Abstract
Phytotherapy can be an alternative for the control of gastrointestinal parasites in human and animals. Coriander (Coriandrum sativum L.) is a medicinal plant which grown as a spice crop all over the world. The seeds of this plant have been used to treat parasitic disease, indigestion, diabetes, rheumatism and pain in the joints. This study was carried out to compare the efficacy of Niclosamid and alcoholic seed extract of C. sativum on Hymenolepis nana infection, in vivo and vitro. For in vivo study, Balb/c mice were used, to compare the efficacy of 50 mg/kg body weight (B.W) of Niclosamid with different doses of alcoholic extracts of C. sativum (250, 500, and 750 mg/kg B.W). It was found that the efficacy of Niclosamid had reached 100 % after 11 days post treatment, while the efficacy of 500 and 750 mg/kg B.W of C. sativum reached to 100 % after 15 days after treatment. For in vitro study, special nutrient broth media was used. It was found that the addition of 1000 mg/ml of Niclosamid had paralyzed and killed worms within 5 min, while C. sativum killed them within 30 min. Our results showed that extract of C. sativum has good effect against H. nana and could be use in traditional medicine for treatment of parasitic disease.
Keywords: Coriandrum sativum, Niclosamid, Anthelmintic activity, Hymenolepis nana
Introduction
Intestinal helminths are one of the most common causes of infections in humans, especially in tropical and subtropical countries. Current estimates suggest that over half’s of the world population is infected with intestinal helminths and that most of these infected people live in remote rural areas in the developing countries (Hotez and Ehrenberg 2010). These diseases which are currently referred to as Neglected Diseases of Neglected Populations, cause enormous hazards to the health of people, particularly of children, by contributing to malnutrition, anaemia and retarded growth (Hotez and Kamath 2009).
Hymenolepiasis is a disease caused by Hymenolepis diminuta (rat tapeworm) and Hymenolepis nana (dwarf tapeworm) (Leder et al. 2013). Hymenolepiasis has a high prevalence in populations in tropical and subtropical climates characterized by poor hygiene and poverty (Alvarez-Fernandez et al. 2012; Kline et al. 2013). H. nana, is a cosmopolitan intestinal cestode helminthes of the warmer climates, whose entire life-cycle is completed in the bowel, so infection can persist for years if left untreated (Parvathi and Karemungikar 2011). H. nana is the most common cause of all cestode infections and is found globally. In temperate zones, its incidence is high in children and institutionalized groups. H. nana infections are typically asymptomatic, but heavy infections can cause headaches, weakness, anorexia, abdominal pain, and diarrhea (Sadaf et al. 2013). This worm is the only cestode capable of completing its cycle without an intermediate host. Infection is most commonly acquired from eggs in the feces of another infected individual, which are transferred by contamination in food (Malheiros et al. 2014).
Since ancient times, plants and herbal preparations have been used as medicine. Traditional system of medicine and folklore claiming that medicinal plants as a whole or their parts are being used in all types of diseases successfully including antibacterial, anthelmintic, and anti-inflammatory (Patel et al. 2011).
Coriandrum sativum L. (Apiaceae) is an erect annual herb 20–70 cm tall with strong smell and it is widespread throughout the world as a result of cultivation for its aromatic seeds. It’s an annual herb in the family Apiaceae. Coriander is native to regions spanning from southern Europe and North Africa to southwestern Asia. The leaves are variable in shape, broadly lobed at the base of the plant, and slender and feathery higher on the flowering stems. The flowers are borne in small umbels, white or very pale pink, asymmetrical, with the petals pointing away from the center of the umbel longer (5–6 mm or 0.20–0.24 in long) than those pointing toward it (only 1–3 mm or 0.039–0.118 in long). The fruit is a globular, dry schizocarp 3–5 mm in diameter (Maroufi et al. 2010). Although sometimes eaten alone, the seeds are often used as a spice or an added ingredient in other foods. It is also used against stomachache. Extracts from seeds of C. sativum have several pharmacological effects such as anti-fertility, anti-diabetic, anti hyperlipidemic, antioxidant, and hypotensive activities (Asgarpanah and Kazemivash 2012; Sahib et al. 2013). In some countries, it is traditionally used for treatment of ascariasis and hepatitis in human (Eguale et al. 2007). Phytochemical screening indicated the presence of chemicals such as quercetin 3-glucoronide, linalool, camphor, geranyl acetate, geraniol and coumarins. The major fatty acid was petroselinic acid (65.7 % of the total fatty acid methyl esters) followed by linoleic acid (Matasyoh et al. 2009). Taniguchi et al. (1996) isolated three isocoumarins, coriandrones C–E, from whole plants of C. sativum. Two types of 2-C-methyl d-erythritol glycosides were also recently isolated from the seed of C. sativum (Sahib et al. 2013). The objective of the current study was therefore to assess the in vitro and in vivo anthelmintic potential of the seeds of C. sativum on H. nana.
