Embodiment:
The present invention is described in detail below in conjunction with embodiment:
The chemical constitution study of first part's Root of Chinese Eriosema petroleum ether extract
1 laboratory apparatus and material
1.1 medicinal material
Root of Chinese Eriosema is available from the place: Longyan, Fujian; Identify: the Wang Zhendeng researcher of Fujian Institute of Traditional Chinese Medicine identifies.
1.2 instrument
Micro-analytical balance (Beijing Sai Duolisi balance company limited); RE-52A Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant); Thermostat water bath; Fraction Collector (Shanghai Hu Xi analytical instrument Co., Ltd., Factory); Ultrasonic cleaning instrument (Kunshan Ultrasonic Instruments Co., Ltd.); Ultraviolet-visible pectrophotometer (VARIAN, Cary 50); AVATAR360 infrared spectrometer (U.S. Thermo Nicoler); GS 20 analysis mode high-speed counter-current chromatographs (Beijing UE Biotechnology Co., Ltd.); GS 10 countercurrent chromatography instrument (Beijing UE Biotechnology Co., Ltd.); High performance liquid chromatograph (U.S. Agilent 1100series); Elemental analyser (German Vario EL III); Mass spectrograph (U.S. LCQ DecaXP Max); Nuclear magnetic resonance analyser (U.S. Unity 500).
1.3 reagent
Normal hexane, vinyl acetic monomer, methyl alcohol, distilled water, sherwood oil (60-90 ℃) are the AR level; Chromatogram methyl alcohol; Nothing is indicated especially and is analytical reagent.
2 experimental techniques
2.1 specimen preparation
Root of Chinese Eriosema piece root is pulverized 1kg, exceed the about 1cm of medicinal material with 95% ethanol submergence medicinal material and liquid level, three times (2h, 2h 2h), merge ethanol extract with normal pressure power refluxing extraction at 82~85 ℃.Ethanol extract is got medicinal extract 140g with the Rotary Evaporators concentrating under reduced pressure, add the water suspendible of about 700~1000mL,, get sherwood oil medicinal extract 30g with the Rotary Evaporators concentrating under reduced pressure with 300~500mL petroleum ether extraction 8~10 times.
Preferred embodiment of the present invention: Root of Chinese Eriosema piece root is pulverized 1kg, exceed the about 1cm of medicinal material with 95% ethanol submergence medicinal material and liquid level, refluxing extraction three times under preferred 84 ℃ of temperature and normal pressure power (2h, 2h, 2h), merge three times ethanol extract, ethanol extract is got medicinal extract 140g with the Rotary Evaporators concentrating under reduced pressure, and medicinal extract adds 850 ml water suspendibles, with the petroleum ether extraction of 0.8 times of above-mentioned water yield 10 times, merge petroleum ether extraction liquid, the Rotary Evaporators concentrating under reduced pressure gets sherwood oil medicinal extract 30g.
2.2HSCCC the selection of solvent system and the preparation of sample solution
High-speed countercurrent chromatography (High-speed Countercurrent Chro-matography is called for short " HSCCC ", down together) is two immiscible solvent countercurrent flows, and sample distributes between two-phase, without the full liquid chromatography method of solid adsorbent; The selection of HSCCC solvent system: the rough polarity according to separated material is selected the separation system classification, in this experiment, select the normal hexane system of low-pole, adopt the proportioning of tlc selective solvent system simultaneously, at first prepare a certain amount of two-phase solvent by selected solvent systems, respectively get 2mL in small test tube, add several milligrams of samples, behind the thermal agitation 1min, standing demix, the two layers of solution up and down of getting approximate equivalent with kapillary treats that the intact back of solvent evaporates launches with the organic layer in the solvent systems, with the colour developing of iodine cylinder on the TLC plate, according to spot colourity and area can observation sample in the distribution condition of each component in two-phase solvent, promptly estimate the size and the difference of partition ratio, judge the suitability of solvent systems thus, determine solvent systems by analysis mode high-speed counter-current instrument again.
The preparation of sample solution: above-mentioned 200mg sherwood oil crude extract can be dissolved in the 2mL volume ratio and be in 1/1 the system of phase solvent up and down, with the membrane filtration of 4.5 μ m organic systems, filter membrane adopts the filter membrane of nylon in the example of the present invention, in order to the HSCCC sample introduction.
