CN106344511B

CN106344511B - Nano liushen pill solution and its preparation method

Info

Publication number: CN106344511B
Application number: CN201610859484.4A
Authority: CN
Inventors: 李玉桑; 陈夕桢; 唐和斌
Original assignee: South Central University for Nationalities; Shenzhen Huazhong University of Science and Technology Research Institute
Current assignee: Shenzhen Huazhong University of Science and Technology Research Institute; South Central Minzu University
Priority date: 2016-09-28
Filing date: 2016-09-28
Publication date: 2020-02-18
Anticipated expiration: 2036-09-28
Also published as: CN106344511A

Abstract

The invention discloses a nanometer Liushen pill solution and a preparation method thereof, the nanometer Liushen pill solution is a nanometer Liushen pill alcohol water-in-water oil preparation, and comprises a high-dose nanometer Liushen pill alcohol-in-water oil preparation or a low-dose nanometer Liushen pill alcohol-in-water oil preparation; the nano Liushen pill solution has a good prevention and treatment effect on liver cancer induced by chemical toxicants, obvious tumor nodules can be seen by naked eyes in liver tissues of 4 (50%) mice in a nano DEN group, obvious tumor nodules can be seen by naked eyes in liver tissues of 3 (37.5%) mice in a high-dose nano Liushen pill group, no obvious tumor nodules can be seen by naked eyes in liver tissues of the mice in a low-dose nano Liushen pill group, and the HE staining result shows that moderate disorder occurs in liver tissue structures of the mice in the nano DEN group, so that the disorder degree of the liver tissue structures of the mice treated by the high-dose and low-dose nano Liushen pill solution is relatively low.

Description

Nano liushen pill solution and its preparation method

Technical Field

The invention belongs to the technical field of biology, and relates to a nano liushen pill solution and a preparation method thereof.

Background

The death rate of primary liver cancer is high, the death order of malignant tumor is second to stomach cancer and esophagus cancer, and the disease condition is rapid, and the life health of human beings is seriously damaged (Hepatocellular cancer. Recentri ProgMed, 2016, 107 (7): 386-394). Most patients with liver cancer suffer from not only malignant diseases but also chronic liver diseases, have poor prognosis and cannot improve the quality of life (Nutrition and hepatocellular carcinoma. gastroenterest turbines, 2016, 2 (4): 188-94). The incidence of liver cancer is The first in China, about 38.3 thousands of deaths per year, which accounts for about 51% of The total number of deaths of liver cancer (The global burden of liver cancer: The major impact of China, hepatology, 2014, 60 (6): 2099-. Therefore, the search for effective means for preventing and treating liver cancer is always the focus of clinical research.

There are many factors to be considered in treating primary liver cancer, such as the number of tumors, the size of tumor volume, whether there is tumor spread, how well the liver function is stored, the age of the patient and whether there are complications, etc., and the existing treatment methods can be divided into surgical treatment, local treatment and traditional Chinese medicine treatment. The current clinical treatment scheme of primary liver cancer is surgical resection, which can completely remove the focus, but has narrow indications and is not suitable for patients with middle and late stage liver dysfunction. Liver transplantation is the only means for patients who cannot be surgically treated radically, especially for patients with severe cirrhosis, but patient selection criteria are strict, liver source is in short supply and is accompanied by high risk of recurrence and metastasis. The local treatment comprises methods such as selective hepatic artery chemoembolization (TACE), local ablation treatment, radiotherapy, biological treatment and the like, has the advantages of simple and convenient operation, few complications, strong repeatability and the like, but has limitations, and the selective hepatic artery chemoembolization can achieve the treatment purpose after being repeated for many times; the application range of local ablation treatment is narrow; radiotherapy equipment is expensive and treatment cost is high; biological treatment seems to be good and promising at present, but the technology is still immature and has some potential risks (new progress of primary liver cancer treatment research, J.Utility medicine, 2016, 14: 2275-.

The traditional Chinese medicine treats primary liver cancer by taking the treatment principle of simultaneous treatment of elimination and reinforcement and strengthening body resistance to eliminate pathogenic factors and the treatment method of detoxifying, removing blood stasis and nourishing liver and kidney. Although the mechanism is unknown, many traditional Chinese medicine preparations show potential value in improving the life quality of patients, prolonging the life cycle of patients, and the like, such as bufadienolides (arenobufaginoidaterials with DNA leading to G2 cell cycle arm ATM/ATR pathway, oncotarget.2015, 6 (33): 34258-75). At the present stage, most of the traditional Chinese medicine preparations are used for clinical assistance, treatment is carried out in a mode of combining traditional Chinese medicine and western medicine, and the traditional Chinese medicine preparation is beneficial to patients in all periods.

