WO2022227890A1

WO2022227890A1 - High-activity and high-safety substance, preparation method therefor and use thereof in medicine

Info

Publication number: WO2022227890A1
Application number: PCT/CN2022/080585
Authority: WO
Inventors: 朱义鑫; 徐爽
Original assignee: 苏州海益生物医药科技有限公司
Priority date: 2021-04-30
Filing date: 2022-03-14
Publication date: 2022-11-03
Also published as: CN115261124A

Abstract

Disclosed are a high-activity and high-safety substance, a preparation method therefor and the use thereof. The substance uses rubber seed crude oil as a raw material, and is obtained by means of neutralization with an alkali, natural settling, centrifugal separation, adsorption filtration and distillation. The substance not only has high activity, which is higher than that of rubber seed crude oil in preventing and treating atherosclerosis, but also has high safety, can meet the requirements of national standards of food safety, can be applied to medicine, and is used for preventing and treating atherosclerosis in patients with cardiovascular and cerebrovascular diseases.

Description

A kind of high activity, high safety substance, preparation method and its application in medicine

technical field

The invention belongs to the field of biomedicine.

Background technique

Drugs can have certain side effects, but it is hoped that the fewer side effects the better, especially drugs for chronic diseases. Because patients with chronic diseases need to take medicines for a long time, the long-term accumulation of minor side effects may also cause major problems. Atherosclerosis leads to cardiovascular and cerebrovascular diseases. It is a typical chronic disease with a long course and patients need lifelong medication. It would be a good thing for patients with cardiovascular and cerebrovascular diseases to develop a drug that has a strong therapeutic effect on atherosclerosis and is also highly safe.

Cardiovascular and cerebrovascular diseases caused by atherosclerosis (As) are the first cause of death in Chinese people, and are harmful. However, there are currently no particularly effective drugs that can prevent and treat atherosclerosis. At present, the drugs for the treatment of atherosclerosis are divided into six categories, including lipid-lowering drugs, antiplatelet drugs, anticoagulant and thrombolytic drugs, antioxidant drugs, and anti-inflammatory drugs. However, these drugs can only slow down the formation rate of atherosclerosis and stabilize the atherosclerotic plaque, and cannot completely reverse and ablate the atherosclerotic plaque, and the therapeutic effect is limited.

Medical research shows (Document 1. Liu Chaoran, Chen Guozhen, Li Yunshan, et al. Research on the regression of atherosclerosis in monkeys [J]. Chinese Science (Series B), 1987(02): 66-74; Document 2. Liu Chaoran, Yang Liang, Chen Guozhen, et al. Study on the effect of rubber seed oil on atherosclerosis - Ⅰ. Effect on the formation and regression of experimental aortic atherosclerosis in rabbits[J]. Journal of Kunming Medical College, 1980(3 ); Document 3. Liu Chaoran, Li Yunshan, Chen Guozhen, et al. Effects of rubber seed oil on changes in blood lipids and cardiac function during atherosclerotic modeling in monkeys [J]. Journal of Tropical Crops, 1985(2): 1. Document 4 .Liu Chaoran,Chen Guozhen,Li Yunshan,Chen Longshun,Tang Chaocai,Zhou Shuyun,Zhang Zhixiong.Study on the preventive effect of rubber seed oil on monkey atherosclerosis[J].Journal of Kunming Medical College,1985(02):8-22.Document 5. Liu Hanjun, Zhang Hongfei, Liu Aiwu, et al. Ultrastructural observation of rubber seed oil in preventing experimental atherosclerosis in monkeys [J]. Journal of Kunming Medical University, 1986(2). Rubber seed oil has obvious preventive and therapeutic effects on atherosclerosis sclerosis-like effect, with the potential to develop into a drug.

Documents 1-5 speculate that rubber seed oil is rich in unsaturated fatty acids and has a hypolipidemic effect, and speculates that the hypolipidemic effect leads to its reversal and regression effect on atherosclerosis. But speculation is only speculation after all, and it is neither certain nor denied that the association between the unsaturated fatty acids contained in rubber seed oil and the effect of reversing and regressing atherosclerosis. To date, no studies have revealed a correlation between specific components contained in rubber seed oil and its biological activity (activity to reverse and regress atherosclerosis).

Documents 1-5, as studies in the medical field, do not mention the preparation method of rubber seed oil. According to the date of publication of the document and the national conditions at that time, as well as our investigation and research, the rubber seed oil in this paper should be the crude rubber seed oil or crude oil in food science, that is, the crude extract of the fat-soluble components of rubber seed in phytochemistry.

The rubber seed crude oil has been confirmed by animal experiments that although it has obvious biological activity of reversing and ablating atherosclerosis, it also contains many components that are unfavorable to health and even toxic and harmful. Document 6 (Document 6: Luo Xiaolan, Zhu Wenxin, He Jian, et al. Research and Practice of Rubber Seed Oil Refining [J]. Grain and Oil Processing, 2008, 000(011): 46-49.), Document 12 (Document 12: Li Linkai, Li Chen, Tao Yin.Research on short-path/molecular distillation process of rubber seed oil[J].Grain and Oil Processing (Electronic Edition),2015(09):26-28.) All pointed out: rubber seed crude oil has high acid value and impurities Many and complex, in addition to the impurities contained in general crude oil such as mechanical impurities, phospholipids, mucus, glycolipids, etc., it also contains some special impurities, such as rubber, resin, lipoprotein, cyanoside, etc., even because of its corrosiveness Strong, but also produce metal ion chelates.

In food science, the process of removing unhealthy components from crude vegetable oil is called oil refining. There are several food science papers that examine various methods of refining crude rubber seed oils. Here are the research papers:

Document 6: Luo Xiaolan, Zhu Wenxin, He Jian, et al. Research and practice of rubber seed oil refining [J]. Grain and Oil Processing, 2008, 000(011): 46-49.

Document 7: Shen Shandeng. Experimental study on physical refining of rubber seed oil [J]. China Oils and Fats, 1992(06):18-20.

Document 8: Hu Xiaohong, Liu Dachuan, Zhang Xincai. Research on the preparation and refining process of rubber seed oil [J]. China Oils and Fats, 2005(11):66-68.