2.3HSCCC separating step
Before each the separation, at first prepare the two-phase solvent system that has chosen, with standing demix behind its thorough mixing.Be separated two before use, will squeeze into mutually on the above-mentioned selected solvent system in the post of HSCCC (cumulative volume is V), earlier until filling with whole pipe with constant flow pump.Open HSCCC then, rotating speed is 700rmin
-1, moving phase is with 2.0mLmin simultaneously
-1Flow velocity squeeze into HSCCC, after by the time the polyfluortetraethylene pipe tail end flows out moving phase, show that two-phase has reached balance among the HSCCC, collect the effusive stationary phase volume V of post tail end
1, by six-way valve sample being injected HSCCC, moving phase outflow end UV-detector wavelength is 254nm.According to manual each the chromatographic peak component of collecting of color atlas.After separating end, two-phase solvent is gone out from polytetrafluoroethylhelix helix tube, measured retention value (SF) SF=V-V of stationary phase with nitrogen
1/ V * 100%.
2.4 the purity check of each component
Root of Chinese Eriosema sherwood oil medicinal extract separates each component that obtains through HSCCC and carries out purity check by HPLC.
2.5 the structure of each component is identified
Separate the compound that obtains and carry out the molecular structure evaluation by method of spectroscopy such as ultimate analysis, MS, IR, UV, NMR.
3 results and analysis
3.1HSCCC solvent system
By " a 2.2 " method screening down, obtain preferable solvent system at last and measure normal hexane by volume: vinyl acetic monomer: methyl alcohol: water=5: 5: 7: 3.Under following optimum experimental condition, experimentize: solvent system normal hexane: vinyl acetic monomer: methyl alcohol: water (5: 5: 7: 3); Stationary phase: go up phase; Moving phase: following phase; Flow velocity: 2.0mLmin
-1Rotating speed: 700rmin
-1, obtain the separation of petroleum ether extract, from experimental result 4 peaks are arranged as can be seen, wherein 2 the peak resolution in back are relatively good, reach baseline separation.
3.2HSCCC separating resulting
Experiment condition is: solvent system normal hexane: vinyl acetic monomer: methyl alcohol: water measures=5 with volume ratio: 5: 7: 3; Stationary phase: go up phase; Moving phase: following phase; Flow velocity: 2.0mLmin
-1Rotating speed: 700rmin
-1The 200mg petroleum ether extract is at certain chromatographic condition (solvent system normal hexane: vinyl acetic monomer: methyl alcohol: water [with the volume ratio metering]=5: 5: 7: 3) separate through preparation type HSCCC down, form chromatographic peak profile, the chromatographic peak that component 1 and component 2 are arranged, after component 1 is manually collected, naturally volatilizing in room temperature has yellow oil to separate out, yellow oil prepares through thin layer, developping agent is: sherwood oil: vinyl acetic monomer is with volume ratio metering=4: 1, obtain yellow prismatic crystal (in the ethanol liquid), obtain 29mg compound 1; After component 2 was manually collected, volatilizing naturally in room temperature had faint yellow needle crystal to separate out, and obtains 4mg compound 2.
3.3 stationary phase retention value
The stationary phase retention value is very big to the separating effect influence, and retention value is big more, and separating effect is good more.The factor that influences the stationary phase retention value is a lot, comprises characteristics, the flow rate of mobile phase of solvent systems itself, serviceability of HSCCC instrument etc.For guaranteeing separating effect, the retention value of stationary phase can not be lower than 50%.
This measuring solvent system is a normal hexane with the volume ratio metering: vinyl acetic monomer: methyl alcohol: water=5: 5: 7: the retention rate of 3 stationary phase is 63.96%.
3.4HSCCC the purity of counter-current separation component
The separating obtained compound 1 of HSCCC is in HPLC chromatographic determination condition in 3.2: water/acetonitrile was with initial ratio 85: 15, be 80: 20 behind the 5min, be 60: 40 behind the 10min, be 50: 50 behind the 20min, 30min is 40: 60, is 20: 80 behind the 40min, is 10: 90 behind the 45min, column temperature: 30 ℃, flow velocity: 1mLmin
-1, analyzing demonstration HPLC collection of illustrative plates under the condition of detection wavelength: 254nm is simple spike, and the peak shape symmetry, is indicated as single component; Compound 2 is in HPLC chromatographic determination condition: water/acetonitrile is 60: 40 with initial ratio 80: 20 behind the 25min, is 50: 50 behind the 30min, and 33min is 40: 60, is 20: 80 behind the 35min, is 10: 90 behind the 40min, column temperature: 30 ℃, and flow velocity: 1mLmin
-1, analyzing demonstration HPLC collection of illustrative plates under the condition of detection wavelength: 254nm is simple spike, and the peak shape symmetry, also is indicated as single component, can carry out structural analysis.
