CN101401830B - Preparation method for extracting flavone from purslane and uses thereof - Google Patents
Preparation method for extracting flavone from purslane and uses thereof Download PDFInfo
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- CN101401830B CN101401830B CN2008101976472A CN200810197647A CN101401830B CN 101401830 B CN101401830 B CN 101401830B CN 2008101976472 A CN2008101976472 A CN 2008101976472A CN 200810197647 A CN200810197647 A CN 200810197647A CN 101401830 B CN101401830 B CN 101401830B
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- herba portulacae
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Abstract
The invention discloses a preparation method for extracting flavone from purslane and application thereof. The medicine is characterized by a purslane alcohol extract. The preparation method comprises the following steps: 1. cleaning and drying the purslane; 2. cutting the purslane into short sticks; 3. extracting the flavone in the purslane by the reflux of alcohol; 4. performing the pumping filtration on the extracting solution; and 5. concentrating the liquid obtained after pumping filtration. The application method of the extract comprises the following steps: 1. inoculating tumor cells; 2. randomly dividing mice into four groups; 3. performing the group treatment; 4. sampling the blood, and weighing tumor; 5. obtaining blood serum; 6. measuring the content of blood urea nitrogen; and 7. measuring the content of serum creatinine. Animal tests prove that the extract can effectively resist kidney damage caused by the anticancer drug cisplatin; moreover, the extract has the advantages of convenient drug application, good effect and no toxic and side effects; and the purslane is wide in source, and low in price, so that the extract also has the advantages of low price and good quality.
Description
Technical field
The present invention relates to medical technical field, more specifically relate to a kind of preparation method of from Herba Portulacae, extracting total flavones, also relate to the purposes of Herba Portulacae total flavones simultaneously.
Background technology
1. antitumor drug easily causes renal damage
Though the application of antitumor drug has bigger help to the control of tumor, its damaging action to kidney is a problem that can not be ignored.Antitumor drug is machine-processed as follows to kidney injury:
At first, the concentrated urine Chinese medicine concentration that makes of crude urine easily produces crystallization far above blood drug level, blocks renal tubules.Simultaneously, kidney weight only percentage of liveweight 0.4%, and blood flow accounts for 25% of heartbeat blood volume.Therefore, just easilier when the deleterious antitumor drug of kidney and metabolite thereof are arrived kidney with blood flow make it to produce pathological changes.
On the other hand, renal metabolism enzymatic activity height.Some antitumor drug produces noxious substance when this transforms.The reaction of urinary system that antitumor drug causes mainly contain reaction of urethra internal stimulus and excess of the kidney matter infringement (Han Tao. the untoward reaction of antitumor drug and control thereof [J]. capital medicine, 2008, (4): 45-48).
More deep experiment shows that antitumor drug causes that the main component of kidney injury is its hydrolysis metabolite.The nucleophilic amino of antitumor drug can produce a large amount of free radicals with the hydrone effect, causes cell membrane and mitochondrial oxidative damage, afunction..Except that causing oxidative damage, antitumor drug can suppress renal tubules brush border and organic anion transport system, particularly Na in the renal metabolism process
+-K
+-atpase activity has reduced the heavily absorption of kidney proximal tubule to glucose, makes the epithelial cell hydropic degeneration.Some scholar thinks that antitumor drug can also suppress the conjugated protein regulin (Ca of renal cortex
+-binding protein regucalcin) mRNA expresses, and the renal cortex calcium content increases, and causes the intracellular Ca2+ stable state unbalance, causes cell injury (KinYK, Byun HS, Kim YH.Toxical Appl.Pharmacol.1995,130 (1): 19).
Nowadays, the use of antitumor drug is more and more, and injury of kidney becomes the problem of everybody growing interest.Therefore, the research of injury of kidney protection medicine becomes now-the big hot topic problem.
