CN105535045A - Orixine hydrochloride as well as preparation method and medical application thereof - Google Patents

Abstract

The invention relates to orixine hydrochloride as well as a preparation method and medical application thereof. In particular, through pumping hydrochloric acid gas into an ethanol solution of radix dichroa total alkaloids, the orixine hydrochloride of which the stability and water solubility are both increased in comparison with orixine is obtained, the anti-malarial activity of the orixine hydrochloride is enhanced and the orixine hydrochloride can be used as novel anti-malarial drugs.

Description

Hemosine hydrochloride and its preparation method and medical application

technical field

The present invention relates to stable halosine hydrochloride and its preparation method and application.

Background technique

At present, the epidemic situation of malaria in the world, especially in some underdeveloped areas, is still very serious. The emergence of multi-drug resistant strains is becoming more and more common, and there is an urgent need to develop new antimalarial drugs and new drug combinations.

Malaria is a well-known protozoan disease and one of the most common infectious diseases in tropical and subtropical countries in Africa, Southeast Asia, and South America. Worldwide, 225 million people are infected with malaria every year, and 781,000 people die from malaria. In most malaria-endemic regions, the increasing prevalence of multidrug-resistant strains has greatly reduced the effectiveness of current antimalarial drugs in preventing and treating the disease. What is particularly worrying is that human falciparum malaria, which poses the greatest threat to human health and life, has not only developed resistance to quinoline antimalarials such as chloroquine and other first-line drugs, but has recently become resistant to the currently commonly used artemisinin antimalarials. Drug resistance has also been reported. If this strain of P. falciparum resistant to artemisinin-based antimalarials spreads, there will be no cure for the disease. Therefore, the clinical treatment of malaria urgently needs the research and development of a new generation of antimalarial drugs.

At present, the research and development of antimalarial drugs mainly relies on the structural modification and empirical screening of the original drugs, and one of the characteristics of multi-drug resistant strains is that they are very prone to resistance to similar antimalarial drugs with similar structures, so that these structurally similar antimalarials lose their therapeutic value. One of the characteristics of artemisinin antimalarials is that their chemical structure is very different from quinoline antimalarials, so they have achieved great success in the treatment of malaria. In view of the above, it should be necessary to carry out research and development of a new generation of antimalarial drugs with new structures as soon as possible, and the research and development of new antimalarial drugs and the rational and standardized implementation of chemotherapy programs will undoubtedly play an important role in the elimination of malaria worldwide.

Changshan is a classic antimalarial drug in the treasure house of traditional Chinese medicine in my country. Its antimalarial effect is outstanding, but its wide clinical application is greatly limited due to its strong emetic side effects.

Changshan is the dry root of the saxifrage plant Huangchangshan. Traditional Chinese medicine believes that Changshan is bitter, pungent, and cold, and returns to the liver, spleen, and heart meridian. The main active ingredients of Changshan are quinazolinone alkaloids, including Changshanine, Changshanine and Changshanine. Modern studies have confirmed that Changshan has anti-amoeba, anti-leptospira, anti-virus, anti-tumor, anti-arrhythmia, antihypertensive, emetic, and exciting uterine smooth muscle effects. It is mainly used clinically to treat malaria, chicken coccidiosis, Giardia lamblia and other diseases.

Changshan is one of the important antimalarial traditional Chinese medicines in traditional Chinese medicine. Before the Ming and Qing Dynasties, most antimalarial prescriptions of traditional Chinese medicine used Changshan as the main drug. It is expected to replace the "Jun Lie" antimalarial traditional Chinese medicine prescriptions based on Changshan. Although Xiao Chaihu Decoction reduces the toxic and side effects, its antimalarial efficacy is obviously not as good as that of Changshan, so Changshan has always played a vital role in the antimalarial clinical practice of traditional Chinese medicine.

Modern pharmacology and chemical studies have shown that Changshan has good antimalarial activity, and its main active ingredients are Changshanine and abnormal mountainine, among which Changshanine is 100 times more active than quinine. Kobayashi et al. found that the EC ₅₀ values of hemosine and anomaline against Plasmodium falciparum were 2.0×10 ^-7 and 1.6×10 ^-7 mol/L respectively; the EC ₅₀ values against Plasmodium were 7.6×10 ^-11 and 2.9 respectively ×10 ^-10 mol/L. The in vitro anti-two kinds of Plasmodium falciparum (FCR-3 and K1) activity of hirhosine, abnormal hsine and its derivatives are all better than the reference drug chloroquine and artemisinin, wherein the _EC50 of hsodosine is 7.0×10 ^-10 (FCR-3 respectively) -3) and 1.2×10 ^-9 (K1) mol/L, the EC ₅₀ values of abnormal mountain alkali are 3.4×10 ^-9 (FCR-3) and 1.8×10 ^-9 (K1) mol/L respectively, while chloroquine The EC ₅₀ values are 1.8×10 ^-9 (FCR-3) and 1.3×10 ^-7 (K1) mol/L, respectively.

