CN109288895B

CN109288895B - Preparation method of pharmaceutical composition

Info

Publication number: CN109288895B
Application number: CN201811300777.4A
Authority: CN
Inventors: 屈长青; 周忍; 葛传慧; 岳少云
Original assignee: Fuyang Normal University
Current assignee: Fuyang Normal University
Priority date: 2018-11-02
Filing date: 2018-11-02
Publication date: 2021-08-03
Anticipated expiration: 2038-11-02
Also published as: CN109288895A

Abstract

The invention discloses a preparation method of a pharmaceutical composition, which comprises the following steps: extracting the cedrela sinensis in ethanol under the assistance of ultrasonic waves to obtain a cedrela sinensis extraction mixed solution; volatilizing the Chinese toon extraction mixed liquor until no ethanol smell exists, centrifuging, and taking precipitate to obtain a crude Chinese toon extract product; preparing the crude product of the Chinese toon extract into a diluent of the crude extract of the Chinese toon by using water; passing the diluted solution of the crude extract of Toona sinensis through a macroporous resin column; washing the macroporous resin column with distilled water until the effluent liquid is colorless; eluting the macroporous resin column with ethanol until the effluent liquid is colorless, and collecting the eluate; volatilizing and concentrating the eluent until no alcohol smell exists to obtain a pure Chinese toon extract product; freeze-drying the pure Chinese toon extract product to obtain pure Chinese toon extract product powder; mixing the Toona sinensis extract with curcumin to obtain the pharmaceutical composition. The pharmaceutical composition prepared by the method can effectively reduce the damage of cisplatin to organisms, increase the content of glutathione in vivo and reduce the content of malondialdehyde in vivo.

Description

Preparation method of pharmaceutical composition

Technical Field

The invention belongs to the field of medicine, relates to a preparation method of a pharmaceutical composition, and particularly relates to a preparation method of a pharmaceutical composition containing a toona sinensis extract and curcumin.

Background

Cisplatin is one of the most commonly used tumor drugs at present due to its characteristics of broad anticancer spectrum, strong action and capability of acting with multiple antitumor drugs. However, cisplatin has no specificity to cell killing, so that cisplatin can damage normal cells while being used for treating cancer, and generates strong toxic and side effects, which greatly limits the clinical application of cisplatin. Micronucleus (MN) assay, a conventional genotoxicity test, has been widely used. The specimens they detect also extend from bone marrow to blood and tissues. Besides the conventional drug genotoxicity detection, the kit plays an important role in the diagnosis, the evaluation and the prevention of the curative effect and the like of gene modification diseases^[2]。

Chinese toon seed can be called asThe toona sinensis flowers and toona sinensis bells are fruits of toona sinensis. Researches show that the toona sinensis seeds contain multiple active ingredients such as polyphenol, volatile oil and alkaloid and have strong effects of reducing blood sugar and the like. Curcumin (Curcumin) is a natural phenolic pigment extracted from Curcuma rhizome (curculin longa) of Curcuma of Zingiberaceae, and has no toxicity and harm^[6]. Researches show that the curcumin not only has excellent colorability, but also has various nutritional activities and pharmacological functions, including antioxidation, hypolipidemic, immunity enhancement and the like^[5]。

Reference documents:

[1]Noha Ibrahim Said Salem,Magda Mohammad Noshy,Azza Ali Said.Modulatory effect of curcumin against genotoxicity and oxidative stress induced by cisplatin and methotrexate in male mice[J].Food and Chemical Toxicology,2017,105.

[2]Agarwal,Rakhi.,Goel,Sudhir K.,Behari,Jai Raj..(2010).Detoxification and antioxidant effects of curcumin in rats experimentally exposed to mercury.,JAppl Toxicol,30(5),457-68.

[3] the rhodiola rosea extract has the protective effect on renal cell toxicity caused by cis-platinum [ J ] canceration, distortion and mutation, 2018,30(03):204 and 208.