Document 9: Wu Weizhong. Industrial use of rubber seed oil [J]. China Oils and Fats, 1988(05):62-63.

Document 10: Jia Wei. Process and Practice of Rubber Seed Oil Production [J]. China Oils and Fats, 2006(02):12-14.

Document 11: Zu Tingyue. Aqueous enzymatic extraction of rubber seed oil and its microemulsion preparation. Jiangnan University, 2013. You

Document 12: Li Linkai, Li Chen, Tao Yin. Research on Short Path/Molecular Distillation Process of Rubber Seed Oil [J]. Grain and Oil Processing (Electronic Edition), 2015(09):26-28.

Document 13: Guo Xiong. Preparation of rubber seed oil and protein [D]. Wuhan University of Light Industry, 2018.

Document 14: Lan Qinmu. Storage test report of refined rubber seed oil [J]. Oil Science and Technology, 1982(05):12-17.

Document 15: Tian Hua, Huang Tao, Su Minghua. Research on degumming and decolorization of rubber seed oil [J]. Journal of Wuhan University of Light Industry, 2007, 26(002): 9-11.

Document 16: Wang Xiaoli, Zhan Lin. Research on the refining of rubber seed oil [J]. China Oil and Fats, 2000, 25(004): 10-11.

These refining methods vary, and the refined oils obtained can be good or bad from a food science point of view. The judgment of food science on the quality of the refining method is often considered from the following aspects:

1. Food safety, whether it can meet the requirements of "GB 2716-2018 National Food Safety Standard Vegetable Oil";

2. The yield of finished products, taking the weight of raw materials as 100%, what percentage of finished products can be finally obtained, of course, the higher the yield, the better;

3. The production cost mainly includes the complexity of the process, the level of equipment requirements, the length of the production cycle, the consumption of energy and auxiliary materials, etc. Of course, the more economical the better.

Regardless of how good or bad these refining methods are in food science, one thing in common is that the rubber seed refined oils prepared by these methods are not linked to their biological activity, that is, the effect of reversing and ablating atherosclerosis. We do not know whether the rubber seed refined oils produced by these different refining methods have the atherosclerotic reversal and ablative effects described in documents 1-5.

In order to study the effect of these refining methods on the biological activity of rubber seed oil (effect of reversing and ablating atherosclerosis), the present invention repeats the protocol in documents 6-15, regardless of whether the safety is up to standard, the yield is high or low, As for the production cost, the present invention finds that they have almost no biological activity through a large number of animal experiments. For details, please refer to Examples 4-9.

Rubber seed crude oil has the biological activity of preventing and treating atherosclerosis, but it contains a lot of substances that are harmful to health, even toxic and harmful; the refined oil after removing the harmful and even toxic and harmful components through oil refining loses it at the same time. Biological activity, which is a major problem restricting the development and utilization of rubber seed oil.

SUMMARY OF THE INVENTION

In order to solve the problem of development and utilization of rubber seed oil, the present invention focuses on the biological activity of rubber seed oil from the perspective of biology and medicine, while taking into account the safety requirements in food science.

One of the objectives of the present invention is to provide a new product that is not available in the prior art and whose biological activity reaches or even surpasses the raw material crude oil while the safety reaches the "GB 2716-2018 National Food Safety Standard Vegetable Oil". rubber seed oil.

The second purpose of the present invention is to provide a preparation method of this highly active and highly safe substance.

The third purpose of the present invention is to provide the application of this highly active and highly safe substance in pharmacy. The substance can be used in medicines for the prevention and treatment of atherosclerosis.

In order to achieve the above object, the present invention provides the following technical solutions:

A substance with high activity and high safety, characterized in that the substance is prepared by the following method: using crude rubber seed oil as a raw material, neutralizing by adding alkali, natural sedimentation, centrifugal separation, adsorption filtration, and distillation to obtain .

The invention also provides a method for preparing the high activity and high safety substance: the crude rubber seed oil is used as a raw material, and is obtained through alkali neutralization, natural sedimentation, centrifugal separation, adsorption filtration and distillation.

In the neutralization step of adding alkali, the alkali is selected from food grade alkali, and the amount of alkali added is equal to the molar mass of the fatty acid contained in the crude oil, and the addition method is an aqueous solution of alkali, and then mixed.

In the natural sedimentation step, the conditions are as follows: the temperature of the mixture is kept at a temperature above 70°C, and after the fatty acid salt and the oil phase are significantly separated, the lower fatty acid salt phase is discarded, and the oil phase is retained.

In the centrifugal separation step, the conditions are: the temperature of the oil phase is above 70°C, and the centrifugal force is greater than or equal to 5000g.

In order to realize the conditions of this centrifugal separation in large-scale industrial production, the present invention also provides a specific realization method: use a disc centrifuge or a tubular centrifuge for centrifugal separation, and the centrifugal separation temperature of the oil phase material is between 70°C- 100°C, centrifugal force 5000-30000g, preferably 6000-18000g, discard the remaining fatty acid salt, and separate the oil phase.

In the distillation step, the distillation temperature is less than or equal to 180° C., water vapor is introduced from the bottom, and the rubber seed oil with high activity and high safety is finally retained.

The present invention also provides a pharmaceutical composition containing a therapeutically effective amount of the rubber seed oil with high activity and safety and a pharmaceutically acceptable carrier.

The present invention also provides the application of the high-activity and high-safety rubber seed oil in the preparation of a medicament for preventing and treating atherosclerosis, as the only active ingredient.

Compared with the existing scheme, the rubber seed oil obtained by this scheme is fundamentally different in activity and also in the method of realization.

According to experimental data, the present invention has the following advantages:

The present invention provides a kind of vegetable oil whose safety reaches "GB 2716-2018 National Food Safety Standard Vegetable Oil" (Example 1-2), and the activity of preventing and treating atherosclerosis can reach or even surpass the raw rubber seed hair. New species of oil (Examples 10-11).

The present invention also provides a method for industrialized and large-scale production of this highly active and highly safe substance, not limited to laboratory preparation (Example 1-2).

The substantive content of the present invention will be further described below through the accompanying drawings and embodiments of the present invention, but the present invention is not limited by this.

Description of drawings

FIG. 1 shows the grades of atherosclerotic lesions in different groups of mice in Example 9. FIG.

