CN108542937A - A kind of preparation method and applications of high-purity Herba Cistanches benzyl carbinol glycosides - Google Patents
A kind of preparation method and applications of high-purity Herba Cistanches benzyl carbinol glycosides Download PDFInfo
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Abstract
The invention belongs to field of traditional Chinese medicine extraction, more particularly to it is prepared from Herba Cistanches with the preparation method of two kinds of benzyl carbinol glycosides high-purity benzyl carbinol glycosides as main component and its application in preparing anti-fibrosis drug, specially a kind of preparation method and applications of high-purity Herba Cistanches benzyl carbinol glycosides.The present invention has developed three kinds of resin column method for combined use, using impurity such as Amberlite XAD16HP macroreticular resins removal sugar, pigment and non-phenylethanoid glycoside mainly are effectively removed using Amberlite FPC22Na (large porous strong acid phenylethylene resin series) and D201 (highly basic I types anion exchange resin) for three kinds of resin column combinations；This method is the preparation process that this research department is formed by various kinds of resin screening, prepared Herba Cistanches benzyl carbinol glycosides extract is faint yellow, wherein total benzyl carbinol glycosides content is more than 95% (weight ratio), the content of echinacoside and acteoside is more than 85% (weight ratio), and finds that this benzyl carbinol extract has preferable effect in aspect of resisting pulmonary fibrosis.
Description
Technical field
The invention belongs to field of traditional Chinese medicine extraction, more particularly to it is main to be prepared with two kinds of benzyl carbinol glycosides from Herba Cistanches
The preparation method of the high-purity benzyl carbinol glycosides of ingredient and its application in preparing anti-fibrosis drug, it is specially a kind of high-purity
Spend the preparation method and applications of Herba Cistanches benzyl carbinol glycosides.
Background technology
Herba Cistanches, be Orobanchaceae Cistanche deserticola perennial herb parasitic plant, its medicinal root, first recorded in《Legendary god of farming's book on Chinese herbal medicine
Through》, it is classified as top grade.It is warm-natured, it is sweet in flavor, salty, return kidney, large intestine channel, has the effect of nourishing liver supports kidney, benefiting essence-blood, relaxes bowel, for controlling
Treat the diseases such as the insufficiency of the kidney yang, blood and essence asthenia, soreness and weakness of waist and knees, muscles and bones inability, dry constipation of intestines.《Chinese Pharmacopoeia》The method that version in 2015 is recorded
It is Desert Herba Cistanches Cistanche deserticola Y.C.Ma and Cistanche tubulosa C.tubulosa (Schenk) to determine kind
Wight(《Chinese Pharmacopoeia》One, version in 2015).Phenylethanoid glycoside in Herba Cistanches is that it plays the main of benefiting action
Ingredient, this constituents are made of benzyl carbinol base and sugar, have higher water solubility, and more with liver protecting, neuroprotection etc.
Kind activity (study, 2013,36 (6) by progress [J] drug evaluations of the Herba Cistanches such as Chen Fei, Chen Zhuo, Xing Xuefei:469-
475.).Therefore, the extraction and purification process research of Herba Cistanches phenylethanoid glycoside, all has its new product development research
Significance.
In recent years, it has been reported and research (the Wu Tao pipes that benzyl carbinol glycosides purify in Herba Cistanches is carried out using macroporous absorbent resin
Extraction, purifying, finger-print and the improvement memory research of flower herba cistanches benzyl carbinol glycosides, Xinjiang University's M Sc thesis,
2010), we have found through testing that the volume containing the sample of these resins and, purity all relatively low to the purification efficiency of benzyl carbinol glycosides
Reach 40% or so.
Lung dimensionization is chronic one kind, progressive, irreversible and the most common type mortality lung disease, main performance
Appearance for fibroblast stove (fibroblastic foci) leads to a large amount of extracellular matrix (extracellular
Matrix, ECM) it deposits, collagenous accumulations, alveolar structure destroys, and eventually leads to normal lung tissue structural damage (Kong Qin, Chen Min
Progress [J] the China comparative medicine magazine of sharp idiopathic pulmonary fibrosis pathogenesis, 2012,08:74-80.).At present
Treatment means mainly based on Western medicine, especially hormone medicine, can only generally improve symptom, cannot control its process；And
Prolonged application, curative effect weakens and toxicity increases, on the whole at present clinically still without suitable medicine.In it is known that
Medicine side effect is low, although the course for the treatment of cured is longer with respect to Western medicine, can fundamentally treat disease.Therefore, from Chinese medicine
The traditional Chinese medicine ingredients for extracting, filtering out effectively treatment pulmonary fibrosis resistant, are clinically particularly important.
Invention content
In view of the problems of the existing technology, the present invention provide a kind of high-purity Herba Cistanches benzyl carbinol glycosides preparation method and
It is applied；The present invention is prepared from Herba Cistanches using echinacoside and acteoside both benzyl carbinol glycosides as main component
High-purity benzyl carbinol glycosides preparation method, which can be applied to prepare in anti-fibrosis drug.The meat being prepared
Desert cistanche extract, purity is higher, and pulmonary fibrosis resistant activity is preferable, and purity is more than 95%, can make injection, meet new Chinese medicine
Exploitation requirement.
To achieve the goals above, the preparation method of high-purity Herba Cistanches benzyl carbinol glycosides provided by the invention, specifically includes
Following steps.
