CN101181336A - A method for refining the effective part of bitter gourd for lowering blood sugar - Google Patents

Abstract

The invention belongs to the technical field of medicine, and discloses a method for refining the effective part of bitter melon for lowering blood sugar. (1) preparing bitter melon medicinal liquid before decolorization and refining: dry and pulverize bitter melon decoction pieces, extract with ethanol reflux, and concentrate to obtain extract; The extract is diluted, adsorbed on a macroporous adsorption resin column, washed, and then eluted with ethanol solution to obtain the bitter gourd liquid before decolorization and refining. (2) Refining process: collect the ethanol eluate from the resin column, add a decolorizer, heat to reflux, filter, concentrate under reduced pressure, and dry in vacuum to obtain Momordica charantrin. The Momordica charantrin extracted in the present invention can be directly made into various preparations, such as tablets, capsules, granules, oral liquids, pills, suspensions, dropping pills, cataplasms, patches and the like. Compared with the traditional technology, the invention has the characteristics of small volume, no moisture absorption and high active ingredient content. Compared with the solvent extraction method, no toxic and volatile organic solvents are used in the process, which is safe and reliable, with low production cost and no pollution to the environment.

Description

A method for refining the effective part of bitter gourd for lowering blood sugar

technical field

The invention belongs to the technical field of medicines, and relates to a method for refining bitter melon effective fractions (KGE) for lowering blood sugar, in particular to a method for refining steroidal components of effective fractions for lowering blood sugar in bitter melon.

Background technique

Bitter gourd is a traditional Chinese medicine, and there are many records in ancient prescriptions. In recent years, domestic and foreign studies and a large number of literature reports have shown that bitter melon has a sustained and efficient hypoglycemic effect without any toxic and side effects. The active ingredients contained in bitter melon can not only regulate blood sugar by promoting the secretion of insulin by β-cells, but also have insulin-like effects, and the oral curative effect is definite. Developing it into a highly efficient, low-toxic, and cheap modern Chinese medicine preparation for treating type II diabetes has broad market prospects.

At present, the methods for preparing bitter melon steroids mainly include extraction processes such as water decoction, alcohol precipitation, and macroporous adsorption resin. The above-mentioned process has many impurities, low active ingredient content, a large amount of sugar, and certain moisture-absorbing ingredients, which affect the efficacy and product quality.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for refining the steroidal glycoside components in the effective hypoglycemic part of the bitter gourd.

The technical solutions adopted are:

1. Prepare bitter gourd medicinal liquid before decolorization and refining: dry and pulverize bitter gourd decoction pieces, extract with 10-95% ethanol under reflux, recycle ethanol, and concentrate to obtain extract; dilute the extract with water, absorb on a macroporous adsorption resin column, and use Washing with water with a pH of 2-8, discarding the water eluent, and then eluting with 5%-95% ethanol solution for 5-20 times the volume of the resin column to obtain the bitter melon liquid before decolorization and purification.

2. Refining process: collect the ethanol eluate from the resin column, add a decolorizer according to the volume of the eluent at 0.1-50%, heat and reflux for 0.1-10 hours, filter, concentrate under reduced pressure, and dry in vacuum to obtain Momordica charantrin. The elution volume of the resin column is adjusted so that the steroidal glycosides can be completely eluted, and the concentration of the drug in the eluate is suitable for decolorization, so that the two steps are closely connected.

The content of momordica glycosides in the effective part of the bitter gourd for lowering blood sugar by the method can reach more than 50%.

The Momordica charantrin extracted in the present invention can be directly made into various preparations, such as tablets, capsules, granules, oral liquids, pills, suspensions, dropping pills, micropills, buccal tablets, capsules, soft capsules, Sprays, aerosols, cataplasms, patches, etc. provide a new way to realize the modernization of traditional Chinese medicine.

Compared with the traditional process, the Momordica glucoside extraction and refining process of the present invention has the following three advantages:

1. Reduce the dose, improve the internal quality and preparation level of traditional Chinese medicine. The refined product obtained after the ethanol extract of bitter gourd is processed by macroporous adsorption resin (RAT) and decolorization can make the medicinal ingredients highly enriched, with few impurities, and the extraction rate is only 1-5% of the original drug (the decoction method is 20- About 30%, alcohol precipitation method is about 15%), is conducive to making the traditional Chinese medicine of modern dosage form, is also convenient for quality control.