Figure 2 shows the atherosclerotic plaque tracings of the typical rabbit aorta in Example 10, from left to right, taken from the normal diet group, the high-fat diet group, the rubber seed crude oil + high-fat diet group, the present Invention + high-fat diet group, simvastatin + high-fat diet group.

FIG. 3 shows the proportions of atherosclerotic plaques in the aorta in different groups of rabbits in Example 10. FIG.

Figure 4 shows the P value of the difference between different groups in Example 10, P less than 0.05 is a significant difference, and P less than 0.01 is a very significant difference.

FIG. 5 shows the proportion of atherosclerotic plaques in the aorta in different groups of rabbits in Example 10. FIG.

Figure 6 shows the atherosclerotic plaque traces of the typical rabbit aorta in Example 11, taken from left to right from the normal diet group, the atherosclerosis modeling group, the modeling + normal feeding group, the modeling group Model + hair oil treatment group, model + treatment group of the present invention, model + simvastatin treatment group.

FIG. 7 shows the proportions of atherosclerotic plaques in the aorta in different groups of rabbits in Example 11. FIG.

Figure 8 shows the P value of the difference between different groups in Example 11, P less than 0.05 is a significant difference, and P less than 0.01 is a very significant difference.

FIG. 9 shows the proportions of atherosclerotic plaques in the aorta in different groups of rabbits in Example 11. FIG.

Detailed ways

Example 1

According to the method of the present invention, high activity and high safety rubber seed oil is prepared.

Taking 1000kg of rubber seed crude oil as raw material, the measured acid value is 30mgKOH/g, and the required NaOH solid is 21.4kg. Add 78.6 kg of water to the solid alkali to dissolve, and prepare 100 kg of lye solution with a concentration of 21.4% by weight. Heat the crude oil to 90°C, then stir at a slow speed, slowly add the lye to the crude oil, continue to stir at a slow speed until the reaction between the crude oil and the lye is complete, then stop stirring, keep the oil temperature, and settle naturally. After 4 hours of natural sedimentation, it was found that the oil phase and the fatty acid salt phase were obviously stratified. The fatty acid salt phase was discarded, and the temperature of the oil phase was adjusted to 90 °C. 1.5 minutes. The oil phase after centrifugation was dehydrated by vacuum heating, vacuum pressure 10kPa, heating temperature 100°C, dehydration for 60 minutes, then adding an adsorbent with an oil weight of 1.2%, the adsorbent was activated clay, stirring for 10 minutes, then filtering, filtering out Clear oil. The clear oil was heated to 180°C, and in a vacuum environment of 1kPa, steam was introduced, and distilled for 30 minutes, then stopped, cooled, and pressure released to obtain 500kg of finished product with a yield of 50%. The product is light yellow in color, is a clear, transparent oily liquid without foreign matter, has the inherent smell and taste of rubber seed oil, and has no peculiar smell.

The obtained finished products were tested according to the standard of "GB 2716-2018 National Food Safety Standard Vegetable Oil", and all of them were qualified. The following are some test results:

This shows that the safety of the rubber seed oil of the present invention has reached the national safety standard of food (edible vegetable oil).

Example 2

Taking 1000kg of rubber seed crude oil as raw material, the measured acid value is 30mgKOH/g, and the required NaOH solid is 21.4kg. Add 78.6 kg of water to the solid alkali to dissolve, and prepare 100 kg of lye solution with a concentration of 21.4% by weight. Heat the crude oil to 80°C, then stir at a slow speed, slowly add the lye to the crude oil, continue to stir at a slow speed until the reaction between the crude oil and the lye is complete, then stop stirring, keep the oil temperature, and settle naturally. After 6 hours of natural sedimentation, it was found that the oil phase and the fatty acid salt phase were obviously stratified, the fatty acid salt phase was discarded, the temperature of the oil phase was adjusted to 70°C, and the temperature of the oil phase was adjusted to 70 °C, and then injected into a tubular centrifuge to continue centrifugation. The centrifugal force was 18,000 g, and the centrifugation retention time was 1.5 minutes. The oil phase after centrifugation was dehydrated by vacuum heating, the vacuum pressure was 10kPa, the heating temperature was 100°C, and the dehydration was performed for 60 minutes. Then an adsorbent with 1.5% oil weight was added. The adsorbent was a mixture of activated clay and diatomaceous earth, and stirred for 10 minutes. , and then filter to filter out the clear oil. The clear oil was heated to 105°C, and in a vacuum environment of 0.2kPa, steam was introduced into it, distilled for 30 minutes, then stopped, cooled, and depressurized to obtain 520kg of finished product with a yield of 52%. The product is light yellow in color and is a clear, transparent oily liquid without foreign matter. It has the inherent smell and taste of rubber seed oil, and has no peculiar smell.

Example 3

Explore the conditions and methods to achieve centrifugal separation in large-scale industrial production.

3.1. The disc centrifuge is directly centrifuged. Taking 1000kg of rubber seed crude oil as raw material, the measured acid value is 30mgKOH/g, and the required NaOH solid is 21.4kg. Add 78.6 kg of water to the solid alkali to dissolve, and prepare 100 kg of lye solution with a concentration of 21.4% by weight. Crude oil and lye were mixed in a ratio of 10:1, heated to 80°C, and then injected into a disc centrifuge for centrifugation, but the separation failed. No matter how to adjust the flow rate, change the ratio of crude oil to lye, adjust centrifugal force, adjust temperature and other technical parameters, it will all fail.

3.2 The tube centrifuge is directly centrifuged. Taking 1000kg of rubber seed crude oil as raw material, the measured acid value is 30mgKOH/g, and the required NaOH solid is 21.4kg. Add 78.6 kg of water to the solid alkali to dissolve, and prepare 100 kg of lye solution with a concentration of 21.4% by weight. Crude oil and lye were mixed in a ratio of 10:1, heated to 80°C, and then injected into a tube centrifuge for continuous separation under the condition of 10,000 g. The flow was adjusted so that the residence time of the mixture in the centrifuge was 1.5 minutes. The separated oil phase was dehydrated by vacuum heating, and then adsorbed and filtered, but the filtration failed. No matter how to change the composition, dosage, filtration temperature, pressure and other conditions of the adsorbent, it fails.

