CN101401830B - Preparation method for extracting flavone from purslane and uses thereof - Google Patents

Abstract

The invention discloses a preparation method and application of extracting flavonoids from purslane. The medicinal feature is the ethanol extract of purslane. The preparation steps are as follows: ①washing and drying the purslane; ②cutting the purslane into short sticks; ③refluxing with ethanol to extract the flavonoids in the purslane; ④suction filtering the extract; . The application method of the extract is as follows: ① Inoculate tumor cells; ② Divide the mice into four groups at random; ③ Group treatment; ④ Take blood and weigh the tumor; ⑤ Obtain serum; Determination. Animal experiments have confirmed that the extract can effectively resist kidney damage caused by the anticancer drug cisplatin. In addition, the extract is convenient to administer, has good effect, and has no toxic and side effects. Purslane has a wide range of sources and low price, so that the extract has the advantages of high quality and low price.

Description

Preparation method and application of extracting flavonoids from purslane

technical field

The invention relates to the technical field of medicine, more specifically to a preparation method for extracting total flavonoids from purslane, and also relates to the application of total flavonoids from purslane.

Background technique

1. Antineoplastic drugs can easily cause kidney damage

Although the application of antitumor drugs is of great help to the control of tumors, its damage to the kidneys is a problem that cannot be ignored. The mechanism of antineoplastic drugs on kidney damage is as follows:

First of all, the concentration of the original urine makes the drug concentration in the urine much higher than the blood drug concentration, which is easy to produce crystals and block the renal tubules. At the same time, the weight of the kidneys accounts for only 0.4% of the body weight, while the blood flow accounts for about 25% of the cardiac blood volume. Therefore, when antineoplastic drugs and their metabolites that are harmful to the kidneys reach the kidneys with the blood stream, they are more likely to cause pathological changes.

On the other hand, kidney metabolic enzyme activity is high. Some antineoplastic drugs produce toxic substances during this transformation. Urinary system reactions caused by antineoplastic drugs mainly include urethral irritation and renal parenchyma damage (Han Tao. Adverse reactions of antineoplastic drugs and their prevention [J]. Capital Medicine, 2008, (4): 45-48).

More in-depth experiments have shown that the main components of antineoplastic drugs causing kidney damage are their hydrolytic metabolites. The nucleophilic amino group of antineoplastic drugs can interact with water molecules to generate a large number of free radicals, causing oxidative damage to cell membranes and mitochondria, and loss of function. In addition to causing oxidative damage, antineoplastic drugs can inhibit the renal tubular brush border and organic ion transport system, especially the activity of Na ⁺ —K ⁺ —ATPase during renal metabolism, and reduce the reabsorption of glucose by renal proximal tubules , so that epithelial cell edema degeneration. Some scholars believe that antineoplastic drugs can also inhibit the expression of renal cortex-binding protein regucalcin (Ca ⁺ —binding protein regucalcin) mRNA, increase the calcium content of renal cortex, cause intracellular calcium homeostasis imbalance, and cause cell damage (KinYK, Byun HS, Kim YH. Toxical Appl. Pharmacol. 1995, 130(1): 19).

Nowadays, with the increasing use of antineoplastic drugs, kidney injury has become a growing concern. Therefore, the study of renal damage protection drugs has become a hot topic today.

2. Special biological activity of flavonoids

Studies have shown that plant-based flavonoids widely present in nature have antioxidant effects, and their antioxidant effects are related to their structures (Wang Shumei. Antioxidant effects and structure-activity relationships of natural flavonoids [J]. Strait Pharmaceutical Journal, 2004 , 16(3), 10-13). At present, some studies have explored the effect and mechanism of flavonoids in some plants against kidney injury caused by antineoplastic drugs. It has been reported in the literature that the total flavonoids of Lycium barbarum can promote blood circulation and remove blood stasis, lower blood fat, lower blood sugar and improve immune function; the total flavonoids of hawthorn leaves can effectively prevent and treat cardiovascular diseases, scavenge oxygen free radicals, reduce lipids, diuresis and enhance deficiency tolerance Oxygen capacity; the protective effect of soybean isoflavones on the kidney is also a relatively popular research topic. Soy protein is beneficial to various kidney diseases such as animal and human polycystic kidney disease, diabetic nephropathy, and nephrotic syndrome (Su Bo, Cai Donglian. Soybean The protective effect of isoflavones on kidney disease [J]. Medical Recapitulate, 2003, 9(10): 611-612). In addition, Xiong Xiaoming et al. confirmed that Ginkgo biloba extract, a compound containing flavonoids and terpene lactones, has anti-cisplatin nephrotoxicity (Xiong Xiaoming et al. Ginkgo biloba extract treats cisplatin-induced renal injury in rats[ J]. Chinese Hospital Pharmacy, 2008, 28(4): 265-268).

