of Cruciferae Isatis indigotica Fort. platymiscium Isatis indigotica Fort. (isantis indigotica fort.), bitter in the mouth
Cold in nature, there is the effects such as heat clearing away, removing toxic substances, removing heat from blood, sore-throat relieving, for maculae caused by virulent heat pathogen, high heat headache, hepatitis, influenza etc., be
The representative medicine of antipyretic and antidotal type Chinese medicine.Radix Isatidis preparation producing and selling amount can be rated as Chinese patent medicine, only single preparation just includes
Radix Isatidis granule, Radix Isatidis tablet, indigowoad root oral liquid, Radix Isatidis capsule, isatis root drops, dispersant isatis root tablet are interior more than ten
Individual kind, nearly 1000 of authentication code.
The development and application of Radix Isatidis preparation is with a long history, and its curative effect obtains the generally accreditation of doctors and patients already.But due to plate
Blue root complex chemical composition, all not understands to its active substance basis and the mechanism of action, so adopting existing preparation in early days
Traditional extraction process, such as decoction and alcohol sedimentation technique (decocting boils ethanol precipitation), ethanol reflux extraction etc., it is numerous to there is technique in these methods
Trivial, time-consuming, the low deficiency of active component extraction ratio, meanwhile, it is inadequate that the difference of the extracting method of each producer also results in curative effect
Stable.
Through research in recent years, it is significantly disease-resistant that people gradually recognize that contained dl- goitrin in Radix Isatidis has
Cytotoxic activity, Chinese Pharmacopoeia version in 2010 also by the content of dl- goitrin ((r, s)-goitrin) as chromatogram of Radix Isatidis and decoction pieces
Quality control index.But due to existing preparation adopt preparation technology be unfavorable for dl- goitrin extraction so that in preparation dl- accuse
Content according to the spring is extremely low, or even has part batch not detect.This has had a strong impact on the drug effect of Radix Isatidis preparation, from another angle
For, it is also the waste to chromatogram of Radix Isatidis.
Content of the invention
For deficiency present in existing Radix Isatidis preparation extracting method, inventor studies through substantial amounts of system experimentation,
Establish a kind of preparation method of Radix Isatidis extract, this preparation method is not only simple, reproducible, significantly more improves
The content of dl- goitrin in extract.
The invention provides a kind of preparation method of Radix Isatidis extract, in described extract, contain dl- goitrin, should
Preparation method comprises the following steps:
(1) Radix Isatidis powder is broken to granule or the powder of 20～80 mesh；
(2) (1) obtain Jia 10 in Radix Isatidis granule or powder the～water of 60 times amount to step, soak 3～6 in 20～60 DEG C little
When, filter, obtain filtrate；
(3) (2) step is obtained filtrate to concentrate, be dried, obtain Radix Isatidis extract.
Dl- goitrin of the present invention is alternatively referred to as (r, s)-goitrin.
Above-mentioned Radix Isatidis can be chromatogram of Radix Isatidis or its decoction pieces, preferably dry product.Described " water of 10～60 times amount " are
Refer to, amount of water is 10～60 times of Radix Isatidis granule or powder weight, similarly hereinafter.
Experiment shows, the impact to dl- goitrin content in Radix Isatidis for the extracting method, with thioglycoside-myrosase
System is relevant.Thioglycoside (abbreviation sulfur glycosides) itself does not possess biological activity, but its enzyme hydrolysis, thermal decomposition or intestinal degradation
Product has multiple biological activities, and d- goitrin and l- goitrin are respectively by Beilstein before the thioglycoside in Radix Isatidis
Produce through Semen Sinapiss enzyme hydrolysiss with front goitrin.Sulfur glycosides and myrosase exist jointly, but mutually isolated on micro-space, therefore
How to activate and keep the activity of myrosase, promote the two reaction so that sulfur glycosides is converted completely, be to improve dl- in Radix Isatidis extract
The key of goitrin content.
Find in inventor's research, the ratio extracting water in solvent is lower, the ratio of organic reagent (as methanol, ethanol etc.)
Higher, enzyme more easily inactivates, dl- goitrin extraction ratio is lower.In addition to extracting solvent and extracting mode, the granularity of Radix Isatidis and
Extracting temperature affects more notable on dl- goitrin extraction ratio.When Extracting temperature is higher than 60 DEG C, myrosase is with temperature rising
Fast deactivation, accordingly, enzymatic hydrolysate dl- goitrin content declines.Because sulfur glycosides is different from the locus residing for myrosase,
When Radix Isatidis granularity is more than 20 mesh, cell breakage not exclusively, the two can not completely dissolution, contact, react to each other, thus affecting
The content of activated product (dl- goitrin), but this granularity is not the smaller the better, when less than 80 mesh, the dl- in extracting solution accuse according to
On the one hand spring content substantially reduces, and this is to be due to surface adsorption effect, causes limited by chemical composition dissolution, be on the other hand because
It is in crushing process, material and blade are acutely conflicted heat production (it is known that the thinner required time of grinding particle size is longer), destroy
Part Semen Sinapiss enzymatic activity.
