Ehrlich ascites carcinoma is a spontaneous murine mammary adenocarcinoma adapted to ascites form and carried in outbred mice by serial intraperitoneal (i/p) passages. The previous work from our laboratory showed that honey having higher phenolic content was potent in inhibiting colon cancer cell proliferation. In this work, we extended our research to screen the antitumor activity of two selected honey samples and eugenol (one of the phenolic constituents of honey) against murine Ehrlich ascites and solid carcinoma models. Honey containing higher phenolic content was found to significantly inhibit the growth of Ehrlich ascites carcinoma as compared to other samples. When honey containing higher phenolic content was given at 25% (volume/volume) intraperitoneally (i/p), the maximum tumor growth inhibition was found to be 39.98%. However, honey was found to be less potent in inhibiting the growth of Ehrlich solid carcinoma. On the other hand, eugenol at a dose of 100 mg/kg i/p was able to inhibit the growth of Ehrlich ascites by 28.88%. In case of solid carcinoma, eugenol (100 mg/kg; i/p) showed 24.35% tumor growth inhibition. This work will promote the development of honey and eugenol as promising candidates in cancer chemoprevention.
The previous work from our laboratory showed that honey could induce apoptosis in colon cancer cell lines. In the present studies, four honey samples named as Sample A, B, C, and D of different origins were investigated for their phenolic content. Among them, Sample C showed higher phenolic content of 65.06 Gallic acid equivalent (GAE), per 100 g of honey followed by Sample A (60 GAE), Sample D (47.10 GAE) and Sample B (29.96 GAE). The apoptotic potential of honey against colon cancer cells was found to vary among the samples depending upon the phenolic content. Sample C showing higher phenolic content was found to be more potent in inhibiting the cancer cell proliferation as compared to other samples [
Ehrlich ascites carcinoma is a spontaneous murine mammary adenocarcinoma [
Several researchers have investigated the effect of crude honey on cancer. Research conducted on the antitumor activity of honey showed that it possesses moderate antitumor and significant antimetastatic effects in five different kinds of rat and mouse tumors. Moreover, the antitumor activity of certain chemotherapeutic drugs such as 5-fluorouracil and cyclophosphamide was facilitated by honey [
This work reports the results of antitumor effects of the selected honey samples and eugenol against Ehrlich ascites and solid carcinoma. This work will determine whether the amount of phenolic constituent will have any effect on the antitumor activity of honey against Ehrlich ascites and solid carcinomas.
Ehrlich ascites carcinoma (EAC) cells were collected from the ascitic fluid of BALB/c mice harbouring 8–10 days old ascitic tumor.
Group I: Control (0.2 mL of saline; i/p) from day 1 to 9,
Group II: 0.2 mL of 25% (v/v) solution of Sample B in normal saline injected consecutively from day 1 to 9 (i/p),
Group III: 0.2 mL of 25% (v/v) solution of Sample C in normal saline injected consecutively from day 1 to 9 (i/p),
Group IV: Eugenol (80 mg/Kg; i/p) injected consecutively from day 1 to 9,
Group V: Eugenol (100 mg/Kg; i/p) injected consecutively from day 1 to 9,
Group VI: Eugenol (125 mg/Kg; i/p) injected consecutively from day 1 to 9,
Group VII: 5-Flurouracil (20 mg/Kg; i/p) injected consecutively from day 1 to 9.
The first group administered normal saline (0.2 mL, i/p) from day 1 to 9 was the tumor bearing control; whereas seventh group treated with 5-fluorouracil served as positive control. On day 12, animals were sacrificed and ascitic fluid was collected from peritoneal cavity of each mouse for the evaluation of tumor growth. Percent tumor growth inhibition was calculated by comparing the total number of tumor cells present in the peritoneal cavity of treated groups and the control group as on day 12 of the experiment. Tumor cell growth in saline-treated control group was taken as 100 percent cell growth.
