The present invention relates to the intravenous injection preparation thereof, especially to the preparation of the saponins family of Radix notoginshen intravenous injection.

       In Chinese traditional medicine, Radix notoginshen is the dry root of Panax notoginshen (Burk.) (F. H.Chen). The plant is harvested, washed, and dried before flowering in the fall. The roots are collected and sorted into root stalks, rootlets, and stem bases. The medicine is mild, has minimal odor and bitter-sweet taste.

       The main chemical component of the Radix notoginshen extract is saponin. It is very effective for treatment of cardiac and cerebral vascular diseases. The Radix notoginshen preparations in today's markets are widely used in clinical practice, for example, Xueshuantong injection and Xuesaitong injection. It invigorates the circulation of blood, removes vascular stases, dilates blood vessels, and improves circulation. However, reactions of fluid infusions constantly occurs in clinical practices because of improper buffer, over prescription of medicines, existence of foreign particles, and improper compatibility of medicines, among others. Those reactions could bring severe pain to the patient and even be life threatening.

       The research shows that foreign particles could cause pyrogen reaction, allergies, and so on when injected in vivo. Pharmacopoeia of many countries have strict requirements on the number of particles in large dose injections. However, there is no requirement for small dose injections and powder injections. Application of multiple medications is commonplace in today's intravenous injections. Research has shown increases in particle numbers in blood vessels after injection, especially when liquid and powder intravenous injections of Chinese traditional medicine are involved. Significant increases of insoluble particles have been observed when many of the Chinese traditional medicine injections are applied with fluid infusion. The main reason lies in the complexity of the Chinese traditional medicine components. Also, different medicinal manufacturing industries have different preparation methods, including the purification and extraction of the effective components. Some components in the medicinal solution, such as pigment, tannin, starch, and protein, exists in colloidal state. Many reactions occur after the medicinal solution is applied with the fluid infusion, including oxidation, polymerization, and precipitation of large amounts of insoluble particles from saponin and alkaloid due to changes in pH. A dilution buffer of 5% or 10% glucose injection, rather than physiological saline, is recommended when applying intravenous injections using Xuesaitong, red rooted salvia compound, acanthopanax, β-aescin sodium and other Chinese traditional medicines. This is because of high occurrence of large amount of insoluble particles from precipitating out if using physiological saline with the large array components of the Chinese traditional medicine extract. This could increase the possibility of fluid infusion reaction and cause medical incidences (Honglan Zhong, Analysis and solutions for fluid infusion reactions, Guangdong Medicine, 2002, vol. 12, number 4).

       Improper compatibility of medicines could affect clarity of the compound solution, cause crystallization, and change pH (Xiujing Luo, 1999, The cause of fluid infusion fever, the preventive measures and thereof, Chinese Nursing Magazine, 34(10)). Reaction of fluid infusion is caused by over-dosage of multiple medicines and improper compatibility. The chances of contamination greatly increase because of repetitive injection of medicine into the medicine bottle in which multiple medicines are added. The clarity of the solution violates standard rules after adding 10% glucose injection with a chloromycetinNitamin C mixture, erythromycinNitamin C mixture, and tetracycline with other compounds. The solution then contains crystals, as well as pigmented and white clumps, among others. The pH of the 10% glucose solution for injection is decreased below the standard value after tetracycline is added. Medicinal particles could be generated if the powder injection is not dissolved thoroughly. The effective phase of penicillin G in glucose solution is only 2 hours. If it sits too long, the penicillin solution could even generate allergens which increase the chances of allergic reaction (Guitian Niu, Research and Experiments of Contamination in Clinic Fluid Infusion, Practical Nuring Magazine, 1993, 9(4), 25-27). On the other hand, chances of contamination greatly increase if any of the diluted solution sits for too long. The solubility, pH, age, and temperature could affect the solution after medicine homogenization and cause quality deterioration. This is especially true when there are large doses and multiple medicinal components involved because the contained pyrogen could accumulate to toxic levels, which will cause pyrogen reaction when injected in vivo.

       The saponin could be hydrolyzed in solution with improper pH. The solubility could then decrease and white particles, clumps, and cotton-like solids could appear in the solution. The quality of the medicine solution could then be compromised. Thus it is very important to choose and maintain an appropriate pH in production and storage of the saponins of Radix notoginshen injection.

       There are not only oral preparations, but also injections (small needle) used in today's clinics for the Chinese traditional medicine Radix notoginshen. The advantages of injection are efficiency and high biological utilization. The injection is especially beneficial to those patients who could not be treated with oral medicines and those who have serious conditions. However, small amounts of injection solution could be contaminated in practice. The operating procedures are complicated, which could add a greater burden to the medical practitioners, and cause the patients to undergo fluid infusion reactions.

       The first object of the invention is to provide a method for intravenous injection of saponins of Radix notoginshen in large doses with a stable pH, which will be used for prevention and treatment of cardiac and cerebral vascular disease and its sequelae.

