Screening of QHF formula for effective ingredients from Chinese herbs and its anti-hepatic cell cancer effect in combination with chemotherapy

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1 Chinese Medical Journal 2008; 121(4): Original article Screening of QHF formula for effective ingredients from Chinese herbs and its anti-hepatic cell cancer effect in combination with chemotherapy CHEN Tao, LI Dan, FU Ya-ling and HU Wei Keywords: effective ingredients; hepatic cell cancer; uniform design; QHF formula; cisplatin H Background Recent studies have shown that effective ingredients of Chinese herbs are used more and more widely in the treatment or co-treatment of cancers, however, they are usually used separately and there has been limited research about joint application of Chinese herbs in multi-modal treatment. The aim of this study was to screen a QHF (Q: Qingrejiedu, H: Huoxuehuayu and F: Fuzhengguben) formula for effective ingredients from Chinese medicines and assess its anti-hepatic cell cancer (HCC) effect in combination with chemotherapy. Methods Six effective ingredients from Chinese medicine were selected based on the previous literature and used in the study. The QHF formula and the best ratio of ingredients were evaluated in H 22 mouse (KM) models with solid tumors and ascites tumors by uniform design and monitoring inhibition of tumor growth and survival. We then observed the anti-hepatic cell cancer (HCC) effect of QHF when combined with cisplatin (DDP) in H 22 mouse (Balb/c) models with solid tumors and ascites tumors. Evaluating of the therapeutic effect included the general condition of the mice, inhibition of tumor growth, survival, changes in body weight, thymus index, spleen index and WBC counts. Results The optimal QHF dose ratio for anti-hepatic cell cancer treatment was: 800 mg/kg Cinobufotalin, 14 mg/kg Ginsenosides Rg3, 5.5 mg/kg PNS and 100 mg/kg Lentinan. Treatment was more efficient in inhibiting the growth of transplanted tumors in H 22 mice when using the QHF formula (55.91%) than using Cinobufotalin (33.25%), Ginsenosides Rg3 (35.11%), PNS (27.12%) or Lentinan (4.97%) separately. QHF also prolonged the life of H 22 ascites hepatic cancer mice more efficiently (38.13%) than Cinobufotalin (25.00%), Ginsenosides Rg3 (27.27%), PNS (23.30%) or Lentinan (24.43%). QHF combined with DDP could reduce DDP-induced leucopenia, spleen and thymus atrophy and other toxic reactions. Combining QHF with DDP the tumor growth inhibition reached 82.54% with a 66.83% increase in survival. Conclusions QHF is more efficient in anti-hepatic cell caner treatment than the single drugs that constitute the formula. QHF combined with DDP can attenuate tumor growth and suppresses the DDP-induced toxic reactions. epatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. Most patients are advanced when diagnosed and their prognosis is poor. 1,2 Chinese medicine plays an important role in the prevention and treatment of HCC in China. 3-5 The prescription is the main form of Chinese medicine. 6,7 Therefore, there is significant in developing new prescriptions with a clear components and mechanism for improving treatment of HCC. Under the guidance of the theories of traditional Chinese medicine we systematically studied the efficiency of combined effective ingredients of Chinese herbs for liver cancer therapy. We applied Qingrejiedu (clear away heat and toxin), Huoxuehuayu (promote blood flow to remove stasis) and Fuzhengguben (strengthen healthy qi and root) as major therapeutic methods (QHF). 