CC BY
0WVJ
World's
Veterinary Journal
World's Vet. J. 3(2): 33-37, 2013
© 2011, Scienceline Publication
Evaluating the Anti-Leech Effects of Methanolic Extracts of Peganum harmala L. and Olea europaea L. on Limnatis nilotica
Mahmoud Bahmani 1*, Mahmoud Rafieian-Kopaei2, Zohre Eftekhari3, Ebrahimkhalil Banihabib1,4, Ghader Hajigholizadeh 1, Farzaneh Bahmani5, Jafar Azadzadeh1, Reza Abdollahi1, Aghakhan Kheyri1 , Abdolsalam
Sotoudeh1, Seyed Ahmad Karamati1 , Mahyar Jelodari6
'Urmia University of Medical Sciences, Urmia, Iran 2Medicinal Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran 3Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 4Tabriz University of Medical Sciences, Tabriz, Iran 5Department of Entomology, Ahvaz Jondishapour University of Medical Sciences, Ahvaz, Iran
6Urmia University, Urmia, Iran
Corresponding author's email: mahmood.bahmani@gmail.com
ABSTRACT
Leeches had several complications such as pain, itching, inflammation, severe anemia, short-term bleeding, hypersensitivity, and anaphylactic reactions in their hosts. Harmal Peganum harmala L. is used as an analgesic and anti-inflammatory agent and it has antibacterial activity. Olive Olea europaea L. has antibacterial, anti-viral, hypoglycemic and the relaxation of blood vessels properties. Antioxidant properties of olive also had been reported. This study was carried out to detect the effects of methanolic extracts of P. harmala L. and O. europaea L. on L. nilotica immature form. In 2011, 55 immature leeches collected from the southern area of Ilam province were prepared. The methanolic extract of O. europaea L and P. harmala L. were compared with levamisole as the control drug. Distilled water was evaluated as the placebo group which investigated L. nilotica using anti-leech assay. Then extract and drugs were added and their effects were screened for 720 min and time to paralyze, kill and death of each leech was recorded. The results showed that olive methanolic extractions (600 and 300mg) could kill the leeches in an average time of 145±77.57 and171±33.28 min, respectively. An average death time for levamisole was found to be 15±7.49 min. The highest effectiveness was found for levamisole at dose 300 mg. Methanol extracts of the Harmal (300 and 600 ^g/m) and springs water showed no anti-leech. In sum, olive plant could use for anti Limnatis nilotica expenditure.
KEY WORDS: Peganum harmala L., Olea europaea L., Levamisole, Limnatis nilotica, Anti-leech Assay
INTRODUCTION
Leeches that cause parasitic pollution in humans are terricolous or aquatic. Terricolous or land leeches include
Haemadipsa zylanica, Haemadipsa sylvestris, and Haemadipsa picta, while aquatic leeches include Limnatis nilotica, Myxobdella africana, Dinobdella ferox, Phytobdella catenifera, and Teromyzon tessulatom ( Haycox et al., 1995; Vera et al., 2005; Wallis, 1988).
Leeches are not true worms, but they are described as parasitic worms. Leeches are segmented worms that belong to the phylum Annelida and comprise the subclass Hirudinea which are external parasites of humans, domestic and wild animals (Chares AND Hendrix, 1981).
They also have two suckers, with each sucker located at the end of each animal, which can suck blood of her host as many as nineteen times of body weight. Leeches as pathogenic parasites cause complications such as pain, itching, inflammation, severe anemia, short-term bleeding, hypersensitivity, and anaphylactic reactions on their host (Ahmadizadeh, 2001; El-Awad and K Patil, 1990; Hadrani, 2000). In some reports in humans, leeches as a foreign body caused acute bleeding of the alimentary system in two children, 2.5 to 3 years old (Hemmati et al., 2002), hematemesis in a 3.5-year-old child (Kazemi and Bajoghli, 2002) or bleeding of the vagina (Yaghmaee, 2002), and one case of
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To cite this papery Bahmani M., Rafieian-Kopaei M., Eftekhari Z., Banihabib E., Hajigholizadeh G., Bahmani F., Azadzadeh J., Abdollahi R., Kheyri A., Sotoudeh A. Karamati S.A., Jelodari M. 2013. Evaluating the Anti-Leech Effects of Methanolic Extracts of Peganum harmala L. and Olea europaea L. on Limnatis nilotica. World's Vet. J. 3(2): 33-37.