Step 1 takes Herba Cistanches coarse powder, and 10 times of amounts and 8 times of amount (weight of medicinal material and the volume ratio of water) decoctings are respectively adopted
Boil extraction twice, 1 hour every time, refluxing extraction merged extracting solution twice, and being concentrated into medicinal material amount volume, (volume after concentration exists
Numerically identical as Herba Cistanches coarse powder weight, i.e., 1 kilogram of medicinal material is concentrated into 1 hydrargyrum oxydatum crudum liquid)；95% ethyl alcohol is added into concentrate,
Concentration of alcohol is set to reach 70%, the alcohol precipitation 12 hours under the conditions of 4 DEG C；Filtering, takes supernatant, is concentrated under reduced pressure into one halfbody of medicinal material amount
Product, until without alcohol taste, medicinal material amount volume is added water into concentrate.
Amberlite XAD16HP macroporous absorbent resins in step 2, the concentrate for obtaining step 1, the volume of the concentrated liquid with
Resin volume ratio is 1:2, elution speed is 0.5 concentrate loading volume per hour；After liquid adds, it is placed at room temperature for 6 hours；
4 times of amount washings of medicine liquid volume are first used, 3 times of 50% ethanol elutions of amount of medicine liquid volume are then used；Ethanol eluate first passes through
(dosage is the bodies such as upper Amberlite XAD16HP macroporous absorbent resin liquids to Amberlite FPC22Na large porous strong acids resin
Product), then (dosage is upper Amberlite XAD16HP macroporous absorbent resin liquids by D201 strong basic type anion-exchange resins
Column in equal volume), the elution speed eluted twice are a medicine liquid volume per hour；It is pure by large porous strong acid type and strong base resin
50% ethanol eluate changed is concentrated under reduced pressure, and 50 DEG C or less are dried under reduced pressure or are lyophilized, and obtain Herba Cistanches benzyl carbinol glycosides extract.
Step 3, cistanche extracts assay show in extract total benzyl carbinol glycosides content not less than 95% (weight
Amount ratio)；The total content of echinacoside and acteoside is not less than 85% (weight ratio) in total benzyl carbinol glycosides.
The cistanche extracts can be applied to prepare in anti-fibrosis drug.
Beneficial effects of the present invention.
The invention discloses a kind of application of high-purity Herba Cistanches benzyl carbinol glycosides in terms of preparing anti-fibrosis drug.It is existing
The preparation method of some Herba Cistanches benzyl carbinol glycosides substantially uses alcohol water to extract, purification with macroreticular resin method；And it is of the invention
The heavy method of -ol is carried using water, has effectively saved cost；In addition, present invention discover that the volume containing the sample of HPD series plastics used at present
It is low, and reuse number is low, general 6-7 times or so, and purification efficiency is low, and the present invention is used by continuous exploration discovery
Amberlite XAD16HP macroporous absorbent resins, not only volume containing the sample is high, and reuses number and obviously increase, up to 20 times；This
It is found in invention research process, using the purity of Herba Cistanches benzyl carbinol glycosides prepared by currently used D101 or AB-8 series plastics
Relatively low, total content reaches 40% or so, and due to the influence of pigment, actual result should be lower.In order to further obtain high-purity
Benzyl carbinol glycosides extract, the present invention has developed three kinds of resin column method for combined use, and three kinds of resin columns combinations are main to be used
Amberlite XAD16HP macroreticular resins remove the impurity such as sugar, using Amberlite FPC22Na (large porous strong acid polystyrenes
Resin) and D201 (highly basic I types anion exchange resin) effectively remove pigment and non-phenylethanoid glycoside；This method is this
Research department by various kinds of resin screen and formed preparation process, prepared Herba Cistanches benzyl carbinol glycosides extract be it is faint yellow,
Wherein total benzyl carbinol glycosides content is more than 95% (weight ratio), and the content of echinacoside and acteoside is more than 85% (weight
Than), and find that this benzyl carbinol extract has preferable effect in aspect of resisting pulmonary fibrosis.Further pass through in vivo studies knot
Fruit shows, it has been found that benzyl carbinol glycosides extract has pulmonary fibrosis effect caused by stronger anti-bleomycin.It can be seen that
Herba Cistanches benzyl carbinol glycosides in the present invention have stronger activity, and Small side effects in aspect of resisting pulmonary fibrosis, cheap, tool
There is preferable druggability.The high-purity Herba Cistanches benzyl carbinol glycosides extract of the present invention can share individually or with other medicines, be used for
The preparation of anti-fibrosis drug.
Description of the drawings
Fig. 1 lung tissue observation charts.
Fig. 2 pathologic state colony sections observation results；CON represents blank group, MOD representative model groups, and POS represents positive drug
Group, TGL represent total glycosides low dose group, and TGH represents total glycosides high dose group.
Specific implementation mode
With reference to specific embodiment, the present invention is described further.
Embodiment 1.
The preparation method of Herba Cistanches benzyl carbinol glycosides extract.
Step 1 takes 1.0 kilograms of Herba Cistanches coarse powder, with 10 liters of water boiling and extractions it is primary after, then with 8 liters of water boiling and extractions one
It is secondary, merge extracting solution, be concentrated into 1 liter, 95% ethyl alcohol is added, concentration of alcohol is made to reach 70%, 4 DEG C of alcohol precipitations 12 hours, filtering takes
Supernatant, is concentrated under reduced pressure into 0.5 liter, and no alcohol taste adds water to 1 liter.
