LETHAL EFFECTS OF PISTACIA ATLANTICA LEAVES EXTRACT AGAINST PROTO-SCOLECES OF HYDATID CYSTS

LETHAL EFFECTS OF PISTACIA ATLANTICA LEAVES EXTRACT AGAINST PROTO-SCOLECES OF HYDATID CYSTS

Mohammad Reza Aflatoonian ^{1, 2}, Mojgan Saki ³, Farnaz Kheirandish ⁴, Hossein Mahmoudvand ³ and Mohammad Reza Nazer^*4

Bam University of Medical Sciences ¹, Bam, Iran.

Research Center for Tropical and Infectious Diseases ², Kerman University of Medical Sciences, Kerman, Iran.

Student Research Committee ³, Razi Herbal Medicines Research Center ⁴, Lorestan University of Medical Sciences, Khorramabad, Iran.

ABSTRACT: Surgery is one of the best options to treat cystic echinococcosis (CE, hydatid cyst). To date, surgeon used different protoscolicidal drugs to eliminate of protoscoleces during CE surgery, but most of these agents have some serious side effects. The current study aims to explore the in-vitro protoscolicidal effects of Pistacia atlantica leave extract against hydatid cyst proto-scolices.  Proto-scolices were collected from livers of infected sheep and were exposed with different concentrations of P. atlantica extract (8.75 - 70 mg/ml) for 10 - 60 min. Finally, to explore the viability of proto-scolices eosin exclusion examination was applied. The major components of leave extract of P. atlantica in gas chromatography/ mass spectrometry were trans-caryophyllene (15.18%), α-amorphene (8.1%), and neoalloocimene (6.21%), respectively. The obtained results revealed that P. atlantica leave extract had remarkable proto-scolicidal effects against proto-scoleces of hydatid cysts; so that killed completely (100%) all of proto-scoleces at the dose of 70 mg/ml after 10 min of treatment. Also, P. atlantica leave extract at the concentration of 35 mg/ml was eliminated 100% of proto-scoleces after 20 min of incubation. These findings indicated that P. atlantica leave extract had remarkable protoscolicidal properties versus hydatid cyst proto-scoleces which indicated the ability of P. atlantica as a natural resource to produce of novel proto-scolicidal drug for CE surgery.

Pistacia, Protoscoleces, Cystic echinococcosis, In-vitro

INTRODUCTION: Historically, the use of medicinal plants to treat illness and keep public health is highly prevalent around the world. Plant materials and their derivates show a central role in the field of novel drugs investigation for treatment or prevention of a wide range of diseases such as infectious ones ¹.

One of these interesting plants is Pistacia atlantica Desf. (Anacardiaceae), which commonly observed in various parts of the world such as Iran. In folk medicine, some parts of the P. atlantica have been extensively applied to treat several disease and illness such as diarrhea, asthma, anemia and infectious diseases ². Reviews have also reported anti-inflammatory, anticancer, antitumor, antialergic and antimicrobial properties of P. atlantica ².

Cystic echinococcosis (CE) or hydatidosis consider as one of the main parasitic diseases in human and animals which caused by a small dog tapeworm, Echinococcus granulosus ³.

Normally, humans acquire infection via consumption of vegetables, food or water contaminated with E. granulosus eggs. CE in human because of its effects on vital organs including liver and lung, and livestock due to considerable economic problems in stock breeding has attracted World Health Organization (WHO) attention to itself ⁴. From last decades, surgery is the most common choice in CE treatment. Surgeons during open cyst removal use some chemical scolicidal agents to decrease the risk linkage of proto-scoleces ⁵.

However, it has been proven that these common proto-scolicidal agents have some side effects such as necrosis of liver ^{6, 7}. Therefore, search for a proper protoscolicidal drug with potent efficacy and lowest side effects is necessary for surgeons.  The present investigation aims to evaluate the protoscolicidal effects P. atlantica on protoscoleces of hydatid cyst on in - vitro.

MATERIALS AND METHODS:

Plant Materials: Leaves of P. atlantica were collected from mountains of Lorestan province, West of Iran, in September 2014. The materials were determined by a botanist of the Botany Department of Lorestan University, Lorestan, Iran. A voucher sample also has been recorded in the herbarium of Research Center for Agriculture Sciences, Khorramabad, Iran (LF 2522).