2. the special biologic activity of flavone compound
Studies show that, extensively be present in natural vegetalitas flavone compound and have antioxidation, and its antioxidant effect relevant with its structure (Wang Shumei. the antioxidation of natural flavone compounds and structure activity relationship [J] .Strait Pharmaceutical Journal, 2004,16 (3), 10-13).At present, some research and inquirement the effect and the mechanism of action thereof of injury of kidney due to the flavone antagonism antitumor drug in some plants.The effect that existing bibliographical information Fructus Lycii total flavones has certain blood circulation promoting and blood stasis dispelling, blood fat reducing, blood sugar lowering and raising immunologic function; Folium Crataegi total flavones can effectively be prevented and treated cardiovascular disease, remove oxygen-derived free radicals, blood fat reducing, diuresis and enhancing hypoxia-bearing capability; Soybean isoflavone also is a research topic of comparison hot topic to the protective effect of kidney; soybean protein is to animal and human's class polycystic kidney; diabetic nephropathy; all useful (the Su Bo of multiple kidney disease such as the nephrotic syndrome; Cai Donglian. soybean isoflavone is to protective effect [J] the .Medical Recapitulate of kidney disease; 2003,9 (10): 611-612).In addition, Bears Xiao Ming etc. has then confirmed Folium Ginkgo extract, a kind of contain flavonoid and terpene lactones chemical compound have the effect of anti-cisplatin nephrotoxicity (Bears Xiao Ming etc. kidney of rats damage [J] due to the extract of ginkgo biloba for treating cisplatin. Chinese Hospitals pharmacy, 2008,28 (4): 265-268).
3. the progress of Herba Portulacae and extract thereof
Herba Portulacae (PortulacaOleraceaL.) has another name called Formica fusca dish, long life dish, Semen Benincasae dish, five elements' grass etc., is Portulacaceae annual herb plant, almost distribution is all arranged the each province in China, aboundresources, and it is convenient to gather.It is the Chinese medicine of China, at applicating history among the people existing thousands of years.Effective ingredient in the Herba Portulacae such as alkaloid, polysaccharide, anthraquinone etc. can be prevented and treated cardiovascular disease; Herba Portulacae also has blood fat reducing, the anti-ageing effect of waiting for a long time, and energy enhancing human body immunity function; Herba Portulacae also contains multivitamin, mineral, protein and aminoacid, has effects such as atherosclerosis, antibiotic, antitumor and antioxidation.
Herba Portulacae ethanol extraction main component is a flavone, and wherein general flavone content is up to 6.37% (Wei Xun, Wang Zhongying. the mensuration of Herba Portulacae general flavone content [J] .Chinese Journal Of Spectroscopy Laboratory, 2003 (1), 20 (1): 128-129).But by retrieval, up to now, do not see the report that injury of kidney due to the anti-cisplatin of Herba portulacae extract is arranged.
On this basis, we study the kidney protective effect of Herba portulacae extract, confirm that by zoopery the present invention has good protective effect really to the injury of kidney of caused by chemotherapeutic medicines.
Summary of the invention
The objective of the invention is to be to provide a kind of preparation method of from Herba Portulacae, extracting total flavones.This method is utilized the characteristics of flavone for liposoluble substance, uses 75% alcohol reflux, and is simple for process; And the concentration technique of circling round of extracting solution is also ripe.And the extraction ratio of using this method total flavones is higher, can also keep its biological activity, with the carrying out of experiment after guaranteeing.In addition, the Herba Portulacae wide material sources, cheap, thereby make extract inexpensive.
Another object of the present invention is to be to provide the application of a kind of total flavones that extracts from Herba Portulacae in the medicine of injury of kidney due to preparation treatment or prevention (opposing) cisplatin.Prove that through zoopery this extract effect is remarkable, administration group mice blood urea nitrogen concentration is starkly lower than not administration group, and high dose group mice blood urea nitrogen concentration also is starkly lower than low dose group.This extract adopts conventional peroral dosage form, convenient drug administration, and no pain does not have sequela, and almost without any side effects.
To achieve these goals, the present invention adopts following technical measures:
A kind of preparation process of extracting total flavones from Herba Portulacae is:
1. new fresh Herba Portulacae is cleaned with clear water and won and remove its leaf and root, drain away the water and dry in the air in the lucifuge ventilation until removing its surface moisture fully, the time is controlled at 20～24h.
2. air dried Herba Portulacae stem is cut into the corynebacterium about 0.8-1.2cm.
3. add 70% ethanol (mass ratio) of 6 times of volumes in Herba Portulacae, the water-bath return time was controlled at 1-3 hour under the 78-82 ℃ of condition, backflow 2-4 time.
4. gained solution sucking filtration stays filtrate after will refluxing, and abandons impurity, then 2-4 time is refluxed and the resulting liquid mixing of sucking filtration.
5. temperature is controlled at and is rotated evaporation above-mentioned steps 4 gained liquid under the 60-65 ℃ of condition in Rotary Evaporators, has obtained a kind of concentrated solution of total flavones.
