A novel antitumor agent was developed from six kinds of herbs containing Rhus verniciflua (Rv-PEM01). The components were traditionally established for each formula for traditional medicine. The formula was designed to affect antitumor effect as well as maintain host immune functions. First, we investigated the antiproliferative activities of Rv-PEM01 on human and canine tumor cell lines in vitro, and on antitumor effects using BALB/cAJcl-nu/nu mice in vivo. Acute oral toxicity of Rv-PEM01 was also investigated in vivo in ddY mice. Rv-PEM01 exhibited antiproliferative activities against PC-3 (IC<sub>50</sub>: 0.328 ± 0.081 mg/ml), A549 (IC 50: 0.520 ± 0.070 mg/ml), D-17 (IC 50: 0.124 ± 0.037 mg/ml) and MRC-5 (IC 50: 0.505 ± 0.058 mg/ml) cells. Luteolin 7- β-D-glucopyranoside and apigenin 7- β-D-glucopyranoside were identified as the main active compounds in Rv-PEM01 by HPLC analysis. The single dose toxicity study of Rv-PEM01 did not result in any deaths or abnor-malities in daily behavior, body weight gain, or anatomical observations at necropsy. Thus, so we could not calculate the 50% lethal dose (LD 50) in mice, but it would be higher than 5.0 g/kg. Treat- ment with Rv-PEM01 at a dose of 2.5 g/kg tended to show antitumor activities on mice bearing Colon26 tumors compared with the control group. It was concluded that the formula was a safe antitumor agent with no side effects on mouse physiological function as judged by survival and organ weight.
Many phytochemicals from fruits, vegetables, and herbs which have antitumor activities may represent promising therapeutic and prophylactic treatment approaches against different types of cancers. The effects of phytochemicals on inhibition of tumor growth are well demonstrated both in vitro and in vivo. Many of these compounds, such as vinca alkaloids, have been reported to kill cancer cells [
The antitumor properties of medicinal herbs may be attributed to their antioxidant [
The popularity of complementary and alternative medicine (CAM) is an international trend in cancer therapy. Due to the severe side effects and limited therapeutic efficacy of cancer chemotherapeutic agents used in conventional cancer treatment, CAM might improve clinical outcome and reduce adverse reactions to anticancer drugs. The World Health Organization has estimated that 80% of people worldwide are interested in traditional medicine [
We have been analyzing the antitumor activity of plant extracts, as well as their ability to maintain host immune capacity. Rhus verniciflua (R. verniciflua) is commonly known as the lacquer tree. The sap of this tree, which is collected by scratching the bark of the tree, has been used as a natural coating substance for wood car- vings for several thousand years. In Korea, the bark, branch and stem of the tree are eaten with chicken and duck soups [
Rv-PEM01 was prepared using method of Hiruma W., et al. [
Herbs | Amount of herbs used for extraction (g) |
---|---|
Rhus verniciflua | 90 |
Ulmus hollandica | 60 |
Polygonatum sibiricum | 50 |
Lycium chinense | 10 |
Ganoderma japonicum | 10 |
Panax ginseng | 10 |
Total | 230 |
The extract of Rv-PEM01 was prepared as follows. Each of the six herbs was ground and mixed to one powder (total volume is 230 g), which was extracted with 10 vol of 70% ethanol in water at room temperature. The extracted solutions were filtered, and the solvents were evaporated, filtered and lyophilized, yielding 60 g of lyophilized Rv-PEM01 from 230 g of herb powder.
ethanol in water at room temperature. The extracted solution was filtered and the solvent evaporated, after which the extract was lyophilized, yielding approximately 60 g of lyophilized Rv-PEM01. Urushiols were not detected in Rv-PEM01 by HPLC analysis (
Human prostate adenocarcinoma cell line PC-3, human lung adenocarcinoma cell line A549, and human normal lung fibroblast cell line MRC-5 were obtained from Health Science Research Resources Bank (Osaka, Japan). The canine osteosarcoma cell line D-17 was purchased from American Type Culture Collection (Virginia, USA). The PC-3 cell line was maintained in RPMI1640 medium containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml). The A549, MRC-5 and D-17 cell lines were maintained in Dulbecco’s Modified Eagle medium (DMEM) containing 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), d-glucose (1 mg/ml), and sodium pyruvate (110 μg/ml). All cells were maintained in a humidified incubator containing 5% carbon dioxide at 37˚C and cultured with various concentration of Rv-PEM01 for 72 hs.