2. Reduce the moisture absorption of the product. Most of the traditional Chinese patent medicines have strong hygroscopicity, which is a long-standing problem in the production and storage of traditional Chinese medicines. However, after RAT and decolorization treatment, it can effectively remove a large amount of moisture-absorbing components such as sugars, inorganic salts, and mucus in the extract, which is beneficial to the production of various traditional Chinese medicine forms and enhances the stability of the product.

3. Improve the appearance and color of the preparation, and increase the content of active ingredients. After adding a certain decolorizing agent to the ethanol eluent of RAT and heating to reflux, the color of the obtained Momordica glycosides is light yellow or yellow, which is more acceptable than the yellowish brown or brown before decolorization, and also further improves the quality of Momordica charantia. The content of Momordica glycosides in effective fractions for lowering blood sugar.

The effective parts extracted and refined by this process are small in size, non-moisture-absorbing, and light in color, and can be easily made into various dosage forms with beautiful appearance, especially suitable for granules, capsules and tablets, making the thick, large and black preparations of traditional Chinese medicine Upgrading to modern formulations.

The Momordica glycoside extracted and refined by this process has a certain degree of hypoglycemic effect on hyperglycemia in different animal models caused by different reasons, and some of its hypoglycemic effects are comparable to or even stronger than positive control drugs.

Compared with the traditional solvent extraction method, the invention has simple process, does not use toxic and volatile organic solvents in the process flow, is safe and reliable, has low production cost, and has no pollution to the environment.

Detailed ways

1. Refining method of effective part of bitter gourd for lowering blood sugar

Example 1

The raw material is balsam pear with impurities removed, dried and crushed, and refluxed with 90% ethanol to extract twice, each time the amount is 7 or 5 times the weight of the raw medicinal material, the extraction time is 2 or 1 hour, the extracts are combined, filtered, and the ethanol is recovered , concentrated to get the medicinal extract, the medicinal extract is diluted with water to 15 times the volume of the original medicinal material, and adsorbed on a resin column. The weight ratio of the original medicinal material and resin is 1: 3, washed with water, and eluted with 50% ethanol solution for 15 resin column volumes , collect the ethanol eluent, add activated carbon according to the volume of the eluent at 10%, heat to reflux for 2 hours, filter, concentrate under reduced pressure, and dry in vacuum to obtain Momordica charantrin. The content of momordica glycosides in the effective part of the bitter gourd for lowering blood sugar by the method can reach more than 50%. It can also be eluted with 5% ethanol solution.

Example 2

The raw material is balsam pear that removes impurities, dried and crushed, and refluxed with 70% ethanol to extract twice, each time the amount is 8 or 6 times the weight of the raw medicinal material, the extraction time is 2 or 2 hours, the extracts are combined, filtered, and the ethanol is recovered , concentrated to obtain medicinal extract, the medicinal extract is diluted with water to 25 times the volume of the original medicinal material, and adsorbed on a resin column. The weight ratio of the original medicinal material and resin is 1: 5, washed with water, and eluted with 70% ethanol solution for 20 resin column volumes , collect the ethanol eluent, add activated carbon according to the volume of the eluent at 7%, ultrasonicate for 1.5 hours, filter, concentrate under reduced pressure, and dry in vacuum to obtain Momordica charantrin. The content of momordica glycosides in the effective part of the bitter gourd for lowering blood sugar by the method can reach more than 50%.

Example 3

The raw material is balsam pear with impurities removed, dried and crushed, and refluxed with 70% ethanol to extract twice, each dosage is 10 or 8 times the weight of raw medicinal materials, and the extraction time is 2 or 1 hour. Combine the extracts, filter, and recover ethanol , concentrated to obtain medicinal extract, the medicinal extract is diluted with water to 25 times the volume of the original medicinal material, and adsorbed on a resin column. The weight ratio of the original medicinal material and resin is 1: 5, washed with water, and eluted with 70% ethanol solution for 20 column volumes, Collect the ethanol eluent, add diatomaceous earth according to the volume of the eluent at 7%, heat to reflux for 2 hours, filter, concentrate under reduced pressure, and dry in vacuo to obtain Momordica charantrin. The content of momordica glycosides in the effective part of the bitter gourd for lowering blood sugar by the method can reach more than 50%.

