Antiparasitic activity of the microalgae Cladophora crispata against the Protoscolices of hydatid cysts compared with albendazole drug

Antiparasitic activity of the microalgae Cladophora crispata isolated from Garmat-Ali River in southern Iraq was studied. Water samples were collected from river in the northern of Basrah, and cultured in chu-10 medium. Supernatants, alkaloidic and ethylacecate extracts from biomass were extracted and screened against the hydatidosis compared with albendazole drug. The present study reveal that 2(N, N-dimethyl hydrazine) cyclohexanecarbointrile and pyridine 2,3,4,5 tetrahyro compounds have activity against the protoscolices of hydatid cysts accordingly to the activity of albendazole based on the mean of weight of mice, mean of hydatid cysts number, mean of diameters and weights of hydatid cysts. The current study concludes that the alkaloidal and ethylacetate extracts of Cladophora crispata have a positive effect on the protoscolices of hydatid cyst in comparison with albendazole activity, related to reduction in the number and weight of hydatid cyst as well as the obstruction of germinated layer which is responsible for proliferation of protoscolices.


INTRODUCTION
Microalgae are a diverse group of photosynthetic microorganisms found in the soil, fresh water and marine environments (Metting and Pyne, 1986).They are able to produce a range of biochemical active compounds such as antibacterial, antifungal, antiviral, enzyme inhibitors, immunostimulants, cytotoxic, atiplasmodial activities (Ghasemi et al., 2004) and antitrypanosomal activities (Lorena et al., 2009).Most of the isolated chemical substances belong to groups of alkaloids, peptides, tannins, saponins, triterpenes and phenols (Molera and Semesi, 1996) as well as proteins (Athbi, 2011).
Diseases such as hydatid disease, hydatidosis, cystic echinococcosis, unilocularhydatid disease and echinococcosis, are referred to as the infections which are caused by cestodes of the genus Echinococcus and mainly by E. granulosus (Dar et al., 1977;Akhan et al., 2002;Georgopoulos et al., 2007).Hydatid cysts remains a significant public health problem in the endemic areas such as Turkey, the Middle East, South America, New Zealand, Mediterranean region, Africa, China, northern Kenya, Australia, and other sheep-raising areas (Morar and Feldman, 2005;McMnus et al., 2003).As an endemic disease, it causes social and economic losses for countries.WHO reports that approximately 100 000 people in the world are infected with these diseases every year which is more common in rural population of underdeveloped countries because of their close association with domestic and wild animals (Parija and Sheeladevi, 1999).Until recently, surgery was the only option for treatment of echinococcal cysts, however, chemotherapy with benzimidazole compounds and, cyst puncture, percutaneous aspiration, injection of chemicals, and reaspiration (PAIR) are increasingly used to as an alternative to replace a surgical method (Morar and Feldman, 2005).
The undesirable side effects associated with this classical drug, as well as the development of resistance, has encouraged researchers to use alternative synthetic or natural compounds for treatment of hydatid disease.In this regard, studies have focused on the bioactivity of natural substances derived from algae, mainly due to their accessibility and use in traditional medicine (Smit, 2004;Kladietal, 2008;Lorena et al., 2009); although many researches have been focused on the extracts from algae as a source of antiparasitic compounds.Early studies on bioactive chemical compounds isolated from chlorophyta have led to the discovery of several compounds, including the diterpenoids, udoteal and halimedatrial isolated from U. flabellum and Halimeda sp.respectively, and the sesquiterpenoids hipocephanal isolated from Rhipocephalus phoenix that inhibited cell division in sea urchin eggs (Fenical and Paul, 1984).Anuomrthine, Pronueiferine, Glaucine, Nuciferine, Yeserpin, Evodianin, Caulerpine, Leptoclinidamin-A, and Halimedin are alkaloidal chemical compounds isolated from the species of chlorophyta and act as antioxidant, antiviral, antibacterial, antifungal, and anticancer (Calixto et al., 2000;Radwan et al., 2007;Carrol et al., 2007;Everton et al., 2009).Al-Nasiri (2010) isolated an alkaloid chemical compound from C. crispata which was similar in structure to calothrixin and had in vitro antibacterial activity.
Recent studies have shown promising antimalarial activity by the alga Laurencia sp.(Topcu et al., 2003) and trypanocidal and leishmanicidal activity by Fucuse vanescens, pelvetiaba bingtonil, Ulva lactuca and Sargassum natans ( Naraetal, 2000;Orhan et al., 2006 ).A study of Leon -Deniz et al. (2009) revealed that the activity of the organic extracts of green algae U. conglutinata and U. flabellum cause a total inhibition of the trypanosomes parasite at 24 h; 48 h and even seven days after growth.The present study was designed to examine the in vivo activity of bioactive chemical compounds (alkaloids and ethylaacetale) extracted from Cladophora crispata (Chlorophyta) against the protoscolices of hydatid cyst of E. granulosus compared with a commercial drug albendazole.

