Paralytic and lethal effects of Trichosanthes dioica root extracts in experimental worms

Abstract

Context: Trichosanthes dioica Roxb. (Cucurbitaceae), called pointed gourd in English, is a dioecious climber and its roots are traditionally used in India as a hydrogouge cathartic, tonic, and febrifuge, and in the treatment of jaundice, anasarca, and ascites.

Objective: To evaluate the in vitro effects of different solvent extracts of T. dioica root in experimental worms, viz. annelids and nematodes.

Materials and methods: The in vitro paralytic and lethal effects of defatted dichloromethane (DCTD), methanol (METD), and aqueous (AQTD) extracts of T. dioica root were evaluated against Pheretima posthuma (Annelida) and Ascaridia galli (Nematoda) by keeping the worms in different concentrations of each test extract under specific experimental conditions followed by determination of mean paralysis and lethal times. Albendazole was used as the reference drug.

Results and discussion: All the extracts demonstrated concentration-dependent paralytic and lethal effects on P. posthuma and lethal effects on A. galli. The DCTD was found to be the most potent followed by the METD and AQTD. A. galli was found to be more sensitive than P. posthuma against all extracts, indicating T. dioica root as an effective nematocide.

Conclusion: The present study establishes the in vitro wormicidal property of T. dioica root extracts against the experimental worms, showing promising nematocidal (and hence anthelmintic) potential.

Keywords::

• Nematocide
• anthelmintic
• Pheretima posthuma
• Ascaridia galli

Introduction

Helminths are parasitic worms and helminth infections are prevalent globally – one-third of the world’s population harbors them – but they are more common in developing tropical and subtropical countries with poorer personal and environmental hygiene (Tripathi, 2008). In developing countries they pose a great threat to public health and contribute to the prevalence of malnutrition, anemia, stunted growth, cognitive impairment, and increased susceptibility to other diseases, particularly in children. Although the majority of incidences of helminth infection are generally limited to tropical regions, they can also occur in travelers who have visited these areas, and some of them can develop in temperate climates (Bundy, 1994). In addition to humans, domestic animals are very susceptible to helminth infection, which adds to the economic burden of developing countries and also poses a problem for agriculture in many developed countries (Panjarathinam, 2007).

The helminths which infect the intestine are cestodes, e.g., tapeworms (Taenia solium), nematodes, e.g. hookworm (Ancylostoma duodenala) and roundworm (Ascaris lumbricoids), and trematodes or flukes (Schistosoma mansoni and Schistosoma haematobolium) (Panjarathinam, 2007). Increasing problems of resistance development in intestinal helminths against synthetic anthelmintics have led to the proposal of screening medicinal plants for their anthelmintic activity. Resistance against synthetic anthelmintics for gastrointestinal worms is a worldwide problem in sheep, goat, and pig breeding, resulting in considerable economic losses (Urban et al., 2008). Diseases due to nematode infections continue to be the greatest constraint in sustainable livestock production worldwide, primarily due to the rapid evolution of drug resistance in these parasites to all classes of synthetic anthelmintics. In addition, the global appreciation and general endorsement of organic farming poses serious restrictions on the prophylactic use of synthetic drugs (Waller, 2003; Stear et al., 2007). Hence, there is an increasing need for natural anthelmintics. A number of medicinal plants have been traditionally used in the Indian subcontinent to treat different helminth infections in man and animals (Akhtar et al., 2000; Mali & Mehta, 2008).

Trichosanthes dioica Roxb. (Cucurbitaceae), called pointed gourd in English, Potol in Bengali, Palval in Hindi, 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. It is also cultivated, particularly in Uttar Pradesh, Bihar, West Bengal, and Assam states of India, for its fruits, a common culinary vegetable in India. In India, all parts of this plant have been used traditionally for various medicinal purposes. According to Ayurveda, the traditional system of Indian medicine, its root is a drastic purgative. The root has traditionally been used as a hydrogouge cathartic, tonic, and febrifuge, and in the treatment of jaundice, anasarca, and ascites (Kirtikar & Basu, 1935; Anonymous, 1976, Nadkarni, 1976; Sharma et al., 2002). The leaves and tender shoots are also used medicinally and as a culinary spinach in West Bengal and Assam, and are called Palta in Bengali.