Although Changshan has such a high antimalarial potency, it has not been developed into a new antimalarial drug. One of the important reasons is the instability of halosine. The inventors have found through research on Changshan and its active ingredients that there are the following important problems in the drug-making properties of Changshan: First, the content of Changshan in Changshan is very low, about 1/10,000. The pure product is extremely difficult; the second, hirhosine is extremely unstable, and it is very easy to convert into abnormal halosine, and the two transform each other under certain conditions. Within 1 hour, part of hibiscatel will be transformed into anomaline. After that, the content of halosine will continue to decrease and the content of anomaline will continue to increase until the two reach a dynamic balance. No change; thirdly, in terms of chemical synthesis, hirsutine is unstable, and the yield of derivatization reaction is too low.

In the research, the present inventor solved the problem of the instability of bishenzine through the salt-forming process, and solved the technical bottleneck of the extremely poor stability of bishenzine which is difficult to be widely used by establishing the preparation process of bishenzine hydrochloride.

Contents of the invention

Therefore, the object of the present invention is to provide stable hosherine hydrochloride. The present inventor has carried out a large amount of researches after encountering the practical problem of the extreme instability of bishenzine, and found that after bishenzine hydrochloride is acidified into bishenzine hydrochloride, the stability of the bishenzine hydrochloride solution is greatly greater than that of the bishenzine solution. Increase, and the water solubility of Changshan alkali hydrochloride is also greatly increased compared with Changshan alkali. Thus, the present inventor has established a set of process for the preparation of halosine hydrochloride with simple process, controllable quality and moderate yield, thereby solving the problem that halosine hydrochloride is difficult to produce on a large scale, and the efficacy of halosine hydrochloride also improved.

One aspect of the present invention provides a halosine hydrochloride.

The present invention further provides the preparation method of described halosine hydrochloride, it comprises the following steps:

Radix Changshan with ethanol preferably 70% ethanol heat reflux extraction to obtain the total alkaloids of Changshan;

Dissolving the total alkaloids of Changshan in ethanol, preferably absolute ethanol, and then passing hydrochloric acid gas into it to obtain a precipitate;

Recrystallize the obtained precipitate with ethanol, preferably 50%-100% ethanol, to obtain halosine hydrochloride.

Dichroa febrifuga Lour. described in the present invention is the dry root of the saxifrage plant Dichroafebrifuga Lour.

The present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of the above-mentioned hosbinine hydrochloride and one or more pharmaceutically acceptable carriers.

Those skilled in the art can directly or indirectly add the halosine hydrochloride described in the present invention to various commonly used pharmaceutically acceptable adjuvants required for the preparation of different dosage forms, such as fillers, disintegrants, lubricants, binders, etc. Preparations, etc., are made into common preparations suitable for oral or parenteral (intravenous or subcutaneous) administration such as tablets, capsules, injections, oral liquids, granules, pills, powders, drops, etc. Pills, lozenges, creams, suspensions, etc. Among them, fillers such as starch, lactose, sucrose, glucose, mannitol and silicic acid; disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates and sodium carbonate, low-substituted hydroxypropyl Cellulose; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate; binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, Sucrose and gum arabic.

The dosage and method of use of the pharmaceutical preparation of the present invention depend on many factors, including the user's age, body weight, sex, natural health status, nutritional status, activity intensity of the compound, use time, metabolic rate, severity of disease course, and diagnosis and treatment. Physician's subjective judgment. Those skilled in the art will vary depending on the nature and severity of the disease, the route of administration and the age and weight of the patient.

The present invention further provides the use of the above-mentioned hoshenine hydrochloride or the pharmaceutical composition containing it in the preparation of medicines for preventing and/or treating diseases caused by Plasmodium. The Plasmodium is in particular a multi-drug resistant strain. Diseases caused by Plasmodium are especially malaria.

The present invention further provides the application of the above-mentioned hoshenine hydrochloride or the pharmaceutical composition containing it in the preparation of medicines for preventing and/or treating malaria, chicken coccidiosis and giardiasis.