Water extraction (decoction) temperature of traditional method is 100 DEG C, the time generally more than 1 hour, heavy damage myrosase
Activity is although also there is research worker to adopt ultrasonic method (about 1 hour), but ultrasonic same heat production, particularly ultrasonic for a long time, test
Prove, water temperature may be up to 70 DEG C within ultrasonic 1 hour, also to the enzymatic activity of myrosase, there is certain destruction it is often more important that,
Ultrasonic method only rests on laboratory and extracts on a small scale at present, and in still can not be applied to produce greatly, practicality is poor.
Through substantial amounts of experimental study, inventor's screening and optimizing goes out preparation method of the present invention, the method effective guarantee
Semen Sinapiss enzymatic activitys, meet conjugation condition, the environment of myrosase and sulfur glycosides, thus substantially increase dl- goitrin precursor
Conversion ratio, i.e. improve the extraction ratio of dl- goitrin in chromatogram of Radix Isatidis, thus improve with medicine prepared by this extract
Curative effect, and taking dose can be reduced.The method greatly simplifies existing Extraction Technology of Radix Isatidis, reduces manpower, energy etc.
Expend, the more amazing content being to considerably improve effective ingredient dl- goitrin in extract.
The (1) described pulverizing of step, can adopt any this area conventional comminution processes.
Described step soaking 3～6 hours in 20～60 DEG C (2), can realize by this area conventional meanses, such as direct
Add 20～60 DEG C of water, and carry out isothermal holding, or add normal-temperature water post-heating to carrying out isothermal holding again after temperature required.
Step concentration (3), drying, are the conventional concentrate drying method in this area, are such as evaporated to dry, heating concentration and are evaporated.
The granularity that described Radix Isatidis are pulverized, preferably 40～65 mesh, the crushing process of this particle size range does not interfere with myrosase
Activity, can substantially meet the reaction condition of sulfur glycosides and myrosase again；Further, preferably it is crushed to 50 mesh, this granularity is to be beneficial to
Sulfur glycosides and the dissolution of myrosase, fully reaction and the best combination keeping enzymatic activity, the extraction of this granularity Radix Isatidis dl- goitrin
The most complete.
Described soaking temperature, it is preferred to use 20～39 DEG C, this preferable temperature is not the warm macerating that this area is generally said
Temperature, warm macerating is defined as 40-50 DEG C, higher than preferable temperature of the present invention, 20～39 DEG C of temperature in " Chinese Pharmacopoeia "
Degree scope is more beneficial for myrosase and plays its activity；More preferably 36～38 DEG C of soaking temperature of the present invention, most preferably 37
DEG C, Semen Sinapiss enzymatic activity highest under this temperature conditions.
Water consumption in preparation method of the present invention is preferably 20～40 times, and test proves, after the water yield is more than 40 times,
Dl- goitrin content tapers into the increase rate that water consumption increases, and after 60 times, ascensional range is less, says
Bright, 40 times amount water sulfur glycosides hydrolyze completely substantially, accordingly, it is considered to arrive water utilization rate problem, preferably 20～40 times of water consumption, more
Preferably 30 times.
Described soak time, preferably 4 hours, this preferred time both ensure that the extraction ratio of dl- goitrin, takes into account again
Extraction efficiency, meanwhile, is also beneficial to ensure the quality of extract.During long-time extraction (immersion), aqueous are very easy to mould
Become, particularly when temperature meets or exceeds 30 DEG C.Test shows, after 37 DEG C are soaked 12 hours, can detect mildew.
(2) described step is preferably: (1) obtains Jia 10 in Radix Isatidis granule or powder the～water of 60 times amount to step, in 36
～38 DEG C are soaked 4 hours, filter, obtain filtrate.The combination of this water consumption, Extracting temperature and time, is remarkably improved dl- goitrin
Extraction ratio.
When needing to obtain Beilstein monomer, the extract that can obtain preparation method of the present invention is further purified point
From e.g., elder generation is through column chromatography purification, then carries out chiral chromatogram fractionation.
In order to accelerate to extract, can add in preparation method step of the present invention (2) immersion process and the step such as shake, stir.