Previous workers have reported different phytochemical and pharmacological studies on T. dioica fruits and seeds in experimental animal models (Sharma & Pant, 1988a, 1988b, 1988c; Sharma et al., 1990; Kabir, 2000; Sultan et al., 2004; Sultan & Swamy, 2005; Sharmila et al., 2007; Ghaisas et al., 2008; Rai et al., 2008a, 2008b). As there are no reports on the anthelmintic activity of T. dioica roots, we found it necessary to evaluate different solvent extracts of the T. dioica root for their in vitro effects against experimental worm models, viz., Pheretima posthuma and Ascaridia galli.

Materials and methods

Plant material

The mature tuberous roots of T. dioica were collected during December 2008 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 (SB-02) was deposited at the Pharmacognosy Research Laboratory, Bengal School of Technology (A College of Pharmacy), Hooghly, India. Just after collection the plant material was washed thoroughly with water and shade dried at room temperature (24–26°C), and ground mechanically into a coarse powder.

Drugs and chemicals

All the chemicals used were of analytical grade, obtained from Ranbaxy Fine Chemicals Ltd., New Delhi, India. The reference drug, albendazole, was obtained as a gift sample from Mepro Pharmaceuticals Pvt. Ltd., Surendranagar, Gujarat, India. Double-distilled water from an all-glass still was employed throughout the study.

Preparation of extracts

The powdered plant material (750 g) was initially macerated with n-hexane (1 L) overnight, and the air-dried marc was macerated separately with dichloromethane (DCM), methanol (MeOH), and distilled water (450 mL each) at room temperature (24–26°C), with frequent shaking, for 4 days followed by re-maceration with the solvents, similarly for 3 days. The pooled extracts were filtered and evaporated to dryness in vacuo at 40°C to yield DCM (3.72%), MeOH (7.22%), and aqueous extracts (11.05%), which were denoted as DCTD, METD, and AQTD, respectively. All the dry extracts were kept in a refrigerator until use. Preliminary phytochemical screening (Harborne, 1998) revealed the presence of flavonoids, triterpenoids, and steroids in the DCTD. The METD revealed the presence of flavonoids, triterpenoids, and steroids, saponins, amino acids, carbohydrates, and reducing sugars, whereas the AQTD indicated the presence of flavonoids, saponins, carbohydrates, reducing sugars, and amino acids.

Experimental worms

Adult Indian earthworms, Pheretima posthuma L. Vaill. (Annelida), were obtained from the waterlogged areas of the State Paddy Research Center, Chinsurah, West Bengal, India. Live infected fowls (Gallus domesticus Linn.) of age 8–9 months, obtained from the local abattoir at Chandernagore, West Bengal, India, were sacrificed, and immediately, live Ascaridia galli Schrank (Nematoda) were recovered from the small intestines. Only the live adult worms with more or less the same length (65–75 mm) were selected, and then collected in phosphate buffered saline (PBS, pH 7.2, 0.15 M). All nematodes were immediately maintained at 39 ± 2°C in an incubator. Both types of worms were identified at the State Paddy Research Center, Chinsurah, West Bengal, India.

Test samples

Test samples for in vitro bioassay were prepared freshly. Varying concentrations of all the test extracts, viz. 50, 25, 12.5, 6.25, 3.13 mg/mL, were prepared by dissolving or suspending in distilled water, for annelids. Similar dilutions were made in PBS (pH 7.2, 0.15 M), supplemented with 2% dimethyl sulfoxide (DMSO), for nematodes.

In vitro bioassay

The bioassay was carried out according to methods reported by previous workers (Ghosh et al., 2005; Lalchhandama, 2008) with requisite modifications. Fresh worms of nearly equal size were selected for the study. Both types of worm were divided into 17 groups (n = 6). The first group served as positive control and was kept in 9 cm Petri dishes (one worm in each) containing 20 mL of albendazole (10 mg/mL) in distilled water (for annelids) or in 2% DMSO in PBS (for nematodes). The second group served as negative control and was kept in distilled water or in 2% DMSO in PBS (pH 7.2, 0.15 M) for annelids and nematodes, respectively. Five groups were kept in Petri dishes containing 20 mL of DCTD at five different concentrations (50 to 3.125 mg/mL). Similarly, the remaining 10 groups were kept in varying concentrations of METD and AQTD as prepared for annelids and nematodes. The Petri dishes were kept at room temperature (25 ± 2°C) for annelids and maintained at 39 ± 2°C for nematodes in an automated glass-chambered incubator. Physical activity of the worms was observed, and the times taken for complete paralysis and death were recorded. The mean paralysis time and mean lethal time for each group were determined. Time for paralysis was noted when no movement of any sort could be observed except when the worms were stimulated gently by a blunt pin or probe to activate them. Death was ascertained when complete immobility was noted upon poking and shaking, and dipping the parasite in tepid water (~50°C), which induced movement in living sentient worms, along with the observation of fading of their body color. Albendazole served as the reference drug in the positive control group.

