Antimicrobial Susceptibility of Euphorbia tirucalli Extracts and the Effect of Latex on Growth and Viability of Mouse Breast Cancer Cells EMT-6

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1 IOSR Journal of Dental and Medial Sienes (IOSR-JDMS) e-issn: , p-issn: Volume 13, Issue 3 Ver. VI. (Mar. 2014), PP Antimirobial Suseptibility of Euphorbia tirualli Extrats and the Effet of Latex on Growth and Viability of Mouse Breast Caner Cells EMT-6 Khalil Lafi Aljabarin, M. Farraj, Bahjat Jabarin, and Zhou Jian Feng Department of Internal Mediine, Tongji hospital, Medial College, Huazhong University of Siene and Tehnology, Wuhan, China Department of Immunology and Mirobiology, Medial Siene ollege, Birzeit University, Palestine Summary: Euphorbia tirualli is a plant presents in many ountries whih also known as Aveloz. This tree used in traditional mediine for treatment different types of disease. In this researh we tested the effets of aveloz latex, stem and leaves extrats on different gram positive and gram negative bateria, and its effets on some types of fungi, also we tested the aveloz effets on mouse breast aner ells. So we did extration for leaves and stems of aveloz by methanol and prepared the latex diretly from the tree by asepti tehnique. Then we prepared different dilutions from the 3 produts, then we prepared the inoulums, ulture and sensitivity tehniques were be performed, tissue ulture for mouse breast aner ells and latex were performed to study its anti-aner effets. Results showed us there were effets of the stem and leaves extrats on some types of bateria where the latex appeared a best sensitivity on some fungi speies. There was an interesting effet of latex on aner ells. Aveloz produts an be used in produing materials to prepare medial produts may be help in treating different diseases. Key words: Euphorbiaeae; Euphorbia tiruali; Aveloz; Latex; EBV (Epestein bar Virus); CFU-GM (olony forming unit- Granuloyte Monoyte) I. Introdution Euphorbia tirualli, a garden plant from Afria, belongs to the family Euphorbiaeae. Other names used inlude Tirualia india, Tithymalus tirualli, and Euphorbia viminalis. Common names used to for this plant inlude penil tree, aveloz. All parts of the tree serete a white milky fluid referred to as latex (1). The latex of Aveloz ontains terpenes inluding phorbol esters and ingenol esters whih ause severe irritation upon diret exposure. It ontains fatty aid frations onsist of palmiti and linolei aids, phytosterol onsist of sitosterol, stigmasterol, ampesterol and holesterol, and also ontain yanidin glyoside (2). The latex, stem and leaves of Aveloz tree are used in traditional mediine in many ountries to treat several diseases, partiularly skin diseases (3). Therefore, we were tested the stem, leaves extrats and latex, on several multidrug resistant miroorganisms. Several gram positive and gram negative bateria, yeasts and molds were obtained and ultured. Faultative gram positive oi suh as Staphyloous aureus, Staphyloous epidermides, miroous lutens, vanomyin resistane enteroous, enteroous faealis, and MRSA. Gram negative bateria suh as Pseudomonas aeruginosa, E. oli, K. pneumoniae Proteus mirabilis, Shigella Sonni, Shigella flaxiniri, Pseudomonas MDR, and Atinobater MDR. Fungi whih inlude Candida sp, Aspergillus spp, Fuzarium, and Rhizopus. Antimirobial suseptibility was performed following the reommendations of CLSI. In addition, the anti tumor properties of the latex were tested in-vitro on the breast aner ells EMT-6 ells. II. Materials and Methods 1.1 Aveloz Aveloz latex was olleted following asepti tehniques diretly from the euphorbia tree in a sterile tube. The latex was then plaed in a ooler and transferred immediately to the laboratory for proper storage and testing. Aveloz was also extrated from the plant's stem and leaves separately: Piees from the stem and leaves were individually mixed in a blender and the homogenate was plaed in a flask with 200 ml methanol for 24 hours. The liquid was then filtered through several layers of sterile gauze and then entrifuged at 5000xg for 15 minutes. The supernatant was then arefully removed and plaed in a white glass plate to evaporate the alohol. The remaining fluid was then olleted and plaed in a sterile tube to be tested. 1.2 Miroorganisms ATCC strains of Gram positive, gram negative bateria and fungus were obtained from the stoks at the laboratory of the Master's Program at Birzeit University. The antimirobial and anti fungal harateristis of 59 Page