Pain is a painful sensation caused by tissue damage or potential injury. It is a complex physiological and psychological activity and is one of the common symptoms of many diseases in clinic. Pain can be used as an early warning to the body, causing a series of defensive protections. On the other hand, however, pain has its limitations as a warning (e.g. when cancer is painful, it is already late). Some long-term severe pain is an intolerable affliction to the body, and the life quality of patients is poor. At present, non-steroidal anti-inflammatory drugs and opioid analgesics are mainly used for treating pain clinically, and although the curative effect of the drugs is remarkable, adverse reactions such as peptic ulcer and addiction occur after long-term use. Therefore, the search for more effective and less toxic analgesics is urgent.

In the early stage of research, the traditional Chinese medicine Liushen pill with the functions of clearing away heat and toxic material, softening hardness and dissipating stagnation, removing blood stasis and relieving pain is found to have good anticancer activity and analgesic effect (invasion of substention P/nookinin-1 receptor in the anagesic and anticancer activities of minor top-filtration from the traditional Chinese medicine Liu-Shen-wain vision. biol phase Bull.2014, 37) (431 and 438; Liushen pill in vitro anti-liver cancer activity research, Chinese hospital pharmacy 2014, 34(19), 1627 and 1630). The reason is considered that the good anticancer and analgesic effects of the Liushen pills can be attributed to: bezoar has the effects of clearing heat and removing toxicity, relieving swelling and dissipating stagnation, musk is pungent and fragrant and disperses, and blood circulation promoting and dissipating stagnation is used as monarch drugs, so that abdominal mass and swelling, stasis and blood stasis dissipating, abdominal stasis and stasis eliminating, carbuncle pertinacious furunculosis treating and cancer swelling phlegm removing; toad venom is used for detoxifying and relieving pain, realgar is used for detoxifying and relieving pain, and both effects of removing heat and toxic materials and dredging are achieved, so that the efficacy of eliminating tumor, detoxifying and relieving pain can be enhanced as ministerial drugs; borneol and pearl are cold in nature, and can clear heat and relieve pain, so the borneol and pearl are used together as adjuvant drugs in a formula, are warm and cool and can be used together to tonify and thoroughly move together, and can enhance the effects of dredging collaterals, dissipating stagnation and removing blood stasis. The medicines are combined to play the effects of reducing phlegm, resolving masses, removing blood stasis, reducing swelling, dredging collaterals, relieving pain, clearing away heat and toxic materials, calming the heart, attacking hard mass, cutting rock and healing sores. The research results can provide new thinking and bedding for the pharmacological application of the traditional Chinese medicine Liushen pill in resisting tumor and easing pain.

The global nanotechnology is rapidly developing, and compared with the traditional medicines, the nanometer material has higher bioavailability, better solubility, targeting property, sustained and controlled release property and the like, so that the medicine effect can be improved, and adverse reactions can be reduced, thus the nanometer material is widely applied to the fields of medicine transmission, gene therapy, cancer therapy and the like (delivery of doxorubicin and decurcumin with lipid nanoparticles in improved efficacy of chemotherapy in cancer. international Journal of Nanomedicine, 2015, 10: 257 and 270). In recent years, a plurality of researches show that the liushen pills have the effects of resisting inflammation, easing pain, resisting tumors and the like (pharmacological research of the liushen pills, Chinese medicine and clinic, 2011, 11 (8): 935) 936; and external anti-liver cancer activity of the liushen pills, Chinese hospital pharmacy, 2014, 34 (19): 11-14), so that the liushen pill solution is designed to be nano, and the liushen pill solution is expected to achieve the effects of slow release and synergy in the aspects of treating primary liver cancer and relieving pain.

Disclosure of Invention

Aiming at the problems of narrow treatment range, high risk, high treatment cost and the like of primary liver cancer indications in the prior art, the invention provides a nano Liushen pill solution which can well prevent and treat liver cancer induced by chemical poisons and a preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a nanometer LIUSHEN pill solution is characterized in that: the solute of the said nanometer LIUSHEN pill solution is LIUSHEN pill alcohol extract freeze-dried powder.

