|Year : 2014 | Volume
| Issue : 1 | Page : 39-43
Evaluation of anthelmintic activity and in silico PASS assisted prediction of Cordia dichotoma (Forst.) root extract
Prasad G Jamkhande, Sonal R Barde
Department of Pharmacology, School of Pharmacy, Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India
|Date of Web Publication||4-Feb-2015|
Prasad G Jamkhande
Department of Pharmacology, School of Pharmacy, Swami Ramanand Teerth Marathwada University, Dnyanteerth, Vishnupuri, Nanded - 431 606, Maharashtra
Source of Support: The present work has been supported by School
of Pharmacy, Swami Ramanand Teerth Marathwada University,
Nanded, Maharashtra, India, Conflict of Interest: None
Background: Worm infection and associated complications are severe problems that afflict a large population worldwide. Failure of synthetic drugs in worm infections because of drug resistance has made alternative drug therapy desirable. Cordia dichotoma (Forst.) is an ethnomedicinal plant which is rich in several secondary metabolites. Traditionally, the plant has been claimed to have high medicinal properties including antimicrobial activity and cytotoxicity.
Materials and Methods: The study begun with an aim to explore plant-based natural anthelmintic agents against Pheretima posthuma, an Indian earthworm. Methanol extract of the drug was obtained by successive soxhlet extraction. The extract was tested for different phytochemicals. Worms were exposed to 10 mg/ml, 25 mg/ml, 50 mg/ml, and 75 mg/ml concentrations of extract and standard drug, albendazole. A software-based tool, prediction of activity spectra for substances was used to estimate anthelmintic efficacy of plant metabolites.
Result: The phytochemical analysis revealed presence of alkaloids, tannins, glycosides, saponins, flavonoids, and phenols. The extract showed dose-dependent effects, affecting worm motility, viability, and mortality. It was also found that the biological activity spectrum of the plant phytoconstituents such as octacosanol, lupeol, caffeic acid, and hentricontanol were >0.5 (probable activity > 0.5).
Conclusion: The findings of the present work suggest that the extract of C. dichotoma significantly interferes with motility pattern of P. posthuma. The paralysis and mortality of P. posthuma might be due to the combined effects different phytoconstituents. The extract of C. dichotoma promises natural sources to control worm infection.
Keywords: Anthelmintic activity, Cordia dichotoma, paralysis and death, Pheretima posthuma, prediction of activity spectra for substances prediction
|How to cite this article:|
Jamkhande PG, Barde SR. Evaluation of anthelmintic activity and in silico PASS assisted prediction of Cordia dichotoma (Forst.) root extract. Ancient Sci Life 2014;34:39-43
| Introduction|| |
Worm infection, particularly helminthiasis is one of the most common chronic infections of humans. Worldwide more than 200 million people harbor these infections.  It is major global health problem mainly in tropical countries. These infections are common risk factor to community health in the developing countries and responsible for several conditions such as pneumonia, anemia, eosinophilia, and malnutrition.  Expelling or killing of helminths is the two primary mechanisms by which drugs produces anthelmintic effects.  Helminths mainly reside in the gastrointestinal tract, but some are known to invade tissues. The harmful effects of infection manifest as food deprivation, blood loss, local organ injury, intestinal or lymphatic obstruction. Sometimes, the same effects are also produced by worm secreted toxins. Although helminthiasis is not fatal, it is a principal etiological factor responsible for morbidity that in turn influences personal and social health and productivity.  The main aim of anthelmintic treatment is to control morbidity and eradicate infection burden, interrupt transmission and complete eradication of the parasite reservoir.  WHO has recommended only four drugs: Albendazole, mebendazole, levamisole, and pyrantel pamoate for soil-transmitted helminthiasis and these have been in use for several decades.  The main challenges in animal and human health are a wide range of parasitic diseases and the emergence of resistance to anthelmintic drugs. Resistance to anthelmintic drugs is a persistent problem worldwide and needs urgent solution. 
Drugs of natural origin have gained attention as a potential source of new therapeutic agents. Most of the clinically active drugs are either natural products or pharmacophore of the natural substance. It indicates the importance of drugs having natural sources in drug discovery process.  The use of plant as a source of medicine is as old as mankind. Traditional Indian medicine system is based on plants. 
