CN106665617A - Botanical mice composite sterilant preparation method - Google Patents
Botanical mice composite sterilant preparation method Download PDFInfo
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- CN106665617A CN106665617A CN201611034180.0A CN201611034180A CN106665617A CN 106665617 A CN106665617 A CN 106665617A CN 201611034180 A CN201611034180 A CN 201611034180A CN 106665617 A CN106665617 A CN 106665617A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N45/00—Biocides, pest repellants or attractants, or plant growth regulators, containing compounds having three or more carbocyclic rings condensed among themselves, at least one ring not being a six-membered ring
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/002—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing a foodstuff as carrier or diluent, i.e. baits
- A01N25/004—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing a foodstuff as carrier or diluent, i.e. baits rodenticidal
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
- A01N35/06—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing keto or thioketo groups as part of a ring, e.g. cyclohexanone, quinone; Derivatives thereof, e.g. ketals
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a preparation method of a botanical mice composite sterilant Nongda-1, which belongs to the technical field of medicine compound. By researching reasonable ratio and synergism of the medicinal materials, the medicines prepared by the pharmaceutical composition of the invention have good effect for controlling mice fertility, alkannin extracted from roots of Macrotomia euchroma is combined with quinsetrol, an unique technology is employed, so that the botanical mice composite sterilant can be prepared, and the botanical mice composite sterilant has good effect for controlling mice fertility.
Description
Technical field
The present invention relates to a kind of rodents sterilant and preparation method, more particularly to a kind of rodents sterilant agricultural university -1
Preparation method, belongs to pharmaceutical compounding techniques field.
Background technology
Anti-fertility control is one of control bandicoot new technique for rising in the world, its principle be by reduce the breeding of muroid with
Control muroid population quantity.By model, find in 10,000 muroid colonies, such as continuous 3 generation, kill 70% individuality, its kind
Group can still return to previous level after 26 generations；If but make for 70% individual continuous 3 generation sterile, can then make population through 19 generations
Extinction.Therefore, the purpose of preventing and treating can more be reached than traditional killing mouse using Anti-fertility control.Anti-fertility control also have safe operation and
The features such as being difficult to environment.Currently, the aspect such as China's screening, drug action, pharmacological mechanism in apholate is entered
The substantial amounts of exploration of row, and obtain the effect for attracting people's attention.
In view of the traditional chemical measure of killing mouse can produce super compensation reproduction (breeding compensation
Effect), carry out control in rodent pests using apholate has become the focus of current scholar's research.Anti-fertility control i.e. by certain technology and
Method makes the male or female sexual sterilization of muroid, or hinders embryo nidation development, or even makes germling block growth promoter, to reduce it
Fertility rate, is substantially exactly to control muroid by controlling fertility rate so as to reduce the purpose of population quantity.Therefore, with
Traditional mouse killing method is compared, and using effective insect sterile technique the purpose of the effectively preventing and treating plague of rats is more likely to be reached, and with operation
Safety, the advantages of being difficult to environment, low cost and lasting effect.
Mouse sterile control existing more research at home and abroad, achieves some achievements, is concentrated mainly on to apholate
Screening, laboratory test and the preliminary control application aspect to Part Wild population, in theory mainly with ecology Marxism
Inquire into bandicoot Regularity of population dynamics under Anti-fertility control.And scientific circles' focus of attention concentrate on how to select it is pollution-free, without public affairs
The apholate of evil environment-friendly type, and continuable control can be realized to bandicoot population quantity.Plant source apholate becomes master
Want selecting object.The country is also by Radix Tripterygii Wilfordii, gossypol, Folium Symplocoris Caudatae, oil tea saponin, Oleum Ricini, Radix Trichosanthis, Colchicine, Semen Ricini, cowherb
The plant source apholate such as art alcohol, neem oil is applied to the Anti-fertility control of bandicoot, is respectively provided with certain effect, but undesirable.
The content of the invention
The purpose of the present invention is the extract shikonin by extracting lithospermum euchromum Royle (Arnebia euchroma) root
(Shikonin) mixed with estrogen quinestrol (Quinestrol), so as to preparing a kind of effect is significant and can holding
The composite drug of continuous control small rodent breeding.
A kind of preparation method of rodents sterilant agricultural university -1, it is characterised in that the apholate is made up of following raw materials：
The weight portion of shikonin 100, the weight portion of quinestrol 10, the shikonin of the apholate, quinestrol are mixed in proportion, according to
Amount proportioning adds edible oil, is heated to 30 DEG C -35 DEG C and shakes up 20 minutes, stands 10 minutes, obtains the apholate.
Further, the preparation method includes：The shikonin derives from the root of lithospermum euchromum Royle, and utilizes organic solvent
Petroleum ether extraction.
Further, the consumption of the petroleum ether is 10 times of the raw material of Chinese medicine weight.
Further, the temperature of the petroleum ether is 50 DEG C, and extraction time is 1 hour.
Further, after the petroleum ether is extracted for the first time, extracting solution is separated, and rejoins the petroleum ether with first time equivalent
Second extraction is carried out, repeats the preparation method of said extracted.
Further, 2% sodium hydroxide is added in the petroleum ether extract for obtaining, at 40 DEG C, is converted 1 hour.
Further, after 1 hour, add concentrated hydrochloric acid to precipitation is produced, stand 2 hours.
Further, precipitation distillation is washed to neutrality, dries at 50 DEG C.
Further, using HZ-806 absorption with macroporous adsorbent resin.
Further, in detached dowel, HZ-g06 macroporous adsorbent resins are added and equivalent to the 95% of its volume 0.4-0.5 times
Ethanol, soaks 24 hours.
Further, after 24 hours, pillar is flow through with 95% ethanol of two times of column volumes, with distilled water flushing effluent to pH
Value is neutral, then repeats the preparation method described in previous step with 5% hydrochloric acid, 2% sodium hydroxide solution.
Further, by the product of the preparation method described in claim 9, solution is made with 95% ethanol, pH value is 5.9,
With desorbing is carried out with the flow velocity of 3BV/h with acetone again after the HZ-806 resin absorptioies for processing, stripping liquid is concentrated in vacuo, obtains pure
The shikonin Chinese medicine sterling of change.
Further, acetone is 2: 1 as the ratio of eluent and the amount of sample liquid.