Materials and methods
Mice, C. sativum, Niclosamid, and parasite collection
White Balb/c mice were obtained from Institute Pasteur, Tehran, Iran. They were offered diet obtained from general company for animal diet, after sterilization in autoclave at 120 °C at 15 lb/in2 pressure (Pal et al. 1984). Niclosamid was obtained from Parsdarou, Tehran, Iran. C. sativum was obtained from local market in Tehran.
The eggs from adult worms were obtained from naturally infected mice, after dissecting their intestine. Experimental mice were infected with 2000 eggs/mouse by stomach tube. Egg counts were performed daily after 10–14 days post administration to ensure the infection. The eggs count was done by modified McMaster method (Levecke et al. 2011).
In vivo study
Twelve hundred male mice was used; they were divided into eight groups (24 mice in each group). Group 1, was as untreated (control), the second group was treated with 50 mg/kg B.W Niclosamid. The third, fourth and fifth groups were treated with 250, 500 and 750 mg/kg of alcoholic extract of C. sativum, respectively. The efficacy of Niclosamid and C. sativum were estimated using the method described previously (Hrckova and Velebny 2013).
In vitro study
To study the effect of Niclosamid, and alcoholic seed extract of C. sativum, 36 adult worms were collected from small intestine of mice which experimentally infected with H. nana. The worms were washed in petridishes containing physiological normal saline solution and 500 IU crystalline penicillin for 30 min to prevent bacterial contamination. The adult worms were divided into 13 groups (four worms in each group). Each group of worm was kept in sterile test tube containing 3 ml of nutrient broth (10 g pepton, 2.5 g yeast extract, 5.0 g NaCl, and 1000 ml distilled water) and antibiotics were added to prevent bacterial growth. The first group was as control without treatment. In groups 2, 3, 4, and 5 added 0.2 ml of 10, 100, 500, and 1000 µg/ml of Niclosamid, respectively. In groups 6, 7, 8 and 9 the same concentration of alcoholic extracts of C. sativum were added, respectively. The viability of worms was followed for 24 h using microscopically technique.
Results
Tables 1 and 2 shows the effect of treatments on the number of eggs passed in the faeces. It is shown that Niclosamid leads to decline the mean number of eggs to 28 per gram faeces from 458 eggs after 8 days after treatment with efficacy 94 % then reached to 100 % after 11 days after treatment.
Table 1.
Average number (mean ± SD) of eggs passed by experimental mice treated with alcoholic seed extract of Coriandrum sativum and Niclosamid
| Groups | Days before treatment | Days after treatment | ||||
|---|---|---|---|---|---|---|
| 3 | 8 | 11 | 15 | 21 | ||
| Control (untreated group) | 413.3 ± 20.3 | 712.6 ± 154.2 | 689.1 ± 186.5 | 716.3 ± 201.4 | 718.6 ± 181.4 | 732.8 ± 195.7 |
| Niclosamid | 534.8 ± 201.4 | 421.1 ± 150.4 | 32.1 ± 28.5 | 0 | 0 | 0 |
| C. sativum (250 mg/kg B.W) | 541.8 ± 174.5 | 482.3 ± 179.4 | 401.5 ± 184.3 | 327.2 ± 202.9 | 208.2 ± 185.2 | 162.8 ± 99.8 |
| C. sativum (500 mg/kg B.W) | 572.4 ± 212.3 | 512.5 ± 191.4 | 231.3 ± 141.9 | 28.4 ± 30.5 | 0 | 0 |
| C. sativum (750 mg/kg B.W) | 594.7 ± 234.1 | 248.9 ± 174.5 | 39.2 ± 31.2 | 4.8 ± 20.1 | 0 | 0 |
Table 2.
Efficacy of alcoholic seed extract of Coriandrum sativum and Niclosamid on H. nana infection In vivo
| Groups | Efficacy percentage in different days | |||
|---|---|---|---|---|
| 8 | 11 | 15 | 21 | |
| Niclosamid | 94 | 100 | 100 | 100 |
| C. sativum (250 mg/kg B.W) | 11.5 | 27.9 | 34.9 | 60.9 |
| C. sativum (500 mg/kg B.W) | 65.8 | 95 | 100 | 100 |
| C. sativum (750 mg/kg B.W) | 92.5 | 98 | 100 | 100 |
The effect of 250 mg/kg B.W of C. sativum leads to decrease in the number of eggs present per gram of faeces to 181 after 21 days post-treatment with efficacy of 60.9 %. Dose of 500 and 750 mg/kg B.W of C. sativum leads to complete disappearance of eggs in faeces after 15 days post-treatment with efficacy of 100 %.
