|Year : 2016 | Volume
| Issue : 1 | Page : 21-24
Lack of anthelmintic activity of Kalanchoe pinnata fresh leaves
Rohan Sharadanand Phatak
Directorate of Research, Krishna Institute of Medical Sciences, Deemed University, Karad, Maharashtra, India
|Date of Web Publication||19-Feb-2016|
Rohan Sharadanand Phatak
Directorate of Research, Krishna Institute of Medical Sciences Deemed University, NH-4, Near Dhebewadi Road, Malkapur, Karad - 415 539, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The main objective was to explore the anthelmintic capacity of Kalanchoe pinnata (K. pinnata). Materials and Methods: Petroleum ether and methanolic extracts of K. pinnata were extracted by maceration methods. Both the extracts were investigated for their anthelmintic activity against Pheretima posthuma. Different concentrations of 25 mg/mL, 50 mg/mL, 100 mg/mL, and 200 mg/mL of both extracts were studied by determining time of paralysis (PT) and time of death (DT) of the earthworms. Results: Both the extracts have exhibited no anthelmintic activity even at the highest concentration of 200 mg/mL. A concentration of 15 mg/mL of albendazole was used as standard reference or positive control while normal saline was used as negative control. Conclusion: This study has revealed that in vitro anthelmintic activity of K. pinnata leaves lacks anthelmintic property. Both the extracts of this plant have displayed no vermicide activity against the earthworms used in the study.
Keywords: Albendazole, anthelmintic, Kalanchoe pinnata, Pheretima posthuma
|How to cite this article:|
Phatak RS. Lack of anthelmintic activity of Kalanchoe pinnata fresh leaves. J Pharm Negative Results 2016;7:21-4
| Introduction|| |
According to the World Health Organization, soil-transmitted helminths is prevalent in more than 24% of the population in the world.  Infections are widely distributed in tropical and subtropical areas, with the greatest numbers occurring in sub-Saharan Africa, America, China, and East Asia. Roundworm (Ascaris lumbricoides), the human whipworm (Trichuris trichiura), and hookworms (Necator americanus and Ancylostoma duodenale) ,, are the main responsible species of helminths.
The World Health Organization has taken various preventive measures to deworming helminth infections through periodic treatment of children, cleaning of water, sanitation, and hygiene training to minimize parasitic infections.  Thus, the anthelmintic effect of plant extracts is needed in a great deal to combat the anthelmintic resistance. Due to increased prevalence of anthelmintic resistance, and less efficacy of conventional anthelmintics, it is the need of the hour to discover naturally occurring anthelmintics as alternative strategies.
Upon reviewing the scientific documents on Kalanchoe pinnata till date,  it was found that majority of the studies are on its phytochemical active principles rich in alkaloids, triterpenes, glycosides, flavonoids, cardenolides, steroids, bufadienolides, lipids, and its pharmacological properties such as hepatoprotective, nephroprotective, neuropharmacological, antimutagenic, antiulcer, antibacterial,  antioxidant,  free radicals scavenging,  antidiabetic, immunosuppressive, antihypertensive, analgesic, anti-inflammatory, wound healing, uterine contractility, insecticidal, fungitoxic, phytotoxic activities,  and other in vitro activities against renal urolithiasis. 
K. pinnata (family Crassulaceae) is grown as a weed in the tropical countries such as India and Bangladesh. The leaves of K. pinnata have complex chemical composition comprising triterpenoids, steroids, polyphenols; flavonoids, squalene, phytol, squalene, and 5-oxotetrahydrofuran-2,3-dicarboxylic acid dimethyl ester.  These leaves possess prominent pharmacological properties. The Latin synonyms of this plant, such as Bryophyllum calycinum Salisb and Bryophyllum pinnatum Linn, are well known. Miracle plant or air plant, panfuti (Hindi), life plant, love plant, air plant (Mexican), good luck or resurrection plant, Zakham-e-hayat, Canterbury bells, and cathedral bells are diverse common folk names for this plant. It is also popularly known as katakataka for the presence of an astonishing taste. 
Costa et al. 1999  have reported that the K. pinnata leaf extract can significantly reduce the parasite load in mice infected with Leishmania amazonensis by stimulating the production of reactive nitric oxide (NO) in the macrophages to effectively instigate indirect inhibition of extracellular promastigotes and to decrease the intracellular amastigote growth. Quercitrin, an unusual flavonoid in the plant K. pinnata, was isolated and evaluated by Costa et al. (2006) and was established as an effective antileishmanial agent. 
However, anthelmintic activity of K. pinnata has not been reported till date during literature survey, as they are well-documented antileishmanial activities. , Thus, in the present study, an effort has been made to explore whether fresh leaves of K. pinnata exhibited anthelmintic activity.
| Materials And Methods|| |
Fresh leaves of K. pinnata were collected in the month of February from Karad, Maharashtra, India. It has already been authenticated by the authority of the Botany Department, Yashwantrao Chavan College of Science, Karad, Maharashtra, India, in a previous study. 
Preparation of extracts
Fresh leaves of K. pinnata were collected and washed under running tap water. About 350 g of chopped leaves of this plant was macerated with petroleum ether for 24 h for defatting process. The filtrate was collected and concentrated on evaporation till a syrupy mass was obtained. Then, the extract was again dissolved with sufficient amount of petroleum ether and was preserved in a suitable container at 4°C. The percentage yield was obtained to be 0.26%.
Defatted leaves of this plant were then extracted with methanol by cold maceration. The collected filtrate was evaporated to dryness. The percentage yield was obtained to be 1.0%.