3. Research progress of purslane and its extract

Purslane (Portulaca Oleracea L.), also known as ant vegetable, long-lived vegetable, melon seed vegetable, five-element grass, etc., is an annual herbaceous plant of the family Portulaca. It is distributed in almost every province in my country. It is rich in resources and easy to collect. It is a traditional Chinese medicine in my country and has been used in the folk for thousands of years. The active ingredients in purslane, such as alkaloids, polysaccharides, anthraquinone, etc., can prevent and treat cardiovascular diseases; Vitamins, minerals, proteins and amino acids, have anti-atherosclerosis, anti-bacterial, anti-tumor and anti-oxidation effects.

The main component of purslane ethanol extract is flavonoids, and the total flavonoids content is as high as 6.37% (Wei Xun, Wang Zhongying. Determination of total flavonoids content in purslane [J]. Chinese Journal Of Spectroscopy Laboratory, 2003 (1), 20 ( 1): 128-129). However, after retrieval, so far, there has been no report of purslane extract resisting cisplatin-induced kidney injury.

On this basis, we study the renal protective effect of purslane extract, and prove that the present invention has a good protective effect on renal injury caused by chemotherapy drugs through animal experiments.

Contents of the invention

The purpose of the present invention is to provide a preparation method for extracting total flavonoids from purslane. The method utilizes the feature that flavonoids are fat-soluble substances, and uses 75% ethanol to reflux to extract, and the process is simple and easy; and the cyclone concentration technology of the extract is also mature. Moreover, the extraction rate of total flavonoids by this method is high, and its biological activity can be maintained to ensure the subsequent experiments. In addition, purslane is widely available and inexpensive, making the extract affordable.

Another object of the present invention is to provide an application of total flavonoids extracted from purslane in the preparation of medicines for treating or preventing (resisting) kidney damage caused by cisplatin. Animal experiments have proved that the extract has a significant effect. The blood urea nitrogen concentration of the mice in the drug-administered group is significantly lower than that of the non-medicated group, and the blood urea nitrogen concentration of the mice in the high-dose group is also significantly lower than that in the low-dose group. The extract adopts a conventional oral dosage form, and the administration is convenient, painless, without sequelae, and almost without any toxic and side effects.

In order to achieve the above object, the present invention adopts the following technical measures:

A preparation step for extracting total flavonoids from purslane is:

1. Wash the fresh purslane with clean water, remove its leaves and roots, drain the water and dry it in a dark and ventilated place until the surface moisture is completely removed. The time is controlled within 20-24 hours.

2. Cut the stem of the dried purslane into a short stick shape of about 0.8-1.2cm.

3. Add 6 times the volume of 70% ethanol (mass ratio) to the purslane, control the reflux time in the water bath at 78-82°C for 1-3 hours, and reflux for 2-4 times.

4. Suction filter the solution obtained after reflux, leave the filtrate, discard impurities, and then reflux and suction filter the obtained liquid for 2-4 times and mix well.

5. Temperature control Rotary evaporation of the liquid obtained in the above step 4 was carried out in a rotary evaporator under the condition of 60-65° C. to obtain a concentrated solution of total flavonoids.

The basic process of the application of total flavonoids in the preparation of medicines for treating or preventing (resisting) cisplatin-induced kidney damage is:

1. Inoculation of tumor cells

40 female Kunming mice, weighing (25±2) g, were injected with 0.2 ml of S ₁₈₀ mouse ascites (containing tumor cell line) in the axillary cavity on the first day of the experiment.

2. Grouping

The inoculated mice were randomly divided into 4 groups (normal saline control group, cisplatin injury model control group, low dose purslane drug treatment group, high dose purslane drug treatment group), 10 in each group.

3. Packet processing

The mice were divided into groups as follows:

1) Normal saline control group: 24 hours after inoculation, 0.5ml of normal saline was administered into the stomach every day for a total of 7 days;

2) Cisplatin injury model control group: 24 hours after inoculation, 0.5ml of normal saline was administered into the stomach every day for 7 consecutive days, and cisplatin (12.0mg/kg) was injected intraperitoneally once on the fourth day;

3) Low-dose administration group of total flavonoids of purslane: the extract of purslane was administered once a day (0.5ml) according to the ratio of mouse body weight 5mg/kg, for 7 consecutive days, and on the 4th day, a one-time intraperitoneal injection was given. Platinum (12.0mg/kg);

4) High-dose purslane extract treatment group: use purslane extract once a day (0.5ml) according to the ratio of mouse body weight 20mg/kg, for 7 consecutive days, and inject cisplatin ( 12.0 mg/kg).