Step 2, by Amberlite XAD16HP large pore resin absorption columns on concentrate, (stem height is than 1:9), resin volume is
It 2 liters, after liquid adds, places 6 hours, first uses 4 liters of washings, then with 3 liter of 50% ethanol elution, ethanol eluate continues through
The glass of the glass column and 1 liter of D201 strong basic type anion-exchange resin of 1 liter of Amberl ite FPC22Na large porous strong acid resin
Column.In three kinds of resin columns, elution speed is 1 liter per hour.By 50% second of large porous strong acid type and strong base purifying resin
Alcohol eluen is concentrated under reduced pressure, and 50 DEG C are dried under reduced pressure 2 hours, obtain Herba Cistanches benzyl carbinol extract 201 grams (yields 20.1%).
The Amberlite XAD16HP that the present invention uses is Rhom and Hass's products and Amberlite FPC22Na trees
Fat is the calm moral bio tech ltd production in Shanghai, and D201 is Hebei Hua Zhong Chemical Co., Ltd.s product.
Step 3, cistanche extracts assay show total 97.5% (weight of benzyl carbinol glycosides content in extract
Than), wherein echinacoside content is the content 15.5% (weight ratio) of 72%, acteoside.
The assay method of benzyl carbinol glycosides：The measurement of benzyl carbinol glycosides is carried out using ultraviolet spectrophotometry, selects echinacoside
For reference substance, due to a length of 330nm of the maximum absorption wave of echinacoside, relatively measured by 330nm dulling luminosity ratios.Reference substance is molten
The preparation of liquid：5.0 milligrams of echinacoside reference substance is taken, accurately weighed, solution of every 1ml containing 0.2mg is made in methanol, shakes up, standby
With.The preparation of test solution：Herba Cistanches powder 0.2g is taken, it is accurately weighed, it sets in 50ml brown volumetric flasks, precision is added 50%
Methanol 45ml, ultrasonic dissolution are shaken up with 50% methanol constant volume, and filtration, filtrate is set spare in brown bottle.
Echinacoside and acteoside assay method are with reference to 2015 editions in benzyl carbinol glycosides《Chinese Pharmacopoeia》It carries out：Chromatostrip
Part：Agilent Zorbax C18 columns (250mm × 4.6mm, 5 μm)；Mobile phase is -0.1% aqueous formic acid of chromatography methanol, ladder
Degree elution：0~17min, 26.5%A；17~20min, 26.5%~29.5%A；20~27min, 29.5%A；Detection wavelength
330nm.Component separating degree to be measured is more than 1.5, and theoretical cam curve is all higher than 3300 with echinacoside, acteoside calculating.
The preparation of reference substance solution：Take echinacoside reference substance, acteoside reference substance 5.0 milligrams suitable, it is accurately weighed,
Add 50% methanol that echinacoside reference substance, acteoside the reference substance respectively mixed solution containing 0.2mg is made in every l ml solution,
To obtain the final product.
The preparation of test solution:Extract 0.2g is taken, it is accurately weighed, it sets in 100ml brown measuring bottles, the dissolving of 50% methanol
(being settled to 100ml), shakes up, spare.
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product；Assay is carried out using one point external standard method.
The screening process of above-mentioned resin is as follows：We are passed through using resins such as D101 and AB-8 commonly used in the art first
Experiment obtains the content of benzyl carbinol glycosides in extract in 40-45%, and uses Amberl ite XAD16HP macroporous absorbent resins
(I), obtained extract purity reaches 65-70%, continues pure with Amberl ite FPC22Na large porous strong acids resins (II)
After change, extract benzyl carbinol glycosides purity reaches 85-89%, further combined with D201 strong basic type anion-exchange resins (III), institute
It obtains extract purity and reaches 95-97%.It is worth noting that three kinds of resins used by this technique must be used according to current sequence,
Arbitrary exchange sequence can all cause purification efficiency to reduce.In experimentation, mode, obtained purity are combined using different resins
Statistics such as table 1.
The different resin of table 1 is combined mode, obtained purity statistical form.
One, Herba Cistanches benzyl carbinol extract pulmonary fibrosis resistant Effect study.
1. material and reagent.
1.1 experimental drugs and instrument.
Herba Cistanches benzyl carbinol extract comes from embodiment 1；Hydrochloride for injection bleomycin (Nippon Kayaku K. K)；
Prednisone acetate (Tian He pharmaceutcal corporation, Ltds)；10% chloraldurate (bio tech ltd Mei Lun)；The poly- second of injection
Glycol (Tianjin great Mao chemical reagent factories)；Tracheal inserting device and rat opener (self-control of this laboratory)；RT-2100C types are more
Function full automatic microplate reader (Shenzhen Lei Du companies)；ESJ220-4A type analysis balance (Shenyang Long Teng electronics corporation).
Used kit in 1.2 experiments.
Hydroxyproline testing cassete (Bioengineering Research Institute, product batch number 20170610 are built up in Nanjing)；Malonaldehyde (MDA) is surveyed
Try box (Bioengineering Research Institute, product batch number 20170611 are built up in Nanjing)；Myeloperoxidase (MPO) testing cassete (build by Nanjing
At Bioengineering Research Institute, product batch number 20170614)；(bio-engineering research is built up in Nanjing to lactic dehydrogenase (LDH) testing cassete
Institute, product batch number 20170615)；(Bioengineering Research Institute, product batch number are built up in Nanjing to total protein quantitative test box
20170616)；TNF-α ELISA detection kit (day and marine growth Science and Technology Ltd., product batch number 201706)；IL-
6ELISA detection kits (day and marine growth Science and Technology Ltd., product batch number 201706)；TNF-beta ELISA detection kit
(day and marine growth Science and Technology Ltd., product batch number 201706)；NF- κ B ELISA detection kits (day and marine growth science and technology
Co., Ltd, product batch number 201706).