Preferably, the nano Liushen pill solution adopted by the invention is a nano Liushen pill alcohol extract oil-in-water preparation.

Preferably, the nano Liushen pill solution adopted by the invention is a high-dose nano Liushen pill alcohol extract oil-in-water preparation or a low-dose nano Liushen pill alcohol oil-in-water preparation; the content of LIUSHEN pill ethanol extract in the high-dose nano LIUSHEN pill ethanol extract oil-in-water preparation is not more than 24.089 mg/ml^-1(ii) a The content of LIUSHEN pill ethanol extract in the low-dose nano LIUSHEN pill ethanol extract oil-in-water preparation is not more than 4.818 mg/ml^-1。

Preferably, the content of the Liushen pill alcohol extract in the high-dose nano Liushen pill alcohol extract oil-in-water preparation adopted by the invention is 12.045 mg/ml^-1-24.089mg·ml^-1(ii) a The content of LIUSHEN pill ethanol extract in the low-dose nano LIUSHEN pill ethanol extract oil-in-water preparation is 2.409 mg/ml^-1-4.818mg·ml^-1。

A process for preparing a nano liushen pill solution as described above, characterized by: the method comprises the following steps:

1) dissolving the lyophilized powder of the alcohol extract of the Liushen pill and the yolk lecithin in sesame oil to prepare an oil phase; the final concentration of the egg yolk lecithin is 6% W/V;

2) dissolving Tween 80 and glycerol in ultrapure water to prepare a water phase; the final concentration of Tween 80 was 1.25% V/V; the final concentration of glycerol was 2.81% V/V;

3) dripping the oil phase prepared in the step 1) into the water phase prepared in the step 2) under the condition of a constant-temperature water bath at 60 ℃, and stirring for 15min under the condition of 1200 r/min; the volume ratio of the oil phase to the water phase is 1: 4;

4) shearing at 10000rpm for 2min to obtain emulsified particles with particle size of 100-200 nm;

5) homogenizing under high pressure at 1000bar for 6 times to obtain oil-in-water preparation of alcoholic extract of nanometer LIUSHEN pill.

Compared with the prior art, the invention has the following advantages and effects:

1) the nano Liushen pill solution has the advantages of low cost of ① medicaments, strong stability of ② medicaments, convenient administration by ③, less pain, long-term use of ④ medicaments, obvious effect and improved life quality of patients.

2) At present, the non-steroidal anti-inflammatory drugs and opioid receptor agonists are used for clinically treating pain, and are generally administrated in a swallowing or injection mode, side effects such as peptic ulcer and addiction can be generated after long-term administration, the effect is usually not lasting, and the life quality of patients is poor. Experimental research shows that the nano Liushen pill solution can relieve pain, has long action time in vivo, has a slow release effect, small side effect, convenient buccal administration and greatly improved life quality of patients.

Drawings

FIG. 1 is a schematic diagram showing the weight changes of mice in each group at weeks 10, 15, 20 and 30 in the experimental process of preventing and treating liver cancer with the nano liushen pill solution in example 1;

FIG. 2 is an eye photograph of liver flesh of each group of mice at thirty weeks in example 1;

FIG. 3 is a photograph showing HE staining of liver of each group of mice at thirty weeks in example 1;

FIGS. 4 a-4 c are graphs showing the number of writhing times of each group of mice injected with 0.6% V/V acetic acid at different time points in example 2.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific examples. It should be understood that the following should not be taken as limiting the scope of the invention in any way.

The invention concept of the invention is as follows:

primary liver cancer is one of the most common malignant tumors of the digestive system, is found in middle and late stages, has poor prognosis, and is considered to be related to viral hepatitis, cirrhosis, chemical carcinogens such as aflatoxin and diethylnitrosamine and environmental factors at present. The etiology and pathogenesis of the disease are not clear, and the health of human beings is seriously threatened. The means for treating primary liver cancer are surgical treatment, local treatment and Chinese medicine treatment. Surgical treatments include surgical resection, liver transplantation; the local treatment comprises methods of selective hepatic artery chemoembolization, local ablation, hepatic artery intervention, radiotherapy, biotherapy and the like. Surgical or local treatment has limitations, which inevitably damages the normal functions of the body, and most patients with primary liver cancer have basic liver diseases, and with the occurrence and development of diseases, most patients are found to be in middle and late stages, often have liver function abnormality, and cannot receive radical operation treatment. The traditional Chinese medicine emphasizes overall regulation, balance regulation, body resistance strengthening and evil elimination for treating the liver cancer, shows potential values in the aspects of improving symptoms, relieving pain, controlling recurrence and metastasis, prolonging life cycle and the like, and can make up for many defects of western medicine, so that the traditional Chinese medicine is increasingly emphasized by doctors and patients.