       Applications of the invention are as follows:

       The provided saponins of Radix notoginshen intravenous injection is composed of saponins of Radix notoginshen with a concentration of 0.1 mg-14.0 mg (calculated as Rg1)/ml, iso-osmotic solution with a concentration of 7.5-8.5 mg/ml, and pH stabilizer with a concentration of 0.1-0.5 mg/ml. Distilled water is used as solvent.

       For said saponins of Radix notoginshen intravenous injection, the preferred concentration is 1.4 mg (calculated as Rg1)/ml. Said iso-osmotic preparation could be sodium chloride, glucose, sorbital and so on, among which the sodium chloride is preferred. The concentration of the sodium chloride falls between 7.5-9.5 mg/ml among which 8.5 mg/ml is preferred. Said pH stabilizer could be sodium citrate, citrate, phosphate, and acetate among which the sodium citrate is preferred. The concentration of the sodium citrate is 0.1-0.5 mg/ml, in which 0.3 mg/ml is preferred.

       The second object of the invention is to provide a method for production of saponins of Radix notoginshen intravenous injection.

       The method of production of the invention involves the following steps:

       The medicine of the invention is a large dose of intravenous injection in which the active component is saponins of Radix notoginshen. This medicine is for prevention and treatment of cardiac and cerebrovascular disease and its sequelae. The concentration of the medicine is 0.1 mg-14.0 mg (calculated as Rg1) saponins of Radix notoginshen per ml.

       The applied amount of the medicine could be adjusted according to the patient's age, weight and seriousness of the disease. The amount for each time is usually 140 mg-350 mg (saponins of Radix notoginshen). It could be intravenously injected 1-2 times per day for 28 days.

       The medicine of the invention is a large dose of saponins of Radix notoginshen intravenously injected. Intravenous injection provides a safe and convenient method of application.

       The animal experiments showed that the saponins of Radix notoginshen intravenous injection is a good protective medicinal treatment of cerebral anemia. It could significantly increase the cerebrovascular circulation and decrease its resistance, invigorate the circulation of blood, significantly improve rabbit eyeball conjunctiva micro-circulation aplastic status, and increase circulation and capillary openings. It has a good effect on treatment of embolismic cerebrovascular disease and could remove stases.

       The method of production for the invention of saponins of Radix notoginshen intravenous injection is simple and convenient. It is also very practical and inspiring. In this method, the pH was stabilized by adding pH stabilizer to the saponins family of Radix notoginshen intravenous injection. This successfully solved the problem of decreasing pH, which could cause saponin hydrolysis and affect clarity of the intravenous injection preparation.

       Further descriptions with experiments and diagrams for the invention follow.

       FIG. 1 is the technological process diagram for the saponins family of Radix notoginshen intravenous injection.

       1. Materials
2. Methods

       The technological processes are shown as FIG. 1. The production steps for the saponins family of Radix notoginshen intravenous injection are as follows. First, 7.5 g of sodium chloride was dissolved in distilled water and diluted to a concentration of 100 mg/ml. 0.4 g of active carbon was added to the solution. The liquid was stirred, boiled, cooled, and filtered to be rid of active carbon. 0.1 g (calculated as Rg1) of the saponins family of Radix notoginshen was then dissolved in the filtrate. Next, 0.3 g of sodium citrate was added to the solution. The pH of the liquid was adjusted to 6.0 by adding sodium hydroxide and diluted to a volume of 1000ml. The solution was then filtered through 0.4 g active carbon, a 0.45 μm filter membrane, and a 0.22 μm filter membrane until clear. The final product of saponins family of Radix notoginshen intravenous injection was put in a 100 ml fluid infusion bottle, capped, pasteurized at 110° C., checked and then packed.

       1. Materials
2. Methods
The technological processes are shown in FIG. 1. The production steps for the saponins family of Radix notoginshen intravenous injection are as follows. First, 8.5 g of sodium chloride was dissolved in distilled water and diluted to a concentration of 100 mg/ml. 0.4 g of active carbon was added to the solution. The liquid was stirred, boiled, cooled, and filtered to be rid of active carbon. 1.0 g (calculated as Rg1) of the saponins family of Radix notoginshen was then dissolved in the filtrate. Next, 0.3 g of sodium citrate was added to the solution. The pH of the liquid was adjusted to 6.0 by adding sodium hydroxide and diluted to a volume of 1000 ml. The solution was then filtered through 0.4 g of active carbon, a 0.45 μm filter membrane, and a 0.22 μm filter membrane until clear. The final product of saponins family of Radix notoginshen intravenous injection was put in a 100 ml fluid infusion bottle, capped, pasteurized at 110° C., checked and then packed.
Experiment 3. Preparation of the Saponins Family of Radix Notoginshen Intravenous Injection

       1. Materials
2. Methods

       sThe technological processes are shown as FIG. 1. The production steps for the saponins family of Radix notoginshen intravenous injection are as follows. First, 8.5 g of sodium chloride was dissolved in distilled water and diluted to a concentration of 100 mg/ml. 0.4 g of active carbon was added to the solution. The liquid was stirred, boiled, cooled, and filtered to be rid of active carbon. 1.4 g (calculated as Rg1) of the saponins family of Radix notoginshen was then dissolved in the filtrate. Next, 0.3 g of sodium citrate was added to the solution. The pH of the liquid was adjusted to 6.0 by adding sodium hydroxide and diluted to a volume of 1000 ml. The solution was then filtered through 0.4 g of active carbon, a 0.45 μm filter membrane, and a 0.22 μm filter membrane until clear. The final product of saponins family of Radix notoginshen intravenous injection was put in a 100 ml fluid infusion bottle, capped, pasteurized at 110° C., checked and then packed.

