Chemo Preventive Activity of Triumfetta rhomboidea in 7, 12-Dimethylbenz (A) Anthracene Induced Breast Cancer in Sprague –Dawley Rat Model

 

R. Suresh1, Dr. D. Benitojohnson2, Dr. C. Maheswari3, Dr. R. Venkatnarayanan4, Dr R.Manavalan5

1Professor, Department of Pharmacology, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore-641402

2Professor and Head, Department of Pharmacology, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore-641402

3Asst. Professor, Department of Pharmacology, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore-641402

4Principal, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore-641402

5Head and Research Coordinator, Department of Pharmaceutics, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore-641402

*Corresponding Author E-mail: rsureshpharma@gmail.com

 

ABSTRACT:

The chemopreventive potential was assessed by monitoring the tumour incidence, total no of tumours and tumour volume and also by analyzing the level of biochemical markers such as 17-β estradiol (E2), TBARS and antioxidants during DMBA induced mammary carcinoma. A single subcutaneous injection of DMBA (25mg/kg) produced mammary carcinoma in female Sprague-Dawley rats. Oral administration of 100mg/kg and 200mg/kg of TRM to DMBA treated rats significantly prevented the tumour incidence, total no of tumours, tumour volume and brought back the above said biochemical markers to normal. The present study confirmed the chemopreventive activity of leaves of Triumfetta rhomboidea in mammary carcinoma

 

KEYWORDS:  Mammary carcinoma, Triumfetta rhomboidea, Leaves, DMBA, Antioxidants.

 

 


INTRODUCTION:

Breast cancer is a malignant tumour that starts in the cells of the breast. A malignant tumour is a group of cancer cells that can grow into (invade) surrounding tissues or spread (metastasize) to distant areas of the body. The disease occurs almost entirely in women, but men can get it, too. The female breast is made up mainly of lobules (milk-producing glands), ducts (tiny tubes that carry the milk from the lobules to the nipple), and stroma (fatty tissue and connective tissue surrounding the ducts and lobules, blood vessels, and lymphatic vessels). Most breast cancers begin in the cells that line the ducts (ductal cancers). Some begin in the cells that line the lobules (lobular cancers), while a small number start in other tissues. The lymph system is important to understand because it is one way breast cancers can spread. This system has several parts.

 

Lymph nodes are small, bean-shaped collections of immune system cells (cells that are important in fighting infections) that are connected by lymphatic vessels. Lymphatic vessels are like small veins, except that they carry a clear fluid called lymph (instead of blood) away from the breast. Lymph contains tissue fluid and waste products, as well as immune system cells. Breast cancer cells can enter lymphatic vessels and begin to grow in lymph nodes. Most lymphatic vessels in the breast connect to lymph nodes under the arm (axillary nodes). Some lymphatic vessels connect to lymph nodes inside the chest (internal mammary nodes) and either above or below the collarbone (supraclavicular or infra clavicular nodes). If the cancer cells have spread to lymph nodes, there is a higher chance that the cells could have also gotten into the bloodstream and spread (metastasized) to other sites in the body. The more lymph nodes with breast cancer cells, the more likely it is that the cancer may be found in other organs as well. Because of this, finding cancer in one or more lymph nodes often affects the treatment plan. Still, not all women with cancer cells in their lymph nodes develop metastases, and some women can have no cancer cells in their lymph nodes and later develop metastases.

 

Treatment options for Breast cancer are problematic.  Because Allopathic drugs are effective against this cancer but exhibit severe toxicity which leads to death.  Physicians and patients are in need of maximum therapeutic value with no or low incidence of side effects to improve the quality of life. Several herbal plants potentially constitute such a group. In recent years, researchers have examined the effects of plants used traditionally by indigenous healers and herbalists to support to treat cancer. Several hundred plants have been reported for use in different types of cancer. Only a handful has been fairly well researched. The plant selected for the study is Triumfetta rhomboidea Jacq, which belongs to the family Tiliaceae. The genus Triumfetta consists of about 150 species out of which 8 are found in India. It is known as Burweed in English.It is a herb or under shrub with stellate pubescence. Roots of Triumfetta rhomboidea have been traditionally used in dysentery, intestinal ulcer and as diuretic. Leaves and stem are used in treatment of bacterial diseases3, tumors, gonorrhoea and leprosy. It is reported that this plant is used as antimicrobial1, anti-inflammatory2, antidiabetic4 and analgesic activity.