3.3. Centrifugal force. Taking 1000kg of rubber seed crude oil as raw material, the measured acid value is 30mgKOH/g, and the required NaOH solid is 21.4kg. Add 78.6 kg of water to the solid alkali to dissolve, and prepare 100 kg of lye solution with a concentration of 21.4% by weight. Heat the crude oil to 80°C, then stir at a slow speed, slowly add the lye to the crude oil, continue to stir at a slow speed until the reaction between the crude oil and the lye is complete, then stop stirring, keep the oil temperature, and settle naturally. After 6 hours of natural sedimentation, it was found that the oil phase and the fatty acid salt phase were obviously stratified, the fatty acid salt phase was discarded, the oil phase was heated to 85°C, and then injected into a tubular or disc centrifuge for centrifugation. From 1000g to 40000g, the centrifugal force is gradually increased according to the interval of 1000g, and it is found that effective separation cannot be achieved when it is less than 5000g; when it is above 5000g (inclusive), it can be effectively separated, the centrifugal force increases, and the processing efficiency of the equipment increases. When it exceeds 30000g, the material requirements of the equipment, processing accuracy requirements, wear speed, and operation difficulty will increase significantly, and the benefits will be limited. When the centrifugal force is 6000-18000g, the overall performance is the best.

Example 4

Refined rubber seed oil was prepared according to the method of document 6. Using 1000kg of rubber seed crude oil as raw material, the measured acid value is 30mgKOH/g. According to the method optimized in document 6, adjust the oil temperature to 40°C, add 3kg of phosphoric acid with a concentration of 85%, fully stir and mix, then heat up to 80°C, stir rapidly, add 80kg of demineralized water at 80°C (remove calcium, magnesium and other ions in the water). water), change to slow stirring after adding water, heat up to 85°C, then continue stirring for 30 minutes, stop stirring, let it settle for more than 6 hours, release oily feet, sewage and dirty oil, and then carry out vacuum dehydration. Vacuum dehydration pressure 0.005MPa (0.08MPa-0.075MPa), temperature) 90°C, after dehydration is completed, the oil temperature is cooled to below 40°C in a vacuum state. The pre-decolorized oil was heated to 110°C, and 25kg of activated clay was added under a vacuum of 0.005MPa (0.08MPa-0.075MPa), and 1.25kg of activated carbon was added to the clay at the same time. When the decolorization filtration is halfway through, it is switched to the pre-decolorization of the raw material oil. When the pressure of the leaf filter reaches the upper limit of the equipment, the filtration is stopped, and the filter is blown to remove the shaking cake to complete a filtration cycle. The whole filtering process is difficult and the filtering speed is very slow. The decolorized oil enters the packed deacidification tower, the pressure is less than or equal to 100Pa, the temperature of the oil entering the tower is heated to 240-250°C, the temperature at the outlet is controlled at ≥230°C, and the acid value at the outlet is controlled at ≤1mgKOH/g. The oil from the tower was cooled to room temperature to obtain 810 kg of the refined oil product prepared by the method of Document 6, with a yield of 81%. The refined oil is red, clear and transparent, and has no peculiar smell.

The refined oil was tested according to the standard of "GB 2716-2018 National Food Safety Standard Vegetable Oil", and it was judged as a standard product.

Example 5

Refined rubber seed oil was prepared according to the method of document 10. Using 1000kg of rubber seed crude oil as raw material, the measured acid value is 30mgKOH/g. According to the method in document 10, the oil temperature was heated to 60°C, 2kg of 85% phosphoric acid was added, reacted for 18 minutes, left for precipitation, the lower layer liquid was released, and water was added for washing once, and a relatively obvious emulsification phenomenon occurred. Heat the oil to 65°C, add 1kg of formic acid, react for 20-25 minutes, let it stand for precipitation, add water to wash 3 times, each time there is obvious emulsification, and the oil becomes milky white. Under the vacuum condition of pressure 0.6kPa, the oil is heated to 112℃ for drying and dehydration. After the dehydration was completed, 40 kg of activated clay and 10 kg of activated carbon were added, and the temperature was kept at 100° C. and stirred for 30 minutes. Then filter it while it is still hot, and filter out the clear oil. Filtration is more difficult and filtration is slow. According to the ratio of oil: alcohol = 1: 1.5, the filtered clear oil was added with 75% ethanol for the first round of extraction, mixed and stirred at a temperature of 58°C, and then left to stand for stratification to separate the oil phase of the lower layer; Under the conditions of the first round, the second round of extraction was performed; 15 rounds of extraction were repeated in this way, and the acid value was reduced to less than 3, and then the extraction was terminated. The oil phase was heated under negative pressure to evaporate the ethanol to obtain 690kg of the refined oil product prepared by the method of Document 10, with a yield of 69%. The refined oil is red, clear and transparent, and can smell ethanol after being placed in the container for a long time.

The refined oil was tested according to the standard of "GB 2716-2018 National Food Safety Standard Vegetable Oil". Two indicators were unqualified, and the rest were qualified. The unqualified indicators are: smell and solvent residue.

Example 6

Refined rubber seed oil was prepared according to the method of document 12. Using 1000g of rubber seed crude oil as raw material, the measured acid value is 30mgKOH/g. According to the method optimized in document 12, adjust the oil temperature to 40°C, add 3g phosphoric acid with a concentration of 85%, stir and mix thoroughly, heat up to 80°C, stir rapidly, add 80g, 80°C demineralized water (remove calcium, magnesium and other ions in the water). After adding water, it was changed to slow stirring, and the temperature was raised to 85 °C, then continued stirring for 30 min, stopped stirring, and allowed to settle for more than 6 hours. Under normal pressure, heat the oil to 110°C until no bubbles emerge. After dehydration is complete, the oil is cooled to below 40°C. Heat the oil to 110°C, add 50 g of activated clay, and at the same time add 2.5 g of activated carbon to the clay, stir for 30 minutes, and then filter with 3 layers of filter paper. In order to speed up the filtration rate, Buchner funnel suction filtration was used for filtration. Still, filtering is slow. The filtered oil enters the molecular distillation unit for molecular distillation. The conditions of molecular distillation are: feed pump rotation frequency 20Hz, wiper rotor speed 300rpm, coolant temperature 25℃, heat transfer oil temperature 200℃, vacuum pump vacuum degree 1.33Pa.