       1 Materials
2 Methods
The technological processes are shown as FIG. 1. The production steps for the saponins family of Radix notoginshen intravenous injection are as follows. First, 8.5 g of sodium chloride was dissolved in distilled water and diluted to a concentration of 200 mg/ml. 0.4 g of active carbon was added to the solution. The liquid was stirred, boiled, cooled, and filtered to remove active carbon. 3.5 g (calculated as Rg1) of the saponins family of Radix notoginshen was then dissolved in the filtrate. Next, 0.3 g of sodium citrate was added to the solution. The pH of the liquid was adjusted to 6.0 by adding sodium hydroxide, then diluted to volume of 1000 ml. The solution was then filtered through 0.4 g active carbon, a 0.45 μm filter membrane, and a 0.22 μm filter membrane until clear. The final product of saponins family of Radix notoginshen intravenous injection was put in a 100 ml fluid infusion bottle, capped, pasteurized at 110° C., checked and then packed.
Experiment 5. Preparation of the Saponins Family of Radix Notoginshen Intravenous Injection

       1. Materials
2. Methods
The technological processes are shown as FIG. 1. The production steps for the saponins family of Radix notoginshen intravenous injection are as follows. First, 9.5 g of sodium chloride was dissolved in distilled water and diluted to a concentration of 200 mg/ml. 0.4 g of active carbon was added to the solution. The liquid was stirred, boiled, cooled, and filtered to be rid of active carbon. 7.0 g (calculated as Rg1) of the saponins family of Radix notoginshen was then dissolved in the filtrate. Next, 0.3 g of sodium citrate was added to the solution. The pH of the liquid was adjusted to 6.0 by adding sodium hydroxide and diluted to volume of 1000 ml. The solution was then filtered through 0.6 g of active carbon, a 0.45 μm filter membrane, and a 0.22 μm filter membrane until clear. The final product of saponins family of Radix notoginshen intravenous injection was put in a 100 ml fluid infusion bottle, capped, pasteurized at 110° C., checked and then packed.
Experiment 6. The Protective Effect of the Saponins Family of Radix Notoginshen Intravenous Injection to Rat Cerebral Ischemia Reperfusion Injury
(1) Experimental medicine: 5% saponins family of Radix notoginshen intravenous injection preparation (manufactured by Lizhu Group Limin Pharmaceutical Industry, packed as 100 ml, batch No. 20000301). The total amount of saponins of Radix notoginshen intravenous injection preparation used in clinical practice is 350 mg/250 ml per treatment. It could be applied 1-2 times per day for 28 days for one treatment phase. The 5% saponins family of Radix notoginshen intravenous injection in the experiment was diluted to the needed concentration with physiological saline.
(2) Experimental animal: 150 SD rats with body weight of 280.45±14.91 g, half male, half female.
(3) Amount of medicament: calculated for the experimental animal according to the one for human by using transform factor mg/Kg-mg/m ². The rat low dosage group was given a single dose of the equivalent amount of medicament for animals. The medium dosage group was given twice the amount as the low dosage group, and the high dosage was given 4 times the amount of the low dosage group. The amount of saponins family of Radix notoginshen intravenous injection for an adult is 250 ml per day (containing 350 mg of saponins family of Radix notoginshen), which is 350 mg/60 kg per day. The equivalent amount medicament for rats is 350 mg/60 kg×35/6 ?34.03 mg of saponins family of Radix notoginshen. High dosage is 34.03×4=136.12 mg/Kg, 34.03×2=68.06 mg/Kg, 34.03×1=34.03 mg/Kg.
The control medicament was the Salvia Miltiorrhiza injection which is well used in present day Chinese clinical practices. The amount of medicament administered temporally for an adult is 10 ml (containing 10 g of crude drug), twice per day, which makes 20 g of crude drug/60 kg per day. The applying amount of Salvia Miltiorrhiza injection for a rat is 20 g/60 kg×35/6?1.94 g/Kg.
(4) Experimental conditions: room temperature (25° C.), relative humidity 60%˜70%
(5) Experimental methods:
150 SD rats were used, of which half were male and half were female, each of them weighting 282.11±14.77 g. They were grouped into 6 groups with 6 rats per group. The first group was a pseudo-operation group. The second group was a model group. The third group was a Salvia Miltiorrhiza injection group. The fourth to the sixth groups were applied, respectively, with low dosage, medium dosage, and high dosage of saponins family of Radix notoginshen intravenous injection. Each group was applied with medicament through lingual veins continuously for 4 days. The first and second groups were injected with same amount of physiological saline. One hour after the last medicament application, the animals were anaesthetized using 1 g/Kg ip of Urethane. Both sides of the a.carotis communis were then separated and threaded for future experiments. The animals were then injected with 50 mg/Kg 5% Evans Blue (EB) and 486 u/Kg heparin through v.femoralis. After 5 minutes, both sides of rat a.carotis communis in the model and other medicament groups were clipped shut. And at the same time, 2.5 ml of blood was drained from the v.femoralis. These two steps above were not included in the pseudo-operation group. The a.carotis communis of rats in the model and other medicament groups were released and reinfused for another 30 minutes after 3 hours. The rats were then decapitated on ice and the brain (the bulbus olfactorius, cerebellum and low truncus encephalicus) carefully removed. Two rat brains were randomly selected from each group and fixed in formaldehyde and glutaraldehyde for morphological observation. The rest were weighed and soaked in 5 ml of formamide, water bathed for 48 hours at 45° C., and regularly stirred. The supernatant was obtained and 1.5 ml of chloroform was added. Next, the liquid was centrifuged and the supernatant was obtained and analyzed using an ultraviolet spectrophotometer (620 nm). The content of Evans Blue in the rat brains was calculated using standard curves. The second rat brain taken was baked in the oven at 110° C. until its weight was constant. The brains were then weighed and water content of the brains were calculated as %=(brain wet weight−brain dry weight)/brain wet weight×100%, and brain index was calculated as (brain wet weight/10 g body weight).
(6) Results
1) The effect of saponins family of Radix notoginshen intravenous injection on experimental rat brain indexes, water content and Evans Blue content.