[4] Study on influence factors of cyclophosphamide induced micronuclei in mouse bone marrow cells [ J ] modern preventive medicine 2007(05): 935) 936+941.

[5] Lelianying, study on curcumin anti-tumor effect and mechanism [ J ] contemporary medicine, 2014,20(32):21-22.

[6] Chenjianping, Lilin, Sujianyu, research on antioxidant and antitumor activities of curcumin [ J ] modern food technology, 2014,30(12):11-15+6.

[7] The influence of the medicine shells of the 6 kinds of gelatin capsules on the micronucleus rate of the bone marrow cells of mice [ J ]. Guangdong medicine, 2015,36(13): 1982) and Yao.

Disclosure of Invention

Toona sinensis seed (a. juss.) is a fruit of Toona sinensis of the family meliaceae, and the protective effect of an Extract of Toona Sinensis Seed (ETSS) on cisplatin (Cis-diammineadipoprolinamiclatum, CDDP) inducing bone marrow cytotoxicity in mice was studied. The experiment comprises the steps of extracting a toona sinensis seed extract to pretreat a mouse, taking the toona sinensis seed extract as an intervener through an in-vivo experiment, simultaneously adopting Curcumin (Curcumin, CMN) as a positive control, carrying out intraperitoneal injection on the toona sinensis seed extract for three consecutive days, then carrying out cisplatin induction treatment, killing the mouse after 24 hours, taking bone marrow cells of the mouse, comparing the micronucleus rate of each group with biochemical index values of kidney tissues GSH and MDA, and researching the protection effect of the toona sinensis seed extract on the cisplatin-induced mouse cytotoxicity. The results show that compared with the cisplatin model group, the micronucleus rates of the toona sinensis extract group, the curcumin group and the toona sinensis extract and curcumin combined group are remarkably reduced, the GSH content is remarkably increased, and the MDA content is remarkably reduced (p <0.05 or p < 0.01). And (4) conclusion: the cedrela sinensis seed extract has a protective effect on cisplatin-induced mouse bone marrow cell and kidney cell toxicity, can reduce the in-vivo oxidative stress degree, improves the self antioxidant capacity, and has a more obvious effect of combining curcumin.

The application provides a preparation method of a pharmaceutical composition, which comprises the following steps:

mixing the cedrela sinensis extract with curcumin to obtain the pharmaceutical composition.

In some embodiments, the step of preparing the toona sinensis extract comprises:

(a) extracting the cedrela sinensis in ethanol under the assistance of ultrasonic waves to obtain a cedrela sinensis extraction mixed solution;

(b) volatilizing the Chinese toon extraction mixed liquor until no ethanol smell exists, centrifuging, and taking precipitate to obtain a crude Chinese toon extract product.

In some embodiments, the step of preparing the toona sinensis extract further comprises:

(c) preparing the crude cedrela sinensis extract into a diluted solution of the crude cedrela sinensis extract by using water;

(d) passing the diluent of the crude extract of the Chinese toon through a macroporous resin column;

(e) washing the macroporous resin column with distilled water until the effluent liquid is colorless;

(f) eluting the macroporous resin column with ethanol until the effluent liquid is colorless, and collecting the eluent;

(g) and volatilizing and concentrating the eluent until no alcohol smell exists to obtain the pure cedrela sinensis extract product.

(h) and freeze-drying the pure Chinese toon extract product to obtain pure Chinese toon extract product powder.