2 experimental methods.
2.1 pulmonary fibrosis model method for building up.
The chloraldurate (3mL/kg) of rats by intraperitoneal injection 10% faces upward after anesthesia and is fixed on the inclined mouse plate of 45-60 degree
On, fixing limbs take an intense light source direct irradiation in rat bottleneck throat skin surface, with wood clamp gently by rat tongue it is outside on
Pull-up, holds spatula with left hand and stretches into oral cavity and jack up tongue abdomen upwards, and visible rat oral cavity is in orange red, tracheae below oral cavity at this time
In a white bright spot, it is seen that valve opens a conjunction with rats breathing one, by No. 14 gavage needle tubings (the external 2.0mm rubber after sterilizing
Gavage pipe (sterilizing)) it is slowly inserted into tracheae to needle tubing mark arrival rat oral cavity, making rat, uprightly push-in is noted rapidly
It penetrates with bleomycin hydrochloride to ensure that drug sprays into rat trachea (5mg/kg, about 0.2 milliliter), it is rapid to take out needle tubing and inject
0.1-0.2mL air, rat vertical rotary make medicaments uniformity be distributed in lung in 3-4 weeks, and several seconds are placed in mouse cage to it
It wakes up from anesthesia；Blank group gives the physiological saline of equivalent.
2.2 dosage.
Herba Cistanches benzyl carbinol glycosides are low, high dose group is administered according to 20mg/kg and 40mg/kg respectively, and positive drug acetic acid sprinkles Buddhist nun
Pine is administered according to 5mg/kg, and Herba Cistanches benzyl carbinol glycosides and positive drug are all made of 20% polyethylene glycol and carry out mixed apolegamy system administration
Liquid.
2.3 animal packets and medication.
Choosing SD rats 50, (by Liaoning, long-living Bioisystech Co., Ltd provides, quality certification number：SCXK (the Liao Dynasty) 2010-
0001) 50 mouse are divided into blank group, model group, positive group (prednisone acetate by, weight 180-220g, animal rank SPF
Group), benzyl carbinol glycosides low dose group and high dose group, every group 10.
Model group：After modeling from the 2nd day, daily gavage gives 20% polyglycol solution (10mL/kg), continuous gavage
27 days, put to death rat within the 28th day.
Blank group：The physiological saline for giving equivalent, with model group administering mode.
Positive group：After modeling from the 2nd day, daily gavage gives prednisone acetate (5mg/kg), successive administration 27 days, and the 28th
It puts to death rat.
Benzyl carbinol glycosides low dose group：After modeling from the 2nd day, daily gavage gives benzyl carbinol glycosides (20mg/kg), successive administration
27 days, put to death rat within the 28th day.
Benzyl carbinol glycosides high dose group：After modeling from the 2nd day, daily gavage gives benzyl carbinol glycosides (40mg/kg), successive administration
27 days, put to death rat within the 28th day.
2.4 apparent index observings.
Observe rat situation, including fur after administration daily, spirit and appetite, nutrition condition, respiratory rate and mode, four
Limb color etc..
Execution, materials and the slice analysis of 2.5 rats.
Abdominal aortic blood：According to rat body weight, in the chloraldurate (3mL/kg) of injection 10% in rat abdominal cavity, anesthesia
It is lain supine upon afterwards on rat operation plate；After 75% alcohol disinfecting, abdominal cavity is cut off along abdomen median line with operating scissors, is stopped blooding
Gauze wipes a small amount of blood of spilling, gently pushes the tissue such as fat aside with cotton fully to expose abdominal aorta, aorta is in shallow
Red has bounce to feel；Disposal vacuum blood taking needle is taken accurately to be pierced into abdominal aorta, vacuum blood collection tube collects abdominal aorta blood
Liquid until not going out completely；The blood of collection is subjected to 4 DEG C of centrifugations (10min, 3000r/min) with refrigerated centrifuge, takes supernatant
Serum after packing is placed in -80 DEG C of ultra low temperature freezers and preserves by liquid, and for measuring albumen in serum, TNF-α and IL-6's contains
Amount.BAL fluid is collected：With 10% chloraldurate (3mL/kg) intraperitoneal injection of anesthesia animal, abdominal aortic blood,
Then thoracic cavity is opened, inferior caval vein is ligatured, below tracheae crotch, ligatures after the right principal bronchus that dissociates, is swum then at neck
Separate out tracheae, cut an inverse-T-shaped mouth at 0.5cm under tracheae cartilagines larynigs, by 2mL syringes (including 4 DEG C of 1mL physiological saline) with
After the gastric perfusion needle of straight peen connects, it is placed in tracheae intracavitary, reaches at 1cm, is fixed with No. 0 suture, is slowly rinsed for several times, per minor tick
30s or so, it is 4 times continuous (rate of recovery ＞ 80%), the bronchoalveolar lavage fluid of recycling pours into the 2mLEP pipes to sterilize in advance, then will
EP pipes are placed on ice bag and preserve, and to after certain amount, carry out 4 DEG C of low-temperature centrifugations (15min, 3000r/min), are taken with pipettor
It is clear to preserve, the content for measuring albumen and LDH in irrigating solution.
