CN100998286A - Bulbus Fritillariae Cirrhosae and its cultivation method - Google Patents
Bulbus Fritillariae Cirrhosae and its cultivation method Download PDFInfo
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- CN100998286A CN100998286A CN 200610172189 CN200610172189A CN100998286A CN 100998286 A CN100998286 A CN 100998286A CN 200610172189 CN200610172189 CN 200610172189 CN 200610172189 A CN200610172189 A CN 200610172189A CN 100998286 A CN100998286 A CN 100998286A
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Abstract
The invention provides a method for cultivating fritillaria cirrhosa, and particularly relates to an artificial cultivation method for simulating the original ecological environment of the fritillaria cirrhosa by adopting intercropping and rotation according to the biological characteristics and life habits of the original plant of the fritillaria cirrhosa. The method has simple steps, and the collected Bulbus Fritillariae Cirrhosae has high yield, good quality, and wide application, and can creatively realize artificial cultivation of Bulbus Fritillariae Cirrhosae, and effectively protect environment and prevent water and soil loss. Experiments prove that compared with the medicine application part of the wild fritillaria cirrhosa medicinal material, the fritillaria cirrhosa medicinal material cultivated by the method disclosed by the invention has the advantages that the appearance, the physical and chemical identification and the like all accord with pharmacopeia standards, the types of main medicinal components are consistent, the content accords with the pharmacopeia standards, the fritillaria cirrhosa medicinal material is an excellent substitute of the wild fritillaria cirrhosa medicinal material, and the market prospect is excellent. Meanwhile, a new identification and detection method of the bulbus fritillariae cirrhosae medicinal material is provided for the public.
Description
Technical field
The present invention relates to the source cultivation of plants method of Bulbus Fritillariae Cirrhosae, belong to the medicinal material field of planting.
Background technology
The Bulbus Fritillariae Cirrhosae that the Pharmacopoeia of the People's Republic of China (version was an one in 2005) records is the dry bulb of liliaceous plant leaf roll bulb of fritillary Fritillaria cirrhosa D.Don, dark violet bulb of fritillary F.unibracteata Hsiao et K.C.Hsia, Gansu bulb of fritillary F.przewalskii Maxim.ex Balal and Bulbus Fritillariae cirrhosae F.delavayi Franch.Bulbus Fritillariae Cirrhosae is divided into " loose shellfish ", " blue or green shellfish ", " lubei " by bulb size and plant origin traditionally.The leaf roll bulb of fritillary, the dark violet bulb of fritillary, the Gansu bulb of fritillary claim " loose shellfish ", " blue or green shellfish " by different habit of shape; Bulbus Fritillariae cirrhosae is practised and is claimed " lubei ".Bulbus Fritillariae Cirrhosae is relieving cough and reducing sputum good medicine, and the tcm prescription consumption is quite big, is that the Chinese patent drug of raw material production reaches more than 100 kinds with the Bulbus Fritillariae Cirrhosae.Wild Bulbus Fritillariae Cirrhosae is because of excessively excavating, and wild resource minimizing rapidly is in Critical Condition, is three grades of wild medical herb species under state protections.The dark violet bulb of fritillary F.unibracteata Hsiao et K.C.Hsia that pharmacopeia is recorded from Songpan, the Hongyuan region contracts to the Ruoergai grassland, leaf roll bulb of fritillary Fritillaria cirrhosa D.Don, Gansu bulb of fritillary F.przewalskii Maxim.ex Balal, Bulbus Fritillariae cirrhosae F.delavayi Franch all belong to rare in commodity.Though the substitute products of some Bulbus Fritillariae Cirrhosae medicinal materials are arranged on the market, as kinds such as Siberian fritillary bulb, fritillary bulbs, these kinds have realized artificial cultivation, and its output is also higher, but all not as Bulbus Fritillariae Cirrhosae, the market acceptance is not high at aspects such as appearance character, physicochemical property, drug effect qualities.
Bulbus Fritillariae Cirrhosae source plant (being the leaf roll bulb of fritillary, the dark violet bulb of fritillary, the Gansu bulb of fritillary and Bulbus Fritillariae cirrhosae) is the herbaceos perennial of Liliaceae Fritillaria, the weather conditions that happiness cools, have cold-resistant, happiness is wet, be afraid of high temperature, characteristic that happiness covers; Temperature reaches 30 ℃ or ground temperature and surpasses 25 ℃, and plant will wither; Can not survive in the area that height above sea level is low, temperature is high.Be distributed in 3200~4700 meters of height above sea level, sunny, humus is abundant, the high and cold meadow of loosing soil, high-cold scrub meadow and high and cold grassy marshland area more.
The artificial cultivation Bulbus Fritillariae Cirrhosae has vegetative propagation and sexual propagation dual mode, carries out vegetative propagation based on bulb, but this method cost height, technical difficulty is big, and is unfavorable for the maintenance of Bulbus Fritillariae Cirrhosae self varietal character.It is that seminal propagation is bred that the sexual propagation of employing is now also arranged, and plantation is opened up wasteland in the Yinshan Mountains that more options are leeward or land for growing field crops, half Yinshan Mountains, needs scaffolding to shade before emerging; But existing cropping pattern is land for growing field crops reclamation of wasteland plantation easily causes adverse consequencess such as water and soil loss, soil weathering, influence ecological environment; Put up a shed to shade simultaneously and need expend a large amount of manpower and materials, cost is higher, because the growth cycle of Bulbus Fritillariae Cirrhosae is longer, such production method is unfavorable for the running of enterprise; The most important thing is that field planting is because of having broken away from original ecotope of Bulbus Fritillariae Cirrhosae and growth conditions, the degeneration that may cause species changes the effective ingredient kind and the content of medicinal material, even cause the variation of species, medical material quanlity can not get guaranteeing, can't reach the medicinal standard of Bulbus Fritillariae Cirrhosae medicinal material, affect the treatment.And does not still have at present when manually cultivating Bulbus Fritillariae Cirrhosae and other plant is planted at interval and in the relevant report of non-agro-farming district artificial cultivation Bulbus Fritillariae Cirrhosae.
Based on the tight demand of market, be badly in need of providing a kind of easy, low-cost, stable yield, product quality to meet the Bulbus Fritillariae Cirrhosae cultivation method that country will use standard to Bulbus Fritillariae Cirrhosae.