8 From the published literature 9-15 and from single drug screening tests, a QHF formula for anti-liver cancer therapy was obtained by uniform design We further observed the anti-hepatic cell cancer (HCC) effect of QHF when combined with chemotherapy. This study was performed to find an effective way to integrate the application of the anti-hepatoma ingredients from Chinese medicine and improve their clinical efficacy. METHODS Chemicals and materials Kunming (KM) mice were used in the screening of QHF. Male g mice were provided by the Experimental Animal Center in Hubei Province (certificate number, SCXK (E) ). Balb/c mice used in the efficacy research, g males, were provided by the Experimental Animal Center in Huazhong University of Science and Technology Tongji University Medical School (certificate number SCXK (E) ). Mouse hepatocellular carcinoma cell line H 22 was purchased from the Shanghai Institute of Material Medica, Chinese Academy of Science. Cinobufotalin was obtained from the Anhui Jinchan biochemical Ltd, Lot No: Ginsenosides Rg3 was from the Jilin Yatai Department of Traditional Chinese Medicine, Three Gorges University Medical College, Yichang, Hubei , China (Chen T, Li D, Fu YL and Hu W) Correspondence to: CHEN Tao, Department of Traditional Chinese Medicine, Three Gorges University Medical College, Yichang, Hubei , China (Tel: Fax: chentao@ctgu.edu.cn)

2 364 Pharmaceutical Co, Ltd, Lot No: PNS was from the Yunnan Weihe Pharmaceutical Co, Ltd, Lot No: Lentinan was purchased from the Zhejiang Jinhua Essen Pharmaceutical Co, Ltd, Lot No: Tanshinone and Norcantharidin were from the Nanjing Qinze Medical Technology Development Co, Ltd (purity: 99%). Cisplatin (DDP) was purchased from the Jinzhou Jiutai Pharmaceutical Co, Ltd, Lot No: Pingxiao Tablets (PX) were from the Shanxi Panlong Pharmaceutical Co, Ltd, Lot No: Animal model H 22 -bearing mice were sacrificed by cervical dislocation 7 days after intra peritoneal inoculation with H 22 tumor cells. The ascitic cells were collected aseptically and stained by Trypan Blue to calculate the number of live tumor cells. H 22 cells were diluted with normal saline and cell concentration was adjusted to /ml. The mice were inoculated with 0.2 ml of tumor cell solution on the right axilla to establish the solid tumor model and the same numbers of the cells were inoculated into the peritoneum to establish the ascites tumor model. Single drug screening Drug selection The drugs used in this study must fulfill the following criteria: chemical constituents and pharmacological mechanism are relatively clear and their efficiency for the treatment of liver cancer has been proven experimentally or clinically. According to these criteria, 6 different drugs were chosen out: Cinobufotalin, Cantharidin, PNS, Tanshinone, Ginsenosides Rg3 and Lentinan. Animal grouping and administration H 22 solid and ascites tumor bearing mice were randomly divided into 7 groups, with 10 mice in each group. Each mouse was given 0.4 ml of drug by ig administration once a day for 10 days after the tumor implantation. (1) NS group: normal saline; (2) Cinobufotalin group: 600 mg/kg; (3) Norcantharidin group: 0.1 mg/ kg; (4) PNS group: 5 mg/kg; (5) Tanshinone group: 9 mg/kg; (6) Ginsenosides Rg3 group: 6 mg/kg; (7) Lentinan group: 120 mg/kg. Inhibition rate of tumor growth Twenty-four hours after the last administration the mice were sacrificed (solid tumor model) by cervical dislocation, the tumors were removed and weighted. Inhibition rate was calculated by the following formula: Inhibition rate (%) = (tumor weight of NS group (g) tumor weight of drug group (g)) / tumor weight of NS group (g) 100%. Survival of mice After continuous administration of therapeutics for 10 days we observed the condition of mice with ascitic tumors and recorded their survival. The survival rate was calculated according to the following formula: Survival (%) = (average survival in days of drug group average survival in days of the NS group)/average survival in days of the NS group 100%. Initially determined composition of the QHF formula According to the results from single drug screening and application of the three treatment principles of Chinese medicine for liver cancer; four drugs with a remarkable efficiency compared with the NS group were selected: one drug for Qingrejiedu (Cinobufotalin), one