first , the condensed solution was prepared by one or several materials of traditional chinese medicine list in table 1 . the preparation process comprising : ( 1 ) six times more amount of water was added into the 360 grams of traditional chinese medicine to made a 2160 ml solution ; ( 2 ) the 2160 ml solution was condensed as 1200 ml by reduce pressure under high temperature ( about 70 ° c . ); ( 3 ) kept the condensed solution stable and cool , and removed 5 % precipitate after two days to obtain a condensed solution . then , an energy - rich liquid , a salt - rich liquid , an assist agent and an anti - oxide agent were added sequentially into above mentioned 200 ml condensed solution which contains 60 grams of solid precipitate . after mixing , the solution was fermented at room temperature ( 25 ° c .) for 3 - 7 days . preferably , fermentation is conduct 3 days . said energy - rich liquid comprising 5 mg of ferric chloride ( fecl 3 ) and 200 ml of distilled water . said salt - rich liquid comprising 125 grams of deep sea salt , 30 grams of magnesium chloride , 20 ml brine , 1 . 1 grams of calcium chloride ( cacl 2 ) and 0 . 55 grams of potassium chloride ( kcl ). said assist agent comprising 20 grams of citric acid , 20 grams of selenium yeast , 10 % concentration 300 mg of coenzyme q10 and 3 grams of vitamin c . said antioxidant comprising 70 % concentration 10 grams of chitin oligosaccharides , 98 % concentration 5 grams of glutathione , 80 % concentration 1 g of pine bark and 80 % concentration 1 gram of fucoidan . the distilled water was added into the above - mentioned fermented solution to make a total volume of solution is 500 ml . after stirring , kept solution stable for 1 day and then centrifuged at 1500 rpm for 15 minutes . the supernatant is the pharmaceutical compositions of the present invention , which contains 25 % deep sea salt , 6 % magnesium chloride . the ph value of the pharmaceutical composition is 1 . 5 ± 0 . 3 in order to maintain the product stability . 2 . the effect of the presented pharmaceutical compositions on tumor growth 7 - week - old male mice ( 20 - 24 grams , cb17 / icr - prkdcscid / icricocrlbltw ) was subcutaneously inoculated with 3 × 10 6 ht - 29 colon cancer cells or 1 × 10 7 a549 lung adenocarcinoma cells , leading to colon cancer or lung adenocarcinoma . ht - 29 colon cancer cells and a549 lung adenocarcinoma cells are dissolve in 100 μl pbs contains 50 % matrigel , respectively . while the volume of tumors is grown to 200 mm 3 , the male mice were divided into three groups including : ( a ) control group , do not give any medication on day 0 to day 14 ; ( b ) contrast group , give the chemotherapeutic agent 5 - fu ( 100 mg / kg ) or cisplatin ( 4 mg / kg ) only on the day 0 and day 7 ; and ( c ) experimental group , administered the presented pharmaceutical composition continuously for 21 days . the presented pharmaceutical composition was administered 7 days before the give chemotherapeutic agent . the administered method is twice daily , and each dose is 0 . 5 - 1 . 67 μl / mouse . on day 0 and day 7 , the chemotherapy drugs 5 - fu ( 100 mg / kg ) or cisplatin ( 4 mg / kg ) was administered while the presented pharmaceutical composition was not administered . on day 14 after administered presented pharmaceutical composition , the tumor was acquired and measured its weight . please refer to fig1 a - 1b . compared to control group , the volume of tumor of contrast group is reduce 6 . 3 % in average . the inhibitory ability of contrast group was 1 times higher than the control group . however , the volume of tumor of experimental group can effectively reduce 34 . 1 %. the inhibitory ability of experimental group was 5 . 4 times higher than the control group . therefore , the pharmaceutical compositions of the present invention can improve the inhibitory ability of chemotherapeutic agent on tumor . 3 . the effect of the presented pharmaceutical compositions on the function of liver many studies have shown that the cancer cells would flow into the liver through the blood , leading to the liver dysfunction . thus , the serum of each group in example 2 was collected at day 14 after administered drugs to analyze the expression of alanine aminotransferase ( alt ). please refer to fig2 a . the rats of normal group were neither implanted colon cancer cells nor administered drugs . compare with normal group , the alt concentration of control group was increased . means that the colon cancer cells would cause liver damage . compare with control group , the alt concentration of contrast group was increased . however , the alt concentration of experimental group was decreased and the expression level is close to the normal group . please refer to fig2 b . the rats of normal group were neither implant lung adenocarcinoma nor administered drugs . compare with normal group and control group , the alt concentration of the contrast group was increased . however , the alt concentration of experimental group was decreased and the expression level is close to the normal group . therefore , the chemotherapeutic agent 5 - fu and cisplatin does not protect against liver cancer cell from destroy , but rather to increase the burden on the liver and accelerate liver damage , however , the pharmaceutical compositions of the present invention has a protection function on liver , and maintain normal liver function , in order to increase the effectiveness against cancer cells . 