The acid value of one round of distillation directly according to the method of document 12 is still as high as 12mgKOH/g, which is higher than the standard 3mgKOH/g of "GB 2716-2018 National Food Safety Standard Vegetable Oil". After repeating 3 rounds of molecular distillation, the acid value dropped below 3 mgKOH/g, and finally 830 grams of the product prepared by the method of Document 12 was finally obtained, with a yield of 83%.

Example 7

Refined rubber seed oil was prepared according to the method of document 14. Using 1000kg of rubber seed crude oil as raw material, the measured acid value is 30mgKOH/g. The crude oil was heated to 70° C., 3kg of 85% concentration phosphoric acid was added, 20kg of saturated NaCl aqueous solution was added, and the mixture was left to stand to separate out the hydrate of the lower layer and retain the upper oil phase. 21.4 kg of NaOH solid was added to 78.6 kg of saturated NaCl solution to prepare 100 kg of NaOH-saturated NaCl solution (hereinafter referred to as alkali-sodium chloride solution) with a concentration of 21.4% by weight. 100 kg of alkali-sodium chloride solution was added to the oil phase, and the entire reaction system was heated to 70°C. At this time, the oil phase and the alkali-sodium chloride solution were divided into upper and lower layers, and no acid-base neutralization reaction occurred. In order to make the oil phase contact and react with the lye solution, the alkali-sodium chloride solution is gradually contacted with the oil phase by means of slow stirring, and an acid-base neutralization reaction occurs, resulting in a large amount of fatty acid sodium, and then an emulsification phenomenon occurs. The oil phase-sodium fatty acid-alkali-sodium chloride solution was emulsified together to form a brown, viscous, homogeneous emulsion system. After being kept for 24 hours, the upper layer of emulsion was taken and washed with saturated brine at 100°C, which was still severely emulsified. After being kept for 24 hours, there was still no obvious stratification. The upper layer of milky liquid was heated to 120 °C for drying and dehydration. Dehydration was extremely difficult, and a large amount of foam overflowed. After dehydration, 120 kg of refined oil according to the method of document 14 was obtained, and the yield was 12%. The refined oil is viscous jelly-like, reddish-black, cloudy, and has a strong odor.

The refined oil was tested according to the standard of "GB 2716-2018 National Food Safety Standard Vegetable Oil", and it was judged as a non-standard product.

Example 8

Refined rubber seed oil was prepared according to the method of document 13. Using 1000g rubber seed crude oil as raw material, the measured acid value is 30mgKOH/g. According to the method optimized in document 13, 1000 g of crude rubber seed oil was dissolved in 1703 g of n-hexane to prepare a 37% mass fraction of crude rubber seed oil-n-hexane solution. A filter membrane with a pore size of 10kDa was selected, and the crude oil-n-hexane solution was allowed to pass through the filter membrane under a forward pressure of 0.23MPa. The filtered crude oil-n-hexane solution was heated, and the n-hexane solvent was removed by negative pressure, to obtain 950 grams of the product prepared by the method of document 13, and the product yield was 95%. The product is a dark red, opaque oily liquid that is not much different from the raw material of crude oil.

The product was tested in accordance with the standard of "GB 2716-2018 National Food Safety Standard Vegetable Oil", and the results of the direct test of crude oil were similar to those of the crude oil. Many indicators were not up to the standard, especially the acid value was as high as 27mgKOH/g, no matter how many times it was filtered through the membrane, The acid values are all above 25 mgKOH/g, and there is no possibility that the acid value falls below 3 mgKOH/g. The substances obtained by this method have little difference in physical and chemical indicators with crude oil.

Example 9

Rubber seed crude oil, rubber seed oil of the present invention, and rubber seed oil pairs prepared in Example 4 (File 6), Example 5 (File 10), Example 6 (File 12), and Example 7 (File 14) Animal experiments on the effects of atherosclerotic plaque formation. The physical and chemical indicators of the rubber seed oil prepared in Example 8 (document 13) are not much different from those of the crude rubber seed oil, so they were not selected for animal experiments.

Twelve 8-week-old male wild-type (wide type) C57BL/6J mice were selected; 84 8-week-old male APOE-/- mice were selected, weighing 24±2g. APOE-/- mice were divided into 7 groups with 12 mice in each group, and 12 wild-type (wide type) C57BL/6J mice were divided into 8 groups in total. The groupings are as follows:

(1) Normal diet group: 12 8-week-old male wild-type (wide type) C57BL/6J mice, fed with basal chow, and gavaged with normal saline;

(2) High-fat diet group: 12 APOE-/- mice, fed with high-fat diet + peanut oil gavage, gavage dose of 6.2g/kg/d (converted according to adult and animal body weight, body surface area and Km factor) ;

(3) Rubber seed crude oil + high-fat diet group: 12 APOE-/- mice, fed with high-fat diet + rubber seed crude oil gavage, the gavage dose was 6.2 g/kg/d (according to the body weight of adults and animals , body surface area and Km factor conversion);

(4) Group of the present invention + high-fat diet group: 12 APOE-/- mice, fed with high-fat diet + preparation of Example 1-2 by gavage, the gavage dose was 6.2 g/kg/d (according to adults and Animal body weight, body surface area and Km factor conversion);

(5) File 6 group + high-fat diet group: 12 APOE-/- mice, fed with high-fat diet + gavage with the preparation of Example 4, gavage dose of 6.2 g/kg/d (according to the body weight of adults and animals , body surface area and Km factor conversion);

(6) File 10 group + high-fat diet group: 12 APOE-/- mice, fed with high-fat diet + gavage of the preparation of Example 5, gavage dose of 6.2 g/kg/d (according to the body weight of adults and animals , body surface area and Km factor conversion);

(7) File 12 group + high-fat diet group: 12 APOE-/- mice, fed with high-fat diet + gavage of the preparation of Example 6, gavage dose of 6.2 g/kg/d (according to the body weight of adults and animals , body surface area and Km factor conversion);

(8) File 14 group + high-fat diet group: 12 APOE-/- mice, fed with high-fat diet + gavage with the preparation of Example 7, gavage dose of 6.2 g/kg/d (according to the body weight of adults and animals , body surface area and Km factor conversion);

The mice in the above groups were given continuous gavage for 8 weeks. High-fat feed formula: basal feed: 70%, lard: 20%, sucrose: 5%, milk powder 4%, bile salts 1%, cholesterol 0.15%.

