CN101468104B - Chinese medicinal compound preparation for treating osteoporosis and method for preparing the same - Google Patents
Chinese medicinal compound preparation for treating osteoporosis and method for preparing the same Download PDFInfo
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Abstract
The invention relates to a traditional Chinese medicine composite preparation for treating osteoporosis, especially relates to effective parts in traditional Chinese medicine composite preparation and preparation method thereof, belonging to the traditional Chinese medicine field. The traditional Chinese medicine composite preparation is composed of barrenwort epimedium, astragalus root, prepared tuber fleeceflower, rhizome of drynaria and medlar, based on Chinese medicine theory 'kidney-nourishing and essence-strengthening'. The traditional Chinese medicine composite preparation for treating osteoporosis is prepared by extracting and purificating the total polysaccharide and total flavone parts as effective parts with respectively molecular weight cutoff of less than 10000 and 10000-50000 from the ram materials using separation and puration technique such as macropore adsorbed resin and ultra-filtration. The vitro experiment of serum containing the above effective parts shows that the effective parts have obvious promotion effect on the in-vitro osteoblast proliferation rate.
Description
Technical field
The invention belongs to the field of Chinese medicines, relate to a kind of compound Chinese medicinal preparation for the treatment of osteoporosis and preparation method thereof.
Background technology
Osteoporosis is because the change of the fine structure of whole body sclerotin and osseous tissue.Cause that bone strength weakens, bone fragility increases, and the systemic multi-pathogenesis skeletal diseases of fracture very easily takes place.Studies show that the U.S., Europe and nearly 7,500 ten thousand people of Japan get involved, wherein 1/3 is postmenopausal women; Domestic situation also allows of no optimist, and 60～69 years old elderly woman incidence of osteoporosis is up to 50%～70%, and the elderly men incidence rate is about 30%.Along with social population's aging, osteoporotic incidence rate also can continue to increase, and brings white elephant for therefrom family and society.
Chinese medicine has curative effect preferably by methods such as the kidney invigorating, spleen invigorating aspect the treatment osteoporosis.Still at the Chinese medicine preparation of development process, mostly be the Chinese medicine compound crude extract from go on the market and report, general taking dose is bigger, and clinical drug compliance is relatively poor, especially the sufferers of osteoporosis face that needs are taken for a long time.Even have indivedual preparations through the modern times extract, purification technique, dose is greatly reduced, but mostly is single medicinal material, can't embody drug matching and use advantages such as the Synergistic that produced, attenuation.
Therefore at the effective Chinese medicine compound of osteoporosis disease, from the effective site aspect, by suitable extracting method and technology, extract as much as possible and keep wherein effective ingredient, to guarantee the drug effect of Chinese medicine compound, be that Chinese medicine (compound recipe) improves curative effect, less dose, a kind of feasible method of increase technology content.
Summary of the invention
The purpose of this invention is to provide a kind of the kidney invigorating and essence nourishing effect that has, be used for a kind of compound Chinese medicinal preparation for the treatment of osteoporosis of osteoporosis treatment.
Further purpose of the present invention provides the preparation method of above-mentioned compound Chinese medicinal preparation, relates to effective site in the described Chinese medicine compound and preparation method thereof more specifically.
The present invention is according to " kidney storing essence; the gas of bone marrow also " of Chinese medicine, and " kidney governing bones " is that kidney has the living marrow of store essential substances, nourishes the intreractive theory of skeleton, adopt Chinese crude drug Herba Epimedii, the Radix Astragali, Radix Polygoni Multiflori Preparata, Rhizoma Drynariae, Fructus Lycii, make the compound preparation of treatment osteoporosis.
Compound preparation of the present invention is made by following bulk drugs component:
2 parts of Herba Epimedii, 1.5 parts of the Radixs Astragali, 1 part of Radix Polygoni Multiflori Preparata, 1 part of Rhizoma Drynariae, 1 part of Fructus Lycii
Herba Epimedii Xin Ganwen in the side goes into the Liver and kidney warp, kidney invigorating and YANG supporting, hard rigidity of soft tissues bone; The Radix Polygoni Multiflori " detailed outline " meaning: " the temperature compensation liver can be restrained vital essence for this thing temperature bitter and puckery flavor, bitter the kidney invigorating, thus the liver benefiting that can nourish blood, the controlling nocturnal emission with astringent drugs kidney tonifying, muscle reinforcing and bone strengthening, not warm not dry, lay particular stress on YANG invigorating with Herba Epimedii and match, positively cloudy, cloudyly positive with length with life, make negative and positive gentle; The sweet tepor of the Radix Astragali is the QI invigorating key medicine, and Radix Astragali QI invigorating lays particular stress on the flesh table, thus by QI invigorating flourish muscles and bones, granulation promoting meat; Rhizoma Drynariae has the double effect of invigorating kidney, promoting blood circulation concurrently, and it is very suitable to control lumbago due to renal deficiency, has both helped the product of all kidney tonifying, and kidney tonifying and negative and positive are taken into account is helped Radix Astragali action QI and blood again, is convenient to medicine to sick institute; The Fructus Lycii sweet in the mouth is flat, the kidney invigorating and essence nourishing, and the merit of YIN nourishing is better than supporing yang, is equipped with the Radix Polygoni Multiflori, the Radix Astragali to reach the effect of flat nourishing YIN sun, replenishing vital essence and invigorating vital QI.
By adopting macroporous adsorbent resin, ultrafiltration etc. to separate purification technique, the total polysaccharides and the total flavone part that extract in the above-mentioned compound preparation of purification are effective site more specifically in the present invention, as follows the compound Chinese medicinal preparation of preparation treatment osteoporosis.Its molecular cut off of described total polysaccharides is:＜1 ten thousand and 1～50,000.