       The experimental results are shown in FIG. 1. It shows that the brain indexes, water content and EB content of pseudo-operation group all decreased, and had significant statistical differences which indicated success in operation modeling. When comparing the model group with the three groups which were applied saponins family of Radix notoginshen intravenous injection, the above indexes all decreased and had significant statistical differences. These facts indicated that the saponins family of Radix notoginshen intravenous injection could improve incomplete ischemia reperfusion injury of the experimental animals, relieve the increase of cerebrovascular penetrating and cerebral edema caused by cerebral ischemia reperfusion injury, and thus protect the brain.

       Results observed under the microscope are as follows. The results were normal for the pseudo-operation group. Occasional capillary hyperemia, slight expansion between vascular and nerve cells were observed. The results for the model group were quite different. Typical cell edema, denaturation and necrosis were observed. Also there was significant expansion between meningeal vascular, neurons, and cerebral cortex cells, expansion of cell nucleus, nucleolus clouding, disappearance of tiger porphyritics, bleeding of cerebral cortex, local congestion of colloidal cells, and occasionally cell ischemia necrosis. The symptoms included decreasing cell count, disappearance of the cell outlines and local hemorrhage. Different degrees of the cell edema and denaturation were also observed from the positive control group and three saponins family of Radix notoginshen intravenous injection groups. However, the symptoms for the positive control group and three medicament groups were far milder than that of the model group. The symptoms included expansion of the cavity between cells and vasculars and vascular hyperemia. Propagation of small colloidal cells was observed in the high dosage treatment group.

       Results observed under the electron microscope are as follows. In the pseudo-operation group, clear cell stuctures, intact axon and cell membranes, evenly distributed chromosomes, clear mitochondria, clear Golgi body and clear rough endoplasmic reticulum were observed. All structures seemed to be normal. In the model group, the abnormal symptoms observed included expansion of neuron cells, decreasing cell count, congestion of nuclear chromosomes, expansion of the Golgi body, appearance of empty bubbles in the Golgi body, expansion of rough endoplasmic reticulum, significant expansion of mitochondrion, obscure cristae, expansion of vascular peripheral cavities, destruction of the endomembrane, and partial dissolution of nuclear chromotin. In the three saponins family of Radix notoginshen intravenous injection groups and the positive control group, there was indication of ischemia edema, which is however, far less severe than that of the model group. The symptoms included congestion of the chromosomes, various degrees of expansion of the rough endoplasmic reticulum, Golgi body and mitochondria, but at levels much less severe than that of the model group.