3.6 the structure of compound 2
3.6.1 the ultra-violet absorption spectrum of compound 2
From the ultraviolet spectrogram of compound 2 as can be known, 2 main absorption bands are arranged, appear at 299nm and 348nm; Most flavonoid compounds; because of the cross conjugation system that exists cinnamyl and benzoyl to form in the molecule; so its at the region memory of 200~400nm two main ultraviolet absorption band; be called peak band I (300~400nm) and peak band II (220~280nm), may be flavonoid compound according to the UV spectrum of compound 2 explanation compound 2.
3.6.2 the infrared absorption spectrum of compound 2
According to the infrared spectra of compound 2 as can be known, main peaks is belonged to as follows: 3469cm
-1Near strong wide absorption peak, showing has due to the formed hydrogen bond that mutually combines of a plurality of hydroxyls 1615cm
-1, 1519cm
-1Absorption peak show phenyl ring and have 825cm
-1Be C-H in-plane bending vibration absorption peak in the benzene, 1650~1615cm
-1There is 2922cm in the C=O structure that near absorption peak shows ketone
-1Near absorption peak is-CH
2The hydrocarbon key stretching vibration of C-H, 1373cm
-1Be-CH
3The C-H flexural vibration, 1256cm
-1Absorption peak shows ether structure and exists, and shows that further compound 2 is flavonoid compound.
3.6.3 the elementary composition and mass spectrometry results of compound 2
Determination of elemental analysis value: N:<0.3%, C:71.14%, H:6.74%, ESI-MS figure shows that the negative ion peak is: 437[M-1]-, illustrate that molecular weight is 438, in conjunction with ultimate analysis, infer that molecular formula is: C
26H
30O
6
3.6.4 the proton nmr spectra of compound 2 and carbon spectrum
Survey compound 2
1H-NMR (400MHZ, CDCl
3) the hydrogen spectrum, can see obviously that from the hydrogen spectrum 30 hydrogen are arranged: δ ppm:3.85 (3H, s, OCH
3), be the hydrogen signal of methoxyl group on the phenyl ring; 1.67-1.754 (12H, s, 4CH
3), showing has four methyl; 3.26 (2H, d, CH
2), 3.51 (2H, d, CH
2) (CH), 5.29 (CH), showing has two-CHCH for 1H, t for 1H, t with 5.17
2-; 7.47 (2H, d, 2 ', 6 '-H), 6.98 (2H, d, 3 ', 5 '-H), show that it is that symmetry replaces that a phenyl ring is arranged; 6.477 (1H, s ,-OH), 11.496 (1H, s ,-OH); 4.50 (1H, d, C-3); 4.98 (1H, d, C-2).
Survey compound 2
13C-NMR (100MHZ, CDCl
3) the carbon spectrogram, δ ppm:196.26 (C-4), 163.19 (C-7), 160.15 (C-5), 158.75 (C-9), 157.80 (C-4 '), 134.90,134.42 (C-3 "; 3 " '), 128.72 (C-1 '), 128.67 (C-2 ', 6 '), 121.57,121.48 (C-2 ", 2 " '), 113.99 (C-3 ', 5 '), 107.82 (C-10), 106.96 (C-6), 100.40 (C-8), 82.89 (C-3), 72.55 (C-2), 55.53 (OCH
3), 21.70,21.27 (C-5 ", 5 " '), 25.85,25.84 (C-4 ", 4 " '), 17.90,17.82 (C-1 ", 1 " ').
Compose as can be known from hydrogen spectrum and carbon, all hydrogen signals and carbon signal all have rational home to return to, above-mentioned hydrocarbon signal and 6,8-(3-methyl-2-butene base)-5-methoxyl group-3,7,4 '-trihydroxyflavone structure conforms to substantially, again in conjunction with uv-spectrogram, and infared spectrum, ultimate analysis, the mass spectrum result, preliminary evaluation compound 2 structural formulas are as follows, are isolated a kind of new compounds from this Root of Chinese Eriosema plant first.