Described effective site prepares by following step:
Above-mentioned bulk drugs adds 10 times in water, soaks 30 minutes, decocts 1 hour for the first time, for the second time add 10 times in water, decocted 1 hour, cross 200 order nets, merge extractive liquid,, channel separator centrifugal (16000r/min) is got supernatant, is evaporated to extracting solution 1ml and contains the 1g crude drug.Twice of the ethanol precipitate with ethanol of adding 95% (containing the alcohol amount is 80%) must precipitate part and pure molten part;
Described precipitation part thin up to 30% (in the medical material amount) is crossed the ultrafilter membrane of 10,000 and 50,000 molecular cut offs with ultrafilter,＜1 ten thousand polysaccharide position, 1～50,000 polysaccharide position, is drying to obtain the total polysaccharides position at concentrating under reduced pressure;
The molten part of alcohol is not distinguished the flavor of to there being alcohol through decompression recycling ethanol, thin up contains the 1g raw medicinal herbs to 1ml, adopt DM301 macroporous adsorbent resin (blade diameter length ratio is 1: 10), with sample on the 0.25BV/h speed (wet resin throwing amount is 2: 1 with the crude drug ratio), use 3～10 times (comparing) distillation washing and 3～10 times of amounts (comparing), 10% ethanol flush away impurity respectively with the wet resin amount with the wet resin amount, (it is 10: 1 with the crude drug ratio that eluting holds the agent consumption to use 70% ethanol elution purification total flavones then, the suction speed of taking off is 1ml/min (being equivalent to 2.3BV/h)), collect to inhale and take off liquid, decompression recycling ethanol, concentrating under reduced pressure, be drying to obtain total flavone part.
Above-mentioned＜10,000 polysaccharide positions, 1～50,000 polysaccharide position and total flavone part extract add appropriate amount of starch through pulverizing, and mixing incapsulates and promptly gets compound preparation of the present invention.
Warp is to the experiment in vitro of the pastille serum of above-mentioned effective site-total flavones and molecular cut off＜10,000 polysaccharide and molecular cut off 1～50,000 polysaccharide, and the result shows that described effective site has obvious facilitation to the osteoblastic proliferation rate of In vitro culture.
Description of drawings
Fig. 1 is the leakage plot of total flavones among the present invention, icariin, stilbene glucoside and naringin.
Fig. 2 is total flavones among the present invention, icariin, stilbene glucoside and naringin elution curve,
Wherein, A-total flavones B-icariin C-stilbene glucoside D-naringin;
The explanation of total flavones elution curve volumetric flask numbering:
1,2-water elution liquid (50ml); 3,4,5,6-water elution liquid (25ml); 7,8,9,10-10% alcohol eluen (25ml); 11,12,13,14-70% alcohol eluen (25ml); 15,16,17,18-90% alcohol eluen (25ml); 19,20,21-95% alcohol eluen (25ml);
Icariin, stilbene glucoside and the explanation of naringin volumetric flask numbering:
1,2,3,4-water elution liquid (50ml); 5,6,7,8-10% alcohol eluen (25ml);
9,10,11,12-70% alcohol eluen (25ml); 13,14,15,16-90% alcohol eluen (25ml);
17,18,19,20-95% alcohol eluen (25ml).
Fig. 3 is the influence of the pastille serum of the different extract parts of the present invention to the osteoblastic proliferation rate.
The specific embodiment
Embodiment 1 extraction and purification process
Get each crude drug by described weight portion proportioning, add 10 times in water, soaked 30 minutes, decocted 1 hour for the first time, for the second time add 10 times in water, decocted 1 hour, cross 200 order nets, merge extractive liquid,, channel separator centrifugal (16000r/min) is got supernatant, is evaporated to extracting solution 1ml and contains the 1g crude drug, twice of the ethanol precipitate with ethanol of adding 95% (containing the alcohol amount is 80%) must precipitate part and pure molten part;
Precipitation part thin up to 30% (in the medical material amount) is crossed the ultrafilter membrane of 10,000 and 50,000 molecular cut offs with the plate and frame ultrafilter,＜1 ten thousand position, 1～50,000 position and＞5 ten thousand positions, are drying to obtain the dry thing of polysaccharide different parts at concentrating under reduced pressure;
To there not being the alcohol flavor, thin up contains the 1g raw medicinal herbs to 1ml to the molten part of alcohol through decompression recycling ethanol.Select the DM301 macroporous adsorbent resin for use, with sample on the 0.25BV/h speed, use 10 times (comparing) distillation washing and 10 times of amounts (comparing), 10% ethanol flush away impurity respectively with the wet resin amount with the wet resin amount, (wet resin throwing amount is 2: 1 with the crude drug ratio to use 70% ethanol elution purification total flavones then, it is 10: 1 with the crude drug ratio that eluting holds the agent consumption, blade diameter length ratio is 1: 10, the suction speed of taking off is 1ml/min (being equivalent to 2.3BV/h)), collect to inhale and take off liquid, decompression recycling ethanol, is drying to obtain the total flavone part extract at concentrating under reduced pressure.
Embodiment 2 sets up the method for measuring total polysaccharides content
1) instrument and reagent
UV-240 ultraviolet spectrophotometer (Tianjin, island), and anhydrous glucose (assay usefulness, purchase in Chinese pharmaceutical biological product and identify institute, lot number: 110833-200302), anthrone (CP).
2) preparing standard solution and developer
The glucose standard solution: precision takes by weighing the anhydrous glucose 22mg of 105 ℃ of constant weights, places the 25ml volumetric flask, and adding distil water also is diluted to scale, shakes up the standard solution that promptly gets C=0.88mg/ml.
0.2% anthrone developer: take by weighing anthrone 0.2g, place the 100ml volumetric flask, add concentrated sulfuric acid dissolution and be diluted to scale, face with stylish and join.
3) select maximum absorption wavelength
The above-mentioned standard solution 0.1ml that makes is in test tube in accurate absorption, adds distilled water 0.9ml to 1.0ml; The accurate 0.2% anthrone developer 3.0ml that adds shakes up, be placed to room temperature after, adding developer 3.0ml with distilled water 1.0ml is blank, carries out absorption spectrum in 500～700nm wave-length coverage and scans, the result is presented at the 625nm place absorption maximum, solution is aeruginous.After showing that total polysaccharides of the present invention adds the colour developing of 0.2% anthraquinone concentrated sulfuric acid solution, maximum absorption band is arranged at the 625nm place.