The mice were sacrificed, the thoracic cavity of the mice was cut open, the blood vessels of the mice were perfused with pre-cooled normal saline, the aorta was stripped, the aorta was completely cut, washed with PBS, and fixed in 10% formaldehyde solution. Degree of disease:

Grade 0: The surface of the intima is smooth and there is no cream change, that is, no plaque;

Grade 0.5: There are extensive cream or milky changes in the intima, but no plaques protruding from the surface;

Grade 1: There are obvious cream-colored raised plaques in the intima, and the plaque area is less than 3mm2;

Grade 2: There are obvious cream-colored raised plaques in the intima, but there is no phenomenon of fusion into sheets, the largest plaque, the area is greater than 3mm2;

Grade 3: There are many plaques of different sizes, some of which are fused into pieces, and the area of large plaques exceeds 3mm2

Grade 4: The arterial intima surface is almost completely covered by fused plaque.

The results of animal experiments are shown in the following table and Figure 1:

Experiments show that:

(1) The rubber seed oil of the present invention has the effect of preventing and inhibiting atherosclerotic plaque. After adding the rubber seed oil of the present invention, the development degree of atherosclerosis in APOE-/- mice on a high-fat diet was 1; Grade 3-4; after adding the rubber seed oil of the present invention, the degree of atherosclerosis development in APOE-/- mice is obviously slowed down, indicating that the rubber seed oil of the present invention has preventive and inhibitory effects on atherosclerotic plaques;

(2) The rubber seed oil prepared in Example 4, Example 5, Example 6, and Example 7 did not have obvious effect of preventing and inhibiting atherosclerotic plaque. Arteries of APOE-/- mice on a high-fat diet after addition of the rubber seed oils prepared in Document 6 (Example 4), Document 10 (Example 5), Document 12 (Example 6), and Document 14 (Example 7) The degree of development of atherosclerosis was grade 3-4; the degree of atherosclerosis in APOE-/- mice with no added high-fat diet was also grade 3-4; whether or not document 6 (Example 4), document 10 were added (Example 5), document 12 (Example 6), and document 14 (Example 7) prepared the rubber seed oil, the degree of atherosclerosis development in APOE-/- mice obviously did not change significantly. The rubber seed oils prepared in Example 4), Document 10 (Example 5), Document 12 (Example 6), and Document 14 (Example 7) did not have obvious preventive and inhibitory effects on atherosclerotic plaque.

Example 10

Animal experiments on the effect of rubber seed crude oil, rubber seed oil of the present invention, and simvastatin on atherosclerotic plaque formation.

Rabbit is an ideal animal model for the study of atherosclerosis induced by diet, and its pathogenesis is closer to the human body than that of genetically deficient mice and rats. Select 40 Japanese white rabbits, 4 months old, male, weighing 2.0 ± 0.2 kg, and randomly divided into 5 groups, the groups are as follows:

(1) Normal diet group: 8 male Japanese white rabbits, fed with basic feed;

(2) High-fat diet group: 8 male Japanese white rabbits, fed with peanut oil + high-fat fortification + basal feed, and the feeding dose of peanut oil was 1.5 g/kg/d (converted according to adult and animal body weight, body surface area and Km factor) ;

(3) Rubber seed crude oil + high-fat diet group: 8 male Japanese white rabbits, rubber seed crude oil + high-fat fortification + basal diet feeding, rubber seed crude oil feeding dose 1.5g/kg/d (according to adults and animal body weight, body surface area and Km factor conversion);

(4) The present invention + high-fat diet group: 8 male Japanese white rabbits, the preparation of Example 1-2 + high-fat fortification + basic feed feeding, the feeding dose of the preparation of Example 1-2 1.5g/kg/d (Converted according to adult and animal body weight, body surface area and Km factor);

(5) Simvastatin + high-fat diet group: 8 male Japanese white rabbits, simvastatin + high-fat fortification + basal diet feeding, simvastatin feeding dose 2.0 mg/kg/d (according to adult and animal body weights) , body surface area and Km factor conversion);

The high-fat fortified feed was as follows: each rabbit was fed with 0.5 grams of cholesterol, 2.0 grams of lard, and 20 grams of egg yolks per day.

The animals were fed for 6 weeks according to the above grouping and feeding conditions and then sacrificed. After the animals were sacrificed, the aorta was removed from the aortic valve orifice to the beginning of the common iliac artery, the aortic wall was cut longitudinally along the midline of the abdomen, fixed with 10% formaldehyde solution, and the distribution of aortic lesions was recorded according to the tracing method. The area of the atherosclerotic plaque, and the total lumen area of the aorta expanded, were determined, and the percentage of plaque area was calculated.

The experimental results are as follows:

The typical rabbit atherosclerotic plaque traces in the experiment are shown in Figure 2, and the typical rabbit aortic atherosclerotic plaque traces in Example 10, from left to right, were taken from the normal diet group, high-fat Diet group, rubber seed crude oil + high-fat diet group, the present invention + high-fat diet group, simvastatin + high-fat diet group;

Figure 4: P value of the difference between different groups in Example 10, P less than 0.05 is a significant difference, P less than 0.01 is a very significant difference;

Figure 5: The proportion of atherosclerotic plaques in the aorta of different groups of rabbits in Example 10.

Experimental results show that:

(1) The rubber seed oil of the present invention has obvious effect of preventing and inhibiting atherosclerotic plaque (the plaque area of the present invention + high-fat diet group is 14.7±4.2%, which is smaller than that of the high-fat diet group 42.6±9.7%, P <0.01, the difference is extremely significant);

(2) The efficacy of the rubber seed oil of the present invention in preventing and inhibiting atherosclerotic plaque is stronger than that of the rubber seed oil (the plaque area of the present invention + high-fat diet group is 14.7±4.2%, which is smaller than that of the rubber seed oil + high-fat diet group). Diet group 19.7±4.7%, P<0.05, significant difference);

(3) The rubber seed oil of the present invention, at a dose of 1.5 g/kg/d, has a stronger effect of preventing and inhibiting atherosclerotic plaques than simvastatin at a dose of 2.0 mg/kg/d (the present invention + The plaque area in the high-fat diet group was 14.7±4.2%, which was smaller than that in the simvastatin+high-fat diet group (20.2±4.2%, P<0.05, significant difference).