Cordia dichotoma is an edible plant belonging to family Boraginaceae. The plant is widely distributed across India with references in traditional literature of Ayurveda and materia medica. In this, the Cordia species has been recommended in the treatment of several diseases. The plant is richly supplied with diverse phytochemicals including flavonoids, glycosides, alkaloids, terpenes and sterols, carbohydrates and proteins, and minerals like chromium. , Various parts of C. dichotoma such as fruit, seed, bark, and leaves have been screened for antidiabetic, antiulcer, anthelmintic, immunomodulatory, hepatoprotective, and antilarvicidal activity. The fruit and pulp have been reported as possessing significant anthelmintic activity. , Although roots of the plant have already screened for different phytochemicals, the anthelmintic activity is yet to be explored.
| Materials and methods|| |
Collection and identification of plant material
The roots of C. dichotoma were collected from Kendra (Budruk) area of Hingoli district, Maharashtra, India in the month of December and it was authenticated by Dr. B. D. Gachande of Department of Botany, N. E. S. Science College, Nanded, Maharashtra, India.
Preparation of extract
The shade-dried root material was powdered with the help of a grinder (sieving was manually done), and coarse powder material was obtained. The coarse powder material (500 g) was extracted with methanol using soxhlet apparatus. The extract was filtered, concentrated by evaporating the solvent in a rotary evaporator and kept in the refrigerator.
Preliminary phytochemical screening
The freshly prepared root extract of C. dichotoma was subjected to preliminary phytochemical screening for the identification of major chemical constituents according to the standard procedures. ,
Collection and authentication of worm
The Indian earthworms Pheretima posthuma (Annelida) employed in the present study were collected from moist and muddy soil of Vishnupuri village, Nanded district, Maharashtra, India. Worms were washed with normal saline to remove all the fecal matter and authenticated by Dr. P. B. Deshmukh, Department of Zoology, N. E. S. Science College, Nanded, Maharashtra, India.
The anthelmintic potential of methanolic root extract was screened using Indian earthworms P. posthuma because they anatomically and physiologically resemble the human intestinal roundworm parasite. , The different extract concentrations (10, 25, 50, and 75 mg/ml) were obtained by dissolving 100 mg, 250 mg, 500 mg, and 750 mg/ml of crude extract in 1 ml of tween 80 and adjusted volume up to 10 ml with normal saline solution. Albendazole was used as a standard drug for this study. , Nine groups each containing six earthworms of approximately of equal size were used. Four groups of earthworms were tested with methanolic extract at concentrations 10 mg/ml, 25 mg/ml, 50 mg/ml, and 75 mg/ml and other four groups were treated with standard anthelmintic drug, albendazole at same concentrations. A group of earthworms was exposed normal saline and tween 80 and considered as control.  Each group was observed for worm motility, and the time required to produce paralysis, or complete inactivity and mortality was recorded. The worms were considered paralyzed when no movement were observed except after vigorous shaking. The death time was recorded after ascertaining that worms neither moved when given external stimuli nor dipped in warm (50°C) water. Fading of body color was considered as a sign of complete mortality. Inhibition of worm motility was the factor considered for anthelmintic activity. ,
Prediction of activity spectra for substances computer program
The software-based program, prediction of activity spectra for substances (PASS) was used to obtain biological activity spectra including anthelmintic activity of phytoconstituents. Software estimates predicted activity spectrum of a compound as probable activity (P a ) and probable inactivity (P i ). The prediction of activity is based on structure-activity relationship analysis of the training set containing more than 205,000 compounds exhibiting more than 3750 kinds of biological activities. The values of P a and P i vary between 0.000 and 1.000. Only activities with P a > P i are considered as possible for a particular compound. If P a > 0.7, the probability of experimental pharmacological action is high and if 0.5 < P a < 0.7, probability of experimental pharmacological action is less. If the value of P a < 0.5, the chance of finding the activity experimentally is less, but it may indicate a chance of finding a new compound. ,,
All data were expressed as the mean ± standard error of mean data were subjected to one-way ANOVA followed by Tukey test. The statistical analysis performed with Graphpad Instat (Version 3, USA) software. P < 0.05 was considered statistically significant.
| Result|| |
Phytochemical screening of the extract
Preliminary phytochemical screening of C. dichotoma root extract revealed presence of several phytoconstituents listed in [Table 1].