Specifically, in the apholate that the present invention is provided, the mechanism of action of each medical material component is：
Shikonin：It is bitter in the mouth, cold, with removing heat from blood, promoting blood circulation, heat clearing away, removing toxic substances.Warm macule is controlled, jaundice due to damp-heat, purpura is burnt,
The effects such as eczema, erysipelas.With the effect such as antibacterial, antiinflammatory, antiviral, antitumor, antifertility.Also can be used for medicine, cosmetics
Coloring agent.
Quinestrol：Quinestrol (quinestrol) is Delestrogen.
Used as a kind of preferred implementation of the present invention, the content of the apholate each component is：The weight of shikonin 100
Part, the weight portion of quinestrol 10.
In the apholate that the present invention is provided, rational proportion, synergism that inventor passes through each medical material component of research so that
The drug mixture that the apholate provided using the present invention is prepared has good effect in terms of control small rodent fertility.
In order that technical problem solved by the invention, technical scheme and beneficial effect become more apparent, below in conjunction with
Implement illustration, the present invention will be described in further detail.It should be appreciated that it is described herein be embodied as illustration only to
The present invention is explained, is not intended to limit the present invention.
Description of the drawings
Accompanying drawing 1 is variable concentrations experimental group female mice uterus organ coefficient.
Accompanying drawing 2 is the seminal vesicle organ coefficient of variable concentrations experimental group male mice.
Accompanying drawing 3 is the sperm concentration of variable concentrations experimental group male mice.
Accompanying drawing 4 is the testis organ coefficient of variable concentrations experimental group male mice.
Accompanying drawing 5 is different disposal group mice breeding young baby's number for the first time.
Accompanying drawing 6 is that different disposal group mice breeds young baby's number second.
Accompanying drawing 7 is the different disposal group MOUSE REPRODUCTION starting period.
Accompanying drawing 8 compares for Meriones meridianus population the first tire litter size.
Accompanying drawing 9 compares for noon gerbil jird population second fetus litter size.
Accompanying drawing 10 compares for noon gerbil jird population triplet litter size.
Accompanying drawing 11 compares for Meriones meridianus Population breeding tire number.
Accompanying drawing 12 compares for the Meriones meridianus Population breeding starting period.
Accompanying drawing 13 is shikonin extraction, conversion, the flow chart of purification.
In figure, Control is matched group, and F-Treatment is female treatment group, and M-Treatment is male treatment group.
Specific embodiment
Practical application, example 1, the impact that shikonin is bred to white mice
For the antifertility action of clear and definite plant extract shikonin, with Kunming white mice as experimental subject, 4 groups are divided first,
15 pairs per group, wherein 1 group is matched group, remaining 3 groups are filled respectively with 5mg/kg, 20mg/kg and 50mg/kg concentration shikonin
Stomach is administered.Dissect and determine 3 organ coefficients such as female uterus, male seminal vesicle, testis and sperm concentration totally 4 indexs.Its
It is secondary, select a kind of ideal concentration of shikonin to carry out breeding experiment, will newly select mice to be divided into 4 groups, 15 pairs per group, 1 group of matched group, its
Remaining 3 groups set 3 kinds of different disposals, male treatment group, female treatment group and male and female treatment group, bred after treatment group gastric infusion
Experiment.As a result show：The female uterus organ coefficient pole of 50mg/kg shikonin concentration experiment groups be substantially less than matched group and remaining
There is obvious atrophy in 2 concentration experiment groups (F=6.29, P ＜ 0.01), uterus, and male seminal vesicle organ coefficient, sperm concentration are equal
Substantially less than matched group (F=6.49, P ＜ 0.01；F=4.60, P ＜ 0.05), 50mg/kg shikonin concentration has antifertility
Expected Results；MOUSE REPRODUCTION experiment shows that 50mg/kg shikonin concentration significantly reduces female treatment group and male and female treatment group
The breeding starting period of natality, female and male significantly postpones, and will effectively reduce the breeding potential of mice whole idiophase.
Material
Radix Arnebiae (Radix Lithospermi) rope (Shikonin, molecular formula C16H16O5, analytical standard >=97%, molecular weight 288.30)
For lithospermum euchromum Royle extract.Laboratory animal is the Kunming white mice that grows up, body weight 33.1-44.6g.
Method
Laboratory animal process
Adult mouse is cultivated at normal temperatures, gives adequately feed and water, and development is normal.4 groups of laboratory mouse point, per group
15 pairs, wherein 1 group is matched group, remaining 3 groups carry out gavage with 5mg/kg, 20mg/kg and 50mg/kg concentration shikonin respectively,
With edible oil dissolving, every Mus single oral gavage oil soluble shikonin is 0.2ml to shikonin, and 1 week gavage 2 times is spaced 3 days, matched group
With equal amount edible oil gavage.Dissected within 1 week after last 1 gavage, determined female uterus organ coefficient, male seminal vesicle dirty
4 indexs such as device coefficient, sperm concentration and testis organ coefficient.Organ coefficient is calculated with below equation：
The preferable shikonin concentration of selection carries out breeding experiment, new 4 groups of mice of selection, 15 pairs per group, wherein 1 group of matched group,
3 groups of experimental group, if 3 kinds of different disposals：Male treatment group (male administration, female is not administered), female treatment group (female administration,
Male is not administered), male and female treatment group (female, male to be all administered).Experimental group is spaced 3 days with 1 week gavage of ideal concentration shikonin 2 times,
Matched group is mated with equal amount edible oil gavage, last 1 gavage after 1 week, record white mice breeding situation.