The present invention has the following advantages:

1. The purslane extract that the present invention relates to is obtained through ethanol extraction and progressive concentration with purslane as raw material matrix. Wide range of sources of raw materials and low prices;

2. The extraction and concentration process of total flavonoids of purslane is simple and easy, and the technology is mature;

3. The development of total flavonoids of purslane as a drug to protect kidney damage caused by chemotherapy drugs is not only an enrichment of the medical theory of the motherland, but also an in-depth study of the effective parts of purslane. Therefore, it has important social and economic benefits ;

4. The total flavonoids of purslane can not only effectively treat kidney damage caused by chemotherapy drugs, but also reduce blood fat, anti-aging and other effects, and can enhance the immune function of the body;

5. The high-efficiency anti-kidney damage activity of the total flavonoids of purslane lies in its multiple effects such as scavenging free radicals, increasing antioxidant enzyme activity, reducing lipid peroxide levels, and immune regulation;

6. After searching, the acute toxicity test of the total flavonoids of purslane shows that it has almost no toxic side effects;

7. The present invention adopts an oral administration mode, which is convenient, painless, and without sequelae.

Detailed ways

One, a kind of flow process that extracts total flavonoids from purslane is:

Fresh purslane processing → reflux extraction → suction filtration → supernatant → concentration → purification

Specific steps are as follows:

1. Wash the fresh purslane with clean water, remove its leaves and roots, drain the water and hang it in a dark and ventilated place until the surface moisture is completely removed. The time is controlled at 20 or 21 or 22 or 23 or 24 hours.

2. Cut the dried purslane stems into 0.8 or 0.9 or 1 or 1.1 or 1.2 cm short sticks.

3. Add 6 times the volume of 70% ethanol (mass ratio) to the purslane, reflux in a water bath at 78 or 79 or 80 or 81 or 82°C for 1 or 2 or 3 hours, and reflux for 4 or 3 or 2 times.

4. Suction filter the solution obtained after reflux, leave the filtrate, discard impurities, and then mix the liquid obtained by reflux and suction filtration three times.

5. Temperature control Rotary evaporation of the liquid obtained in step 4 at 60 or 61 or 62 or 63 or 64 or 65° C. to obtain a concentrated solution.

2. Determination of total flavonoid content in purslane

1. Identification of purslane flavonoids

Add magnesium powder and hydrochloric acid color reaction: take a little sample, add 1-3ml of 95% ethanol, put it in a test tube with a ground plug, shake at room temperature (23°C-27°C), add a little magnesium powder to shake, then drop Add a few drops of concentrated hydrochloric acid, and it turns red after 1-2 minutes, which proves that the extract contains flavonoids.

2. Determination of total flavonoids in purslane

① Preparation of standard curve

Accurately weigh 200 mg of the rutin reference substance, dissolve it with 70% ethanol (mass ratio), set the volume in a 100 ml volumetric flask, and shake well. Accurately draw 10ml, place it in a 100ml volumetric flask, and dilute to volume with water to obtain a 0.2mg/ml standard solution. Accurately draw 0.4, 0.8, 1.2, 1.6, 2.0, 2.4ml of the standard solution, place them in a 10ml volumetric flask, add water to 2.4ml, add 0.4ml of sodium nitrite with a mass fraction of 5%, mix well, place for 6min, add The mass fraction is 0.4ml of 10% aluminum nitrate, mix well, stand for 6min, add 4.3% sodium hydroxide 4.0ml, dilute with water to the mark, shake well, stand for 15min, use a blank tube as a control, use a UV spectrophotometer , Measure the absorbance at a wavelength of 500nm to obtain a calibration curve of concentration C and absorbance A: C=72.714A-0.5268, r=0.9991.

② Determination of total flavonoid content

Precisely draw 1.0ml of flavonoid extract and place it in a 10ml volumetric flask, add water to 2.4ml, add 0.4ml of sodium nitrite with a mass fraction of 5%, shake well, let stand for 6min, add 0.4ml of aluminum nitrate with a mass fraction of 10%, shake well, Stand for 6 minutes, add 4.0ml of sodium hydroxide with a mass fraction of 4.3%, add water to the mark, shake well, let stand for 15 minutes, and use a blank reagent tube as a control. Using a UV spectrophotometer, measure the absorbance at a wavelength of 500nm (full-wavelength scanning, showing an absorption peak at 500nm), and substitute it into the regression equation to calculate the total flavonoid content.