For honey samples, 25% (volume/volume) solutions were prepared in normal saline and 0.2 mL of these solutions was administered to each mouse intraperitoneally every day at 2.00 PM from days 1 to 9. For Eugenol, its dose (in mg) was divided by its specific gravity (1.06) to arrive at its volume to be used in dose preparation. The required volume was emulsified in distilled water by using Tween 80 (0.5% of the total volume). The emulsion was prepared in such a way that the required daily dose was contained in 0.2 mL of the emulsion. 0.2 mL of this emulsion was administered to each mouse intraperitoneally, daily at 2.00 PM from day 1 to 9.
Ehrlich ascites carcinoma (EAC) cells were collected from the ascitic fluid of BALB/c mice harbouring 8–10 days old ascitic tumor.
Group I: Control (normal saline (0.2 mL, i/p) injected consecutively from day 1 to 9),
Group II: 0.2 mL of 50% (v/v) solution of Sample C in normal saline injected consecutively from day 1 to 9 (i/p),
Group III: Eugenol (100 mg/Kg; i/p) injected consecutively from day 1 to 9,
Group IV: 5-Flurouracil (20 mg/Kg; i/p) injected consecutively from day 1 to 9.
The first group administered normal saline (NS) (0.2 mL, i/p) was the tumor bearing control and the fourth group treated with 5-fluorouracil served as positive control. On days 9 and 13, tumor bearing thigh of each animal was shaved and longest and shortest diameters of the tumor were measured with the help of vernier caliper. Tumor weight of each animal was calculated using the following formula:
For honey sample, 50% (volume/volume) solution was prepared in normal saline and 0.2 mL of this solution was administered to each mouse intraperitoneally daily at 2.00 PM from days 1 to 9. For Eugenol, its dose (in mg) was divided by its specific gravity (1.06) to arrive at its volume to be used in dose preparation. The required volume was emulsified in distilled water by using Twin 80 (0.5% of the total volume). The emulsion was prepared in such a way that the required daily dose was contained in 0.2 mL of the emulsion. 0.2 mL of this emulsion was administered to each mouse intraperitoneally, daily at 2.00 PM from day 1 to 9.
Body weights of animals were measured regularly on predetermined days. The body weight at the end of day 12 was more compared to the day 1 in all the experimental animals except in case of 5-FU treatment where it showed a decrease. Moreover 125 mg/kg of eugenol dose was found to be toxic to animals. While comparing the body weight of saline control on day 12 with Sample C and Eugenol treated animals, a decrease in body weights was observed indicating these samples to have effect on the body weights of treated animals (Table
Body Weights of the Animal after the Treatment.
Treatment | Dose | Body weight (g) | |||
Day 1 | Day 5 | Day 9 | Day 12 | ||
Control | 0.2 mL NS i/p | 22.75 | 25.37 | 25.62 | 27.00 |
Sample B | 25% (v/v) i/p | 22.71 | 24.57 | 24.57 | 27.28 |
Sample C | 25% (v/v) i/p | 22.85 | 24.66 | 24.83 | 25.33 |
Eugenol | 80 mg/kg i/p | 22.71 | 23.28 | 24.66 | 25.91 |
Eugenol | 100 mg/kg i/p | 22.85 | 23.82 | 24.28 | 25.33 |
Eugenol | 125 mg/kg i/p | 22.57 | 19.00 | 19.66 | Animals died |
5-FU | 20 mg/kg i/p | 23.00 | 22.57 | 21.85 | 20.42 |
Further more, average volume of ascitic fluid was measured by scarifying all the animals at the end of day 12. Volume collected from the Sample C treated animals (3.41 mL) was found to be less compared to other treatment groups. The number of tumor cells present in the ascitic fluid of each treatment group was also counted. It was observed that Sample C treated animals had only
Percentage of Tumor Inhibition of Ehrlich Ascites after Treatment.