The present invention adopts the new tool HSCCC of separation, purifying to separate to come the chemical ingredients of separation and purification Root of Chinese Eriosema petroleum ether part.HSCCC utilizes multi-layer helical pipe planetary motion to form special centrifugal force vectors field, realize the one-way hydrokinetics distribution of two solvent phase in tubing string, in this case, can guarantee that stationary phase reached more than 50% at the reservation percentage in the tubing string when moving phase was passed tubing string at a high speed, simultaneously promote two alternate thorough mixing and adverse current transmission greatly.This system be owing to can adopt various system condition and operating method, provides good condition to the effective constituent of separation and Extraction different qualities from the natural product crude extract, thereby, more meet compartment analysis and preparation purifying requirements of one's work.
The selection of HSCCC solvent system also is the most scabrous problem for ten fens keys of HSCCC simultaneously, and the difficulty that solvent is selected has restricted the application of adverse current chromatogram.Usually, two phase solvent system should satisfy following requirement: (1) does not cause the decomposition or the sex change of sample; (2) sufficiently high sample dissolution degree; (3) on-following biphase volume about equally, in order to avoid the waste solvent; (4) sample suitable partition ratio value in solvent systems; (5) separation time of solvent system is less than 30s, and making fixedly is on good terms realizes sufficiently high reservation.In general, first three point can judge more easily, back 2 to the high-speed counter-current chromatograph particularly important that seems, need through measuring or experiment is judged.Measuring method comprises TLC, HPLC and analysis mode HSCCC method etc.
The accuracy of TLC method is lower, but easily and fast, can make easily the suitability of solvent systems and tentatively judging.According to spot colourity can observation sample in the relative content of each component and the distribution condition in two-phase solvent thereof, promptly estimate the size and the difference of partition ratio, can determine the elution order of suitability and each component of solvent systems thus.Therefore the TLC method is adopted in this experiment, but the TLC method can only be made preliminary judgement to the suitability of solvent systems, to the estimation roughly of partition ratio do, under the situation of lacking experience, is difficult to hold solvent systems in view of the above and is used for the isolating feasibility of adverse current chromatogram.Therefore this experiment by the TLC method detect solvent upper and lower all target product is arranged mutually after, screen the best solvent system by analysis mode HSCCC method again.
The result shows:
1. the selection of solvent system is a most key link during HSCCC separates, and it is whether suitable that solvent system is selected, and is directly connected to separating resulting and gets fine or not.The polarity of petroleum ether part a little less than, therefore should select the low-pole system for use.Normal hexane/vinyl acetic monomer/methanol is one of separated from solvent system that has been widely used, is mainly used in the separation of low-pole material.In solvent system, normal hexane and water become two-phase respectively, add the polarity that methyl alcohol, vinyl acetic monomer are regulated solvent systems more as required, to reach good separating effect.For the system of normal hexane/vinyl acetic monomer/methanol, if target components is soluble in the last phase of this system, increase the content of methanol-water, target components can be pulled to down phase.Otherwise, then can reduce the ratio of methyl alcohol.Reason is that when the amount of methyl alcohol increased, it can reduce the polarity of water on the one hand, and the while can increase the polarity of organic phase again, can reach the purpose that changes the partition ratio of composition to be separated in two-phase.It is normal hexane that the optimum solvent matching system (with the volume ratio metering) that separates petroleum ether part is explored in the present invention's research: vinyl acetic monomer: methyl alcohol: water=5: 5: 7: 3, these solvent systems not only have the ratio of suitable upper and lower phase, suitable stationary phase retention value, and target component had more satisfactory distribution.
With traditional pass through solvent extraction, the method for chromatography is compared repeatedly, HSCCC not only can simplify many steps greatly, save time, and can not cause the loss of solvent and sample, target product can reach higher purity.And in the HSCCC operating process, as long as debugged instrument, after sample introduction finished, subsequent separation process then can realize automatization fully, and sepn process can detect in conjunction with ultraviolet.These characteristics has shown the clear superiority of HSCCC aspect separation, purification compound.Certainly, HSCCC can not be omnipotent aspect separation, the purification compound yet.The compound that has then can not separate by the means of HSCCC owing to be difficult to select suitable solvent systems.Other has, and is used for carrying out the isolating sample of HSCCC target component purity is improved.So both be convenient to seek suitable solvent systems, can have improved separating effect effectively again.Therefore, when HSCCC purifies in the separation that is applied to chemical ingredients, should carry out with other separation method complementation commonly used as far as possible.This experiment is with traditional solvent refluxing extracting method, combines with traditional thin-layer chromatography preparation by HSCCC and carries out separation and purification, separates obtaining 2 kinds of new compounds first from this medicinal material petroleum ether part.