       According the results observed under the electron microscope, the most severe injuries were observed in model group. This indicated that the model is constructed successfully. On the other hand, much less severe damage was observed in the three saponins family of Radix notoginshen intravenous injection groups and the positive control group. Furthermore, the cell damage decreased while the amount of saponins family of Radix notoginshen intravenous injection increased. This indicated that the saponins family of Radix notoginshen intravenous injection could protect animal from acute incomplete ischemia reperfusion injury.
The above results prove that the saponins family of Radix notoginshen intravenous injection has certain protective effects on experimental rat acute incomplete ischemia reperfusion injury. It is worthy to have further application on clinical practices.
Experiment 7, the Effect of the Saponins Family of Radix Notoginshen Intravenous Injection on the Cerebrovascular of Anaesthetized Dog
(1) Experimental medicament and its dosage: 5% saponins family of Radix notoginshen intravenous injection preparation (manufactured by Lizhu Group Limin Pharmaceutical Industry, packed as 100 ml, batch No. 20000301).). The total amount of saponins of Radix notoginshen intravenous injection preparation used in clinical practice is 350 mg/250 ml per treatment. It could be applied 1˜2 times per day for a 28 day treatment phase. In the experiment, the dosage of the medicament for the experimental animal was calculated according to that for a human by using the transform factor mg/Kg-mg/m ². A single dose of the equivalent amount of medicament for animals was applied to the dog low dosage group. The medium dosage group received twice the equivalent medicament and high dosage received 4 times the equivalent medicament of the low dosage group. The dosage of saponins family of Radix notoginshen intravenous injection for an adult is 250 ml once per day. That is, 350 mg of saponins family of Radix notoginshen is applied per day. In the case of dog, 1) the equivalent amount of medicament is 350 mg/60 kg×35/19?10.75 mg saponins family of Radix notoginshen 1 kg, 2) low dosage is 10.75×1=10.75 mg/Kg, 3) medium dosage is 10.75×2=21.5 mg/Kg, 4) high dosage is 10.75×4=43 mg/Kg. 5% of total amount of saponins of Radix notoginshen intravenous injection and Mailuoning injection were diluted before usage to a needed concentration using physiological saline.
The control medicament was Mailuoning injection (manufactured by Nanjing Jinglin Pharmaceutical Corp., medicine content of 10 g/ml, batch No. 960812). The adult dosage was 10 ml (crude drug of 10 g), twice per day, that is, 20 g/60 kg per day. For the dog, 1) equivalent dosage is 20 g/60 kg×35/19?0.61 g/Kg, 2) dosage of medicament is 0.61×4=2.44 g/Kg. Physiological saline was manufactured by Southern Hospital Pharmacy, batch No. 20000926.
(2) Experimental animals: 30 healthy adult hybrid dogs, each weights 11˜15kg, half male and half female.
(3) Experimental conditions: room temperature 25° C., relative humidity 60%˜70%, and regular pasteurization of experimental equipments.
(4) Equipment: electromagnetic flowmeter, model FM-27, manufactured by Japanese Photoelectric Ltd. The [8] Physiological Recording Instrument of model RM-6000, manufactured by Japanese Photoelectric Ltd and Chengdu Instrument Industry.
(5) Method of medicaments application: intravenous injection with a concentration of 30 ml/Kg.
(6) Experimental control: the negative control group was given same amount of physiological saline while the positive control group was given 4 times of the equivalent amount of the Mailuoning injection. (
7) Experimental data statistics
The experimental data was analyzed by statistical t-testing. The physiological statistics were used for data analysis for comparison among all groups and the negative control group before and after medicament applications.
(8) Experimental methods
The experimental animals were randomly grouped into 5 groups including the negative and positive control groups, and the low dosage, medium dosage and high dosage of saponins family of Radix notoginshen intravenous injection. The dogs were first anaesthetized via an intravenous injection of 30 mg/Kg 3% pentobarbital sodium, and then fixed on the operation platform. 1 mg/ml of pentobarbital sodium diluted in physiological saline was intravenously infused to maintain the anesthesia. The dog neck was cut for about 10cm to separate the left a.carotis communis, and thread on the same side the a.carotis externa and the a.vertebralis. The No. 4 sensor of the electromagnetic flowmeter was put in the a.carotis communis. The blood flow of the a.carotis communis, calculated by the electromagnetic flowmeter, could be considered as cerebral blood flow. At the same time, the a.fermoralis and v.fermoralis on one side were separated. The a.fermoralis was connected to the pressure transducer and v.fermoralis was connected to the 3-way pipe for medicament application. All four legs were connected to the sensors for the electrocardiogram monitor. Meanwhile, blood pressure of the a.fermoralis, II lead electrocardiogram, and the average blood flow of the a.carotis communis were recorded by the [ 8 ] Physiological Recording Instrument. When the average blood flow, electrocardiogram and blood pressure were indicated stable by the equipment, the data were recorded and the medicaments were applied through intravenous injection. The average amount of medicament application was 30 ml/Kg for all groups. At the same time, the cerebral blood flow, blood pressure and cardiac rhythm were recorded at 0 min, 5 min, 15 min, 30 min and 60 min after medicament application. At the end of the experiment, the skull was opened and the whole brain was removed from above the medulla oblongata. The brain was then weighed. The brain weight of one side was obtained by dividing the resulting data by 2. The cerebral blood flow per minute for each gram of brain tissue was obtained by dividing the recorded cerebral blood flow by the brain weight of one side.
(9) Result
1) The effect of the Saponins Family of Radix Notoginshen Intravenous Injection on the Cardiac Rhythm of Anaesthetized Dog
The experimental results indicated statistically meaningful fluctuations of the cardiac rhythm in the low dosage group recorded after medicament application 5 min and 15 min, and the medium dosage group recorded after 0 min. There were no statistical differences in other groups and time phases (table 2).
2) The Effect of the Saponins Family of Radix Notoginshen Intravenous Injection on the Blood Pressure of Anaesthetized Dog

       As in tables 3, 4, 5, systolic pressure decreases (p<0.05) in the anaesthetized dog was observed in the saponins family of Radix notoginshen intravenous injection groups 15 min after application, while the effect on diastolic pressure was not obvious (the only statistical meaningful blood pressure decrease lay in the low dosage group at 5 min after application). Also, the only statistical meaningful decrease of the average blood pressure was observed in medium and low dosage groups while no obvious change was observed in the high dosage group after medicament application.

