CN107260810A

CN107260810A - The Preparation method and use of high-purity clove leaf total iridoid glycoside extract and its preparation

Info

Publication number: CN107260810A
Application number: CN201610214670.2A
Authority: CN
Inventors: 刘欣; 孙晓君; 马慧媛; 张凤鸣; 庞海军; 庞素艳
Original assignee: Harbin University of Science and Technology
Current assignee: Harbin University of Science and Technology
Priority date: 2016-04-08
Filing date: 2016-04-08
Publication date: 2017-10-20

Abstract

The present invention provides the Preparation method and use of a kind of high-purity clove leaf total iridoid glycoside extract and preparation.Extracted or heating and refluxing extraction using dynamic ultrasound adverse current, dynamic axial compression process-scale chromatography, or prepared by high speed adverse current chromatogram separation, freeze or be spray-dried, produce high-purity clove leaf total iridoid glycoside extract.The inventive method completes to extract compared with the short time under low temperature, and separation cycle is short, efficiency high, saves and extracts volume consumption, reduces production cost, reduces energy consumption and environmental pollution.Detected through HPLC, clove leaf total iridoid glycoside purity is up to more than 75%.Thin membrane coated tablet, capsule, dispersible tablet, pill, granule, oral sustained-release preparation is made by adding various pharmaceutic adjuvants, and the various preparations such as spray, aerosol, injection, for treating the infectious diseases such as the infection of the upper respiratory tract, acute and chronic tonsillitis, acute/chronic bronchitis, acute gastroenteritis, acute icterohepatitis, acute mastitis and bacillary dysentery.

Description

The preparation method of high-purity clove leaf total iridoid glycoside extract and its preparation and Purposes

Technical field

The invention belongs to plant and Chinese medical extract field, more particularly to high-purity clove leaf total iridoid glycoside extract and The Preparation method and use of its preparation.

Background technology

Bacterial infection inflammation is a kind of frequently-occurring, generality disease, and clinic mainly takes the method for anti-inflammation to enter Row treatment.As treatment bacterial infection disease key agents, antibiotic is that most widely used, with fastest developing speed, kind is most in the world Many class medicines.Investigation result is shown, in European and American developed countries, and the usage amount of antibiotic substantially accounts for the 10% of all medicines Left and right, and China's Grade A hospital accounts for 30%, basic hospital may be up to 50%, it can be seen that frequency of use of the antibiotic in China It is very high【http://drug.sosyao.com】.However, with the extensive use of antibiotic, bacterial resistance sex chromosome mosaicism also day Benefit is prominent.Especially doctor's aphalangia requisition medicine, administration time length, selection antibiotics are unreasonable etc., cause bacterial resistance, The phenomenons such as Multidrug resistant bacteria, superbacteria, cause the highest attention of medical field.Chinese medicine antibacterial have be not likely to produce drug resistance, Adverse reaction is low, the advantages of antibiotic resistance can be reversed is combined with antibiotic, from antibacterium, fungi, mycoplasma, Chlamydia etc. Aspect seeks Chinese medicine natural antibiotics turns into the new way for solving bacterial resistance.Thus, exploitation high-efficiency antimicrobial, toxic side effect are low Chinese herbal medicine resource, and then be developed into anti-inflammation new drug, it is significant.

Clove leaf Folium syringae are from the purple fourth of Oleaceae (Oleaceaceae) Genus Syringa (Syringa) plant Fragrant (Syringa oblate Lindl), Korea cloves (Syringa diatata Nakai), Syring vulgaris（Syringa Vulgaris L.）Dried leaf, bitter is cold in nature, with clearing heat and detoxicating, the effect of anti-inflammtory anti-dysentery, has recorded in Chinese Pharmacopoeia One annex of version in 2005【Mono- Beijing of Chinese Pharmacopoeia Commission Pharmacopoeias of People's Republic of China：Chemical Industry Press, 2005】.Clove leaf is wide in the Northeast's distributed pole, it is easy to cultivate, aboundresources, is the chief species of courtyard greening.It is modern Pharmacological research shows that clove leaf has broad-spectrum antiseptic anti-inflammatory, and antiviral, hepatic cholagogic strengthens the humoral immunity of body, cooled, Decompression, the effect such as kobadrin, to staphylococcus aureus, staphylococcus albus, proteus, Pseudomonas aeruginosa, shigella dysenteriae, The bacterium of 22 kinds such as typhoid bacillus, first Bacillus paratyphosus B, bacillus typhi murium, food poisoning salmonella has stronger Antibacterial action, by clinical observation, to 314 bacillary dysentery cure rates be more than 90.5%, to 30 Western medicine antibiotic therapies Ineffective Cases effective percentage is 79.0%【Qi Chen Chinese patent drug recipe pharmacology and clinic Beijing：People's Health Publisher, 1998.； Wangdan is red, Liu Shengquan, Chen Yingjie, the research Acta Pharmaceutica Sinicas .1982 of the Active Constituents of Syringa Oblata Lindl such as Wu Lijun, 17 (12): 951-954；Lu Dan, the chemical composition and Advance on Pharmacological Activities Changchun Traditional Chinese Medical College journal of Li Pingya genus syringas, 2001, 17 (4): 58-59】.Using the anti-inflammation medicine piece of clove leaf single preparations of ephedrine, 400 clinics for the treatment of children's acute infection The Curative effect of case, is controlled for the infection of the upper respiratory tract, tonsillitis, pneumonia caused by most of viruses using anti-inflammation medicine piece After treatment, total effective rate is up to 100%【Bai Qing, Liang Wei anti-inflammation medicines piece treatment children's acute infect 400 observation of curative effect and clinical right Magazine, 2005,12 (1) are write according to Chinese medicals: 32-33】.Confirmation separately is had been reported that, Folium Caryophylli extract is to herpe simplex Virus, adenovirus (Afv3, Afv7), parainfluenza virus, Respiratory Syncytial Virus(RSV) (RSV), Coxsackie virus (CoxB1, CoxB2, CoxB3, CoxB4, CoxB5, CoxB6) and hepatitis B institute cytopathogenic effect have obvious inhibiting effect, and and antibiosis Element compares with antiviral drugs, and there were significant differences for curative effect, and then it is definitely reliable to demonstrate the antibacterial of clove leaf, antivirus action 【The practical ophthalmology of immunologic assay before and after Yang Weiwei, Xing Meiyu leaf of Syringa oblata Lindl preparation are treated to herpes simplex keratitis is miscellaneous Will, 1990,8 (4): 225-226；Li Bo, Zhu Jun visit the species and pharmacological research of contained effective ingredient in clove leafs Be in progress Heilungkiang medicine, 2009,22 (4): 510-511；Profound scholar is strange, Niu Junqi, the syringa extrcts such as Wang Feng and The comparative studies cells and molecular immunology magazine of interferon, hepatitis medicine resistance of hepatitis B, 2003,19 (4): 385- 386】。