Lung tissue detaches：After having collected bronchoalveolar lavage fluid, rat or so lungs are isolated, physiological saline washes away part-blood
After liquid, blotting paper suck dry moisture carefully separates extra tissue, and assay balance is weighed, accurate weighing lung weight, and records.It takes
Right lung, which is weighed, is divided into three parts, after part homogenate packing, takes supernatant for the kit measurements such as ELISA and MDA, a part
Measurement of the lung tissue for MPO contents, a part of weighed tissue wet is taken to be used for the measurement of HYP contents；Three parts are stored in-
In 80 DEG C of refrigerators.
Lung tissue section analyzes：The right lung of rat is taken to be put into 4% paraformaldehyde fixed.Routinely pathology method packet
It buries, be sliced.Pathological section is subjected to HE dyeing, is then analyzed.
2.6 indicators in vivo measure.
2.6.1 the measurement of paragonimus cyst.
Paragonimus cyst=lung weight (mg)/weight (g) × 100%.
2.6.2 total protein content measures.
The measurement of total protein uses BCA methods, is specifically carried out according to kit specification；Process is as follows.
(1) tissue samples pre-treatment：Accurately weigh tissue weight.By weight (g)：Volume (mL)=1:9 ratio is added
The physiological saline of 9 times of volumes, mechanical homogenisation under the conditions of ice-water bath, 2500r/min centrifuge 10min, supernatant are taken to use physiology salt again
Water is diluted to the homogenate of 1% tissue, to be measured.
Serum sample pre-treatment：By serum：Physiological saline=1:99 dilution proportion, it is to be measured.
Bronchoalveolar lavage fluid Sample pretreatment：It takes 2 samples to carry out preliminary experiment, dilutes in proportion, measurement range 20-2000
μg/mL。
(2) 20 μ L of distilled water are added in blank tube, and 20 μ L of standard items are added in standard pipe, measure pipe and 20 μ L of sample to be tested are added,
250 μ L of working solution, whirlpool mixing is added into each pipe respectively, 37 DEG C of incubation 30min take out to be added to terminate and apply 750 μ L of liquid, whirlpool
Whirlpool mixing stands 5 minutes, wavelength 562nm, optical path 0.5cm, and distilled water zeroing measures each pipe absorbance value.
Calculation formula：
2.6.3 the measurement of lung permeation index.
The measurement of total protein concentration uses BCA methods in this experiment；Total protein concentration in lung permeation index=bronchoalveolar lavage fluid/
Total protein concentration in serum.
2.6.4 lung tissue hydroxyproline (HYP) assay.
Assay method is carried out according to kit specification, and specific method is：The fritter lung tissue of freezen protective is put into examination
In pipe, accurately add hydrolyzate 0.1mL, mixing, 95 DEG C of water-baths hydrolysis 20min after capping, by each test tube liquid pH value tune after cooling
To 6.0-6.8 or so, add distilled water to 10mL, mixing, the solution after taking 3-4mL to dilute adds proper amount of active carbon, mixing, 3500r/
Min centrifuges 10min, takes supernatant 1mL, after sequentially adding three kinds of reagents, mixing, and 60 DEG C of water-bath 15min, mixing, 60 DEG C of water-baths
15min, 3500r/min centrifuge 10min, take supernatant in wavelength 550nm, 1cm optical paths, and distilled water zeroing measures the extinction of each pipe
Degree.
Calculation formula：
2.6.5 myeloperoxidase (MPO), malonaldehyde (MDA) assay in lung tissue.
It is measured according to chemical kit specification.
2.6.6ELISA method measures TNF-α, the content of NF- κ B, IL-6, TGF-β.
The measurement of these indexs is carried out according to kit operational manual, and substantially operating process is as follows.
(1) lath needed for being taken out from the aluminium foil bag after equilibrium at room temperature 20min, remaining lath valve bag sealing put back to 4
℃。
(2) standard sample wells is set and sample aperture, standard sample wells respectively add the 50 μ L of standard items of various concentration.
(3) sample aperture first adds 10 μ L of sample to be tested, then adds 40 μ L of Sample dilution；Blank well is not added with.
(4) in addition to blank well, the detection of horseradish peroxidase (HRP) label is added in standard sample wells and sample aperture per hole
100 μ L of antibody seal reacting hole with sealing plate film, and 37 DEG C of water-baths or insulating box incubate 60min.
(5) liquid is discarded, is patted dry on blotting paper, cleaning solution is filled it up with per hole, stands 1min, gets rid of cleaning solution, on blotting paper
It pats dry, so repeats board-washing 5 times (can also be machine-washed plate with board-washing).
(6) substrate A, each 50 μ L of B are added per hole, 37 DEG C are protected from light incubation 15min.
(7) it is added in terminate liquid 50 μ L, 15min per hole, each hole is measured at 450nm wavelength.
3. experimental result.
The apparent index observing of 3.1 rats.
Rat after modeling, occurs at 3 days different degrees of apathetic, and diet declines with amount of drinking water, later appetite by
Gradually restore normal；Rat is short of breath within 7 days, and abdominal respiration effect is apparent, and happiness, which is closed one's eyes, rolls up；There is abdomen in partial rat within 14 days
Portion swells, and hair is at random, and the universal build of rat is become thin；There is cyanosis in rat hindlimb at 25 days, and breathing is very rapid, and spirit is not
It shakes, amount of exercise is reduced.
3.2 lung tissues are observed.
Blank group lung pinkiness, no oedema, surface is smooth, full, and soft texture is flexible, and alveolar wass is easier to,
See the figure A in Fig. 1.