Preparation of test sample
Samples for in vitro study of anthelmintic activity were prepared by dissolving 0.5 mL of petroleum ether and methanolic extracts of K. pinnata in 10 mL of distilled water to a stock solution with the concentration of 50 mg/mL. From this stock solution, different working solutions of different concentrations of 25 mg/mL, 50 mg/mL, 100 mg/mL, and 200 mg/mL were prepared. The anthelmintic assay was carried as per the methods of Ajaiyeoba et al. and Mulla et al. ,
Adult Indian earthworm-Pheretima posthuma (Annelida: Megascolecidae)-was chosen due to its anatomical and physiological resemblance with the intestinal roundworm parasite of human beings.  Because of easy availability, earthworms have been used widely for the initial evaluation of anthelmintic compounds in vitro.  These earthworms (P. posthuma) were collected from moist soil and were washed with normal saline to remove all fecal matter before using them for the anthelmintic study. The earthworms of 3-5 cm in length and 0.1-0.2 cm in width were used for all the experimental protocols. Bioassay was performed on these earthworms.
In 10 experimental groups, 6 earthworms were taken in Petri dish More Details containing different concentrations of petroleum ether and methanolic extracts. Earthworms were served normal saline as negative control in group I. Albendazole in dose of 10 mg/mL was given to earthworms for positive control in group II. Different doses of petroleum ether extract of K. pinnata (25 mg/mL, 50 mg/mL, 100 mg/mL, and 200 mg/mL) were given to earthworms in groups III, IV, V, and VI. Methanolic extract of K. pinnata was given to earthworms in groups VII, VIII, IX, and X with various doses (25 mg/mL, 50 mg/mL, 100 mg/mL, and 200 mg/mL) correspondingly.
In the first set of experiments, three groups of six earthworms were released into 50 mg/mL of solutions of petroleum ether and methanolic extracts of K. pinnata each in 25 mg/mL, 50 mg/mL 100 mg/mL, and 200 mg/mL. Albendazole was used as standard reference while normal saline was used as control.
All the test solutions and standard drug solutions were freshly prepared before starting the experiments. Observations were made for the time taken for paralysis (PT), no movement of any sort was observed except when the worms were shaken vigorously. The time of death (DT) of worms was recorded after ascertaining that worms did not move even when shaken vigorously. The death was confirmed when the worms lost their motility, followed by fading away of their body colors.
Mean and standard deviation (Mean ± SD) was applied as statistical tools.
| Results And Discussion|| |
Petroleum ether and methanolic extracts of K. pinnata were investigated for its anthelmintic activity against P. posthuma. Various concentrations (25 mg/mL, 50 mg/mL, 100 mg/mL, and 200 mg/mL) of each extract were tested in the bioassay, which involved determination of PT and DT of the earthworms. Albendazole was included as standard reference or positive control and normal saline was included as negative control. All the results were shown in [Table 1] and expressed as mean ± SD of six earthworms in each group.
|Table 1: Anthelmintic activity of Kalanchoe pinnata fresh leaves extract|
Click here to view
Albendazole in a dose of 25 mg/mL caused the paralysis to earthworms within 1.99 ± 0.005 min and lead to death within 5.14 ± 0.005 min. Since results are negative, so statistical tools like t value or P value cannot be applied.
The outer layer of the earthworm is made up of complex polysaccharides with a mucilaginous layer, which enables ciliary movement. Any leakage of mucopolysaccharide membrane leads to the loss of movement and irritation leading to spastic paralysis. Damage to the mucopolysaccharide layer may cause the death of the worm.  To evaluate anthelmintic assay, earthworms were chosen from different types of worm species as it is more vulnerable to all anthelmintics. 
From the results shown in [Table 1], the predominant effect of albendazole on worms is to cause a flaccid paralysis that result in expulsion of the worm by peristalsis. Albendazole leads to hyperpolarization in the worm muscle membrane by increasing chloride ion and this in turn results in flaccid paralysis of worms.  Therefore, it is better to use albendazole as positive standard in the study.
Several modes of action of certain anthelmintic drugs have been explored in different ways such as causing spastic paralysis by acting on nicotinic acetylcholine receptors in muscle, antagonism effect on cholinesterase receptors, enzymes involved in arachidonic acid metabolism, phosphoglycerate kinase and mutase, opening of glutamate-gated chloride (GluCl) channels, pharyngeal pumping, increasing permeability of calcium, binding selectively to beta-tubulin, inhibiting microtubule formation, and inducing proton ionophores.  Anthelmintic resistance is widespread among cattle and humans, which is a major concern for public health. Benzimidazole, levamisole/morantel, and ivermectin resistances are detected in nematodes of cattle. 
Some researchers  have explained four contributing factors for developing anthelmintic resistance such as initial resistance allele frequency, treatment frequency, refugia, and possible underdosing. Improved studies in biochemistry for anthelmintic actions and molecular genetics for DNA probes in benzimidazole resistance/susceptibility are needed for developing newer sensitive assays in the detection of anthelmintic resistance.  To overcome benzimidazole resistance, some researchers have developed one compound of aminobenzylated mannich bases of piperazine that was found to be more significant than standard drugs. 
Negative result in the current anthelmintic study indicates that it should be further investigated in studies apart from earthworm in vitro anthelmintic assay. Larval development and egg hatch assays are needed to detect the anthelmintic activity of the plant in the future studies. Our study is in line with other studies in this area. 
| Conclusion|| |
This study has revealed that in vitro anthelmintic activity of K. pinnata leaves is insignificant, therefore, it is an ineffective anthelmintic agent. Both extracts of this plant have displayed no vermicide activity against the earthworms used in the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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