2 Antimirobial Suseptibility of Euphorbia tirualli Extrats and the Effet of Latex on Growth and the aveloz latex, leaves and stem extrats were evaluated on Mueller-Hinton agar following the reommendations of CLSI. The optimal dilution was determined by applying several dilutions of the aveloz to filter paper disks plaed on the surfae of agar plates seeded with the organism to be tested. 1.3 Effets of Aveloz Latex on Mouse Breast Caner Cells TEM-6 The mouse breast aner ells EMT-6 were seeded in 24 wells plates. A total of 1X10 4 ells were seeded in eah well and the plate was inubated for 24 hours. The medium was then removed, the ells washed with HBSS and the aveloz latex was applied in tripliate at different dilutions. RPMI-1640 medium supplemented with 10% FBS was added to three wells and used as positive. After 48 hours of inubation, the medium from eah well was removed and plaed in 15 ml onial entrifuge tube. Trypsin EDTA was added to eah well (200 ul) and the plate was inubated at 37 o C for 5 minutes. RPMI-1640 was then added to eah well (200 ul) to stop the ation of the trypsin and the ontents of eah well were plaed in the respetive onial entrifuge tube. After entrifugation for 7 minutes at 700xg, the supernatant was removed and the ells were suspended in 500 ul of RPMI-1640 medium. Total and viability ounts were then performed and reorded. The distribution of the different aveloz latex dilutions in the 24 well plates as well as the positive wells is shown in (Table 1). Tube A 1 2 Tubes numbers B C D o Table 1. Distribution of the aveloz latex dilutions in the wells and the positive wells Counting of Cells and Estimation of Viability The ells (200 ul) were mixed with HBSS (300 ul) and trypan blue stain (500 ul). The tube was briefly vortexed and allowed to stand at room temperature for 5 minutes. A hemoytometer hamber was then properly filled with the stained ells. Four orner squares were then ounted. The total number of ells was alulated aording to the following formula: Total number of ells = Average number / square X dilution fator X 10 4 Viability was determined aording to the following formula: Viability = Total number of ells blue or dead ells Total number of ell 1.4 Inoulums Preparations The bateria was inoulated on blood agar plates and inubated for 18 to 24 hours, while fungus was inoulated on Sabaraud Dextrose agar until growth ours on room temperature. One or two olonies were then removed and a suspension equivalent to 0.5 MaFarland was prepared. A sterile otton swab was then dipped in the suspension, the exess fluid removed by gently rotating the swab against the inside of the tube ontaining the suspension. The swab was then used to inoulate the surfae of Mueller-Hinton agar plates. Sterile filter paper diss were then aseptially plaed on the surfae of the agar plates and 5 ul of eah aveloz dilution was plaed in dupliate to the filter paper. The plates were inubated for 18 to 24 hours in a non-co2 inubator; the zone diameters were aurately measured using Vernier aliper and reorded. Several strains of ATCC strains of Gram positive and negative bateria were used inluding: Gram ve bateria: Proteus mirabilis, Shigella Sonni, shigella flaxiniri, E. oli, Pseudomonas MDR, Pseudomonas earugenoza, Klebseilla Pneumonia, Atinobater MDR. Gram + bateria: Staphyloous aureus, Staphyloous epidermides, miroous lutens, vanomyin resistane enteroous, enteroous faealis, MRSA. Fungus isolates: Aspergillus, Rhizopus, Fuzarium, Yeast and Candida speies And Mold. III. Results 2.1 Effets of Aveloz Latex on Mouse Breast Caner Cells (EMT-6) EMT-6 ells (1x10 4) taken during the logarithmi phase of growth were plaed in eah well of a 24 well plate in tripliate. Different dilutions of the aveloz were added to the wells, and the plates were inubated for 24 hours. The results obtained are summarized in (Table 2). There were omplete lyses of the ell up to a 60 Page