Page 100 and the 20th page 176 of Drug Standard of Ministry of Public Health of the Peoples Republic of China the 9th copy, is recorded respectively The two kinds of formulations of anti-inflammation medicine tablet and quick-acting anti-inflammation capsule agent produced by raw material of clove leaf【Chinese in Pharmacopoeia Commission of the Ministry of Public Health The 9th, Beijing of people republic the Sanitation Ministry medicine standard：People's Health Publisher, 1990；In Pharmacopoeia Commission of the Ministry of Public Health Magnificent the 20th, Beijing of people's republic's the Sanitation Ministry medicine standard：People's Health Publisher, 1990】, clinically for controlling Treat the infection of the upper respiratory tract, acute/chronic bronchitis, bacillary dysentery, acute gastroenteritis, acute icterohepatitis, acute mastitis Deng infectious diseases, preferable curative effect is received【Lv Zhu You, edits northeast medicinal plant will Heilungkiang Science Press, 1989, 4 (1): 883；Xin Liu, Jianming Wang. Anti-inflammatory effects of iridoid glycosides purified from Folium syringae leaves on TNBS-induced colitis in rats. Journal of Ethnopharmacology 2011, 133 (2): 780-787；Xin Liu, Jianming Wang. Iridoid glycosides of Folium syringae leaves modulates NF-κB signal pathway and intestinal epithelial cells apoptosis in experimental colitis. Plos one. 2011, 6 (9): e24740, 1-12】, novel form enteric-coated micro-pill and colon location preparation are to enteric infection Disease has good efficacy【A kind of Preparation method and use of clove leaf total iridoid glycoside of Liu Xin, the new of girth, China, ZL200910100317.1, on December 21st, 2011；Liu Xin doctor research paper, the preparation of anti-inflammation medicine site specific DDS for colon coating tablet With Study on mechanism Harbin：Heilongjiang University of Chinese Medicine .2008】.Chemical research proves, three kinds of cloves leaf effective-parts Chemical composition it is essentially identical, the maximum iridoid glycosides of predominantly iridoid glycosides and liposoluble ingredient, wherein content Composition is clove leaf anti-inflammation, antiviral main active component【Xin Liu, Jianming Wang, Changxin Zhou, Lishe Gan. Preparative separation and enrichment of syringopicroside from Folium syringae leaves with macroporous resins. Journal of Biomedicine and Biotechnology. 2010, Article ID 572570, doi:10.1155/2010/572570】.Clove leaf total Iridoid glycoside treats bacterial infectious disease, and determined curative effect proves effective fast, the course for the treatment of is relatively short, without any side effects, is A kind of preferable broad-spectrum antiseptic antiphlogistic, has high practical value and wide market prospects in terms of new drug development.

The content of the invention

Extracted or heating and refluxing extraction, inhaled with reference to macropore using dynamic ultrasound adverse current it is an object of the invention to provide one kind Attached resin column chromatography, dynamic axial compression process-scale chromatography or high speed adverse current chromatogram separation prepare high-purity clove leaf total iridoid The method of glucoside extract, the technical process is simple, quality controllable.The high-purity clove leaf total cyclenes ether prepared in this way Terpene glycosides, can be made medically acceptable various formulations, for the clinical treatment infection of the upper respiratory tract, urgency by adding pharmaceutic adjuvant The infectious diseases such as chronic bronchitis, bacillary dysentery, acute gastroenteritis, acute icterohepatitis, acute mastitis.

The inventive method is realized by following steps：Clove leaf is crushed to 10~20 mesh, plus 6~20 times of volume of water or body The alcoholic solution that product percent concentration is 10%~80%, or acetone soln, or aqueous isopropanol, dynamic ultrasound adverse current are extracted or heated Refluxing extraction 2~3 times, 0.5~2h, filtering, merge extract solution, Extraction solvent are recovered under reduced pressure, being suitably concentrated into concentration is every time 0.4~2.0 g crude drugs/ml, centrifugation or filtering after, plus watery hydrochloric acid regulation pH to 2.0~6.0 upper prop solution, upper prop solution with 2BV/h~4BV/h flow velocity is 1 according to the ratio of solid content and dried resin by macroporous adsorption resin chromatography post, upper prop solution： 5~10, first with 4~6BV distilled water washing resin post bed, elution flow rate is 2BV/h~4BV/h, discards water elution；Use again Concentration of volume percent is 10%~80% alcohol eluent, and elution flow rate is 2BV/h~4BV/h, merges eluent decompression Alcohol is reclaimed, it is 1.05~1.30 to be concentrated under reduced pressure into relative density, adds the column chromatography silica gel filler of 200~300 mesh, plus methanol is mixed Dried after even, cross the reverse industrial preparation chromatographic column of dynamic axial compression, methylene chloride-methanol, chloroform-methanol or methanol-water (20：1~10：1) eluted for eluent gradient, the principal piece eluent of the constituents containing clove leaf total iridoid glycoside collected respectively, Merge, be concentrated under reduced pressure, filter, solvent is recovered under reduced pressure, is freeze-dried, is spray-dried or is dried under reduced pressure, high purity butylene spiceleaf is produced Total iridoid glycoside（HPLC detects purity >=80%）.