drug for Huoxuehuayu (PNS) and two drugs for Fuzhengguben (Ginsenosides Rg3 and Lentinan). These four drugs comprised the initial QHF formula. Drug proportion Factors and levels The four drugs were evaluated, each at five levels (ten groups in total). The range of doses depends on the literature 19,20 and the clinical dose range (Table 1). Table 1. Evaluation factors and levels of the 4 drugs Levels (mg/kg) Cinobufotalin Ginsenosides Rg3 PNS Lentinan Uniform design As shown in Table 2, we choose uniform design table U10* (10 8 ) and take the first, third, fourth and fifth lines to arrange the factors and levels. 16 Groups Table 2. Uniform design table Cinobufotalin Ginsenosides Rg3 PNS (mg/kg) (mg/kg) (mg/kg) Lentinan (mg/kg) 1 1(600) 2(8) 2(7) 3(120) 2 1(600) 3(10) 4(11) 5(160) 3 2(800) 5(14) 1(5) 2(100) 4 2(800) 1(8) 3(9) 5(160) 5 3(1000) 2(10) 5(13) 2(100) 6 3(1000) 4(12) 1(7) 4(140) 7 4(1200) 5(14) 3(9) 1(80) 8 4(1200) 1(8) 5(13) 4(140) 9 5(1400) 3(10) 2(7) 1(80) 10 5(1400) 4(12) 4(11) 3(120) Inhibition of tumor growth and survival of mice Mice bearing solid tumor were randomly divided into 11 groups and ten groups were given drugs by ig administration once a day for 10 days according to the uniform design above. One negative control group was given saline by ig administration. Tumor growth was calculated as described above. Another experiment with the same 11 groups was used to assess survival. Verification test of QHF formula The QHF formula components and the optimal dose ratio obtained above were used for a verification test. Mice bearing both solid and ascitic tumors were randomly divided into 6 groups: NS, QHF formula, Cinobufotalin (800 mg/kg), Ginsenosides Rg3 (14 mg/kg), PNS (5.5 mg/kg), and Lentinan groups (100 mg/kg). The drugs were diluted to the required concentration with NS and 0.4 ml administrated ig once a day for 10 days. The tumor

3 Chinese Medical Journal 2008; 121(4): growth inhibition and survival were assayed the same as above. Studies on anti-hepatic cell cancer effect of QHF combined with DDP Animal grouping and administration H 22 solid and ascites tumor bearing Balb/c mice were both randomly divided into 5 groups: NS group (Control group), DDP group, PX group, QHF group and QHF+DDP group. The DDP group was given 0.1 ml DDP by intraperitoneal injection every other day a total of five times and given 0.4 ml saline by ig administration once a day for 10 days. The remaining drug groups and the control group were given 0.4 ml of drug by ig administration once a day for 10 days concurrently they were given 0.1 ml saline by intraperitoneal injection every other day. Drug doses were as following: DDP 3 mg/kg, Cinobufotalin 800 mg/kg, Ginsenosides Rg3 14 mg/kg, PNS 5.5 kg and Lentinan 100 mg/kg. Inhibition of tumor growth and survival of mice We closely observed the tumor growth and general condition of mice during the entire experiment. The inhibition of tumor growth and survival were calculated as described above. Weight of mice Twenty-four hours after the last administration of drugs the ascites tumor implanted mice were weighted, and their changes of weight during treatment were computed. Counting of WBC Before killing the mice with ascites tumors we took blood from the tail vein to perform a conventional white blood cell count. Thymus and spleen index Twenty-four hours after the last drug administration, the ascites tumor bearing mice were sacrificed by cervical dislocation. We removed the thymus and spleen and weighed them on an electronic balance. The thymus or spleen index was calculated by the following formula: thymus (spleen) index = weight of thymus (spleen) (mg)/weight of mouse (g). Data processing and statistical analysis The data were analyzed by SPSS 10.0 software. Measurement data used single-factor analysis of variance and the enumeration data used the chi-square test. The DPS data processing system was used to carry out the quadratic polynomial regression