4) preparation standard curve
Accurate standard solution 0.15,0.25,0.50,0.75,1.00 and the 1.20ml of drawing places the 10ml volumetric flask respectively, and thin up is to scale.Get 0.0132,0.022,0.044,0.066,0.088 respectively, the 0.1056mg/ml standard solution; Other gets the standard solution 5ml of C=0.0132mg/ml, places the 10ml volumetric flask, adds distilled water diluting to scale, gets C=0.0066mg/ml.The standard solution 1ml that gets above-mentioned 7 kinds of concentration respectively is in test tube, the anthrone developer 3.0ml of accurate adding 0.2%, shake up, after being placed to room temperature, measure trap at the 625nm place, and to add developer 3.0ml with distilled water 1.0ml be blank, regression equation be: Y=0.0112x+0.0898, r=0.9949, the range of linearity is 6.6～105.6 μ g/ml.
Table .1 is the standard curve of preparation.
Table .1
| Standard substance concentration (μ g/ml) | Absorbance (A) |
| 105.6 88 66 44 22 13.2 6.6 | 1.258 1.066 0.837 0.608 0.417 0.197 0.117 |
5) precision test
Precision is measured standard solution (C=0.0352mg/ml) 1ml, and totally 6 parts, according to the anthrone developer that adds 0.2% under the standard curve item from precision, measure its absorbance, the result shows that its mean light absorbency is 0.437, RSD is 1.98%.Table 2 is Precision test result.
Table 2
| Tested number | 1 | ?2 | ?3 | ?4 | ?5 | ?6 | Meansigma methods (A) | RSD(％) |
| Measured value | 0.438 | ?0.448 | ?0.442 | ?0.435 | ?0.422 | ?0.437 | 0.437 | 1.98 |
6) replica test
Precision is measured same sample solution 1ml, totally 6 parts, according to the anthrone developer that adds 0.2% under the standard curve item from precision, measures its absorbance.The result shows that its mean light absorbency is 0.4836, and RSD is 3.08%.Table 3 is replica test results.
Table 3
| Tested number | 1 | ?2 | ?3 | ?4 | ?5 | ?6 | Meansigma methods (A) | RSD(％) |
| Measured value | 0.499 | ?0.494 | ?0.494 | ?0.466 | ?0.465 | ?0.484 | 0.484 | 3.08 |
7) PSPP content Determination of Polysaccharide
Precision is measured the sectional polysaccharide solution of PSPP, behind the dilution standardize solution, and extracting sample solution 1ml, the anthrone developer 3.0ml of accurate adding 0.2% shakes up, and picks up counting in adding sulphuric acid, measures its absorbance behind 30min, and the mensuration wavelength is 625nm.
Embodiment 3 sets up the total flavones assay method
1.) instrument and reagent
UV-240 ultraviolet spectrophotometer (Tianjin, island), icariin (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number: 0737-9910).
2.) preparing standard solution and developer
The icariin reference substance solution: precision takes by weighing icariin reference substance 1.4mg, places the 10ml volumetric flask, adds 60% ethanol dilution and is settled to scale, shakes up the standard solution that promptly gets C=0.14mg/ml.
3.) select maximum absorption wavelength
Making icariin respectively is the UV absorption spectrum scintigram of reference substance and flavone partial purification of the present invention front and back, the absorption spectrum comparative result of icariin reference substance and described total flavones sample shows, they all have an absworption peak at the 270nm place, bigger through purification with macroreticular resin post-absorption peak, the macroporous adsorbent resin enrichment has been described flavones ingredient, this conforms to the general characteristic ultraviolet absorption of flavones ingredient, it is reference substance that the present invention adopts with the icariin, measure its ultraviolet absorptivity at the 270nm place, in order to measure content of total flavone.
4.) preparation standard curve
Precision takes by weighing 1.4mg icariin standard reference material, place the 10ml volumetric flask, add 60% ethanol dilution and be settled to scale, shake up the standard solution that promptly gets C=0.14mg/ml, accurate respectively absorption 1ml places the 10ml volumetric flask, 1ml places the 5ml volumetric flask, 2ml place the 5ml volumetric flask concentration be 0.014,0.028, the 0.056mg/ml standard solution; The standard solution 1ml that gets C=0.014mg/ml more respectively places the 5ml volumetric flask, and 2ml places the 5ml volumetric flask, add 60% ethanol standardize solution after, concentration be 0.0028, the 0.0056mg/ml standard solution; Get the standard solution 2ml of C=0.056mg/ml again, place the volumetric flask of 5ml, add 60% ethanol and be settled to scale, get the standard solution of C=0.02246mg/ml; Get the standard solution of above-mentioned 6 kinds of concentration respectively, measure trap at the 270nm place, and be blank with 60% ethanol, regression equation be: Y=38.456x+0.0172, r=0.9997, the range of linearity is 0.0028～0.056mg/ml.Table 4 is standard curves of preparation.
Table 4
| Standard substance concentration (mg/ml) | Absorbance (A) |
| 0.0028 0.0056 0.014 0.0224 0.028 0.056 | 0.110 0.235 0.560 0.901 1.120 2.149 |
5., after respectively sample suitably being diluted, be that the 270nm place measures content of total flavone in extractive of general flavone and the purification with macroreticular resin sample at wavelength) according to above-mentioned condition determination.
Embodiment 4 macroporous adsorbent resin type Selection Test
1) test apparatus, material and reagent
UV-240 ultraviolet spectrophotometer (Tianjin, island), HP1100 high performance liquid chromatograph, electronic balance, vacuum drying oven;
Icariin, stilbene glucoside, naringin reference substance (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute), model are D101, DA201, DM301 elaboration grade resins (available from Tianjin sea light chemical industry company limited), 95% medical ethanol;
This compound preparation water extract (1ml contains 1 gram medical material) is a mother solution, and precipitate with ethanol 2 times (add 95% ethanol and reach 80% to containing the alcohol amount) merges alcoholic solution according to a conventional method, is concentrated into 1ml and is equivalent to the 1g medical material, and through assay, its content of total flavone is 17.56mg/ml.
