CN103751792B - Radix Angelicae Sinensis extract is as the application of natural intestinal absorption enhancer - Google Patents

Abstract

The invention relates to the technical field of medicine, and discloses the application of an angelica extract as a natural intestinal absorption accelerator. In the present invention, the angelica extract is used as a natural intestinal absorption promoter, which can be compatible with the drug to be promoted or used as a compound drug, which can exert a significant synergistic effect, improve the bioavailability of the drug to be promoted, and will not affect the absorption site. mucous membrane damage. The application of angelica extract as a natural intestinal absorption enhancer has great application value.

Description

Application of Angelica Sinensis Extract as Natural Intestinal Absorption Promoter

technical field

The invention relates to the technical field of medicine, and more specifically, relates to the application of angelica extract as a natural intestinal absorption promoter.

Background technique

The study found that in drug research, 39% of new compounds were eliminated due to gastrointestinal absorption problems after oral administration or needed to be formulated to improve absorption. Traditional Chinese medicine also has absorption problems. Compared with chemical drugs, the absorption mechanism of traditional Chinese medicine or the active ingredients of traditional Chinese medicine is more unclear. Traditional Chinese medicine in the motherland is our treasure, but how Chinese medicine enters the blood from the stomach is the most interesting problem for Chinese medical workers, but the current research progress on the above-mentioned problem is not great.

Intestinal absorption enhancers refer to substances that can promote the transmembrane transport of drugs in the gastrointestinal tract. Its main mechanism of action includes: (1) enhancing the fluidity of cell membranes, promoting the formation of cell membrane pores, reducing the viscosity of the mucosal layer, and improving membrane permeability ; (2) Inhibit the action of hydrolytic enzymes and enhance the ability of drugs to exert their efficacy; (3) Temporarily change the tight junction state between epithelial cells at the drug site, which is conducive to the passage of drugs; (4) Prevent protein aggregation and enhance the thermodynamic movement of drugs; (5) ) Increase the blood flow per unit time at the drug site, and increase the gradient of drug concentration inside and outside the cell membrane.

At present, intestinal absorption enhancers mainly include surfactants, bile salts, fatty acids and fatty acid salts, and bioadhesive polymers.

Angelica sinensis , a perennial herb, is distributed in Gansu, Yunnan, Sichuan, Qinghai, Shaanxi, Hunan, Hubei, Guizhou and other places in China, and is cultivated in various places. The root of Angelica sinensis can be used as medicine and is one of the most commonly used traditional Chinese medicines. Angelica can also be used in the ingredients of stewed products. Its main feature is to remove the fishy smell and increase the aroma, and increase the aroma of meat products and medicines. Angelica can be combined with various traditional Chinese medicines to form a drug pair. However, the compatibility of traditional Chinese medicines and the formation mechanism of the drug pair are relatively complicated. Existing studies have not provided the specific mechanism and specific application effect of Angelica to promote drug absorption. The unclear mechanism and effect will eventually affect Drug development and application. The Angelica sinensis extract prepared by conventional methods such as water extraction, alkaline water extraction, ethanol immersion method, ethanol thermal reflux method, ethanol percolation method, etc. contains volatile oil components and organic acids, mainly succinic acid and ligustilide , ferulic acid, etc.

At present, there is no technical report on the use of Angelica sinensis extract as an intestinal absorption enhancer for related drugs.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiency of the application technology of the existing natural intestinal absorption enhancer, and provide the application of the angelica extract as the natural intestinal absorption enhancer.

The purpose of the present invention is achieved through the following technical solutions:

Provided is the use of an angelica extract as a natural intestinal absorption enhancer for medicines.

The drugs include, but are not limited to, drugs absorbed in the duodenum, jejunum, colon and/or ileum, especially drugs absorbed in the duodenum.

As one of the preferred application schemes, the Angelica sinensis extract is used in combination with medicines.

As the second preferred application scheme, the Angelica sinensis extract is prepared together with medicines for pharmaceutical application.

Angelica sinensis extract has significant intestinal absorption-promoting effect on many drugs and active ingredients of drugs, and more preferably, angelica extract has significant intestinal absorption-promoting effect on astragaloside IV, gastrodin or paeoniflorin.

The Angelica sinensis extract is prepared together with medicines for pharmaceutical application, and the preferred dosage form is an oral administration dosage form. Including but not limited to tablet, capsule, granule, oral powder, oral solution, oral emulsion or oral suspension.

The present invention further provides the application of the active component ferulic acid in the angelica extract as a natural intestinal absorption accelerator of medicine.

