Effect of Euphorbia thymifolia Linn. on Lipid Parameters of Wistar Albino Rats with Transplanted Fibrosarcoma

Introduction

Malignancy that starts from the connective fibrous tissue of bones of lower and upper limbs, and spreads towards the surrounding tissues is called fibrosarcoma.¹ Fibrosarcoma is of two types, Congenital and Adult. Congenital or infantile fibrosarcoma is the most recurrent form of soft tissue sarcoma occurring in infants that gradually becomes more benign in older children (11-16 years of age) and is almost similar to the fibrosarcoma seen in adults. The other form is the adult form which occurs in grown-up children and young adults, between the ages of 10 to 15 years. It is a more aggressive form and the treatment is more complicated.

The major treatment modality for cancer is chemotherapy. Certain plants have provided active principles which can be used to control advanced stages of malignancies in the clinical setting. These plants are Catharanthus roseus, Podophyllum emodii, Taxus brevifolia, Ochrosia elliptica, Campototheca acuminate and Podophyllum peltatum. Reports suggest that most of the chemotherapeutic agents inhibit some or the other normal tissue toxicities resulting in undesirable side effects. Another drawback of various potent antineoplastic drugs is that they are highly expensive, mutagenic, teratogenic and carcinogenic.²

Chhotidudhi, laghudugdhikaa are the other names for Euphorbia thymol Linn. (Euphorbiaceae). It is an annual weed which is a small prostate, hispid pubescent found in India and other tropical countries. It possesses a wide range of activities which include laxative, diuretic, anti-allergic and anti-hemorrhoidal activities.³ Dudhika was prescribed by Charaka as an ingredient of vegetable soup for diarrhea and painful bleeding piles.⁴ Euphorbia thymifolia possesses antioxidant and anti-viral properties.⁵ The plant is also used to treat eye swellings and discharge.⁶

Among the Euphorbia species, only E. tirucalli and E. thymifolia are capable of crassulacean acid metabolism (CAM) pathway. Since CAM pathway is a strategy for harsh climate resistance, these plants always show deviation in biosynthetic pathways leading to the synthesis of variant forms of phytochemicals. A few plants that belong to the same family are Manihot esculenta, Ricinus communis, Genus acalypha, and Codiaeum variegatum. The chemical structure of the major constituents of the plant are Diterpenoids ent-abietane and Jatrophane (Figure 1).

However, there is no scientific evidence on the antitumor activity of Euphorbia thymifolia against transplanted fibrosarcoma in rats. Therefore, the present study was planned to assess the effect of Euphorbia thymifolia Linn. on lipid parameters of Wistar albino rats with transplanted fibrosarcoma.

Materials and methods

Plant material

Euphorbia thymifolia was collected as a whole plant from an area in close vicinity to Bengaluru city and was identified by Prof. Jawahar Raveendran, in-charge, Raw Drug Repository, Foundation for Revitalization of Local Health Traditions, Bengaluru, Karnataka, where a voucher specimen was deposited. The plant material was shade dried, powdered, and passed through sieve no. 40, and stored in an airtight container.

Extraction

Extraction of the powdered plant material was done using Soxhlet apparatus with ethanol (95% v/v). After extraction, the filtrate was concentrated under reduced pressure and stored in an airtight container at 4ºC in the refrigerator.

Phytochemical screening

Preliminary phytochemical screening of Euphorbia thymifolia disclosed the existence of tannins, steroids, flavonoids, triterpenoids, glycosides, and phenolic compounds.^7,8

Animals

Wistar albino rats of both sexes (150 g to 200 g) and Swiss albino mice (25 g to 35 g) were obtained from Raghavendra Enterprises, Bengaluru. The rats were sorted into groups and accommodated in polypropylene cages under controlled environment (12-hour dark/ light cycle and temperature of 25±2 ºC) and were given pellet diet while water was allowed at will. Institutional Animal Ethical Committee (CPCSEA No. 997/C/06/ CPCSEA) has reviewed and approved all the procedures used in the research.

Acute toxicity study

The acute oral toxicity study was conducted according to the guidelines (Guideline no. 423) set by the organization for Economic Co-operation and Development (OECD).9 For the study, healthy adult Swiss albino mice with a body weight of 25 to 35 gm were selected. Four sets of three animals were grouped and were fasted overnight. Different doses of the extract (5, 50, 300, 2000 mg/kg body weight) were administered, and the experimental animals were observed. No mortality was noted. To further optimize the study, dose levels of 250 mg/kg and 500 mg/kg were selected for evaluation.

Fibrosarcoma

Induction of fibrosarcoma¹⁰

Methylcholantherene-induced fibrosarcoma in rats were removed, minced in saline and prepared as a 10% suspension by homogenizing in a tissue homogenizer. 0.2 mL of this was injected into the inguinal region of the experimental animals. After 28 days, once the tumor reached a palpable stage, the treatment was initiated. Five groups were put together with each group comprising of six animals. Normal rats were allocated to the first group, while tumor-bearing control animals were grouped under the second, which received only the vehicle. The third and fourth groups (tumor-bearing) were administered orally 250 and 500 mg/kg of Euphorbia thymifolia, respectively. The fifth group of animals received 5-Fluorouracil (20 mg/kg, orally), continuously for 20 days.