 

Based on the literature review there is no established work in  the Chemo preventive activity of  Triumfetta rhomboidea in 7, 12-Dimethylbenz (A) Anthracene induced Breast cancer in Sprague –Dawley Rat model.  The present study is planned to evaluate the Chemo preventive effect of methanol extract of leaves of Triumfetta rhomboidea in 7, 12-Dimethylbenz (A) Anthracene induced Breast cancer in Sprague –dawley rat model.

 

MATERIALS AND METHODS:

Drugs and Chemicals:

DMBA was obtained from Sigma-Aldrich Chemicals Pvt. Ltd., Bangalore, India.  All other chemicals were obtained from local sources and were of analytical grade.

 

Collection and Authentication of plant:

The fresh healthy plant leaves of Triumfetta rhomboidea   was collected from Irular society, Thandarai, TamilNadu, India during the month of December 2013. The plant was identified and authenticated by Botanical Survey of India, Coimbatore and a voucher specimen has been preserved in the Department of Pharmacognosy, R.V.S. College of Pharmaceutical sciences, sulur, Coimbatore. After authentification, the fresh, healthy plant leaves of Triumfetta rhomboidea   was properly dried in shade for 2-3weeks. It was pulverized in a blender, sieved and used for further studies.

 

Preparation of the Extracts :

About 2 kg of shade dried plant leaves of Triumfetta rhomboidea   extracted in soxhlet successively extracted with n-hexane, chloroform, ethyl acetate and methanol. Each extract was evaporated by using rotary vacuum evaporator. The extract obtained with each solvent was weighed and the percentage yield was calculated in terms of dried weight of the plant leaves. The Consistency and Colour of the extract were noted. All the solvents used for this work were of analytical grade.

 

Plant extract:

The therapeutically active extract  of Triumfetta rhomboidea Methanol extract (TRM)  to carry out the pharmacological studies in animals was selected on the basis of in- vitro studies like in-vitro free radical scavenging activity and phytochemical analysis 5,6,7

 

Experimental animals:

Adult male Wister rats weighing 100-150g were obtained from R.V.S. College of Pharmaceutical Sciences, Sulur, Coimbatore. They were maintained at standard housing conditions and fed with commercial diet and provided with water and libitum during the experiment. The institutional animal ethics committee (Reg.no 1012/c/06/CPCSEA) permitted the study. The animals were kept in clean and dry polycarbonate cages and maintained in a well ventilated animal house with 12hrs light – 12 hrs dark cycle.

 

Acute toxic class method:8,9

Acute toxicity study was done according to OECD guidelines 423 (Organization of Economic Co-Operation and Development). It is a procedure with three animals of single sex per step. Based on the mortality and morbidity status of the animal, average of 2-4 steps necessary to allow decision on the test substance. The procedure is to fix a minimal number of animals, which allows acceptable database scientific conclusion. The method uses different doses (5, 50, 500, 2000mg/kg body weight) Procedure Three healthy, Wistar Albino rats weighing 150-200gms were selected for the study. The rats were fasted over-night and provided with water ad libitum. Following the period of fasting, the animals were treated with the methanolic extract at the dose of 2000mg/kg body weight, orally. As most of the crude extracts possess LD50 value more than 2000mg/kg body weight and this was used as starting dose. After oral administration, the rats were observed for 24hrs to access mortality and to observe any changes in the autonomic or behavioral responses viz. aggressiveness, alertness, irritability, spontaneous activity, corneal reflex, tremor, salvation, urination, convulsion and respiration etc. The rats were observed regularly for 14 days to note the mortality or toxic symptoms. Since there was no death as per the guidelines, the study was repeated with the same dose to confirm the results.  The TRM was found devoid of mortality of animals at the dose of 2000 mg kg-1 body weight. Hence (100 mg kg-1, p.o.) and (200 mg kg-1, p.o.) of the dose selected for the screening of Chemopreventive activity

 

Screening of 7,12  dimethyl benz anthracene induced breast cancer:

Experimental design:

Forty rats were divided into four groups and each group contained ten rats.