The extract exhibited significant dose-dependent anthelmintic activity at all concentrations as compared to standard, albendazole [Table 2]. At higher concentration, loss of motility and mortality was more pronounced against P. posthuma. The time required for causing paralysis was (3.00 ± 0.11) min and death (9.00 ± 0.46) min at 75 mg/ml by the extract which was almost equal to the results obtained with albendazole. Among all the concentrations tested, the significant results for time to produce paralysis were observed at 10, 25 and 50 mg/ml whereas significant mortality was shown at all concentrations.
|Table 2: Anthelmintic activity of control, methanolic extract and albendazole |
Click here to view
Prediction of activity spectra for substances computer program
The phytoconstituents of C. dichotoma root were evaluated for their biological activity spectra, and results were used in a flexible manner. All the compounds showed greater P a than P i [Table 3]. The P a of caffeic acid, lupeol, and hentricontanol was found to be highest as 0.674, 0.622, 0.600, respectively. The P a of octacosanol and epigenine was more than 0.5 whereas taxifolin, rutin, hesperidin, α-amyrin, β-sitosterol glycoside, β-sitosterol, botulin, and chlorogenic acid showed less P a < 0.5.
|Table 3: PASS predictions of main phytoconstituents for anthelmintic activity |
Click here to view
| Discussion|| |
Several bioactive phytoconstituents such as alkaloids, tannins, glycosides, saponins, flavonoids, and phenolics were found predominantly during preliminary phytochemical screening of the extract of C. dichotoma which have been associated with an anthelmintic potential. , The plant, C. dichotoma has been already found to be rich in various secondary metabolites. ,
Infection of helminths is a major problem in human as well as in animals that in turn adversely affects the health and also causes drug resistance to other diseases. , To overcome these problems, there is a need for studies focusing on natural sources such as plants which give new biologically active agents having no or fewer side effects and more compatible with human physiology.  Phytochemical screening of the extract of C. dichotoma revealed the presence of different phytochemicals that produces anthelmintic effects by acting on metabolic pathways or physiological functioning of the worms. Alkaloids have ability to produce paralysis by acting on central nervous system whereas polyphenols and tannins selectively bind to free proteins present in the gastrointestinal tract and eventually cause mortality. Anthelmintic potential of saponin is due to its membrane permeabilizing property. , The anthelmintic efficacy of the methanolic extract of C. dichotoma may be due to a single compound or combined effect of these phytochemicals.
Previous studies of C. dichotoma reported the presence of caffeic acid, β-sitosterol, taxifolin, rutin, octacosanol, lupeol, hesperidin, α-amyrin, and hentricontanol.  PASS computer program was used to screen biological activity of these compounds as anthelmintics. Caffeic acid, lupeol, and hentricontanol showed highest P a value indicating a high probability of these compounds acting as anthelmintic agents. In humans, caffeic acid produces reactive oxygen species , whereas epigenine shows healing and platelet aggregation effect.  Lupeol is a multi-target agent with immense anti-inflammatory potential and also has an inhibitory effect on microtubule formation while octacosanol rapidly blocks the intracellular gap junction channels. 
In the present study, methanolic root extract of C. dichotoma has demonstrated potent anthelmintic effect on P. posthuma. C. dichotoma may offer an alternative drug source to control worm infection. However, more detailed studies are needed to confirm the results of anthelmintic activity and PASS predictions by various in vitro and in vivo methods. Further, there is a need to identify and evaluate the active components, their mechanisms of action, and toxicity profile so as to explore C. dichotoma as an anthelmintic agent.
| Acknowledgments|| |
We are very thankful to Director Prof. S. G. Gattani and Head of Department Prof. S. C. Dhawale School of Pharmacy, Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India for providing laboratory facilities for this research work.
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[Table 1], [Table 2], [Table 3]
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