Mouse sperm density measurement
Under an optical microscope, counted using red blood cell count(RBC) plate, in 1mm2Counting chamber in, count 25 grids in four
Sperm quantity in week and central totally 5 grids.Computational methods：
Sperm concentration (sperm sum/ml)=sperm count × extension rate × 5mm2×10um×1000ul
Data processing
Using the SAS9.0 one-factor analysiss of variance (One-way ANOVA) to different disposal mice female uterus internal organs system
Number, male seminal vesicle, testis organ coefficient and sperm concentration are compared analysis, and each group raettin new life young baby's number is compared
Analysis.As a result with analysis
Impact of the shikonin to MOUSE REPRODUCTION organ
The female uterus organ coefficient, male of tri- concentration experiment groups of 5mg/kg, 20mg/kg and 50mg/kg and matched group
Relative analyses result such as Fig. 1-4 of seminal vesicle organ coefficient, sperm concentration and testis organ coefficient.As shown in Figure 2, female uterus
Organ coefficient 50mg/kg concentration experiment groups pole is substantially less than matched group and remaining 2 concentration experiment group (F=6.29, P ＜
0.01).From the point of view of anatomical results, the uterus of three experimental mices does not find in shape such as blackening, edema, hyperemia device
Matter changes and pathological changes, after testing uterus organ coefficient, and only 50mg/kg concentration experiments group has pole significance difference with remaining 3 groups
Different, i.e., there is obvious atrophy in 50mg/kg concentration experiments group uterus.From Fig. 2-3,50mg/kg concentration experiment group male mices
Seminal vesicle organ coefficient, sperm concentration are substantially less than matched group (F=6.49, P ＜ 0.01；F=4.60, P ＜ 0.05).By scheming
4 understand, testis organ coefficient and the matched group of variable concentrations experimental group male mice are not significantly different from (F=1.09, P ＞
0.05).The above results show that 50mg/kg shikonin concentration has the Expected Results of antifertility action.
Impact of the shikonin to MOUSE REPRODUCTION
We select 50mg/kg concentration shikonin as ideal concentration apholate, and reselecting mice carries out breeding experiment
Contrast.Mice is divided into 4 groups, wherein 1 matched group, 3 experimental grouies, 15 pairs per group.Experimental group is respectively：Female treatment group is (male
Property be not administered), male treatment group (female is not administered), male and female all treatment groups (female, male to be all administered).New individual to per group of birth
Number is counted, analysis result such as Fig. 5.As shown in Figure 5, from the point of view of the germling quantity of first time birth, at matched group and male
Reason group difference is not notable, and this 2 groups (F=14.78, P ＜s extremely notable with female treatment group and male and female treatment group difference
0.001), i.e., the number of individuals of rear 2 experimental grouies birth is significantly less than matched group and male treatment group.And female treatment group and male and female
The individual amount difference for the treatment of group birth is not notable.Show that 50mg/kg concentration shikonin significantly reduces female treatment group and female
The natality of male treatment group.
Mice breeds young baby number such as Fig. 6 second.From in figure, breeding germling quantity variance is with first time not
Together, matched group is not notable with male treatment group difference, (F=4.23, P extremely notable with female treatment group and male and female treatment group difference
=0.0103), i.e., after the birth of 2 experimental grouies number of individuals significantly less than matched group, and male treatment group and female treatment group and
Male and female treatment group difference is not notable.Therefore, 50mg/kg concentration shikonin continuous and effective reduces female treatment group and male and female are processed
The natality of group.
Mice from male and female mate to the first nest young baby be born natural law for breeding the starting period (Wang Taotao etc., 2015).
The breeding starting period such as Fig. 7 of different disposal group mice.From the point of view of the breeding starting period, the pole of 3 treatment groups noticeably greater than compares
Group (F=11.83, P ＜ 0.001), the i.e. breeding starting period for the treatment of group substantially postpones.Male treatment group and male and female treatment group with
The difference of matched group quite, than matched group averagely postponed 10-12 days by the breeding starting period.The difference of female treatment group and matched group
Maximum, its breeding starting period has averagely postponed 16 days compared with matched group.At second nest germling date of birth, female treatment group and male
Reason group is postponed 10-11 days than matched group, and male and female treatment group is postponed 18-20 days than matched group.So shikonin extends little
The breeding cycle of Mus, the year breeding number of times and breeding potential of the direct effect mice that has been relative reduction.Show that 50mg/Kg concentration is purple
Careless element is defined to the normal reproductive process of female, male mice and substantially interfered with, and to female, male mice antifertility action is respectively provided with.
Practical application, example 2, the impact that apholate of the invention is bred to Meriones meridianus
Meriones meridianus (Meriones meridianus) is divided into into 3 groups, if matched group, female treatment group and male are processed
Group, to the action effect of apholate experimentation has been carried out.As a result show, Meriones meridianus is bred for the first time in annual idiophase,
The Mean litter size of male treatment group significantly reduces (F=12.76, P ＜ 0.01), is 1.42 ± 0.12, and antifertility rate reaches
42.86%；Breed for second, female treatment group and male treatment group Mean litter size significantly reduce (F=11.71, P ＜
0.01), respectively 1.42 ± 0.13 and 0.14 ± 0.04, antifertility rate respectively reaches 57.14% and 85.78%；It is right in annual
According to group breeding number of times 2-3 time, 2 treatment groups breed number of times 0-2 time, substantially less than matched group (F=11.87, P ＜ 0.01)；2
The breeding starting period obvious postpone of individual treatment group, female treatment group postpones 15-22d, and male treatment group postpones 32-157d.Infertility
Agent serves sterile effect to both sexes.
Materials and methods
Material
Shikonin (Shikonin, molecular formula C16H16O5, analytucal standard >=97%, molecular weight 288.30)
For lithospermum euchromum Royle extract, main component is naphthoquinone compound；Quinestrol (Quinestrol, Assay 99.27%) is by north
Capital black bamboo work of nature Science and Technology Ltd. provides (product observes U.S. license passport USP22.)；Experiment Meriones meridianus
(Meriones meridianus) catches from Alashan of Alashan Area, inner Mongolia Autonomous Region alliance south Typical Desert habitat in October, 2014
(E104 ° 10 ' -105 ° 30 ', N37 ° 24 ' -38 ° 25 ').Individually raised in Inner Mongol agriculture with the mouse cage of 40cm × 40cm × 60cm
Desert ecology and rodent controlling study base laboratory (being located at locality) are learned by sparetime university, and well-ventilated, natural lighting, feedstuff is sufficient certainly
By taking food.After raising 5 months safe overwinterings, choose health in April, 2015 and sexual maturity Meriones meridianus male and female are each 45, totally 90
It is only stand-by.