The 2 bioactive researchs of second section Root of Chinese Eriosema flavone extract---compound
1 laboratory apparatus and material
1.1 medicinal material
Root of Chinese Eriosema is available from the place: Longyan, Fujian; Identify: the Wang Zhendeng researcher of Fujian Institute of Traditional Chinese Medicine identifies.Root of Chinese Eriosema flavone extract---compound 2 is obtained by first part of the present invention experiment.
1.2 instrument
Ultraviolet-visible pectrophotometer (VARIAN, Cary 50); RE-52A Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant); BS210S type micro-analytical balance (Beijing Sai Duolisi balance company limited); Electrically heated drying cabinet (Shanghai sunlight laboratory apparatus company limited); HH-2 digital display thermostat water bath (Shanghai state China plant and instrument company limited); Ultralow Temperature Freezer (Sanyo); Ultrasonic cleaning instrument (Kunshan Ultrasonic Instruments Co., Ltd.); TGL-16G high speed freezing centrifuge (going up Hai'an booth instrument plant); Low speed large capacity centrifuge (going up Hai'an booth instrument plant); Water distillation apparatus (Shanghai Yarong Biochemical Instrument Plant); SSB-1 Bechtop (Shanghai treating plant factory).
1.3 reagent
Bacterial isolates: streptococcus aureus (GIM1.55), intestinal bacteria (GIM1.115), salmonella typhi (ATCC6539), withered grass bud pole bacterium (O901) are provided by Microbiological Lab of University Of Agriculture and Forestry In Fujian; Plain agar substratum (medical), Xinhua's middling speed filter paper, erythromycin, glycyrrhiza compound oral liquid (Guangxi Medical Univ. Pharmaceutical Plant); Ammoniacal liquor (25%~28%); Dexamethasone sodium phosphate (Fujian Sanai Pharmaceutical Co., Ltd); Phenol red; Sherwood oil (AR, boiling range 60-90 ℃), dehydrated alcohol (AR), NaOH, NaCl, NH
4Cl is homemade analytical pure.
1.4 laboratory animal
Kunming mice, mean body weight be (18~22g), available from Medical University Of Fujian's Experimental Animal Center.
2 experimental techniques
2.1 the research of Root of Chinese Eriosema flavone extract---compound 2 bacteriostatic activities
2.1.1 detect the preparation of bacteria suspension
To detect bacterium and be connected to the inclined-plane, and cultivate 24h, with oscillator lawn is shaken dispersion in physiological saline (sterilizing), and it is diluted to required OD value for 37 ℃ with nutrient agar.
2.1.2 the filter paper method is measured bacteriostatic activity
With punch tool filter paper (thick about 4mm) is broken into the disk of diameter 5mm, stand-by behind the autoclaving.After soaking aseptic cotton carrier with the above-mentioned bacteria suspension for preparing, allow cotton swab extruding gently on test tube wall, squeeze out unnecessary bacteria suspension, with the side of cotton swab rather than top from three close plates that are coated with of different angles, each angle differs about 60 °, smears a week along the plate inner edge at last.The sterilization filter paper of cut-off footpath 6mm, every dropping content is
250mgmL
-1Compound 2 extracting solutions 20 μ l as the experiment print, the negative control print drips 20 μ l sterilized waters, the positive control print drips the 10 μ gmL of 20 μ l
-1Erythromycin solution, 50 ℃ of oven dry take out filter paper with aseptic nipper and be attached to and contain on the bacterium flat board, and applying light fully contact it.Put 37 ℃ of constant temperature culture 18-24h.Measure the inhibition zone size of filter paper, relatively fungistatic effect.