Analysis of tumor cell growth in vitro was carried out using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo- phenyl)-2H-tetrazolium, monosodium salt (WST-1, DOJINDO, Kumamoto, Japan) and 1-methoxy-5-methyl- phenazinium methylsulfate (1-Methoxy PMS, DOJINDO, Kumamoto, Japan) colorimetric assay [
The Rv-PEM01 was analyzed by HPLC using a Shimadzu LC6A system (Kyoto, Japan) equipped with a SPD- 6AV UV-visible spectrophotometric detector and TSK gel ODS-80TM (7.8 i.d. × 300 mm) column (Tosoh, Tokyo, Japan). The mobile phase consisted of 50% methanol, and flow rate was 0.8 ml/min with UV detection at 254 nm. Measurement of urushiols was performed using the method of Du Y., et al. [
Six male and female seven-week-old ddY mice, were used for the acute oral toxicity study [
BALB/cAJcl-nu/nu mice were used to investigate the antitumor effects of Rv-PEM01 in vivo using the method of Kiyama S., et al. [
The data are expressed as the mean ± standard deviation (SD.). The significance of differences between groups was assessed using the Student’s t-test. P < 0.05 was considered to indicate statistical significance.
The Rv-PEM01 treatment resulted in a dose-dependent decrease in cell growth in all cell lines (
Cell lines | IC50 (mg/ml)* | Cell origin | Reference |
---|---|---|---|
MOLT-3 | 0.208 ± 0.022 | Human tumor cell | Hiruma, et al. [ |
KG-1 | 0.293 ± 0.007 | Human tumor cell | Hiruma, et al. [ |
HeLa | 0.433 ± 0.043 | Human tumor cell | Hiruma, et al. [ |
DLD-1 | 0.510 ± 0.030 | Human tumor cell | Hiruma, et al. [ |
MCF-7 | 0.580 ± 0.054 | Human tumor cell | Hiruma, et al. [ |
K-562 | 0.610 ± 0.141 | Human tumor cell | Hiruma, et al. [ |
Colon26 | 0.389 ± 0.093 | Mouse tumor cell | Hiruma, et al. [ |
B16 | 0.565 ± 0.028 | Mouse tumor cell | Hiruma, et al. [ |
PC-3 | 0.328 ± 0.081 | Human tumor cell | This work |
A549 | 0.520 ± 0.070 | Human tumor cell | This work |
D-17 | 0.124 ± 0.037 | Canine tumor cell | This work |
MRC-5 | 0.505 ± 0.058 | Human normal diploid fibroblast cell | This work |
*Results are means ± SD. (n = 3).
The HPLC chromatogram of Rv-PEM01 is illustrated in
No deaths or abnormalities of body weight, water and food consumption, or coat condition were observed in the treated mice. Necropsy evaluation of the mice did not reveal any significant differences in thymus, liver, spleen, kidney, adrenal gland and testicle weights between the control group and the Rv-PEM01 treatment groups, or between males and females (
No deaths or significant differences in the body weight gain, tumor volume, or tumor size were observed between the control group and Rv-PEM01 treatment group (
In this study, Rv-PEM01 exhibited antitumor activities against human, canine and mouse tumor cell lines in vitro. These results confirmed our previous data, which suggested the main active herb in Rv-PEM01 was R. verniciflua. The branches and the sap from R. verniciflua contain active compounds such as urushiol, fustin, quer- cetin, butein and sulfuretin, and the antioxidant [
Peak No. | MOLT-3 IC50 (mg/ml) | KG-1 IC50 (mg/ml) | Colon26 IC50 (mg/ml) | PC-3 IC50 (mg/ml) | D-17 IC50 (mg/ml) |
---|---|---|---|---|---|
1 | 0.035 ± 0.009 | >0.750 | >0.750 | >0.750 | >0.750 |
2 | 0.010 ± 0.001 | 0.022 ± 0.003 | 0.030 ± 0.005 | 0.023 ± 0.004 | 0.011 ± 0.004 |
3 | 0.063 ± 0.025 | 0.089 ± 0.032 | 0.184 ± 0.025 | 0.182 ± 0.034 | 0.162 ± 0.028 |
4 | 0.013 ± 0.005 | 0.007 ± 0.003 | 0.013 ± 0.002 | 0.011 ± 0.003 | 0.009 ± 0.004 |
5 | 0.055 ± 0.012 | 0.046 ± 0.014 | 0.127 ± 0.030 | 0.073 ± 0.011 | 0.096 ± 0.033 |
*Results are means ± SD. (n = 3).