Preparation of Extract: One hundred g of powdered dried leaves were isolated using percolation method with methanol (80%) for 72 h. in 21 °C. The extract was crossing through filter paper (Whatman No. 3, Sigma, Germany) to delete artifacts. Finally, the extract was concentrated at 50 °C using a rotary evaporator (Heidolph, Germany) and kept at -20 °C, until use ^{8, 9}.

Gas Chromatography/Mass Spectrometry (GC/ MS) Analysis of Extracts: Chemical compounds of P. atlantica extract were identified by extracting them using a solid phase micro extraction (SPME) technique. Initially, the samples were first ground into a fine powder using a household mill. Two grams of the sample was weighed and transferred to a 20 ml vial. Then the vial containing the sample was transferred to the ultrasonic device to extract the volatile substances. The temperature of the ultrasonic device was set on 50 °C for 15 min. In the next step, the SPME fiber was placed on the upper space of the sample for 40 min to extract the volatile compounds. Immediately after the extraction, the SPME fiber was injected into the GC-Mass device for desorbing and identifying the compositions of sample. Desorbing was performed in the GC column for 2 min.

The SPME fiber holder for manual use and the 100 µm Polydimethyl siloxane (PDMS) fibers were obtained from Supelco (Bellefonte, PA, USA) ¹⁰. GC/MS was carried out by an Agilent 6890N coupled with a HP-5MS column (30m × 0.25 mm, film thickness 0.25 mm). The column temperature was maintained at 40 °C for 12 min and programmed to 180 °C at a rate of 10 °C per min, and kept constant at 180 °C for 4 min. Injector and interface temperatures were 250 and 280 °C, respectively. The flow rate of Helium as carrier gas was (1 mL/min C.F). The percentages were calculated by electronic integration of FID peak areas without the use of response factors correction. Linear retention indices for all components were determined by co-injection of the samples with a solution containing homologous series of C8-C24 n-alkanes.

Identification of Components: The constituents of the extracts were determined in comparison with their standards Wiley 2001 library data of the GC/MS system or compared with those of reported in the previous studies ¹¹.

Collection of Proto-scoleces: E. granulosus proto-scolexes were obtained from the livers of naturally infected livestock slaughtered at Kerman abattoir, Iran. The cyst fluid was aseptically aspirated and was left to set for 30 min. After washing of proto-scoleces for two times with PBS (pH 7.2) solution the number of protoscoleces/ml was adjusted as 2× 10³ protoscoleces in 0.9 % NaCl solution with at least 90 % viability rate ¹⁶.

Proto-scolicidal Effects: To determine the lethal effects P. atlantica on hydatid cyst protoscoleces, various doses of P. atlantica extract were applied for 10, 20, 30 and 60 min. The protocol was performed according to the method described by Mahmoudvand et al., (2014). Initially, 0.5 ml of the protoscoleces (2× 10³/ml) and 0.5 ml of different doses of tested extract was added to each test tube. Test tubes were slowly mixed and then kept at 37°C for 10 - 60 min. After the time of incubation, the supernatant was discarded and 50 µl of 0.1 % eosin stain was added to protoscoleces and smeared on a glass slide and tested under a light microscope ¹⁷. The percentages of dead proto-scoleces were counted by counting 100 proto-scoleces ^{18, 19}. Moreover, normal saline and hypertonic saline of 20% were applied as negative and positive controls.

Evaluation of Viability of Protoscoleces: In this study, eosin exclusion assay (eosin solution 0.1%) was applied to determine the viability of proto-scoleces of hydatid cysts ¹⁴. The live proto-scoleces are colorless and displayed characteristic muscular movements and flame cell activity after exposure to the stain, whereas dead proto-scoleces are red color.

Statistical Analysis: Statistical analysis was performed using SPSS software (17.0). Moreover, t-test was applied to determine the differences between groups. P<0.05 was also considered statistically significant ¹⁵.

RESULTS:

GC/MS Analysis of Extracts: Table 1 shows the identified compounds of leave extract of P. atlantica using GC/MS.