Group I rats received the excipient (single dose of 1 mL of emulsion of sunflower oil and physiological saline, s.c.) and 1 mL of 2% DMSO (p.o.) throughout the experimental period served as vehicle treated control.

Groups II, III and IV rats were induced mammary carcinogenesis by providing single subcutaneous injection of 25 mg of DMBA. Group 2 rats received no other treatment.

 

Group III and IV rats were orally administered with TRM 100 and 200 mg kg-1 b.wt., dissolved in 2% DMSO starting one week before the exposure of the carcinogen respectively and continued till the experimental period.

 

The experiment was terminated at 16th week to evaluate the chemopreventive effect of TRM during DMBA-induced mammary carcinogenesis. All rats were sacrificed by cervical dislocation at the end of experimental period to evaluate the tumor incidence and tumor volume in control and experimental rats in each group.

 

Biochemical Estimation:

Blood samples were collected into heparinized tubes. Plasma was separated by centrifugation at 1000x g for 15 min. Tissue samples from rats were washed with ice cold saline and dried between folds of filter paper, weighed and homogenized using appropriate buffer in an All-glass homogenizer with teflon pestle and used for biochemical estimations Lipid peroxidation was estimated as evidenced by the formation of thiobarbituric acid reactive substances (TBARS). TBARS in plasma were assayed by the method of Yagi (1987) 13. Tissue lipid peroxidation was done by the method of Ohkawa et al. (1979). The activities of superoxide dismutase (SOD), catalase (CAT) and Glutathione Peroxidase (GPx) were assayed by the method of Kakkar et al. (1984)10, Sinha (1972) 11 and Rotruck et al. (1973) 12 respectively. The GSH level in plasma and mammary tissues was determined by the method of Beutler and Kelley (1963)14. Chemiluminescent immunoassay (CLIA) was used for the estimation of serum 17 β-estradiol (E2)15

 

Histopathology:

For histopathological studies, tumor tissues and normal mammary gland tissues were fixed in 10% formalin and were routinely processed and paraffin embedded, 2-3 μm sections were cut in a rotary microtome and were stained with hematoxylin and eosin.

 

Statistical Analysis:

The results were expressed as mean ± SEM of six animals from each group. The statistical analysis were carried out by one way analysis of variance (ANOVA) P values < 0.05 were considered significant.

 

RESULTS AND DISCUSSION:

Pharmacological screening of 7,12  dimethyl benz anthracene induced breast cancer:

Table 1 shows the incidence of mammary tumors in DMBA, DMBA+TRM 100mg/kg and DMBA+TRM 200mg/kg treated rats. The present study observed 100% tumor incidence in rats treated with DMBA alone. The tumors were histopathologically confirmed as moderately and poorly differentiated adenocarcinoma. Oral administration of TRM 100mg/kg and 200mg/kg to DMBA-treated rats reduced the tumor incidence (40% and 70%, respectively) and tumors in this group (60% and 30%) (Figure 1) were histopathologically confirmed as well-differentiated adenocarcinoma. The size of the tumor and tumor volume observed in DMBA+  200mg/kg treated rats   were very small as compared to rats treated with DMBA alone ( Figures 2 and 3).

 

Figures 4 to 6 show histological features in Control, DMBA, and DMBA+200mg/kg  treated rats. Control Rat shows normal ductal epithelium (Figure 4) DMBA alone treated rats showing ductal carcinoma and abnormal cellular proliferation(Figure 5) TRM 200mg/kg treated rats showed  ductal dysplasia(Figure 6 )


 

Table 1. Incidence of mammary tumor in control and experimental rats in each group

Groups

Treatment

Tumour incidence

Total no of Tumours

Tumour Volume

I

Control

0

0

0

II

DMBA

100%

10/10

15.68±1.2

III

DMBA + TRM  (100mg/kg)

60%

06/10

2.14±0.12

IV

DMBA+TRM (200mg/kg)

30%

03/10

1.09±0.07

 

 

Figure 1. Tumour Incidence

 

 

Figure 2. Total Number of Tumours

 

 

Figure 3. Tumour Volume

 

 

Figure 7. Effect  of plasma 17-β estradiol (E2) in control and experimental rats in each group

