Schistosomicidal and molluscicidal activities of two Junipers species cultivated in Egypt and the chemical composition of their essential oils

In the present study, in vitro bioassay screening of total methanolic extracts of two Juniperus species (Juniperus horizontalis Moench. and Juniperus communis L.) cultivated in Egypt for schistosomicidal and molluscicidal activities was carried out. Schistosoma mansoni Sambon worms and Biomphalaria alexandrina (Ehrenberg) snails were used. The screening results showed that both tested extracts had almost similar schistosomicidal activity (LC50 ≈ 91 μg/ml, in 3 days) while J. communis extract possess more potent molluscicidal activity than J. horizontalis (LC50 = 22.9 and 38.9 ppm, after one day, respectively). Analysis of the chemical composition of the essential oils obtained by hydro-distillation of the aerial parts of the two Junipers species was done by GC/MS. Sixty seven (94.32%) components were identified in J. communis oil with homogeraniol (36.95%) being the major constituent, while sixty (95.39%) components were identified in J. horizontalis oil with the main component, bronyl acetate representing 41.17%.


INTRODUCTION
The Cupressaceae or Cypress family is a conifer family of cosmopolitan distribution.The family includes about 70 genera (17 monotypic) with about 130-142 species.One of the important genus is Juniperus which has been well known as a source of cedarwood oil and widely used in folk medicine (Seca et al., 2007;Gumral et al., 2015).In the present study, the chemical composition of the essential oils of two Juniperus species was investigated: Juniperus horizontalis Moench known as creeping juniper and Juniperus communis L. known as juniper berry cultivated in Egypt and the antiparasitic effect of the methanolic extract of these two species were investigated.Several biological activities has been tested for both species under investigation such as hepatoprotective (Manvi et al., 2010), anti-inflammatory (Tunon et al., 1995), analgesic (Banerjee et al., 2012), antibacterial (Sati et al., 2010;Eryiğit et al., 2014), antihypercholesterolemic (Akdogan et al., 2012), antioxidant Stoilova et al., 2014) and antimalarial (Milhau et al., 1997).These biological activities may be attributed to the *Corresponding author.E-mail: suzanmina@yahoo.com.Tel: 01147311226.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License diversity of chemical constituents found in this two species e.g.volatile oils (Hoferl et al., 2014;Chandra et al., 2007), flavonoids (Ilyas et al., 1990;Lamer 1975) glycosides, sterols and di and triterpenes (Souravh et al., 2014).One of the biggest challenges in Egypt is the control of bilharzias or Schistosomiasis.Schistosomiasis is a parasitic disease caused by the digenetic trematodes of the genus Schistosoma which are commonly known as blood flukes.It is well documented that Schistosoma haematobium was endemic in Ancient Egypt.It was first diagnosed in mummies by Ruffer in 1910 (Rashida, 2013).Schistosomiasis comes after malaria among parasitic diseases with regard to the number of people infected and those at risk of infection (Chitsulo et al., 2000).In the continuous search for a control of this parasitic infection, the total methanolic extract of the aerial parts of both J. horizontalis Moench and J. communis L were screened for their schistosomicidal and molluscicidal activities.

Plant
Non-flowering aerial parts of J. communis L. and J. horizontalis Moench were collected on April 2013 from the International Garden at Cairo, Egypt.Identification of the plants was confirmed by Dr. Therese Labib, specialist of plant identification in El Orman Garden, Cairo, Egypt.Two voucher specimens (No.JH-36 J. horizontalis and JC-37 J. communis) were deposited in the herbarium of Pharmacognosy Department, Faculty of Pharmacy, Helwan University.

Preparation of plant extracts and oil extraction
Fresh aerial parts of both J. horizontalis and J. communis, 200 g each, were suspended in twice their volumes of distilled water and subjected to steam distillation for 6-8 h using volatile oil distillation apparatus.Prior to distillation, the samples were chopped into about 2 cm long pieces.The distillate was allowed to cool at room temperature, volatile oil was allowed to separate from water and each essential oil sample was weighed on analytical scale (1.5 ml (0.75% v/w) from J. horizontalis and 1.7 ml (0.85 % v/w) from J. communis).The oils were collected, dried over anhydrous Na2SO4 and kept in a freezer at -5°C until the GC-MS analysis can be performed.10 g of the concentrated total alcoholic extract of the aerial part of each of J. horizontalis and J.communis prepared in 80% methanol were reserved for schistosomicidal and molluscicidal study.