Isolation of microalga
C. crispata was isolated from Garmat -Ali River in Basra city, southern Iraq, from January to April 2012.Primary culturing was done in Chu -10 medium.After incubation, pure cultures of the living specimens were prepared by sub culturing with agar plate method in Chu -10 medium (Stein,1975).Preserved specimens were prepared and the living specimens were inoculated in 100 ml conical flasks.Constant illumination was used at 60 µE m -2 Sec -1 intensity using white fluorescent lamps and incubated at 25 ± 2°C.Algal cultures were identified based on thier morphological characteristic following the taxonomy schemes of Prescott (1975) and Sant 'Anna (2004).

Prepration of algal extracts
Preparation of extracts was made according to Reichelt and Borowitazka (1984) using ethylacetate extract. 1 g of C. crispata biomass were extracted by soxhlet continuously with 250 ml of ethylacetate as solvent for 24 h.The alkaloidal extract was prepared using 0.5 g of dried culture extracted with acidic ethanol (ethanol absolute with 2% acetic acid) for 24 h in a continuous extraction by soxhlet apparatus.The extracts were filtered, and ethanol was evaporated on a rotary evaporator under vacuum at a temperature of 45°C to a small volume (about a quarter), then a small amount of NH 3 (25%) was added to make pH of 9. Subsequently, 100 ml of chloroform was added and slowly shacked for 10 min.until the alkaloidal compounds were separated from water and enter to the chloroform phase.This process was repeated three times, then total chloroform phase was evaporated, yielding a total alkaloid extract and dried under a reduced pressure and stored in -20°C for further studies.

Identification of biochemical active compounds
Ultra-violet (UV) spectrum (LKB-Sweden UV), Infra-red spectrum (IR) (Pye-Unicam Sp3-3005 UK), and gas Chromatography Mass (GC-mass) (Agilent Technologies GC-mass 7890 AGC System) methods were applied for the identification and determination of the molecular weights and chemical structure of the isolated biochemical active compounds.

Parasite materials
E. granulosus hydatid cysts containing protoscolices were removed under aseptic conditions from livers and lungs of naturally infected sheep (The outer surfaces of the cysts were sterilized with70% ethanol before being dissected).Protoscolices were extracted according to Smyth (1980) (Figure 1).

Estimation of viable protoscolics
200 μL of hydatid fluid and 200 μL of 0.1% eosin staining solution  were combined in a microtube.After 20 min of incubation, the viability of protoscolices was assessed by microscopic observation.Stained protoscolices were considered as nonviable (Figure 3) and the protoscolices which have been stained with eosin were considered as viable (Figure 2) according to conventional (Taran et al., 2009).

The counting of viable protoscolices
Protoscolices were counted according to the method of Rounno et al. (1974) cited by Al-Humairy (2010).After estimating the viability of protoscolices, 10 µL of the hydatid fluid was taken by a micropipette, the count was done under dissecting microscope, and  Viability in 1 ml = number of protoscolices in 10 µL × 100

Injection of mice with protoscolices
Male Mus musculus mice Balb/C strain was injected with 0.2 ml 480/ L (2400/5 ml rate of viability) of protoscolices intraperitoneally (I.P) using 1 ml volume syringes.The region of injection was sterilized with 70% of ethyl acohol.

