The triterpenoid fraction from Trichosanthes dioica root suppresses experimentally induced inflammatory ascites in rats

Abstract

Context. Trichosanthes dioica Roxb. (Cucurbitaceae), called pointed gourd in English, is a dioecious climber found wild throughout the plains of the Indian subcontinent and traditionally used in India for several medicinal purposes.

Objective: The present study evaluated the protective effect of the triterpenoid enriched fraction from T. dioica root (CETD) against experimentally induced acute inflammatory ascites in Wistar albino rats.

Materials and methods: The CETD was administered orally at the different doses (25, 50 and 100 mg/kg body weight) to overnight fasted rats, and then ascites was induced by intraperitoneal administration of formalin solution. After 7 h, the rats were sacrificed and the volume of ascitic fluid was measured.

Results: The CETD demonstrated significant (p < 0.01) reduction of ascitic fluid formation in a dose-dependent manner as compared with control.

Conclusion: The CETD produced significant and dose-dependent inhibition of experimentally induced inflammatory ascites in Wistar albino rats.

• Anti-inflammatory
• ascitic fluid
• cucurbitacins
• formalin

Introduction

The major merits of traditional medicine seem to be their perceived efficacy, low incidence of serious adverse effects and comparatively low cost. Traditional or herbal medicine worldwide is being re-evaluated by extensive research on different plant species and their therapeutic principles. The ethnopharmacological approach in this context is based on the traditional knowledge of medicinal plant use.

Trichosanthes dioica Roxb. (Cucurbitaceae), called pointed gourd in English, Potol in Bengali, and Patola in Sanskrit, is a dioecious climber found wild throughout the plains of North and North-east India from Punjab to Assam and Tripura states. It is also grown and commercially cultivated in India, Pakistan, Bangladesh and Sri Lanka for its fruits, a common culinary vegetable in the Indian subcontinent. In India, all parts of this plant have been traditionally used for medicinal purposes. According to Ayurveda, the traditional system of Indian medicine, its root is a strong purgative. The root has been traditionally used in India as cathartic, tonic, febrifuge; in treatment of jaundice, anasarca and ascites (Anonymous, 1976; Kirtikar & Basu, 1935; Nadkarni, 1976; Sharma et al., 2002). In our previous course of studies, we have reported anthelmintic, antibacterial, antimitotic, antiproliferative, antitumor, analgesic, laxative, cancer chemopreventive, arsenic toxicity ameliorative and antileishmanial activities of the root of T. dioica (Bhattacharya & Haldar, 2010a,b, 2012a–h; Bhattacharya et al., 2010, 2011a,b, 2013). In the present study, we have evaluated the triterpenoid enriched fraction from T. dioica root (CETD) for its possible protective effects on experimentally induced inflammatory ascites in Wistar albino rats to justify the traditional and folkloric attributes.

Materials and methods

Collection and authentication of plant material

The mature tuberous roots of T. dioica were collected during December 2009 from Majdia, Nadia district, West Bengal, India. The species was identified by Dr. M. S. Mondal, at the Central National Herbarium, Botanical Survey of India, Howrah, West Bengal, India, and a voucher specimen (CNH/I-I/57/2009/Tech.II/493) was deposited at the Pharmacognosy Research Laboratory, Bengal School of Technology, A College of Pharmacy, Delhi Road, Sugandha, Hooghly 712102, West Bengal, India for future reference.

Drugs and chemicals

Formalin was from SISCO Research Laboratories, Mumbai, India. Ibuprofen was form Perk Indus Pharmaceuticals, Faridabad, India. Doubled distilled water from all-glass still was employed throughout the present study.

Preparation of triterpenoid fraction (CETD)

Just after collection, the fresh roots were washed thoroughly with water, cut into moderate pieces and immediately crushed thoroughly in tepid water (∼50 °C) using a mechanical grinder. After cooling to room temperature (23 ± 2 °C), the extract was separated from the remaining vegetable debris by pressing the material through muslin cloth. The resulting liquid was filtered and extracted once with n-hexane and the aqueous phase was further extracted successively with dichloromethane. The organic phases (i.e., dichloromethane extracts) were pooled and evaporated to dryness in vacuo (at 35 °C and 0.8 MPa) in a Buchi evaporator, R-114. The dry extract, i.e., triterpenoid enriched fraction (CETD, yield: 6.55% w/w) was kept in a vacuum desiccator until use in the study.

Standardization of CETD

Qualitative phytochemical analysis revealed the presence of triterpenoids in CETD (Harborne, 1998). Abundance of triterpenoids and presence of cucurbitacin type aglycones in CETD were further ascertained by planar chromatography on silica gel pre-coated high performance thin layer chromatography (HPTLC) plates (Silica gel 60 F₂₅₄ Merck, Germany) detected with vanillin-phosphoric acid reagent (Wagner & Bladt, 1996). CETD was dispersed in isotonic saline as per required concentrations and sonicated for 10 min immediately prior to administration.

Experimental animals

Adult Wistar albino rats of either sex weighing 180–220 g were obtained from Laboratory Animal Centre, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India. The rats were maintained under standard laboratory conditions (temperature 25 ± 2 °C, relative humidity 48% with dark and light cycle 12/12 h). They were allowed free access to standard dry pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. The rats were acclimatized to laboratory conditions for 10 days before commencement of the experiment. All experimental procedures described were thoroughly reviewed and approved by the University Animal Ethical Committee, Jadavpur University (Regn. no. 367001/C/CPCSEA).