Method
Drug level and examination Mus packet：It is that 100mg/kg shikonin and quinestrol are equal according to the mixing of 10: 1 ratios to choose concentration
It is even, it is configured to shikonin-quinestrol oil solution apholate (15ml/kg) with edible oil as solvent.Experimental mouse be divided into matched group,
Raettin treatment group, male Mus treatment group, every group of male and female are each 15, individually raise.Weight 62.78 ± 10.07 (SE).Raettin process
Group is spaced 3 days raettin gavages twice in one week, and last time gavage one week after and normal male Mus are mated in 1: 1 ratio.Equally, it is male
Mus treatment group is spaced 3 Radix Aconiti Mus gavages twice in one week, and last time gavage one week after and normal raettin are mated in 1: 1 ratio.
One week interior female, male Mus of matched group are spaced 3 days gavages twice with equivalent edible oil, and last time gavage one week after is female, male Mus press 1: 1
Ratio is mated.
Record index：The experimental mouse observation period be April-October, the farrowing day of daily inspection record each group Mus within the observation period
Phase, every tire litter size and breeding tire number.
Data analysiss：One factor analysis of variance is made to per group of breeding tire number, the litter size per tire, breeding starting period difference
(the One-Way ANOVA), data adopt SAS9.0 software analysis, significance level P ＜ 0.05.Antibiosis to Meriones meridianus
Educate rate to calculate using following formula：
Antifertility rate=matched group fertility rate-test group fertility rate.
As a result
First tire litter size compares
The tire of Meriones meridianus first breeds litter size one factor analysis of variance result such as Fig. 8.2 treatment groups are same with matched group
Breeding is occurred in that, the litter size of each group breeding is different, and matched group Mean litter size is 4.28 ± 0.72, and female treatment group is average
Litter size is 3.71 ± 0.28, and male treatment group Mean litter size is below matched group for 1.42 ± 0.12,2 treatment groups.It is female
Property treatment group it is not notable with matched group difference, and the litter size of male treatment group is aobvious with matched group and female treatment group inequality heteropole
Write (F=12.76, P ＜ 0.01), i.e., the litter size pole of male treatment group is substantially less than matched group and female treatment group.Illustrate not
Agent is educated to the infertility effect of male apparently higher than female.Male treatment group antifertility rate is 42.86%.
Second fetus litter size compares
Number of individuals the results of analysis of variance such as Fig. 9 of Meriones meridianus population second fetus breeding.Matched group Mean litter size is
3.57 ± 0.43, female treatment group Mean litter size is 1.42 ± 0.13, and male treatment group Mean litter size is 0.14 ± 0.04.
It can be seen that 2 treatment group Mean litter size poles are substantially less than matched group (F=11.71, P ＜ 0.01), and 2 treatment groups
Difference it is not notable, breeding potential is substantially reduced, female treatment group be 42.86%, male treatment group be 14.28%, matched group
For 100%.Show that apholate significantly reduces number of individuals and the breeding potential that 2 treatment groups are bred for second.Female treatment group resists
Fertility rate is 57.14%, and male treatment group antifertility rate is 85.78%, significantly raised.
Triplet litter size compares
Number of individuals difference analysis result such as Figure 10 of Meriones meridianus population triplet breeding.Matched group is bred,
Treatment group is not bred, it can be seen that although the breeding litter size of matched group and 2 treatment groups is without significant difference (F=2.36, P
＞ 0.05), but the adult that matched group still has 28.57% has carried out third time breeding, and treatment group participates in breeding without individual.
The annual internal reference group Meriones meridianus last time breeding germling date of birth that this research recorded is September 20, although being close to
Its breeding rest period, but treatment group still can not completely exclude the effect of apholate without the individual phenomenon for participating in breeding.
Meriones meridianus Population breeding tire number compares
Meriones meridianus Population breeding tire number difference analysis such as Figure 11 during experiment.Understand matched group breeding 2-3 tires, breeding
3 tires account for 28.57%；Female treatment group breeds 1-2 tires, and that breeds 2 tires accounts for 42.86%；Male treatment group breeds 0-2 tires, numerous
That grows 2 tires accounts for 14.29%.It can be seen that the difference between 2 treatment groups and matched group, and 2 treatment groups reaches and extremely shows
Write (F=11.87, P ＜ 0.01).Show that apholate pole significantly reduces treatment group reproductive frequency, it is relative to extend its breeding week
Phase, the effect especially for male treatment group becomes apparent from.
The Meriones meridianus breeding starting period compares
The breeding starting period refers to from Meriones meridianus the natural law mated pairing starts to the first nest young baby to be born.This research
Observation period is 183d (5-October 5 April), if not farrowing in the statistics phase, breeds day of the starting period for observation period
Number.Meriones meridianus Population breeding starting period such as Figure 12.As can be seen that matched group breeding starting period 26.43 ± 1.13d of average out to,
41.57 ± 2.43d of female treatment group average out to, 119 ± 6.37d of male treatment group average out to.Difference analysis show, at female
Reason group is not notable with matched group difference, and male treatment group is with matched group difference extremely significantly (Fig.5, F=14.46, P ＜ 0.001).
It can be seen that apholate has been postponed the breeding starting period of 2 treatment groups, female treatment group postpones 15-22d, and male treatment group postpones 32-
157d, therefore cause change and passage of 2 treatment groups follow-up trimester of pregnancy within year, and also 2 treatment group triplets are not
Can breed, hence it is evident that reduce the effective breeding potential in Meriones meridianus population year.
The extraction of shikonin, conversion and purification flow process
Principle active component is shikonin and its derivant in lithospermum euchromum Royle root, and shikonin and its derivant are naphthoquinone class
Compound, mostly coloured crystallization, and there are two phenolic hydroxyl groups 5,8- positions in structure, therefore in purple.Shikonin molecular structure of chemistry formula
For
Chinese：Shikonin；
Alias name：Shikonin, Rhizoma Seu Herba Bergeniae pigment, Ou Zicao；
English name：Shikonin；
Chemical name：5,8- dihydroxy -2- [(1R) the amyl- 3- thiazolinyls of -1- hydroxy-4-methyls] naphthalene-Isosorbide-5-Nitrae-diketone；
English language Chemical title：5,8-Dihydroxy-2- [(1R) -1-hydroxy-4-methyl-pent-3-enyl]
Naphthalene-1,4-dione；
Molecular formula：C16H16O5；
Molecular weight：288.30；
There is phenolic hydroxyl group in shikonin molecule, therefore have acidity, alkali liquor can be dissolved in, be acidified with acid and can separate out, this property is normal
For extracting.