Journal homepage: http ://wvj.science-line. com/
ophthalmorrhagia that was caused by leech attachment has been reported (Davari, 2008). The parasite in the respiratory tract has been reported in animals. Some reports have mentioned that hirudiniasis may cause severe anemia with hemoglobin lesser than 5g/dl (Hemmati et al, 2002). Nowadays, hirudiniasis categorized as emerging and reemerging diseases (Bahmani et al., 2010).
According to World Health Organization (WHO), plants are supplier of medicines for human resources. Factors such as lack of access to the majority chemical drugs, cost of drugs, reduce the therapeutic effects of chemical drugs because of its resistance to pathogens and cause side effects, leading up to the day on traditional medicine and treatment plant societies to the positive more (Ghasemi pirbalouti, 2009).
Types of medicinal plants used in the treatment of various diseases are the olive and P. harmala L. Harmal is a plant of the family Nitrariaceae, native from the eastern Mediterranean region east to India. It is a perennial plant, which can grow to about 35-100 cm tall. The roots of the plant can reach a depth of up to 6.1 m, if the soil it is growing in is very dry. The flowers are white and are about 2.5-3.8 cm in diameter .The round seed capsules measure about 11.5 cm in diameter, have three chambers and carry more than 50 seeds. In Turkey Peganum harmala is called yuzerlik or uzerlik and ispand in Iran, Afghanistan, Azerbaijan, Uzbekistan and Tajikistan, harmal in Iraq and alharma in Spain is called (Zarzuelo et al., 1991). P. harmala is used as an analgesic and anti-inflammatory agent and it was used to treat depression. It has antibacterial activity against drug-resistant bacteria and used treatment of syphilis in India, fever in North Africa, hysteria, neuralgia, Parkinson's disease, uterine prolapsed, rheumatism, asthma and eye irritation. P. harmala is an abortifacient and effective against protozoa including malaria (Markin et al., 2003).
The active alkaloids of Harmal seeds are the MAOI-A (monoamine oxidase inhibitor A) compounds. Studies in Pakistan and Egypt shows that the P. harmala extract revealed antifungal and antimicrobial effects and its effects are related to harmine alkaloids (El-Awad and Patil, 1990; Ahmad et al., 1992; Ross, 1980).
Olive tree or shrub of the family Oleaceae and evergreen stands, native to the coastal areas of the eastern Mediterranean Basin as well as northern Iran at the south end of the Caspian Sea, up to 20-10 cm high, often branching ramose and, with bayonets rectangular with oval leaves, white flowers, immature fruit is green and the shaft it is purple when ripe ( Mozaffarian, 2008). The relaxation of blood vessels [14] and antioxidant properties of olive also had been recorded (Briante, 2002; Visioli and Galli, 1997).
Levamisole was originally used as an anthelminthic to treat worm infestations in both humans and animals. Most current commercial preparations are intended for veterinary use as a de wormer in cattle, pigs, and sheep. Levamisole stimulates the parasympathetic and sympathetic Ganglia in susceptible worms and interferes with nematode carbohydrate metabolism by blocking fumarate reduction and succinate oxidation. The net effect is a paralyzing effect on the worm which is then expelled alive. Levamisole's effects are considered to be nicotine-like in action.
Because the usefulness of medicinal plants must be proven through scientific assessments and research, in present study tried to evaluating the anti-leech effects of methanolic extracts of Peganum harmala L. and Olea europaea L. on Limnatis nilotica immature form.
MATERIAL AND METHODS
In this study, a number of immature form L. nilotica leeches (55 in total) were selected from the springs of the southern region of Ilam province (West of Iran). The dark green color surface with rows of green spots on the dorsal surface, and yellowish-orange and dark green bands on either side were the main signs for detection of L. nilotica species. The methanolic extractions of Peganum harmala L. and Olea europaea L. were prepared by adding a 1:3 ratio of olive and methanol, respectively, and this was subjected to Soxhlet extraction for 72 h according to the prescribed method of Eidi et al. (2006). After extraction, the solvent was filtered. The information studied for the plants has been determined in Table 1. Levamisole (Sinadaru, Iran) tablets (an anthelmintic and immunomodulator) were investigated as a control and compared with distilled water. These tablets were powdered and diluted in 10 ml distilled water. For the anti-leech assay, the leeches were located individually in a glass container with 600 ml spring water. The extract and drugs were then added, their effects were screened for 720 min, and time to paralyze, kill, and death of each leech was recorded.