Or take the macroreticular resin crude extract of clove leaf, add chloroform-n-butanol-methanol/ethanol-water system or n-hexane- In the lower phase of ethyl acetate-n-butanol-water system, concussion makes to be completely dissolved, and carries out high speed adverse current chromatogram (HSCCC) separation system It is standby.High speed adverse current chromatogram separation condition：Clove leaf anti-inflammation active ingredient UV absorption wavelength cloves is bitter according to the literature Glycosides and oleuropein (221nm~223nm), protocatechuic acid (258 nm), tyrosol (278 nm), liposoluble ingredient (323 nm), Therefore Detection wavelength gradient is 210~360 nm, main frame temperature is 25 DEG C~30 DEG C, chloroform-n-butanol-methanol/ethanol-water（5： 4：2：4~6）Or n-hexane-ethyl acetate-n-butanol-water system（1：2：1：3~5 or 2：1：2~4：2~4）For solvent system System, chloroform-n-butanol or n-hexane-ethyl acetate（Stationary phase）For upper phase, methanol/ethanol-water or n-butanol-water（Mobile phase） For lower phase.Stationary phase is filled into chromatography column with 10~40 ml/min flow velocitys, adjustment high-speed counter-current chromatograph engine speed is 600~900 rpm/min, while entering mobile phase with 2.0~6.0 ml/min flow pump, treat that two-phase reaches dynamic equilibrium, open Sampling valve is opened, sample liquid sample introduction, gathered data simultaneously records ultraviolet chromatogram, purpose flow point is reclaimed under 210~360 nm wavelength, Solvent, freezing or spray drying is recovered under reduced pressure, high-purity clove leaf total iridoid glycoside extract is obtained（HPLC detections purity >= 80%）.

Macroporous absorbent resin of the present invention is polarity, low pole and non-polar macroporous resin in polystyrene type, Including AB-8, ADS-17, ADS-750, LSA-21, LX-26, XDA-6, D101, D113, D141, D151, D152, D3520, H103、HPD100A、HPD-300、HPD400、HPD450、HPD-600、HPD-650、HPD-700、HPD-750、HPD-800、 HPD-820, DM301, X-5, S-8, SP825, CAD45,860021, NKA- II or NKA-9 one or more, preferably macropore are inhaled Attached resin D141, HPD-400 or HPD-600 type.During centrifugation, centrifugal speed is in the range of 3000~10000 revs/min. Dynamic axial compression (DAC) preparing chromatograph in industry filler is the column chromatography silica gel of 100~300 mesh, and elution mobile phase is dichloromethane Alkane-methanol, chloroform-methanol or methanol-water (25：1~10：1) gradient elution.High speed adverse current chromatogram (HSCCC) elution used is molten Agent is chloroform-n-butanol-methanol/ethanol-water system or n-hexane-ethyl acetate-n-butanol-water system.

It is an advantage of the invention that：

1. the present invention utilizes dynamic ultrasound adverse current extraction process first, with reference to macroporous adsorbent resin column chromatography and dynamic axial compression Preparing chromatograph in industry or high speed adverse current chromatogram isolation technics, prepare the extraction of the clove leaf total iridoid glycoside of high purity more than 80% Thing, and a variety of formulations can be made by adding various pharmaceutic adjuvants, to develop the treatment infection of the upper respiratory tract, acute and chronic bronchitis The kind new medicine of Chinese medicine five of the infectious diseases such as inflammation, bacillary dysentery, acute gastroenteritis, acute icterohepatitis, acute mastitis There is provided material base；

2. the present invention is reasonable in design, technique is simple, is followed the example of using dynamic ultrasound alcohol extracting, and macroporous adsorbent resin column chromatography combines dynamic Process-scale chromatography or high speed adverse current chromatogram isolation technics are compressed axially, the clove leaf total iridoid glycoside extract of high-purity is obtained, Non-environmental-pollution, simple and efficient to handle, cost is relatively low, is especially suitable for industrialized production；

3. the purification with macroreticular resin method selected by the present invention, big with adsorbance, desorption efficiency is high, can be anti-after resin regeneration The advantages of using again；Dynamic axial compression (DAC) preparing chromatograph in industry is a kind of efficient isolation technics, and chromatogram is prepared with tradition Compare, filling filler time short, chromatogram column efficiency high, separating rate fast the features such as big with load sample amount can reach close point Analyse the post effect of post；There is high speed adverse current chromatogram isolation technics (HSCCC) carrier-free rapidly and efficiently to separate, the rate of recovery is high, fractional dose Prepared by greatly, simple operation and other advantages, the separation that active skull cap components are widely used in recent years, therefore answered with good Use prospect.

Resulting high-purity clove leaf total iridoid glycoside extract, can be made into commonly by adding various pharmaceutic adjuvants The solid orally ingestibles such as tablet, thin membrane coated tablet, capsule, dispersible tablet, pill, granule, sustained-release preparation, and spraying The formulations such as agent, aerosol, injection freeze-dried powder.

Brief description of the drawings

Fig. 1 is the preparation technology flow chart of high-purity clove leaf total iridoid glycoside extract of the present invention.

Fig. 2 is the HPLC chromatogram of clove leaf total iridoid glycoside in sample after polishing purification.

Fig. 3 is the tablet for using high-purity clove leaf total iridoid glycoside to be prepared for raw material.

Fig. 4 is the site specific DDS for colon tablet for using high-purity clove leaf total iridoid glycoside to be prepared for raw material.

Embodiment

The invention will be further described with reference to embodiments, but the invention is not limited in these embodiments.