ED50 for eggs passed by experimental mice was estimated. ED50 for C. sativum when used for 8, 11, 15, 21 days were 520, 455, 350 and 175 µg/kg B.W. The effects of treatments on the number of adult worms are shown in Table 3. According to results Niclosamid leads to disappearance of worms from the gut on day 11 post-treatment. Treatment with 250 mg/kg B.W of C. sativum had no effect on the adult worms. On the other hand 500 mg/kg B.W of C. sativum lead to disappearance of worms after 15 days, while 750 mg/kg B.W of C. sativum lead to disappearance of parasite after 11 days of treatment. Addition of 10, 100, 500, and 1000 µg/ml of Niclosamid lead to death of adult worms within 21, 10, 6, and 4 min, respectively. While addition of the same concentration of C. sativum lead to death of worms after 240, 120, 65 and 25 min, respectively (Table 4).
Table 3.
Average number (mean ± SD) of worms in the intestine of experimental mice treated with alcoholic seed extract of Coriandrum sativum and Niclosamid
| Groups | Days before treatment | Days after treatment | ||||
|---|---|---|---|---|---|---|
| 3 | 8 | 11 | 15 | 21 | ||
| Control (untreated group) | 32.5 ± 1.8 | 24.1 ± 2.1 | 24.7 ± 2.3 | 29.5 ± 3.9 | 30.1 ± 2.8 | 32.5 ± 2.1 |
| Niclosamid | 19.4 ± 2.7 | 17.1 ± 4.1 | 2.1 ± 3.5 | 0 | 0 | 0 |
| C. sativum (250 mg/kg B.W) | 24.0 ± 1.2 | 22.1 ± 2.1 | 20.1 ± 5.5 | 20.0 ± 2.8 | 17.0 ± 5.8 | 15.0 ± 3.6 |
| C. sativum (500 mg/kg B.W) | 24.2 ± 2.5 | 24.1 ± 3.8 | 14.3 ± 2.5 | 2.0 ± 3.1 | 0 | 0 |
| C. sativum (750 mg/kg B.W) | 20.1 ± 4.2 | 9.0 ± 3.1 | 2.0 ± 2.8 | 0 | 0 | 0 |
Table 4.
Viability of H. nana adult worms in different concentrations of C. sativum and Niclosamid
| Concentration (mg/ml) | Type of treatment | ||
|---|---|---|---|
| Niclosamid | C. sativum | Control | |
| 10 | 21 min | 240 min | Over 24 h |
| 100 | 10 min | 120 min | – |
| 500 | 6 min | 65 min | – |
| 1000 | 4 min | 25 min | – |
Discussion
Using doses of 250, 500 and 750 mg/kg B.W of C. sativum in this study is due to lack of references, about using the proper doses of these plant extracts. This is the first report of effect of C. sativum extract on helminthes parasites in our country.
The doses of C. sativum were repeated for 7 days, to be sure about reaching the treatment on all stages of the parasite. The treatment started 14 days post infection, due to parasites become adults and start laying eggs after 12–14 days after infection (4). The efficacy of C. sativum with doses of 250, 500 and 750 mg/kg B.W reached 60.9, 100, and 100 % after 21 days, respectively. This indicates that doses of 500 and 750 mg/kg B.W of C. sativum had significant effect on the worm. The effect of C. sativum as anthelmintics might be due to its content of some medical component including volatile fats (5). The efficacy of Niclosamid in this study is in agreement with that reported by other workers (6, 7, 8 and 9), who showed the efficacy of Niclosamid were 100 % on immature and adult worms. According to our results, it is possible to say that plants will remain an important source to find new materials, which have great efficacy as chemically produced drugs (10). It is recommended to encourage people to use medical plants as substitute for chemical drugs because they have neither side effects nor toxicity.
In conclusion, the plant evaluated in the current study had already been reported as anthelmintic agents. Therefore, the current finding is the first step to justify their use in folk medicine. Further investigation of isolated fractions at different dose levels should be pursued. In addition, tests with animals can be performed with C. sativum essential oil or extract to evaluate the toxicity and to confirm their anthelmintic activity in target species.
Acknowledgments
We would like to thank all the people who cooperated in this study especial researcher and worker in Institute Pasteur, Tehran, Iran.
Conflict of interest
The authors have no financial or personal relationship with other people or organizations that could inappropriately influence or bias this paper.
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