The evaluation of death of a leech was based on immobility after stimulation with a needle. The low average paralyzing and killing time of these compounds reflects anti-leech properties (Bahmani et al., 2012). The severity of effect of these compounds/drugs based on time was categorized into five groups (Bahmani et al., 2012).
Classification of the intensity effect treatment
4+ is paralysis and death of each leech within 1-60 min after addition of the drug
3+ is paralysis and death of each leech within 61-120 min after addition of drug
2+ is paralysis and death of each leech within 121-180 min after addition of drug
1+ is paralysis and death of each leech within 181-240 min after addition of drug
Is negative paralysis and death of each leech within 241-720 min after addition of drug
The efficacy of the drugs which were able to kill leeches within 1 -60 min after addition reflects the anti-leech properties of these compounds, and therefore, they may be used in the treatment of infestation with L. nilotica in the future (Bahmani et al., 2012). The differences between the control and treated groups were analyzed using one-way ANOVA using Sigma State 2 program.
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To cite this papery Bahmani M., Rafieian-Kopaei M., Eftekhari Z., Banihabib E., Hajigholizadeh G., Bahmani F., Azadzadeh J., Abdollahi R., Kheyri A., Sotoudeh A.
Karamati S.A., Jelodari M. 2013. Evaluating the Anti-Leech Effects of Methanolic Extracts of Peganum harmala L. and Olea europaea L. on Limnatis nilotica. World's Vet
J. 3(2): 33-37.
Journal homepage: http ://wvj.science-line. com/
RESULTS
The anti-leech activities for treatments on L. nilotica are shown in Table 1. Paralysis and death properties of olive extraction according to criteria revealed that the methanol extract at different doses have different potential which increasing with dose. Methanol extracts of this plant had no effect L. nilotica. The highest affectivity was found for levamisole. The exact timing of drug treatments for paralysis and death are shown in Table 1. Statistical analysis shows the different drug treatments for paralysis and death factors, there is significant difference between the leeches.
Table 1. The anti-leech activity of used compounds compared to levamisole as positive control.
Extract/Drug Mean ± SD Dose Severity
Levamisole 15±7.49 300mg/ml 4+
Spring water 720±0.00 100 ml -
Methanol extract of the Harmal 720±0.00 300 mg /ml -
Methanol extract of the Harmal 720±0.00 600 mg /ml -
Methanol extract of the Olive 171±33.28 300 mg /ml 2+
Methanol extract of the Olive 145±77.57 600 mg /ml 2+
DISCUSSION
The results of this study showed that olive methanolic extractions (600 and 300mg / ml) could kill the leaches in an average time of 145±77.57 and171±33.28 min while average death time for levamisole was found to be 15±7.49 min. The highest effect was found for levamisole (300 mg/ml). Methanol extracts of the Harmal (300 and 600 mg) and spring water showed no anti-leech effect. Therefore, we recommend that the other plant extracts (ethanol, hexane, hydroalcoholic) be examined on leech species.
Olive leaf contains choline compounds, unsaturated lactones, elenolide, oleuropeic acid , sugar, tannin, wax, manit and gallic acid have been identified (Sanei, 1970). The oleuropine components contain biological properties, barrier properties and the ability to activate free radical oxidation (Visoli et al., 1997 and 1998). Oleuropein, have pharmacological properties, which have an antibiotic and antiviral effects. Other, compounds of olive contains oleoropein, caffeic acid, elenolic acid glucoside and luteolin 7-glucoside (Amiot, 1998).
In present study, increasing doses of methanol extracts of the olive from 300 mg to 600 mg caused reduction in the average death time by 26 minutes, which revealed that ability of anti-leech effects enhance with increasing doses or concentrations of metabolic substances. This result- suggested that another components of olive be identified and examine on mature form of L.nilotica.
Bahmani et al. (2011) reported that methanolic extract of Allium sativum L. exhibited anti leech activities on l. nilotica immature form. They added that the average time of leech death (Immature form) for Allium sativum L was 68.44±28.39 min and for niclosomide was 6.22±2.94 min (Bahmani et al, 2011). Gholami-Ahangaral et al. (2012) studied effects of the methanolic extract of Vitis vinifera L., niclosamide and ivermectin on l. nilotica and found that mean death time of leeches treated with niclosamide and ivermectin for mature and immature forms were 15.4 and 11.2 and 10.1 and 11.2 minutes, respectively.The doses of 300 and 600 mg of methanol extract of V. vinifera L. against L. nilotica mature worm were ineffective but they exhibited death time with 260±63 and 200±50 minutes, respectively against the immature form of L. nilotica.