analysis of the data to determine the optimal dose ratio. A P value < 0.05 was considered statistically significant. RESULTS Single drug screening Inhibition rates of tumor growth in mice by 6 single drugs The results showed that Cinobufotalin, Norcantharidin, PNS and Ginsenosides Rg3 could significantly inhibit the growth of tumor. The tumor growth was inhibited by 34.90%, 27.93%, 24.25% and 36.46%, respectively (Table 3). Table 3. Inhibition of tumor growth in mice (mean ± SD) Groups Dose (mg/kg) n Weight of tumor(g) Inhibition (%) NS ± Cinobufotalin ± ** Norcantharidin ± ** PNS ± ** Tanshinone ± Ginsenosides Rg ± ** Lentinan ± ** P<0.01 vs the NS group. NS: normal saline group. Survival of mice treated with 6 single drugs Cinobufotalin, PNS, Ginsenosides Rg3 and Lentinan significantly extended the survival of the mice. Increased survival compared to NS treated control animals was 32.92%, 29.81%, 32.30% and 35.40%, respectively (Table 4). Table 4. Survival time of the tumor-bearing mice (mean ± SD) Groups Dose (mg/kg) n Survival time (days) % survival NS ±2.3 Cinobufotalin ±1.6 ** Norcantharidin ± PNS ±1.7 ** Tanshinone ± Ginsenosides Rg ±1.6 ** Lentinan ±1.8 ** ** P<0.01 vs the NS group. NS: normal saline group. As shown in the Tables 3 and 4, 4 out of the 6 drugs proved to be efficient in inhibiting tumor growth and prolonging the survival of tumor bearing animals. They were Cinobufotalin for Qingrejiedu, PNS for Huoxuehuayu, Ginsenosides Rg3 and Lentinan for Fuzhengguben. These four drugs were used to comprise the initial QHF formula in the following study. Drug proportion Using inhibition of tumor growth as evaluating factor Uniform Design was used to work out the optimal drug proportion for QHF when inhibition of tumor growth was used as the evaluating factor. The results in Table 5 were assayed by the DPS data processing system for quadratic polynomial regression and the following regression equation was obtained: Y= X X X X 1 X X 1 X X 2 X X 3 X 4 ; correlation coefficient: R= , F= , P=0.0242, S= , R a = According to this equation the optimal drug doses in QHF was determined to be: Cinobufotalin mg/kg, Ginsenoside Rg3 314 mg/kg, PNS mg/kg, Lentinan mg/kg, the inhibition of tumor growth for this QHF formula was 56.98%. The sequence of correlation was X 2 >X 1 > X 4 >X 3. Using survival as the evaluating factor Uniform Design was also used to work out the optimal drug doses for QHF when survival was used as evaluating factor. The results in Table 6 were assayed by the DPS

4 366 Table 5. Uniform design for drug proportion using inhibition rate as evaluating factor Groups Cinobufotalin Ginsenosides Ginsenoside Lentinan Inhibition (mg/kg) Rg3 (mg/kg) (mg/kg) (mg/kg) (%) 1 1(600) 2(8) 2(7) 3(120) (600) 3(10) 4(11) 5(160) (800) 5(14) 1(5) 2(100) (800) 1(8) 3(9) 5(160) (1000) 2(10) 5(13) 2(100) (1000) 4(12) 1(7) 4(140) (1200) 5(14) 3(9) 1(80) (1200) 1(8) 5(13) 4(140) (1400) 3(10) 2(7) 1(80) (1400) 4(12) 4(11) 3(120) data processing system for quadratic polynomial regression and the following the regression equation was obtained: Y= X X X X 1 X X 1 X X 3 X X 2 X 3 ; correlation coefficient: R= , F= , P=0.0226, S= , R a = According this equation the optimal drug doses for QHF was determined to be: Cinobufotalin mg/kg, Ginsenoside Rg3 314 mg/kg, PNS mg/kg and Lentinan mg/kg. The increased survival associated with this formula was 37.99%. The sequence of correlation was X 2 >X 1 > X 4 >X 3. Table 6. Uniform design for drug proprotion using survival as the evaluating factor Groups Cinobufotalin Ginsenosides Ginsenoside Lentinan (mg/kg) Rg3 (mg/kg) (mg/kg) (mg/kg) % Survival 1 1(600) 2(8) 2(7) 3(120) (600) 3(10) 4(11) 5(160) (800) 5(14) 1(5) 2(100) (800) 1(8) 3(9) 5(160) (1000) 2(10) 5(13) 2(100) (1000) 4(12) 1(7) 4(140) (1200) 5(14) 3(9) 1(80) (1200) 1(8) 5(13) 4(140) (1400) 3(10) 2(7) 1(80) (1400) 4(12) 4(11) 3(120) Determination of the optimal dose ratio (Ratio 1) Combining the results in the