The traditional Chinese medicine considers that the primary liver cancer belongs to the categories of diseases such as mass, tympanites, accumulation syndrome, qi and blood hypertrophy, and the like, and is a malignant disease which is formed by spleen dysfunction, body fluid qi and blood circulation imbalance, phlegm coagulation, qi stagnation, heat toxin, damp turbidity and other pathological changes caused by seven-emotion stagnation, yin-yang imbalance, viscera damage and other reasons, is accumulated in viscera, is mutually gambled, and is deposited for a long time (the research progress of the traditional Chinese medicine for treating the primary liver cancer, the journal of clinical hepatobiliary diseases, 2016, 32 (1): 174-177). The Liushen pill contains six medicines of musk, toad venom, borneol, realgar, pearl and bezoar, is a classic traditional Chinese medicine famous prescription, has the effects of clearing away heat and toxic materials and relieving swelling and dispersing stasis, and has better curative effects on sore throat, carbuncle, sore and furuncle, innominate toxic swelling and the like. Researches in recent years show that the Liushen pill also has the effects of resisting inflammation, easing pain, resisting tumors and the like.

The applicant proves that Liushen pills and different extracts and separated components thereof can inhibit the survival of HepG2 cells, wherein alcohol extraction supernatant is the best effective part, and the toxicity of the alcohol extraction supernatant to L02 cells is small, which shows that Liushen pills have the effect of specifically inhibiting the survival of cancer cells and resisting the liver cancer activity in vitro. Meanwhile, early experiments prove that the alcohol extract supernatant of the Liushen pill can reduce the release of P substances in DRG cells of dorsal root ganglia induced by bradykinin, and the Liushen pill has the function of easing pain. Therefore, a large amount of scientific experimental researches on the prevention and treatment of primary liver cancer and the pain relief of the nano liushen pills are carried out in the later period, and the treatment effect of the nano liushen pills on corresponding diseases is researched by establishing a primary liver cancer model and a pain model. The research result shows that the nanometer liushen pill after being administrated can not only have obvious treatment effect on the primary liver cancer induced by the chemical cancer-inducing agent, but also relieve pain.

Based on the theory, the invention provides a nano liushen pill solution, the solute of the nano liushen pill solution is liushen pill alcohol extract freeze-dried powder, and the nano liushen pill solution is nano liushen pill alcohol extract water-in-water oil preparation; wherein the nanometer LIUSHEN pill alcoholic extract oil-in-water preparation is high-dose nanometer LIUSHEN pill alcoholic extract oil-in-water preparation or low-dose nanometer LIUSHEN pill alcoholic extract oil-in-water preparation; the content of LIUSHEN pill ethanol extract in the high-dose nanometer LIUSHEN pill ethanol extract oil-in-water preparation is 12.045 mg/ml^-1-24.089mg·ml^-1(ii) a The content of LIUSHEN pill ethanol extract in the low-dose nanometer LIUSHEN pill ethanol extract oil-in-water preparation is 2.409 mg/ml^-1-4.818mg·ml^-1. The preparation method of the freeze-dried powder of the alcohol extract of the liushen pills comprises the steps of taking 120 Liushen pill particles (purchased from Shanghai Leyun Shang pharmaceutical industry Co., Ltd.) (0.375g), grinding the Liushen pill particles, putting the Liushen pill particles into a 15ml centrifuge tube, adding 20ml of absolute ethanol, soaking for 3h, carrying out ultrasonic treatment for 30min, uniformly mixing the Liushen pill particles and the ultrapure water in a mixer for 10min, standing the mixture at 4 ℃ for overnight, centrifuging the mixture for 15min at 1000rpm, taking supernatant, mixing the obtained supernatant and the ultrapure water according to a volume ratio of 1:1, standing the mixture at-86 ℃ for overnight, and carrying out freeze-drying on the next day to obtain the freeze-.