Preferably, ferulic acid, the active ingredient of the Angelica sinensis extract, can significantly promote the absorption of the drug in the duodenum without causing damage to the absorption site.

Ferulic acid, the active ingredient of Angelica extract, can significantly promote the absorption of astragaloside IV in the duodenum. And the promotion of absorption is achieved by increasing the absorption rate constant Ka and apparent absorption coefficient Papp of the duodenum.

Beneficial effects of the present invention:

The invention provides the application of Angelica sinensis extract as a natural intestinal absorption promoter, said Angelica sinensis extract has a good intestinal absorption promotion effect, and after the promoted drug enters the human body, it can exert a synergistic effect and improve the absorption of the drug. bioavailability. The application of the Angelica sinensis extract can reduce the dosage of drugs and the use of other intestinal absorption promoters, and reduce the toxic and side effects of drugs and the absorption of other invalid chemical foreign substances.

The study of the present invention concludes that the extract of Angelica sinensis has significant intestinal absorption promoting effect on related drugs, and provides a strong technical basis for exploring the principle of compatibility of Angelica sinensis and other traditional Chinese medicines to form a drug pair from the perspective of intestinal absorption.

Further, the present invention provides the application of ferulic acid, the active ingredient of Angelica sinensis extract, as an intestinal absorption promoter of astragaloside IV. Ferulic acid has a good intestinal absorption promoting effect on astragaloside IV, and astragaloside IV enters the human body. After in vivo, it can exert a synergistic effect, promote the absorption of astragaloside IV in the intestine, increase the blood concentration, reduce the toxic side effects of the drug and the absorption of other invalid chemical foreign substances, and will not cause damage to the mucosa of the intestinal absorption site, making full use of it. Ferulic acid has great application value as an intestinal absorption promoter of astragaloside IV.

Description of drawings

Fig. 1 is the Ka-osmotic ratio of different absorption enhancers to paeoniflorin in the duodenum;

Fig. 2 is the comparison of absorption enhancer to paeoniflorin in duodenal Papp;

Figure 3 is the effect of ferulic acid on the absorption of astragaloside IV in different intestinal segments ( ±s, n=5);

Figure 4 is the promotion of ferulic acid on the intestinal transport of astragaloside IV;

Figure 5 is the promotion of ferulic acid on the intestinal transport of astragaloside IV Papp;

Figure 6 is a section of rat duodenum intestine (HE staining, 400 times).

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the raw materials and methods used in the examples of the present invention are conventional commercially available raw materials and conventionally used methods in the art.

Example 1 Experiment of the intestinal absorption-promoting effect of angelica extract on paeoniflorin

The Angelica sinensis extract used in this example was purchased from Nanjing Zelang Plant Extract Biotechnology Co., Ltd.; paeoniflorin was purchased from Xi'an Kailai Bioengineering Co., Ltd., but this does not limit the present invention. Those skilled in the art can use angelica extract and paeoniflorin from other sources, and can also use conventional extraction methods to make their own.

Using the rat model of one-way intestinal perfusion in vivo, the content of paeoniflorin in the perfusate was determined by HPLC, and the promoting effect of angelica extract on the intestinal absorption of paeoniflorin was investigated respectively.

Carry out the test of rat in vivo one-way intestinal perfusion model according to the following steps:

S1. Rats were fasted for 12 hours, free to drink water, anesthetized by intraperitoneal injection of 1% pentobarbital sodium solution (5 mL/kg), fixed on the back, and maintained body temperature;

S2. Open the abdominal cavity about 3 cm along the midline of the abdomen, take about 10 cm of the duodenum, and place it 1 cm away from the pylorus. Rinse the material, then empty the physiological saline with air, and install the device;

S3. Perfuse with perfusate preheated to 37°C at a flow rate of 0.2 mL/min for 30 minutes to balance the pipeline and intestine;

S4. Start timing, collect the perfusate at 0-15, 15-30, 30-45, 45-60, 60-75, and 75-90 min respectively, weigh, and record the weight of perfusate and collected solution;

S5. At the end of the experiment, measure the length and inner diameter of the intestinal segment.

The content of paeoniflorin in the perfusate was determined by HPLC, and the absorption rate constant Ka and apparent absorption coefficient Papp of the drug were calculated according to the following formula:

                        

Among them, Q _in and Q _out are the volumes of the perfusate and collection fluid infused into the intestinal segment (mL), l and r are the length (cm) and cross-sectional radius (cm) of the perfused intestinal segment, respectively, and Q is the perfusion velocity , C _in and C _out are the concentration of the perfusate and the collected solution (μg/mL), and V is the volume of the perfused intestinal segment (cm ³ ).