It is a further object of the invention to provide the extract that a kind of above-mentioned preparation method obtains, and contain this extraction
The Pharmaceutical composition of thing, this pharmaceutical composition can make the medicine of oral form or injection form by this area common process
Preparation, described oral form, such as tablet, granule, oral liquid, capsule etc., injection form, such as injection, freeze-dried powder
Deng.This pharmaceutical composition also contains customary adjuvant, and described customary adjuvant is the acceptable pharmaceutic adjuvant in this area.System of the present invention
Effective ingredient dl- goitrin content in the Radix Isatidis extract that Preparation Method is obtained is higher, and curative effect is more secure.
Inventor obtains preparation method of the present invention by following experiment to optimize.
Reiterate: experiment is the illustrative experiment in numerous experiments in development process of the present invention below, is not covered by
Be exhausted all experiments that the inventor present invention is done, purpose is only in that and illustrates present invention preparation side with those data
The impact of the extraction efficiency to dl- goitrin for the method.
The impact to dl- goitrin in Radix Isatidis for 1 extracting method
1.1 instruments and reagent
Waters2695 chromatograph of liquid, waters2996 diode array detector, climacell climatic chamber.
Trifluoroacetic acid aqueous solution is purchased from merck, and the pure methanol of analysis, triethylamine, glacial acetic acid are purchased from Beijing chemical reagents corporation, and water is ultra-pure water.
Dl- goitrin ((r, s)-goitrin) reference substance is derived from National Institute for Food and Drugs Control.
Tertiary industry ground chromatogram of Radix Isatidis is purchased from medicinal material market.
1.2 chromatographic condition
Chromatographic column: alltima c18Post (4.6mm × 250mm, 5 μm).Mobile phase: water-acetonitrile-triethylamine (90:10:
0.2), with glacial acetic acid tune ph value to 5.2, flow velocity: 0.8ml/min.Detection wavelength: 241nm.Sample size: 10 μ l.
The impact of 1.3 extracting modes
Weigh chromatogram of Radix Isatidis powder 1g, the solubilizer 30ml of different sources 50 mesh, press the preparation of method shown in table 1 respectively
Need testing solution, every group parallel 3 parts, sample introduction measures, and external standard method calculates dl- goitrin content, and (extraction ratio is to convert extraction ratio
The dl- goitrin referring to extract accounts for the weight percent content of the medical material that feeds intake, similarly hereinafter), result shows, different sources Radix Isatidis medicine
The dl- goitrin extraction ratio rule of material is consistent, and that is, water soaks > water is ultrasonic > alcohol reflux > decocting boils > methanol eddy > EtOH Sonicate
> methanol is ultrasonic.
The extraction ratio % of the different extracting mode dl- goitrin of table 1
2nd, the impact to dl- goitrin extraction ratio in Radix Isatidis for the extraction process
2.1 instruments and reagent
Waters aquity Ultra Performance Liquid Chromatography instrument, climacell climatic chamber.
Chromatographic Pure Methanol, isopropanol, n-hexane are purchased from merck, and the pure methanol of analysis, triethylamine, acetic acid are purchased from Beijing chemistry
Reagent Company.
Dl- goitrin ((r, s)-goitrin) reference substance is derived from National Institute for Food and Drugs Control.Goitrin before table, front
Goitrin reference substance is that laboratory voluntarily separates preparation.
2.2 chromatographic condition
2.2.1uplc chromatographic condition 1 (for the mensure of goitrin before table and front goitrin)
Chromatographic column: waters acquity uplc hss t3 (2.1mm × 100mm, 1.8 μm), column temperature: 40 DEG C, flowing
Phase: methanol-water (2:98) (containing 0.1% triethylamine, with acetic acid tune ph value to 5.5), flow velocity 0.1ml/min, Detection wavelength:
245nm, sample size: 5ul.
2.2.2uplc chromatographic condition 2 (for the mensure of dl- goitrin)
Chromatographic column: waters acquity uplc hss t3 (2.1mm × 100mm, 1.8 μm), column temperature: 40 DEG C, flowing
Phase: methanol-water (5:95), Detection wavelength: 245nm, sample size: 2ul.