In some embodiments, in step (a), the ethanol concentration used is 60-80%, preferably 70%; and/or

In step (a), the temperature of the ultrasonic-assisted extraction is 65-75 ℃, preferably 70 ℃; and/or

In the step (a), the power of the ultrasonic-assisted extraction is 200-; and/or

In step (a), the time for ultrasonic-assisted extraction is 30-120min, preferably 60min, 45 min; and/or

In step (a), the ratio of the extraction solution to the extraction solution in the ultrasonic-assisted extraction is 1:10-50, preferably 1:30 or 1: 25; and/or

In the step (a), the Chinese toon is extracted in ethanol by ultrasonic wave assistance for two times or more, and the obtained Chinese toon extraction mixed liquor is mixed and used; and/or

In step (b), the speed of the centrifugation is 3500-; and/or

In step (b), the time of the centrifugation is 10-30min, preferably 20 min; and/or

In step (c), the weight ratio of the water to the toona sinensis raw material used in step (a) is 1:2-10, preferably 1: 5; and/or

In step (d), the macroporous resin is AB-8 macroporous resin; and/or

Before performing step (d), soaking 12-36 hr packed column with 90-98% ethanol, washing with water until no alcohol smell, preferably soaking 24 hr packed column with 95% ethanol, washing with 95% ethanol, and washing with water until no alcohol smell; and/or

In step (d), passing the dilution of crude extract of Toona sinensis seed through the macroporous resin column at a rate of 5-10mL/min, preferably 8 mL/min; and/or

In step (f), the ethanol is used in a concentration of 60 to 80%, preferably 70%; and/or

Freezing the purified product of Toona sinensis extract in a refrigerator at-80 ℃ for 5-10 hours, preferably 8 hours, prior to step (h); and/or

Putting the pure Chinese toon extract product into a freeze dryer for 12-36 hours, preferably 24 hours to obtain Chinese toon extract powder; and/or

The cedrela sinensis is selected from cedrela sinensis seeds; and/or

The Toona sinensis is in powder form.

In some embodiments, a pharmaceutically acceptable carrier is added to the mixture of the cedrela sinensis extract and the curcumin.

In some embodiments, the weight ratio of the toona sinensis extract to the curcumin is 1:0.01 to 100, preferably selected from 1:0.1 to 1:10, 1:0.2 to 1:5 and 1:0.5 to 1:2, more preferably 1: 1.

The pharmaceutical composition prepared by the method can effectively reduce the damage of cisplatin to organisms, increase the content of glutathione in vivo and reduce the content of malondialdehyde in vivo.

Drawings

FIG. 1 is a photograph of a blank control bone marrow cell micronucleus staining;

FIG. 2 is a photograph of the micronucleus staining of the marrow cells treated with the Toona sinensis seed extract;

FIG. 3 is a photograph of micronucleus staining of curcumin-treated bone marrow cells;

FIG. 4 is a photograph of micronucleus staining of Toona sinensis seed extract + curcumin treated bone marrow cells;

FIG. 5 is a photograph of the micronucleus staining of Toona sinensis seed extract + cisplatin extract treated bone marrow cells;

FIG. 6 is a photograph of micronucleus staining of curcumin + cisplatin-treated bone marrow cells;

FIG. 7 is a photograph of micronucleus staining of Toona sinensis seed extract + curcumin + cisplatin-treated bone marrow cells;

FIG. 8 is a photograph of micronucleus staining of cisplatin-treated bone marrow cells.

Detailed Description

In order to better explain the technical scheme of the invention, the following detailed description of the embodiment of the invention is combined with the accompanying drawings. The following examples are intended to further illustrate the invention but should not be construed as being limitations or restrictive thereon. Unless otherwise specified, technical features used in the embodiments may be replaced with other technical features known in the art having equivalent or similar functions or effects without departing from the inventive concept.

1 materials and methods

1.1 materials

Chinese toon seeds purchased in the Anhui Bozhou medicinal material market; 80 SPF-level Kunming adult male mice are 6-8 weeks old and 25-30 g in weight, and are freely eaten and drunk by the anti-aging Chinese herbal medicine engineering center of Anhui province.