Schistosomicidal activity (WHO, 1985)
The schistosomicidal in vitro effect of each plant was determined according to Yousif et al. (2007).The schistosome material used in the present study was supplied by the Schistosome Biological Supply Centre (SBSC) at Theodor Bilharz Research Institute, Cairo, Egypt.Adult male and female Schistosoma mansoni Sambon worms were collected 7 weeks post exposure of laboratory bred Syrian golden hamsters (Mesocricetus auratus) to cercariae by perfusion technique.Worms were cleaned from blood in small sieves of 20 µ mesh size using phosphate buffer (pH 7.4).A stock solution (500 µg/ml) of each plant extract was prepared in 100% dimethyl sulfoxide (DMSO) and 0.1 ml of this solution was made to 2 ml with RPMI 1640 containing 20% fetal calf serum, 300 mg streptomycin, cc 300 units pencillin and 160 µg gentamicin/100 ml medium.The obtained stock solution was diluted by the same medium to give the following concentrations: 100, 80, 60, 40 and 20 µg/ml.Three pairs of worms, males and females equally represented were placed in each well using sterilized forceps, they were exposed to these concentrations for 3 days and two replicates were done for each concentration.Praziquantel, the most effective schistosomicidal drug was used as a positive control (0.2 µg/ml) and a clean medium was used as a negative one to allow critical comparison of the effect.Test and control wells were maintained in an incubator at 37°C examined daily for 3 days for worm viability using stereomicroscope.Worms which did not show any sign of motility for one minute were considered dead.The activity of the plant extract was measured by calculating the number of dead worms relative to the total number of worms and compared with the negative (DMSO) and positive (praziquantel) controls.
For determination of LC50 (lethal concentration that kills 50% of the worms) and LC90 (lethal concentration that kills 90% of the worms), the same experiment was repeated six times.The worm mortality was recorded in each case and the LC50 and LC90 was determined using SPSS statistical program (version 20, Chicago, IL, USA).

Molluscicidal activity
The molluscicidal efficacy of the plant extracts was determined against the snails using standard method (WHO, 1965;El Bardicy et al., 2012).Adult Biomphalaria alexandrina (Ehrenberg) (Planorbidae) snails were obtained from the laboratory colony at the Schistosome Biological Supply Centre (SBSC) at Theodor Bilharz Research Institute, Cairo, Egypt.A stock solution of 1 L of the dechlorinated water with a concentration 100 ppm of each extract was prepared and the following concentrations (20, 30, 40, 50 and 60 ppm) were tested and ten snails were added to each concentration.They were maintained in the solution for 24 h at room temperature (25 ± 1°C).After the exposure period, the snails were washed thoroughly with dechlorinated water and maintained in fresh water for another 24 h for recovery.In each case, two replicates were performed and two groups of snails were used as negative and positive control groups.The conventional molluscicide (niclosamide) at the same concentrations was used as a positive control; dead snails were counted in each case.Snails that were killed either during exposure or recovery period were counted and recorded.
For determination of LC molluscicidal effect, the same method using descending concentration was performed and LC50 and LC90 were determined by SPSS statistical program (version 20).

Chemical composition study
Gas chromatography/mass spectroscopy [GC/MS] analysis was performed using a Shimadzu GC-17A gas chromatograph equipped with a DB5-MS fused silica capillary column (30 m x 0.25 mm; film thickness 0.25 μm) and coupled to GCMS-QP 5050 mass analyzer.Operating conditions were: carrier gas helium, flow rate 0.9 ml/min; oven temperature program: 40-240°C at 3°C/minute; sample injection port temperature, 240°C; detector temperature, 230°C; ionization voltage and ionization current were according to tuning result; scanning speed, 0.5 s; split, 1:54.Essential oil components peaks were first deconvoluted using AMDIS software.Compounds were identified by their Kovates indices (KI) relative to n-alkanes (C6-C20) and through matching mass spectra and retention indices with those deposited in the NIST, WILEY library database and

RESULTS
The screening results of schistosomicidal and molluscicidal activities of the total methanolic extracts of the two Juniperus species under investigation showed nearly equivalent schistosomicidal activity after 3 days exposure (Table 1).However this activity is less than that of praziquantel used as a positive control but still under 100 ppm.As for the molluscicidal activity against Biomphalaria alexandrina snails, J. communis extract showed higher activity with an LC50 equivalent to 22.9 ppm as compared to 38.9 ppm after one day exposure for J. horizontalis (Figure 1).The chemical composition of the oils of the two Juniperus species under investigation analyzed by GC/MS (Table 2) showed a total of 60 compounds for J. horizontalis oil and 67 components for J. communis oil.
The percentage of component identification for both plants was more than 90%.