Example 4

The raw material is balsam pear with impurities removed, dried and crushed, and refluxed with 95% ethanol to extract twice, each time the amount is 8 or 6 times the weight of the raw medicinal material, the extraction time is 2 or 1 hour, the extracts are combined, filtered, and the ethanol is recovered , concentrated to obtain medicinal extract, the medicinal extract is diluted with water to 20 times the volume of the original medicinal material, and adsorbed on a resin column. The weight ratio of the original medicinal material and resin is 1: 3, washed with water, and eluted with 90% ethanol solution for 12 resin column volumes , collect the ethanol eluent, add Al ₂ O ₃ according to the volume of the eluent at 7%, ultrasonicate for 1.5 hours, filter, concentrate under reduced pressure, and dry in vacuo to obtain Momordica charantrin. The content of Momordica glucoside in the active parts of Momordica glucoside for lowering blood sugar by this method can reach more than 50%.

Example 5

The raw material is balsam pear with impurities removed, dried and crushed, and refluxed with 90% ethanol to extract twice, each dosage is 8 or 8 times the weight of raw medicinal materials, and the extraction time is 2 or 1 hour. The extracts are combined, filtered, and the ethanol is recovered , concentrated to get the medicinal extract, the medicinal extract is diluted with water to 15 times the volume of the original medicinal material, and adsorbed on a resin column. The weight ratio of the original medicinal material and resin is 1: 4, washed with water, and eluted with 90% ethanol solution for 15 resin column volumes , collect the ethanol eluate, add magnesium oxide according to the volume of the eluent at 10%, heat to reflux for 1 hour, filter, concentrate under reduced pressure, and dry in vacuo to obtain Momordica charantrin. The content of momordica glycosides in the effective part of the bitter gourd for lowering blood sugar by the method can reach more than 50%.

Example 6

The raw material is balsam pear that removes impurities, dried and crushed, and refluxed with 70% ethanol to extract twice, each time the amount is 8 or 6 times the weight of the raw medicinal material, the extraction time is 2 or 2 hours, the extracts are combined, filtered, and the ethanol is recovered , concentrated to get the medicinal extract, the medicinal extract is diluted with water to 15 times the volume of the original medicinal material, and adsorbed on a resin column. The weight ratio of the original medicinal material and resin is 1: 7, washed with water, and eluted with 70% ethanol solution for 20 resin column volumes , collect the ethanol eluent, add magnesium oxide according to 5% of the volume of the eluent, heat it at 80°C for 4 hours, filter, concentrate under reduced pressure, and dry in vacuum to obtain Momordica charantrin. The content of momordica glycosides in the effective part of the bitter gourd for lowering blood sugar by the method can reach more than 50%.

Example 7

The raw material is balsam pear that removes impurities, dried and crushed, and refluxed with 70% ethanol to extract twice, each time the amount is 8 or 6 times the weight of the raw medicinal material, the extraction time is 2 or 2 hours, the extracts are combined, filtered, and the ethanol is recovered , concentrated to obtain medicinal extract, the medicinal extract is diluted with water to 15 times the volume of the original medicinal material, and adsorbed on a resin column. The weight ratio of the original medicinal material and resin is 1: 7, washed with water, and eluted with 90% ethanol solution for 20 resin column volumes , collect the ethanol eluent, add magnesium oxide according to 10% of the volume of the eluent, heat it at 80° C. for 4 hours, filter, concentrate under reduced pressure, and dry in vacuum to obtain Momordica charantrin. The content of momordica glycosides in the effective part of the bitter gourd for lowering blood sugar by the method can reach more than 50%.

2. The hypoglycemic effect of refined Momordica glycosides

Example 8

(1) Hypoglycemic effect on hyperglycemia induced by glucose mice, streptozotocin and alloxan in diabetic mice

1 Test material

1.1 Instruments: 7230 Spectrophotometer (Shanghai Analytical Instrument Factory), 80-2 Centrifugal Precipitator (Shanghai Surgical Instrument Factory), DK-98-I Electronic Constant Temperature Water Bath (Tianjin Test Instrument Co., Ltd.), adjustable trace Pipette (10-50ul), (Shanghai Yarong Biochemical Instrument Factory), adjustable dosing device (Shanghai Qiujing Glass Instrument Factory), GT-1640 super blood glucose tester (Kyoto, Japan).

1.2 Medicinal materials and reagents: The dry bitter melon medicinal material was purchased in Guangzhou, and was identified as the dried fruit of Momordica Charantia L. by Professor Wang Bing of the Botanical Teaching and Research Office of Liaoning University of Traditional Chinese Medicine; Momordica Charantia L. The subject group extracts and purifies it (total steroid glycoside content is greater than 50%). Streptozotocin (Sigma Company, Beijing subpackaging, specification 100mg); Alloxan (produced by Fluka Company): glucose kit (Beijing Beihua Kangtai Clinical Reagent Co., Ltd., batch number 050508); Jiangtangling (Tianjin Pacific Pharmaceutical Co., Ltd., Batch No. 030809); glucose (Shenyang Chemical Reagent Factory, batch No. 20010306); blood glucose test strips (made in Japan) general distributor of Shanghai Yiwei Economic and Trade Development Co., Ltd.; column chromatography silica gel and TLC silica gel G are products of Qingdao Ocean Chemical Factory, others Reagents were all analytically pure.