Meanwhile, the invention also provides a method for preparing the nano liushen pill solution, which comprises the following steps:

1) dissolving the lyophilized powder of the alcohol extract of the Liushen pill and the yolk lecithin in sesame oil to prepare an oil phase; the final concentration of egg yolk lecithin was 6% W/V (g/ml);

3) dripping the oil phase prepared in the step 1) into the water phase prepared in the step 2) under the condition of a constant-temperature water bath at 60 ℃, and stirring for 15min under the condition of 1200 r/min; the volume ratio of the oil phase to the aqueous phase was 1: 4;

The technical scheme provided by the invention is further explained in detail by combining the specific implementation mode as follows:

after one week of adaptive feeding of 128 male kunmin KM mice, they were randomized: four groups, namely a normal group, a DEN group (model group), a nano liushen pill solution low-dose group (administration dose of 1.927mg/kg, administration volume of 4 mu l/10g) and a nano liushen pill solution high-dose group (administration dose of 9.639mg/kg, administration volume of 4 mu l/10g), and 32 mice in each group. The mice in the other three groups except the normal group were given 16.5mg/kg DEN once a week for twenty weeks; meanwhile, the nano Liushen pill solution low-dose group gives the low-dose nano Liushen pill alcohol extract oil-in-water preparation twice per week for buccal administration, and the nano Liushen pill solution high-dose group gives the high-dose nano Liushen pill alcohol extract oil-in-water preparation twice per week for buccal administration for thirty weeks continuously. The prevention and treatment effect of the nano Liushen pill alcohol extract oil-in-water preparation on primary liver cancer is researched.

After 90 male Kunming KM mice are adaptively fed for one week, the mice are randomly divided into three groups, namely 1/3h group, 1h group and 6h group according to time points, and each group comprises 30 mice; each large group was divided randomly into five groups, namely a model group, a morphine treatment group (drug concentration 500. mu.g/ml, administration dose 10mg/kg), a sodium dichlorophenolate treatment group (drug concentration 1.5mg/ml, administration dose 15mg/kg), a nano Liushen pill solution low dose group (administration dose 1.927mg/kg, administration volume 4. mu.l/10 g) and a nano Liushen pill solution high dose group (administration dose 9.639mg/kg, administration volume 4. mu.l/10 g), and 6 groups. Except for a model group, a morphine treatment group in a positive control group is injected with morphine in the abdominal cavity, a diclofenac sodium group is administered with diclofenac sodium by gastric gavage, a nano Liushen pill low-dose group is orally taken with a low-dose nano Liushen pill alcohol extract oil-in-water preparation, a nano Liushen pill solution high-dose group is orally taken with a high-dose nano Liushen pill alcohol oil-in-water preparation, 0.6 percent V/V acetic acid (10ml/kg) is injected in the abdominal cavity after 1/3h, 1h and 6h of administration respectively, the times of writhing of each group of mice within 30min are observed, and the relieving effect of the nano Liushen pill solution on pain is researched.

Example 1 prevention and treatment of Primary liver cancer in mice with Nano liushen pill solution

Animals: 128 tested SPF-grade Kunming KM male mice (provided by research center of laboratory animals in Hubei province) had weights of 18-22g, were fed with water freely, and were kept at 25 + -1 deg.C in the rearing room with a light-dark cycle of 12 hours.

Preparing a nano liushen pill solution: the oil phase is prepared by dissolving LIUSHEN pill ethanol extract lyophilized powder and yolk lecithin in oleum Sesami, wherein the final concentration of yolk lecithin is 6% W/V. The aqueous phase was prepared by dissolving Tween 80 and glycerol in ultrapure water, the final concentration of Tween 80 was 1.25% V/V and the final concentration of glycerol was 2.81% V/V. Dropping the oil phase into the water phase under the condition of a constant temperature water bath at 60 ℃, stirring for 15min under the condition of 1200r/min, wherein the volume ratio of the oil phase to the water phase is 1: 4. shearing at 10000rpm for 2min to obtain emulsified particles with particle size of 100-200 nm, and homogenizing at 1000bar under high pressure for 6 times to obtain oil-in-water preparation of alcohol extract of nanometer LIUSHEN pill. The content of LIUSHEN pill ethanol extract in high-dose nanometer LIUSHEN pill ethanol extract oil-in-water preparation is not more than 24.089 mg/ml^-1(ii) a The content of LIUSHEN pill ethanol extract in the low-dose nanometer LIUSHEN pill ethanol extract oil-in-water preparation is not more than 4.818 mg/ml^-1。