Shikonin is naphthalene Quinone Pigments, fat-soluble strong, so being soluble in petroleum ether, chloroform, dissolves in vegetable oil, ethanol, insoluble
Yu Shui.Alkannin derivant is respectively provided with active group, double bond and ester bond such as on 5,8- positions phenolic hydroxyl group, side chain etc., so carrying
All should prevent constituent structure from changing when taking, prepare and storing or destroyed.Naphthoquinone constituents are in 60 DEG C of temperature ＜, acidity-basicity ph ＜
7th, it is less to composition influence under the conditions of ultrasound ＜ 30min；In 60 DEG C of light direct projection, temperature ＞, acidity-basicity ph ＞ 7, ultrasound ＞
It is larger to composition influence under the conditions of 30min.
Radix Arnebiae (Radix Lithospermi) total pigment extraction process
1. Extraction solvent is preferred
In order to choose optimum solvent, Radix Arnebiae (Radix Lithospermi) 50g is weighed respectively, according to the data of table 1, be separately added into petroleum ether (40~80
DEG C), 95%7 alcohol, ethyl acetate, chloroform, acetone, vibration, place certain hour, filter, filtrate constant volume in 1000ml measuring bottles,
Shake up, precision measures 10ml, in being placed on 100ml measuring bottles, coordinative solvent constant volume is used respectively, shake up, with visible ultraviolet spectrometry light
Degree instrument determines trap at 520nm wavelength, by Shikonin (C16H16O5) absorptance (E1cm 1%) calculate purple for 242
Careless hydroxyl naphthoquinone total pigment amount, by formula (1), calculates Radix Arnebiae (Radix Lithospermi) total pigment yield, the results are shown in Table 1.
The different solvents of table 1 extract the experimental data of Radix Arnebiae (Radix Lithospermi) total pigment
From the point of view of different solvent extraction effects, petroleum ether, chloroform are more satisfactory.Naphthoquinone in Zicao class pigment for it is fat-soluble into
Point, due to the readily soluble characteristic of similar person, it is dissolved in the stronger solvent of lipotropy.Chloroform has certain toxicity, should use up in the industrial production
Amount avoids using the solvent.Petroleum ether (40~80 DEG C) and water, the equal Fails To Respond of alkali liquor, it is preferable with alkali dose effect, and stone
Oily ether (40~80 DEG C) is also preferable to the extractability of Radix Arnebiae (Radix Lithospermi) total pigment, therefore selects (40~80 DEG C) of petroleum ether to be preferably extraction
Solvent.
2. dynamic extraction method extracts Radix Arnebiae (Radix Lithospermi) total pigment
Using dynamic extraction method, Radix Arnebiae (Radix Lithospermi) total pigment is converted into into shikonin, experimental design synthesis Radix Arnebiae (Radix Lithospermi) total pigment leach with
Time, the investigation result of temperature relation and produce reality, select extraction time, the amount of each solubilizer and extracting times to investigate
Factor, each factor sets three levels again, according to factor level table, selects orthogonal table L9(34) tested, and with each reality
The Radix Arnebiae (Radix Lithospermi) total pigment yield of extraction is tested as inspection target, colorimetric method for determining content, statistical analysiss, determines that optimum extraction extracts work
Skill.
(1) orthogonal array selects L9(34) factor level table, it is shown in Table 2.
The factor level table of table 2
(2) test method and test data
Radix Arnebiae (Radix Lithospermi) 50g is accurately weighed, is tested according to the experimental condition of table 3 respectively, united extraction liquid is filtered, and is settled to
1000ml, shakes up, and precision measures 10ml, in placing 100ml volumetric flasks, uses petroleum ether constant volume, shakes up, with visible ultraviolet spectrometry light
Degree instrument determines its trap at 516nm.By Shikonin (C16H16O5) absorptance (E1cm 1%) it is 242, calculate Radix Arnebiae (Radix Lithospermi)
Hydroxyl naphthoquinone total pigment amount, and total pigment extraction ratio is calculated by formula (1), the results are shown in Table 3.
The orthogonal test arrangement of table 3 and result
(3) the results of analysis of variance
The analysis of variance table of table 4
According to the results of analysis of variance, it is not significantly different between each level of A factors, can select any water in A factors
It is flat, according to production cycle more short better requirement, from extracting twice, extract 1 hour every time.And between each level of B, C factor
There is significant difference, due to B1＜ B2＜ B3, C1＜ C2＜ C3, therefore optimised process should be A1B3C3.It is straight from orthogonal
Observation is connect it can be seen that extracting the higher experiment of Radix Arnebiae (Radix Lithospermi) total pigment content also has A2B2C3, i.e., No. 5 experiment, now by both the above
Extraction process repeatedly test three times respectively, it is as a result as follows：
The preferred process contrast table of table 5
Result above shows that two kinds of preferred process are extracted Radix Arnebiae (Radix Lithospermi) total pigment yield and are more or less the same, but from reduction production cost,
Shorten the factors such as production cycle to consider, from optimised process A1B3C3.Extract secondary, each extraction time is 1 hour, every time
Plus petroleum ether amount is 10 times, Extracting temperature is 50 DEG C.
Extract Radix Arnebiae (Radix Lithospermi) total pigment optimised process：Petroleum ether (40~80 DEG C) dynamic extraction 2 times, every time plus petroleum ether (40~
80 DEG C) to measure as 10 times of crude drug, every time 1 hour, Extracting temperature is 50 DEG C, obtains Radix Arnebiae (Radix Lithospermi) total pigment petroleum ether liquid.By formula
(1), Radix Arnebiae (Radix Lithospermi) total pigment yield is calculated.Extracted using this technique, Radix Arnebiae (Radix Lithospermi) total pigment extraction rate is 2.16%.
The technique that Radix Arnebiae (Radix Lithospermi) total pigment is converted into shikonin
Radix Arnebiae (Radix Lithospermi) total pigment is converted into the process of shikonin：
(1) under the effect of shikonin total pigment alkali liquor, there are hydrolysis ,-R is converted into-H in most of pigment molecular, i.e.,
It is converted into shikonin sodium salt.
(2) conversional solution acid adding again, shikonin sodium salt is changed into shikonin.