Total flavones as the application basic process in the medicine of injury of kidney due to preparation treatment or prevention (opposing) cisplatin is:
1. inoculation oncocyte
40 of female kunming mices, body weight (25 ± 2) g tested first day, every the equal axil of mice chamber injection S
180Mouse ascites (containing tumor cell strain) 0.2ml.
2. grouping
Postvaccinal mice is divided into 4 groups (normal saline matched group, cisplatin damage model matched group, low dosage Herba Portulacae medication therapy groups, high dose Herba Portulacae medication therapy groups), 10 every group at random.
3. packet transaction
The mice group disposition is as follows:
1) normal saline matched group: inoculate after 24 hours, irritate stomach 0.5ml with normal saline every day, 7 days altogether;
2) cisplatin damage model matched group: inoculate after 24 hours, irritate stomach 0.5ml with normal saline every day, and continuous 7 days, in the 4th day disposable celiac injection cisplatin (12.0mg/kg);
3) Herba Portulacae total flavones low dosage administration group: irritate stomach once (0.5ml) with Herba portulacae extract according to the ratio of mice body weight 5mg/kg every day, and continuous 7 days, in the 4th day disposable celiac injection cisplatin (12.0mg/kg);
4) high dose Herba portulacae extract treatment group: irritate stomach once (0.5ml) with Herba portulacae extract according to mice body weight 20mg/kg ratio every day, and continuous 7 days, in the 4th day disposable celiac injection cisplatin (12.0mg/kg).
The present invention has the following advantages:
1. the Herba portulacae extract that the present invention relates to is to obtain through ethanol extraction and progressive concentrating as the raw material parent with Herba Portulacae.Raw material sources are extensive, and are cheap;
2. the extraction of Herba Portulacae total flavones, concentration technology are simple, technology maturation;
3. the Herba Portulacae total flavones is as the drug development of protection caused by chemotherapeutic medicines injury of kidney, and this is not only abundant to motherland's theory of medicine, and is the further investigation to Herba Portulacae effective site, therefore, has important society and economic benefit;
4. the Herba Portulacae total flavones can not only effectively be treated the injury of kidney that chemotherapeutics causes, and blood fat reducing is arranged, the anti-ageing effect of waiting for a long time, and energy enhancing body's immunological function;
5. the efficient anti-injury of kidney activity of Herba Portulacae total flavones is that it has the removing free radical, improves the polyphenoils enzymatic activity, reduces many-sided effects such as lipid peroxide level and immunomodulating;
6. by retrieval, the acute toxicity test of Herba Portulacae total flavones shows its almost non-toxic side effect;
7. the present invention adopts the oral administration mode, convenient drug administration, and no pain does not have sequela.
The specific embodiment
One, a kind of flow process of extracting total flavones from Herba Portulacae is:
New fresh Herba Portulacae processing → reflux, extract, → sucking filtration → supernatant → concentrate → purification
Concrete steps are as follows:
1. new fresh Herba Portulacae is cleaned with clear water and won and remove its leaf and root, drain away the water and dry in the air in the lucifuge ventilation until removing its surface moisture fully.Time is controlled at 20 or 21 or 22 or 23 or 24h.
2. air dried Herba Portulacae stem is cut into 0.8 or 0.9 or 1 or 1.1 or the corynebacterium of 1.2cm.
3. 70% ethanol (mass ratio) that adds 6 times of volumes in Herba Portulacae, water-bath refluxed 1 or 2 or 3 hour under 78 or 79 or 80 or 81 or 82 ℃ of conditions, refluxed 4 or 3 or 2 times.
4. gained solution sucking filtration stays filtrate after will refluxing, and abandons impurity, then three times is refluxed and the resulting liquid mixings of sucking filtration.
5. temperature is controlled at rotation evaporation step 4 gained liquid under 60 or 61 or 62 or 63 or 64 or 65 ℃ of conditions, obtains concentrated solution.
Two, the mensuration of Herba Portulacae general flavone content
1. the Herba Portulacae flavone is identified
Add magnesium powder and hydrochloric acid chromogenic reaction: sample thief a little, add 95% ethanol 1-3ml, put in the test tube of its grinding port plug, room temperature (23 ℃～27 ℃) is vibration down, adds a little magnesium powder jolting, drips several concentrated hydrochloric acid again, take on a red color behind 1～2min, prove in institute's extract and contain flavone compound.