1.3 Animals: healthy Kunming mice, weighing 23±1g, provided by the Animal Experiment Center of Liaoning University of Traditional Chinese Medicine, license: SCX (Liao) 2004-0018. Animals in each group were given water, food, and natural light in the same environment. The temperature is 25°C and the humidity is 60-70%. Pellet feed was provided by Animal Experimental Feed Factory in Yuhong District, Shenyang City.

2 methods

2.1 Preparation of Momordica charantia glycosides: take Momordica charantia medicinal material, and reflux extraction with 70% ethanol for 3 times. The filtrates were combined, passed through a D-101 macroporous adsorption resin column, washed with water until colorless, and then eluted with 70% ethanol. The ethanol eluate was collected, decolorized, filtered, and the filtrate was concentrated under reduced pressure to obtain Momordica glycosides. As determined by colorimetry, the content of Momordica glycosides was 55.2%.

2.2 Effect of Momordica charantrin on hyperglycemia in glucose-induced mice: Take 50 healthy mice, half male and half male, and after feeding for 3 days, they were randomly divided into 5 groups (control group, model group, high-dose group, low-dose group, Jiangtangling group), 10 per group. Before the experiment, they were fasted without water, and 12 hours later, they were administered by intragastric administration. The high-dose and low-dose groups were given Momordica glycosides 200 and 100 mg/kg respectively, the Jiangtangling group was given Jiangtangling 100 mg/kg, and the control and model groups were given the same volume of distilled water. After 30 minutes, each group was given glucose solution 4.0g/kg by intragastric administration, and the control group was given the same volume of distilled water. At 60 and 120 minutes after the administration, blood was collected from the orbital sinus of the mice, and the blood samples were centrifuged (3000 rpm/10 min) to separate the serum, and the blood sugar content was measured according to the instructions of the glucose kit. The experimental results were statistically analyzed using the t-test method to compare the differences between each administration group and the model group.

2.3 Effect of Momordica charantrin on hyperglycemia in streptozotocin-induced diabetic mice: 92 qualified mice (male) were selected, and after feeding for 3 days, 12 mice were taken as the control group, and normal saline was injected intraperitoneally, and the remaining 80 mice were injected intraperitoneally. Streptozotocin 150mg/kg, test the fasting blood glucose content of the mice after 72h, take the mice with blood glucose values between 16-24mmol/L, and divide them into 4 groups according to the blood glucose content (model group, high-dose group, low-dose group) , Jiangtangling group) 12 rats in each group. The high-dose and low-dose groups were given 200 and 100 mg/kg Momordica glucoside by intragastric administration, the Jiangtangling group was given 100 mg/kg Jiangtangling, and the control group and model group were given the same volume of distilled water. Oral gavage once a day, continuous administration 14d. At 3, 7, and 14 days after the administration, the blood sugar content of the mice was tested respectively. The mice were fasted for 12 hours before measuring the blood sugar. During the test, the mice were put into a glass bell jar, and the tail was exposed. The super blood sugar tester measures the blood sugar content and records the blood sugar value. The t test was used to compare the differences between the control group and the model group, and between each administration group and the model group.

2.4 Effects of Momordica charantrin on hyperglycemia in alloxan-induced diabetic mice: 92 qualified mice (male) were selected, and after feeding for 3 days, 12 mice were taken as a control group, and normal saline was injected intraperitoneally, and the remaining 80 mice were injected four times intraperitoneally. Oxypyrimidine 220mg/kg, test the fasting blood glucose content of the mice after 72h, take the mice with blood glucose values between 16-24mmol/L, and divide them into 4 groups according to the blood glucose levels (model group, high-dose group, small-dose group, Jiangtangling Group) 12 per group. The high-dose and low-dose groups were given 200 and 100 mg/kg Momordica glucoside by intragastric administration, the Jiangtangling group was given 100 mg/kg Jiangtangling, and the control group and model group were given the same volume of distilled water. Oral gavage once a day, continuous administration 14d. At 3, 7, and 14 days after the administration, the blood sugar content of the mice was tested respectively. The mice were fasted for 12 hours before measuring the blood sugar. During the test, the mice were put into a glass bell jar, and the tail was exposed. The super blood sugar tester measures the blood sugar content and records the blood sugar value. The t test was used to compare the differences between the control group and the model group, and between each administration group and the model group.