Determination of lethal dose (LD50)
Males of the M. musculus Balb/C strain were dosed orally to determine the LD 50 using a stomach tube with bioactive chemical compounds extracted from C. crispata (ethylacetate and alkaloid extracts).The animals were monitored for 72 h and weakness, unstable walking, loss of balance and death was checked during this period.Injection started with low dose then continued to high dosages based on Litchfield and Wilcoxon (1949) equation: Where, LD 50 is the lethal dose 50, highest dosage is the dose with 100% mortality of mice, a is the value of difference between the previous and next dose, b is the summation of dead animal for each dose (previous dose + next dose / 2) and n is the number of animals used for each dose .

Experimental design
Male mice of M. musculus Balb/c albino strain aged 6 to 8 weeks were used in this study.They were injected interperitoneal (IP) with viable protoscolices of the E. granulosis and left for six months before treatment.
A positive control group was infected without treatment and the negative control group was left without infection and treatment.The in vivo study included two parts (Table 1).

Treatment
The treatment included two groups of 24 infected male albino mice which were treated with bioactive chemical compounds.Eight infected mice were treated with albendazole and 8 more left as a positive control.Eight intact male mices were used as a negative control.More details as follows:

Treated 1 (T1) group
In this group, 24 infected male mice were dosed orally daily for one month with concentrations (280, 300, 330 µg/ml) of the alkaloidal bioactive compound extracted from C. crispata with 8 mices for each concentration.

Treated 2 (T2) group
This group consists of 24 infected male mice which were treated orally daily for one month with (100, 110, 120 µg/ml) concentration of ethyl acetate extracted from C. crispata with four pairs for each concentration.

Albendazole group
In this group, 8 of infected male mice were daily treated orally with 500 µg/ml of albendazole for 30 days.
The weight of mices and their organs were checked before and after treatment.Diameters of cysts were measured by the ruler and the number of hydatid cysts was counted based on the following formula to calculate the effective dose:

Statistical analysis
The statistical analysis was conducted in SPSS 9.0; T-test at 0.05 level was used to analyze variation of the mean of viable protoscolices treated with bioactive chemical compounds and albendazole in vitro and in vivo experiments.

Alkaloid extract
In the present study, two peaks were detected by the GC-Mass analysis as a results of alkaloidal component analysis (Figure 4): Pyridine, 2, 3, 4, 5 -tetrahydro with R.T. 26.187 min and molecular weight 83 killo Dalton (kd), and the other compound was N-methyl-propylamine with R.T 26.289 min and amolecular weight of 73 kd (Figure 5).

Ethylacetate extract of C. crispata
The GC-mass spectrum of ethylacetate extract revealed the presence of 17 peaks as shown in Figure 6.The results indicate that the compounds were arranged as 2-(N, N-dimethylhydrazino) cyclohexanecarbonitrile which consist of 8.77% (R.T. 20.59 min) of the total extract followed by Triazolo [1, 5-a] pyrimidine carboxylic acid (7.25%) (Figure 7).

Experimental infection with hydatid cysts
The examination of experimentally infected males Balb/C mice with protoscolices, 1,2,3,4, and 6 months-post infection revealed the presence of hydatid cysts in liver, spleen, mesenteries, kidneys and lungs (Plates 1 and 2).

In vivo effects of the extracts on the weight of infected and treated mice:
Weight of the negative control group, positive group, and the treated groups of mice were checked.Results show that the weight of the positive control group weighted about 40.2 g, while the weight of the negative control group was 32.64 g and the weight of treated group with albendazole was 31.4 g.The T1 group showed 34 g of weight compared with T2 (33.8g) groups.Table 2 shows the weight of organs for each group.

In vivo activity of extracts on the number of hydatid cysts compared with albendazole
The number of hydatid cysts was decreased after the treatment with extracts of C. crispata.The number of hydatid cysts in spleen, lung and kidney was decreased to zero; this result is similar to those of other groups of mice treated with albendazole.Mesenteries and liver had a higher number of hydatid cysts than other organs for the positive control group (6 and 7.1 hydatid cysts) and they decreased after treatment with bioactive chemical compounds from C. crispata and albendazole (Table 3 and Plate 3A, B and C).
The results of the in vivo activity of the extract on the number of hydatid cysts of infected mice showed significant differences between groups treated with extracts and the positive control group compared with the group treated with albendazole.In T2 group, the percent number of hydatid cysts recorded 59.86% while the T1 group it was 48.6%, since the number of hydatid cysts reduced from 15.2 in the positive control group to 6.1 and 7.8 respectively in T2 and T1 groups.These results almost corresponded with the effective dose of albendazole (61.18%) compared with the T2 group and are slightly different from T1 group (Table 3).