2. the mensuration of Herba Portulacae total flavones
1. the preparation of standard curve
Accurately take by weighing control substance of Rutin 200mg, with 70% ethanol (mass ratio) dissolving, standardize solution shakes up in the 100ml volumetric flask.Accurately draw 10ml, place the 100ml volumetric flask, the water standardize solution obtains the standard solution of 0.2mg/ml.Accurately draw standard solution 0.4,0.8,1.2,1.6,2.0,2.4ml, place the 10ml volumetric flask respectively, add water to 2.4ml, the adding mass fraction is 5% sodium nitrite 0.4ml, and mixing is placed 6min, adding mass fraction is 10% aluminum nitrate 0.4ml, mixing is placed 6min, and adding mass fraction is 4.3% sodium hydroxide 4.0ml, be diluted with water to scale, shaking up, place 15min, is contrast with blank pipe, use ultraviolet spectrophotometer, measure absorbance at 500nm wavelength place, obtain the calibration curve of concentration C and absorbance A: C=72.714A-0.5268, r=0.9991.
2. the mensuration of general flavone content
The accurate flavone extractive 1.0ml that draws places the 10ml volumetric flask, add water to 2.4ml, adding mass fraction is that 5% sodium nitrite 0.4ml shakes up, and places 6min, adding mass fraction is 10% aluminum nitrate 0.4ml, shake up, place 6min, adding mass fraction is 4.3% sodium hydroxide 4.0ml, add water to scale again, shaking up, place 15min, is contrast with the blank reagent pipe.Use ultraviolet spectrophotometer, measure absorbance (full wavelength scanner shows absworption peak at the 500nm place) at 500nm wavelength place, the substitution regression equation calculates general flavone content.
Three, zoopery
1. inoculation oncocyte
40 of female kunming mices, body weight (25 ± 2) g tested first day, every the equal axil of mice chamber injection S
180Mouse ascites (containing tumor cell strain) 0.2ml.
2. grouping
Postvaccinal mice is divided into 4 groups (normal saline matched group, cisplatin damage model matched group, low dosage Herba Portulacae medication therapy groups, high dose Herba Portulacae medication therapy groups), 10 every group at random.
3. packet transaction
The mice group disposition is as follows:
1) normal saline matched group: inoculate after 24 hours, irritate stomach 0.5ml with normal saline every day, 7 days altogether;
2) cisplatin damage model matched group: inoculate after 24 hours, irritate stomach 0.5ml with normal saline every day, and continuous 7 days, in the 4th day disposable celiac injection cisplatin (12.0mg/kg);
3) Herba Portulacae total flavones low dosage administration group: irritate stomach once (0.5ml) with Herba portulacae extract according to the ratio of mice body weight 5mg/kg every day, and continuous 7 days, in the 4th day disposable celiac injection cisplatin (12.0mg/kg);
4) high dose Herba portulacae extract treatment group: irritate stomach once (0.5ml) with Herba portulacae extract according to mice body weight 20mg/kg ratio every day, and continuous 7 days, in the 4th day disposable celiac injection cisplatin (12.0mg/kg).
4. get blood, claim tumor heavy
Irritated stomach the 7th day, eyeball of mouse is got blood in test tube, put to death mice then and take out the tumor piece of its oxter, weigh, record.
5. obtain serum
After eyeball of mouse is got blood, will adorn the test tube of blood immediately and put into refrigerator, take out centrifugal 10min behind the 30min, draw the upper strata light yellow transparent liquid, i.e. serum.
6. blood urea nitrogen (BUN) assay
In test tube, add reagent according to table 1
Table 1 BUN assay scheme
| Blank pipe | Standard pipe | Measure pipe | |
| Distilled water (ml) | 0.05 | ||
| BUN titer (ml) | 0.05 | ||
| Serum (ml) | 0.05 | ||
| 1g/L oxime solution (ml) | 2.5 | 2.5 | 2.5 |
| Acid solution (ml) | 2.5 | 2.5 | 2.5 |
Reagent that mixing adds is put accurate water-bath 15min in the water, cools off with tap water immediately.Use wavelength 520nm, 1cm optical path glass cuvette, the OD value is measured in blank pipe or distilled water zeroing.Then, calculate the content of blood urea nitrogen according to the OD value.