inhibition of tumor growth and survival in mice, the optimal dose ratio of QHF formula was determined to be: Cinobufotalin 800 mg/kg, Ginsenosides Rg3 14 mg/kg, PNS 5.5 mg/kg, Lentinan 100 mg/kg. Validation of the QHF formula with optimal dose ratio As shown in Tables 7 and 8 the inhibition of tumor growth and survival were higher in the QHF group than in the groups treated with the single drugs that comprise the QHF formula (P<0.01). Anti-hepatic cell cancer effect of QHF combined with DDP General condition of mice (solid tumor model) and tumor growth inhibition Within 5 days of tumor inoculation there were no significant changes in the appearance of the mice: except for 1 hour daily after gavage when the mice were still, Table 7. Validation of the QHF formula by inhibition of tumor growth in mice as the evaluating factor (mean ± SD) Groups Dose (mg/kg) n Weight of tumor Inhibition (%) NS ± QHF Ratio ± ** Cinobufotalin ± ** Ginsenosides Rg ± ** PNS ± ** Lentinan ± ** P<0.01 vs NS group. Table 8. Validation of QHF formula with survival as the evaluating factor (mean ± SD) Groups Dose (mg/kg) n Survival time % Survival NS ±1.08 QHF Ratio ±1.34 ** Cinobufotalin ±1.76 ** Ginsenosides Rg ±2.22 ** PNS ±2.06 ** Lentinan ±1.60 ** ** P<0.01 vs NS group. crowded together and then gradually returned to normal. One week later we could palpate the subcutaneous tumor mass on the right axillary of mice in each group. The tumor in control group grew rapidly with obvious local swelling and pressure symptoms. Tumors in mice in the treated group grew slowly, matt elliptical, a significant vertical hair phenomena, with a reduction of activity, but clearly better than the control group. Mice in the DDP group had obvious toxic reactions such as anorexia, diarrhea, emaciation, superficial peeling of hair. Mice in QHF+DDP group had no obvious toxicity or side effects, they had no significant emaciation and their activity and feeding appeared normal. QHF, QHF+DDP and DDP treatment could significantly inhibit tumor growth (P<0.01). The tumor growth inhibition was 55.53%, 82.54%, and 74.28%, respectively (Table 9). Table 9. Inhibition of tumor growth in mice (mean ± SD) Groups Dose (mg/kg) n Weight of tumor (g) Inhibition (%) NS ±0.118 ## PX ±0.098 **## QHF Ratio ±0.113 **## QHF+DDP Ratio ±0.061 ** DDP ±0.056 ** ** P<0.01 vs NS group; ## P<0.01 vs DDP group. General condition and survival of mice After 6 days of tumor inoculation, we observed the formation of abdominal ascites, the presence of significant vertical hair phenomena and for reduced and slow activity in the NS and PX groups. Symptoms steadily increased from day 6 with the PX group showing fewer symptoms than the NS group. After 8 days of inoculations mice in the DDP and QHF groups were also observed to have developed abdominal ascites and tumor grew more slowly. Mice in the DDP group became emaciated and lacked energy. Ascites formed in mice of the QHF+DDP group only after stopping treatment. Tumors grew slowly, the mice had no significant weight

5 Chinese Medical Journal 2008; 121(4): loss and their activity and feeding appeared normal. But 5 days after the treatment was stopped, they began to appear less active and food intake decreased, significant vertical hair phenomena was observed and fast increase of the ascites was seen. The QHF and QHF+DDP groups had extended survival (P<0.01). Survival increased by 38.46% and 87.02%, respectively. The survival of the QHF+DDP group was better than in the DDP group (P<0.01, Table 10). Table 10. Survival of mice in each group (mean ± SD) Groups Dose(mg/kg) n Survival time (days) % Survival NS ±2.440 ## PX ±2.846 ** QHF Ratio ±2.821 **## QHF+DDP Ratio ±1.969 **## DDP ±1.564 ** ** P<0.01 vs NS group; ## P<0.01 vs DDP group. Influence of treatment on body weight of mice The body weight of mice was significantly increased in the QHF group (P<0.01), but lower in the DDP group (P<0.01). The average body weight of the QHF+DDP group