1.2 instruments and reagents

The instrument comprises the following steps: motic BA400 type biological microscope (with digital camera and image analysis system), traditional Chinese medicine grinder (Yangxi hardware medical apparatus factory of Yongkang city, Zhejiang), electronic balance (Shanghai Aohaus apparatus Co., Ltd.), desk centrifuge (Shanghai An Ting scientific apparatus factory), constant temperature water bath (Beijing Oriental Jingrui science and technology development Co., Ltd.), enzyme labeling instrument (Thermo), freeze drying machine (SIM International Group), drying box (Shanghai Jingjinhong experiment equipment Co., Ltd), -80 ℃ refrigerator (Thermo), ultrapure water for experiment prepared by Millipore ultrapure water machine, and ultrasonic instrument (Kunshan ultrasonic apparatus Co., Ltd.).

Reagent: giemsa pigment, cisplatin (CDDP, lyophilized) were obtained from Qilu pharmaceutical (Hainan) Inc. (batch number: 2WA2A1307047B,20 mg/bottle), curcumin (Biyuntian Biotech Co., Ltd., product number SC0299-25mg), GSH kit and MDA kit, all purchased from Nanjing institute of bioengineering.

1.3 methods

1.3.1 extraction of Toona sinensis seed extract

Weighing 20g of Chinese toon seed powder in a beaker, performing ultrasonic assisted extraction (70 ℃,320W) by 70% ethanol twice, wherein the extraction material liquid ratio is 1:30 and 1:25 respectively, the extraction time is 60min and 45min respectively, merging the filtrate, volatilizing and concentrating until no ethanol smell exists, centrifuging at 4000r/min for 20min, taking the precipitate, namely the crude product of the Chinese toon extract, and adding 100mL of distilled water for dilution for later use, thus obtaining the diluent of the crude extract of the Chinese toon seed.

Taking 100g of AB-8 macroporous resin, firstly soaking the AB-8 macroporous resin in 95% ethanol for 24 hours, then loading the AB-8 macroporous resin into a column, finally washing the column with 95% ethanol until no alcohol smell exists, enabling the diluent of the toona sinensis fruit crude extract to pass through an AB-8 macroporous adsorption resin column at the speed of 8mL/min, firstly washing the column with distilled water after adsorption is finished, washing off redundant pigments and impurities until an effluent liquid is colorless, then eluting the column with 70% ethanol until the effluent liquid is colorless, finally volatilizing and concentrating the eluent until no alcohol smell exists, freezing the eluate for 8 hours in a refrigerator at the temperature of minus 80 ℃, and then placing the eluate in a freeze dryer for 24 hours to obtain toona sinensis fruit extract powder, namely a pure product of the toona sinensis fruit extract, and storing the toona sinensis fruit extract in the refrigerator at the temperature of 4 ℃ for later use.

The following tests were carried out on the Toona sinensis seed extract using the pure Toona sinensis seed extract.

1.3.2 toxicity preliminary experiments of Cedrela sinensis seed extract and curcumin injected into abdominal cavity of mice

Taking 32 male mice, and randomly dividing the mice into 4 groups, wherein the grouping condition is (1) blank control group; (2) a Toona sinensis seed extract group; (3) curcumin group; (4) toona sinensis seed extract group and curcumin combined group. After 1 week of acclimation to feeding, each group was treated as follows:

(1) group was given intraperitoneal injection of physiological saline 0.1ml/kg bw for 3 consecutive days; (2) group was administered with 150mg/kg bw of Cedrela sinensis extract for 3 consecutive days; (3) group was given intraperitoneal injections of curcumin 150mg/kg bw for 3 consecutive days; (4) the group was administered with the intraperitoneal injection of cedrela sinensis seed extract and curcumin at 150mg/kg bw (ratio of cedrela sinensis seed extract to curcumin was 1: 1) for 3 consecutive days. The intraperitoneal injection of each group is performed once a day.

Each mouse is killed after the last administration for 24 hours, femurs on both sides are taken out, bone marrow is washed by PBS (phosphate buffer solution) with the pH value of 6.8, bone marrow cells are taken out and are respectively used for the following micronucleus test, kidney tissues are taken to be prepared into 10% homogenate for standby, and the content of Malondialdehyde (MDA) and Glutathione (GSH) in the kidney tissues is measured.