The dynamic ultrasound of embodiment 1 adverse current extracts combination macroporous adsorbent resin column chromatography and dynamic axial compression is industrially prepared Chromatographic separation technology prepares high-purity clove leaf total iridoid glycoside extract：

The kg of clove leaf medicinal material 10 is crushed to 20~30 mesh, plus the ethanol that 8 times of amount concentration of volume percent are 70%, in frequency 30 KHz, the kW Continuous Countercurrent Extractions 3 of ultrasonic power 20,40 min, filtering, merge extract solution, Extraction solvent are recovered under reduced pressure every time, fit It is 0.6 g crude drugs/ml when being concentrated into concentration, plus 1 mol/L hydrochloric acid tune pH value is to 4, it is big by HPD650 with 2 BV/h flow velocity Macroporous adsorbent resin chromatographic column, sample solution is 1 in the ratio of solid quality and dried resin：6, blade diameter length ratio is 1：8, first with 4 BV's Distilled water washing resin post bed, elution flow rate is 2 BV/h, discards water elution；Again with the second that concentration of volume percent is 50% Alcohol is eluted, and elution flow rate is 2 BV/h, and ethanol is recovered under reduced pressure in eluent, is concentrated into relative density for 1.15 (50 DEG C measure), plus Enter the column chromatography silica gel filler of 200~300 mesh, plus methanol mix thoroughly after dry, cross the reverse preparing chromatograph in industry of dynamic axial compression Post, methylene chloride-methanol (15：1~10：1) eluted for eluent gradient, the master containing clove leaf total iridoid glycoside is collected respectively Section eluent, merges, is concentrated under reduced pressure, and filters, and solvent, freeze-drying or spray drying is recovered under reduced pressure, clove leaf total cyclenes is measured Ether terpene glycosides content >=80%（HPLC methods, percentage by weight）.

The heating and refluxing extraction combination macroporous adsorbent resin column chromatography of embodiment 2 and high speed adverse current chromatogram separation prepare high-purity Spend clove leaf total iridoid glycoside extract：

In 2 kg clove leaf medicinal materials of 20~30 mesh are crushed to, plus the ethanol that 15 times of amount concentration of volume percent are 80%, heating Refluxing extraction 3 times, 1.5 h, filtering, merge extract solution every time, be concentrated into concentration for 1.0 g crude drugs/ml, 1 mol/ is added after filtering L hydrochloric acid adjusts pH value to 4~5, and with 3 BV/h flow velocity by D101 macroporous adsorption resin chromatography posts, sample solution presses solid quality Ratio with dried resin is 1：8, blade diameter length ratio is 1：10, first with 6 BV water washing resin, elution flow rate is 4 BV/h, discards water Eluent；Eluted again with concentration of volume percent for 60% methanol, elution flow rate is 3 BV/h, methanol is recovered under reduced pressure in eluent, Relative density is concentrated into for 1.25 (50 DEG C measure), is dried under reduced pressure, chloroform-n-butanol-methanol/ethanol-water system or just is added In the lower phase of hexane-ethylacetate-n-butanol-water system, concussion makes to be completely dissolved, and carries out high speed adverse current chromatogram (HSCCC) point From preparation.High speed adverse current chromatogram separation condition：The nm of Detection wavelength 210~360, main frame temperature is 30 DEG C, n-hexane-acetic acid second Ester-n-butanol-water is solvent system（1：2：1：3~5 or 2：1：2~4：2~4）, n-hexane-ethyl acetate（Stationary phase）To be upper Phase, n-butanol-water（Mobile phase）For lower phase.Stationary phase is filled into chromatography column with 10 ml/min flow velocitys, high-speed counter-current is adjusted Chromatograph engine speed is 900 rpm/min, while entering mobile phase with 2.0 ml/min flow pump, treats that two-phase reaches that dynamic is flat Weighing apparatus, opens sampling valve, and sample liquid sample introduction, gathered data simultaneously records ultraviolet chromatogram, purpose is reclaimed under 210~360 nm wavelength Flow point, is recovered under reduced pressure solvent, freezing or spray drying, measures clove leaf total iridoid glycoside content 78%（HPLC methods, weight hundred Divide ratio）.

The heating and refluxing extraction combination macroporous adsorbent resin column chromatography of embodiment 3 and dynamic axial compression preparing chromatograph in industry point High-purity clove leaf total iridoid glycoside extract is prepared from technology：

The kg of clove leaf 5 is moderately crushed, plus 10 times of 60% methanol measured, and in the kHz of frequency 30, ultrasonic power 20kW continuous ultrasounds are inverse Stream is extracted 3 times, and 0.5 h, filtering, merge extract solution, methanol is recovered under reduced pressure every time, is added water and is adjusted concentration to 1.5 g crude drugs/ml, Plus 1 mol/L hydrochloric acid adjust pH value to 6, with 2BV/h flow velocity by HPD400 macroporous adsorption resin chromatographies post 2 times, sample solution is pressed The ratio of solid quality and dried resin is 1：9, blade diameter length ratio is 1：8, first with 4 BV water washing resin, elution flow rate is 2 BV/h, discards water elution；Again with the ethanol elution that concentration of volume percent is 70%, elution flow rate is 3 BV/h, and eluent subtracts Receipts ethanol is pushed back, relative density is concentrated into for 1.15 (50 DEG C measure), the column chromatography silica gel filler of 200~300 mesh is added, plus Methanol is dried after mixing thoroughly, crosses the reverse industrial preparation chromatographic column of dynamic axial compression, chloroform-methanol (20：1~10：1) it is mobile phase Gradient elution, collects the principal piece eluent containing clove leaf total iridoid glycoside respectively, merges, is concentrated under reduced pressure, and filters, is recovered under reduced pressure Solvent, freeze-drying or spray drying, measure clove leaf total iridoid glycoside content 82%（HPLC methods, percentage by weight）.