1. concentration of sodium hydroxide solution is preferred
The Radix Arnebiae (Radix Lithospermi) total pigment petroleum ether liquid 850ml of the preferred process extraction for preferably going out accurately is measured (equivalent to crude drug amount
50g) totally 5 parts, Jia 0 respectively, 1%, 2%, 3%, 4% sodium hydroxide solution 500ml extractions, place 24 hours, point take alkaline extraction
Liquid, with concentrated hydrochloric acid generation precipitation is acidified to, and concentrated hydrochloric acid can be excessive, stands 2 hours, Bush's funnel sucking filtration, precipitation distillation washing
To neutral, dry at 50 DEG C, weigh, and with alkannin content in high effective liquid chromatography for measuring Radix Arnebiae (Radix Lithospermi) dry extract, calculate
Go out shikonin amount, and shikonin yield is calculated by formula (2), the results are shown in Table 6.
Impact of the concentration of sodium hydroxide solution of table 6 to shikonin proposition amount
As shown in Table 6,2% sodium hydroxide solution extraction effect is best, therefore selectes 2% sodium hydroxide solution most preferably to carry
Take solvent.
2. sodium hydroxide solution consumption is preferred
Totally 5 parts of the Radix Arnebiae (Radix Lithospermi) total pigment petroleum ether liquid 850ml extracted using the preferred process for preferably going out accurately is measured, is added respectively
2% sodium hydroxide solution 400,500,600,700,800ml is extracted, and is placed 24 hours, is divided and is taken alkali extracting solution, is acidified with concentrated hydrochloric acid
To precipitation is produced, concentrated hydrochloric acid can be excessive, stands 2 hours, and buchner funnel sucking filtration, precipitation distillation is washed to neutrality, at 50 DEG C
Drying, weighs, and with alkannin content in high effective liquid chromatography for measuring Radix Arnebiae extract, calculates shikonin by formula 2 and obtain
Rate, the results are shown in Table 7.
Impact of the sodium hydroxide solution consumption of table 7 to shikonin proposition amount
As shown in Table 7, shikonin proposition amount is improved with the increase of sodium hydroxide solution consumption, and 700ml and 800ml
It is more or less the same.
3. impact of the temperature to shikonin yield
Totally 4 parts of the petroleum ether liquid 850ml extracted using the preferred process for preferably going out accurately is measured, 2% hydroxide is added respectively
Sodium solution 700ml is extracted, and respectively in 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C of couveuses, stands 2 hours, buchner funnel sucking filtration, precipitation
Neutrality is washed to distillation, is dried at 50 DEG C, weighed, and contained with shikonin in high effective liquid chromatography for measuring Radix Arnebiae extract
Amount, calculates shikonin amount, and calculates shikonin yield by formula 2, the results are shown in Table 8.
Impact of the Extracting temperature of table 8 to shikonin yield
As shown in Table 8, shikonin extraction rate is with the different and different of Extracting temperature.Start with the rising of temperature,
Shikonin proposition amount also increases, the proposition amount highest at 40 DEG C.And continue improve temperature to 50 DEG C, shikonin proposition amount on the contrary under
Drop.Because it is a hydrolysis that Radix Arnebiae (Radix Lithospermi) total pigment is converted into shikonin, increase temperature can make its reaction accelerate, but with when
Between extend, Radix Arnebiae (Radix Lithospermi) total pigment is hydrolyzed to after shikonin, is further added by hydrolysis time, and shikonin may proceed to decompose, and be converted into other things
Matter.Therefore 40 DEG C of hydrolysis of design alkalization hydrolysis technique of 1,2,3,4,5,6 hours, is shown in Table 9.As can be seen from Table 9 40 DEG C when, water
Solution 1 hour, shikonin yield is higher.
The contrast experiment of the different alkalization times of 9 40 DEG C of table
4. the preferred Radix Arnebiae (Radix Lithospermi) total pigment of orthogonal experiment is converted into shikonin process conditions
(1) experimental design：The investigation result and produce reality of conversion process influence factor, choosing are extracted with reference to above-mentioned shikonin
Concentration of sodium hydroxide solution, consumption, alkalization time are selected as investigation factor, three levels are set respectively, according to factor level table,
From L9(34) factor level table, tested, it is shown in Table 10.
The factor level table of table 10
(2) test method and test data：Accurately measure petroleum ether liquid 850ml to enter by the experimental condition of the design of table 11 respectively
Row test, plus the alkali liquor of variable concentrations, after the different times are placed in alkalization, alkali extracting solution concentrated hydrochloric acid is acidified to generation precipitation,
Concentrated hydrochloric acid can be excessive, stands 2 hours, and buchner funnel sucking filtration, precipitation distillation is washed to neutrality, dries at 50 DEG C, weighs, and
With alkannin content in high effective liquid chromatography for measuring Radix Arnebiae (Radix Lithospermi) dry extract, by formula 2 shikonin yield is calculated.The results are shown in Table
11。
The orthogonal test arrangement of table 11 and result
(3) the results of analysis of variance
The analysis of variance table of table 12
As can be seen here factor A, C is extracted conversion ratio on shikonin and is affected extremely significantly, and factor B extracts conversion ratio shadow to shikonin
Ring notable, each factor is followed successively by C ＞ A ＞ B, therefore the optimal ginseng of tri- factors of A, B, C to the influence degree for extracting changing effect
Number is A2B3C2, i.e., shikonin conversion optimised process be：At 40 DEG C, concentration of sodium hydroxide solution is 2%, and consumption is 700ml
(equivalent to 14 times of medical material amount), transformation time is 1 hour.
By this optimised process arrangement test, 3 parts of operation repetitive, shikonin proposes that conversion ratio is respectively 1.760%,
1.776%, 1.765%.Average out to 1.767%；Shikonin purity is respectively 75.12%, 76.05%, 75.84%, average out to
75.67%.
Shikonin conversion process result of study shows that optimised process is：When 40 DEG C, concentration of sodium hydroxide solution is 2%, is used
Measure as 700ml (equivalent to 14 times of medical material amount), hydrolyze 1 hour, now shikonin yield is 1.767%；Radix Arnebiae (Radix Lithospermi) dry extract
Middle shikonin purity is 75.67%, is now also not up to object of experiment and requires.