2.1.3 test tube serial dilution
According to the result of bacteriostatic experiment, choose sensitive organism and measure the minimum inhibitory concentration of each extract it.Adopt double dilution method, as solubility promoter, the concentration of tween 80 is 2% with tween 80.Get 7 in small size test tube, numbering.The 1st step, every arm added the 1mL broth culture, added the 1mL soup at the 1st pipe then, with suction pipe at the 1st pipe pressure-vaccum for several times, make soup and broth culture mixing, take out 1mL and be added on the 2nd pipe, the taking-up 1mL that is mixed is added on the 3rd pipe, so be diluted to the 6th pipe and discard 1mL, the 7th pipe does not add soup in contrast.The 3rd step, every pipe added bacterium liquid 50 μ l, and mixing is put in 37 ℃ of incubators and cultivated 24h.From every pipe, take out culture 20 μ l then and be inoculated on the plain agar flat board, continue to cultivate 24h, observations again.Highly diluted concentration with no bacterial growth is the minimum inhibitory concentration of this soup.Parallel doing 3 times is as the criterion with 2 times or 3 identical results, and sets up tween 80 contrast and sterilized water to contrast.Because the difference of the muddiness of herb liquid itself and artificial vision, behind the bacterial growth, make naked eyes to observe, or the experimental result of observing is prone to deviation, therefore, gets culture 20 μ l again from each pipe, transferred species is to Agar Plating, with L rod coating, put 37 ℃ of incubators and cultivate 18-24h, be the minimal inhibitory concentration (MIC) of this medicine with the highest drug dilution degree of the bacterium colony of not growing to this kind bacterium.
2.2 the research of compound 2 anti-inflammatory activities
2.2.1 the preparation of test liquid
The petroleum ether extract that first part obtains---compound 2 with an amount of dissolve with ethanol after, add the water heating and make the ethanol volatilization, at last with the water constant volume, the concentration of preparation is 4.680mgmL
-1(dosage of small white mouse is according to the dosage conversion of body surface area by the people).
2.2.2 the influence of p-Xylol induced mice auricle edema
30 small white mouses are divided into 3 groups at random, 10 every group; Negative control group is irritated physiological saline, and positive controls is pressed 5mgkg
-1Through the administration of subcutaneous injection dexamethasone sodium phosphate, experimental group is irritated petroleum ether extract---compound 2, gives the morning every day feeding animal preceding administration, and administration liquid is long-pending to be 10mLkg
-1, successive administration 10d.Auris dextra shell melted paraxylene 20 μ l behind 10d administration 1h, cause the 30min dislocation of scorching back and put to death mouse, gently wipe left and right sides ear left drug away with absorbent cotton, cut two ears along the auricle baseline, with diameter is that the punch tool of 8mm is laid round auricle at the same position of two ears respectively, scales/electronic balance weighing is obtained two ear weight differences, carries out the t check with two ear weight differences as the index of inflammation swelling degree.
2.3 the active research of cough-relieving
2.3.1 the preparation of test liquid
Root of Chinese Eriosema flavone extract---compound 2 with an amount of dissolve with ethanol after, add water heating and make the ethanol volatilization, at last with the water constant volume, the concentration of preparation is 4.680mgmL
-1(dosage of small white mouse is according to the dosage conversion of body surface area by the people).
2.3.2 experiment grouping and medication
30 small white mouses are divided into 3 groups at random, 10 every group; Negative control group is irritated physiological saline, positive controls is irritated glycyrrhiza compound oral liquid (dosage of small white mouse is according to the dosage conversion of body surface area by the people), experimental group is irritated Root of Chinese Eriosema flavone extract---compound 2, give the morning every day feeding animal preceding gastric infusion, administration liquid is long-pending to be 10mLkg
-1, successive administration 10d.
2.3.3 mouse ammoniacal liquor draws the method for coughing
This is tested behind last administration 1h and carries out.Put a cotton balls that is marked with 80 μ l ammoniacal liquor in the 250mL wide-necked bottle, with the stopper that ventpipe is housed beyond the Great Wall, after treating that ammoniacal liquor is saturated, tested small white mouse is put into wide-necked bottle one by one, the record small white mouse is placed in the cup and shrinks time (i.e. the time of coughing for the first time) and the interior cough number of times of 1.5min open one's mouth to abdominal muscle occurring, observed and recorded ammoniacal liquor causes cough latent period and the cough number of times of animal, the ammoniacal liquor cotton balls that animal of every test all will more renew.