2) the resin pretreatment is by prior art: (Pan sees, Chen Qiang, and Xie Huiming, etc. Transactions of the Chinese Society of Agricultural Engineering, 1999,15 (1): 236-240),
(determination of total flavonoids-with the icariin is a standard reference material to analytical method with reference to prior art; Icariin, stilbene glucoside and naringin HPLC assay; Shi Wanzhong, Zhu Lan, Xu Desheng, etc. Chinese herbal medicine, 2000,31 (5): 341; Shi Wanzhong, Huang Lihong, Xu Desheng, etc. new Chinese medicine and clinical pharmacology, 2001,12 (5): 359; Shi Wanzhong, Xu Desheng, Kong Deyun, etc. the time precious traditional Chinese medical science traditional Chinese medicines, 2000,11 (6): 500).
3) measure static parameter
With the resin quality is that unit measures adsorbance: take by weighing through each 3 parts of pretreated resins, each 2.5g puts in the volumetric flask of 100mL, accurate adding sample liquid 25mL of the present invention vibrates on electronic vibrating machine, frequency is 120rmin-1, after vibration 20h fully adsorbs, filter, measure the concentration of residue total flavones, be calculated as follows each resin adsorbance (mgg-1) at room temperature.
Q＝(C1-C2)×V/W
In the formula: Q is adsorbance (mgg-1); C1 is initial concentration (mgmL-1); C2 is residual concentration (mgmL-1); V is liquor capacity (mL); W is weight resin (g).
Retinue accurately takes by weighing each 1g of pretreated resin in addition, place the drying bottle of recording quality respectively, 100 ℃ dry to constant weight, draw the quality that every gram aqueous resins is equivalent to dried resin, the result shows, with total flavones and icariin is index, the adsorbance DM301 of 3 kinds of adsorbent resiies＞D101＞DA201; With stilbene glucoside and naringin is index, DM301＞DA201＞D101 then, and wherein DM301 is to the adsorbance and the adsorption rate maximum of 3 kinds of index components.With DM301 quality coefficient maximum, infer its intensity in 3 kinds of macroporous adsorbent resins, be difficult for broken and enter medicine greater than other 2 kinds.
Table 5 is the adsorption effect comparisons to index components of the present invention of 3 kinds of macroporous adsorbent resins.
Table 6 is 3 kinds of aqueous resins and dried resin mass conversion coefficient
Table 5
| Index components | D101 | DA201 | DM301 | |||
| Adsorbance/mg | Adsorption rate/% | Adsorbance/mg | Adsorption rate/% | Adsorbance/mg | Adsorption rate/% | |
| Total flavones icariin stilbene glucoside naringin | 64.70 18.43 8.14 1.57 | 19.99 29.29 14.22 8.77 | 59.22 12.28 11.30 3.18 | 16.33 19.52 19.74 17.76 | 81.18 21.63 17.56 3.70 | 21.81 34.38 30.68 20.64 |
Wherein: mother solution is that 1ml is equivalent to 1g crude drug amount
Table 6
| The resin model | Every gram aqueous resins is equivalent to the dried resin quality coefficient |
| D101 DA201 DM301 | 0.2548 0.2213 0.2825 |
4) mensuration of resin desorption rate
Get according to 3) each 3 parts of saturated resins of method absorption, add each 50mL of ethanol of 50%, 70% and 90% respectively, soak vibration 24 hours, filter, the concentration of total flavones and icariin, stilbene glucoside and naringin in the mensuration filtrate, and calculate desorption efficiency (%), the result shows, DM301 type resin all has desorption preferably to total flavones, icariin, stilbene glucoside and naringin, and eluting concentration is advisable with 70% or 90% concentration of alcohol.
Table 7 is the desorption efficiency comparisons to index components of the present invention of 3 kinds of macroporous adsorbent resins.
Table 7
| Index components | ?D101 | DA201 | DM301 | ||||||
| ?50％ | 70％ | ?90％ | 50％ | ?70％ | ?90％ | 50％ | 70％ | 90％ | |
| Total flavones icariin stilbene glucoside naringin | ?72.63?62.91?51.70?75.31 | 85.92 81.44 68.56 91.58 | ?85.07?88.93?66.80?88.92 | 87.57 95.14 92.38 52.51 | ?89.43?95.51?90.75?64.02 | ?97.82?100.02?88.71?53.97 | 87.90 89.29 97.11 79.65 | 90.91 83.85 91.77 76.91 | 98.91 91.16 83.34 68.34 |
Wherein: mother solution is the crude drug amount that 1ml is equivalent to 1g
5) mensuration of static adsorption kinetic curve
Experimental technique is with 3), in the time of 25 ℃, when the mother solution general flavone content was 17.56mg/ml, resin demand was 2g, when adding mother solution amount is 40ml, measuring the rate of adsorption of DM301 resin to total flavonoid chemical compound in the mother solution with static adsorptive method, was 0 moment when contacting with mother solution with adsorbent resin, and 0.5ml at regular intervals takes a sample, measure wherein content of total flavone, calculate the adsorbance of resin this moment,, get the static adsorption rate curve with the adsorption rate mapping of moment t to resin.The result shows, the adsorbance when adsorbing 7 hours is 100%, and then in the time of 0.5 hour, its adsorbance only is 61%; Adsorption time is more than 4 hours the time, and its adsorbance is approaching substantially, 4 hours adsorbance be 7 hours 96%.Determine with 4 hours serve as the time that the assurance sample fully adsorbs, the speed of last sample is defined as 0.25BV/h with this.
The absorption with macroporous adsorbent resin rate of the different sampling time points of table 8.