Taking the drug absorption rate constant Ka and apparent absorption coefficient Papp as indicators, the experimental results showed that hydroxypropyl-β-cyclodextrin and angelica extract significantly improved the absorption rate constant of paeoniflorin in the duodenum of rats and Apparent absorption coefficients, showing the possibility of their use as absorption enhancers in oral preparations of paeoniflorin.

Table 1 Effects of absorption enhancers on the Ka of paeoniflorin in the duodenum

Table 2 Effects of absorption enhancers on paeoniflorin Papp in duodenum

Using the same method as above to carry out the experiment, taking the absorption rate constant Ka and the apparent absorption coefficient Papp as indicators, the effect of Angelica sinensis extract on promoting intestinal absorption of drugs such as astragaloside IV, gastrodin, paeoniflorin is better than that without adding Angelica sinensis Extract medicine, and stronger than the corresponding concentrations of sodium caprate and sodium deoxycholate.

Example 2

Experimental animals: SD rats, half male and half female, weighing 160-220 g, provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine, certificate number: SCXK (Guangdong) 2008-0020.

Instruments: SHIMADZU high-performance liquid chromatography (Japan); TGL-20B high-speed centrifuge (Shanghai Anting Scientific Instrument Factory).

Drugs and reagents: Astragaloside IV reference substance (National Institute for the Control of Pharmaceutical and Biological Products, batch number: 110781-200613); acetonitrile and methanol (Dima Company, chromatographically pure); water is double distilled water. All other reagents were analytically pure.

Krebs-Ringer nutrient solution: hereinafter referred to as KR solution, containing 7.8g NaCl, 0.35g KCl, 1.37g NaHCO ₃ , 0.32g NaH ₂ PO ₄ , 0.02g MgCl ₂ , 1.4g glucose per 1000 ml, make up 1000mL with water; Prepare and use on the same day.

In this example, ferulic acid was purchased from Nanjing Zelang Plant Extract Biotechnology Co., Ltd. (batch number 20110903), but this does not limit the present invention. Those skilled in the art can use ferulic acid from other sources, and can also use the prepared angelica extract Ferulic acid obtained by separating and purifying the product. The extract of Astragalus membranaceus can be commercially purchased or homemade (containing 13.9% astragaloside IV). The self-made method is as follows: take the dried Astragalus root raw material, wash, dry, crush, add 10 times the amount of 70% ethanol, heat and reflux extraction twice, each time for 3 hours, combine the secondary extracts, recover ethanol under reduced pressure, and add the residue Dissolve in an appropriate amount of water so that the concentration of astragaloside IV is 0.8~1.1mg/mL, take D101 macroporous adsorption resin, load the sample at 2 BV/h, wash with water until there is no Molish reaction, elute with 5 BV 70% ethanol, collect and wash Deliquification, recovery of ethanol under reduced pressure, drying, that is.

1. Medicine solution preparation by eversion intestinal sac method

Astragalus group: An appropriate amount of Astragalus extract was taken, dissolved in Krebs-Ringer nutrient solution, and the volume was adjusted to 100 mL to prepare a solution containing 34.75 μg·mL ^-1 of astragaloside IV.

Compatibility group: according to the dose of Angelica-Astragalus 1:1 (the mass ratio of Angelica and Astragalus is 1:1), with reference to the concentration of Astragalus group, prepared in the same way.

2. Experiment of eversion intestinal sac method

Healthy SD rats (provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine, certificate number: SCXK (Guangdong) 2008-0020) were fasted overnight (free drinking water), and injected intraperitoneally with 1% pentobarbital at 400 mg·kg-1 For sodium solution anesthesia, different intestinal segments were divided, the intestinal tract was turned over, and 1 mL of blank Krebs-Ringer nutrient solution was injected into the intestinal sac, and the segment of intestinal sac was placed in the preheated everted intestinal sac method liquid, and poured into the liquid The mixed gas of carbon dioxide: oxygen (5:95) was continuously fed into the medium, and kept at 37°C for 0.5 h. After the experiment, the serosal fluid was collected to determine the drug content and the intestinal absorption area to investigate the absorption of astragaloside IV by different intestinal segments and the effect of ferulic acid on the absorption of astragaloside IV.