The active research 2.4 reduce phlegm
2.4.1 the preparation of test liquid
Root of Chinese Eriosema flavone extract---compound 2 with an amount of dissolve with ethanol after, add water heating and make the ethanol volatilization, at last with the water constant volume, the concentration of preparation is 4.680mgmL
-1(dosage of small white mouse is according to the dosage conversion of body surface area by the people).
2.4.2 the drafting of standard phenol red colouring agent for food, also used as a Chinese medicine line
Accurately take by weighing a certain amount of phenol redly with analytical balance, add 5% sodium hydrogen carbonate solution dissolving, be made into 1mL and contain 100 μ g, be diluted to every milliliter then in turn and contain phenol red 0.050,0.100,0.125,0.250,0.500,1.000,1.250,2.500,5.000,10.000 μ g surveys light absorption value A with spectrophotometer in the 546nm place, be X-coordinate with phenol red dosage, light absorption value A is an ordinate zou, the drawing standard curve.
2.4.3 the phenol red method of tracheae section
Get 30 of mouse, be divided into 3 groups at random, 10 every group.Negative control group is irritated physiological saline, presses 10mLkg
-1Irritate stomach distilled water, positive controls is pressed 1gkg
-1Irritate stomach ammonium chloride, experimental group is irritated Root of Chinese Eriosema flavone extract---compound 2, presses 10mLkg
-1Irritate stomach, administration gives feeding animal preceding gastric infusion morning every day, successive administration 10d.
Behind the last administration 1h, press 0.5gkg
-1Dosage give respectively every mice by intraperitoneal injection phenol red-physiological saline, after injecting phenol red 0.5h, put to death small white mouse, carefully peel off tracheae reticular tissue on every side, cut tracheae from thyroid cartilage to the segmental bronchus crotch, put into the test tube that fills 2mL physiological saline, add 0.1mL sodium hydroxide solution (1molL again
-1), shake up, survey light absorption value A in the 546nm place.Calculate phenol red content according to phenol red typical curve, organize a t check between administration group and the negative control group.
Proofread and correct phenol red content=phenol red content (mg)/mouse body weight (kg)
The rate of reducing phlegm (%)=(the phenol red content of the phenol red content/model group of administration group) * 100%
3 results and analysis
3.1 fungistatic effect
3.1.1 the fungistatic effect of compound 2
For sample Root of Chinese Eriosema flavone extract---2 pairs of intestinal bacteria of compound, salmonella typhi, the subtilis restraining effect is not obvious, and staphylococcus aureus is had outside certain restraining effect, and fungistatic effect sees the following form:
The fungistatic effect of table 1 compound 2
3.1.2 the minimal inhibitory concentration of 2 pairs of staphylococcus aureus of compound
From the minimal inhibitory concentration experimental results of 2 pairs of staphylococcus aureus of compound as can be seen the minimal inhibitory concentration of 2 pairs of staphylococcus aureus of compound be 20mgmL
-1, the minimal inhibitory concentration experimental result sees the following form:
Each extract of table 2 is to the minimal inhibitory concentration experimental result of staphylococcus aureus
"-" expression is aseptic in the table drops out now, and "+" expression has bacterium colony to occur.
3.2 antiphlogistic effects
As can be seen from Table 3, the mouse auricular concha swelling due to Root of Chinese Eriosema flavone extract---compound 2 p-Xylol all has utmost point significant inhibitory effect, with the poor heteropole of negative control group significantly (P<0.01).
The influence of table 3 Root of Chinese Eriosema flavone extract p-Xylol induced mice auricular concha swelling
Annotate: compare with negative control group,
*P<0.01
3.3 cough suppressing effect
As can be seen from Table 4, the Root of Chinese Eriosema flavone extract can prolong the small white mouse cough latent period and reduce the interior cough number of times of per minute, with the negative control group comparing difference significance is arranged, the poor heteropole of Root of Chinese Eriosema flavone extract and negative control group is (P<0.01) significantly.
Flavone extracts such as table 4 porkling cause the influence of coughing mouse to strong aqua
Annotate: compare with physiological saline,
*P<0.01
The effect 3.4 reduce phlegm
With phenol red dosage is X-coordinate, and light absorption value A is an ordinate zou, and the drawing standard curve by Regression Equations is: y=0.1727x-0.0011; The experimental result of reducing phlegm sees Table 5, and the Root of Chinese Eriosema flavone extract has very strong resolve phlegm effect to small white mouse, compares with negative control group, and difference is extremely remarkable, has the stronger activity of reducing phlegm.