4 . the effect of the presented pharmaceutical compositions on immune system it is well known that the immune cells and cytokines are involved in inhibit cancer growth . to understand the effect of presented pharmaceutical compositions on immune system , the blood of each group in example 2 was collected at day 14 after administered drugs to analyze the numbers of immune cells and cytokines . please refer to fig3 a - 3e . the rats of normal group were neither implant colon cancer cells nor administered drugs . compare with control group and contrast group , present pharmaceutical composition can effectively increase the numbers of white blood cell ( fig3 a ), lymphocyte ( fig3 b ), monocyte ( fig3 c ), granulocyte ( fig3 d ) and platelet ( fig3 e ). please refer to fig4 a - 4e . the rats of normal group were neither implant with lung adenocarcinoma nor administered drugs . compare with control group and contrast group , present pharmaceutical composition can effectively increase the numbers of white blood cell ( fig4 a ), red blood cell ( fig4 b ), monocyte ( fig4 c ), granulocyte ( fig4 d ) and platelet ( fig4 e ). please refer to fig5 a - 5e . the rats of normal group were neither implant colon cancer cells nor administered drugs . compare with control group and contrast group , present pharmaceutical composition can decrease the expression of human cytokine il - 8 ( fig5 a ), il - 6 ( fig5 b ), il - 1ra ( fig5 c ), tnf - α ( fig5 d ), and can increase the expression of murine cytokine g - csf ( fig5 e ), il - 12 ( p70 ) ( fig5 f ) and human cytokine il - 12 ( p70 ) ( fig5 g ). please refer to fig6 a - 6d . the rats of normal group were neither implant lung adenocarcinoma nor administered drugs . compare with normal group and control group , present pharmaceutical composition can decrease the expression of human cytokine il - 6 ( fig6 a ), tnf - α ( fig6 b )), and can increase the expression of murine cytokine g - csf ( fig6 c ) and human cytokine il - 12 ( p70 ) ( fig6 d ). according to above results , the pharmaceutical compositions of the present invention have ability to improve the autoimmunity of cancer patients . furthermore , a common side effect of chemotherapy is a drop in the number of white blood cells , which leads to an increased risk of getting an infection . currently , granulocyte colony stimulating factor ( g - csf ) treatment can make white blood cell levels go up faster . however , patients may have one or more side effects of using g - csf . to understand the effect of present pharmaceutical composition on white blood cells , cyclophosphamide - induced leukopenia experiment execute as follows : 6 to 7 week - old female rats ( 20 - 24 grams , icr ) were divided into normal group , disease group , contrast group and experimental group . in normal group , rats were not gave any medication . in disease , contrast and experimental group , rats were administered cyclophosphamide on day 0 to induce leukopenia . besides , in contrast group , rats were also administered g - csf on day 2 to day 4 . in experimental group , rats were treated with presented pharmaceutical composition for 5 days before administered cyclophosphamide , and continuously treated for 10 days after administered cyclophosphamid . therefore , the total days of presented pharmaceutical composition administered in experimental group is 15 days . the administered method of presented pharmaceutical composition is twice daily , and the dosage was 0 . 5 - 1 . 67 ul / mouse . the rats in each group were phlebotomized on day 0 before chemotherapy , and on day 4 , day 7 and day 10 after chemotherapy . please refer to table 1 . compared to normal group , the numbers of white blood cell are extremely reduced in disease group , means cyclophosphamid can induce leucopenia . in contrast group , the numbers of white blood cell increase on day 7 and day 10 . however , compared to contrast group , the experimental group recover white blood cells quickly after chemotherapy . also , the mice in experimental group not observe have any side effects . the results show that the pharmaceutical composition of the present invention is better than g - csf , and have great positive effects on mice . cancer pain is a kind of pain caused by cancer , which is most common symptoms and most feared symptoms for patients . if the pain is not controlled properly , the life quality of patients would be affected severely . to understand the effect of presented pharmaceutical composition on cancer pain , the experiment was following : 6 to 7 week - old female rats ( 20 - 24 grams , icr ) were divided into control group , contrast group and experimental group . in control group , rats were not gave any medication . in contrast group and experimental group , rats were administered taxol ® ( 20 mg / kg ) from day 0 to day 5 to induce the pain . besides , the experimental group were also treated with presented pharmaceutical composition for 7 days before administered taxol ®, and continuously treated for 14 days after administered taxol ®. therefore , the total days of presented pharmaceutical composition administered in experimental group is 21 days . the administered method is twice daily , and the dosage was 0 . 5 - 1 . 67 ul / mouse . on day 3 , 7 and 14 during the drug administration , the tail flick latency ( tail - flick unit 37360 ) was conducted . the tail flick latency is used to evaluate the degree of change of pain threshold by heating the tail of rat through infrared . please refer to fig7 . the seconds of the contrast group were lower , means taxol ® would leading increase of the pain sensitivity of rats . however , compared to contrast group , the seconds of the experimental group were increase in day 3 , day 7 and day 14 . accordingly , the pharmaceutical compositions of the present invention have ability to reduce the cancer pain . organ hypertrophy is common side effect induced by chemotherapy drugs . to understand the impact of the presented pharmaceutical compositions on organs , the female rats of example 5 were sacrificed on day 15 . the heart , liver , spleen and kidney acquired from rats were weighted respectively . please refer to fig8 a - 8d . in contrast group , the heart ( fig8 a ), liver ( fig8 b ), spleen ( fig8 c ) and kidney ( fig8 d ) was significantly heavier than control group . however , in experimental group , the weight of heart ( fig8 a ), liver ( fig8 b ), spleen ( fig8 c ) and kidney ( fig8 d ) was significantly decreased , even lower than control group . thus , the pharmaceutical compositions of the present invention could protect the organs from the side - effects caused by chemotherapeutic drugs . although the present invention has been described in terms of specific exemplary embodiments and examples , it will be appreciated that the embodiments disclosed herein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims .
the present invention provides a pharmaceutical composition comprising a mushroom , a rhizome , a fruit , a leaf , a flower , an alga , an energy - rich liquid , a salt - rich liquid , an assist agent and an anti - oxide agent . said pharmaceutical composition has ability to protect liver , improve autoimmunity , reduce pain caused by cancer , protect an organ from the side - effects caused by chemical cancer drugs and increase the functions of chemical cancer drugs .