1. Establishment of primary liver cancer model and nano liushen pill solution administration

One week after adaptive feeding of 128 male Kunming mice, randomized: four groups, namely a normal group, a DEN group (model group), a nano liushen pill solution low-dose group (administration dose of 1.927mg/kg, administration volume of 4 mu l/10g) and a nano liushen pill solution high-dose group (administration dose of 9.639mg/kg, administration volume of 4 mu l/10g), and 32 mice in each group. The mice in the other three groups except the normal group were given 16.5mg/kg DEN once a week for twenty weeks; meanwhile, the nano Liushen pill solution low-dose group is orally taken twice by giving a low-dose nano Liushen pill alcohol extract oil-in-water preparation every week, the nano Liushen pill solution high-dose group is orally taken twice by giving a high-dose nano Liushen pill alcohol extract oil-in-water preparation every week for thirty weeks continuously, then the rest mice in each group are dislocated and killed, the abdominal cavity is opened, and the liver is taken out.

2. Mouse ethology and gross liver view

Mice were observed daily for mental status, coat changes, dietary status, and weekly weight records. At the tenth week, fifteen weeks, twenty weeks and thirty weeks of the experiment, 8 mice are taken from each group to be killed, liver tissue samples are taken, 10% paraformaldehyde solution is fixed for 24 hours, and the liver tissue samples are embedded in normal paraffin and prepared into 2-micron continuous paraffin sections by a slicer; the slices were synchronously baked, then deparaffinized, gradient alcohol hydrated, and stained with hematoxylin-eosin (H & E). Gradient alcohol dehydration, xylene transparency and resin sealing; the cytoplasm was stained red under the light mirror and the nucleus was stained blue.

3. Results of the experiment

The mice in the normal group are lively and motile, flexible in response and glossy in fur, and compared with the mice in the normal group, the mice in each group have different degrees of yellow, withered, lusterless and slow hair color, and the weight of the mice in each group changes, as shown in figure 1. HE results showed that at week fifteen of the experiment, the liver tissues of the model group showed more inflammatory cell infiltrates compared with the normal group, while the inflammatory cell infiltrates of each group treated with liushen pills were relatively less; in the twentieth week of the experiment, HE results show that a model group has more inflammatory cell infiltrates, is partially accompanied by adiposity and fibrosis, and has light and moderate disorder of liver tissue structure; the liver tissues of the low-dose group and the high-dose group of the nano Liushen pill solution only have inflammatory cell infiltration foci without obvious adiposis and fibrosis; thirty weeks after the experiment, no tumor was observed in liver tissue of the normal group of mice, as shown in fig. 2 (a); the liver tissue of 4 (50%) mice in the model group was visually observed for obvious tumor nodules, see fig. 2 (b); in the high-dose group of the nano liushen pill solution, tumor nodules were found in liver tissues of 3 mice (37.5%), as shown in fig. 2 (d); the liver tissues of the mice in the low-dose nano liushen pill solution group have no obvious tumor nodules seen with naked eyes as shown in fig. 2 (c).

In fig. 3, a: a normal group; b: a model group; c: a low-dose group of nano liushen pill solution; d: the nanometer LIUSHEN pill solution is used in high dose group. The scale is 100 μm and the upper right corner is an enlarged image (400 times) of the partial image (100 times) shown in black box. Observed under a light microscope, the liver cells of the mice in the normal group are arranged regularly, the sizes of the nuclei are consistent and are uniformly distributed, the liver cell cables surround the central vein and are distributed in a radial manner, and liver sinuses are clear, which is shown in a figure 3 (a). In the model group mice, nodule foci formed by the capsule can be clearly seen, the cell sizes and the forms are different, the cell nucleus forms are irregular, the cell cells of the tumor can be seen, and the nuclear division image is obvious, which is shown in figure 3 (b). The liver tissue microscopic examination of two groups of mice treated by the nano liushen pills can show that the liver cells are degenerated and necrotic and inflammatory cell infiltration; mild and moderate disturbance of liver cord, liver sinus distortion, and no tumor formation yet, as shown in fig. 3(c, d).