Summary of the invention
First technical problem to be solved by this invention is: the cultivation method that Bulbus Fritillariae Cirrhosae is provided.Wherein, the source plant of Bulbus Fritillariae Cirrhosae is one or more among dark violet bulb of fritillary F.unibracteata Hsiao et K.C.Hsia, leaf roll bulb of fritillary Fritillaria cirrhosaD.Don, Gansu bulb of fritillary F.przewalskii Maxim.ex Balal or the Bulbus Fritillariae cirrhosae F.delavayi Franch.And the source plant of these several product Bulbus Fritillariae Cirrhosaes also is Bulbus Fritillariae Cirrhosae in this area, also all is called Bulbus Fritillariae Cirrhosae in the present invention, and the bulb of Bulbus Fritillariae Cirrhosae becomes the Bulbus Fritillariae Cirrhosae medicinal material.
This method comprises the steps:
A, in height above sea level 2800~4700m area Bulbus Fritillariae Cirrhosae and other plant planting district are set, described other plant is one or more in legume, high crude drug, the plateau scrub plant;
Wherein, other plant described in the step a is one or more in legume, high crude drug or the plateau scrub plant.
Further, described legume is lima bean, pea, at least a in leguminous forage red clover, alfalfa, sainfoin, the butch clover; High crude drug is at least a in the Radix Astragali, Radix Angelicae Sinensis, conic gymnadenia tuber, bark of ash, notopterygium root, rheum officinale, the Radix Codonopsis; Described plateau scrub plant is common scrub plants such as bush cinqefoil group, Yin Lumei group, meadow sweet group, arborvitae shrubbery, cuckoo group, gold thread chrysanthemum group, two gland willow group, willow shrubbery, and these scrub plants are the common distribution coenotypes of Bulbus Fritillariae Cirrhosae.
B, in growing area Bulbus Fritillariae Cirrhosae and described other plant of a step are cultivated at interval, wherein Bulbus Fritillariae Cirrhosae growth belt railway carriage or compartment is wide is 0.3~2m, the wide 0.3~5m in other plant planting band railway carriage or compartment; Width between the growth belt is 0.1~0.5m;
Wherein, the cultivation spaced apart among the step b is at interval banded or at interval block.
Further preferred, Bulbus Fritillariae Cirrhosae growth belt railway carriage or compartment is wide to be 0.5~1.5m, and described other plant planting band railway carriage or compartment is wide to be 0.5~2m, and the width between the growth belt is 0.2~0.3m.
C, fertilising in the growth belt railway carriage or compartment, sowing, blinding get final product; Wherein thickness of sowing is 2000~4000/square metre, and blinding thickness is 2~3cm.
Gather after d, the Bulbus Fritillariae Cirrhosae maturation.
The present invention also provides a kind of cultivation method of Bulbus Fritillariae Cirrhosae, also be optimization and improvement to aforementioned Bulbus Fritillariae Cirrhosae cultivation method, it comprises the steps: promptly after Bulbus Fritillariae Cirrhosae is gathered, with Bulbus Fritillariae Cirrhosae and legume crop rotation, promptly at former legume growth belt plantation Bulbus Fritillariae Cirrhosae, at former Bulbus Fritillariae Cirrhosae growth belt plantation legume.
Cultivation method of the present invention is source phytobiology feature and the life habit according to Bulbus Fritillariae Cirrhosae, and itself and other plant (as legume, high crude drug, scrub plant etc.) is planted at interval.Because of other plant growing vigorous, look high than the Bulbus Fritillariae Cirrhosae of the sowing same period, can be for poky Bulbus Fritillariae Cirrhosae growth shading, cover the moon, preserve moisture, it is easy to reduce the scaffolding cost and the cultivation method that shade like this, and has avoided adverse consequencess such as the water and soil loss that causes because of the non-agro-farming of a large amount of reclamation of wastelands district and environmental deterioration; Plant at interval with legume that to also have an advantage with crop rotation promptly be that legume root system root nodule has nitrogen fixation, the Bulbus Fritillariae Cirrhosae of former leguminous plant growth belt high-quality fertile soil after for crop rotation provides profuse fertilizer.Plant Bulbus Fritillariae Cirrhosae and other plant at interval, simulated the original ecotope of Bulbus Fritillariae Cirrhosae as high crude drug, scrub plant etc., near wild Bulbus Fritillariae Cirrhosae growth conditions, the primary characteristic that can keep kind, the Bulbus Fritillariae Cirrhosae medicinal material of plantation gained is compared with wild Bulbus Fritillariae Cirrhosae medicinal material, the consistent standards of pharmacopoeia that meets of agents area appearance character, every physics and chemistry detection index such as moisture, total ash, sour insoluble composition, extract all meet standards of pharmacopoeia, and active constituent content measuring also meets the regulation of pharmacopeia.
Second technical problem to be solved by this invention is: the Bulbus Fritillariae Cirrhosae medicinal material by the cultivation method gained of above-mentioned Bulbus Fritillariae Cirrhosae is provided.
Bulbus Fritillariae Cirrhosae medicinal material of the present invention detects gained HPLC-ELSD finger-print by high performance liquid chromatogram, wherein, the HPLC-ELSD finger-print of the dark violet bulb of fritillary of cultivation gained as shown in Figure 5, the HPLC-ELSD finger-print of the leaf roll bulb of fritillary of cultivation gained as shown in Figure 6, the cultivation gained the Gansu bulb of fritillary the HPLC-ELSD finger-print as shown in Figure 7, the cultivation gained Bulbus Fritillariae cirrhosae the HPLC-ELSD finger-print as shown in Figure 8;
The chromatogram testing conditions of above-mentioned four kinds of HPLC-ELSD finger-prints is:
Detector is: EISD ELSD, 40 ℃ of gasification temperatures, nitrogen pressure 1.7~3.5bar; Preferred nitrogen pressure is 3.5bar.
Flowing phase is: acetonitrile-water (containing 0.1% diethylamine), gradient elution, overall flow rate: 1.0ml/min; Linear gradient: 0~8min wherein, the volume fraction of acetonitrile keeps 30%, 8~35min, and acetonitrile rises to 60% by 30% linearity, keeps 10min, 45~65min, acetonitrile rises to 90% by 60% linearity, keeps 5min;
Chromatographic column is: Kromasil ODS C18 150 * 4.6mm, particle diameter 5 μ m, sample size: 20ul;
Reference substance is: imperialine oxynitrides, different peimine, different peimine oxynitrides, bulb of fritillary suffering, chuanbeinone, imperialine, bulb of fritillary first element and verticinone;
Need testing solution preparation: pulverize the Bulbus Fritillariae Cirrhosae medicinal material, cross 40 mesh sieves, 80 ℃ of dryings 12 hours, precision takes by weighing the about 2.0g of Tendrilled Fritillaria Bulb respectively, put in the 100ml conical flask and alkalize with 3.0ml ammoniacal liquor earlier, add chloroform-methanol (4: 1) mixed solution 40ml then, 65~70 ℃ of water-bath refluxing extraction 4 hours; Extract put cold after, filter in conical flask, with an amount of mixed solvent washing dregs of a decoction, filter, filtrate merges; Behind the recovered under reduced pressure partial solvent, surplus solution is quantitatively transferred to porcelain steam in the ware, water-bath volatilizes; Use dissolve with methanol, constant volume shakes up in the 2ml measuring bottle, promptly gets need testing solution.