Table 8
| Sample time | Adsorbance (the every g resin of mg/) |
| 0.5h 1h 2h 3h 4h 5h 6h 7h | 52.95 67.51 72.78 79.41 83.15 84.53 86.35 86.35 |
Wherein: adsorption rate is in wet resin
The experimental study of embodiment 5 effective site total flavones purifying process
1) leakage plot
According to the static adsorption parameter, estimate and select to be fit to the DM301 adsorbent resin of this compound recipe total flavones purification, take by weighing wet resin 23g dress post (1.5cm * 15cm, column volume is 26ml), the accurate sample liquid 60mL of the present invention (upper prop of 1g crude drug/ml) of drawing, the control flow velocity, 0.10mLmin-1 (being equivalent to 0.25BV/h) flows through resin column, the quantitative collection effluent, working sample liquid and mistake post liquid total flavones concentration and icariin, the content of stilbene glucoside and naringin, applied sample amount (volume) is an abscissa in the leakage process to take place, with the concentration of index components in the effluent and the ratio of sample liquid concentration of the present invention is vertical coordinate, makes the DM301 leakage plot, with icariin, stilbene glucoside and naringin are evaluation index, it leaks (applied sample amount: when wet resin is 20/23=0.87, begin to occur leaking) when appearing at 20ml approximately; And be index with the total flavones, its leakage appears at (applied sample amount of this moment: wet resin is about 0.5) about 10ml, shows that the sensitivity of total flavones index is better than icariin, stilbene glucoside and naringin index.
2) desorption curve
According to steady-state solution adsorption experiment result, the ethanol of employing 70% and 90% is eluent, carries out the desorption curve preparation.
Get DM301 wet resin 10g, wet method dress post (column diameter 1.5cm, the high about 7cm of post), accurate absorption sample liquid 10mL upper prop of the present invention, use distilled water 200mL then respectively, each 100mL gradient elution of 10% ethanol, 70% ethanol, 90% ethanol and 95% ethanol, eluent flow rate is 0.5mLmin-1, the distilled water eluent is collected 4 parts of 50mL, and 10% ethanol, 70% ethanol, 90% ethanol and 95% ethanol elution are collected 4 parts of 25mL respectively.Measure the content (Fig. 2) of total flavones, icariin, stilbene glucoside and the naringin of each eluent section respectively.
According to above-mentioned elution curve, adopt with the distilled water of 10 times of amounts (comparing) and remove impurity with 10 times of amount 10% ethanol (comparing) eluting with the wet resin amount with the wet resin amount.
3) condition optimizing of DM301 purification on adsorbent resins total flavones
Orthogonal Experiment and Design: according to the physicochemical property of flavone and the absorption characteristics of macroporous adsorbent resin, investigate medicine upper column quantity, the blade diameter length ratio of pillar, the consumption of eluting solvent, 4 factors of elution speed, and selected 3 horizontal design experiment schemes in conjunction with the trial test result.Table 9 is each factor and level design table.
Table 9
| Level | A amount of resin: applied sample amount/g: g | B blade diameter length ratio diameter: highly | C eluting solvent consumption/mL: applied sample amount | D elution speed/mLmin -1 |
| 1 2 3 | 2∶1 1∶1 0.5∶1 | 1∶5 1∶10 1∶20 | 5∶1 10∶1 20∶1 | 0.5 1.0 1.5 |
Adopt the analytical column of 1.5cm * 50cm, according to the factor level table, with L9 (3
4) the orthogonal table experiment arrangement, each test precision is got a certain amount of sample liquid upper prop of the present invention, with fixed 10 times of amounts (comparing) distilled water and 10 times of amounts (comparing), 10% ethanol flush away impurity with the wet resin amount with the wet resin amount, use 70% ethanol elution total flavones of the present invention then, collect 70% eluent, be evaporated to a certain amount of, and standardize solution.Quantitatively measure sample segment liquid, measure the content of wherein total flavones, icariin, stilbene glucoside and naringin, all the other samples are concentrating under reduced pressure and be dried to constant weight again, pale brown toner end.Calculate yield and purity respectively.
With total flavones yield and purity and icariin, stilbene glucoside and naringin yield summation and purity summation is index, estimates and definite optimum process condition.
Table 10 is to be index, Orthogonal experiment results and variance analysis with general flavone content and purity.
Table 11 is to be index Orthogonal experiment results and variance analysis with total content (icariin+stilbene glucoside+naringin) and purity.
Table 10
| Factor | ?A | B | C | D | General flavone content (%) | Purity (%) | |
| Tested number | 1 2 3 4 5 6 7 8 9 | ?1?1?1?2?2?2?3?3?3 | 1 2 3 1 2 3 1 2 3 | 1 2 3 2 3 1 3 1 2 | 1 2 3 3 1 2 2 3 1 | 11.7735 14.4344 12.0041 10.6195 11.3825 10.5121 9.1783 9.0285 8.1808 | 50.1466 58.4490 58.8689 57.8787 61.3106 52.5034 62.4559 58.4947 66.9602 |
| General flavone content | I II III R S P | ?38.2120?32.5140?26.3876?11.8244?23.3130?＜0.05 | 31.5713 34.8453 30.6970 4.1483 3.1880 --- | 31.3142 33.2346 32.5649 1.9204 0.6334 --- | 31.3368 34.1248 31.6522 2.7880 1.5540 --- | G＝97.1137 CT＝G2/9＝1047.8966 SJ＝(K12+K22+K32)/3-CT | |
| Purity | I II III R S P | ?167.4645?171.6927?187.9108?20.4464?77.6620?--- | 170.4812 178.2543 178.3325 7.8513 13.5634 --- | 161.1448 183.2878 182.6355 22.1431 105.8435 ＜0.05 | 178.4174 173.4083 175.2423 5.0091 4.2818 --- | G＝527.0680 CT＝G 2/9＝30866.7442 J＝(K12+K22+K32)/3-CT | |
Table 11
| Factor | A | B | C | D | Total content (%) | Purity (%) | |
| Tested number | 1 2 3 4 5 6 7 8 9 | 1 1 1 2 2 2 3 3 3 | 1 2 3 1 2 3 1 2 3 | 1 2 3 2 3 1 3 1 2 | 1 2 3 3 1 2 2 3 1 | 8.0961 9.5723 8.5718 7.5817 8.1436 8.1571 6.6263 6.6613 5.5183 | 34.4832 38.7609 42.0365 41.3222 43.8650 40.7415 45.0903 43.1578 45.1676 |
| Total content | I II III R S P | 26.2401 23.8825 18.8059 7.4342 9.6219 ＜0.05 | 22.3041 24.3772 22.2472 2.1300 0.9820 --- | 22.9145 22.6723 23.3417 0.6695 0.0766 --- | 21.7580 24.3557 22.8148 2.5977 1.1377 --- | G＝68.9285 CT＝G 2/9＝527.9047 SJ＝(K12+K22+K32)/3-CT | |
| Purity | I II III R S P | 115.2806 125.9286 133.4157 18.1351 55.3685 ＜0.05 | 120.8956 125.7837 127.9456 7.0499 8.6965 --- | 118.3825 125.2507 130.9918 12.6093 26.5698 --- | 123.5158 124.5926 126.5165 3.0006 1.5405 --- | G＝374.6249 CT＝G 2/9＝15593.7591 SJ＝(K12+K22+K32)/3-CT | |
The result shows, from total flavones yield and purity, factor A (the wet resin amount: applied sample amount) the total flavones yield there is significance influence (P＜0.05), and amount of resin in 3 levels: applied sample amount is 2: 1 o'clock the bests, therefore selects 1 level for use; And factor C (eluting solvent consumption) influences significantly (P＜0.05) to purity, and the 2nd level the best, therefore selects 10 times of amounts for use; Factor B (blade diameter length ratio) is all not remarkable to general flavone content and purity, examines that to filter the eluting solvent consumption too big, strengthen follow-up spissated workload, so the B factor is selected 2 levels for use; Factor D (elution speed) is not remarkable to the influence of general flavone content and purity yet, examines to filter elution speed and can shorten elution time greatly, be convenient to the practical operation of full-page proof, so factor D (elution speed) selects 2 levels for use.The purification condition of determining optimized macroporous adsorbent resin is: A1B2C2D2.