The dynamic ultrasound of embodiment 4 adverse current, which is extracted, combines macroporous adsorbent resin column chromatography and high speed adverse current chromatogram separation preparation High-purity clove leaf total iridoid glycoside extract：

The kg of clove leaf 5 is moderately crushed, plus 12 times of 50% ethanol measured, and in the kHz of frequency 30, ultrasonic power 20kW continuous ultrasounds are inverse Stream is extracted 3 times, and 1.5 h, filtering, merge extract solution, ethanol is recovered under reduced pressure every time, is added water and is adjusted concentration to 1.2 g crude drugs/ml, Plus 1 mol/L hydrochloric acid adjust pH value to 3~5, with 3BV/h flow velocity by D141 macroporous adsorption resin chromatographies post 2 times, sample solution is pressed The ratio of solid quality and dried resin is 1：10, blade diameter length ratio is 1：8, first with 4 BV water washing resin, elution flow rate is 2 BV/h, discards water elution；Again with the ethanol elution that concentration of volume percent is 50%, elution flow rate is 3 BV/h, and eluent subtracts Receipts ethanol is pushed back, relative density is concentrated into for 1.15 (50 DEG C measure), is dried under reduced pressure, add chloroform-n-butanol-methanol/second In the lower phase of alcohol-water system, concussion makes to be completely dissolved, and carries out high speed adverse current chromatogram (HSCCC) separation and prepares.High speed adverse current chromatogram Separation condition：Detection wavelength is 210~360 nm, and main frame temperature is 25 DEG C, and chloroform-n-butanol-methanol/ethanol-water is solvent System（5：4：2：4~6）, chloroform-n-butanol（Stationary phase）For upper phase, methanol/ethanol-water（Mobile phase）For lower phase.Will be fixed Chromatography column is filled with 20 ml/min flow velocitys, adjustment high-speed counter-current chromatograph engine speed is 600 rpm/min, simultaneously Mobile phase is entered with 3.0 ml/min flow pump, treats that two-phase reaches dynamic equilibrium, sampling valve is opened, sample liquid sample introduction gathers number According to and record ultraviolet chromatogram, purpose flow point is reclaimed under 210~360 nm wavelength, solvent is recovered under reduced pressure, freezing or spraying are dry It is dry, measure clove leaf total iridoid glycoside content 75%（HPLC methods, percentage by weight）.

Embodiment 5

The kg of clove leaf 5 is moderately crushed, plus 13 times of 30% ethanol measured, heating and refluxing extraction 3 times, and 2.0 h, is filtered every time, is merged Extract solution, is recovered under reduced pressure ethanol, and the regulation concentration that adds water is to 0.8 g crude drugs/ml, plus 1 mol/L hydrochloric acid adjusts pH value to 6.5, with 2BV/h flow velocity is in the ratio of solid quality and dried resin by HPD700 macroporous adsorption resin chromatographies post 2 times, sample solution 1：6, blade diameter length ratio is 1：8, first with 4 BV water washing resin, elution flow rate is 2 BV/h, discards water elution；Volume hundred is used again Divide the ethanol elution that specific concentration is 70%, elution flow rate is 3 BV/h, and ethanol is recovered under reduced pressure in eluent, and being concentrated into relative density is 1.20 (50 DEG C measure), the column chromatography silica gel filler of 200~300 mesh of addition, plus methanol are dried after mixing thoroughly, cross dynamic axial pressure Contract reverse industrial preparation chromatographic column, methylene chloride-methanol (25：1~8：1) eluted for eluent gradient, collect contain cloves respectively The principal piece eluent of leaf total iridoid glycoside, merges, is concentrated under reduced pressure, and filters, and solvent is recovered under reduced pressure, and freeze-drying or spraying are dry It is dry, measure clove leaf total iridoid glycoside content 85%（HPLC methods, percentage by weight）.

Embodiment 6

The kg of clove leaf 5 is moderately crushed, plus 10 times of 20% ethanol measured, heating and refluxing extraction 2 times, each 2h, filtering, is merged and is extracted Liquid, is recovered under reduced pressure ethanol, and the regulation concentration that adds water to 0.5 g crude drugs/ml, plus 1 mol/L hydrochloric acid adjust pH value to 7.0, with 3BV/h's Flow velocity is 1 in the ratio of solid quality and dried resin by AB-8 macroporous adsorption resin chromatographies post 2 times, sample solution：6, footpath is high Than for 1：8, first with 4 BV water washing resin, elution flow rate is 2 BV/h, discards water elution；Concentration of volume percent is used again For 60% ethanol elution, elution flow rate is 3 BV/h, and ethanol is recovered under reduced pressure in eluent, and it is 1.20 (50 to be concentrated into relative density DEG C measure), add the column chromatography silica gel filler of 200~300 mesh, plus methanol mix thoroughly after dry, cross the reverse work of dynamic axial compression Industry prepares chromatographic column, methylene chloride-methanol (20：1~6：1) eluted for mobile phase, collect contain clove leaf total iridoid respectively The principal piece eluent of glycosides, merges, is concentrated under reduced pressure, and filters, and solvent, freeze-drying or spray drying is recovered under reduced pressure, total cyclenes is measured Ether terpene glycosides content 81%（HPLC methods, percentage by weight）.

The preparation of high-purity clove leaf total iridoid glycoside extract formulation

The preparation of the clove leaf total iridoid glycoside dripping pill of embodiment 7

The g of high-purity clove leaf total iridoid glycoside extract 25, adds 75 g melting mixing matrix PEG6000：PEG4000 (1:1) it is transferred to after, stirring in reservoir, it is closed and be incubated at 75 DEG C, it is cold with constant displacement pump pill dripping machine dripping from top to bottom But agent is dimethicone, water dropper bore（Inside/outside footpath）：2.1/2.5 mm, drips speed：30 balls/minute, drop away from：8 cm, per ball 25mg, collects dripping pill, sucks surface cooling agent, dries, produces.