Table 2. Effect  of plasma 17-β estradiol (E2) in control and experimental rats in each group

Groups

17-β estradiol (pg/ml)

Control

45.1±0.12

DMBA

58.3±0.34

DMBA+TRM(100mg/kg)

51.1±0.16

DMBA+TRM(200mg/kg)

48.2±0.21

Values are expressed as Mean±SEM. Group II is compared with Groups I, and  IV P<0.01

 

Table 3. Effect  of plasma TBARS and antioxidants in control and experimental rats in each groups

Groups

TBARS (nmols/ml)

SOD (U/mL)

CAT (U/mL)

GPx (U/mL)

GSH (mg/dl)

Control

1.84±0.12

2.89±0.01

1.59±0.11

154±0.09

31±0.12

DMBA

3.45±0.21

1.73±0.05

1.09±0.24

109±0.04

19±0.16

DMBA+TRM(100mg/kg)

2.59±0.09

2.31±0.09

1.21±0.32

139±0.07

24±0.09

DMBA+TRM(200mg/kg)

1.34±0.11

2.74±0.12

1.45±0.19

146±0.03

29±0.20

Values are expressed as Mean±SEM. Group II is compared with Groups I,III and  IV P<0.05

          

Table 4. Effect of mammary tissues TBARS and antioxidants in control and experimental rats in each group

Groups

TBARS

(nmols/100g of tissue)

SOD

(U/mg of protein)

CAT

(U/mg of protein)

GPx

(U/mg of protein)

GSH

(mg/100mg of tissue)

Control

0.74±0.11

12.69±0.21

41.59±0.10

18±0.19

    08±0.32

DMBA

1.15±0.31

07.30±0.05

28.09±0.24

29±0.08

13±0.46

DMBA+TRM(100mg/kg)

0.99±0.19

10.31±0.19

35.01±0.12

21±0.17

11±0.69

DMBA+TRM(200mg/kg)

0.78±0.12

12.74±0.20

39.25±0.14

19±0.13

09±0.42

Values are expressed as Mean±SEM. Group II is compared with Groups I,III and  IV P<0.05

 


HISTOPATHOLOGY

 

Fig 4 : Control Rat shows normal ductal epithelium

 

 

Fig 5: DMBA induced rats showing ductal carcinoma and abnormal cellular proliferation

 

 

Fig:6  DMBA+TRM(200mg/kg) rats showing ductal dysplasia

 

17-β Estradiol the predominant circulating ovarian steroid, is carcinogenic in human breast epithelial cells involved in the initiation of breast cancer. Estrogens have been implicated in the initiation and promotion stages of breast cancer and lifetime estrogen exposure is a major risk factor for breast cancer development. It is reported that lower serum estrogen levels leads to lower incidence of DMBA-induced mammary tumors in rats. In our study the level of plasma E2 was significantly increased in DMBA treated rats as compared to control rats (Table 2 and Figure 7 ). Oral administration of TRM 200mg/kg to DMBA-treated rats as well as control rats significantly (p<0.05) decreased the level of E2 which indicate the antiestrogenic activity of the plant during DMBA-induced mammary carcinogenesis.

 

 

The level of plasma TBARS serve as an index to assess the extent of tissue damage. Elevated levels of TBARS in plasma of mammary cancer could be related to overproduction and diffusion from the mammary tumor tissues and damaged host tissues. Abnormalities in the level of antioxidants lead to many disorders including cancer. Decreased level of plasma SOD, CAT and GPx were well experimented in mammary cancer. Decreased level of plasma glutathione is probably due to utilization by mammary tumor tissues to reduce reactive oxygen species during carcinogenic process.

 

In our study the plasma levels of TBARS were significantly increased whereas GSH content and activities of SOD, CAT and Gpx in plasma were decreased in DMBA treated rats as compared to control rats. Oral administration of 200mg/kg of TRM to DMBA-treated rats significantly (p<0.05) decreased the levels of TBARS and increased the level of GSH and activities of SOD, CAT and GPx. (Table 3)

 

In mammary tissues the levels of TBARS, GSH and activity of GPx were increased whereas the activities of SOD and CAT were decreased in the tumor tissues as compared to normal tissues of control rats. Oral administration of 200mg/kg of TRM to DMBA-treated rats significantly (p<0.05) restored the status to near normal.(Table 4)

 

In histopathological studies, control rats showed normal ductal epithelium. DMBA induced rats showing ductal carcinoma and abnormal cellular proliferation. Rats treated with higher dose of TRM (200mg/kg) rats showed ductal dysplasia. Results confirmed the protective activity against DMBA indued mammary cancer.