Example 11

Animal experiments on the effects of rubber seed crude oil, rubber seed oil of the present invention, and simvastatin on established atherosclerotic plaques.

Rabbit is an ideal animal model for the study of atherosclerosis induced by diet, and its pathogenesis is closer to the human body than that of genetically deficient mice and rats. Select 40 Japanese white rabbits, 4 months old, male, weighing 2.0±0.2kg, and randomly divided into 2 groups. The groups are as follows:

(1) Normal diet group: 8 male Japanese white rabbits, fed with basic feed;

(2) Atherosclerosis modeling group: 40 male Japanese white rabbits, fed with high-fat fortification + basal feed, and fed with peanut oil at a dose of 1.5 g/kg/d (converted according to adult and animal body weight, body surface area and Km factor) );

The high-fat fortified feed was as follows: each rabbit was fed 0.5 g cholesterol, 4.0 g lard, and 20 g egg yolk per day. The animals were fed according to the above grouping and feeding conditions for 6 weeks, during which 1 rabbit died in the normal diet group.

After 6 weeks, 8 atherosclerosis model rabbits were randomly selected and sacrificed to test whether the atherosclerosis model was successfully established. The inspection method is as follows: after the animals are sacrificed, the aorta is removed from the aortic valve orifice to the beginning of the common iliac artery, the aortic wall is cut longitudinally along the midline of the abdomen, fixed with 10% formaldehyde solution, and the aorta is recorded according to the tracing method. Distribution map of arterial lesions, measuring the area of atherosclerotic plaque, and the total lumen area of the aorta, and calculating the percentage of plaque area. As a comparison, 8 rabbits in the normal diet group were also all sacrificed, and the area percentage of aortic atherosclerotic plaques was detected.

After confirming that the rabbits in the atherosclerosis model group were successfully established, the remaining 32 rabbits in the modeling group were randomly divided into 4 groups with 8 rabbits in each group, and the groups were as follows:

(3) Modeling + normal feeding group: 8 male Japanese white rabbits with successful atherosclerosis modeling, peanut oil + basal diet feeding, peanut oil feeding dose 1.5g/kg/d (according to adult and animal body weight, Body surface area and Km factor conversion);

(4) Modeling + rubber seed crude oil treatment group: 8 male Japanese white rabbits with successful atherosclerosis modeling, rubber seed crude oil + basic diet feeding, rubber seed crude oil feeding dose 1.5g/kg/d (Converted according to adult and animal body weight, body surface area and Km factor);

(5) Modeling + treatment group of the present invention: 8 male Japanese white rabbits with successful atherosclerosis modeling, fed with the preparation of Example 1-2 + basic feed, and fed with the preparation of Example 1-2 at a dose of 1.5 g /kg/d (converted according to adult and animal body weight, body surface area and Km factor);

(6) Modeling + simvastatin treatment group: 8 male Japanese white rabbits with successful atherosclerosis modeling were fed with simvastatin + basal diet, and the feeding dose of simvastatin was 2.0 mg/kg/d (according to adult Human and animal body weight, body surface area and Km factor conversion);

Continue to feed for 3 months according to the above grouping and feeding conditions, during which 1 rabbit died in each of the modeling + normal feeding group and the modeling + simvastatin treatment group. After continuing to feed for 3 months, all animals were sacrificed.

After the animals were sacrificed, the aorta was removed from the aortic valve orifice to the beginning of the common iliac artery, the aortic wall was cut longitudinally along the midline of the abdomen, fixed with 10% formaldehyde solution, and the distribution of aortic lesions was recorded according to the tracing method. The area of the atherosclerotic plaque, and the total lumen area of the aorta expanded, were determined, and the percentage of plaque area was calculated.

The experimental results are as follows:

The typical rabbit atherosclerotic plaque trace in the experiment is shown in Figure 6: the typical rabbit aortic atherosclerotic plaque trace in Example 11, taken from left to right in the normal diet group, atherosclerotic plaque Hardening modeling group, modeling + normal feeding group, modeling + crude oil treatment group, modeling + treatment group of the present invention, modeling + simvastatin treatment group;

Figure 7: The proportion of atherosclerotic plaques in the aorta of different groups of rabbits in Example 11;

Figure 8: P value of the difference between different groups in Example 11, P less than 0.05 is a significant difference, P less than 0.01 is a very significant difference;

Figure 9: The proportion of atherosclerotic plaques in the aorta in different groups of rabbits in Example 11.

Experimental results show that:

(1) The rubber seed oil of the present invention can not only inhibit the increasing trend of atherosclerosis (27.5±6.8% of the plaque area in the modeling + the present invention group, less than 59.9 ± 8.9% in the modeling + normal feeding group, P<0.01 , the difference is very significant), and it can ablate the already formed atherosclerotic plaque (the plaque area of the modeling + the present invention group is 27.5±6.8%, less than the modeling group 45.8±9.9%, P<0.01, the difference is extremely significant) ;

(2) The efficacy of the rubber seed oil of the present invention in inhibiting atherosclerotic plaque is better than that of the rubber seed oil (the plaque area of the modeling + the present invention group is 28.1±6.4%, which is smaller than that of the modeling + crude oil group 27.5 ± 6.8% , P<0.05, significant difference);

(3) At the dose of 1.5 g/kg/d, the rubber seed oil of the present invention has a stronger effect on inhibiting atherosclerotic plaque than that of simvastatin at the dose of 2.0 mg/kg/d (the plaque area of the present invention group). 27.5±6.8%, less than 47.1±8.3% in simvastatin group, P<0.01, the difference is extremely significant).

Example 12:

According to the method in Example 1-2, the high-activity and high-safety substance of the present invention was prepared, and the excipient was added in a ratio of 1:1 or 1:2 by weight of the part to the excipient, and granulated and pressed into tablets. . Tablets can be used as medicines.