The characteristic peak retention time is that 17~24min goes out the peak, and the peak area of this time period accounts for more than 50% of its gross area, and this chromatogram feature can be used as the important diagnostic characteristics of Bulbus Fritillariae Cirrhosae medicinal material of the present invention.Test shows that Bulbus Fritillariae Cirrhosae medicinal material of the present invention differentiates that the specificity and the reappearance of collection of illustrative plates all meet the requirements.
There is following defective in the detection method of Bulbus Fritillariae Cirrhosae medicinal material promptly at present: because of bulb of fritillary Alkaloid does not structurally have conjugated double bond, be difficult to directly measure with the HPLC-UV method; Also there is certain problem in the content of measuring bulb of fritillary Alkaloid with the HPLC-UV method and gas-chromatography (GC) analytical method of pre-column derivatization; And with total alkaloid content in the Bulbus Fritillariae Cirrhosae medicinal material as the method for quantitative target, also have the not strong defective of specificity.
The 3rd technical problem to be solved by this invention is: the discriminating measuring method of Bulbus Fritillariae Cirrhosae medicinal material is provided,
A, preparation reference substance, need testing solution:
Reference substance is: imperialine oxynitrides, different peimine, different peimine oxynitrides, bulb of fritillary suffering, chuanbeinone, imperialine, bulb of fritillary first element and verticinone; Dilute with methyl alcohol respectively;
Need testing solution preparation: pulverize the Bulbus Fritillariae Cirrhosae medicinal material, cross 40 mesh sieves, 80 ℃ of dryings 12 hours, precision takes by weighing the about 2.0g of Tendrilled Fritillaria Bulb respectively, put in the 100ml conical flask and alkalize with 3.0ml ammoniacal liquor earlier, add chloroform-methanol (4: 1) mixed solution 40ml then, 65~70 ℃ of water-bath refluxing extraction 4 hours; Extract put cold after, filter in conical flask, with an amount of mixed solvent washing dregs of a decoction, filter, filtrate merges; Behind the recovered under reduced pressure partial solvent, surplus solution is quantitatively transferred to porcelain steam in the ware, water-bath volatilizes; Use dissolve with methanol, constant volume shakes up in the 2ml measuring bottle, promptly gets need testing solution;
B, chromatographic condition is set
Detector is: EISD ELSD, 40 ℃ of gasification temperatures, nitrogen pressure 1.7~3.5bar; Nitrogen pressure 3.5bar preferably is set;
Flowing phase is: acetonitrile-water (containing 0.1% diethylamine), gradient elution, overall flow rate: 1.0ml/min; Linear gradient: 0~8min wherein, the volume fraction of acetonitrile keeps 30%, 8~35min, and acetonitrile rises to 60% by 30% linearity, keeps 10min, 45~65min, acetonitrile rises to 90% by 60% linearity, keeps 5min;
Chromatographic column is: Kromasil ODS C18 150 * 4.6mm, particle diameter 5 μ m;
C, sample introduction;
D, must detect data.
The present invention adopts HPLC-ELSD coupling discrimination method, and the compound that does not have absorption for the ultra-violet (UV) band is a kind of good detection method, has remedied the deficiency of UV and RI detector.Because the response of ELSD does not rely on the optical property of sample, can detect the component (no matter which kind of functional group it has) that any volatility is lower than flowing phase, the detection of all samples is almost had identical response factor.Signal response value and the concentration of ELSD are not simple linear relation, and in the finite concentration scope, the natural logrithm of its peak area and concentration has better linearity.Therefore HPLC and common detector ELSD (EISD) coupling analysis bulb of fritillary Alkaloid is a kind of outstanding detection method.
The cultivation method step of Bulbus Fritillariae Cirrhosae of the present invention is easy, and cost is low, simultaneously can protect environment effectively, prevents erosion, the soil weathering; Especially Bulbus Fritillariae Cirrhosae and scrub plant and high crude drug are planted the original ecotope of also having simulated the wild bulb of fritillary at interval.The experiment proved that the inventive method cultivation gained Bulbus Fritillariae Cirrhosae medicinal material meets pharmacopeia quality control standard regulation, is outstanding wild Bulbus Fritillariae Cirrhosae medicinal material substitute, has fabulous market prospects.But also provide a kind of discriminating detection method of new Bulbus Fritillariae Cirrhosae medicinal material for the public.
Description of drawings
The banded growth belt diagrammatic sketch at interval of Fig. 1
The block growth belt diagrammatic sketch at interval of Fig. 2
The circular interval of Fig. 3 growth belt diagrammatic sketch
Among Fig. 1~Fig. 31 expression Bulbus Fritillariae Cirrhosae growth belt; 2 other plant planting bands of expression, other plant can be single leguminous plant, high crude drug or scrub plant, also can be two or three in mixed planting leguminous plant, high crude drug or the scrub plant; 3 expression intervallums.
Fig. 4 reference substance HPLC-ELSD chromatographic fingerprinting
The mark peak is represented respectively among the figure: 1, imperialine oxynitrides, 2, different peimine and different peimine oxynitrides, 3, bulb of fritillary suffering, 4, imperialine, 5, bulb of fritillary first element, 6, verticinone, 7, chuanbeinone.
The dark violet bulb of fritillary HPLC-ELSD of Fig. 5 chromatographic fingerprinting
Fig. 6 leaf roll bulb of fritillary HPLC-ELSD chromatographic fingerprinting
Fig. 7 Gansu bulb of fritillary HPLC-ELSD chromatographic fingerprinting
Fig. 8 Bulbus Fritillariae cirrhosae HPLC-ELSD chromatographic fingerprinting
Embodiment
Rotary tillage is ploughed with rake burring root, tree root and turf with planting area by a, selection agro-farming district, the height above sea level 2800m left and right sides, plants the dark violet bulb of fritillary and lima bean at interval:
Dark violet bulb of fritillary growth belt railway carriage or compartment is wide to be 0.3~0.5m, and lima bean growth belt railway carriage or compartment is wide to be 0.5~0.7m, and the high 0.2~0.3m in railway carriage or compartment, the width between the growth belt are 0.3~0.5m;
B, with the dark violet bulb of fritillary and lima bean cultivating and growing as shown in Figure 2 spaced apart:
Face is executed an amount of fertilizer in dark violet bulb of fritillary growth belt railway carriage or compartment, and in the railway carriage or compartment face, thickness of sowing is 2000~3000/square metre, covers fine earth again with dark violet fritillaria thunbergii uniform broadcasting, and blinding thickness is 2~3cm.