The present invention further provides the use of the above-mentioned halosine hydrochloride or the pharmaceutical composition containing it in the preparation of medicines, and the medicines are used for anti-inflammatory, antibacterial, antiviral, antitumor, antiarrhythmic, or antihypertensive Wait.

Description of drawings

Fig. 1 is the ¹ H-NMR spectrum of halosine hydrochloride prepared in the present invention.

Fig. 2 is the HPLC chart (measured at 0h after preparation) of halosine hydrochloride prepared by the present invention.

Fig. 3 is the HPLC figure of fushenine (measured at 0h after preparation).

Fig. 4 is the HPLC graph of the halosine hydrochloride prepared in the present invention (determined 288h after preparation).

Fig. 5 is the HPLC figure of halosine (determined 288h after preparation).

Fig. 6 is a chart showing the change of the percentage content of femospine over time.

Fig. 7 is a graph showing the change of the percentage content of halosine hydrochloride prepared in the present invention with time.

detailed description

The present invention will be described in detail below through examples, but these examples do not limit the scope of the present invention.

The Changshan medicinal material was purchased from the Hebei Anguo medicinal material market, and was identified as the dried root of the saxifrage plant Dichroafebrifuga Lour. by Li Chun, an associate researcher at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

The preparation of embodiment 1 halosine hydrochloride

Take the coarse powder of Changshan medicinal materials, extract by 70% ethanol heat reflux, recover the solvent from the extract until it has no alcohol smell, first add concentrated hydrochloric acid to adjust the pH value to about 2.5, then add chloroform to extract to remove acidic impurities, add concentrated ammonia water to the raffinate phase to adjust the pH value to about 9.0, then add chloroform for extraction, and recover the solvent from the chloroform extract to obtain the total alkaloids of Changshan.

Add absolute ethanol to the obtained total alkaloids of Changshan to dissolve and filter to obtain a solution, and directly pass hydrochloric acid gas into the absolute ethanol solution of total alkaloids of Changshan for 4 hours to obtain a precipitate. The precipitate was recrystallized three times with absolute ethanol to obtain halosine hydrochloride. Detected by HPLC, its content is more than 96%. Table 1 shows the elemental analysis results of felindine hydrochloride.

Table 1 The elemental analysis results of halosine and halosine hydrochloride

The instrument used for NMR is Bruker AvanceIII600-NMRspectrometer (internal standard is TMS, Bruker Company, Germany). The hydrogen spectrum of Changshan alkali is shown in Figure 1.

Embodiment 2 studies on the stability of halosine and halosine hydrochloride

The stability of halosine and halosine hydrochloride was determined by HPLC.

The chromatographic conditions are as follows: Agilent1200 high performance liquid chromatography; chromatographic column: YWG-C ₁₈ (4.6mm×250mm, 10μm); mobile phase: acetonitrile-water-triethylamine-glacial acetic acid (9:91:0.35:0.75); Detection wavelength: 225nm; column temperature: 30°C; flow rate: 1mL/min. Sample concentration: 1.0 mg/mL; injection volume: 10 μL.

Preparation of the reference substance solution: Precisely weigh the reference substance of Changshan alkali (references: Zhang Ya, Li Chun, Lei Guolian. Research on the chemical constituents of Changshan. Chinese Journal of Experimental Formulas, 2010,16(5):77-79. preparation) and the as-prepared hirshenine hydrochloride (put into a decompression desiccator to dry for 24 hours after the hirsutine hydrochloride is prepared) each 10mg, respectively add methanol to dissolve and settle to two 10mL volumetric flasks, to obtain .

After sample configuration, 0, 1, 2, 4, 8, 12, 24 ... 288 hours of sample injection measurement, calculate the relative percentage content of halosine and halosine hydrochloride according to the relative peak area, the obtained chromatogram is shown in Figure 2 -5. In addition, the abscissa is the storage time of the sample after configuration, and the relative percentage of halosine or halosine hydrochloride is the ordinate, and the results are shown in Figures 6 and 7. The total alkaloids of Changshan mainly include Changshanine and abnormal mountain alkali. Therefore in the HPLC determination of halosine hydrochloride, can see two chromatographic peaks of halosine hydrochloride and anomaline hydrochloride at the same time, the ratio of the relative peak area of the two to the total peak area is respectively 96.3% and 3.7%.

The results showed that the stability of halosine hydrochloride was significantly higher than that of halosine, and within 288 hours (12 days) of the measurement time, the mass fraction of halosine hydrochloride hardly changed, while the mass fraction of halosine dropped by nearly half .