Statistical analysis

The data are presented as mean ± standard error of the mean (SEM).

Results

The results for in vitro effects of different test extracts from T. dioica root in P. posthuma and A. galli are summarized in Tables 1 and 2, respectively. Against P. posthuma, all the test extracts exhibited significant paralytic and lethal actions in a concentration-dependent manner (Table 1). The DCTD was the most potent, showing the shortest paralysis and lethal times, followed by METD and AQTD, which was least active and only at higher concentrations, exhibiting the most prolonged paralytic and lethal times. In two cases (Table 1), lethal times were not detected during 72 h of observation. Neither paralysis nor death of test worms was found in the negative control group, or with lower concentrations of the AQTD during 72 h of observation.

Table 1.  Effects of T. dioica root extracts against P. posthuma.

Treatment	Concentration (mg/mL)	Mean paralysis time (min) ± SEM	Mean lethal time (min) ± SEM
Positive control (albendazole)	10	35.56 ± 0.85	62.75 ± 0.43
Negative control (vehicle^a)	—	—	—
DCTD	50	38.33 ± 0.66	102.00 ± 0.72
	25	54.77 ± 0.92	81.64 ± 1.09
	12.5	98.13 ± 1.66	123.65 ± 0.87
	6.25	126.73 ± 1.13	211.84 ± 1.71
	3.125	154.93 ± 1.54	261.12 ± 1.78
METD	50	56.12 ± 0.90	70.28 ± 0.78
	25	66.84 ± 1.11	106.36 ± 0.95
	12.5	78.56 ± 1.36	120.23 ± 1.25
	6.25	160.18 ± 1.16	304.50 ± 1.14
	3.125	243.47 ± 1.62	—
AQTD	50	148.32 ± 1.04	226.04 ± 1.31
	25	237.68 ± 1.83	378.15 ± 2.19
	12.5	327.06 ± 2.24	—
	6.25	—	—
	3.125	—	—

Note. Number of worms per group (n) = 6. SEM, standard error of mean. ^aDistilled water.

Table 2.  Effects of T. dioica root extracts against A. galli.

Treatment	Concentration (mg/mL)	Mean lethal time (min) ± SEM
Positive control (albendazole)	10	26.08 ± 0.79
Negative control (vehicle^a)	—	67.94^b ± 0.83
DCTD	50	53.06 ± 0.63
	25	64.28 ± 0.82
	12.5	80.61 ± 1.17
	6.25	104.54 ± 1.41
	3.125	119.84 ± 1.09
METD	50	66.23 ± 0.86
	25	83.17 ± 1.04
	12.5	117.13 ± 0.98
	6.25	144.37 ± 1.26
	3.125	176.45 ± 1.41
AQTD	50	93.19 ± 0.75
	25	112.33 ± 0.93
	12.5	140.73 ± 1.13
	6.25	186.56 ± 1.08
	3.125	261.03 ± 1.63

Note. Number of worms per group (n) = 6. SEM, standard error of mean. ^a2% DMSO in PBS solution (pH 7.2, 0.15 M). ^bTime in hours.

Also in the case of A. galli, all the test extracts demonstrated concentration-dependent lethal effects (Table 2). Once the movement of worms ceased, i.e. they were paralyzed, death occurred instantaneously; hence, the paralysis times were not detected in this case. The DCTD was the most active, followed by METD and AQTD, which was the least active. The DCTD showed the shortest lethal time (17.06 min), as compared against P. posthuma at the same concentration (50 mg/mL). At all concentrations of all test extracts, mortality of worms was detected. In this case, however, mortality was found in the negative controls during 72 h of observation.

