|Year : 2014 | Volume
| Issue : 4 | Page : 1071-1075
Antitumor activity of Pogostemon benghalensis Linn. on ehrlich ascites carcinoma tumor bearing mice
Manish S Patel1, Bhavesh V Antala1, Ena Dowerah2, Raju Senthilkumar3, Mangala Lahkar1
1 Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Guwahati, India
2 Department of Pathology, Gauhati Medical College and Hospital, Guwahati, Assam, India
3 Department of Pharmaceutical Chemistry, Swamy Vivekanandha College of Pharmacy, Elayampalayam, Tiruchengodu, Namakkal, Tamil Nadu, India
|Date of Web Publication||9-Jan-2015|
Manish S Patel
NIPER-Ahmedabad, C/O B. V. Patel, PERD Centre, SG Highway, Thaltej, Ahmedabad - 380 054, Gujarat
Source of Support: None, Conflict of Interest: None
Background: Pogostemon benghalensis has been used traditionally as anticancer in India.
Aim of the Study: The present study was undertaken to investigate the antitumor activity of hydroethanolic extract of P. benghalensis (HEEPB) and aqueous extract of P. benghalensis (AEPB) on ehrlich ascites carcinoma (EAC) tumor bearing mice.
Materials and Methods: For antitumor effect, treatments with HEEPB and AEPB at doses of 250 mg/kg and 500 mg/kg/day orally were started after 24 h of intraperitoneal inoculation of EAC cells. After the treatment period, median survival time (MST), hematological parameters and solid tumor volume were used to evaluate antitumor activity of the extracts.
Results: Both, HEEPB and AEPB, have increased MST (P < 0.05), reduced solid tumor volume (P < 0.05) and normalized hematological parameters (P < 0.05) significantly.
Conclusion: As HEEPB and AEPB were effective in reducing the severity of morphological and biochemical parameters induced by EAC cells, this study justifies the traditional use of this plant in cancer.
广鹬（Pogostemon benghalensis Linn）对艾氏腹水癌荷瘤小鼠的抗肿瘤活性
材料和方法：腹腔接种EAC细胞后24小时开始，分别予每天250 mg/kg和500 mg/kg 的剂量口服HEEPB 和 AEPB来对抗肿瘤。治疗后，中位生存时间（MST），血液学参数和实体肿瘤体积被用来评价提取物的抗肿瘤活性。
结果：HEEPB 和 AEPB都显著增加了MST（P＜0.05），减小了肿瘤体积（P＜0.05）和正常化了血液学参数（P＜0.05）。
结论：HEEPB 和 AEPB减少了EAC细胞诱导的形态学严重程度和生化参数，这项研究证明这种植物的在癌症的传统作用。
Keywords: Ehrlich ascites carcinoma, hook-loti, median survival time, Pogostemon benghalensis, solid tumor
|How to cite this article:|
Patel MS, Antala BV, Dowerah E, Senthilkumar R, Lahkar M. Antitumor activity of Pogostemon benghalensis Linn. on ehrlich ascites carcinoma tumor bearing mice. J Can Res Ther 2014;10:1071-5
|How to cite this URL:|
Patel MS, Antala BV, Dowerah E, Senthilkumar R, Lahkar M. Antitumor activity of Pogostemon benghalensis Linn. on ehrlich ascites carcinoma tumor bearing mice. J Can Res Ther [serial online] 2014 [cited 2020 Feb 28];10:1071-5. Available from: http://www.cancerjournal.net/text.asp?2014/10/4/1071/138014
| > Introduction|| |
Cancer chemoprevention can be defined as the prevention, inhibition or reversal of carcinogenesis by administration of one or more chemical entities, either as individual drugs or as naturally occurring constituents of the diet. The ideal anticancer drug should minimally effective for normal cells. Usage of natural sources as an alternative cancer therapy is thought to have a great value for cancer control. Pogostemon benghalensis (Burm. F.) Ktze. (Lamiaceae) known as "Hook-loti" in Assam, India. Preliminary phytochemical screening showed the presence of flavonoids, alkaloids, saponins, phenolic compounds, terpenoids and steroids in both extracts. The methanol extract of aerial parts of P. benghalensis Ktze syn. Pogostemon plectrathoides Desf. (Lamiaceae) was found to be active concentration of 2 g/ml of dried plant material in disc diffusion assay with chloramphenicol as a positive control against Mycobacterium phlei. Traditionally, leaves of this plant are widely used to treat cancer.  A methanol extract from Pogostemon cablin showed antimutagenic activity against mutagen furylfuramide.  However, no studies to date have been done to demonstrate antitumor activity of P. benghalensis. Ehrlich ascites carcinoma (EAC) model has been used as a transplantable tumor model to investigate the antineoplastic effects of several chemical compounds.  In the present study, the antitumor activity of the leaves of P. benghalensis on to EAC in mice has been evaluated.