The quadrature result of total content (icariin+stilbene glucoside+naringin) and purity is similar to total flavones.
The separation purifying technique parameter of the macroporous adsorbent resin of the total flavones that the present invention determines is: preferred applied sample amount is wet resin amount/applied sample amount=2: 1 (being equivalent to 0.5BV), last sample speed is 0.25BV/h, blade diameter length ratio (1: 10), eluting solvent consumption 10: 1, elution speed 1ml/min (being equivalent to 2.5BV/h).
4) quadrature repeated experiments
According to above-mentioned Orthogonal experiment results, adopt the purifying process parameter of optimizing, carry out the quadrature repeated experiments, the result shows, adopts above-mentioned technology, can make the extraction ratio of total flavones and total content bigger, guarantees that simultaneously the purity of total flavones in the purification thing reaches more than 50%.The ratio of described three index components in the purification thing is icariin: stilbene glucoside: naringin=3.32: 5.63: 1.00; The yield of total flavones purification thing is 2.25% (in the medical material that feeds intake).Table 12 is quadrature repeated experiments results.
Table 12
| Index | Measured value (mg/g) | Yield average (mg/g) | RSD(％) | Measured value (g/g, %) | Purity (%) average (g/g, %) | ?RSD(％) |
| Total flavones | ?12.77?12.02?12.00 | 12.26 | 3.57 | 57.04 53.84 52.87 | 54.58 | ?4.00 |
| Total content | 9.69 9.27 9.16 | 9.37 | 2.99 | 43.29 41.51 40.49 | 41.72 | ?3.55 |
Wherein: yield is the medical material amount of the content/last sample of mensuration gained; Purity is the amount of the content/solid content of mensuration gained
Embodiment 6 polysaccharide ultrafiltration technology condition optimizings
1) material and instrument
The present invention extracts the precipitation part in the purification step; HPM template basket formula ultrafilter (Shanghai nuclear research institute), vacuum desiccator, electronic balance, thermometer.
2) method and result
The precipitation part that will make by embodiment 1 method, being converted to concentration by its crude drug amount is 40%, 30%, 20% and 10% liquor strength (medical material amount/quantity of solvent), carry out ultrafiltration respectively, pressure is controlled at 0.2Mpa, and temperature control (logical condensed water) is being not more than 60 ℃, after the ultrafiltrate that does not have filter membrane flows out, add 2 times of distilled water of measuring the medical material amounts with the medicinal liquid ultrafiltration again 2 times, be not the ultrafiltration end to there being the ultrafiltrate outflow.
Collect molecular weight＜10,000 respectively, three molecular weight sections of 1～50,000 and＞5 ten thousand, concentrate, after water-bath is evaporated to and does, press official method, measure the amount and the yield of dry extract, the result shows that liquor strength is more little, then＜10,000 and 1～5 the dry extract yield of son section is high more very much, illustrate that the ultrafiltration effect is good more, from＜1 ten thousand and 1～5 yield evaluation of son section very much, the dry extract yield of the medicinal liquid of 30% concentration is lower than the dry extract yield of 10% and 20% concentration, but gap is little, consider follow-up super work simultaneously, liquor strength is more little, has then increased follow-up spissated cost and cycle, therefore selects 30% ultrafiltrate concentration for use.
According to The above results, factor in conjunction with practical operation, the technology of determining super rate is: 30% compound recipe total polysaccharides liquor strength (in medical material), super rate pressure is controlled at 0.2Mpa, temperature control (logical condensed water) is not more than 60 ℃, after the ultrafiltrate that does not have filter membrane flows out, add 2 times of distilled water of measuring the medical material amounts with the medicinal liquid ultrafiltration again 2 times, be not the ultrafiltration end, collect the ultrafiltrate of different molecular weight section to there being the ultrafiltrate outflow.
Table 13 is the influences to 3 molecular weight section yield of ultrafiltration solution concentration.
Table 13
| Ratio | ＜1 ten thousand | 1～50,000 | ＞5 ten thousand | |||
| Dried cream amount | Yield | Dried cream amount | Yield | Dried | Yield | |
| 40％ 30％ 20％ 10％ | 25.38 27.33 27.94 28.05 | 1.95 2.10 2.15 2.16 | 30.05 34.61 35.03 35.16 | 2.31 2.66 2.69 2.70 | 59.22 54.37 53.98 52.14 | 4.56 4.18 4.15 4.01 |
The development test of embodiment 7 extract part quality control indexs
The present invention carries out carrying out preliminary Quality Control development test before ALENDRONATE FOSAMAX is imitated research to flavonoid and the polysaccharide extract that extracts the purification gained, understanding the relation of experimental drug amount and drug effect better, and provide the basis for the different component of further compatibility and the drug effect of estimating various dose.