3. Determination of drug content in serosal fluid by eversion intestinal sac method

Chromatographic column: Diamonsil C18 column (4.6 mm×250 mm, 5 μm); mobile phase: acetonitrile-water (32.4:67.6); column temperature: 25°C; flow rate: 1.0 mL·mL ^-1 ; injection volume: 20 μL. The specificity of the method was investigated, and the results showed that the blank intestinal juice had no interference on the determination of astragaloside IV. Take an appropriate amount of stock solution of astragaloside IV reference substance, add the blank intestinal juice of the eversion intestinal sac method, and prepare solutions with a series concentration of 8.03, 16.06, 24.06, 32.12, 40.15, 48.18, 56.21 μg·mL ^-1 , and adjust the concentration of astragaloside IV to (C) Regression was performed on the peak area (A), and the standard curve was obtained: A=716.8C–108.53, R=0.9994, and the linear range: 8.03～56.21 μg·mL-1. The lowest detection limit was 1.5 μg·mL ^-1 . The recoveries of high, medium and low concentrations were 93.9%-97.4%, the RSDs were 1.40%-2.81%, the intra-day precision RSDs were 0.54%-0.78%, and the inter-day precision RSDs were 0.58%-0.99%, all of which met the requirements.

4. Experimental results

The rat model of everted intestinal sac was used to investigate the absorption of Astragalus membranaceus in different intestinal segments and the effect of ferulic acid on its absorption. The results showed that astragaloside IV was absorbed in the entire intestinal segment of the small intestine, and the Papp order of different intestinal segments was: twelve Dentum > jejunum > colon > ileum, with the best absorption in the duodenum. After adding ferulic acid, the absorption in the duodenum and jejunum increased significantly, as shown in Figure 3.

5. Experimental conclusion

In the everted intestinal sac experiment, Zhu Jingyun et al. measured the activity of the intestinal sac by the glucose oxidase method, and confirmed that the activity of the isolated intestinal sac can only be maintained for 1 hour. Absorption and transfer, and through the constant temperature of 37 ℃, the introduction of oxygen-enriched air ensures the authenticity and reliability of the data. According to the summary of the experiment of the present invention, it is found that ferulic acid can well promote the absorption of astragaloside IV, especially the absorption of astragaloside IV in the duodenum.

Example 3

1. In vivo one-way intestinal perfusion liquid preparation

Astragalus group: Take an appropriate amount of Astragalus extract, dissolve it with sodium chloride injection and dilute to 100 mL, and prepare astragaloside IV concentrations of 10.5, 15.75, 21.00, 31.50, 36.75 μg·mL ^-1 , a total of five gradient concentrations Perfusate.

Compatibility group: according to the dose of Angelica-Astragalus 1:1 (the mass ratio of Angelica and Astragalus is 1:1), with reference to the concentration of Astragalus group, prepared in the same way.

2. In vivo one-way intestinal perfusion experiment

Healthy SD rats (provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine, certificate number: SCXK (Guangdong) 2008-0020) were fasted overnight (free drinking water), and injected intraperitoneally with 1% pentobarbital at 400 mg·kg ^- 1 Anesthetized with sodium solution, the abdominal cavity was opened, intubation and ligation were made at both ends of the intestinal segment under investigation (approximately 10 cm) to form a circulatory circuit. The intestinal contents were washed clean with 37°C normal saline, and 100 mL of in vivo one-way intestinal perfusion solution was taken at 37°C, and perfused at a flow rate of 1.0 mL·min ^-1 for 10 min, and then the flow rate was adjusted to 0.2 mL·min min ^-1 , after pre-equilibrating for 30 min, collect and weigh the perfusate at 30-45, 45-60, 60-75, 75-90, 90-105, 105-120 min. After the experiment, the intestinal segments were cut and the absorption area was measured. The concentration of the perfusate was measured, and the absorption of astragaloside IV in the intestinal segment and the effect of ferulic acid on the absorption of astragaloside IV were investigated respectively.

3. Determination of drugs in in vivo intestinal perfusate

According to the chromatographic conditions under "Determination of drug content in serosal fluid by eversion intestinal sac method", the specificity of the method was investigated, and the results showed that the impurities in the intestinal perfusate did not interfere with the determination of astragaloside IV. Take an appropriate amount of astragaloside IV reference substance, add in vivo intestinal perfusion blank intestinal juice, prepare solutions with a series of concentrations of 8.71, 17.42, 26.13, 34.84, 43.55, 52.26, 60.97 μg·mL ^-1 , centrifuge at 12000 rpm for 10 min, and take The supernatant was filtered with a 0.45 μm microporous membrane, and 20 μL of the filtrate was injected into the sample for determination. The astragaloside concentration (C) was regressed against the peak area (A) to obtain a standard curve: A=829.3C-76.24, R = 0.9995, linear range: 8.71～60.97 μg·mL ^-1 . The lowest detection limit was 1.7 μg·mL ^-1 . The recoveries of high, medium and low concentrations were 92.9%-99.3%, RSD were 1.62%-3.53%, intra-day precision was 0.64%-1.86%, and inter-day precision was 0.38%-1.99%, all of which met the requirements.