Example 13:

According to the method in Example 1-2, the high activity and high safety substance of the present invention is prepared, and according to this part, it is wrapped in the soft capsule material, and the soft capsule is prepared according to the conventional capsule preparation method. Softgels can be used as medicines.

Example 14:

According to the method in Example 1-2, the high-activity and high-safety substance of the present invention was prepared, and the part was added with emulsifier, osmotic pressure regulator and water, and emulsified by high pressure homogenization to obtain an emulsion for injection.

Example 15:

The high-activity, high-safety rubber seed oil of the present invention is obtained by the method in Example 1-2, and then tablets are made according to the following method according to this part:

Preparation method: the rubber seed oil of the present invention is mixed with auxiliary agents, sieved, and uniformly mixed in a suitable container, and the obtained mixture is granulated and pressed into tablets.

Example 16:

The rubber seed oil of high activity and high safety of the present invention is obtained by the method in Example 1-2, and by this part, the soft capsule is made by the following method:

Preparation method: the gelatin and glycerin are soaked in distilled water to swell and soften the gelatin, and then stirred and mixed evenly to obtain a capsule material glue. Take out the prepared capsule material glue, apply it on the flat surface of the board to make the thickness uniform, and then heat it at a temperature of about 90 ℃ to evaporate the surface water, and become a soft film with certain toughness and elasticity. Finally, the rubber seed oil (1000 mg) of the present invention is filled into the soft capsule skin by using a pill pressing die or an automatic rotary capsule rolling machine to produce a soft capsule.

Example 17:

The rubber seed oil with high activity and high safety of the present invention is obtained by the method in Example 1-2, and according to this part, the injection emulsion is made by the following method:

Preparation method: according to the formula, mix the rubber seed oil of the present invention, lecithin and glycerin, add water to 1000mL, add an appropriate amount of sodium chloride to adjust the osmotic pressure, homogeneously emulsify at high pressure, sterilize at 121°C, and cool to room temperature to obtain injection Emulsion.

Example 18:

Tablet: 10 mg of the rubber seed oil, 180 mg of lactose, 55 mg of starch, and 5 mg of magnesium stearate of the present invention were prepared according to the method in Example 1-2;

Preparation method: Mix the rubber seed oil, lactose and starch of the present invention obtained by the method in Example 1-2, wet evenly with water, sieve and dry the wetted mixture, sieve again, and add magnesium stearate , and then the mixture was compressed into tablets, each tablet weighing 250 mg, and the rubber seed oil content of the present invention was 10 mg.

Example 19:

Capsules: 2000g of high-activity, high-safety rubber seed oil, 1000g of gelatin, 500g of glycerin, and 1000g of water of the present invention were prepared according to the method in Example 1-2;

Preparation method: 1000 g of gelatin and 500 g of glycerin are soaked in 1000 g of water to swell and soften the gelatin, and then stirred and mixed evenly to obtain a capsule material glue. Take out the prepared capsule material glue, apply it on the flat surface of the board to make the thickness uniform, and then heat it at a temperature of about 90 ℃ to evaporate the surface water, and become a soft film with certain toughness and elasticity. Finally, 1000 mg of the rubber seed oil of the present invention is put into the soft capsule skin by using a pill pressing die or an automatic rotary capsule rolling machine to obtain a soft capsule. Each soft capsule weighs 1.4 g, and the content of the gum seed oil of the present invention is 1000 mg.

Example 20:

Injection emulsion: 200g of high-activity, high-safety rubber seed oil of the present invention, 25g of glycerol, 12g of lecithin, 1000g of water, and 1g of sodium chloride were prepared according to the method in Example 1-2;

Preparation method: Accurately weigh 200 g of rubber seed oil, 25 g of glycerol, and 12 g of lecithin of the present invention according to the method in Example 1-2, mix, add water to 1000 mL, add sodium chloride, and adjust the osmotic pressure to 350 mosm/ kg H2O. High-pressure homogeneous emulsification to obtain an emulsion, sterilized at 121° C., lowered to room temperature, and filled into a 250 mL sterile glass bottle under aseptic conditions for packaging. The specification of each bottle of injection emulsion is 250 mL, and the content of the rubber seed oil of the present invention is 50 g.

Claims

A substance with high activity and high safety, characterized in that the substance is prepared by the following method: using crude rubber seed oil as a raw material, neutralizing by adding alkali, natural sedimentation, centrifugal separation, adsorption filtration, and distillation to obtain .
The method for preparing a high-activity and high-safety substance according to claim 1, wherein the crude rubber seed oil is used as a raw material, and is obtained through alkali neutralization, natural sedimentation, centrifugal separation, adsorption filtration, and distillation.
preparation method as claimed in claim 2, described adding alkali and neutralizing step, it is characterized in that: alkali selects the alkali of food grade for use, the addition amount of alkali is equal to the molar mass of the fatty acid contained in crude oil, and the mode of addition is alkali aqueous solution, and then mixed.
The preparation method according to claim 2, wherein the natural sedimentation step is characterized in that: the temperature of the mixture is kept at a standstill above 70°C, and after the fatty acid salt and the oil phase are obviously layered, the lower fatty acid salt phase is discarded, and the oil is retained. Mutually.
The preparation method according to claim 2, wherein the centrifugal separation step is characterized in that: the temperature of the oil phase is above 70°C, and the centrifugal force is greater than or equal to 5000g.
The preparation method according to claim 5, wherein the centrifugation step is characterized in that: centrifugation is performed with a disc centrifuge or a tubular centrifuge, the centrifugation temperature of the oil phase material is 70-100°C, and the centrifugal force is 5000-30000g , preferably 6000-18000g, discard the fatty acid salt, and separate the oil phase.
The preparation method according to claim 2, wherein the distillation step is characterized in that: the distillation temperature is less than or equal to 180°C, water vapor is introduced from the bottom, and the last remaining substance is the finished product.
A pharmaceutical composition comprising a therapeutically effective amount of the highly active and safe substance of claim 1 and a pharmaceutically acceptable carrier.
The application of the highly active and highly safe substance of claim 1 in the preparation of a medicament for preventing and treating atherosclerosis.
The application according to claim 9, wherein the highly active and safe substance is used as the only active ingredient in the medicine.