1) experiment medicine and instrument
Experiment medicine: extractive of general flavone of the present invention and total polysaccharide extractive;
Experimental apparatus: adopt the HPLC instrument, ultraviolet spectrometry range instrument, vacuum desiccator.
2) experimental technique and result
Measure the ratio of 3 index components in the total flavones:
Adopt the HPLC method to measure before the purification and the content of icariin, stilbene glucoside and the naringin in the total flavones sample behind the purification respectively, and be benchmark with the naringin of content minimum, calculate 3 components in proportions in the total flavones purification thing, the result shows, icariin, stilbene glucoside, naringin are before and after purification with macroreticular resin, and its ratio does not have significant change.
Table 14 is 3 index components content and ratios thereof in total extract.
Table 15 is 3 index components content and ratios thereof in total extract in the former extracting solution.
Table 14
| Index | Icariin | Stilbene glucoside | Naringin |
| Content (mg/g) ratio | 3.13 3.32 | 5.30 5.63 | 0.94 1.00 |
Table 15
| Index | Icariin | Stilbene glucoside | Naringin |
| Content (mg/ml) ratio | 2.66 3.33 | 4.11 5.13 | 0.80 1.00 |
The solid yield of different parts polysaccharide, ratio and assay:
In order to determine the solid content ratio of 3 different parts in the total polysaccharides different parts, crude drug inventory with 13kg, behind the crude polysaccharides of extraction and purification, be mixed with 30% liquor strength, according to above-mentioned technological process, obtain the 3 kinds of different polysaccharide section of holding back polysaccharide respectively, and with official method, measure its polysaccharide solid yield (solid content/crude drug amount), and calculate the ratio of 3 different solid contents in total polysaccharides, simultaneously according to the assay method of total polysaccharides (with glucose as a standard product, sulphuric acid-anthrone method), the content of the polysaccharide of 3 different molecular weights of mensuration;
The present invention carried out the mensuration of the yield of 3 batches of total flavones dry extracts, determined to adopt the dry extract yield of the total flavones behind the purification with macroreticular resin.The result shows, total flavones solid yield (in raw medicinal herbs) is 2.25%, (in icariin (spectrophotography) is 54.58% to content of total flavone in the compound recipe total flavones purification thing, total content (HPLC method) in icariin, stilbene glucoside and naringin is 41.72%), the total flavones yield of crossing macroporous adsorbent resin is 76.30% (spectrophotography).
Table 16 is the yield of the difference section of holding back polysaccharide solid content and the ratio in total polysaccharides thereof.
Table 17 total flavones solid yield and assay result thereof.
Table 16
| The polysaccharide molecule section | Solid yield (%) | Ratio | Content (%) |
| ＜1 1～5 ＞5 | 2.10 2.70 4.00 | 1.00 1.28 1.90 | 29.86 41.87 49.97 |
Table 17
| Quantitative criterion | Content (%) | The yield of solid content (%) |
| 3 index components total amounts of general flavone content (icariin is the mark product) | 54.58 41.72 | 2.25％ |
Different influence tests of extracting the pastille serum at chemical position in embodiment 8 compound recipes of the present invention to the osteoblastic proliferation rate of In vitro culture
1) materials and methods
Main agents and instrument: MEM and Meduium199 culture medium, hyclone (FBS), calf serum (FBS) are Gibco company product, trypsin is a Difco company product, II Collagen Type VI enzyme is a Sigma company product, glutaraldehyde, toluidine blue are E.Merk company product, and 96 well culture plates are Costar company product; 723. the type spectrophotometer is Shanghai instrument plant product.
Osteoblast separates and cultivates: reference literature (Tan Zhilong, Xing Guosheng, Wang Shuyun, Deng. Chinese osteoporosis magazine, 2004,10 (2): 194-96), get 1 age in days SD rat (providing), 75% soak with ethanol 15min, conventional pretreatment by Rui Jin, Shanghai hospital orthopedics department institute Experimental Animal Center, PBS flushing 3 times, with 0.25% trypsin predigestion 25min, the enzymic digestion of reuse 0.1%II Collagen Type VI, 37 ℃ of vibrations, 60 times/minute, 1h * 2 time, centrifugal 1000rpm, 10min.Cell adds and to contain in 10% the NCS-MEM culture fluid after PBS washes, and cultivates in 37 ℃, 5%CO2 environment, changes liquid next day, changes liquid 1 time in later per 2～3 days.
The preparation rat blood serum: reference literature (Han Limin, Liu Bo, Xu Peng. Chinese medicine academic periodical, 2003,21 (5): 678-680; Shao Min, village flood .. China osteoporosis magazine, 2003,9 (2): 117-119), get normal SD rats (Shuguang Hospital's Animal House, the quality certification number: SCXK (Shanghai) 2003-0002,) 10 every group, body weight 300g ± 30, male and female half and half, press the body surface area method and calculate dosage, with the dose,equivalent gastric infusion.The present invention respectively organizes extract 5.47g crude drug amount/kg, guizhi decoction 4.21g crude drug amount/kg (Chinese medicine is provided by the SHUGUANG HOSPITAL Drug Manufacturing Room), different compound recipe extract parts and guizhi decoction (extracting) by described method, JINGUI SHENQI WAN (abbreviation shenqi pill) (LanZhou FoCi Pharmacy Co., Ltd's product, lot number: 20030310,1.68/kg), above medicine, respectively with physiological saline solution or suspendible, successive administration 2 times, each 2 hours at interval, blood sampling in 1 hour behind the last filling stomach, preparation pastille serum, animal is with etherization, and ventral aorta is taken a blood sample under aseptic condition, and 4 ℃ centrifugal, 3000rpm, 30min extracts serum, 56 ℃, 30min water-bath deactivation complement activity, through the degerming of 0.22 μ m filter membrane filter sucking filtration, packing ,-20 ℃ of preservations are standby.