The impact to dl- goitrin extraction ratio for 2.3 extraction temperatures
Take 50 mesh chromatogram of Radix Isatidis powder, precision weighs 1g in 100ml triangular flask, accurate addition water 30ml, mix, claim
Determine weight, extract 4h (a copy of it decocts 1h) respectively by temperature in table 2, take out, let cool, supply the weight of less loss with water, fast
Speed filtration, measures precursor and enzymatic hydrolysate (dl- goitrin) content immediately, and converts extraction ratio, from table 2, temperature exceedes
After 20 DEG C, sulfur glycosides major part is hydrolyzed, and 37 DEG C of hydrolysis completely, afterwards, rise high extraction with temperature and reduce, particularly when temperature liter
To after 70 DEG C, the downward trend of dl- goitrin extraction ratio becomes apparent from, and myrosase serious inactivation is described.Extracted with dl- goitrin
Rate is index, primarily determines that myrosase optimum temperature is 37 DEG C.Medical material boils through 100 DEG C of decoctings, myrosase almost complete deactivation, surveys
Obtain dl- goitrin extraction ratio minimum, be 0.017%.
The different impact to dl- goitrin extraction ratio for the Extracting temperature of table 2
Note: the precursor content in table 2 accounts for the percentage by weight of the medical material that feeds intake for precursor；Conversion ratio is: not detect precursor
When (that is, when precursor converts completely) dl- goitrin content conversion precursor content be precursor total amount, deducted with this precursor total amount
Precursor content is detected to obtain divided by precursor total amount again.(similarly hereinafter)
2.4 the impact to dl- goitrin extraction ratio for the soak time
Take with portion chromatogram of Radix Isatidis, pulverize 50 mesh, precision weighs 1g in 100ml triangular flask, accurate addition water 30ml,
Mix, weighed weight, extract different time (being shown in Table 3) at 37 DEG C respectively, take out, let cool, supply the weight of less loss with water, fast
Speed filtration, measures precursor and enzymatic hydrolysate content immediately, and calculates conversion ratio and the extraction ratio of the two respectively, 6 hours and after
Dl- goitrin content no longer rises, precursor content does not detect, and illustrates, soaks 6 hours sulfur glycosides and substantially hydrolyzes completely, and 4 hours
Extraction efficiency highest.
The different impact to precursor and enzymatic hydrolysate for the soak time of table 3
The impact to dl- goitrin extraction ratio for 2.5 grinding particle sizes
Chromatogram of Radix Isatidis (3～5cm length, diameter 0.5～1cm) the 10g 300ml that adds water is taken to extract 4h at 37 DEG C；Separately take plate
Blue root herb, is crushed to 10～120 mesh respectively, respectively takes 1g, and add water 30ml, 37 DEG C of extraction 4h, supplies the weight of less loss with water, fast
Speed filtration, measures enzymatic hydrolysate dl- goitrin content immediately, and converts extraction ratio, result shows, the conversion ratio of 20～80 mesh is equal
It is not less than 80%, refer to table 4.
Table 4 different grain size is to the impact extracting dl- goitrin in Radix Isatidis
| Corresponding granularity/mesh | Precursor content | Conversion ratio % | Extraction ratio % |
| Do not pulverize | 1.007 | 10 | 0.037 |
| 10 | 0.422 | 62 | 0.2264 |
| 20 | 0.219 | 80 | 0.2935 |
| 30 | 0.128 | 89 | 0.3281 |
| 40 | 0.036 | 97 | 0.3582 |
| 50 | 0 | 100 | 0.3693 |
| 65 | 0.055 | 95 | 0.3492 |
| 80 | 0.123 | 89 | 0.3277 |
| 100 | 0.309 | 72 | 0.2640 |
| 120 | 0.615 | 45 | 0.1643 |
The above results show, this preparation method makes the conversion ratio of the dl- goitrin precursor in chromatogram of Radix Isatidis about reach
80%, preparation method particularly preferably, precursor almost all converts, and substantially increases the utilization rate of medical material, for preparing curative effect
Significantly more Radix Isatidis preparation is laid a good foundation.
In addition to above-mentioned experiment, inventor also to the step of described preparation method (3), extract water consumption dl- goitrin contained
The impact of amount is investigated, and result shows, concentrate drying process does not have any impact to the content of dl- goitrin；5～80
Between times amount water, the content of dl- goitrin increases with water consumption and raises, but this rising trend (amplitude) after 40 times amount gradually
Slow down, after 60 times amount, elevation amplitude is less.
Because length is limited, the method for above-mentioned experiment, step and related data will not be described here.
Specific embodiment
Embodiment 1:
A kind of preparation method of Radix Isatidis extract comprises the following steps:
(1) the 500g Radix Isatidis powder being dried is broken to the powder of 50 mesh；
(2) (1) obtain in Isatis Root, adding the water of 30 times amount to step, soak 4 hours in 37 DEG C, filter, obtain filtrate；
(3) (2) step is obtained filtrate reduced in volume to dry, obtains Radix Isatidis extract 255g, in this extract dl- accuse according to
The content in spring is 0.68% (g/g).