Shikonin purifying process
1. resin pretreatment
In clean detached dowel, be put into the macroporous adsorbent resin for going the removal of impurity, constant volume, add equivalent to
The ethanol that 0.4~0.5 times of resin volume, soaks 24 hours.Then pillar is flow through with the ethanol of two times of column volumes, uses water rinse flow
Go out liquid pH to neutrality, then the hydrochloric acid with 5%, 2% sodium hydroxide solution repeat above step.
2. sample solution is prepared and pretreatment：
Radix Arnebiae (Radix Lithospermi) 50g is weighed, shikonin is prepared into and is slightly carried according to the extraction of optimal dynamic extraction process, the conversion of optimal alkalization process
Thing.Then 95% ethanol is used, the Radix Arnebiae (Radix Lithospermi) crude extract solution of variable concentrations is made, it is standby.
3. dynamic adsorption is processed：
In order to more reasonably design productionization process route, first its dynamic column separation condition is examined using duckpin
Examine, for the foundation that production provides science.
When dynamic adsorption is carried out, the impact of 6 factors is investigated, respectively different eluant, separation column internal diameter, sample solution
Concentration, the pH value of medicinal liquid, loading flow velocity, temperature etc..
(1) impact of the different eluant to shikonin purity and yield
Resin used is 30g in experiment, the concentration of medicinal liquid used be the 0.801mg/ml of crude extract containing shikonin (equivalent to
50g crude drugs/ml medicinal liquids), pH value is 5.0, and medicine liquid volume is 100ml, and post footpath is 22.5mm, different with 200ml after resin absorption
Solvent carries out desorbing with the flow velocity of 1.5BV/h, and stripping liquid is concentrated in vacuo, obtains the shikonin of purification, and shikonin precision is weighed about
0.5g, in putting 100ml measuring bottles, plus ethanol is to scale, constantly shakes in 4 hours, and filtration, precision measures subsequent filtrate 5ml, puts 25ml
In measuring bottle, plus ethanol is to scale, shakes up.According to spectrophotography, trap is determined at the wavelength of 516nm, by Shikonin
(C16H16O6) absorptance (E1cm 1%) calculate for 242, alkannin content is determined, shikonin purity is calculated by formula 3, and press
Formula 4 calculates shikonin yield, the results are shown in Table 13.As shown in Table 13, preferably, ethanol elution effect is poor for acetone desorption effect.
The result of the different solvents desorbing of table 13
(2) internal diameter for selecting pillar is investigated
Medicinal liquid pH value is 5.0, and loading flow velocity is 1.5BV/h, and post footpath is respectively tetra- kinds of 15mm, 20mm, 22.5mm, 25mm
The pillar of different size, the data obtained is as follows, and the data from table can be seen that the shikonin yield of the internal diameter for 20mm of pillar
With purity highest, therefore select internal diameter for 20mm pillar.
The data of shikonin purity in the different post footpath Radix Arnebiae (Radix Lithospermi) rope yields of table 14 and product
(3) concentration of medicinal liquid is investigated
For 20.0mm, amount of resin is 30g to the pillar internal diameter for adopting, and the flow velocity of sample solution is 1.5BV/h, the concentration of sample solution
Respectively 0.801mg/ml, 1.63mg/ml, 3.25mg/ml, medicine liquid volume is respectively 150ml, 75ml, 36ml, that is, keeps identical
Crude drug amount, it is as shown in the table for the data obtained.Data in table, with liquor strength as 1.63mg/ml shikonin yield and product
Shikonin purity highest in product.Therefore liquor strength is selected to be optimal sample concentration for the medicinal liquid of 1.63mg/ml.
Impact of the sample solution concentration of table 15 to shikonin purity in shikonin yield and product
(4) pH value of sample solution is investigated
In general, the pH value of medicinal liquid affects larger to the adsorption rate of resin, therefore investigates not for impact of the pH value to absorption
With impact of the medicinal liquid of pH to Adsorbent rate.As seen from Table 16, with solution ph as 5.9, in shikonin yield and product
Shikonin purity is highest.
Adsorpting data after the medicinal liquid upper prop of the different pH value of table 16
(5) loading flow velocity is investigated
Post footpath：20mm, the dense of sample solution is respectively 1.63mg/ml, and sample solution volume 75ml, pH value is 5.9, resin demand
30g, uses acetone eluting, flow velocity to select 1.5BV/h respectively；3.0BV/h；5.0BV/h；7.5BV/h, the data obtained such as following table.Reason
By upper, the flow velocity of sample solution is slower, and the absorption of shikonin is more abundant, but can be seen that from table 17, the desorbing effect of flow velocity 3.0BV/h
Fruit is suitable with flow velocity 1.5BV/h, and the effect than 5.0BV/h, 7.5BV/h is good.And flow velocity can shorten the production cycle soon,
So it is optimum flow rate to select flow velocity 3.0BV/h.
During the different loading flow velocity of table 17 in the yield and product of shikonin shikonin purity data
(6) impact of temperature is investigated
In general, absorption is divided into physical absorption and chemisorbed.Physical absorption is typically carried out at low temperature, and chemistry is inhaled
It is attached to need certain heat, it is physical absorption or chemisorbed to shikonin to determine punching resin, therefore determine not
Adsorption effect of the synthermal lower resin to shikonin, and washing and alcohol are washed and carried out at normal temperatures.Data from table can
Go out, the yield highest of shikonin adsorbed at room temperature, the yield of shikonin has declined at 35 DEG C, has risen at 51 DEG C,
Decline at 75 DEG C, this absorption of explanation resin to shikonin is both physical absorption, is again chemisorbed, is entirely being adsorbed
During be physical absorption and chemisorbed competition result.
The data of resin absorption shikonin under the different temperatures of table 18
Above test result indicate that, the optimum condition of dynamic adsorption is：The preferable eluting solvent of shikonin be acetone, sample solution
Concentration is 1.63mg/ml, and the optimal pH value of sample solution is 5.9, and post footpath selects to be 20mm that loading flow velocity is 3BV/h, and temperature adopts room
Temperature is lower.
Adsorb at optimum conditions, gained shikonin yield is 1.34%, and shikonin purity reaches 96.6% in product, reaches
Target is arrived.