| > Materials and Methods|| |
The fresh leaves of the P. benghalensis were obtained from the botanical garden in the month of April-May and authenticated by the Curator of Botanical Museum of the Department of Botany. A voucher specimen, representing this collection, has been retained in our laboratory for future reference.
Preparation of extracts
The leaves were cleaned, shade dried in the open air for 8-10 days and pulverized before extraction. A hydroethanolic extract was prepared by extracting hand crushed hook-loti leaves (210 g) by hot continuous percolation method in a soxhlet apparatus with a mixture of water: ethanol (70:30) at 80°C. This process was repeated 15-16 times for complete exhaustion of the constituents, after which ethanol and water were evaporated by rotary evaporator and all traces of ethanol and water were eliminated by leaving the extract under vacuum overnight and dried in a dessicator (yield 36.9 g, 17.57% w/w).
Aqueous extract was prepared by extracting grinded powder of hook-loti leaves (180 g) in a stoppered container with 500 ml water at room temperature and allowed to stand for a period of at least 3 days in a dark place with frequent agitation, until soluble matter is dissolved. The mixture is filtered and after most of the liquid has drained, the residue on the filter is washed with sufficient quantity of water. At last the filtrates are combined. After which water was evaporated by rotary evaporator and all traces of water were eliminated by leaving the extract under vacuum overnight (yield 29.4 g, 16.33% w/w).
The phytochemical screening were performed to check the presence of flavonoids, alkaloids, saponins, phenolic compounds, terpenoids, steroids, glycosides, and tannins. , Both extracts showed the positive test for flavonoids, alkaloids, saponins, phenolic compounds, terpenoids, and steroids.
Swiss albino mice, 6-8 weeks old weighing 20-25 g, were procured and housed in microlon boxes in a controlled environment (temperature 25°C ± 2°C with 45% relative humidity and 12 h dark/light cycle) with standard laboratory diet and water ad libitum. The project Protocols has been approved by the Institutional Animal Ethical Committee (Date: 10/08/09; Approval No. 03/NIPER/CPCSEA/351).
Acute toxicity studies
The acute oral toxicity study was carried out as per guidelines set by Organization for Economic Co-operation and Development (OECD) guideline 423 (adopted in December 2001). The purpose of the acute toxicity study is to allow selection of the appropriate starting for the main study. Here female Swiss albino mice (8-12 weeks old) were taken for acute toxicity study. The hydroethanolic extract of P. benghalensis (HEEPB) (suspended in 0.3% sodium carboxymethyl cellulose solution, [NaCMC] solution) and aqueous extract of P. benghalensis (AEPB) were administered to a group of three female Swiss albino mouse in a sequential manner (increasing dose levels from starting dose 5 mg/kg to 50, 300, 2000 mg/kg body weight) as per OECD 423. Based on the results, the extracts did not produce any mortality at the doses tested. To optimize the dose levels, 1/8 th (250 mg/kg body weight) and 1/4 th (500 mg/kg body weight) of the maximum dose (2000 mg/kg body weight), given for the acute toxicity study as per OECD 423, were selected for the evaluation.