Example 2 nanometer liushen pills for pain relief

Preparing a nano liushen pill solution: the oil phase is prepared by dissolving LIUSHEN pill ethanol extract lyophilized powder and yolk lecithin in oleum Sesami, wherein the final concentration of yolk lecithin is 6% W/V. The aqueous phase was prepared by dissolving Tween 80 and glycerol in ultrapure water, the final concentration of Tween 80 was 1.25% V/V and the final concentration of glycerol was 2.81% V/V. Dropping the oil phase into the water phase under the condition of a constant temperature water bath at 60 ℃, stirring for 15min under the condition of 1200r/min, wherein the volume ratio of the oil phase to the water phase is 1: 4. shearing at 10000rpm for 2min to obtain emulsified particles with particle diameter of 100-200 nm, and homogenizing at 1000bar under high pressure for 6 times to obtain nanometer LIUSHEN pill alcohol extract water packetAn oil preparation. The content of LIUSHEN pill ethanol extract in high-dose nanometer LIUSHEN pill ethanol extract oil-in-water preparation is not more than 24.089 mg/ml^-1(ii) a The content of LIUSHEN pill ethanol extract in the low-dose nanometer LIUSHEN pill ethanol extract oil-in-water preparation is not more than 4.818 mg/ml^-1。

1. Establishment of pain model and nano liushen pill solution administration

After 90 male Kunming KM mice are adaptively fed for one week, the mice are randomly divided into three groups, namely 1/3h group, 1h group and 6h group according to time points, and each group comprises 30 mice; each large group was divided randomly into five groups, a model group, a morphine treatment group (drug concentration 500. mu.g/ml, administration dose 10mg/kg), a sodium dichlorophenolate treatment group (drug concentration 1.5mg/ml, administration dose 15mg/kg), a nano Liushen pill solution low dose group (administration dose 1.927mg/kg, administration volume 4. mu.l/10 g) and a nano Liushen pill solution high dose group (administration dose 9.639mg/kg, administration volume 4. mu.l/10 g), 6 per group. Except for the model group, the morphine solution is injected into the abdominal cavity of the morphine treatment group in the positive control group, diclofenac sodium is administrated by the diclofenac sodium treatment group through intragastric administration, and the nano Liushen pill solution low-dose group contains the low-dose nano Liushen pill alcohol extract water-in-water oil preparation; the nanometer LIUSHEN pill solution high dose group is administered with high dose nanometer LIUSHEN pill alcohol extract oil-in-water preparation. The mice in each group were observed for the number of writhing within 30min by intraperitoneal injection of 0.6% V/V acetic acid (10ml/kg) at 1/3h, 1h, and 6h after administration, respectively.

2. Behavioral observations of mice

Mice were observed for writhing within 30min after injection of 0.6% acetic acid at each time point and counted.

3. Results of the experiment

The oral administration of the high-dose nano Liushen pill solution and the low-dose nano Liushen pill solution, the drenching of diclofenac sodium and the intraperitoneal injection of morphine can obviously reduce the times of writhing of mice. With the prolonged administration time, the therapeutic effects of morphine and diclofenac sodium are weakened (the number of writhing times is 14 + -2 and 23 + -9 times at 20min, and 39 + -9 and 44 + -11 times at 6 h), while the therapeutic effect of the low-dose nanometer LIUSHEN pill solution group is gradually strengthened (the number of writhing times is 45 + -3 times at 20min, and the number of writhing times is 33 + -8 times at 6 h). Referring to FIG. 4, in which FIGS. 4a to 4c are graphs showing the number of writhing times of each group of mice injected with 0.6% V/V acetic acid at different time points in example 2, respectively. This shows that the action time of the nano Liushen pill solution in vivo is long and has the sustained release effect, wherein the twisting times are calculated in the Mean + -SEM mode.

Claims

1. A method for preparing nano liushen pill solution is characterized by comprising the following steps: the method comprises the following steps:

5) homogenizing under high pressure at 1000bar for 6 times to obtain oil in water package preparation of nanometer LIUSHEN pill alcohol extract;

the nanometer LIUSHEN pill solution is LIUSHEN pill ethanol extract with content of 4.818 mg/ml^-1The oil-in-water preparation of the alcohol extract of the nano Liushen pill.