Conclusion
Efficiently purification is that Radix Arnebiae (Radix Lithospermi) active component plays the key point of curative effect to shikonin, and extract, the pass of purification shikonin
Key technology problem is how to remove invalid impurity, improves shikonin purity in the yield and product of shikonin, shortens the production cycle.
Author utilizes the physicochemical characteristicss of Naphthoquinone in Zicao constituents, and dynamic extraction, solution extraction process and advanced resin are inhaled
Attached technique combines, and the technological parameter being separately optimized in extraction, conversion, three steps of resin absorption purification so that Radix Arnebiae (Radix Lithospermi)
The ultimate yield of element reaches 1.34%, and shikonin purity reaches 96.6% in product.And substantially reduce the extraction purification time.Knot
By as follows：
Radix Arnebiae (Radix Lithospermi) total pigment extraction process
1st, Extraction solvent is selected：By to petroleum ether (40~80 DEG C), 95% ethanol, acetone, ethyl acetate, five kinds of chloroform
Comparison of the different solvents to the extractability of shikonin, as a result shows that the extraction effect of petroleum ether (40~80 DEG C) is molten better than other
Agent.
2nd, the selection of extracting mode：(40~80 DEG C) of petroleum ether extracts Radix Arnebiae (Radix Lithospermi) total pigment using different extracting modes, such as soaks
Carry, dynamic extraction, compared by experiment, finally determine convenient extracting method for dynamic extraction, i.e., Radix Arnebiae (Radix Lithospermi) adds petroleum ether
(40~80 DEG C) dynamic extraction twice, make a living 10 times of medical material amount by each solubilization dosage, and Extracting temperature is 50 DEG C, when extracting every time
Between be 1 hour, under the conditions of this, the yield of Radix Arnebiae (Radix Lithospermi) total pigment is up to 2.16%.
Conversion process
This step is that Radix Arnebiae (Radix Lithospermi) total pigment is converted into into shikonin, and Orthogonal experiment results show, the optimised process of conversion is：It is purple
Careless total pigment petroleum ether liquid with 2% sodium hydroxide solution, consumption is 14 times of crude drug amount, and alkalization time is 1 hour, alkalization temperature
Spend for 40 DEG C, divide and take alkali extracting solution, with concentrated hydrochloric acid generation precipitation is acidified to, concentrated hydrochloric acid can be excessive, stands 2 hours, buchner funnel
Sucking filtration, precipitation distillation is washed to neutrality, dries at 50 DEG C, obtains shikonin crude extract.The receipts of shikonin under the conditions of this
, up to 1.767%, the purity of shikonin is up to 75.67% in product for rate.
Purifying process
1st, resin is screened：The macroporous resin of four kinds of opposed polarities is screened by static adsorption, as a result show this four
In planting resin, HZ-806 Adsorbent rates are most fast, and desorption efficiency is also high, therefore are set to the adsorbent resin of this research project.
2nd, dynamic adsorption：The flow velocity of sample solution has been investigated respectively, separated column internal diameter, sample solution concentration, temperature and pH value etc.
Impact of the factor to dynamic adsorption.As a result show：The flow velocity of sample solution be 3BV/h, separations column internal diameter be 20mm, sample solution it is dense
It is room temperature to spend for 1.63mg/ml, temperature, and adsorption effect is best when pH value is 5.9.Optimal petroleum ether (50 DEG C) dynamic extraction-
Under the conditions of alkali liquor conversion-macroporous resin adsorption, Jing one way extract, conversion, after purification shikonin ultimate yield be 1.34%, produce
The purity of shikonin is 96.6% in product, has exceeded the reported values of document.
Above example is only the preferred embodiment of the present invention, it is noted that to those skilled in the art, not
On the premise of departing from the principle of the invention, done some improvement also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of rodents sterilant agricultural university -1 is standby, it is characterised in that the apholate is made up of following raw materials：
The weight portion of shikonin 100, the weight portion of quinestrol 10.
2. a kind of preparation method of rodents sterilant agricultural university -1 according to claim 1, it is characterised in that the infertility
The shikonin of agent, quinestrol are mixed in proportion, and according to consumption proportion edible oil is added, and are heated to 30 DEG C -35 DEG C and are shaken up 20
Minute, 10 minutes are stood, obtain the apholate.
3. a kind of preparation method of rodents sterilant agricultural university -1 according to claim 1, it is characterised in that the preparation
Method includes：The shikonin derives from the root of lithospermum euchromum Royle, and using organic solvent Petroleum ether extraction, the petroleum ether
Consumption is 10 times of the raw material of Chinese medicine weight, and the temperature of the petroleum ether is 50 DEG C, and extraction time is 1 hour.
4. preparation method according to claim 3, it is characterised in that after the petroleum ether is extracted for the first time, extracting solution point
Go out, rejoin carries out second extraction with the petroleum ether of first time equivalent, repeat the extracting method described in claim 3.
5. preparation method according to claim 4, it is characterised in that add in the petroleum ether extract for obtaining
2% sodium hydroxide, at 40 DEG C, converts 1 hour.
6. preparation method according to claim 5, it is characterised in that after 1 hour, adds concentrated hydrochloric acid to precipitation is produced, quiet
Put 2 hours.
7. preparation method according to claim 6, it is characterised in that precipitation distillation is washed to neutrality, dries at 50 DEG C
It is dry.
8. the preparation method according to any one of claim 3-8, it is characterised in that inhaled using HZ-806 macroporous adsorbent resins
It is attached, in detached dowel, add HZ-806 macroporous adsorbent resins and 95% ethanol equivalent to its volume 0.4-0.5 times, immersion 24
Hour.
9. preparation method according to claim 11, it is characterised in that after 24 hours, with 95% ethanol of two times of column volumes
Pillar is flow through, it is neutral to pH value with distilled water flushing effluent, then repeat claim with 5% hydrochloric acid, 2% sodium hydroxide solution
Preparation method described in 8.
10. preparation method according to claim 9, it is characterised in that by the product of the preparation method described in claim 9
Thing, solution is made with 95% ethanol, and pH value is 5.9, with after the HZ-806 resin absorptioies for processing again with acetone with the stream of 3BV/h
Speed carries out desorbing, and stripping liquid is concentrated in vacuo, obtains the Chinese medicine sterling of the claim 3 of purification, the acetone as eluent with
The ratio of the amount of sample liquid is 2: 1.
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