A, selection non-agro-farming district, the height above sea level 3000m left and right sides, open up wasteland the scrub plant band as shown in Figure 3 according to concrete landform:
Leaf roll bulb of fritillary planting area is ploughed rotary tillage with rake burring root, tree root and turf, it is 0.3~0.8m that scrub plant keeps railway carriage or compartment, the zone of vegetation wide, and in the scrub plant reservation zone of vegetation, intert and plant lima bean, leaf roll bulb of fritillary growth belt railway carriage or compartment is wide to be 0.5~1m, high 0.2~the 0.3m in railway carriage or compartment, the width between the growth belt are 0.2~0.3m;
Face is executed an amount of fertilizer in leaf roll bulb of fritillary growth belt railway carriage or compartment, and in the railway carriage or compartment face, thickness of sowing is 3000~4000/square metre, covers fine earth again with the uniform broadcasting of leaf roll fritillaria thunbergii, and blinding thickness is 2~3cm;
B, this is at interval in planting patterns: railway carriage or compartment, the zone of vegetation is wide is limited by step a for the leaf roll bulb of fritillary and scrub plant reservation, and the length of growth belt can be according to topographic features with the random length setting.
A, selection non-agro-farming district, the height above sea level 3200m left and right sides, open up wasteland the scrub plant band as shown in Figure 2 according to concrete landform:
With rake burring root, tree root and turf, it is 1~1.5m that scrub plant keeps railway carriage or compartment, the zone of vegetation wide with the rotary tillage of Gansu bulb of fritillary planting area plough, and bulb of fritillary growth belt railway carriage or compartment, Gansu is wide to be 1~1.5m, the high 0.2~0.3m in railway carriage or compartment; Width between the growth belt is 0.2~0.3m;
B, with the Gansu bulb of fritillary and scrub plant interband every the cultivating and growing as shown in Figure 2 that distributes:
Face is executed an amount of fertilizer in bulb of fritillary growth belt railway carriage or compartment, Gansu, and in the railway carriage or compartment face, thickness of sowing is 2500~3500/square metre, covers fine earth again with Gansu fritillaria thunbergii uniform broadcasting, and blinding thickness is 2~3cm.
Embodiment 4 plants the dark violet bulb of fritillary and Radix Angelicae Sinensis at interval with the inventive method
Rotary tillage is ploughed with rake burring root, tree root and turf with planting area by a, selection agro-farming district, the height above sea level 3400m left and right sides, plants the dark violet bulb of fritillary and Radix Angelicae Sinensis at interval:
Dark violet bulb of fritillary growth belt railway carriage or compartment is wide to be 0.8~1m, and Radix Angelicae Sinensis growth belt railway carriage or compartment is wide to be 1~1.2m, and the high 0.2~0.3m in railway carriage or compartment, the width between the growth belt are 0.2~0.35m;
B, with the dark violet bulb of fritillary and Radix Angelicae Sinensis cultivating and growing as shown in Figure 2 spaced apart:
Face is executed an amount of fertilizer in dark violet bulb of fritillary growth belt railway carriage or compartment, and in the railway carriage or compartment face, thickness of sowing is 3000~4000/square metre, covers fine earth again with dark violet fritillaria thunbergii uniform broadcasting, and blinding thickness is 2~3cm.
A, selection non-agro-farming district, the height above sea level 3800m left and right sides, open up wasteland the scrub plant band as shown in Figure 4 according to concrete landform, the Bulbus Fritillariae cirrhosae planting area is ploughed rotary tillage with rake burring root, tree root and turf, it is 1.5~2m that scrub plant keeps railway carriage or compartment, the zone of vegetation wide, and in the scrub plant reservation zone of vegetation, intert and plant Astragalus membranacus, Bulbus Fritillariae cirrhosae growth belt railway carriage or compartment is wide to be 1.5~2m, and the high 0.2~0.3m in railway carriage or compartment, the width between the growth belt are 0.2~0.35m;
B, with Bulbus Fritillariae cirrhosae and Astragalus membranacus cultivating and growing as shown in Figure 4 spaced apart:
Face is executed an amount of fertilizer in Bulbus Fritillariae cirrhosae growth belt railway carriage or compartment, the Bulbus Fritillariae cirrhosae seed is evenly removed retouched in the railway carriage or compartment face, and thickness of sowing is 2000~3000/square metre, covers fine earth again, and blinding thickness is 2~3cm.
Embodiment 6 plants the dark violet bulb of fritillary and leguminous forage alfalfa, bark of ash at interval with the inventive method
Rotary tillage is ploughed with rake burring root, tree root and turf with planting area by a, selection agro-farming district, the height above sea level 4700m left and right sides, plants the dark violet bulb of fritillary and alfalfa, bark of ash at interval:
Alfalfa growth belt railway carriage or compartment is wide to be 1.5~2m, and at the interspersed plantation of alfalfa growth belt traditional Chinese medicine bark of ash, dark violet bulb of fritillary growth belt railway carriage or compartment is wide to be 1~1.5m, and the high 0.2~0.3m in railway carriage or compartment, the width between the growth belt are 0.3~0.5m;
B, with the dark violet bulb of fritillary and alfalfa, bark of ash cultivating and growing as shown in Figure 2 spaced apart:
Face is executed an amount of fertilizer in dark violet bulb of fritillary growth belt railway carriage or compartment, and in the railway carriage or compartment face, thickness of sowing is 2500~3500/square metre, covers fine earth again with dark violet fritillaria thunbergii uniform broadcasting, and blinding thickness is 2~3cm.
Rotary tillage is ploughed with rake burring root, tree root and turf with planting area by a, selection agro-farming district, the height above sea level 3400m left and right sides, plants the leaf roll bulb of fritillary and scrub plant, pea at interval:
Transplant natural scrub plant in the agro-farming district, scrub plant growth belt railway carriage or compartment is wide to be 0.8~1m, and at the interspersed plantation of scrub plant growth belt pea, leaf roll bulb of fritillary growth belt railway carriage or compartment is wide to be 0.8~1m, and the high 0.2~0.3m in railway carriage or compartment, the width between the growth belt are 0.2~0.3m;
B, with the leaf roll bulb of fritillary and scrub plant, pea cultivating and growing as shown in Figure 2 spaced apart:
Face is executed an amount of fertilizer in leaf roll bulb of fritillary growth belt railway carriage or compartment, and the leaf roll fritillaria thunbergii is evenly removed the railway carriage or compartment face that is sowed at, and thickness of sowing is 2500~3500/square metre, covers fine earth again, and blinding thickness is 2~3cm.