3 results

3.1 Effect of Momordica glucoside on hyperglycemia in glucose-induced mice: After intragastric administration of mice with glucose for 30 minutes, the blood sugar level in the model group increased significantly, and the blood sugar level in the large and small dose groups of Momordica glucoside and Jiangtangling group was significantly lower than that in the model group. In the model group, the hypoglycemic rates were 30.2%, 28.3%, and 39.6% at 60 minutes after drug administration, and the results are shown in the table below.

Table 1-1 Effect of Momordica glucoside on hyperglycemia in glucose-induced mice (X±s, n=10)

group Dose (mg·kg-1) Blood sugar (mmol·L-1) 60(min) after taking medicine 120(min) after medicine Control group, model group, bitter gourd, big bitter gourd, small Jiangtangling --200100100 5.73±1.1023.04±7.6016.09±4.11*16.52±5.2013.92±5.40* 5.96±1.048.25±1.447.39±1.337.96±1.744.14±0.72**

Compared with the model group, *p<0.05, **p<0.01

3.2 Effect of Momordica charantrin on hyperglycemia in streptozotocin-induced diabetic mice: 72 hours after intraperitoneal injection of streptozotocin, the blood glucose concentration was significantly higher than that in the control group. Give Momordica glucoside 200, 100mg/kg, continuous gavage for 14 days. After 3 days of administration, the test blood sugar content decreased slightly, and the hypoglycemic rates of the large and small dose groups and the Jiangtangling group were 18.7%, 12.5%, and 25.4%, respectively. The hypoglycemic rates were 27.2%, 29.8%, and 28.5% when administered for 7 days, and the hypoglycemic effect was significant when administered for 14 days, and the hypoglycemic rates were 46.5%, 41.4%, and 44.1%. The results showed that Momordica glucuronide also had a good hypoglycemic effect on streptozotocin-induced diabetic mice, and the results are shown in the table below.

Table 1-2 Effect of Momordica glucoside on hyperglycemia in streptozotocin-induced diabetic mice (X±s, n=12)

group Dose mg·kg-1 Blood sugar (mmol·L-1) Premedication 3d after medicine 7 days after treatment 1 4d after medicine control group model group -- 5.37±0.4222.80±4.03ΔΔ 5.33±0.5321.76±4.29ΔΔ 5.21±0.6120.40±7.07ΔΔ 4.89±0.4217.22±3.60ΔΔ

Bitter Gourd Big Bitter Gourd Small Hypoglycemic Spirit 200100100 22.80±4.3022.56±5.0222.76±3.54 17.69±6.1519.05±5.0218.33±7.22 16.62±5.2516.00±4.7216.31±2.43* 9.54±5.25**10.02±5.99*9.60±3.84**

Compared with the control group, ΔΔp<0.01; compared with the model group, *p<0.05, **p<0.01. 3.3 Effect of Momordica charantrin on hyperglycemia in alloxan-induced diabetic mice: intraperitoneal injection of alloxan After 72 hours, the blood glucose concentration was significantly higher than that of the control group. Give Momordica glucoside 200, 100mg/kg, continuous gavage for 14 days. After 3 days of administration, the test blood sugar content decreased slightly, and the hypoglycemic rates were 53.5%, 33.9%, and 56.7% respectively at the 7th day of administration. %, 60.2%. The results showed that Momordica glucuronide also has a good hypoglycemic effect on alloxan-induced diabetic mice, and the results are shown in the table below.

Table 1-3 Effect of Momordica glycosides on hyperglycemia in alloxan-induced diabetic mice (X±s, n=12)

group Dose (mg·kg ^-1 ) Blood sugar (mmol·L-1) Pre-drug 3d after medicine 7d after medicine 14d after medicine Control group, model group, bitter gourd, big bitter gourd, small Jiangtangling --200100100 6.42±2.3118.30±4.09ΔΔ18.53±4.0217.93±4.0017.52±3.67 6.31±2.0520.40±3.51ΔΔ18.00±3.2119.45±2.8517.40±3.05 6.00±3.2421.52±4.20ΔΔ10.34±2.99**14.27±2.20**9.32±2.45** 5.86±2.4120.34±3.01ΔΔ8.25±2.16**10.33±2.67**8.08±2.31**

Compared with the control group, ΔΔp<0.01; compared with the model group, **p<0.01.