Embodiment 2:
A kind of preparation method of Radix Isatidis extract comprises the following steps:
(1) 500g Radix Isatidis powder is broken to the powder of 40 mesh；
(2) (1) obtain in Isatis Root, adding the water of 40 times amount to step, soak 6 hours in 39 DEG C, filter, obtain filtrate；
(3) (2) step is obtained filtrate reduced in volume to dry, obtains Radix Isatidis extract 253g, in this extract dl- accuse according to
The content in spring is 0.62%.
Embodiment 3:
A kind of preparation method of Radix Isatidis extract comprises the following steps:
(1) Radix Isatidis powder 500g being dried is broken to the powder of 65 mesh；
(2) (1) obtain in Isatis Root, adding the water of 20 times amount to step, soak 3 hours in 20 DEG C, filter, obtain filtrate；
(3) (2) step is obtained filtrate reduced in volume to dry, obtains Radix Isatidis extract 231g, in this extract dl- accuse according to
The content in spring is 0.59%.
Embodiment 4:
A kind of preparation method of Radix Isatidis extract comprises the following steps:
(1) Radix Isatidis powder 500g being dried is broken to the granule of 20 mesh；
(2) (1) obtain in Radix Isatidis granule, adding the water of 10 times amount to step, soak 4 hours in 20 DEG C, filter, obtain filtrate；
(3) (2) step is obtained filtrate reduced in volume to dry, obtains Radix Isatidis extract 222g, in this extract dl- accuse according to
The content in spring is 0.58%.
Embodiment 5:
A kind of preparation method of Radix Isatidis extract comprises the following steps:
(1) 500g Radix Isatidis powder is broken to the powder of 80 mesh；
(2) (1) obtain in Isatis Root, adding the water of 60 times amount to step, soak 3 hours in 60 DEG C, filter, obtain filtrate；
(3) (2) step is obtained filtrate reduced in volume to dry, obtains Radix Isatidis extract 245g, in this extract dl- accuse according to
The content in spring is 0.57%.
Embodiment 6:
A kind of preparation method of Radix Isatidis extract comprises the following steps:
(1) 500g Radix Isatidis powder is broken to the powder of 40 mesh；
(2) (1) obtain in Isatis Root to step plus 60 times amount water, soak 6 hours in 36 DEG C, in immersion process, often
Every stirring in 2 hours once, filter, obtain filtrate；
(3) (2) step is obtained filtrate reduced in volume to dry, obtains Radix Isatidis extract 253g, in this extract dl- accuse according to
The content in spring is 0.67%.
Embodiment 7:
A kind of preparation method of Radix Isatidis extract comprises the following steps:
(1) 500g Radix Isatidis powder is broken to the powder of 65 mesh；
(2) (1) obtain in Radix Isatidis granule or powder, adding the water of 40 times amount to step, soak 5 hours in 38 DEG C, filter, obtain
Filtrate；
(3) (2) step is obtained filtrate reduced in volume to dry, obtains Radix Isatidis extract 249g, in this extract dl- accuse according to
The content in spring is 0.68%.
The present invention is not limited to above-mentioned embodiment, anyone draw under the enlightenment of the present invention other any with this
Bright same or like as product, be all not precluded within outside protection scope of the present invention.
Claims (5)
1. a kind of preparation method of Radix Isatidis extract, contains dl- goitrin it is characterised in that described preparation in this extract
Method comprises the following steps:
(1) Radix Isatidis powder is broken to granule or the powder of 40～65 mesh；
(2) (1) obtain in Radix Isatidis granule or powder, adding the water of 30 times amount to step, soak 3～6 hours in 36～38 DEG C, filter,
Obtain filtrate；
(3) (2) step is obtained filtrate to concentrate, be dried, obtain Radix Isatidis extract.
2. preparation method as claimed in claim 1 is it is characterised in that described step Radix Isatidis powder (1) is broken to the powder of 50 mesh
End.
3. preparation method as claimed in claim 1 is it is characterised in that described step soak time (2) is 4 hours.
4. preparation method as claimed in claim 1 is it is characterised in that (2) described step is: (1) obtains Radix Isatidis to step
Add the water of 30 times amount in granule or powder, soak 4 hours in 36～38 DEG C, filter, obtain filtrate.
5. a kind of Pharmaceutical composition containing dl- goitrin is it is characterised in that said composition contains any one of claim 1-4
Extract and pharmaceutically acceptable adjuvant that described preparation method is obtained.
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