DISCUSSION
Screening of the biological activity of the methanolic extracts of the two Juniperus species under investigation (Table 1 and Figure 1), showed that the schistosomicidal activity against Schistosoma monsoni worms of the total alcoholic extract of both species is nearly equivalent as seen in their LC 50 values which is equal to 91.4 and 91.6 μg/ml for J. communis and J. horizontalis, respectively.In both cases, it is less than 100 ppm and consequently considered to posses schistosomicidal activity according to the recommendation of the world health organization (WHO, 1985).However, their effect is lesser than that of praziquantel (standard positive control used) showing an LC 50 equal to 0.27 μg/ml.In the case of molluscicidal activity against B. alexandrina snails, J. communis extract showed higher activity than that of J. horizontalis with an LC 50 = 22.9 ppm.These results are similar to that of    (Yousif et al., 2007).
It is worth mentioning that molluscicidal and schistosomicidal activity of several plants rich in volatile constituents were previously studied, such as Thymus capitatus Marrubium vulgare and Chrysanthemum viscidehirtum, showing promising activities against different parasites (Khallouki et al., 2000;Salama et al., 2012).
The oil content obtained by hydro-distillation of the aerial parts of J. horizontalis representing 0.75% v/w was analyzed using GC/MS (Table 2) showing a total of 60 compounds with a percentage of 95.39% of identified compounds.Bornyl acetate was seen as a major component representing 41.17% followed by 10-epielemol representing 8.44% and β-myrcene 5.94%, sabenine 5.51% and thujone 4.8%.This data greatly differs from that previously published on J. horizontalis essential oil composition (Eryiğit et al., 2014) and also differed from the composition stated by Cantrell et al. (2014) who showed that the major constituents present in the oil of J. horizontalis were alpha-pinene, sabinene and limonene.As for J. communis essential oil composition showed 67 component representing 0.85% v/w of which 1,7-Nonadien-4-ol, 4,8-dimethyl (homogeraniol) (36.95%) was the major component followed by 10epielemol (9.45%) and β-Myrcene (5.15%), sabenine (4.5%), also this data differs from that already published on J. communis obtained from other sources mainly predominated by monoterpene constituents (Stoilova et al., 2014).Differences in essential oil composition may be expected due to geographical occurrences, climatic differences, the source of the oil either of commercial or of natural origin and may be due to the well proved relation between the time of collection and the percentage of active constituents and finally may be due to genetic variability among species (Khanzadi et al., 2015;Mammen et al., 2010).The high content of oxygenated compounds in the two species under investigation may explain the strong characteristic odor of these plants as referred to by Lund et al. (1981).However, Bornyl acetate was found to be absent in the aerial part of J. communis, while homogeraniol was found to be absent in the aerial part of J. horizontalis.It is worth mentioning that several compounds identified in the oil composition of the two studied species by the GC/MS analysis belongs to other classes such as hydrocarbons, fatty acids, minor alkaloids, amides and coumarins.

Conclusion
Both plants are good candidates for further studies on their schistosomicidal activity to determine the best effective doses to be used in the control of such dangerous parasite.The chemical composition of the essential oils of the two tested species of Juniperus differs greatly from the same species previously studied in different world zones and analyzed by similar analytical technique (GC/MS) which may be attributed to the difference in both climatic conditions and genetic characters of the studied species.

Figure 1 .
Figure 1.Results of screening of the molluscicidal activity of the methanolic extracts of both J. horizontalis and J. communis.Y-axis represent the concentration in ppm, X-axis represent LC50 and LC90 (% mortality) for different test fractions

Table 1 .
Results of screening of schistosomicidal activity of total alcoholic extracts of both J.communis and J. horizontalis on schistosoma mansoni worms.

Table 2 .
Chemical composition of J. communis and J. horizontalis oils analyzed using GC/MS.