The preparation of the clove leaf total iridoid glycoside dispersible tablet of embodiment 8

Scattered tablet recipe：

The g of bulk drug 100 (25%)

The g of lactose 50（12.5%）

The g of amylum pregelatinisatum 82.5 (20.63%)

Microcrystalline cellulose（MCC） 100 g (25%)

Sodium carboxymethyl starch（CMS-Na） 27.5 g (6.87%)

The g (9.38%) of PVPP (PVPP) 37.5

The g of superfine silica gel powder 2.5 (0.63%)

95% ethanol solution is appropriate

It is made 1000 (0.4 g/ pieces)

Take the g of high-purity clove leaf total iridoid glycoside extract 100, with the g of amylum pregelatinisatum 82.5, the g of microcrystalline cellulose 100, The g of lactose 50, the g of sodium carboxymethyl starch 27.5, the g of PVPP 37.5 are mixed, plus softwood, 24 mesh is made in 95% appropriate amount of ethanol Sieve series grain, 60 DEG C of dryings are added after the g of superfine silica gel powder 2.5 mixings, 22 mesh sieve whole grains, tabletted, piece weighs 400 mg, and every contains The mg of iridoid glycoside 100.

The preparation of the clove leaf total iridoid glycoside injection freeze-dried powder of embodiment 9

The g of high-purity clove leaf total iridoid glycoside 15, adds 20g mannitol, injects water to 200ml dissolvings, uses 1mol/L NaOH solution adjust pH to 7.5, add 0.5% activated carbon, keep pH to 7.5, heating micro-boiling 15 minutes, in the process constantly Stirring, is cooled to 50 DEG C or so filtrations, is cooled to after room temperature through 0.45mm filtering with microporous membrane, is sub-packed in 5ml's by every bottle of 2ml In cillin bottle, 115 DEG C of flowing steam sterilizations 30 minutes, freeze-drying is taken out rear pressing cover packaging and produced.

The preparation of the clove leaf total iridoid glycoside capsule of embodiment 10

High-purity clove leaf total iridoid glycoside extract 40g is taken, is mixed with lactose 20g, starch 60g, plus 95% ethanol is made Softwood, after being pelletized with 14 mesh sieves, puts 60 DEG C~70 DEG C dryings after 12 mesh sieve whole grains, is inserted in No. 1 Capsules and produces, every The mg of capsule medicated 80.

The preparation of the clove leaf total iridoid glycoside conventional tablet of embodiment 11

The g of high-purity clove leaf total iridoid glycoside 25 is taken, is formed sediment with the g of starch 45, microcrystalline cellulose 15g, the g of lactose 12, carboxymethyl Powder sodium 8g is mixed, plus softwood is made in 10% starch slurry in right amount, adds superfine silica gel powder 0.5g, and 30 mesh sieves are pelletized after mixing, 24 mesh sieves Whole grain, tabletted, theoretical piece weight 400mg, the every mg of pastille 100.

The preparation of the clove leaf total iridoid glycoside enteric coatel tablets of embodiment 12

Tablet prepared by embodiment 11 separately takes the g of EudragitⅡ 20, the g of Eudragit Ⅲ 8 as label, plus Enter the dissolving of 95% ethanol, be configured to the solution that concentration is 5%, add appropriate diethyl phthalate, talcum powder respectively as increasing Agent and antiplastering aid are moulded, enteric coating liquid is made.Label is preheated to 40~45 DEG C, 50 revs/min of rotating speed, spraying bag in coating pan Clothing, coating weight gain 5%~7%.

The pharmacology activity research of high-purity clove leaf total iridoid glycoside extract

Protective effect of the clove leaf total iridoid glycoside of embodiment 13 to rat inflammatory enteropathy (IBD)

1. experiment material

1.1 Experimental agents

Clove leaf total iridoid glycoside (self-control, purity ＞ 75%), salicylazosulfapyridine SASP（The good magnificent medicine company of upper Hisense's friendship is limited Company）Lot number：20070204.0.25g/kg concentration is made into 0.5% CMC solution using preceding；

1.2 experimental animal

Healthy SD rat, body weight：180~220 g；（Shanghai Slac Experimental Animal Co., Ltd. provides）

1.3 experiment reagent

Chloraldurate (upper seamount Pu chemical company)

NaCl parenteral solutions (Pharmaceutical Factory No.6, Harbin Pharmaceutical Group)

10% chloraldurate is prepared：10 g chloraldurates are weighed, are dissolved in 100 ml physiological saline；

Dextran sulfate sodium (DSS) (MW=36~50 kDa), MP Biomedicals companies of the U.S.

Myeloperoxidase (MPO) kit, superoxide dismutase (SOD) kit, MDA (MDA) kit, an oxygen Change nitrogen (NO) kit（Bioengineering Research Institute is built up in Nanjing）

TNF (TNF-α) kit, interleukin 8 (IL-8) kit, cyclooxygenase-2 (COX-2) examination Agent box, NF- к Bp65 kits（Beijing bioengineering Co., Ltd of Zhong Shan Golden Bridge）

2. experimental animal is grouped：

Healthy SD rat 50, body weight：180~220 g（Shanghai Slac Experimental Animal Co., Ltd. provides）.After buying Adaptability is raised 7 days, ad lib drinking-water, and feed, drinking-water are changed daily, rat living environment, ventilation and sanitation and hygiene are kept. Feed, drinking-water, behavioral activity, stool and urine situation are observed, confirms as after healthy rat, is randomly divided into 5 groups, i.e. model group, willow nitrogen Sulphur pyridine group, iridoid glycoside high dose group（1.0 g/kg）, iridoid glycoside middle dose group（0.5 g/kg）, iridoid glycoside Low dose group（0.25 g/kg）, every group 10, male and female half and half, each group body weight statistics compares without significant difference（P>0.05）；

3. model is set up：

Reference literature [1-3], rat was raised under the normal condition of laboratory after one week, and model group gives 4% dextran sulfate sodium (DSS) solution substitutes drinking water inducing colitis, and control group drinks filtration sterilization water.Daily detection mouse weight before and after drinking, Stool and situation of occulting blood；

4. the measure of MPO, SOD, MDA, NO content in colon

MPO, SOD, MDA, NO content in colon are determined according to kit specification.SOD measure is aoxidized with xanthine Enzyme process is carried out by operating procedure, at 550 nm, and absorbance is measured with spectrophotometric.MDA measure thiobarbituricacidα- Method is carried out by operating procedure, at 532 nm, and absorbance is measured with spectrophotometric.NO measure nitrate reductase method, in At 550 nm, absorbance is measured with spectrophotometric；