EAC cells were obtained through the courtesy of Chittaranjan National Cancer Institute, Kolkata, India. They were maintained by weekly intraperitoneal inoculation of 10 6 cells/mouse in female Swiss albino mice.  Ascitic fluids were withdrawn from different tumor bearing Swiss albino mouse and the viability of tumor cells were analyzed by Trypan blue dye (0.4%) exclusion assay method.  Cell samples showing above 90% viability were injected intraperitoneally (10 6 cells/mouse) and were kept for 24 h for the multiplication of EAC cells.
Effect of the HEEPB and AEPB on survival time
Mice were divided into six groups. They were inoculated with 1 × 10 6 cells/mouse on day '0' intraperitoneally and treatments with HEEPB (suspended in 0.3% NaCMC solution to administered orally) and AEPB were started after 24 h of inoculation, at doses of 250 mg/kg and 500 mg/kg (dose selected on the basis of acute toxicity study)/day orally. The control was treated with the same volume of 0.9% sodium chloride solution. All the treatments were given for 9 days. The median survival time (MST) and average body weight changes of each group consisting of 6 mice (n = 6) were noted. The antitumor efficacy of the HEEPB and AEPB was compared with that of 5-fluorouracil (FU), procured from Sigma Aldrich, India (20 mg/kg/day, i.p. for 9 days).  The MST of the treated groups was compared with that of the control group using following calculation. 
T: Number of days treated animals survived
C: Number of days control animals survived.
Effect of the HEEPB and AEPB on hematological parameters
There were six groups (n = 6) (I) normal mice (II) tumor bearing mice (EAC cells 1 × 10 6 cells/mouse) (III and IV) tumor bearing mice treated with HEEPB (250 and 500 mg/kg/day respectively, p.o. for 9 days) and (V and VI) tumor bearing mice treated with AEPB (250 and 500 mg/kg/day respectively, p.o. for 9 days). HEEPB was suspended in 0.3%, NaCMC solution at a dose of 250 mg/kg and 500 mg/kg to administer orally. To detect the influence of the HEEPB and AEPB on hematological status of EAC bearing mice, a comparison was made among six groups of mice on the 14 th day after intraperitoneal inoculation. On the 14 th day, blood was withdrawn from each mouse by retro-orbital plexus method, stored in ethylenediaminetetraacetic acid vials and the white blood cell count (WBC), red blood cells (RBC), hemoglobin (Hb), differential count were determined ,,, by using cell counter (Sysmex).
Effect of the HEEPB and AEPB on solid tumor
Mice were divided in six groups (n = 6). Tumor cells (2 × 10 6 cells/mouse) were injected into the right hind limb of all the animals intramuscularly. HEEPB suspended in 0.3%, NaCMC solution at a dose of 250 mg/kg and 500 mg/kg to administer orally. There were five groups (I) tumor bearing mice (II) tumor bearing mice treated with 5-FU (20 mg/kg/day, i.p.) for 5 alternative days (III and IV) tumor bearing mice treated with HEEPB (250 and 500 mg/kg/day, respectively, p.o.) and (V and VI) tumor bearing mice treated with AEPB (250 and 500 mg/kg/day respectively, p.o.) for 5 alternative days. Tumor mass was measured from the 11 th day of tumor induction using Vernier Caliper, the measurement of was carried out every 5 th day for a period of 30 days. The volume of tumor mass was calculated using formula V = 4/3 πr 2 , where "r0" is the mean of "r1" and "r2" which are the two independent radii of the tumor mass. ,
The statistical test applied for the analysis of the study were one way analysis of variance followed by Tukey Kramer multiple comparison test (for comparison between different groups) or Dunnetts' t-test (for comparison of different groups vs. control). The significance in both test is expressed by "F-ratio" and P values, as mentioned in the tables. The values were considered abnormal if it fell outside the limits formed by the mean values for control group. P ≤ 0.05 was considered to be statistically significant (P < 0.01 is taken as highly significant).
| > Results|| |
The MST of tumor bearing mice were increased significantly by treatment with 5-FU (20 mg/kg), HEEPB (500 mg/kg) and AEPB (250 and 500 mg/kg) (P < 0.001 and P < 0.05 vs. tumor control) [Table 1]. The average weight gain of tumor bearing mice was decreased by treatment with 5-FU (20 mg/kg), HEEPB (500 mg/kg) and AEPB (500 mg/kg) (P < 0.001 and P < 0.01 vs. tumor control).