7. serum creatinine assay
Get serum 0.2ml in test tube, add wolframic acid albumen precipitation reagent 2ml, abundant mixing, 3000r/min, centrifugal 10min gets supernatant and measures by table 2
Table 2 serum creatinine assay scheme
| Measure pipe | Standard pipe | Blank pipe | |
| Serum albumin filtrate (ml) | 1.6 | ||
| 10umol/L creatinine standard substance (ml) | 1.6 | ||
| Distilled water (ml) | 1.6 | ||
| Picric acid (ml) | 0.5 | 0.5 | 0.5 |
| 0.75mol/L?NaOH | 0.5 | 0.5 | 0.5 |
Reagent that mixing adds, 37 ℃ of water-bath 10min take out back flowing water cooling, the 1cm optical path, the 510nm wavelength, the OD value is measured in blank pipe or distilled water zeroing.At last, calculate the content of serum creatinine according to the OD value.
Four, experimental result
1. tumor is heavy
Dissect mice, take out whole tumor pieces and take by weighing weight, unit is " gram ".As a result, Herba Portulacae high and low dose extract group tumor is heavy compares no significant difference with cisplatin damage model group, and prompting high and low dose Herba portulacae extract does not have tangible assosting effect (table 3) to the plus cisplatin in treatment tumor.
Table 3 is respectively organized (x ± s) of mouse tumor anharmonic ratio
Compare with the normal saline group
*P＜0.05
2. serum urea nitrogen (BUN), creatinine (Cr) concentration determination
As a result, cisplatin damage model group blood urea nitrogen has been compared marked difference with the normal saline group, points out the disposable cisplatin that gives heavy dose that kidney is had damaging action.High and low dose Herba portulacae extract group serum creatinine is compared no significant difference with cisplatin damage model group; but blood urea nitrogen is compared remarkable reduction than cisplatin damage model group, and the injury of kidney that prompting high and low dose Herba portulacae extract causes cisplatin has protective effect (table 4).
Table 4 respectively organize mice blood urea nitrogen value relatively (x ± s, mmol/L)
Compare with the normal saline group,
*P＜0.01; Compare with cisplatin damage model group,
#P＜0.05.
Five conclusions
Above-mentioned experimental result shows that Herba portulacae extract does not have tangible assosting effect to the plus cisplatin in treatment tumor, but the injury of kidney that cisplatin caused is had significant protective effect.
Claims (1)
1. the application of the total flavones that from Herba Portulacae, extracts in the medicine of injury of kidney due to the preparation prevention cisplatin, the extraction step of described total flavones is as follows:
1. new fresh Herba Portulacae is cleaned with clear water and won and remove its leaf and root, drain away the water and dry in the air in the lucifuge ventilation until removing its surface moisture fully, the time is controlled at 20～24h;
2. air dried Herba Portulacae stem is cut into the corynebacterium of 0.8-1.2cm;
3. 70% ethanol that in Herba Portulacae, adds 6 times of volumes, water-bath refluxes under the 78-82 ℃ of condition, and the time was controlled at 1-3 hour, backflow 2-4 time;
4. gained solution sucking filtration stays filtrate after will refluxing, and abandons impurity, then 2-4 time is refluxed and the resulting liquid mixing of sucking filtration;
5. temperature is controlled under the 60-65 ℃ of condition 4. gained liquid of rotary evaporation above-mentioned steps, obtains concentrated solution.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101708199A (en) * | 2009-11-13 | 2010-05-19 | 段洪东 | Purification method of purslane seed total flavonoid resin |
| CN102526138B (en) * | 2012-01-19 | 2016-02-24 | 贾晓斌 | Composition of active components from fresh purslane for decreasing blood sugar and preparation method |
| JP5763146B2 (en) * | 2013-10-10 | 2015-08-12 | 花王株式会社 | IGFBP-5 expression inhibitor |
| JP5763145B2 (en) * | 2013-10-10 | 2015-08-12 | 花王株式会社 | IGFBP-5 expression inhibitor |
| CN104059774B (en) * | 2014-06-30 | 2016-05-11 | 新疆大学 | A kind of method of preparing purslane polyunsaturated fatty acid from purslane slag |
| CN105640842B (en) * | 2016-02-24 | 2017-05-31 | 广州市禾基生物科技有限公司 | A kind of preparation method of Purslane extract |
| CN107998113B (en) * | 2018-01-08 | 2019-12-10 | 武汉大学 | Application of flavonoid compound CH625 in preparation of anti-glioma drugs |
| CN108785341A (en) * | 2018-08-27 | 2018-11-13 | 维康腾达生物科技有限公司 | A kind of preparation method of purslane extract |
| CN109432144A (en) * | 2018-11-26 | 2019-03-08 | 张世华 | A kind of method that purslane extracts flavones |
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