4 Conclusion

After a mouse was fed with glucose and injected with streptozotocin or alloxan, the blood sugar concentration increased significantly, and the increase rate increased by 4 times compared with the control group. After administration, each administration group was different from the model group. level of blood sugar drop. When the mice were fed with glucose for 30 minutes, the blood sugar content decreased significantly. Compared with the model group, the hypoglycemic rates of the large and small doses and Jiangtangling group were 30.2%, 28.3%, and 39.6%, respectively. After 14 days of continuous intragastric administration in mice, the hypoglycemic effect on streptozotocin-induced diabetic mice is particularly obvious. The hypoglycemic rates of the large and small dose groups and Jiangtangling group were 46.5%, 41.4%, and 44.1% respectively. . The hypoglycemic effect on alloxan-induced diabetic mice is particularly obvious, and the hypoglycemic rates of the high-dose and low-dose groups and the Jiangtangling group were 59.4%, 49.3%, and 60.2%, respectively. The results of the hypoglycemic effect of Momordica glucoside on mice show that Momordica glucoside has a better hypoglycemic effect.

(2) Effects on blood sugar levels in normal mice and adrenaline-induced hyperglycemia mice, blood sugar levels and insulin levels in alloxan-induced diabetic rabbits

1 Instruments and materials:

1.1. Instruments: 7230 Spectrophotometer (Shanghai Analytical Instrument Factory), 80-2 Centrifugal Precipitator (Shanghai Surgical Instrument Factory), Adjustable Micropipette (10-50u1), (Shanghai Yarong Biochemical Instrument Factory), can Adjusting volume dosing device (Shanghai Qiujing Glass Instrument Factory), electric heating constant temperature water bath (Hebei Huanghua City Qiwu Hardware Electric Appliance Factory).

1.2 Medicine: Bitter melon extract, each gram is equivalent to 71.9 grams of crude drug, produced by Liaoning Provincial Traditional Chinese Medicine Modernization Engineering Technology Center, batch number: 20030506. Distilled water was used to make the required concentration for the test; Tang Niao Ling Tablets, produced by Jiangxi Tianshikang Traditional Chinese Medicine Co., Ltd., batch number: 20040212; glyburide, produced by Beijing Taiyang Pharmaceutical Co., Ltd., batch number: 20030806. Jiangtangling, produced by Shandong Boshan Pharmaceutical Co., Ltd., batch number: 20030815.

1.3 Reagents: Alloxan, produced by Fluka Company, UK; epinephrine, produced by Tianjin Amino Acid Company; insulin radioimmunoassay kit, produced by Beijing North Biotechnology Research Institute.

1.4 Animals: White mice, Kunming species, 22-24g, male and female, provided by the Experimental Animal Center of Shenyang Pharmaceutical University, certificate number: SCXK (Liao) 2003-008. Japanese big-eared white rabbits, 2.7-3.8kg, male, provided by the Medical Experimental Animal Center of the Second Clinical College of China Medical University, certificate number: SCXK (Liao) 2003-0013. Pellet feed was purchased from the feed branch of Northeast Sixth Pharmaceutical Factory.

2 Methods and results:

2.1 Preparation of Momordica charantia glycosides: take bitter gourd medicinal material, and reflux extraction with 70% ethanol for 3 times. The filtrates were combined, passed through a D-101 macroporous adsorption resin column, washed with water until colorless, and then eluted with 90% ethanol. The ethanol eluate was collected, decolorized, filtered, and the filtrate was concentrated under reduced pressure to obtain Momordica glycosides. As determined by colorimetry, the content of charantrin was 65.2%.

2.2 Effects on the blood sugar content of normal mice: Take 60 Kunming mice, half male and half male, and randomly divide them into 5 groups according to body weight and sex, 12 mice in each group: blank control group, equal volume water; glyburide group 50mg/ kg; Momordica glycosides high-dose group 300mg/kg; medium-dose group 150mg/kg; low-dose group 75mg/kg. After the animals were fasted for 8 hours, blood was collected from the retro-orbital venous plexus, and the normoglycemia value before administration was determined by the glucose oxidase method. Immediately after blood collection, intragastric administration was performed, with a volume of 0.25ml/10g. after administration

Blood was collected at 3, 6 and 9 hours to measure blood sugar levels. The results are shown in the table below.