Treatment | Dose | Day 12 | |||||
Avg. volume of ascitic fluid (mL) | Avg. weight of ascitic fluid (g) | Avg. No. of tumor cells | % Tumor cell growth | % Tumor growth inhibition | Mortality | ||
Control | 0.2 mL NS i/p | 7.64 | 8.16 | 94.47 | 100.00 | 00.00 | 0/10 |
Sample B | 25% (v/v) i/p | 7.01 | 7.33 | 82.97 | 87.83 | 12.17 | 0/7 |
Sample C | 25% (v/v) i/p | 3.41 | 3.66 | 56.70 | 60.02 | 0/7 | |
Eugenol | 80 mg/kg i/p | 7.02 | 7.46 | 84.09 | 79.44 | 20.56 | 0/7 |
Eugenol | 100 mg/kg i/p | 9.88 | 9.86 | 75.28 | 71.12 | 0/7 | |
Eugenol | 125 mg/kg i/p | All animals died by 10th day | Intolerable | 7/7 | |||
5-FU | 20 mg/Kg i/p | 0.40 | 0.38 | 05.23 | 05.54 | 94.46 | 0/7 |
Similar to ascites model, the body weights of animals were measured on predetermined days. The body weights taken on day 12 did not show any decrease in any of the experimental groups as compared to day 1 weights. However, day 12 body weights of animals treated Sample C and Eugenol were quite lesser as compared to saline treated control indicating these samples to have an effect on the body weights. Further more, average tumor weights calculated on day 12 were found to be considerably less in Eugenol (1412.64 mg) and Sample C (1786.14 mg) treated groups as compared to 1867.35 mg in saline-treated control. The growth inhibition of Ehrlich tumor (solid) was found to be 24.35 and 4.34% for Eugenol and Sample C treated animals, respectively (Table
Average Weights and Tumor Inhibition of Ehrlich Solid Tumor after Treatment.
Treatment Groups | Avg. Body weights (g) of animals on days | Day 13 | %Tumor growth inhibition | Mortality | |||
1 | 5 | 9 | Avg. body weights (g) | Avg. tumor weights (mg) | |||
Eugenol (100 mg/kg i/p) | 19.57 | 21.0 | 19.71 | 20.57 | 1412.64 | 24.35 | 0/7 |
Sample C (50% v/v i/p) | 19.85 | 20.71 | 20.71 | 21.57 | 1786.14 | 4.34 | 0/7 |
Positive control 5 FU (22 mg/kg i/p) | 21.14 | 21.71 | 21.16 | 21.33 | 1275.5 | 31.69 | 0/7 |
Normal Control NS (0.2 mL i/p) | 21.25 | 22.71 | 22.42 | 22.71 | 1867.35 | — | 0/10 |
Epidemiological surveys and experimental studies have provided evidence that environmental factors, including dietary substances, play a major role in the incidence of cancer. Our previous studies reported that the apoptotic potential of honey varied according to the source and phenolic content [
We used 25% v/v honey solution against Ehrlich ascites carcinoma model. This concentration was chosen arbitrarily and also to overcome the highly viscous nature of honey. Results showed that honey having higher phenolic content (Sample C) inhibited the growth of Ehrlich ascites more effectively as compared to other sample (Sample B). These findings corroborate well with our earlier results obtained in in vitro apoptosis studies conducted on the colon cancer cells where it was observed that the honey containing higher phenolic content was able to inhibit the cancer cell proliferation remarkably as compared to other samples [
In case of Eugenol, we had chosen three arbitrary concentrations of 80, 100, and 125 mg/kg based on the recent anticancer work done against melanoma [
Our studies confirmed that the honey with higher phenolic content inhibits the growth of Ehrlich ascites more effectively. Hence antitumor effect against Ehrlich ascites could be attributed to its phenolic content and its antioxidant ability, since Sample C showed significant effect as compared to Sample B. In case of solid Ehrlich tumor honey was found to be less potent. This may occur because of more resistant nature of Ehrlich tumor (solid) and its inhibition requiring systemic effect. Eugenol had shown remarkable inhibition of both Ehrlich ascites and solid tumor. We surmise that mitochondrial and ROS-mediated apoptotic mechanism observed in the antiproliferative activity of honey might have a role in this antitumor activity [
Jaganathan acknowledges I.I.T Kharagpur for his research fellowship for doing this study. All authors acknowledge Ms. Bhuvaneswari S. for formatting and checking this paper.