Table 5 Root of Chinese Eriosema flavone extract is to the influence of the phenol red excretion amount of mouse tracheae
Annotate: compare with negative control group,
*P<0.01
4 this part brief summary and discussion
4.1 bacteriostatic experiment
Filter paper method and tube dilution method are adopted in this test, select for use common Gram-positive and Gram-negative to represent bacterium, and the bacteriostatic activity of Root of Chinese Eriosema flavone extract is studied.Natural phant has formed the meticulous antibacterial defence system of a cover in secular natural selection process, it is rich and varied to show as antimicrobial component, as volatile oil, fat-soluble pigment, flavonoid compound, soap two, polyphenolic compound and alkaloid etc.The power of the outer bacteriostatic action of plant materials and the composition and the concentration of extract have substantial connection, when most of plant crude extracts are concentrated to higher crude drug concentration, thickness often becomes, this extract component complexity, often contain multiple antibiotic or antipathogenic composition, the composition of no bacteriostatic action such as polysaccharide, starch, resin, protein is also arranged, and the composition of no bacteriostatic action tends to influence fungistatic effect.The extract that this experiment utilization extracts Root of Chinese Eriosema carries out the segmentation separation, obtains the Root of Chinese Eriosema flavone extract---compound 2, carry out extracorporeal bacteria inhibitor test again, and making has the composition of antibacterial or fungicidal activity more concentrated, and consumption is few, and effect is more obvious.
By to the Root of Chinese Eriosema flavone extract---the extracorporeal bacteria inhibitor test of compound 2 is discovered, the Root of Chinese Eriosema flavone extract has bacteriostatic activity preferably to staphylococcus aureus, the Root of Chinese Eriosema flavone extract also has certain inhibition effect to some bacterium, but fewer active very high compound 2 materials of amount.
4.2 anti-inflammatory experiment
Along with development of life science, people recognize the pathogenesis of various inflammation gradually, and the exploitation anti-inflammatory drug has become the focus of pharmaceutical field research.And anti-inflammatory drug is mainly derived from the big chemicals of side effect at present, and Chinese medicine is in the research of anti-inflammatory drug, because of its good drug efficacy, and advantage such as the few and source of untoward reaction is abundant, more and more be subject to people's attention, the Chinese medicine that research and development has the anti-inflammatory activity composition also becomes focus gradually.
This experiment selects for use inflammatory model commonly used that it is studied, and is clinical in the hope of therefrom finding the component with anti-inflammatory action to be applied to.Be that Root of Chinese Eriosema flavone extract---compound 2 can suppress the small white mouse ear swelling effect due to the dimethylbenzene, has anti-inflammatory action preferably in the main acute inflammation model experiment that changes with the vascular permeability.
4.3 cough-relieving experiment
Cough is a kind of important guarding reflex of the upper respiratory tract, is the common sympton in the respiratory system disease.There are some researches show that the generation of cough is mainly relevant with C-fibriloceptor acceptance stimulation with the RARs acceptor, the RARs acceptor is mainly to the mechanical stimulus sensitivity, and the C-fibriloceptor is then mainly to the chemical stimulation sensitivity, as capsaicine, Citric Acid etc.
On one's body animal, cough can be produced by machinery, chemical stimulation and electricity irritation tunica mucosa tracheae, vagus nerve, wherein with chemistry and mechanical stimulus near the normal physiological situation.So drawing with chemical stimulation, this experiment employing coughs the antitussive effect of investigating medicine.Root of Chinese Eriosema flavone extract---compound 2 can prolong cough latent period, reduces the cough number of times, has good antitussive effect.
The experiment 4.4 reduce phlegm
This experiment adopts phenol red tracheae to drain method, utilizes phenol redly can measure the phenol red excretion of tracheae from tracheae excretory characteristics to injected in mice, is subjected to the influence of reagent thing to tracheorrhaphy bleeding amount with judgement.This method is easy, but washes out in the tracheae the phenol red energy loss pipe mucous membrane of feeling frustrated with highly basic.Experimental result shows, Root of Chinese Eriosema flavone extract---compound 2 obviously increases the phenol red excretion amounts of mouse tracheaes, shows to promote glandular secretion, makes the sputum viscosity degradation and is easy to expectoration, thereby play phlegm-dispelling functions.