Embodiment 8 plants the dark violet bulb of fritillary and scrub plant at interval with the inventive method
A, selection non-agro-farming district, the height above sea level 3000m left and right sides, open up wasteland the scrub plant band as shown in Figure 2 according to concrete landform:
With rake burring root, tree root and turf, it is 1~1.5m that scrub plant keeps railway carriage or compartment, the zone of vegetation wide with the rotary tillage of dark violet bulb of fritillary planting area plough, and dark violet bulb of fritillary growth belt railway carriage or compartment is wide to be 1~1.5m, the high 0.2~0.3m in railway carriage or compartment; Width between the growth belt is 0.2~0.3m;
B, with the dark violet bulb of fritillary and scrub plant interband every the cultivating and growing as shown in Figure 2 that distributes:
Face is executed an amount of fertilizer in dark violet bulb of fritillary growth belt railway carriage or compartment, and in the railway carriage or compartment face, thickness of sowing is 2500~3500/square metre, covers fine earth again with dark violet fritillaria thunbergii uniform broadcasting, and blinding thickness is 2~3cm.
Embodiment 9 plants at interval with the inventive method crop rotation
Will be according to embodiment 1,2,5,7 described kinds of method for planting, the Bulbus Fritillariae Cirrhosae maturation is gathered after 3 years, crop rotation two growth belt plants, method is with former embodiment.
Below plant the field observation of Bulbus Fritillariae Cirrhosae, and 3 years living Bulbus Fritillariae Cirrhosae bulb quality of cultivation gained are to illustrate the beneficial effect of cultivation method of the present invention by employing cultivation method of the present invention.
Test example 1 cultivation method field observation record of the present invention sees Table 1
Table 1 Bulbus Fritillariae Cirrhosae growth field observation record
The planting site type | Seedling strain number | Begin the withered seedling date | Whole withered seedling dates |
Standard-sized sheet is cultivated |
154 strains, 160 strains, 158 strains, 160 strains, 156 strains, 155 strains, 160 strains | August 3 August 2 July 31 July 31 August 1 August 4 July 26 | August 28 August 28 August 24 August 23 August 26 August 29 August 20 |
Annotate: standard-sized sheet is cultivated planting site and is promptly all planted Bulbus Fritillariae Cirrhosae, does not plant other plant at interval.
The above results shows: standard-sized sheet is cultivated bulb of fritillary seedling ground, owing to there is not the side of other plant to shade, only shades by sunshade net front, and it covers shady effect and the soil moisturizing effect is good not as the Bulbus Fritillariae Cirrhosae of cultivation method plantation of the present invention; Late about 5~10 days of the seedling ground bulb of fritillary withered seedling time is cultivated than standard-sized sheet in the Bulbus Fritillariae Cirrhosae species seedling ground of cultivation method plantation of the present invention, and plant strain growth is more healthy and stronger.
The living Bulbus Fritillariae Cirrhosae bulb quality contrast in 3 years of test example 2 pharmacopeia Bulbus Fritillariae Cirrhosaes and cultivation method gained of the present invention.
Regulation about Bulbus Fritillariae Cirrhosae quality standard etc. in one one of the version Pharmacopoeia of the People's Republic of China in 2005 sees the 25th and 26 page for details.
[processing of gathering] get embodiment 9 adopt 3 years dark violet bulb of fritillary bulbs of life, remove fibrous root, tertia and washing and remove silt, dry or low temperature drying, excavate the record see Table 2:
The dark violet bulb of fritillary of table 2 cultivation method of the present invention institute is excavated record for test agent
Kind | Commercial specification | Acquisition time | Lot number |
The dark violet bulb of fritillary | The blue or green shellfish of the blue or green Bei Songbeisongbei of the blue or green Bei Songbeisongbei of Song Beisongbei | In August, 2005 in August 2005 August of 2005 July, 2004 July of 2004 July, 2004 July in 2003 July, 2003 July in 2003 | 030701 030702 030703 040701 040702 040703 050801 050802 050803 |
Excavate the place: applicant's Bulbus Fritillariae Cirrhosae standardized planting base |
Remove fibrous root, tertia after the sample collection, silt is removed in washing, dries.
[proterties] cultivation method gained of the present invention bulb meets standards of pharmacopoeia.
[discriminating] cultivation method gained of the present invention bulb meets standards of pharmacopoeia.
[inspection]
1, moisture is got the dark violet bulb of fritillary bulb of totally 9 lot numbers of continuous 3 years cultivation and production of cultivation method of the present invention, measure according to aquametry (" an appendix IX of Chinese pharmacopoeia version in 2005 H first method), each two samples, moisture in the calculating bulb measured.Determination of moisture the results are shown in table 3.
Table 3 determination of moisture is table as a result
Kind | Commercial specification | Lot number | Moisture (%) | On average (%) | ||
1 | 2 | On average | ||||
The dark violet bulb of fritillary | The blue or green shellfish of the blue or green Bei Songbeisongbei of the blue or green Bei Songbeisongbei of Song Beisongbei | 030701 030702 030703 040701 040702 040703 050801 050802 050803 | 9.62 8.68 8.89 7.86 9.83 9.53 8.92 9.58 8.27 | 9.65 8.67 8.94 7.86 9.86 9.55 8.90 9.56 8.29 | 9.64 8.68 8.92 7.86 9.85 9.54 8.91 9.57 8.28 | 9.03 |
According to this experimental result, bulb moisture average content is 9.03%, and all less than 10%.Be lower than " Bulbus Fritillariae Cirrhosae moisture inspection moisture (must not cross 15%) limit of stipulating down of Chinese pharmacopoeia version in 2005.
2, total ash is got the dark violet bulb of fritillary bulb of totally 9 lot numbers of continuous 3 years cultivation and production of cultivation method of the present invention, according to " an appendix IX of Chinese pharmacopoeia version in 2005 K method is measured, and measures two samples, calculates total ash content in the bulb at every turn.The total ash measurement result sees Table 4.
Table 4 total ash measurement result table
Kind | Commercial specification | Lot number | Total ash content (%) | On average (%) | ||
1 | 2 | On average | ||||
The dark violet bulb of fritillary | The blue or green shellfish of the blue or green Bei Songbeisongbei of the blue or green Bei Songbeisongbei of Song Beisongbei | 030701 030702 030703 040701 040702 040703 050801 050802 050803 | 2.50 2.25 1.85 1.71 2.12 1.66 2.31 2.04 1.91 | 2.51 2.28 1.89 1.71 2.13 1.69 2.32 2.04 1.93 | 2.51 2.27 1.87 1.71 2.13 1.68 2.32 2.04 1.92 | 2.05 |
According to this experimental result, bulb total ash average content is 2.05%, and all less than 3%.Be lower than " Bulbus Fritillariae Cirrhosae total ash inspection total ash content (must not cross 5%) limit of stipulating down of Chinese pharmacopoeia version in 2005.