In a study by Bahmani et al. (2012) It was found that J. regida and N. oleander have no anti leech effect while Piperazine, Praziquantel, Chlorquine, Levamisole, Niclosamid, Copper sulfate and Chlor killed leeches at 720±0, 40.22±19.05, 720±0, 19.22=8.82, 17.11-4.13, 16.25±6.49 and 6.75±2.98 minutes after exposure, repectively. Bahmani et al. (2012) in another study evaluated effects of the methanolic extract of Peganum harmala L. and some of the anti parasite drugs on L. nilotica (Mature forme).The mean death time of leeches in groups treated with niclosamide, sulfadimidine, furazolidone and pyrvinium were 14.77±3.66, 58.33±22.17, and 137.11±37.84 and 320.44±300 min, respectively. Different doses of P. harmala L. methanolic extract (300, 600, 900, 1200, 1500 and 1800 mg) were ineffective on mature leeches in 720 min of experiment (Bahmani et al., 2012). In this study, Harmal also not any effect in immature forme of leeches. In another study by Gholami-Ahangaran et al., 2012 the results that study was indicated which Olea europaea L. plant and levamisole have more effect on the mature worm of L. nilotica. The mean death time of leech for levamisole and Olive determined 10±0.98 and 210±24.1 minutes, respectively. In our study, Olea europaea L. methanol extract had also good effect of anti leech of immature worm of L. nilotica.
It seems that Iranian Medicinal Plants, Sometimes may be having anti leech effects which due to the active ingredients in these herbs.
CONCLUSION
The survey results showed that methanol extracts of the harmal had no anti leech effects, and Olive has a weak effect on L.nilotica and using of it as a powerful and effective medicinal plant natural product is not recommended but can use as an effective supplement for medicinal products.
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To cite this papery Bahmani M., Rafieian-Kopaei M., Eftekhari Z., Banihabib E., Hajigholizadeh G., Bahmani F., Azadzadeh J., Abdollahi R., Kheyri A., Sotoudeh A. Karamati S.A., Jelodari M. 2013. Evaluating the Anti-Leech Effects of Methanolic Extracts of Peganum harmala L. and Olea europaea L. on Limnatis nilotica. World's Vet. J. 3(2): 33-37.
Journal homepage: http ://wvj.science-line. com/
ACNOWLEDGEMENT
The authors wish to express their gratitude to the research council of Food and Beverages Safety Research Center,
Urmia University of Medical Sciences, Urmia, Iran for their financial supports. This work was used of facilities and
equipment from Islamic Azad University of Dehloran Branch, Ilam Province.
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To cite this paper: Bahmani M., Rafieian-Kopaei M., Eftekhari Z., Banihabib E., Hajigholizadeh G., Bahmani F., Azadzadeh J., Abdollahi R., Kheyri A., Sotoudeh A. Karamati S.A., Jelodari M. 2013. Evaluating the Anti-Leech Effects of Methanolic Extracts of Peganum harmala L. and Olea europaea L. on Limnatis nilotica. World's Vet. J. 3(2): 33-37.
Journal homepage: http ://wvj.science-line. com/
Gholami-Ahangaran, M, Bahmani, M and Zia-Jahrom, N, 2012. In vitro Anti-Leech Effects of Vitis vinifera L.,Niclosamide and Ivermectin on Mature and Immature Forms of Leech Limnatis nilotica. Global Veterinaria.; 8: 229-232.
Bahmani, M, Avijgan, M, Gholami-Ahangaran, M and Rafieian, M, 2012. The comparison of anti- Limnatis nilotica
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To cite this paper: Bahmani M., Rafieian-Kopaei M., Eftekhari Z., Banihabib E., Hajigholizadeh G., Bahmani F., Azadzadeh J., Abdollahi R., Kheyri A., Sotoudeh A. Karamati S.A., Jelodari M. 2013. Evaluating the Anti-Leech Effects of Methanolic Extracts of Peganum harmala L. and Olea europaea L. on Limnatis nilotica. World's Vet. J. 3(2): 33-37.
Journal homepage: http ://wvj.science-line. com/