1.3.3 Effect of Abdominal injection of Toona sinensis extract and curcumin in combination with cisplatin on mice

The male mice with 32 mice are divided into 4 groups randomly, and the groups are (1) a compound treatment group of the cedrela sinensis extract and cisplatin; (2) curcumin and cisplatin composite treatment group; (3) toona sinensis extract, curcumin combination and cisplatin treatment group; (4) cisplatin model group.

After 1 week of parallel acclimation feeding, each group was treated as follows:

(1) after the Chinese toon seed extract is injected into the abdominal cavity for 150mg/kg bw continuously for 3 days, cisplatin 6.5mg/kg bw is injected into the abdominal cavity once; (2) after the curcumin is injected into the abdominal cavity for 3 consecutive days and 150mg/kg bw of the curcumin is injected into the abdominal cavity, 6.5mg/kg bw of cisplatin is injected into the abdominal cavity once; (3) the group is continuously administered with 150mg/kgbw of the toona sinensis extract and the curcumin in an intraperitoneal injection way (the ratio of the toona sinensis extract to the curcumin is 1: 1) for 3 days, and then cisplatin is injected into the peritoneal cavity for 6.5mg/kg bw once; (4) group was given a single intraperitoneal injection of cisplatin 6.5mg/kg bw after intraperitoneal injection of physiological saline (0.9% sodium chloride solution) for 3 consecutive days. The intraperitoneal injection of each group is performed once a day.

1.4 micronucleus test

Taking a proper amount of bone marrow in a concave hole porcelain plate, uniformly mixing with inactivated fetal calf serum, dropwise adding the mixture to a glass slide to push a slide, and naturally drying. 2 pieces were made for each specimen. And (3) putting the push sheet into methanol for fixing for 10min, taking out the push sheet, putting the push sheet into Giemsa dye liquor for dyeing for 15-30 min, washing with pure water, and drying in the air. The area with intact cells, uniform smear and appropriate staining was selected and observed under an oil-scope. Observing under a low power microscope, selecting an area with uniform distribution and good dyeing, and observing and counting under an oil microscope. Polychromophil erythrocytes (PCE) are grayish blue, and one or more micronucleus may appear in one cell^[7]. PCE with multiple microkernels is calculated according to one calculation, the number of PCE (MnPCEs/1000PCEs) with microkernels in 1000PCE is counted for each mouse, andpercentage of PCE with microkernels (% MNPCEs).

1.5 statistical methods

Analysis was performed using SPSS 12.0 statistical software. The count data was checked by chi-square and the metric data was compared by analysis of variance. The difference is significant when P is less than 0.05, and the difference is significant when P is less than 0.01.

2 results of the experiment

2.1 Effect of Toona sinensis seed extract and curcumin on micronucleus rate of mouse bone marrow cells

The PCE micronucleus rate of normal mice is less than 5 per mill, and if the micronucleus rate is more than 5 per mill, the mice are positive^[4]. Regarding the toxicity of the medicine, according to the relevant regulations of Chinese herbal medicine preparations, each Chinese medicine is recorded in more than two ancient books without toxicity, so that the Chinese herbal medicine is regarded as non-toxic, and the Zingiberaceae plant and the Chinese toon of the single Chinese medicine selected in the experiment are not recorded with toxicity in the documents of materia medica, materia medica compendium and bibliography, which shows that the selected medicine is safe and reliable^[3]. As can be seen from table 1, compared with the blank control group, the increase of micronucleus rate is not caused by the simple injection of curcumin or toona sinensis seed extract, no significant difference is generated, and the average value of each group is less than 5 per thousand, which is negative. The Chinese toon seed extract and curcumin do not produce toxic effect on mice. FIGS. 1 to 4 show the micronucleus staining of partial bone marrow cells in each group, indicating that the micronucleus is present at a very low or even no micronucleus, and FIGS. 1 to 4 show photographs of each group of PCE micronucleus (Giemsa,. times.1000 oil-glasses).