3, sour insoluble composition is got the dark violet bulb of fritillary bulb of totally 9 lot numbers of continuous 3 years cultivation and production of cultivation method of the present invention, according to " an appendix IX of Chinese pharmacopoeia version in 2005 K method is measured, each two samples, sour insoluble composition content in the calculating bulb measured.Acid insoluble composition measurement result sees Table 5.
The sour insoluble composition measurement result of table 5 table
Kind | Commercial specification | Lot number | Acid insoluble composition content (%) | On average (%) | ||
1 | 2 | On average | ||||
The dark violet bulb of fritillary | The blue or green shellfish of the blue or green Bei Songbeisongbei of the blue or green Bei Songbeisongbei of Song Beisongbei | 030701 030702 030703 040701 040702 040703 050801 050802 050803 | 0.104 0.126 0.093 0.115 0.131 0.098 0.128 0.12 0.103 | 0.102 0.123 0.091 0.115 0.134 0.096 0.127 0.121 0.105 | 0.10 0.12 0.092 0.12 0.13 0.097 0.13 0.12 0.10 | 0.11 |
According to this experimental result, bulb acid insoluble composition average content is 0.11%, and all less than 0.2%.Be lower than " Bulbus Fritillariae Cirrhosae an acid of Chinese pharmacopoeia version in 2005 insoluble composition inspection sour insoluble composition content (must not cross a 0.5%) limit of stipulating down.
[extract] cultivation method gained of the present invention bulb extract yield is according to " hot dipping under an appendix XA of Chinese pharmacopoeia version in 2005 the ethanol soluble extractives determination method item is measured, and makes solvent with Diluted Alcohol.The dark violet bulb of fritillary bulb of totally 9 lot numbers of continuous 3 years cultivation and production of cultivation method of the present invention is measured by hot dipping, measures two samples at every turn, calculates bulb extract yield.The extract yield determination the results are shown in Table 6.
Table 6 extract yield determination is table as a result
Kind | Commercial specification | Lot number | Extract yield (%) | On average (%) | ||
1 | 2 | On average | ||||
The dark violet bulb of fritillary | The blue or green shellfish of the blue or green Bei Songbeisongbei of the blue or green Bei Songbeisongbei of Song Beisongbei | 030701 030702 030703 040701 040702 040703 050801 050802 050803 | 25.13 23.50 21.61 23.98 22.91 21.97 23.59 22.98 21.11 | 25.39 23.26 21.67 24.26 23.21 22.27 23.23 22.74 21.49 | 25.26 23.38 21.64 24.12 23.06 22.12 23.41 22.86 21.30 | 23.02 |
According to this experimental result, the average yield of bulb extract is 23.02%.Be higher than " Bulbus Fritillariae Cirrhosae extract inspection extract yield (must not be less than 9.0%) limit of stipulating down of Chinese pharmacopoeia version in 2005.
[assay]
1, detected object is selected
The Bulbus Fritillariae Cirrhosae medicinal part is a dry bulb, its main active ingredient alkaloid stable in properties, its content can reflect the inherent quality of this product, is the common counter composition of bulb of fritillary class medicinal material and patent medicine, thus this product with it as detected object.
2, the selection of assay method
Acid-dye colorimetry: bromocresol green in the buffer solution of pH4.4, adopts the 416nm wavelength to detect as acid dye.
3,1. instrument UV-754 ultraviolet specrophotometer (200w, Shanghai Precision Scientific Apparatus Co., Ltd) of experimental instrument and reagent; CS202B type electric heating thermal insulation drying box (Chongqing testing equipment factory of the People's Republic of China (PRC)); SENCOR-201 type rotary evaporator (Shen, Shanghai is along bio tech ltd); W201B type thermostat water bath (Shen, Shanghai is along bio tech ltd); Ultrasonic cleaning machine (80w, 40kHz, Shanghai boats and ships electronic equipment research institute); Automatic dual pure water distiller (D1810C type, Zhengzhou Greatwall Scientific Industrial ﹠ Trading Co., Ltd.).
2. reagent bromocresol green (analyze pure, Shanghai reagent three factories); It is pure that reagent such as chloroform, methyl alcohol, ammoniacal liquor, sodium hydroxide, potassium dihydrogen phosphate are commercially available analysis; Distilled water (self-control); Kashmirine, Bulbus Fritillariae Cirrhosae control medicinal material and test sample source are described with [discriminating].
4, the preparation precision of reference substance and reference substance solution takes by weighing at 105 ℃ of drying under reduced pressure to constant weight kashmirine reference substance 5.0mg, puts in the 10ml measuring bottle, adds chloroform 7ml, puts that low-grade fever makes dissolving in the water-bath, puts coldly, adds chloroform to scale, shakes up.The accurate 2ml that draws puts among the 10ml in the measuring bottle, adds chloroform to scale, shakes up, and promptly gets (every 1ml contains kashmirine 0.1mg).
5, the preparation of need testing solution
1. the preparation precision of sample solution takes by weighing the about 1.0g of dark violet bulb of fritillary bulb (030702) powder, adds liquor ammoniae fortis 1.5ml, soaks into difference chloroform-methanol (4: 1) 40ml, water-bath heating and refluxing extraction 4 hours 1 hour
6, sample determination and content limit determines
Get the dark violet bulb of fritillary bulb of totally 9 lot numbers of continuous 3 years cultivation and production of cultivation method of the present invention, press the sample preparation of need testing solution method, measure, measure two samples at every turn, calculate total alkaloid content in the bulb.Measurement result is listed in table 7.
Table 7 sample determination result
Kind | Lot number | Total alkaloid content (%) | On average (%) | ||
1 | 2 | On average | |||
The dark violet bulb of fritillary | 030701 030702 030703 040701 040702 040803 050801 050802 050803 | 0.0514 0.0472 0.0378 0.0479 0.0497 0.0370 0.0464 0.0443 0.0398 | 0.0517 0.0471 0.0360 0.0479 0.0498 0.0369 0.0463 0.0442 0.0398 | 0.052 0.047 0.037 0.048 0.050 0.037 0.046 0.044 0.040 | 0.045 |
According to this experimental result, dark violet bulb of fritillary bulb total alkaloids average content is 0.045%, and 80% of average content is 0.045% * 80%=0.036%, and 9 batches of bulb total alkaloids minimum contents are 0.037%.
Account form: total alkaloid content in the sample (%)=C * b/W * 100%, C are the sample liquid concentration that colorimetric method (calibration curve method) records, and b is the sample liquid extension rate, and W is an example weight.