In vivo activity of extracts on dimiter and weight of hydatid cysts compared with albendazole
The mean of hydatid cysts diameters was 8.2 mm for the positive control group and was reduced to 4.2 mm in the group treated with albendazole, 4.6 mm in the T2 group, and 6.1 mm for the T1 group (Table 4, Plates 4A, B, C and 5).The weight of hydatid cysts was also studied for each group treated with the bioactive chemical compounds extracted from C. crispata as well as the group treated with albendazole.These results show a decrease in weight of hydatid cysts of the T2 group (0.55 g) and (0.63 g ) in the T1 group compared with the group treated with albendazole recording 0.53 g where positive control group was 1.22 g in mean.

DISCUSSION
The current study demonstrated that the weights of infected experimental mice examined at 6 th months -post infection, were increased compared with the negative control group.The weights of liver and spleen were also increased.On the other hand, there were slight increases in the weight of kidneys and lungs (Table 2).These results agree with those of other studies (Al-Nasiri, 2006;Al-Mobarek, 2006;Barzanjietal, 2009 andAl-Humairy, 2010).There was a significant difference of weights and diameters among the hydatid cysts of organs (livers, spleens, kidneys, lungs and mesenteries).Hepatospleenomegaly caused by the parasite lead to an increase in weights of the experimental animals and their organs.However, many studies have explained the reasons of the increase in the weight of the organs infected with the hydatid cyst.concluded that the increase in liver and lungs weight was due to the formation of granuloma the increase in the immune cell and its migration to the target organ, so the ability of spleen to produce lymphocyte to secrete specific antibody had led to the increase of the spleen weights.
The weights and diameters of hydatid cysts reported in the present study were affected by parasite infection because the weight decreased and approached the values of the negative control group especially in T2 group.The decrease in weight can be explained in relation to the decrease of number of hydatid cysts and calcification with the sloughing of the germinal layer and the disintegration of laminated layer (Maizeles and Yazdanbakhsh, 2003).However the complex layer of cyst has an important role in the transformation of nutritional material from serum to cyst.The knowledge of the parasite nutrition behavior can help for a drug treatment of the inoperative cyst via the selection of the effective drug and adhere them to biological material that promote distribution of drug to the cyst (Rahda et al., 2008).
Compared to the bioactive chemical compounds, albendazole decreases the weights of infected testes animals more than those of the negative control group.
It is difficult to speculate the mechanism by which these bioactive compounds act as parasitic agents.In this regard, Sepulveda-Boza and Cassels (1996) suggested that many bioactive chemical compounds exhibited their parasitecidal activity by virtue of their interference with the redox balance of the parasites, acting either on the respiratory chain or the cellular defenses against oxidative stress.

Conclusions
Overall results of the present study concluded that ethyl acetate extract is more active than alkaloid extract.The bioactive chemical compounds and albendazol decreased the weight of infected animals, and further the weight of organs (liver, spleen, kidney, lung, mesentery), and weight, number and diameter of hydatid cysts more than that of the negative control groups.

Figure 1 .
Figure 1.Hydatid sand containing the daughter cyst, brood capsule and protoscolex aspired from the hydatid cyst.
Number of cysts in positive control group -Number of cysts in treated Effective dose group= Number of cysts in positive control group

Plate 4 .
Pictures of treated organs of infected mice with hydatid cysts.(A) Liver from mice treated with 500 µg/ml of albendazole.(B) Liver from mice treated with 120 µg/ml of T4 group.Pictures of hydatid cysts.(A) Positive control.(B) Hydatid cyst treated with albendazole.(C) Hydatid cyst of T2 group.
repeated three times.The viable protoscolices were counted in 1 ml based on the formula:

Table 2 .
Mean of weight of mice and organs treated with extracts compared with albendazole.

Table 3 .
Mean of hydatid cysts number in treated mice and the effective dose.

Table 4 .
Mean of diameter and weight of hydatid cysts of treated mice with bioactive chemical compound.