5. TNF-α, the measure of IL-8, COX-2, NF- к Bp65 contents in colon

According to kit specification, TNF-α, IL-8, COX-2, NF- к in brain tissue are determined using SABC ImmunohistochemistryMethods Methods Bp65 contents；

6. statistical procedures

Statistical analysis is carried out using statistics software bag SPSS 17.0.Data is represented with ± s, is compared between two groups and is examined with t, Multigroup is compared and uses variance analysis；

7. experimental result：

7. influence of 1 clove leaf total iridoid glycoside to the clear condition of ulcerative colitis in rats Traumatic Colon

Modeling the 7th day, rat is fasted after 24 h, with 10% chloraldurate intraperitoneal injection of anesthesia, and back of the body position is fixed, and splits belly, Total colectomy (sigmoid portion, is operated on ice from ileocecus to pubic symphysis) is taken out, longitudinally cuts off, is rushed with ice physiological saline Wash enteral dirt, measurement colon lengths and Breadth Maximum off, visually observe the situation of Traumatic Colon, colon is weighed, investigate total ring Influence of the alkene ether terpene glycosides to ulcerative colitis in rats Traumatic Colon situation；

Influence (± s) of the clove leaf total iridoid glycoside of table 1 to the clear condition of ulcerative colitis in rats Traumatic Colon

Note:Compared with normal group, △ P<0.05, △ △ P<0.01;

Compared with model group, * P<0.05, * * P<0.01

7. influence of 2 clove leaf total iridoid glycosides to ulcerative colitis in rats MPO, SOD, MDA, NO

MPO contents in neutrophil leucocyte are higher, and its content increases the increasing that can reflect neutrophil leucocyte in a certain tissue Height, MPO activity can point out the infiltration degree of tissue inflammatory cell.SOD, MDA, NO are that measurement degree of inflammation is most sensitive and reliable Index, the judgement of assessment and medication effect for ulcerative colitis disease development, with potential directive significance；

Influence (± s) of the clove leaf total iridoid glycoside of table 2 to Colitis rat SOD, MDA and NO content

Note:Compared with normal group, △ P<0.05, △ △ P<0.01;

Compared with model group, * P<0.05, * * P<0.01

7. influence of 3 clove leaf total iridoid glycosides to Colitis rat TNF-α, IL-8, COX-2, NF- к Bp65

Test result indicates that, compared with normal group, TNF-α, IL-8, COX-2, NF- к in colitis model group rat colon tissue Bp65 contents substantially increase.Compared with model group, the rat colon tissue of the high, medium and low dosage group of clove leaf total iridoid glycoside Middle TNF-α, IL-8, COX-2, NF- к Bp65 contents are substantially reduced；Compared with positive control salicylazosulfapyridine (SASP) group, fourth Spiceleaf total iridoid glycoside is high, middle dose group rat cerebral tissue MDA, NO content is significantly reduced；

The clove leaf total iridoid glycoside of table 3 is to Colitis rat TNF-α, the influence (± s) of IL-8, COX-2, NF- к Bp65 contents

Note:Compared with normal group, △ P<0.05, △ △ P<0.01;

Compared with model group, * P<0.05, * * P<0.01

【Bibliography】

[1] Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathlolgic study of dextran sulfate sodium experimental murine colitis. Lab Invest. 1993；69: 238-249

[2] Xin Liu, Jianming Wang. Iridoid glycosides of Folium syringae leaves modulates NF-κB signal pathway and intestinal epithelial cells apoptosis in experimental colitis. Plos one. 2011, 6 (9): e24740, 1-12.

[3] Zhao Ping, Dong Lei, Luo Jinyan, wait foundation [J] of dextran sulfate sodium-induced ulcerative colitis model in rats Four army medical university's journals, 2005,26 (19): 1738-1740.

Claims

1. the Preparation method and use of high-purity clove leaf total iridoid glycoside extract and its preparation, it is characterised in that extract work Skill process includes：Dynamic ultrasound adverse current is extracted or heating and refluxing extraction；Separation process includes：It is macroporous adsorbent resin column chromatography, dynamic State is compressed axially (DAC) process-scale chromatography or prepared by high speed adverse current chromatogram (HSCCC) separation.

2. the preparation method of high-purity clove leaf total iridoid glycoside extract according to claim 1, it is characterised in that Realized by following steps：Clove leaf is crushed, plus 6~20 times of volume of water or concentration of volume percent be molten for 10%~80% alcohol Liquid, or acetone soln, or aqueous isopropanol, dynamic ultrasound adverse current are extracted or heating and refluxing extraction 2~3 times, every time 0.5~3h, Filtering, merges extract solution, Extraction solvent is recovered under reduced pressure, and is suitably concentrated into concentration for 0.4~2.0 g crude drugs/ml, centrifugation or filtering Afterwards, plus watery hydrochloric acid regulation pH to 2.0~6.0 upper prop solution, upper prop solution inhaled with 2BV/h~4BV/h flow velocity by macropore Attached resin chromatography post, upper prop solution is 1 according to the ratio of solid content and dried resin：5~10, first with 4~6BV distillation water washing Resin column bed, elution flow rate is 2BV/h~4BV/h, discards water elution；Again with the alcohol that concentration of volume percent is 10%~80% Eluent, elution flow rate is 2BV/h~4BV/h, merges eluent and alcohol is recovered under reduced pressure, being concentrated under reduced pressure into relative density is 1.05~1.30, add 200~300 mesh column chromatography silica gel filler, plus methanol mix thoroughly after dry, cross dynamic axial compression it is reverse Industrial preparation chromatographic column, methylene chloride-methanol, dichloromethane-acetone, chloroform-methanol or chloroform-acetone (25：1~10：1) it is Eluent gradient is eluted, and the principal piece eluent of the composition containing total iridoid glycoside is collected respectively, is merged, is concentrated under reduced pressure, and is filtered, decompression Recycling design, is freeze-dried, is spray-dried or is dried under reduced pressure, and produces high-purity clove leaf total iridoid glycoside（HPLC detections are pure Degree >=80%）；Or take the macroreticular resin crude extract of clove leaf, add chloroform-n-butanol-methanol/ethanol-water system or n-hexane- Ethyl acetate-n-butanol-water system, concussion makes to be completely dissolved, and carries out high speed adverse current chromatogram（HSCCC）It is prepared by separation；It is inverse at a high speed Flow chromatography separation condition：Clove leaf anti-inflammation active ingredient UV absorption wavelength syringopicroside and olive are bitter according to the literature Glycosides (221nm~223 nm), protocatechuic acid (258 nm), tyrosol (278 nm), liposoluble ingredient (323 nm), therefore Detection wavelength Gradient is 210~360 nm, and main frame temperature is 25 DEG C~30 DEG C, chloroform-n-butanol-methanol/ethanol-water（5：4：2：4~6）Or N-hexane-ethyl acetate-n-butanol-water system（1：2：1：3~5 or 2：1：2~4：2~4）It is solvent system to clove leaf Macroreticular resin crude extract is separated, upper as stationary phase, lower to be used as mobile phase；By stationary phase with 10~40 ml/min Flow velocity fills chromatography column, and adjustment high-speed counter-current chromatograph engine speed is 600~900 rpm/min, while with 2.0~ 6.0 ml/min flow pump enters mobile phase, treats that two-phase reaches dynamic equilibrium, opens sampling valve, sample liquid sample introduction, gathered data And ultraviolet chromatogram is recorded, purpose flow point is reclaimed under 210~360 nm wavelength, solvent, freezing or spray drying is recovered under reduced pressure, Obtain high-purity clove leaf total iridoid glycoside extract（HPLC detects purity >=80%）.