Dose (g/kg) | Male (n = 6) | Female (n = 6) | ||||
---|---|---|---|---|---|---|
0 | 2.5 | 5.0 | 0 | 2.5 | 5.0 | |
Body weight (g) | 38.0 ± 1.0 | 38.0 ± 1.7 | 38.0 ± 0.9 | 30.3 ± 0.4 | 31.3 ± 1.1 | 30.8 ± 0.5 |
Thymus (mg) | 59.8 ± 13.0 | 68.0 ± 17.4 | 66.7 ± 21.1 | 82.6 ± 20.6 | 78.3 ± 6.7 | 81.2 ± 15.6 |
Liver (g) | 2.1 ± 0.3 | 1.9 ± 0.2 | 2.0 ± 0.2 | 1.6 ± 0.1 | 1.6 ± 0.2 | 1.6 ± 0.1 |
Spleen (mg) | 135.0 ± 15.3 | 159.4 ± 12.6 | 161.7 ± 31.9 | 147.1 ± 28.8 | 170.3 ± 23.2 | 154.6 ± 17.8 |
Left kidney (mg) | 335.1 ± 76.9 | 338.1 ± 41.5 | 333.7 ± 28.2 | 227.0 ± 42.6 | 219.9 ± 23.9 | 206.6 ± 18.7 |
Right kidney (mg) | 280.7 ± 21.0 | 310.6 ± 42.8 | 340.3 ± 52.5 | 227.0 ± 42.6 | 210.1 ± 20.6 | 205.8 ± 18.9 |
Adrenal gland (mg) | 17.4 ± 6.2 | 18.7 ± 6.3 | 15.6 ± 4.3 | 19.7 ± 2.7 | 21.3 ± 3.0 | 20.6 ± 1.5 |
Testicles (mg) | 264.6 ± 25.1 | 273.2 ± 16.3 | 281.5 ± 18.3 | 33.8 ± 12.2 | 41.9 ± 13.2 | 36.7 ± 6.9 |
*Results are means ± SD. (n = 6).
vatives from R. verniciflua were not contained in Rv-PEM01 (
Rv-PEM01 had antiproliferative activities on PC-3, A549, D-17 and MRC-5 cell lines (
HPLC analysis showed five major peaks (
In the acute oral toxicity study, there were no changes attributable to Rv-PEM01 administration at doses of 2.5 or 5.0 g/kg. The 50% mortality rate LD50 could not be calculated, but must be higher than 5.0 g/kg. In the chronic study, we found no adverse effects of Rv-PEM01 at the doses used, suggesting longer-term safety at these doses. Unfortunately, no statistically significant difference of antitumor efficacy from the control group was found, although Rv-PEM01 treatment at dose of 2.5 g/kg resulted in a trend toward antitumor activity compared with control. Additional studies, using different doses and model systems, as well as a study of potential prophylactic antitumor effects of Rv-PEM01 in vivo currently are in progress.
Biological activities of Rv-PEM01, as well as its antitumor effects, safety, and toxicity, were investigated. Rv-PEM01 exhibited antiproliferative activities against PC-3, A549, D-17 and MRC-5 in vitro, and tendency toward tumor growth inhibition in vivo. The main active compounds of Rv-PEM01 were luteolin 7-β-d-gluco- pyranoside and apigenin 7-β-d-glucopyranoside. The safety studies did not identify any adverse reactions in mice. Therefore, it could be useful as a novel functional food material and/or nutritional supplement. Additional studies of the antitumor effects of Rv-PEM01 in vivo are needed.
We are grateful to Chairman and C.E.O. Masahito Hoashi, Kibun Foods Inc., for supporting the present work.