PHYTOCHEMICAL INVESTIGATION OF THE EUCALYPTUS GLOBULUS LEAF EXTRACT AND PIPER NIGRUM SEED EXTRACT AND SCREENING THE NEMATICIDAL ACTIVITY OF THEIR COMBINATION AGAINST NEMATODE PARASITES OF GALLUS GALLUS DOMESTICUS *Dama L.B., *Masuldar A. A. and **Bangale P.P.

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PHYTOCHEMICAL INVESTIGATION OF THE EUCALYPTUS GLOBULUS LEAF EXTRACT AND PIPER
NIGRUM SEED EXTRACT AND SCREENING THE NEMATICIDAL ACTIVITY OF THEIR
COMBINATION AGAINST NEMATODE PARASITES OF GALLUS GALLUS DOMESTICUS
*Dama L.B., *Masuldar A. A. and **Bangale P.P.
*Department of Zoology, D.B.F. Dayanand College of Arts and Science, Solapur, (M.S.), India.
**Department of Zoology, Rashtramata Indira Gandhi College, Jalna (M.S.), India.
Corresponding author: E-mail: damalaxmikant@gmail.com)
ABSTRACT
Nematode parasites causes major health hazard in domestic animals and birds which are responsible for reduction
in livestock productivity. Nowadays different plants and their parts are used as alternate source of anthelmintics
because of high cost of chemical anthelmintics and their unavailability in remote areas. Eucalyptus globulus and
piper nigrum are valuable medicinal plants which are useful and without any side effects. There are certain reports
given by different researchers that plant extracts used in combination against parasites shows better results due to
their synergetic action therefore in present investigation, combination of Eucalyptus globulus leaf extract and piper
nigrum seed extracts were used and screened In vitro against nematode parasite (Ascaridia galli) from host Gallus
gallus domesticus. Activity was found to be good and dose dependent. Phytochemical investigations of these plant
extracts show different bioactive compounds which may be responsible for Nematicidal activity and shows better
results due to their use in combination.
KEY WORDS: Anthihelminthic activity, Ascaridia galli, Eucalyptus globulus, Gallus gallus domesticus, Nematicidal
activity, phytochemical investigation, piper nigrum, synergetic activity.
INTRODUCTON
Livestock productivity has been decreased due to various infectious diseases like protozoal diseases, parasitic diseases
viral and bacterial diseases. Parasitic diseases are due to various ectoparasites and endoparasites. Ectoparasites are
different mice, ticks, flies etc, while endoparasites are cestodes nematodes and trematodes. Among which nematode
infection is also more abundant due to its known pathogenicity. The gastrointestinal nematode infection causes severe
weight loss, anemia, diarrhea which leads to production loss in livestocks. Ascaridiosis due to Ascaridia galli has
become the major cause of economic loss in poultry production system specially free-range floor production system
which causes reductions in growth rate, induces damage to the intestinal mucosa, and leads to blood loss, weight loss of
fowl, as well as it causes complete or partial obstruction of the intestine which increases mortality due to secondary
infections. (Lalchhandama, 2010). To combat these problems, various anthelmintic drugs have been used by different
researchers. The nematode parasites found in small ruminants has got resist to certain synthetic anthelmintics (Badar et
al., 2011). Hence use of medicinal plants to solve these problems is having a great importance. From ancient time
various plants have been used for treating various diseases in human and animals as they provides rich sources of
antibacterial, insecticides and anthelmintics (Dama and Jadhav, 1998; Dama et al., 1998; Dama and Kirdak, 2000;
Jadhav and Dama, 1997; Lateef et al ., 2013). Medicinal plants are worldwide used to combat various diseases because
they are easily available and without any side effect. several different herbs are used in combination by different
Practitioners and they says that using of combining herbs improves efficacy and leads to reduction in adverse effect
which proves the principles of synergy and buffering system of combinations of plants. (Pal et al., 2003).
Therefore, the present study focused on nematicidal activity of combination of Eucalyptus globulus cold methanolic
extract (CME) of leaf as well as cold methanolic extract of piper nigrum seeds, against nematodes of Gallus gallus
domesticus. Gallus gallus domesticus is the important livestock poultry bird and nowadays various diseases are
responsible for loss of poultry production. During present work nematode parasites (Ascaridia galli) were abundantly
found in the intestine of freshly slaughtered host than cestodes. Therefore study is focused on nematode parasites only.
The potential of preparing nematicidal drugs from valuable medicinal plants would play a vital role in treating
Nematadiasis for controlling nematode parasites as well as it may leads to formation of new phytomedicines which will
have more importance.
MATERIALS AND METHODS
Collection of plant material: Leaves of Eucalyptus globulus were collected from trees of local areas of Solapur
District Maharashtra India while seeds of piper nigrum were purchased from local ayurvedic shop of city. The leaves