3. Animal experiments

1. Inoculation of tumor cells

40 female Kunming mice, weighing (25±2) g, were injected with 0.2 ml of S ₁₈₀ mouse ascites (containing tumor cell line) in the axillary cavity on the first day of the experiment.

2. Grouping

The inoculated mice were randomly divided into 4 groups (normal saline control group, cisplatin injury model control group, low dose purslane drug treatment group, high dose purslane drug treatment group), 10 in each group.

3. Packet processing

The mice were divided into groups as follows:

1) Normal saline control group: 24 hours after inoculation, 0.5ml of normal saline was administered into the stomach every day for a total of 7 days;

2) Cisplatin injury model control group: 24 hours after inoculation, 0.5ml of normal saline was administered into the stomach every day for 7 consecutive days, and cisplatin (12.0mg/kg) was injected intraperitoneally once on the fourth day;

3) Low-dose administration group of total flavonoids of purslane: the extract of purslane was administered once a day (0.5ml) according to the ratio of mouse body weight 5mg/kg, for 7 consecutive days, and on the 4th day, a one-time intraperitoneal injection was given. Platinum (12.0mg/kg);

4) High-dose purslane extract treatment group: use purslane extract once a day (0.5ml) according to the ratio of mouse body weight 20mg/kg, for 7 consecutive days, and inject cisplatin ( 12.0 mg/kg).

4. Take blood and weigh the tumor

On the seventh day of gavage, blood was taken from the eyeballs of the mice in a test tube, and then the mice were sacrificed and the tumor mass in the armpit was taken out, weighed and recorded.

5. Get the Serum

After taking blood from the mouse eyeball, immediately put the test tube containing the blood into the refrigerator, take it out after 30 minutes and centrifuge it for 10 minutes, and absorb the upper layer of light yellow transparent liquid, namely serum.

6. Determination of blood urea nitrogen (BUN) content

Add reagents to test tubes according to Table 1

Table 1 BUN content determination scheme

blank tube standard tube Assay tube Distilled water (ml) 0.05 BUN standard solution (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

Mix the added reagents, place in water for 15 minutes, and immediately cool with tap water. Use a glass cuvette with a wavelength of 520nm and an optical path of 1cm, a blank tube or distilled water to adjust to zero, and measure the OD value. Then, the blood urea nitrogen content was calculated according to the OD value.

7. Determination of blood creatinine content

Take 0.2ml of serum into a test tube, add 2ml of tungstic acid protein precipitation reagent, mix well, centrifuge at 3000r/min for 10min, take the supernatant and measure it according to Table 2

Table 2 Determination scheme of blood creatinine content

Assay tube standard tube blank tube Serum protein filtrate (ml) 1.6 10umol/L creatinine standard (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

Mix the added reagents, 37 ° C water bath for 10 minutes, take it out and cool it with running water, 1 cm optical path, 510 nm wavelength, zero the blank tube or distilled water, and measure the OD value. Finally, the content of blood creatinine was calculated according to the OD value.

4. Experimental results

1. Tumor weight

The mice were dissected, all the tumors were taken out and the weight was weighed, and the unit was "gram". As a result, there was no significant difference in tumor weight between the high and low doses of purslane extract group and the cisplatin injury model group, suggesting that high and low doses of purslane extract had no significant auxiliary effect on cisplatin treatment of tumors (Table 3) .

Table 3 Comparison of tumor weight of mice in each group (x±s)

Compared with normal saline group ^* P<0.05

2. Determination of serum urea nitrogen (BUN) and creatinine (Cr) concentrations

As a result, the blood urea nitrogen of the cisplatin injury model group was significantly different from that of the normal saline group, suggesting that one-time administration of large doses of cisplatin can damage the kidneys. Compared with the cisplatin injury model group, the serum creatinine of the high and low doses of purslane extract group had no significant difference, but the blood urea nitrogen was significantly lower than that of the cisplatin injury model group, suggesting that the high and low doses of purslane extract had a significant effect on Cisplatin-induced renal injury was protective (Table 4).

Table 4 Comparison of blood urea nitrogen values of mice in each group (x±s, mmol/L)

Compared with normal saline group, ^** P<0.01; compared with cisplatin injury model group, ^# P<0.05.

Five conclusions

The above experimental results show that the extract of purslane has no obvious adjuvant effect on cisplatin treatment of tumors, but has obvious protective effect on kidney damage caused by cisplatin.

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.