Table 2-1 Effects on blood sugar levels in normal mice (X ± SD)

group Dose (mg/kg) Number of animals (only)   Blood sugar value mmol/L before administration After administration 3 6 9 (h) Blank glyburide bitter gourd large -50300 121212 4.98±0.595.19±0.634.84±0.44 5.17±0.532.84±0.71*4.92±0.49 5.17±0.563.89±0.89*5.10±0.64 4.96±0.773.84±0.655.08±0.68

Bitter gourd small 15075 1212 5.04±0.644.99±0.51 4.88±0.615.06±0.59 4.75±0.525.04±0.56 4.64±0.535.08±0.54

Compared with blank control group *p<0.05

The results showed that momordica glucoside had no obvious effect on lowering blood sugar in normal mice, and the glyburide group had hypoglycemic effect at 3 and 6 hours after the drug, and compared with the control group, p<0.05. 2.3 For adrenergic hyperglycemia mice Effect of blood sugar content: 84 mice, 20-22g, half male and half female, were randomly divided into 7 groups according to body weight and sex, 12 mice in each group: blank control group; model group; Jiangtangling group 100mg/kg; Tangniailing tablet group 1600mg/kg; Momordica glycosides high-dose group 300mg/kg; medium-dose group 150mg/kg; low-dose group 75mg/kg. After fasting for 8 hours, the animals were fasted for 8 hours to take blood to measure the blood glucose level, and then administered intragastrically, once a day, for 7 consecutive days. Animals were fasted for 12 hours before the last drug, and 1 hour after the last drug, 240 mg/kg of epinephrine was injected intraperitoneally (except for the blank control group), and blood was taken 30 minutes later to measure blood sugar. The results are shown in the table below.

Table 2-2 Effect on blood sugar content of adrenaline hyperglycemia mice (X±SD)

group Dose (mg/kg) Number of animals (only) Blood glucose level (mmol/L) Hypoglycemic rate (%) Blank control model Jiangtangling Diabetic Tablets Bitter Gourd Big Bitter Gourd Medium Bitter Gourd Small --100160030015075 12121212121212 5.34±0.6618.6±1.28 ^▲▲ 8.61±0.82**12.1±1.10**10.8±1.39**11.7±1.50**17.4±2.42 -53.734.941.937.16.45

^▲▲ p<0.01 compared with the blank control group; **p<0.01 compared with the model group

The results showed that Momordica glycosides had obvious hypoglycemic effect on mice with adrenaline hyperglycemia, and its effect was stronger than that of Tangniaoling Tablets.

2.4 Effects on the blood sugar content and insulin level of the rabbit diabetic model induced by alloxan: take 80 healthy male rabbits, weighing 2.5-3.0kg, and inject 10% alloxan normal saline solution into the ear vein after fasting for 32 hours 150mg/kg, 72 hours later, blood was taken from the ear vein to measure the blood sugar and insulin content, and 60 hyperglycemic rabbits (14.9-28.8mmol/L) with successful modeling were selected and randomly divided into 6 groups, 10 rabbits in each group. Set up a blank control group: namely: blank control group; model group; Jiangtangling group 80mg/kg; Tangguanling group 640mg/kg; Momordica glycosides high-dose group 120mg/kg; medium-dose group 60mg/kg; low-dose group 30mg/kg . The drug was given by intragastric administration once a day for 7 consecutive days, and 24 hours after the last dose, the animals were fasted and blood was taken to measure the blood sugar and insulin levels. The results are shown in the table below.

Table 2-3 Effects on Alloxan Rabbit Blood Glucose Content and Insulin Level (X±SD)

group Dose mg/kg Number of animals (only) Blood sugar (mmol/L) Insulin (u/ml) Before administration after administration Before administration after administration Blank Model Jiangtangling Diabetic Diabetic Bitter Gourd Big Bitter Gourd Medium Bitter Gourd Small --806401206030 10101010101010 5.46±1.1522.9±3.71 ^▲▲ 22.5±3.5423.1±2.8423.0±3.5623.1±3.0722.0±3.49 5.53±1.03 2.2±3.57 ^▲▲ 10.1±3.13**13.2±3.18**10.9±3.12**13.9±3.08**18.5±3.16* 67.8±8.6345.9±7.45 ^▲▲ 46.2±6.0354.9±8.1252.2±6.2844.9±6.0545.2±6.53 70.1±9.6542.5±8.29 ^▲▲ 60.9±7.1949.0±8.2945.1±6.9246.4±5.5943.1±8.17

Compared with the blank control group ^▲▲ p<0.01; compared with the model group *p<0.05 **p<0.01

The results showed that there was a significant difference between the model group and the blank control group, p<0.01, indicating that the model was successfully established. Momordica glucoside can significantly reduce the blood sugar level of rabbits with alloxan-induced hyperglycemia (p<0.05, 0.01), and has obvious hypoglycemic effect, which is better than Tangniaoling Tablets, but has no obvious effect on insulin level.