Model group lung be in kermesinus, the part lobe of the lung have it is different degrees of whiten, there is fibrous texture on surface, and quality is harder,
There is a certain amount of blutpunkte, has the caking of white, elasticity is poor, sees the figure B in Fig. 1.
3.3 paragonimus cyst.
Model group Earlier period of inflammation cell largely oozes out, and a large amount of hyperplasia of late period fibroblast, collagen over-deposit can all be led
Cause paragonimus cyst to increase, therefore, paragonimus cyst this index can indirect reaction lung fibrosis in rats inflammation and fibrosis degree；Lung system
Several litres height can indirect reaction fibroblast proliferation degree it is big.2 are shown in Table, compared with blank group, the paragonimus cyst of model group is significantly raised
(P<0.05), positive group paragonimus cyst significantly increases (P<0.01).Compared with model group, low, the high dose group of benzyl carbinol glycosides have
It is apparent to decline, but no difference of science of statistics.
2 paragonimus cyst of table influence (N=10).
| Group | n | Paragonimus cyst |
| CON | 10 | 5.50±0.71* |
| MOD | 10 | 7.15±1.01△ |
| POS | 10 | 7.75±2.21△△ |
| TGL | 10 | 6.15±1.37 |
| TGH | 10 | 6.22±1.90 |
Note：CON represents blank group, MOD representative model groups, and POS represents positive group, and TGL represents benzyl carbinol glycosides low dose group,
TGH represents benzyl carbinol glycosides high dose group.Data are examined using one-way analysis of variance LSD, compared with model group, * P<0.05, * *
P<0.01；Compared with blank group, △ P<0.05, △ △ P<0.01.
The influence of 3.4 pairs of lung permeability coefficients.
After lung's extensive injuries, inflammatory cell infiltration, which consumes a large amount of oxygen, can cause oxygen relative deficiency, to adapt to tissue generation
The needs thanked stimulate release angiogenesis factor by hypoxia inducible factor, cause capillary compensatory hypertrophy and lead to blood
Pipe abnormal excessive generates and reconstruct.Meanwhile angiogenesis can promote to stick and increase vasopermeability during Inflammatory Lesions
And aggravate inflammatory reaction.Lung permeability coefficient size can reflect the size of vasopermeability indirectly.3 are shown in Table, compared with blank group, mould
Type group, positive group, benzyl carbinol glycosides are low, the high dose group significant difference (P of lung permeability coefficient<0.01)；With model group ratio
Compared with benzyl carbinol glycosides are low, high dose group all has lung permeation index and is substantially reduced effect (P<0.05).
Table 3 to lung permeability coefficient influence (N=10).
| Group | n | Lung permeability coefficient |
| CON | 10 | 0.62±0.06** |
| MOD | 10 | 0.93±0.08△△ |
| POS | 10 | 0.85±0.08△△ |
| TGL | 10 | 0.77±0.15* |
| TGH | 10 | 0.78±0.14* |
Note：CON represents blank group, MOD representative model groups, and POS represents positive group, and TGL represents benzyl carbinol glycosides low dose group,
TGH represents benzyl carbinol glycosides high dose group.Data are examined using one-way analysis of variance LSD, compared with model group, * P<0.05, * *
P<0.01；Compared with blank group, △ P<0.05, △ △ P<0.01.
Relevant enzyme Index Influence in 3.5 pairs of lung tissues.
3.5.1 the influence of hydroxyproline (HYP).
HYP is one of the main component of body collagen, accounts for about the 13% of its total amino acid content, other remove elastin laminin
Outside containing a small amount of HYP (about 1%), HYP is free of, therefore HYP can be used as the important indicator for weighing collagen content.Because fibrosis is formed
The reason is that the collagen self-regeneration of lung, therefore HYP is increased.
4 are shown in Table, compared with blank group, the HYP content apparent increases (P of benzyl carbinol glycosides high dose group<0.05), model group with
The HYP contents of positive group significantly increase (P<0.01).Compared with model group, benzyl carbinol glycosides high dose group is substantially reduced (P<
0.05), benzyl carbinol glycosides low dose group and arctigenin low dose group significantly reduce (P<0.01).
3.5.2 myeloperoxidase (MPO) active influence.
The principle and meaning of MPO Activity determinations：Myeloperoxidase is one of heme peroxidases superfamily member,
It is primarily present in the aniline blue particles of neutrophil leucocyte.Its horizontal and active variation represents the function of neutrophil leucocyte
And activated state.The enzyme has the ability for making hydrogen-peroxide reduction, is capable of the vigor of enzyme analysis using this feature and determines indirectly
Measure the number of neutrophil leucocyte.MPO activity is bigger, illustrates that inflammatory reaction is more serious.
4 are shown in Table, compared with blank group, the MPO activity of model group significantly increases (P<0.01).It is positive compared with model group
Group, benzyl carbinol glycosides high dose group MPO activity are substantially reduced (P<0.05), benzyl carbinol glycosides low dose group significantly reduces (P<0.01).
3.5.3 the influence of malonaldehyde (MDA) content.
Superoxide dismutase (superoxide dismutase, SOD) plays the oxidative and anti-oxidative balance of body
Vital effect, this enzyme can remove ultra-oxygen anion free radical and protect cells from damage.Body by enzyme system with it is non-
Enzyme system generates oxygen radical, and the latter can attack the polyunsaturated fatty acid (polyunaturatedfatty in biomembrane
Acid, PUFA), cause lipid peroxidation, and form lipid peroxide.Therefore the amount of MDA can reflect body inner lipid
The degree of peroxidating reflects the degree of cellular damage indirectly.It is bigger that MDA increases reflection cell oxidative damage degree.