TABLE 1: LEAVE EXTRACT COMPOSITION OF P. ATLANTICA IDENTIFIED BY GC/MS

The major components of leave extract of P. atlantica were trans-caryophyllene (15.18%), α-amorphene (8.1%), and neoalloocimene (6.21%), respectively.

Protoscolicidal Effects: As shown in Table 2 P. atlantica leave extract demonstrated protoscolicidal activity against hydatid cysts protoscoleces. The current finding revealed that P. atlantica leave extract had remarkable protoscolicidal effects against protoscoleces of hydatid cysts; so that killed completely (100%) all of proto-scoleces at the dose of 70 mg/ml after 10 min of treatment. Also, P. atlantica leave extract at the concentration of 35 mg/ml was eliminated 100% of protoscoleces after 20 min of incubation. The protoscolicidal effect of P. atlantica leave extract at the concentration of 17.5 mg/ml was 14.3, 37.6, 63.3 and 100 % after 10, 20, 30 and 60 min of incubation, respectively.

The mean dead of proto-scoleces in the normal saline control group was 5.3 % after 60 min of treatment, while the proto-scolicidal effects of 20% hypertonic saline as the positive controls was 100% after and 10 min of application. These findings exhibited that the scolicidal activity of P. atlantica leaves extract was significant higher (p<0.05) in comparison with the control group at all exposure times.

TABLE 2: SCOLICIDAL EFFECTS OF P. ATLANTICA LEAVE EXTRACT AGAINST PROTO-SCOLECES OF HYDATID CYST AT VARIOUS CONCENTRATIONS FOLLOWING VARIOUS EXPOSURE TIMES

DISCUSSION: Medicinal plants and their pure compounds show proper opportunities for new drug searches in order to the having many benefits such as higher efficacy and availability as well as lower toxicity ¹⁶. Here, we found that P. atlantica leave extract had remarkable protoscolicidal effects against proto-scoleces of hydatid cysts; so that killed completely (100%) all of protoscoleces at the dose of 70 mg/ml after 10 min of treatment. Also, P. atlantica leave extract at the concentration of 35 mg/ml was eliminated 100 % of protoscoleces after 20 min of incubation. This scolicidal effects of P.

atlantica is comparable with scolicidal activity of hypertonic saline, silver nitrate and mannitol, cetrimide, ethyl alcohol (95%), H₂O₂ and 10% providone iodine, albendazole, chlorhexidine gluconate, Selenium NPs and some plant extracts ^{17- 24}. Reviews have reported antibacterial, antiviral, antifungal and antiparasitic effects of P. atlantica ³.

We found that the major components of leave extract of P. atlantica were trans-caryophyllene (15.18%), α-amorphene (8.1%), and neoalloo-cimene (6.21%), respectively. However, according to the previous investigations chemical composition of extracts depend on species, climate, and time of collection along with growth stage, thereby altering the biological activities studied ²⁵.

Previous studies also revealed the presence of terpenoids, phenols, flavonoids, fatty acids and sterols in phytochemical screening of the crude extract P. atlantica ³. In several investigations biological especially antimicrobial activities of these components have been proven ^{24 - 27}. Thus, we can suggest that these components are responsible for the scolicidal activity of P. atlantica; however their exact action mechanism is not clearly understood. Regarding the some mechanism of antimicrobial terpenoids compounds such as monoterpens, investigations have demonstrated that these compounds diffuse into pathogen and destroy cell membrane. Other studies also reported that anti-microbial effects of these components interfering with vital intracellular sites when cross the cell membranes and penetrate into the cell ^{28, 29}.

CONCLUSION: These findings indicated that P. atlantica leave extract had remarkable proto-scolicidal properties versus hydatid cyst protos-coleces which indicated the ability of P. atlantica as a natural resource to produce of novel proto-scolicidal drug for CE surgery.

ACKNOWLEDGEMENT: Nil.

CONFLICT OF INTEREST: The author declares that there is no conflict of interests in this study.

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    How to cite this article:

    Aflatoonian MR, Saki M, Kheirandish F, Mahmoudvand H and Nazer MR: Lethal effects of Pistacia atlantica leaves extract against proto-scoleces of hydatid cysts. Int J Pharm Sci Res 2017; 8(11): 4653-58.doi: 10.13040/IJPSR.0975-8232.8(11).4653-58.

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