 

At a dose of 100mg/kg the activity was marginal where as at higher dose (200mg/kg) the extract effectively inhibited the activity of DMBA in mammary gland.

 

CONCLUSION:

From the present study, it can be concluded that Triumfetta rhomboidea leaves possesses anti-carcinogenic and anti-oxidative potential against chemically induced mammary gland carcinogenesis in mammals. Chemopreventive activity may be antioxidant/free radical-scavenging constituents present in the plant leaf extract.

 

REFERENCES:

1.        CD Odimegwu, IF Uche, CL Ozioko, CE Ogbuanya, HT Gugu, OC Esimone In vitro antimicrobial evaluation of methanol extract of Triumfetta rhomboidea leaves against some clinical bacterial isolates International Journal of Biological and Chemical Sciences;2015; 5(5); 245-256

2.        Uche, F. I.; Okunna, B. U Phytochemical constituents, analgesic and anti-inflammatory effects of methanol extract of Triumfetta rhomboidea leaves in animal models. Asian Pacific Journal of Tropical Medicine :2009: 2(5); 26-29

3.        Prasad Ekanath Funde Antibacterial activity of water extract and active constituents were isolated of butanol from a Triumfetta rhomboidea and analytical study by HPLC Der Chemica Sinica; 2011; 2 (1): 8-14

4.        N Duganath, D Rama Krishna , Deepak Reddy G, B Sudheera, M Mallikarjun, Pavani Beesetty Evaluation of anti-diabetic activity of Triumfetta rhomboidea in alloxan induced Wistar rats Research Journal of Pharmaceutical, Biological and Chemical Sciences; 2011;2(1);475-77

5.        Takeda, Y.; Matsumoto, T.; Terao, H.; Shingu, T.; Futatsuishi, Y.; Nohara, T.; Kajimoto, T. Phytochemistry, 33, 411, 1993.

6.        Sanchez-Moreno, C., Larrauri, J.A. and Saura-Calixto, F. Free radical scavenging capacity and inhibition of lipid oxidation of wines, grape juices and related polyphenolic constituents, Food Res. Int., 1999; 32: 407-412

7.         Ilhami Gulcin. Haci Ahmet Alici. and Mehmet Cesur., Determination of in vitro antioxidant and radical scavenging activities of propofol. Chem Pharm Bull. 2005;53, 281 – 85.

8.        Ecobichon DJ. The basis of toxicology testing. 2nd ed, CRC Press: New York; 1997, pp. 43-60.

9.         Lorke, D. A new approach to practical acute toxicity testing. Arch. Toxicol., 1983; 54; 275-287

10.     Kakkar, P., B. Das and P.N. Viswanathan, 1984. A modified spectrophotometric assay of superoxide dismutase. Indian J. Biochem. Biophys., 21: 130-132.

11.     Sinha, A.K., 1972. Colorimetric assay of catalase. Anal. Biochem., 47: 389-394

12.     Rotruck, J.T., A.L. Pope, H.E. Ganther, A.B. Swanson, D.G. Hafeman and W.G. Hoekstra, 1973. Selenium: Biochemical role as a component of glutathione peroxidase. Science, 179: 588-590

13.     Yagi, K., 1987. Lipid peroxides and human diseases. Chem. Physiol. Lipids, 45: 337-351

14.     Beutler, E. and B.M. Kelley, 1963. The effect of sodium nitrate on RBC glutathione. Experientia, 29: 96-97.

15.     Buscarlet, L., H. Volland, J. Dupret-Carruel, M. Jolivet and J. Grassi et al., 2001. Use of free radical chemistry in an immunometric assay for 17β-estradiol. Clin. Chem., 47: 102-109

 

 

 

 

Received on 03.12.2016             Modified on 18.01.2017

Accepted on 30.01.2017           © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(3): 687-692.

DOI: 10.5958/0974-360X.2017.00128.7