4 are shown in Table, compared with blank group, the MDA contents of model group have raising trend.Compared with model group, benzyl carbinol glycosides are low
Dosage group is substantially reduced, but is not statistically significant.
3.5.4 in bronchoalveolar lavage fluid lactic dehydrogenase (LDH) influence.
The principle and meaning of LDH activity detection:LDH is to be present in a kind of intracellular catalyzing enzyme, participates in pyruvic acid and breast
Mutually converting between acid.It can be leaked to when body tissue cell is damaged extracellularly and arrive extracellular LDH contents and work
Property increase.Therefore LDH contents or active measurement can reflect the impaired degree of histocyte indirectly, can be used to diagnose sometimes certain
Disease or the evaluation for carrying out curative effect of medication.The content for measuring two ketone acids at wavelength 440nm by colorimetric method, can extrapolate
The activity of LDH and reflect the degree that histocyte is damaged indirectly.LDH activity is bigger, and it is bigger that cell damage hinders degree.
4 are shown in Table, compared with blank group, the LDH activity of model group, positive group significantly increases (P<0.01), with model group ratio
Compared with two dosage group LDH activities of benzyl carbinol glycosides significantly reduce (P<0.01).
Relevant enzyme and albumen Index Influence in 4 lung tissue of table (N=10).
Note：CON represents blank group, MOD representative model groups, and POS represents positive group, and TGL represents benzyl carbinol glycosides low dose group,
TGH represents benzyl carbinol glycosides high dose group.Data are examined using one-way analysis of variance LSD, compared with model group, * P<0.05, * *
P<0.01；Compared with blank group, △ P<0.05, △ △ P<0.01.
The influence of correlation factor index in 3.6 pairs of lung homogenates.
3.6.1 the influence of transforming growth factor β (TGF-β).
Transforming growth factor β (the transforming growth factor near repairing phase, fibrosis lesion
Beta, TGF-β) raising is expressed, TGF-β is important fibrosis growth factor, can stimulate fibroblast proliferation and collagen
Synthesis promotes collagen, fibronectin, hyaluronic acid, proteoglycans etc. to be deposited in interstitial lung, reduces the degradation of ECM.
5 are shown in Table, compared with blank group, the TGF-β content of model group significantly increases (P<0.01).Compared with model group, sun
Property group, benzyl carbinol glycosides low dose group, the TGF-β content of benzyl carbinol glycosides high dose group significantly reduce (P<0.01).
3.6.2 the influence of nuclear Factor-Kappa B (NF- κ B).
TNF-α itself can stimulate NF- kB activations, the NF- κ B of activation to increase the formation of TNF-α again in turn, and the two is mutual
Promote.The expression of lung tissue TNF-α and NF- κ B increase, the common pathologic process for participating in causing pulmonary fibrosis.The expression of NF- κ B is stagnant
Afterwards in the expression of TNF-α, show that TNF-α may be one of the initiating agent of NF- kB activations.
5 are shown in Table, compared with blank group, the NF- κ B contents of model group and positive group significantly increase (P<0.01), and model
Group compares, and benzyl carbinol low dose group is substantially reduced (P with high dose group NF- κ B contents<0.01).
3.6.3 the influence of tumor necrosis factor-alpha (TNF-α).
5 are shown in Table, compared with blank group, the TNF-α content of model group significantly increases (P<0.01).Compared with model group, benzene
The TNF-α content of ethyl alcohol glycosides high dose group is substantially reduced (P<0.05), positive group, the TNF-α content of benzyl carbinol glycosides low dose group
Significantly reduce (P<0.01).
3.6.4 the influence of interleukin-6 (IL-6).
5 are shown in Table, compared with blank group, the IL-6 content apparent increases (P of the positive group and benzyl carbinol glycosides low dose group<
0.05), the IL-6 contents of model group and benzyl carbinol glycosides low dose group significantly increase (P<0.01).Compared with model group, benzyl carbinol
The IL-6 contents of glycosides high dose group are substantially reduced (P<0.05).
Correlation factor Indexs measure result in 5 lung homogenate of table (N=10).
| Group | TGF-β(ng/ml) | NF-κB(ng/ml) | TNF-α(ng/ml) | IL-6(ng/ml) |
| CON | 1.85±0.11** | 7.50±0.65** | 0.92±0.17** | 1.02±0.10** |
| MOD | 2.07±0.08△△ | 9.76±1.60△△ | 1.23±0.13△△ | 1.25±0.11△△ |
| POS | 1.89±0.07** | 9.80±1.70△△ | 0.93±0.15** | 1.17±0.08△ |
| TGL | 1.79±0.03** | 8.01±0.60** | 0.95±0.13** | 1.18±0.06△ |
| TGH | 1.87±0.06** | 7.23±1.01** | 1.01±0.15* | 1.09±0.05* |
Note：CON represents blank group, MOD representative model groups, and POS represents positive group, and TGL represents benzyl carbinol glycosides low dose group,
TGH represents benzyl carbinol glycosides high dose group.Data are examined using one-way analysis of variance LSD, compared with model group, * P<0.05, * *
P<0.01；Compared with blank group, △ P<0.05, △ △ P<0.01.
3.7 pathologic state colony sections observations.
Each group lung tissue HE stained slices (10 × 10), sections observation result are shown in Fig. 2.
