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95]

Original Article

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

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
25mg/mL, 50mg/mL, 100mg/mL, and 200mg/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 200mg/mL. Aconcentration of 15mg/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 invitro 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.
Key words: Albendazole, anthelmintic, Kalanchoe pinnata, Pheretima posthuma

INTRODUCTION
According to the World Health Org anization,
soiltransmitted helminths is prevalent in more than
24% of the population in the world.[1] Infections are
widely distributed in tropical and subtropical areas, with
the greatest numbers occurring in subSaharan Africa,
America, China, and East Asia. Roundworm(Ascaris
lumbricoides), the human whipworm(Trichuris trichiura), and
hookworms(Necator americanus and Ancylostoma duodenale)[13]
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.[2] Thus, the
Address for correspondence:
Mr. Rohan Sharadanand Phatak, Directorate of Research, Krishna
Institute of Medical Sciences Deemed University, NH4, Near
Dhebewadi Road, Malkapur, Karad415539, Maharashtra, India.
Email:phatak.rohan1983@gmail.com
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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,[4] it was found that majority of the studies
are on its phytochemical active principles rich inalkaloids,
triterpenes, glycosides, flavonoids, cardenolides, steroids,
bufadienolides, lipids, and its pharmacological properties such
as hepatoprotective, nephroprotective, neuropharmacological,
antimutagenic, antiulcer, antibacterial,[4] antioxidant,[5] free
radicals scavenging,[6] antidiabetic, immunosuppressive,
antihypertensive, analgesic, antiinflammatory, wound healing,
uterine contractility, insecticidal, fungitoxic, phytotoxic
activities,[4] and other invitro activities against renal urolithiasis.[7]
K.pinnata(family Crassulaceae) is grown as a weed in the
tropical countries such as India and Bangladesh. The leaves
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DOI:
10.4103/0976-9234.177056

How to cite this article: Phatak RS. Lack of anthelmintic activity

of Kalanchoe pinnata fresh leaves. J Pharm Negative Results
2016;7:21-4.

2016 Journal of Pharmaceutical Negative Results | Published by Wolters Kluwer -Medknow

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Phatak: Lack of anthelmintic activity of K. pinnata

of K.pinnata have complex chemical composition comprising

triterpenoids, steroids, polyphenols; flavonoids, squalene,
phytol, squalene, and 5oxotetrahydrofuran2,3dicarboxylic
acid dimethyl ester.[8] 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, Zakhamehayat,
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.[4]
Costa etal. 1999[9] 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 etal.(2006)
and was established as an effective antileishmanial agent.[10]
However, anthelmintic activity of K.pinnata has not been
reported till date during literature survey, as they are
welldocumented antileishmanial activities.[9,10] Thus, in the
present study, an effort has been made to explore whether
fresh leaves of K.pinnata exhibited anthelmintic activity.

MATERIALS AND METHODS

Plant material

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.[5]
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 4C. 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%.

22

Preparation of test sample

Samples for invitro study of anthelmintic activity were

prepared by dissolving 0.5mL of petroleum ether and
methanolic extracts of K.pinnata in 10mL of distilled
water to a stock solution with the concentration of
50mg/mL. From this stock solution, different working
solutions of different concentrations of 25mg/mL,
50mg/mL, 100mg/mL, and 200mg/mL were prepared.
The anthelmintic assay was carried as per the methods of
Ajaiyeoba etal. and Mulla etal.[11,12]
Animals

Adult Indian earthwormPheretima posthuma(Annelida:

Megascolecidae)was chosen due to its anatomical and
physiological resemblance with the intestinal roundworm
parasite of human beings.[11] Because of easy availability,
earthworms have been used widely for the initial
evaluation of anthelmintic compounds invitro.[12] 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 35cm in length and 0.10.2cm 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 containing different concentrations of petroleum ether
and methanolic extracts. Earthworms were served normal
saline as negative control in groupI. Albendazole in dose
of 10mg/mL was given to earthworms for positive control
in groupII. Different doses of petroleum ether extract of
K.pinnata(25mg/mL, 50mg/mL, 100mg/mL, and 200mg/
mL) were given to earthworms in groupsIII, IV, V, and VI.
Methanolic extract of K.pinnata was given to earthworms in
groupsVII, VIII, IX, and X with various doses(25mg/mL,
50mg/mL, 100mg/mL, and 200mg/mL) correspondingly.
Anthelmintic activity

In the first set of experiments, three groups of six

earthworms were released into 50mg/mL of solutions of
petroleum ether and methanolic extracts of K.pinnata each
in 25mg/mL, 50mg/mL 100mg/mL, and 200mg/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.

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Phatak: Lack of anthelmintic activity of K. pinnata

Statistical analysis

Mean and standard deviation(MeanSD) 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(25mg/mL, 50mg/mL,
100mg/mL, and 200mg/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 Table1 and expressed as meanSD of six earthworms
in each group.
Albendazole in a dose of 25mg/mL caused the paralysis
to earthworms within 1.990.005min and lead to death
within 5.140.005min. Since results are negative, so
statistical tools like t value or Pvalue 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.[12] To evaluate anthelmintic assay,
earthworms were chosen from different types of worm
species as it is more vulnerable to all anthelmintics.[13]

muscle, antagonism effect on cholinesterase receptors,

enzymes involved in arachidonic acid metabolism,
phosphoglycerate kinase and mutase, opening of
glutamategated chloride(GluCl) channels, pharyngeal
pumping, increasing permeability of calcium, binding
selectively to betatubulin, inhibiting microtubule formation,
and inducing proton ionophores.[15] 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.[16]
Some researchers[3] 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.[16] 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.[17]
Negative result in the current anthelmintic study indicates
that it should be further investigated in studies apart from
earthworm invitro 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.[18]

From the results shown in Table1, 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.[14] Therefore, it is better to use
albendazole as positive standard in the study.

CONCLUSION

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

Financial support and sponsorship

This study has revealed that invitro 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.
Nil.

Conflicts of interest

There are no conflicts of interest.

Table1: Anthelmintic activity of Kalanchoe

pinnata fresh leaves extract
Test group
Normal saline
Petroleum ether extract
Methanolic extract

Concentration(mg/mL)
25

50

100

200

0
0

0
0

0
0

0
0

Values were taken in meanSD, PT: Time of paralysis(in minutes), DT: Time of
death(in minutes)

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Journal of Pharmaceutical Negative Results | Jan-Dec 2016 | Vol 7 | Issue 1

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