4. Calculation of drug absorption rate constant Ka and drug apparent absorption coefficient Papp Use the gravimetric method to correct the inflow and outflow volumes of the perfusate to eliminate the influence of volume changes, and calculate the absorption rate constant Ka and drug apparent absorption according to the following equation Coefficient Papp:

                    

In the above formula: C _in and C _out are the drug concentration of the perfusate at the entrance and exit of the intestinal tract (μg mL ^-1 ); Q _in and Q _out are the volumes of the perfusate at the entrance and exit of the intestinal tract (mL); is the length (cm) and cross-sectional radius (cm) of the perfused intestinal segment; is the perfusion velocity (mL·min ^-1 ); is the volume of the perfused intestinal segment (cm ³ ).

5. Cytomorphological examination

Take 1 cm of duodenum before and after the in vivo intestinal perfusion experiment, and make tissue sections with HE staining method, and conduct morphological examination of the epithelial cell layer to test whether the duodenal epithelial cells maintain activity after administration.

6. Experimental results

The duodenum of rats was selected, and the in vivo one-way intestinal perfusion model was used to investigate the absorption of astragaloside IV at different concentrations, and to investigate the absorption-promoting effect of ferulic acid on astragaloside IV. The results are shown in attached drawings 4 and 5 Shown, and shown in Table 3, Table 4.

Table 3 Absorption rate constant Ka and osmotic enhancement ratio ER of different concentrations of astragaloside IV in the duodenum of rats

Note: Compared with the Astragalus group, the compatibility group has significant difference, *P<0.05.

Table 4 Apparent permeability coefficient Papp and enhanced permeability ratio ER of different concentrations of astragaloside IV in rat duodenum

The results showed that different concentrations of astragaloside IV were absorbed in the duodenum, and after adding ferulic acid, both Ka and Papp increased.

7 Cell Morphological Examination

After the administration, the small intestinal villi were basically complete without obvious damage, and were basically consistent with those before the administration, as shown in Figure 6. It is proved that astragaloside IV and the combined use of ferulic acid and astragaloside IV of the present invention did not cause damage to the intestinal segment, and the intestinal tract maintained absorption activity during the experiment.

8. Experimental conclusion

In the one-way intestinal perfusion experiment in vivo, the absorption of Astragalus membranaceus in the duodenum showed an increase with the increase of the concentration at low and medium concentrations, showing the characteristics of passive absorption; but at higher concentrations, both Ka and Papp decreased, suggesting that the intestinal transport of astragaloside IV may be carried by carriers.

Under the action of ferulic acid, the Ka and Papp of astragaloside IV increased, and the ER values were greater than 1, and there was a significant difference (P<0.05); among them, the medium concentration (21.00 μg·mL ^-1 ) The effect of promoting absorption is the most obvious: ERKa=1.42, ERPapp=1.47, showing that the ability of ferulic acid to promote intestinal absorption of Astragalus is related to the concentration of astragaloside IV.

Claims (8)

1. the Radix Angelicae Sinensis extract containing ferulic acid is preparing as the natural intestinal absorption enhancer of medicine astragaloside, gastrodine or peoniflorin the application had in the pharmaceutical preparation of the intestinal absorption of improvement.

2. application according to claim 1, it is characterized in that, the described Radix Angelicae Sinensis extract containing ferulic acid is preparing as the absorption enhancer at the duodenum of medicine astragaloside, gastrodine or peoniflorin, jejunum, colon or ileum one of them or several position the application had in the pharmaceutical preparation of the intestinal absorption of improvement.

3. application according to claim 2, it is characterized in that, the described Radix Angelicae Sinensis extract containing ferulic acid is preparing as the duodenal absorption enhancer of medicine astragaloside, gastrodine or peoniflorin the application had in the pharmaceutical preparation of the intestinal absorption of improvement.

4. application according to claim 1, is characterized in that, the described Radix Angelicae Sinensis extract containing ferulic acid and compatibility of drugs are applied.

5. application according to claim 1, is characterized in that, described Radix Angelicae Sinensis extract containing ferulic acid and medicine are prepared jointly to be become medicament and apply.

6. application according to claim 5, is characterized in that, described medicament is oral administered dosage form.

7. application according to claim 6, is characterized in that, described oral administered dosage form is tablet, capsule, granule, oral powder, oral solution, Orally taken emulsion or oral suspensions.

8. application according to claim 1, is characterized in that, described medicine is astragaloside.