TABLE 1 influence of Toona sinensis seed extract, curcumin on micronucleus rate of mouse bone marrow cells

2.2 influence of Toona sinensis seed extract and curcumin on cisplatin-induced micronucleus rate of mouse bone marrow cells

As shown in Table 2, compared with the blank control group, the micronucleus rate of the cisplatin model group is significantly increased, and if the micronucleus rate is greater than 5 per mill, the micronucleus rate is positive, and the difference is very significant (P < 0.01). The other administration groups also show positive micronucleus rates with different degrees, which indicates that a certain amount of cisplatin can induce mice to generate cytotoxicity. However, compared with the cisplatin model group, the micronucleus rate of the mice treated by the cedrela sinensis seed extract and the curcumin is obviously reduced, the difference between the single cedrela sinensis seed extract and the curcumin treated group is obvious (P <0.05), and the difference between the cedrela sinensis seed extract and the curcumin treated in a combined way is extremely obvious (P < 0.01). The results show that mice treated by the toona sinensis seed extract and the curcumin have a certain protection effect on cisplatin-induced cytotoxicity, and the combined treatment effect of the toona sinensis seed extract and the curcumin is better than that of single treatment. FIGS. 5 to 8 are photographs showing the micronuclear staining of each group of bone marrow cells.

TABLE 2 influence of Toona sinensis seed extract and curcumin on cisplatin-induced micronucleus rate of mouse bone marrow cells

Note: p <0.05, P <0.01 compared to control; compared with the model group, # P <0.05, # P < 0.01. The arrows indicate the micro-cores, and each set of PCE micro-cores (Giemsa, x 1000 oil mirror) in FIGS. 5-8

Combining the results of section 2.1 and section 2.2, it can be known that the combination of the toona sinensis seed extract and the curcumin or the combination of the toona sinensis seed extract and the curcumin has no obvious influence on the proportion of micro-nuclei in the bone marrow tissue of the common mouse in PCE, which indicates that the physiological function of the healthy mouse is slightly influenced. And in cisplatin-treated mouse bone marrow tissues, micronucleus content can be significantly reduced. Therefore, the combination of the toona sinensis seed extract and the turmeric or the combination of the toona sinensis seed extract and the turmeric can be at least used for preventing, treating or relieving the damage of the mouse body caused by the cisplatin.

2.3 Effect of Toona sinensis seed extract and curcumin on GSH and MDA content in mouse kidney tissue

As can be seen from table 3, compared to the blank control group, the injection of curcumin or toona sinensis seed extract alone did not result in abnormal GSH and MDA contents, and there was no significant difference compared to the control group. Further proves that the cedrela sinensis seed extract and the curcumin do not produce toxic effect on mice.

TABLE 3 influence of Toona sinensis seed extract, curcumin on mouse kidney tissue GSH, MDA content

2.4 influence of Toona sinensis seed extract and curcumin on cisplatin-induced GSH (glutathione) and MDA (mean serum cholesterol) values of kidney tissues of mice

As can be seen from table 4, compared to the blank control group, the cisplatin model group had significantly reduced GSH content, significantly increased MDA content, and very significant difference (P < 0.01). Compared with a cisplatin model group, the mouse has the advantages that the GSH content of the mouse is obviously increased after the mouse is treated by the cedrela sinensis seed extract and the curcumin, the difference between the independent cedrela sinensis seed extract and the curcumin treatment group is obvious (P is less than 0.05), and the difference between the cedrela sinensis seed extract and the curcumin combined treatment is extremely obvious (P is less than 0.01); the MDA content of the mice treated by the Chinese toon seed extract and the curcumin is obviously reduced, the difference between the single Chinese toon seed extract and the curcumin treated group is obvious (P <0.05), and the difference between the Chinese toon seed extract and the curcumin combined treatment is extremely obvious (P < 0.01). The mouse treated by the toona sinensis seed extract and the curcumin has a certain protection effect on cisplatin-induced cytotoxicity, nephrotoxicity and renal injury, and the combined treatment of the toona sinensis seed extract and the curcumin has a better effect than the single treatment, so that the toona sinensis seed extract and the curcumin can generate a synergistic effect.