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were washed with tap water and air-dried in the shade at the room temperature for about one week and made in
powdered form while seeds were directly powdered.
Extraction of plant materials:
250 grams of powdered leaves and seeds were placed separately in round bottom flask and soaked with methanol and
kept for about one week. By using cold maceration method, Cold Methanolic extract (CME) of Eucalpyptus globulus
and piper nigrum were prepared separately. After that column chromatography of both the extracts were performed for
obtaining different eleuents. Those eleuents further used in combination for observing nematicidal activity against
nematode parasites (Ascaridia galli) of Gallus gallus domesticus.
Collection of nematodes:
Total of 40 freshly slaughtered intestines of Gallus gallus domesticus were collected from different localities in the
month of June 2015 and studied for their gastrointestinal parasites. Most of the nematodes were observed in small and
large intestine of Gallus gallus domesticus after dissection (Figure 1). The collected nematodes were washed with
distilled water and kept in control media (PBS) phosphate buffer solution and proceed for nematicidal activity.
A B
Figure 1. Nematodes infection in small intestine (A) and in rectum (B) of Gallus gallus domesticus.
Use of different medicinal plants against nematode parasites Ascaridia galli and earthworm pheretima posthuma were
studied by Kosalge et al., (2009); Haque et al., (2011) and Subash et al.,(2012).
In vitro Nematicidal activity of Eucalyptus globulus leaf extract and piper nigrum seed extract:
Different concentrations (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/ml) of both plant extracts dissolved in DMSO, were used in
combination for observing nematicidal activity against nematode parasites of Gallus gallus domesticus. Mean values of
paralysis time and death time are given in the observation table 1. All values are expressed as Mean± SEM. Table 2.
Shows presence of phytoconstituents in methanolic extract of both plants.
RESULTS AND DISCUSSION
Methanolic extract of both plant extracts in combination shows significant dose dependent in vitro nematicidal activity
against Ascaridia galli. The results are shown in Table 1 , 2 and in Figure 2, 3).
Table1. Mean values (±SEM) of paralysis and death time of nematodes after Treatment of Eucalyptus globulus
leaf and Piper nigrum seed extract in combination
Concentrations of extracts in
combination (mg/ml)
Time of paralysis in
min(Mean± SEM)
Time of Death in min (Mean±
SEM)
0.05 186.5±0.43 212.0±0.98
0.1 139.4±0.62 189.6±1.23
0.2 140.8±0.58 150.6±0.87
0.3 127.6±0.88 143.0±1.21
0.4 97.1±1.02 140.4±1.33
0.5 85.6±1.15 123.0±1.58
Values represents Mean ± SEM values (for N=6).

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A B
Figure 2. Nematode parasites in Gallus gallus domesticus before testing (A) and after testing (B)
Table 2. Preliminary Phytochemical analysis of Eucalyptus globulus leaf and piper nigrum seed extract
Sr. No. Phytoconstituents in methanolic extract Eucalyptus globulus Piper nigrum
1 Alkaloids ++ +
2 Flavonoids + ++
3 Saponins ++ +
4 Phenols + ++
5 Cumarins + +
6 Tannins + +
7 Quinines + -
8 Glycosides - +
9 Terpenoids + +
10 Phlobatannins - -
11 Anthraquinones - -
12 Steroids + +
A B
Figure 3 Phytochemical Analysis
(Eucalyptus globulus Leaf Methanolic extract (A); Piper nigrum Seed Methanolic extract (B) ).

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Volume 4 Issue 3 (2015) ISSN: 2319 – 314X (Print); 2319 – 3158 (Online) © 2015 DAMA International. All rights reserved. 4
Methanolic extract at the concentration of (0.5mg/ml) shows the potent activity which gives minimum Paralysis and
Death time (85.6, 123.0 min respectively). Preliminary Phytochemical analysis shows presence of Alkaloids, Saponins,
Phenols, Cumarins, Tannins, Quinines, and Terpenoids in methanolic extract of Eucalyptus globulus leaf extract where
as Alkaloids, Flavonoids, Saponins, Phenols, Cumarins, Tannins, Terpenoids and Steroids Glycosides are present in
piper nigrum seed extract. Phytochemical investigations of Piper nigrum seed extract are in resemblance with the
report given by (Patil et al., 2014). While the Phytochemical investigations of eucalyptus globulus leaf extract shows
presence of phytoconstituents resembles as earlier reviewed by (Hardel et al., 2011).
CONCLUSION
From these results it can be concluded that medicinal plants used in combination shows significant Nematicidal activity
(Figure 2) . This may be useful for researchers to use combination of plant extracts against different parasites to reduce
adverse effect. This will provide basic platform for the researchers in formation of anthelmintic drugs by using
medicinal plants in combination for their synergetic activity. Further studies are recommended with use of medicinal
plants in combination for producing anthelmintic drugs for better results.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge University Grants Commission (UGC) New Delhi (India) for financial assistance
provided to carry out this work through major research project entitled “Screening of medicinal plants for in vitro
anthelminthic activity on parasites of Gallus gallus domesticus”, (F. No- 39-645/2010 (SR) Dated 10-01-2011). We
also thank to Dr. G. B. Shinde, Ex-Professor, and Late Shri. Dr. Baba V. Jadhav, Head, Department of Zoology, Dr.
Babasaheb Ambedkar Marathwada University, Aurangabad, for their suggestions and comments on various drafts of
this paper. We also thank to Principal, Dayanand College of Arts and Science, Solapur for providing laboratory
facilities
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