Estimation of lipids

After 20 days of treatment, blood was collected after 24 hours by cardiac puncture before sacrificing the animal. The kidney and liver were removed, washed with ice-cold saline, following which they were preserved immediately. Biochemical kits were procured (Span diagnostics Ltd.) and all the lipid parameters like total cholesterol, high-density lipoprotein (HDL), very-low-density lipoprotein (VLDL) and triglycerides was evaluated in liver, serum and kidney.

Statistical analysis

Assessment was performed by means of variance one-way analysis, and Dunnett’s test. The resultant values were mentioned in terms of mean±SD and p <0.05 was considered statistically significant.

Results

Lipid levels like VLDL, HDL, triglycerides, total cholesterol were assessed (Table 1). The serum lipid levels were observed to have decreased significantly in tumor bearing rats. This confirmed that treatment with Euphorbia thymifolia (500 mg/kg) lowered the levels to normal. The decrease in these parameters to normal was also noted in the kidney and liver of rats with tumor. Normal levels were restored with a dose of 500 mg/kg of Euphorbia thymifolia, while the lower dose (250 mg/kg) resulted only in a limited restoration (Table 2 & Table 3).

Discussion

The present study involving fibrosarcoma-bearing rats observed the contributing factor as increase in total cholesterol which could be because of the concentration of low-density lipoprotein-cholesterol, a major transporter of cholesterol. In the untreated fibrosarcoma-bearing rats, an increase in the total cholesterol concentration was noted which could be due to the underutilization of cholesterol, a state caused in conditions such as carcinoma due to the reduced catabolic rate.¹¹

By reducing the activity of lecithin:lipoprotein lipase, cholesterol acetyltransferase (LCAT), the enzyme responsible for splitting of fats, the increase in total cholesterol and triglycerides levels can be explained. While LCAT is directly and indirectly accountable for the esterification of free cholesterol in plasma, controlling free cholesterol levels in cells and tissues respectively, lipoprotein lipase acts as the clearing factor for triglycerides in plasma by splitting it into glycerol and free fatty acids.¹²

In fibrosarcoma-bearing rats, the raised levels of triglycerides could have been caused due to enhanced VLDL cholesterol synthesis. In untreated fibrosarcomabearing rats, the decreased levels of HDL cholesterol could be due to the simultaneous decrease in LDL cholesterol. Whenever HDL cholesterol reduces, it is balanced out by a rise in LDL cholesterol, and vice versa.¹³

Increased triglyceride levels in fibrosarcoma-bearing animals can be attributed to the heightened VLDL cholesterol synthesis and further to the surplus lipid peroxides formed in fibrosarcoma condition whichmay have led to hyperlipidemia^.14 The hyperlipidemia condition found in cancer patients is caused due to the increased rate of lipid peroxidation.¹⁵

By boosting the activity of cholesterol-7-hydroxylase, Euphorbia thymifolia exerts an hypocholesterolemic effect, leading to increase in cholesterol. An increase in lipolytic activity associated with hyperlipidemia was reported in tumor bearing animals.¹⁶ The state of hyperlipidemia associated with the tumor burden was reversible with tumor removal or remission.

Significant depletion of VLDL, HDL, cholesterol and triglycerides from the animal’s liver and kidney was noticed indicating an increase in lipolytic activity in tumor-bearing animals. Correspondingly, these lipids were seen in elevated levels in serum. The treatment of Euphorbia thymifolia significantly attenuated these variations of lipid levels, both in serum as well as in tissues. The depletion of fat stores associated with tumor growth indicates high energy requirement and hence increased lipolytic activity.¹⁷ Lipid level normalization in tissue and serum with the use of extract of Euphorbia thymifolia may be due to enhanced lipogenesis or decreased lipolysis or both.

These changes were significantly reversed in animals treated with 500 mg/kg of plant extract orally. Results indicate that ethanolic extract of Euphorbia thymifolia exhibited significant reversal of altered lipid levels close to normal values in rats with experimentally induced fibrosarcoma.

Conclusion

Euphorbia species are rich in diterpenoids ent-abietane, jatrophane, lathyrane, myrsinane, premyrsinane, daphnane, tiglane and ingenane derivatives, while the members of ent-Atisane, kaurane, ent-pimarane, easbane, segetane, paraliane, euphoractine and pepluane type occur in traces. These terpenes have exhibited selectivity against cell lines in cytotoxic assays. Rats treated with Euphorbia thymifolia exhibited normalization of altered levels of lipids. Results of the present study demonstrated effective antitumor activity of ethanolic extract of Euphorbia thymifolia Linn.

Acknowledgment

The authors would like to express their gratitude and thanks to RGUHS for financial support and gratitude to Mr. Premanath Reddy, Chairman Acharya Institutes, and Dr. Diwakar Goli, Principal, Acharya and B.M. Reddy College of Pharmacy, Bangalore, India, for providing the necessary facilities to carry out the research work. The authors also express their sincere thanks to the management of Harsha Institutions, Bangalore, for their support to publish this research article.

Conflicts of Interests

Nil