TABLE 4 Toona sinensis seed extract, curcumin content in cisplatin-induced mouse kidney tissue GSH, MDA

Note: p <0.05, P <0.01 compared to control; compared with the model group, # P <0.05, # P < 0.01.

Combining the results of the sections 2.3 and 2.4, it can be known that the combination of the toona sinensis seed extract and the curcumin or the combination of the toona sinensis seed extract and the curcumin has no obvious influence on the content of GSH and MDA in the kidney tissue of the common mouse, which indicates that the physiological function of the healthy mouse is slightly influenced. In cisplatin-treated mouse kidney tissues, the GSH content was significantly increased and the MDA content was significantly decreased.

The significant increase of the content of the GSH can activate or improve a plurality of biochemical reactions which depend on the GSH, such as detoxification, oxidation resistance and the like and play a role in protecting the organism. The MDA content is obviously reduced, so that the harm of MDA to the cross-linking denaturation of biomacromolecules such as promotion protein and the like of an organism can be reduced.

Therefore, the combination of the toona sinensis seed extract and the turmeric or the combination of the toona sinensis seed extract and the turmeric can be at least used for preventing, treating or relieving the damage of the mouse body caused by the cisplatin.

Discussion of 3

Research has shown that oxidative stress is one of the major mechanisms of cisplatin toxicity, and that Reactive Oxygen Species (ROS) -mediated oxidative stress plays an important role in cisplatin toxicity. Cisplatin is mainly excreted through the kidney in vivo, and metal platinum ions can be accumulated in the kidney, so that the nephrotoxicity is the most obvious. Cisplatin is toxic to different parts of the kidney, and can cause glomerular and tubular functions and morphological changes. Cisplatin can interact with an antioxidant system to influence the antioxidant system and an antioxidant enzyme system, so that different tissues are subjected to oxidative damage, and the degree of oxidative stress reaction and the degree of tissue structure loss are in an obvious positive correlation.

The micronucleus test of mouse bone marrow cells is considered the first method to perform in vivo genotoxicity test for detecting the damage of chromosome breaking agent and aneuploid inducer to chromosome^[1]. The micronucleus occurrence rate of the bone marrow PCE is a very useful index for detecting the genetic damage of cells, and all genetic damage substances which can break chromosomes or damage the connection of the chromosomes and spindles can be detected by micronucleus tests.

Researches show that the curcumin structure contains phenolic hydroxyl, and the phenolic hydroxyl can generate oxidation reaction and effectively stop free radical reaction when lipid peroxidation reaction occurs on cell membranes^[1]Thus it shows various physiological activities such as antioxidant, antitumor, anti-aging, etc^[6]Has become a hot research point in natural medicines in recent years. The protective effect of the toona sinensis seed extract on cytotoxicity caused by cisplatin may be related to the antioxidant and free radical scavenging activities of the toona sinensis antioxidant components.

The experiment takes the bone marrow cells of a mouse as a research object, establishes a CDDP induced mouse oxidative damage model, proves the protective effect of the toona sinensis seed extract and the curcumin on cytotoxicity, further proves that the toona sinensis seed extract and the curcumin have a synergistic effect according to the experimental result, and provides a new thought and resource for developing a new natural harmless antioxidant.