      

      

      
3) The Effect of the Saponins Family of Radix Notoginshen Intravenous Injection on the Cerebral Blood Flow of Anaesthetized Dog

       Data of table 6 indicated that blood flow immediately increased (P<0.05) immediately after medicament application in all dosage groups of the saponins family of Radix notoginshen intravenous injection. The blood flow increase was maintained for 5 min in the low and medium dosage group while it was maintained for 15 min in the high dosage group. The blood flow dropped to the level before medicament application afterwards.
4) The Effect of the Saponins Family of Radix Notoginshen Intravenous Injection on the Cerebral Vascular Resistance of Anaesthetized Dog

       Data from table 7 indicates that the cerebrovascular resistance decreased (P<0.01) immediately after medicament application in all dosage groups. This effect was maintained for 15 min after medicament application and then dropped to the level before application afterwards (P>0.05).

       (10) Conclusion

       The saponins family of Radix notoginshen intravenous injection caused decreases in the cardiac rhythm and blood pressure for the anaesthetized dog within a short period of time after application. However, this effect did not become more significant when amount of medicament increased. The saponins family of Radix notoginshen intravenous injection also significantly increased the cerebral blood flow and decreased the cerebral vascular resistance for the experimental animals. The two effects mentioned above also recovered to the level before medicament application afterwards.

       5% of the saponins family of Radix notoginshen intravenous injection preparation, manufactured by Lizhu Group Limin Pharmaceutical Industry, packed as 100 ml, batch No. 20000301. The application amount in clinical practices is an intravenous infusion of 350 mg/250 ml (calculated as the saponins family of Radix notoginshen). It was applied 1˜2 times per day for 28 days to complete one phase. The experimental medicaments were diluted with physiological saline to a concentration of 2.5%, 1.25% and 0.63%. Medicaments were stored in the refrigerator at 4° C. The positive medicament (the saponins family of Radix notoginshen intravenous injection) contained 2 ml/shot. Each shot contained lg of root of red rooted salvia and Lignum Dalbergiae Odoriferae respectively, manufactured by Guangdong Yongkang Pharmaceutical Ltd., batch No. 00070001. The experimental physiological saline was prepared to the desired concentration and stored at 4° C. in the refrigerator.

       The main preparations were polymerized dextranum with molecular weight of 500,000, made in Sweden, Sodium adenosine diphosphate (ADP) manufactured by FARCO, Sodium heparin provided by Chinese Pharmaceutical Corp. Beijing Branch, Urethane provided by Shanghai Chemical Preparation Store, and pentobarbitol sodium provided by Guangzhou Chemical Industry.

       Kunming breed mice, each weighing 18-24 g, in a 1:1 male to female ratio; SD rats, each weighing 190-260 g, in a 1:1 male to female ratio; New Zealand rabbit, each weighing 2.0-2.5 kg, both male and female. The males and females were separated. Each cage contained one rabbit/5 mice/5 rats. The animals were kept 3 days for adaptation. They were only used in experiments after no abnormal physical behaviors or physiological symptoms were observed. The animals were well fed and the room was well lighted and aired. Temperature was maintained at 20-25° C. Humidity was 45-65%. Professional management and regular pasteurization were applied to the room.

       The drug form of the saponins family of Radix Notoginshen intravenous injection in clinical practices is sodium chloride intravenous instillation. The dosage for adult is 350 mg (the saponins family of Radix Notoginshen) per treatment, 1-2 treatments per day. According to the LD ₅₀ and toxicity experiment results of this drug, the adult dosage applied in this experiment was 350 mg per day. A transform factor was used to calculate the dosage for experimental animals according to that for humans. The equivalent dosage for animals was calculated using this transform factor. The low dosage group received a single dose of the equivalent dosage. The medium dosage group received twice the equivalent dosage. And the high dosage group received 4 times the equivalent dosage. The salvia miltiorrhiza injection was used as the positive control with the equivalent dosage.

       4) Dosage of salvia miltiorrhiza injection for positive control group: adult dosage in clinical practice=20 ml per day using intravenous instillation; equivalent dosage for mice=20/60×35/3=3.9 ml/Kg?4 ml/Kg; equivalent dosage for rats=20/60×35/6=1.94 ml/Kg?2 ml/Kg; equivalent dosage for rabbits=20/60×35/12=0.97 ml/Kg?1 ml/Kg.