Example 3 Research on the Water Solubility of Hemosine and Hemosine Hydrochloride

Grind the hematine hydrochloride prepared above into a fine powder, accurately weigh 10 mg at room temperature, add 50 μL of deionized water, shake vigorously for 30 seconds every 5 minutes, and there will be no visible solute particles within 30 minutes , which is completely dissolved. According to the regulations on solubility in the 2015 edition of "Chinese Pharmacopoeia", hirsutine hydrochloride is easily soluble in water [easy soluble: means that 1 g (mL) of solute can be dissolved in 1 to less than 10 mL of solvent].

Grind firdosine into a fine powder, accurately weigh 10 mg at room temperature, gradually add deionized water, shake vigorously for 30 seconds every 5 minutes, until nearly 100 mL of water is added, firdosine is completely dissolved, so firdosine is in water The solubility is very slightly soluble [very slightly soluble: means that 1g of solute can be dissolved in 1000-less than 10000mL of solvent].

The antimalarial potency comparison of embodiment 4 halosine and halosine hydrochloride

Antimalarial test method: Kunming mice, male, weighing 18-20 g, provided by Beijing Weitong Lihua Experimental Animal Breeding Center. Plasmodium berghei (Plasmodium berghei) K173 strain was introduced by the Department of Medical Protozoa, London School of Hygiene and Tropical Medicine in 1983, and kept in our laboratory by cryogenic freezing and blood transfusion alternately. Using the Peters 4-day inhibition test method to evaluate the antimalarial potency of halosine and halosine salt, obtain Plasmodium-infected red blood cells through Plasmodium inoculation in mice, count the Plasmodium-infected red blood cells under the microscope, and count the malaria parasite content in each milliliter of mouse blood The number of erythrocytes infected by the protozoa, and finally the experimental model animals were inoculated with ¹ ×107 parasitized erythrocytes intraperitoneally. After the inoculation of the protozoa, D0-D3 was administered continuously for 4 days, and the mice were administered intragastrically once a day, with a volume of 0.2 mL, D4 Quantitative blood was taken from animals in each group, coated with a thin blood film, and fixed with methanol. The test was divided into administration groups with different doses of Plasmodium-infected animals (0.16mg/kg, 0.26mg/kg, 0.43mg/kg, 0.72mg/kg, 1.2mg/kg, 2mg/kg fusine and 0.16mg/kg, 0.26 mg/kg, 0.43mg/kg, 0.72mg/kg, 1.2mg/kg, 2mg/kg bispine salt) and the control group (0mg/kg) of the Plasmodium infection model without administration, 10 mice in each group, respectively Gierma staining was performed, and the malaria parasites were examined under a light microscope at a magnification of 1000 times.

Table 2 Comparison of antimalarial potency of halosine and halosine salt

The results of the antimalarial drug efficacy experiment show that the antimalarial potency of halosine hydrochloride is significantly improved compared with halosine because of its improved stability, and the number of effective groups converted into ineffective groups is greatly reduced. .

Through a simple and reliable preparation process, the present invention obtains halosine hydrochloride whose stability and water solubility are greatly increased compared with halosine, which is conducive to being absorbed by the body and greatly increasing the antimalarial activity of halosine. Therefore, it is expected to be developed into New antimalarial drugs.

Claims (9)

1. A halosine hydrochloride. 2. the preparation method of halophylline hydrochloride according to claim 1, it comprises that hydrochloric acid gas is passed in the ethanolic solution of the total alkaloids of hilophyllum, and the step of recrystallization with ethanol. 3. The preparation method according to claim 2, wherein the ethanol solution of the total alkaloids of Changshan is the absolute ethanol solution of the total alkaloids of Changshan. 4. The preparation method according to claim 2 or 3, wherein 50%-100% ethanol is used for recrystallization. 5. A pharmaceutical composition, which contains a therapeutically effective amount of holosine hydrochloride according to claim 1 and one or more pharmaceutically acceptable carriers. 6. hemosine hydrochloride according to claim 1 or the pharmaceutical composition according to claim 5 are in the medicine that prevents or treats the disease that is caused by Plasmodium, chicken coccidia, Giardia lamblia the use of. 7. The use according to claim 6, wherein the Plasmodium is a multi-drug resistant strain. 8. The use according to claim 6 or 7, wherein the disease caused by Plasmodium is malaria. 9. hemosine hydrochloride according to claim 1 or the pharmaceutical composition according to claim 5 are used in the preparation of medicine, and the medicine is used for anti-inflammatory, antibacterial, antiviral, antitumor, antiarrhythmic abnormality, or hypotension.