3. a kind of preparation method of high-purity clove leaf total iridoid glycoside extract according to claim 2, used to carry It is water, ethanol, methanol, acetone, isopropanol one or more of mixture therein to take solvent.

4. a kind of preparation method of high-purity clove leaf total iridoid glycoside extract according to claim 2, its feature It is, described macroporous absorbent resin is polystyrene type middle polarity, low pole and nonpolar macroporous adsorption resin, model choosing With AB-8, ADS-17, ADS-750, LSA-21, LX-26, XDA-6, D101, D113, D141, D151, D152, D3520, H103, HPD100A、HPD-300、HPD400、HPD-600、HPD-650、HPD-700、HPD-750、HPD-800、HPD-820、DM301、 X-5, S-8, SP825, CAD45,860021, NKA- II or NKA-9 one or more.

5. a kind of preparation method of high-purity clove leaf total iridoid glycoside extract according to claim 2, its feature It is, eluting solvent used in macroporous adsorbent resin column chromatography is ethanol-water system or methanol-water system, preferred alcohol-water system System；Mobile phase used in dynamic axial compression process-scale chromatography is methylene chloride-methanol, dichloromethane-acetone, chloroform-methanol or chlorine Imitative-acetone system (25：1~10：1) gradient elution；Eluting solvent used in high speed adverse current chromatogram is chloroform-n-butanol-methanol/second Alcohol-water system or n-hexane-ethyl acetate-n-butanol-water system.

6. according to claim 1 method prepare high-purity clove leaf total iridoid glycoside extract and preparation preparation method and Purposes, it is characterised in that medically acceptable various pharmaceutical preparations are made such as by adding various pharmaceutic adjuvants：Film coating The solid orally ingestibles such as piece, capsule, dispersible tablet, pill, granule, sustained-release preparation, and spray, aerosol, note Penetrate the formulations such as agent.

7. high-purity clove leaf total iridoid glycoside extract formulation according to claim 6, including：Thin membrane coated tablet, Dispersible tablet, sustained release or Dospan, it is characterised in that in preparation the mass percent of main ingredient be 10%~80%, the label by The main ingredient of 10%~70% mass percent, the dilution auxiliary material of 10%~60% mass percent, 10%~50% mass percent it is slow Release or controlled-release excipient, the disintegration auxiliary material of 1%~15% mass percent, the bonding auxiliary material of 1%~20% mass percent, 0.3%~5% The lubrication auxiliary material composition of mass percent.

8. high-purity clove leaf total iridoid glycoside extract formulation according to claim 6, it is characterised in that label institute It is one or more of mixed in starch, lactose, dextrin, sucrose, microcrystalline cellulose, calcium monohydrogen phosphate, calcium sulfate with dilution auxiliary material Compound；Disintegration auxiliary material refers to that sodium carboxymethyl starch (CMS-Na), low-substituted hydroxypropyl cellulose (L-HPC), cross-linked carboxymethyl are fine One or more in the plain sodium (cCMC-Na) of dimension, PVPP (PVPP), PVPP, hydroxypropul starch Mixture；Bonding auxiliary material refers to microcrystalline cellulose, hydroxypropyl methylcellulose ketone（HPMC）, sodium carboxymethylcellulose（CMC-Na）、 Percent by volume is 50%~95% polyvinylpyrrolidone (PVP K₂₅) ethanol solution, starch slurry, the one or more of syrup Mixture；Lubrication auxiliary material refers to one or more of mixtures in talcum powder, magnesium stearate, superfine silica gel powder.

9. high-purity clove leaf total iridoid glycoside extract formulation according to claim 6, it is characterised in that the drop The matrix of pill be poloxamer-188 (Poloxamer-188), PEG-4000 (PEG-4000), polyethylene glycol- It is one or more of mixed in 6000 (PEG-6000), xylitol, medical starch, odium stearate, polyoxyethylene monostearate Compound；Instillation cooling agent is one or more of mixtures in dimethicone, atoleine, vegetable oil；Dripping pill prepares bar Part：Medicine is 1 with substrate composition：4~1:2,70 DEG C~90 DEG C of fluid temperature takes insulation dripping, water dropper bore（Inside/outside Footpath）：2.1/2.5 mm~3.0/5.0 mm, drip speed：The d/min of 20 d/min~30, drop away from：4~10 cm.