The research result of the test shows that the combination administration of two natural plant phenol active ingredients, namely the toona sinensis seed extract and the curcumin, can generate a synergistic effect and can effectively reduce the cytotoxicity induced by the cisplatin and the genetic damage of mouse somatic cells. The Chinese toon seed extract is preliminarily judged to be capable of antagonizing mouse chromosome mutation caused by cisplatin, and the Chinese toon seed extract and curcumin have a synergistic effect. In conclusion, the toona sinensis seed extract with a certain concentration can obviously improve the in-vivo oxidation resistance of mice and has good regulation effect on the genotoxicity caused by cisplatin anticancer drugs.

The above embodiments are only used for further illustration of the present invention, and are not intended to limit the scope of the present invention, and all equivalent changes made based on the concept of the present invention and obvious modifications of various technical solutions of the present invention fall within the scope of the present invention.

Claims

1. A process for the preparation of a pharmaceutical composition for reducing cisplatin-induced genetic damage, said process comprising the steps of: mixing the cedrela sinensis extract with curcumin to obtain the pharmaceutical composition;

wherein the toona sinensis extract is from toona sinensis seeds, and the weight ratio of the toona sinensis extract to the curcumin is 1: 0.01-100;

the preparation method of the Chinese toon extract comprises the following steps: (a) extracting the cedrela sinensis in ethanol under the assistance of ultrasonic waves to obtain a cedrela sinensis extraction mixed solution; (b) volatilizing the Chinese toon extraction mixed solution until no ethanol smell exists, centrifuging, and taking precipitate to obtain a crude Chinese toon extract product; (c) preparing the crude cedrela sinensis extract into a diluted solution of the crude cedrela sinensis extract by using water; (d) passing the diluent of the crude extract of the Chinese toon through a macroporous resin column; (e) washing the macroporous resin column with distilled water until the effluent liquid is colorless; (f) eluting the macroporous resin column with ethanol until the effluent liquid is colorless, and collecting the eluent; (g) volatilizing and concentrating the eluent until no alcohol smell exists to obtain a pure cedrela sinensis extract product; (h) freeze-drying the pure Chinese toon extract product to obtain pure Chinese toon extract product powder;

wherein, in the step (a), the concentration of the ethanol is 60-80%; in step (a), the temperature of the ultrasonic-assisted extraction is 65-75 ℃; in the step (a), the power of the ultrasonic-assisted extraction is 200-; in the step (a), the time of ultrasonic-assisted extraction is 30-120 min; in the step (a), the ratio of the extracting materials to the extracting solutions extracted by the ultrasonic auxiliary extraction is 1: 10-50; in the step (a), the Chinese toon is extracted in ethanol by ultrasonic wave assistance for two times or more, and the obtained Chinese toon extraction mixed liquor is mixed and used; in step (b), the speed of the centrifugation is 3500-; in step (b), the time of centrifugation is 10-30 min; in the step (c), the weight ratio of the water to the toona sinensis raw material used in the step (a) is 1: 2-10; in step (d), the macroporous resin is AB-8 macroporous resin; before step (d), soaking the column in 90-98% ethanol for 12-36 hr, washing with 90-98% ethanol, and washing with water until there is no alcohol smell; in the step (d), enabling the diluent of the crude extract of the toona sinensis seeds to pass through the macroporous resin column at the speed of 5-10 mL/min; in step (f), the concentration of the ethanol is 60-80%; before the step (h), freezing the pure Chinese toon extract product in a refrigerator at-80 ℃ for 5-10 hours; putting the pure Chinese toon extract product into a freeze dryer for 12-36 hours to obtain Chinese toon extract powder; the Toona sinensis is in powder form.

2. The process for the preparation of a pharmaceutical composition according to claim 1, wherein a pharmaceutically acceptable carrier is added to the mixture of the cedrela sinensis extract and the curcumin.

3. A process for the preparation of a pharmaceutical composition as claimed in claim 1 or 2, wherein the weight ratio of the cedrela sinensis extract to the curcumin is 1:0.1-1:10, 1:0.2-1:5, 1:0.5-1:2 or 1: 1.