       50 Kunming breed mice were. chosen in a 1:1 male to female ratio. Each weighed 18-24 g. Animals were randomly grouped into 5 groups: bland control group, positive control group and 3 medicament groups respectively applied with low dosage, medium dosage and high dosage of the saponins family of Radix notoginshen intravenous injection. Mice of each group received medicament through tail intravenous injection of the indicated amount continuously for 3 days. 30 min after the last application of medicament, two drops of blood were obtained from the plexus venosus behind the eyeball. Each blood drop was set on a slide and has a diameter of 5 mm. Time was monitored immediately after blood dropped on the slide. Every 30 s, a clean No. 4 needle was used to stir the blood drop once from the edge to the center, to observe whether there appeared any blood filiform. The time period from the obtaining of blood to the appearance of blood filiform was considered as blood coagulation time. The second drop of blood was used for retesting. The experimental results are shown in Table 8. The data indicates that the blood coagulation time for the medicament application groups was significantly longer than that for the blank control group. The statistic analysis results indicated remarkable differences: P<0.05 or P<0.01. The anti-coagulation effect of the saponins family of Radix notoginshen intravenous injection increased with increasing dosage. However, there was no significant difference between the 3 different dosage groups.

       2) Rat Thrombosis Experiment In Vivo

       50 Kunming breed rats were chosen in a 1:1 male to female ratio. Each weighed 220-280 g. Animals were randomly grouped into 5 groups: bland control, positive control, and 3 medicament groups applied with low dosage, medium dosage and high dosage, respectively, of the saponins family of Radix notoginshen intravenous injection. Rats of each group received medicament through tail intravenous injection of the indicated amount continuously for 2 days. Thrombosis in vivo experiments were conducted on the 3 ^rd day after medicament application. Animals were anaesthetized by 20% Urethane (1 g/Kg, IP). The neck was cut and the right side a.carotis commubis and the left side of v.jugularis externa were separated. A No. 4 operation thread of 5 cm was set in the middle pipe of the three connected polyethylene pipes. Among the 3 pipes, two at both ends had an internal diameter of 1 mm and a length of 10 cm while the middle one had an internal diameter of 2 mm and a length of 8 cm. The pipes were filled with heparin physiological saline (50 u/ml). The heparin (50 u/Kg) was filled from the left side of v.jugularis externa through the pipe. Then the pipe was clipped and inserted from the other end to right side of a.carotis commubis. Said amount of medicament was injected through the v.jugularis externa after the operation. The circulation was released 5min after medicament application. The blood was then shunted from the right side of a.carotis commubis to the left side of v.jugularis externa. Circulation was again halted after 15 min. The pre-set operation thread was removed and weighed. The thrombus wet weight was obtained by subtracting thread weight from the total weight. The resistant ratio was calculated according to the following equation: Resistant ratio (%)=(thrombus weight from the control group−thrombus weight from medicament application group)÷thrombus weight from control group×100%

       The experimental results were shown as in Table 9. The data indicates that the thrombus wet weight from the medicament application group was obviously lower than that from the blank control group. The statistical analysis showed significant differences of P<0.05 or P<0.01. The anti-coagulation effect of the saponins family of Radix notoginshen intravenous injection increased with increasing dosage. However, there was no obvious difference between 3 different dosage groups.

       3) Testing of Rat Blood Viscosity, Hematocrit and Platelet Aggregation Ratio

       50 SD rats were chosen in a 1:1 male to female ratio. Each weighed 220-280 g. Animals were randomly grouped into 5 groups: bland control, positive control, and 3 medicament groups applied with low dosage, medium dosage, and high dosage, respectively, of the saponins family of Radix notoginshen intravenous injection. Rats of each group were applied medicament through tail intravenous injection of the indicated amount continuously for 3 days. The animals were anaesthetized after 15 min. by 20% Urethane (1 g/Kg, IP). Blood was obtained from rat aorticus abdominalis. 4 ml of blood was first obtained together with 1% heparin for anti-coagulant (150 μl heparin:4 ml blood). The two above mentioned preparations were used in testing of whole blood viscosity, plasma viscosity, and hematocrit. Another 4 ml of blood was obtained together with 3.8% sodium citrate anti-coagulant (1:9). The above mentioned two preparations were used in platelet aggregation ratio test.

       The instrument used in testing of blood viscosity was LG-R-80A automatic washing blood viscosity instrument manufactured by Beijing Shidi Scientific Instrument Corp. The whole blood viscosity and plasma viscosity were tested respectively at 150, 30, 5, 1 S ⁻¹. The operation was carried out according to the instrument description.

       For hematocrit testing, 1 ml of whole blood was filled in RBC cuvette and centrifuged at 3000 rmp for 30 min. The height of the red blood cell column was recorded. The formula for determining percent hematocrit follows: hematocrit=height of red blood cell column÷height of whole blood column×100%

       For platelet aggregation ratio testing, the blood with 3.8% sodium citrate added was centrifuged at 800 rpm for 6 min. The supernatant was platelet replenish plasma (PRP). The rest of the blood was re-centrifuged at 3000 rpm for 10 min after the supernatant was removed. This time the supernatant was platelet poor plasma (PPP). The blood was induced using ADP (5×10 ⁻⁶ mol/L). The rat platelet aggregation ratio within 5min was tested using PABER-1 platelet aggregation instrument manufactured by Beijing Shidi Scientific Instrument Corp. The operation was carried out according to the instrument descriptions.