was obviously higher than in the DDP group (P<0.01). It showed that QHF had a significant role in maintaining the body weight of mice during chemotherapy (Table 11). Table 11. Influence of treatment on the body weight of tumor bearing mice (mean ± SD) Groups Dose (mg/kg) n Increase in body weight (g) NS ±0.393 ## PX ±0.426 *## QHF Ratio ±0.348 **## QHF+DDP Ratio ±0.465 *## DDP ±0.213 ** * P<0.05, ** P<0.01 vs NS group; ## P<0.01 vs DDP group. Influence on WBC in peripheral blood The WBC counts in the PX, DDP and QHF+DDP treatment groups were significantly lower than in the NS group (P<0.01), while WBC count in QHF+DDP group was higher than that in DDP group (P<0.05, Table 12). Table 12. Influence on the number of WBC of mice (mean ± SD) Groups Dose (mg/kg) n Number of WBC ( /L) NS ±1.815 ## PX ±1.821 ** QHF Ratio ±0.948 ## QHF+ DDP Ratio ±0.976 **# DDP ±0.936 ** ** P<0.01, vs NS group; # P<0.05, ## P<0.01 vs DDP group. Influence on the weight of immune organs of mice Only the QHF group showed a raise in the weight of the thymus and spleen in tumor-bearing mice (P<0.05), while the DDP group had a significantly lower thymus weight (P<0.01) and spleen weight (P<0.05). From the comparison between the DDP and QHF+DDP groups we found that QHF could increase the weight of the thymus (P<0.01) and spleen (P<0.05) in mice during chemotherapy (Table 13). Table 13. The index of thymus and spleen of mice (mean ± SD) Groups Dose Index of thymus Index of spleen n (mg/kg) (mg/g) (mg/g) NS ±0.120 ## 7.960±1.826 ## PX ±0.194 ## 9.020±2.076 ## QHF Ratio ±0.156 *## ±1.833 *## QHF+DDP Ratio ±0.130 ## 9.910±1.986 # DDP ±0.080 ** 5.598±1.059 * * P<0.05, ** P<0.01 vs NS group; # P<0.05, ## P<0.01 vs DDP group. DISCUSSION According to the etiology, pathogenesis and clinic experience combined with pharmacological research of effective ingredients in Chinese traditional medicine for liver cancer treatment, 8,21-23 the first step is screening multiple drugs with the function of Q (Qingrejiedu), H (Huoxuehuayu) and F (Fuzhengguben) to compose an anti-cancer QHF formula. In order to perform a more comprehensive and full analysis for the efficiency of the formula we used Uniform Design to optimize the program. Polynomial regression and optimization results showed that the best components and ratios for QHF was 800 mg/kg Cinobufotalin, 14 mg/kg Ginsenosides Rg3, 5.5 mg/kg PNS and 100 mg/kg Lentinan; with the inhibition of tumor growth and survival as evaluation factors. Correlation analysis showed that Ginsenosides Rg3 and Cinobufotalin had the greater impact on the results. It can also be seen from the regression equation that there were positive interactions between Ginsenosides Rg3 and Cinobufotalin, PNS and Lentinan and there was negative interactions between PNS and Cinobufotalin, PNS and Ginsenosides Rg3. On the basis of the above and in order to find the best therapeutic effect on tumors, we studied the anti-hcc effect of QHF combined with the chemotherapy drug DDP. In vivo, we found QHF combined with DDP could reduce the DDP-induced toxicity; such as anorexia, diarrhea, emaciation, weight loss, leucopenia and the shrinkage of the thymus and spleen. QHF increased the anti-tumor effect of DDP; tumor growth inhibition was increased by 8.26% and reached 82.54%, survival increased by 66.83% and reached 87.02%. These results show a potential synergistic anti-tumor effect of QHF combined with DDP that is worthy of further study to design a safe and effective clinical trial for the human application of QHF. REFERENCES 1. Yu AS, Keeffe EB. Management of hepatocellular carcinoma. Rev Gastroenterol Disord 2003; 3: Di Maio M, De Maio E, Perrone F, Pignata S, Daniele B. Hepatocellular carcinoma: systemic treatments. J Clin Gastroenterol 2002; 35(5 Suppl 2): S109-S Tang ZY. Current situation and prospects of management of hepatocellular carcinoma. Basic Clin J Chin Gen Surg (Chin)

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