3 Conclusion

After mice were injected with adrenaline and rabbits were injected with alloxan modeling, the blood sugar concentration increased significantly, which was significantly different from that of the control group. After administration, each drug group had different degrees of blood sugar drop compared with the model group. After 7 days of continuous administration in mice, the blood sugar of adrenaline diabetic mice was significantly reduced. The hypoglycemic rates of the large and medium dose groups of momordica glycosides were 41.6% and 37.1%, respectively, and that of the Tangniaoling group was 34.9%. Its hypoglycemic effect is better than that of Diabetic group. The hypoglycemic effect on alloxan-induced diabetic rabbits is particularly obvious. The hypoglycemic rates of momordica glycosides in large and medium dose groups are 50.9%, 37.4%, and there is no significant difference in hypoglycemic effect compared with Jiangtangling (54.5%), strong in the Tang Niao Ling group (40.5%). The above results show that: Momordica glucoside significantly reduces the blood sugar level of adrenaline-induced hyperglycemia mice; it also has a significant reduction effect on the sugar level of alloxan-induced diabetes rabbits, but has no significant effect on its insulin value; The value was not significantly affected.

(3) To sum up, Momordica glucoside has a certain degree of hypoglycemic effect on hyperglycemia in different animal models caused by different reasons, and some of its hypoglycemic effects are comparable to or even stronger than positive control drugs. The above experiments prove that the total steroidal glycosides of bitter melon prepared by the optimal process has a hypoglycemic effect, and the hypoglycemic effect is relatively obvious, and it is one of the effective parts of bitter melon for hypoglycemic.

Claims (7)

1. the process for purification of a valid target of bitter gourd for dropping blood sugar is characterized in that: comprise the steps:

(1) the Fructus Momordicae charantiae medicinal liquid of preparation before the decolorizing and refining: with Fructus Momordicae charantiae decoction pieces drying, pulverizing, use the 10-95% alcohol reflux, reclaim ethanol, concentrated extractum; To finite concentration, the water washing of pH2-8 is used in last macroporous adsorptive resins absorption, discards water elution liquid with the extractum thin up, reuse 5%-95% alcoholic solution eluting, and eluting 5-20 times of resin column volume obtains the preceding Fructus Momordicae charantiae medicinal liquid of decolorizing and refining.

(2) subtractive process: collect the ethanol elution of resin column, press effluent volume 0.1-50% and add depigmenting agent, reflux 0.1-10 hour, filter, concentrating under reduced pressure, vacuum drying promptly gets the Fructus Momordicae charantiae sterioside.

2. the process for purification of a kind of valid target of bitter gourd for dropping blood sugar according to claim 1, it is characterized in that: the solution carrier that the valid target of bitter gourd for dropping blood sugar decolorizing and refining is used is the alcoholic solution of 20%-90%.

3. the process for purification of a kind of valid target of bitter gourd for dropping blood sugar according to claim 1 is characterized in that: the concentration of medicinal liquid is equivalent to the alcoholic solution of 1g crude drug/40-80mL90% before the decolorizing and refining.

4. the process for purification of a kind of valid target of bitter gourd for dropping blood sugar according to claim 1, it is characterized in that: the kind of described depigmenting agent is a magnesium oxide, the consumption of depigmenting agent is the 0.1-50% of medicine liquid volume before the decolorizing and refining.

5. the process for purification of a kind of valid target of bitter gourd for dropping blood sugar according to claim 1, it is characterized in that: the using method of described depigmenting agent is a heating reflux method.

6. the process for purification of a kind of valid target of bitter gourd for dropping blood sugar according to claim 1 is characterized in that: the valid target of bitter gourd for dropping blood sugar after refining is the sterioside constituents.

7. the valid target of bitter gourd for dropping blood sugar after according to claim 6 the making with extra care is characterized in that: described sterioside constituents can be mixed and made into tablet, capsule, granule, oral liquid, pill, suspensoid, drop pill, pellet, buccal tablet, soft gelatin capsule, soft capsule, spray, aerosol, cataplasma, the patch of accepting clinically with pharmaceutically acceptable carrier.