The normal bronchus of rat for organizing lung tissue, alveolar epithelial cells and alveolar septum institutional framework are normal, and iuntercellular connects
It connects closely, vascular endothelial cell and basement membrane are intact, the changes such as no congested, oedema and active chronic inflammation.
Model group rats pulmonary capillaries are congested, there is inflammatory cell aggregation around bronchus and blood vessel, alveolar space atrophy,
It disappears, alveolar wall is broken, the apparent broadening and congestion and edema of alveolar septum, vessel wall thickening, visible a large amount of neutral grains in interstitial lung
Cell and mononuclear macrophage infiltration, collagenous fibres and fibroblast proliferation and with lymphocytic infiltration, fibroblast increases
It is more, and have a large amount of irregular, disorganized Collagen fiber depositions.
The accidental interstitial Mild edema of positive drug prednisone acetate group, inflammatory cell infiltration are reduced, and Collagen fiber deposition is compared with mould
Type group has certain mitigation, and the obvious alveolar structure in part can be observed.
Benzyl carbinol glycosides high and low dose group group part pulmonary capillaries are congested, and slight oedema occurs in small part alveolar cell, scorching
Property cellular infiltration it is less, have few Collagen fiber deposition, alveolar structure is more apparent clear.
The above result shows that Herba Cistanches benzyl carbinol glycosides can be from anti-inflammatory, anti-oxidant and Immune-enhancing effect etc. to pulmonary fibrosis
Therapeutic effect is played, can be applied in the drug of pulmonary fibrosis resistant disease.
Claims (3)
1. the preparation method of high-purity Herba Cistanches benzyl carbinol glycosides, which is characterized in that specifically include following steps：
Step 1 takes Herba Cistanches coarse powder, 10 times of amounts and 8 times to measure (weight of medicinal material and the volume ratio of water) water boiling and extractions twice, often
Secondary 1 hour, refluxing extraction merged extracting solution twice, be concentrated into medicinal material amount volume (volume after concentration numerically with Herba Cistanches
Coarse powder weight is identical, i.e., 1 kilogram of medicinal material is concentrated into 1 hydrargyrum oxydatum crudum liquid)；95% ethyl alcohol is added into concentrate, concentration of alcohol is made to reach
70%, the alcohol precipitation 12 hours under the conditions of 4 DEG C；Filtering, takes supernatant, is concentrated under reduced pressure into medicinal material amount half volume, until without alcohol taste, to
Medicinal material amount volume is added water in concentrate.
Amberlite XAD16HP macroporous absorbent resins, the volume of the concentrated liquid and resin in step 2, the concentrate for obtaining step 1
Than being 1:2, elution speed is 0.5 concentrate loading volume per hour；After liquid adds, it is placed at room temperature for 6 hours；First use liquid
Then 3 times of 50% ethanol elutions of amount of medicine liquid volume are used in 4 times of amount washings of volume；Ethanol eluate first passes through Amberlite
FPC22Na large porous strong acids resin (dosage is that upper Amberlite XAD16HP macroporous absorbent resin liquids are isometric), then pass through
D201 strong basic type anion-exchange resins (dosage is that upper Amberlite XAD16HP macroporous absorbent resin liquids are isometric) column,
The elution speed eluted twice is a medicine liquid volume per hour；By 50% second of large porous strong acid type and strong base purifying resin
Alcohol eluen is concentrated under reduced pressure, and 50 DEG C or less are dried under reduced pressure or are lyophilized, and obtain Herba Cistanches benzyl carbinol glycosides extract；
Step 3, cistanche extracts assay show in extract that total benzyl carbinol glycosides content is not less than 95% (weight
Than)；The total content of echinacoside and acteoside is not less than 85% (weight ratio) in total benzyl carbinol glycosides.
2. the preparation method of high-purity Herba Cistanches benzyl carbinol glycosides as described in claim 1, which is characterized in that specifically include following
Step：
Step 1 takes 1.0 kilograms of Herba Cistanches coarse powder, with 10 liters of water boiling and extractions it is primary after, then it is primary with 8 liters of water boiling and extractions, close
And extracting solution, it is concentrated into 1 liter, 95% ethyl alcohol is added, concentration of alcohol is made to reach 70%, 4 DEG C of alcohol precipitations 12 hours, filtering takes supernatant
Liquid, is concentrated under reduced pressure into 0.5 liter, and no alcohol taste adds water to 1 liter；
Step 2, by Amberlite XAD16HP large pore resin absorption columns on concentrate, (stem height is than 1:9), resin volume is 2 liters,
After liquid adds, places 6 hours, first use 4 liters of washings, then with 3 liter of 50% ethanol elution, ethanol eluate continues through 1 liter
The glass column of the glass column and 1 liter of D201 strong basic type anion-exchange resin of Amberlite FPC22Na large porous strong acid resins.
In three kinds of resin columns, elution speed is 1 liter per hour.By 50% ethanol elution of large porous strong acid type and strong base purifying resin
Liquid is concentrated under reduced pressure, and 50 DEG C are dried under reduced pressure, and obtain 201 grams of Herba Cistanches benzyl carbinol extract, yield 20.1%；
Step 3, cistanche extracts assay show total benzyl carbinol glycosides content 97.5% in extract, wherein Echinacea purpurea
Glycosides content is the content 15.5% of 72%, acteoside.
3. the high-purity Herba Cistanches benzyl carbinol glycosides as described in claim 1-2 can be used for preparing anti-fibrosis drug.
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