       The experimental results are shown in Tables 10 and 11. Table 10 indicates that rat whole blood viscosity, hematocrit and plasma viscosity were significantly decreased in the saponins family of Radix notoginshen intravenous injection high dosage group. However, rat whole blood viscosity, hematocrit, and plasma viscosity showed no obvious changes in the saponins family of Radix notoginshen intravenous injection low and medium dosage groups. The salvia miltiorrhiza injection only had effect on whole blood viscosity at low shear rate (5 s ⁻¹ and 1 s ⁻¹) while it had no effect on other indexes. Table 11 indicates that the rat platelet aggregation ratio obviously decreased in the three different experimental medicament groups and the positive control group compared to that in the blank control group. The differences showed high significance (P<0.01) according to statistic analysis. The anti-coagulation effect of the saponins family of Radix notoginshen intravenous injection increased with the increase in dosage. There were significant differences between the high dosage and low and medium dosage group (P<0.01). However, there were few differences between the low and medium dosage group.

      
4) Observation of Rabbit Eyeball Conjunctiva Microcirculation

       30 New Zealand rabbits were chosen in a 1:1 male to female ratio. Each weighed 2.0-2.5 kg. Animals were randomly grouped into 5 groups (6 in each group): blank control, positive control, and 3 medicament groups applied with low dosage, medium dosage and high dosage, respectively, of the saponins family of Radix notoginshen intravenous injection. Rabbits were anaesthetized using 3% Sodium Pentobarbitol (30 mg/Kg, IV). The animals' left eye lids were then opened and the eyeball conjunctiva microcirculation was observed using MTV-3801 CB microcirculation micro video recorder system (provided by Shanghai Laser Technology Research Institute). The magnifying factor used was 2800× after the recorder was standardized using 0.01 mm objective lens micrometer (made by Shanghai 3 ^rd Industry of Optical Instruments). The general flow of the normal eyeball conjunctiva microcirculation was observed. Then 10% polymer dextranum (6 ml/Kg) was intravenously injected through rabbit ear vein. The injection induced the acute microcirculation obstacle and changes in the microcirculation were observed. After 15 min, one of three solutions was applied through intravenous injection: physiological saline, the Saponins family of Radix notoginshen injection, or the salvia miltiorrhiza injection. The applied medicament volume was 1.3 ml/Kg for each medicament or saline. The same area of microcirculation was observed 15 min after medicament application.

       Observation Indexes

       Red blood cell fluid state: According to relative articles and experimental results from this invention, the red blood cell fluid state was classified into 6 levels: linear flow, linear granular flow, granular linear flow, granular flow, stasis, and stop.

       Capillary net conjunction: The edge of the observation area was composed of small arteries and veins. The number of conjunctions between capillaries in this area and the capillaries on the edge was calculated. The non-conjunctive capillaries were not counted.

       The results are shown in Table 12. The data indicated that the blood flow in rabbit eyeball microcirculation changed from normal linear or linear granular flow into granular or granular linear flow after being injected with 10% polymer dextranum. Also, the flow speed significantly decreased. Stasis and stop occurred in a few cases. However, no obvious thrombus or blood vessel distortion was observed. There was no significant blood flow changes observed when injected with physiological saline. After being injected with the saponins family of Radix notoginshen or the salvia miltiorrhiza, the microcirculation flow speed was greatly increased, flow state was mostly linear granular, and capillaries conjunctions were also increased. The increases were significant (P<0.05 or P<0.01) in the high dosage group of saponins family of Radix notoginshen and positive control group comparing the states before and after medicament application.
(6) Statistical Analysis
All experimental data was presented as x±s, and analyzed using t test and one way analysis of variance.
(7) Results
The results of this experiment indicated that the saponins family of Radix notoginshen intravenous injection could significantly invigorate the circulation and remove stases. It could resist rat thrombosis and elongate the coagulation. The high dosage group had relatively greater remarkable effects on rat blood viscosity and hematocrit. It also could greatly resist rat platelet aggregation, resist the rabbit eyeball microcirculation obstacles, increase the capillary openings, and speed up the blood flow. The effect of the saponins family of Radix notoginshen intravenous injection showed certain dosage dependence within the experiment application dosage. However, no obvious differences were observed within high, medium, and low dosage groups.
Experiment 9, the pH Stability Test for the Intravenous Injection of this Invention
The Experiment results of pH are shown in Table 13.
Sample A: Xueshuangtong injection, manufactured by Neimenggu Ganqika Pharmaceutical Industry.

       Sample B: Intravenous injection of this invention, manufactured by Lizhu Group Liming Pharmaceutical Industry

       The Chinese Medicine standard specified that the qualified pH range of Xueshuantong injection was 5.0˜7.0. The experimental results indicated that the pH of sample A was related to the batch No. This showed the instability of sample A pH, which decreased after storage. On the other hand, the pH of Sample B was more stable.