Acute toxicity

The acute oral toxicity of CETD in male Swiss albino mice was studied as per reported method (Lorke, 1983). The CETD was found to be safe in male Swiss albino mice up to the dose of 2000 mg/kg body weight p.o. The LD₅₀ could not be determined because physical factors were limiting for administration of a greater amount of CETD.

Treatment protocol

The overnight fasted rats were divided into five groups (n = 8). The first group of animals (which served as control) received normal saline at the dose of 5 ml/kg body weight p.o. The second group of animals (which served as reference) received ibuprofen at the dose of 50 mg/kg body weight p.o. The remaining three groups received CETD at the doses of 25, 50 and 100 mg/kg body weight, p.o., respectively. Sixty minutes after administration of normal saline, ibuprofen and CETD, formalin solution were administered (1.5% v/v, 1 ml, irrespective of body weight, i.p.) to rats of all groups. After 7 h of formalin administration, all the animals were sacrificed by cervical dislocation (Turner, 1971). The rats were dissected and the ascitic fluid was collected from the peritoneal cavity into a clean dry graduated centrifuge tube. The volume of collected ascitic fluid was measured there. The mean volumes of ascitic fluid in each group were calculated and expressed the protection by using the following formula:

Statistical analysis

The data were expressed as mean ± SEM. The results were analyzed for statistical significance by one-way analysis of variance followed by Dunnett’s multiple comparisons test for significance. The p values less than 0.05 (p ≤ 0.05) were considered as statistically significant.

Results and discussion

The results of the present study revealed that the triterpenoid enriched extract from T. dioica root (CETD) offered significant protection from experimentally induced acute inflammatory ascites in Wistar albino rats in a dose-related fashion.

Table 1 demonstrates the effect of CETD on formalin-induced ascites in rats. Oral administration of CETD at the test dose of 25 mg/kg body weight showed significant (p < 0.05) inhibition of ascitic fluid formation as compared with control. CETD orally at the doses of 50 and 100 mg/kg body weight more significantly (p < 0.01) suppressed ascites when compared with the control group. The effect was dose dependent. The results at the higher doses are comparable to the effect of the reference anti-inflammatory agent ibuprofen (50 mg/kg, p.o.).

Table 1. Effect of CETD on formalin-induced ascites in rats.

Treatment	Dose	Volume of ascetic fluid (ml)	Percentage of protection
Normal saline	5 ml/kg	2.13 ± 0.32	–
CETD	25 mg/kg	1.71 ± 0.14*	19.71
CETD	50 mg/kg	1.46 ± 0.30**	31.46
CETD	100 mg/kg	1.41 ± 0.22**	33.80
Ibuprofen	50 mg/kg	1.32 ± 0.17**	38.03

Data are expressed as mean ± SEM (n = 8). *p < 0.05, **p < 0.01, compared to saline control group.

Ascites is the accumulation of fluid within the peritoneal cavity. The fluid is then known as ascitic fluid. The causes of ascites include infections (such as tuberculosis), cardiac failure, portal hypertension, cirrhosis and some cancers (Anonymous, 2005). Acute inflammatory ascites in human beings can occur as a complication of trauma, perforated ulcer, appendicitis or inflammation of the colon or other tube-shaped organ (diverticulitis). This condition can also develop when intestinal fluids or bacteria invade or inflame the membrane that lines the inside of the abdomen, i.e., peritoneum. Acute inflammatory ascites can be experimentally induced in mice and rats by intraperitoneal administration of certain phlogistic agents such as carrageenan, zymosan, formalin and plant lectins, and may be used to assess acute anti-inflammatory property of drugs and chemicals (Baintner, 2009; Pradhan et al., 2010; Turner, 1971).

Recently, the present authors have reported anti-inflammatory effects of CETD in experimental acute (carrageenan, histamine and serotonin-induced hind paw edema) and chronic models (cotton pellet-induced granuloma) in Wistar albino rats (Bhattacharya & Haldar, 2012i). There, CETD at the doses of 50 and 100 mg/kg body weight exhibited significant and dose-dependent anti-inflammatory activity in all the tested models. The present study corroborates the role of CETD as an acute anti-inflammatory agent. This correlation is further strengthened by the fact that the present study was performed in a different experimental model in which it was not studied earlier. It is therefore suggested that the anti-inflammatory effect of CETD could be further evaluated in other experimental models.

The abundance of triterpenoids along with cucurbitacin type aglycones was affirmed in CETD by phytochemical and planar chromatographic studies (HPTLC). Cucurbitacins are known to possess several important biological activities including anti-inflammatory activity (Chen, 2005; Miro, 2006). The presence of putative cucurbitacins could provide the chemical basis of its observed actions against inflammatory ascites.

From the present preliminary investigation, it can be concluded that the triterpenoid enriched fraction from T. dioica root was found to produce marked suppressive effect on formalin-induced inflammatory ascites in Wistar albino rats. To the best of our knowledge, this is the first report of anti-ascitic effect of T. dioica root. The outcome of the present study suggests that T. dioica root can be used in peritoneal acute inflammatory ascitic conditions and may substantiate the traditional uses of T. dioica root in the Indian subcontinent for the management of ascites. Further definitive studies are necessary to ascertain the mechanism and constituents responsible for this activity.

Declaration of interest

The authors report no declaration of interest. The authors alone are responsible for the content and writing of the paper.

Acknowledgements

The authors are thankful to the authorities of the Bengal School of Technology, A College of Pharmacy, Sugandha, Hooghly 712102, West Bengal, India, and Jadavpur University, Kolkata 700032, West Bengal, India for providing necessary facilities related to the present study.