In sum: the Bulbus Fritillariae Cirrhosae medicinal material of cultivation method gained of the present invention meets the quality standard of pharmacopeia regulation, no matter be appearance character, or physics and chemistry differentiate that project all meets the requirements.
The HPLC-ELSD finger-print research of the test example 3 dark violet bulbs of fritillary, the Gansu bulb of fritillary, Bulbus Fritillariae cirrhosae, the leaf roll bulb of fritillary
This experiment detects (HPLC-ELSD) method with high performance liquid chromatogram-evaporative light-scattering and the dark violet bulb of fritillary, the Gansu bulb of fritillary, Bulbus Fritillariae cirrhosae, the leaf roll bulb of fritillary and the reference substance of cultivation method of the present invention ( embodiment 1,2,3,7) gained compared (sees Fig. 4~Fig. 8).
Method: LC-10AT high performance liquid chromatograph (day island proper Tianjin), SEDEX75 EISD (France), N2000 chromatographic work station; With kromasil ODS C18 (150 * 4.6mm, 5 μ m) is analytical column; With acetonitrile-water (containing 0.1% diethylamine) is flowing phase, gradient elution, overall flow rate 1mlmin
-1, flowing phase condition: linear gradient: 0~8min, the volume fraction of B keeps 30%, 8~35min, and B rises to 60% by 30% linearity, keeps 10min, 45~65min, B rises to 90% by 60% linearity, keeps 5min; Detect the testing conditions of ELSD: 40 ℃ of gasification temperatures, nitrogen pressure 1.7bar with evaporative light-scattering device (ELSD).
The preparation of reference substance and reference substance solution: imperialine oxynitrides, different peimine, different peimine oxynitrides, bulb of fritillary suffering, chuanbeinone separate preparation for the applicant, and structure identifies that through spectrum purity is analyzed greater than 98% through HPLC-ELSD.Imperialine, bulb of fritillary first element and verticinone are all available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
Precision takes by weighing each about 5mg of reference substance, puts in the 25ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, and promptly gets and mixes reference substance solution.
Need testing solution preparation: pulverize each year of utilizing cultivation method gained of the present invention and give birth to the cultivation bulb of fritillary, cross 40 mesh sieves, 80 ℃ of dryings 12 hours, precision takes by weighing the about 2.0g of each year living cultivation bulb of fritillary medicinal powder respectively, put in the 100ml conical flask and alkalize with 3.0ml ammoniacal liquor earlier, add chloroform-methanol (4: 1) mixed solution 40ml then, 65~70 ℃ of water-bath refluxing extraction 4 hours; Extract put cold after, filter in conical flask, with an amount of mixed solvent washing dregs of a decoction, filter, filtrate merges; Behind the recovered under reduced pressure partial solvent, surplus solution is quantitatively transferred to porcelain steam in the ware, water-bath volatilizes.Use dissolve with methanol, constant volume shakes up in the 2ml measuring bottle, promptly gets need testing solution.
Get need testing solution, each 20 μ l sample introductions analysis of reference substance solution (HPLC-ELSD chromatographic fingerprint spectrum is seen Fig. 4~8) respectively.
It is that reference substance carries out assay to Bulbus Fritillariae Cirrhosae of the present invention that this experiment begins to intend with the stronger composition of specificities such as kashmirine.But the alkaloid analysis result in the dark violet bulb of fritillary is found that dark violet bulb of fritillary Chinese and Western shellfish alkali content is low with the HPLC-ELSD method.Simultaneously, be reference substance with chuanbeinone, the alkaloidal analysis result of this product is shown that the chuanbeinone specificity is strong and content is very low, so the applicant is definite the total alkaloids of this product is carried out assay, as the quantitative target of this product with the HPLC-ELSD method.
Conclusion: the Bulbus Fritillariae Cirrhosae medicinal part is a dry bulb, its main active ingredient alkaloid stable in properties, its content can reflect this product quality, is the common counter composition of bulb of fritillary class medicinal material and patent medicine, thus with it as detected object.As seen, the Bulbus Fritillariae Cirrhosae medicinal material of the inventive method cultivation gained meet " medicinal standard of a Bulbus Fritillariae Cirrhosae that records of Chinese pharmacopoeia version in 2005, in addition as can be known according to each Bulbus Fritillariae Cirrhosae sample chromatogram figure, the chromatogram collection of illustrative plates fairly similar of this product and other Bulbus Fritillariae Cirrhosae medicinal material sample.Main component goes out the peak at 17~24min, the peak area of this time period accounts for more than 50% of its gross area, this chromatogram feature can be used as the common diagnostic characteristics of four Bulbus Fritillariae Cirrhosae kinds) though main in the test what measure is detection index by the dark violet bulb of fritillary of cultivation method gained of the present invention, but because growing environment, the association group of other Bulbus Fritillariae Cirrhosae kind and the dark violet bulb of fritillary are basic identical, show according to of the experiment in cultivation of this base that in addition cultivation method of the present invention is suitable equally to other Bulbus Fritillariae Cirrhosae kinds to each Bulbus Fritillariae Cirrhosae.
Test shows, compares with wild product, and total alkaloid content of this product and HPLC-ELSD chromatogram feature all do not have significant difference.The present invention adopts HPLC-ELSD coupling discrimination method, and the compound that does not have absorption for the ultra-violet (UV) band is a kind of good detection method, has remedied the deficiency of UV and RI detector.Because the response of ELSD does not rely on the optical property of sample, can detect the component (no matter which kind of functional group it has) that any volatility is lower than flowing phase, the detection of all samples is almost had identical response factor.Signal response value and the concentration of ELSD are not simple linear relation, and in the finite concentration scope, the natural logrithm of its peak area and concentration has better linearity.Therefore HPLC and common detector ELSD (EISD) coupling analysis bulb of fritillary alkaloid is a kind of outstanding detection method.
The cultivation method step of Bulbus Fritillariae Cirrhosae of the present invention is easy, and cost is low, simultaneously can protect environment effectively, prevents erosion, the soil weathering; Especially Bulbus Fritillariae Cirrhosae and scrub plant and high crude drug are planted the original ecotope of also having simulated the wild bulb of fritillary at interval.The experiment proved that, the Bulbus Fritillariae Cirrhosae that the inventive method cultivated is compared with the agents area of wild Bulbus Fritillariae Cirrhosae, appearance character, physics and chemistry differentiate, the kind and the content basically identical of main medicinal ingredient, meet pharmacopeia quality control standard regulation, be outstanding wild Bulbus Fritillariae Cirrhosae substitute, have fabulous market prospects.But also provide a kind of discriminating detection method of new Bulbus Fritillariae Cirrhosae for the public.