Mtt assay is measured osteoblastic proliferation rate (red legend cyanines, Jin Weifang, Zhang Lili, Deng. Shanghai Medical Univ's journal, 1995,22 (4): 254-255): cell is inoculated in 96 orifice plates with 5 * 103/ holes, is replaced by the culture fluid that contains dosing serum on the 2nd day, cultivate after 3～4 days, the PBS flushing, the MEM culture fluid of replacing serum-free adds MTT solution (5mg/ml) 20ul simultaneously, 37 ℃ are continued to hatch 4h, stop cultivating, inhale and abandon culture supernatant in the hole, every hole adds 150ul DMSO, vibration 10min measures each hole OD value at wavelength 490nm place.
Adopt SPSS11.5 software to carry out variance analysis.
The result shows, the extract of different parts of the present invention is to the influence of the cultured osteoblasts in vitro rate of increase, with the normal saline group is criterion, the osteoblastic proliferation rate of polysaccharide＜10,000, polysaccharide 1～50,000, polysaccharide＞5, total polysaccharides, pure molten part, the full side of compound recipe of the present invention group (being called for short full side group) and shenqi pill group is greater than the normal saline group, certain promotion proliferation of osteoblast is described, its action intensity is descending to be followed successively by polysaccharide＜10,000, full side group, shenqi pill group, total polysaccharides group, polysaccharide 1～50,000 group and total flavones group; A little less than normal saline, illustrate does not have the proliferation of osteoblast of promotion to guizhi decoction to the osteoblastic rate of increase; Centrifugal part group and polysaccharide＞5 osteoblastic proliferation rates are starkly lower than the normal saline group.The statistical disposition result shows: wherein significant difference (P＜0.05) group has been compared in polysaccharide＜10,000 with the normal saline group, and other groups comprise that positive control drug group-JINGUI SHENQI WAN forms osteocyte rate of increase numerical value and be higher than the normal saline group, but no difference of science of statistics.
The present invention sets the threshold value of osteoblastic proliferation rate for judging of normal saline group, determines polysaccharide＜10,000 in the compound recipe of the present invention, polysaccharide 1～50,000, and the total flavones group is an effective site.
Table 18 is the influences (n=8) to the osteoblastic proliferation rate of the pastille serum of different extract parts.
Table 18.
| Group | The osteoblastic proliferation rate |
| Polysaccharide＜10,000 polysaccharide 1～50,000 polysaccharide＞5 total polysaccharides total flavones groups | 0.595±0.195 *0.466±0.1200.421?±0.0760.468±0.0670.465±0.089 |
| The full side's guizhi decoction of centrifugal part shenqi pill normal saline | 0.305±0.048 0.512±0.236 0.437±0.110 0.501±0.125 0.442±0.077 |
Wherein
*Expression P＜0.05
Claims (7)
1. compound Chinese medicinal preparation for the treatment of osteoporosis is characterized in that being made by following bulk drugs component:
2 parts of Herba Epimedii, 1.5 parts of the Radixs Astragali, 1 part of Radix Polygoni Multiflori Preparata, 1 part of Rhizoma Drynariae, 1 part of Fructus Lycii.
2. effective ingredient in Chinese for the treatment of osteoporosis, it is characterized in that, described effective site is total polysaccharides and the total flavone part extract in the described bulk drugs of claim 1, described total polysaccharides molecular cut off is＜1 ten thousand position and 1～50,000 position, and the general flavone content of described total flavone part is greater than 50%.
3. by the effective ingredient in Chinese of the described treatment osteoporosis of claim 2, it is characterized in that described total flavone part contains icariin, naringin and stilbene glucoside.
4. by the effective ingredient in Chinese of the described treatment osteoporosis of claim 3, it is characterized in that the contained icariin of described total flavone part, naringin and stilbene glucoside content ratio are 3～4: 5～7: 1.
5. the preparation method of the compound Chinese medicinal preparation of the described treatment osteoporosis of claim 1 is characterized in that, comprises the steps:
The weighting profit requires 1 described bulk drugs, adds 10 times in water, soaks 30 minutes, decocted 1 hour for the first time, for the second time add 10 times in water, decocted 1 hour, cross 200 order nets, merge extractive liquid,, the centrifugal 16000r/min of channel separator gets supernatant, is evaporated to extracting solution 1ml and contains the 1g crude drug, adding the ethanol precipitate with ethanol is 60～90% to containing the alcohol amount 1～3 time, must precipitate part and pure molten part;
Precipitation part thin up to 10～40% is in the medical material amount, crosses the ultrafilter membrane of 10,000 and 50,000 molecular cut offs with the plate and frame ultrafilter, molecular cut off＜10,000 positions and molecular cut off 1～50,000 positions, concentrating under reduced pressure, dry must the total polysaccharides position;
The molten part of alcohol is not distinguished the flavor of to there being alcohol through decompression recycling ethanol, thin up contains the 1g raw medicinal herbs to 1ml, adopt the DM301 macroporous adsorbent resin, blade diameter length ratio is 1: 10, with sample on the 0.25BV/h speed, uses the distillation washing of comparing 3～10 times with the wet resin amount and the 10% ethanol flush away impurity of comparing 3～10 times of amounts with the wet resin amount respectively, use 70% ethanol elution purification total flavones then, collect eluent, decompression recycling ethanol, concentrating under reduced pressure, the dry total flavone part that gets;
Above-mentioned make＜10,000 polysaccharide positions, 1～50,000 polysaccharide position and total flavone part extract add starch through pulverizing, mixing incapsulates promptly.
6. by the described method of claim 5, wherein, in the sample step, wet resin throwing amount is 2: 1 with the crude drug ratio on the described macroporous adsorbent resin.
7. by the described method of claim 5, in the wherein said usefulness 70% ethanol elution purification total flavones step, the eluting solvent consumption is 10: 1 with the crude drug ratio, and elution speed is 1ml/min.
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