|Table 1: Effects of HEEPB and AEPB on survival time and average increase in body weight |
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Tumor bearing mice showed significant changes in hematological parameters when compared to normal mice [Table 2]. The RBC count and Hb (g %) were decreased and total WBC count were increased in tumor bearing mice when compared to normal mice. Result of differential count of WBC showed percentage of lymphocytes decreased while that of neutrophils increased (P < 0.001 vs. normal mice) in tumor control group. At the same time interval, treatment with HEEPB (250 and 500 mg/kg) and AEPB (250 and 500 mg/kg) showed increase in the level of RBC count and Hb (g %) near to normal (P < 0.001 and P < 0.05 vs. tumor control). Total WBC count was decreased in the groups treated with HEEPB (500 mg/kg) and AEPB (250 and 500 mg/kg) (P < 0.001 and P < 0.01 vs. tumor control). For differential count of WBC, groups treated with HEEPB (500 mg/kg) and AEPB (250 and 500 mg/kg) showed increase in percentage of lymphocytes while that of neutrophils decreased versus tumor control (P < 0.001).
Treatment with 5-FU (20 mg/kg), HEEPB (250 and 500 mg/kg) and AEPB (250 and 500 mg/kg) showed a significant reduction in tumor volume of mice when compared to tumor bearing mice [Table 3]. On the 30 th day, tumor volume of control group was 3.00 ± 0.19 ml whereas it was 1.62 ± 0.05 for 5-FU group (P < 0.01 vs. tumor control), 2.38 ± 0.18 and 1.90 ± 0.05 for the groups treated with HEEPB (250 and 500 mg/kg) respectively (P < 0.01 vs. tumor control). Tumor volume for the groups treated with AEPB (250 and 500 mg/kg) was 1.95 ± 0.15 and 1.67 ± 0.05 respectively (P < 0.01 vs. tumor control).
| > Discussion|| |
The primary therapy for tumors includes surgery, radiation therapy and chemotherapy. The reliable criteria for judging the value of any anticancer drug are prolongation of life span, inhibition of gain in average body weight, decrease in WBC and maintain other hematological parameters such as RBC, Hb, and differential count at normal level. , The result of present study also showed similar effects on these parameters against EAC in Swiss albino mice. The reduction in solid tumor volume indicated that HEEPB and AEPB play a preventive role in tumor cell proliferation.
Colchicine, an alkaloid has powerful antimitotic activity.  Taxol, a diterpenoid used in ovarian and breast tumors. ,, Ginsenoside Rh (2), a panaxadiol saponins, possesses various antitumor properties  and decreases genotoxic effect of cyclophosphamide when given orally in combination with cyclophosphamide.  Phenolic acids (gallic acid) and flavonoids (luteolin, apigenin, quercetin, rutin) possessing antioxidant  and anticancer activity. 
Plant derived extracts containing antioxidant principles may showed cytotoxicity toward tumor cells  and antitumor activity in experimental animals.  According to the results of preliminary phytochemical screening flavonoids, alkaloids, saponins, phenolic compounds, terpenoids, steroids etc., are present in HEEPB and AEPB.
In cancer chemotherapy, the major problems are of myelosuppression and anemia. The anemia encountered in tumor bearing mice is mainly due to reduction in RBC or Hb percentage and this may occur either due to iron deficiency or due to hemolytic or myelopathic conditions. , Treatment with HEEPB and AEPB brought back the Hb content, RBC and WBC cell count near to normal values. This indicates that both HEEPB and AEPB possess protective action on the hematopoietic system.
| > Conclusion|| |
HEEPB and AEPB showed promising antitumor activity against EAC in Swiss albino mice. Further investigations will be carried out to identify the active principles and mechanism of action involved in this antitumor activity.
| > Acknowledgment|| |
The authors are gratefully acknowledge to Dr. Amitava Chatterjee, Chittaranjan National Cancer Institute, Kolkata, India for kindly providing us EAC cells bearing Swiss albino mice.
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[Table 1], [Table 2], [Table 3]