Claims (9)
1, the cultivation method of Bulbus Fritillariae Cirrhosae, it comprises the steps:
A, in height above sea level 2800~4700m area Bulbus Fritillariae Cirrhosae and other plant planting district are set, described other plant is one or more in legume, high crude drug, the plateau scrub plant;
B, in growing area Bulbus Fritillariae Cirrhosae and described other plant of a step are cultivated at interval, wherein Bulbus Fritillariae Cirrhosae growth belt railway carriage or compartment is wide is 0.3~2m, the wide 0.3~5m in other plant planting band railway carriage or compartment;
C, fertilising in the growth belt railway carriage or compartment, sowing, blinding get final product;
Gather after d, the Bulbus Fritillariae Cirrhosae medicinal material maturation.
2, the cultivation method of Bulbus Fritillariae Cirrhosae according to claim 1, it is characterized in that the source plant of described Bulbus Fritillariae Cirrhosae is one or more among dark violet bulb of fritillary F.unibracteata Hsiao et K.C.Hsia, leaf roll bulb of fritillary Fritillaria cirrhosa D.Don, Gansu bulb of fritillary F.przewalskii Maxim.ex Balal or the Bulbus Fritillariae cirrhosae F.delavayi Franch.
3, the cultivation method of Bulbus Fritillariae Cirrhosae according to claim 1 is characterized in that, after the d step promptly after Bulbus Fritillariae Cirrhosae is gathered, with Bulbus Fritillariae Cirrhosae and legume crop rotation.
According to the cultivation method of each described Bulbus Fritillariae Cirrhosae of claim 1~3, it is characterized in that 4, the Bulbus Fritillariae Cirrhosae growth belt railway carriage or compartment described in the step a is wide to be 0.5~1.5m, described other plant planting band railway carriage or compartment is wide to be 0.5~2m.
According to the cultivation method of each described Bulbus Fritillariae Cirrhosae of claim 1~3, it is characterized in that 5, the described cultivation spaced apart of step b is at interval banded or at interval block.
6, the Bulbus Fritillariae Cirrhosae medicinal material that obtains of the cultivation method of each described Bulbus Fritillariae Cirrhosae of claim 1~5.
7, Bulbus Fritillariae Cirrhosae medicinal material according to claim 6, it is characterized in that, the HPLC-ELSD finger-print of the dark violet bulb of fritillary of cultivation gained as shown in Figure 5, the HPLC-ELSD finger-print of the leaf roll bulb of fritillary of cultivation gained as shown in Figure 6, the cultivation gained the Gansu bulb of fritillary the HPLC-ELSD finger-print as shown in Figure 7, the cultivation gained Bulbus Fritillariae cirrhosae the HPLC-ELSD finger-print as shown in Figure 8;
The chromatogram testing conditions of above-mentioned four kinds of HPLC-ELSD finger-prints is:
Detector is: EISD ELSD, 40 ℃ of gasification temperatures, nitrogen pressure 1.7~3.5bar;
Flowing phase is: acetonitrile-water (containing 0.1% diethylamine), gradient elution, overall flow rate: 1.0ml/min; Linear gradient: 0~8min wherein, the volume fraction of acetonitrile keeps 30%, 8~35min, and acetonitrile rises to 60% by 30% linearity, keeps 10min, 45~65min, acetonitrile rises to 90% by 60% linearity, keeps 5min;
Chromatographic column is: Kromasil ODS C18 150 * 4.6mm, particle diameter 5 μ m, sample size: 20ul;
Reference substance is: imperialine oxynitrides, different peimine, different peimine oxynitrides, bulb of fritillary suffering, chuanbeinone, imperialine, bulb of fritillary first element and verticinone;
Need testing solution preparation: pulverize the Bulbus Fritillariae Cirrhosae medicinal material, cross 40 mesh sieves, 80 ℃ of dryings 12 hours, precision takes by weighing the about 2.0g of Tendrilled Fritillaria Bulb respectively, put in the 100ml conical flask and alkalize with 3.0ml ammoniacal liquor earlier, add chloroform-methyl alcohol (4: 1) mixed solution 40ml then, 65~70 ℃ of water-bath refluxing extraction 4 hours; Extract put cold after, filter in conical flask, with an amount of mixed solvent washing dregs of a decoction, filter, filtrate merges; Behind the recovered under reduced pressure partial solvent, surplus solution is quantitatively transferred to porcelain steam in the ware, water-bath volatilizes; Use dissolve with methanol, constant volume shakes up in the 2ml measuring bottle, promptly gets need testing solution;
The characteristic peak retention time is: 17~24 minutes; The peak area of this time period accounts for more than 50% of its gross area.
8, a kind of method of measuring the Bulbus Fritillariae Cirrhosae medicinal material of differentiating is characterized in that,
A, preparation reference substance, need testing solution:
Reference substance is: imperialine oxynitrides, different peimine, different peimine oxynitrides, bulb of fritillary suffering, chuanbeinone, imperialine, bulb of fritillary first element and verticinone; Dilute with methyl alcohol respectively;
Need testing solution preparation: pulverize the Bulbus Fritillariae Cirrhosae medicinal material, cross 40 mesh sieves, 80 ℃ of dryings 12 hours, precision takes by weighing the about 2.0g of Tendrilled Fritillaria Bulb respectively, put in the 100ml conical flask and alkalize with 3.0ml ammoniacal liquor earlier, add chloroform-methanol (4: 1) mixed solution 40ml then, 65~70 ℃ of water-bath refluxing extraction 4 hours; Extract put cold after, filter in conical flask, with an amount of mixed solvent washing dregs of a decoction, filter, filtrate merges; Behind the recovered under reduced pressure partial solvent, surplus solution is quantitatively transferred to porcelain steam in the ware, water-bath volatilizes; Use dissolve with methanol, constant volume shakes up in the 2ml measuring bottle, promptly gets need testing solution;
B, chromatographic condition is set
Detector is: EISD ELSD, 40 ℃ of gasification temperatures, nitrogen pressure 1.7~3.5bar;
Flowing phase is: acetonitrile-water (containing 0.1% diethylamine), gradient elution, overall flow rate: 1.0ml/min; Linear gradient: 0~8min wherein, the volume fraction of acetonitrile keeps 30%, 8~35min, and acetonitrile rises to 60% by 30% linearity, keeps 10min, 45~65min, acetonitrile rises to 90% by 60% linearity, keeps 5min;
Chromatographic column is: Kromasil ODS C18 150 * 4.6mm, particle diameter 5 μ m;
C, sample introduction;
D, must detect data.
9, the method for Bulbus Fritillariae Cirrhosae medicinal material is measured in discriminating according to claim 8, it is characterized in that, nitrogen pressure 3.5bar is set among the described step b.
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