Discussion

In the present investigation, the in vitro effects of the defatted DCM, MeOH, and aqueous extracts of T. dioica root were evaluated by their paralytic and lethal actions against the annelid, free-living earthworm P. posthuma, and the parasitic nematode, A. galli. The adult Indian earthworm P. posthuma, although not a helminth, has been extensively employed by previous workers as an experimental model for initial in vitro anthelmintic evaluation because of the easy availability and anatomical and physiological resemblance to the intestinal roundworm parasites in humans (Chatterjee, 1967; Vidyarthi, 1967; Thorn et al., 1977; Vigar, 1984). A. galli is a roundworm parasitizing the small intestine of birds, and is by far the most prevalent of all helminths infecting poultry. A. galli infections continue to be the most debilitating factor impeding poultry productivity, resulting in retarded growth, weight loss, diarrhea, poor absorption of nutrients, death, and even the spread of fatal bacterial infections (Permin et al., 1999; Gauly, 2007). This worm is also an easily available and suitable experimental model for in vitro anthelmintic evaluation (Kaushik et al., 1974; Singh & Nagaich, 1999, 2002).

In the present study, P. posthuma were found to be paralyzed and eventually killed by all the test extracts in a clear-cut concentration-dependent manner, showing differential toxic activities that diminished with lowering concentration. In the case of A. galli, indeed there was no definitive identifying sign of paralysis, unlike with P. posthuma, in which a considerable time always elapsed between paralysis and actual death. Once the nematodes indicated paralysis (being motionless) they could not be resuscitated, and death was inevitably simultaneous. The DCTD exhibited maximum potency, i.e., shortest paralysis and lethal times in both test worms. The METD also exhibited significant effects. The AQTD was found to be least potent against the test worms, showing comparatively prolonged survival. In fact, all the test extracts were active against the experimental worms. Against A. galli, lethal times for all test extracts were found to be comparatively shorter than those against P. posthuma, thereby indicating the relative sensitivity of nematodes to the extracts and thus confirming their marked nematocidal potential. However, at lower concentrations, paralysis and/or mortality of P. posthuma was not observed in some cases during the observation period (see the ‘Results’ section).

In the case of A. galli, the exceptionally long time (67.94 h) of in vitro survival in the negative control group plausibly revealed the nature of the vehicle used in the experiment with the nematodes. In the vehicle control group the vehicle itself may have affected the activity in an enhancing way. In this group also, the nematodes were persistently active during their long survival time, but once their movement ceased, death ensued. In the case of P. posthuma, neither paralysis nor death was found in negative controls during the 72 h of observation. Hence, statistical analysis for significance was not carried out with respect to the vehicle control group.

Preliminary phytochemical screening revealed the presence of various compounds in METD, whereas the DCTD mainly contained triterpenoids and steroids. It appears that the presence of triterpenoids and/or steroids was responsible for the enhanced activity of DCTD. In this connection it is noteworthy to mention that the METD also contained triterpenoids and steroids, along with several other constituents, but the expected synergistic effect, however, was not observed here, as DCTD was more active than METD. The AQTD exhibited the lowest effect, possibly because of the absence of triterpenoids and steroids. Some synergy was obvious, indicating METD to be more active than AQTD, because of the presence of triterpenoids and steroids in METD. Nevertheless, activity in the AQTD indicated that not only triterpenoids and steroids were responsible for the activity. Dereplication strategies based on these findings could be helpful for isolation of the active constituents.

Thorough histological examination and enzymatic and elemental studies of normal (negative control) and treated worms may give an insight into the structural and biochemical alterations of the organ, tissue, and cellular systems during paralysis and death, in pursuit of the plausible mechanism of paralytic and lethal actions of T. dioica roots.

In the present investigation, a free-living annelid model, P. posthuma, was selected to observe the toxic potential of the extracts on higher organisms. This model was also advocated by previous workers for preliminary anthelmintic screening. The second model used was a parasitic intestinal nematode, A. galli, as prototype helminth worm to confirm the possible anthelmintic potential. The present study confirms the in vitro paralytic and lethal (wormicidal) effects of T. dioica root extracts in both experimental worms, and vermicidal (anthelmintic) activity in parasitic nematodes. The results indicate that the METD and the DCTD, especially, possess potential in vitro wormicidal effects, including remarkable nematocidal property, but more positive results are needed in other groups of parasitic helminths, especially in vivo, for prospective broad-spectrum anthelmintic property. The present preliminary investigation provides comprehensive in vitro evidence that T. dioica root is an effective vermicide, supporting the feasibility of its usage as a natural anthelmintic. However, further in vivo studies are necessary to confirm its anthelmintic potential in the host body.

Acknowledgement

The authors are thankful to the Director of the State Paddy Research Center, Chinsurah, West Bengal, India, for providing and identifying the experimental worms for the present study.

Declaration of interest

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