3 Antimirobial Suseptibility of Euphorbia tirualli Extrats and the Effet of Latex on Growth and dilution of 1:100. The viability was drastially redued to a 22% at 1:1000 dilutions. At lower dilution the ell ounts and viability were omparable to the (fig. 1). aner ells Aveloz latex onentrations 0() No of viable X X X X X 10 4 ells/ml No of dead 1x10 4 1x X X X X X 10 4 ells/ml Total no. of 1x10 4 1x X X X X X 10 4 ells/ml % of viability 0% 0% 22% 94% 95% 98% 99% Table 2. Results of aveloz latex effets on mouse breast aner ells viability 10 ¹ 10 ² 10 ³ µ 10 ont viability Fig.1 viability (%) of ells at different dilutions ( ) of Aveloz, without aveloz. 2.2 Effets of Aveloz Latex Stem and Leaves Extrats on Bateria Aveloz latex was tested on different strains of gram positive and gram negative bateria. Different dilutions were added onto sterile filter papers plaed on a lawn of bateria spread on a Mueller Hinton agar following the reommendations of CLSI. The results obtained indiate that the aveloz latex had no antimirobial ativity. There was growth up to the edge of the filter paper diss with all dilutions used inluding a pure extrat. Extrats from the stem and leaves demonstrated anti-baterial ativity for both gram positive and gram negative bateria. Multidrug resistant non-typhoid Salmonella speies and Pseudomonas aeruginosa were resistant to all dilutions inluding the original extrat. The zones of inhibition are summarized in (Table 3). Aveloz latex onentrations stem extrats leaves extrats Mirobial speies onentrations onentrations Orig 1:1 1:2 1:4 Orig 1:1 1:2 1:4 Orig 1:1 1:2 1:4 on on on Staphyloous aureus Staphyloous epidermidis Miroous lutens MRSA Enteroous Faealis Vanomyin resistane enteroous Proteus mirabilis Shigella sonnei Shigella flexneri Salmonella Ainitobater baumanii E. oli Pseudomonas aeruginosa Pseudomonas aeruginosa Klebseilla pneumonia Table 3. Zone diameter of inhibition in mm of latex, and stem and leaves extrats on Bateria. 61 Page

4 Antimirobial Suseptibility of Euphorbia tirualli Extrats and the Effet of Latex on Growth and 2.3 Effets of Aveloz Latex Stem and Leave Extrats on Fungus Isolates Although aveloz latex had no antibaterial ativity, it showed antifungal ativity at high onentration against Aspergillus, Muor, Rhizopus and Candida spp. The effets obtained were either due to the pure extrat or a 1:10 dilution. The results are shown in (Table 4). The extrats obtained from the stem and leaves had limited or no ativity against fungi, the results is summarized in Table 6 below. Table 4. Diameter of inhibition zones in mm, aused by aveloz materials on fungi. IV. Disussion Our results indiated that aveloz latex suppressed and prevented the growth of mouse breast aner ells. At high onentration, it aused ell lyses and drastially redued ell viability at lower onentration. A study speulated that the mehanism of the antitumor ativity of euphorbia tirualli may be related to the regulation of granuloyte and marophage prodution and expression of funtional ativities (4). Another study revealed that latex has indued suppressed ativity on CD4+ and CD8+ T-ells, inhibited interleukin-2 and gama- interferone prodution and affeted immunomodulation (5). It was finding that latex taken internally aused redution in lymphoytes number (6). Researh onduted on tumor bearing mie with myelosuppression and inreased number of spleen CFU-GM, latex redued these ells (spleen olony formation), redued prostaglandin E2 that is normally elevated in tumor bearing mie and aused redution in tumor growth in the peritoneal avity. This indiates the presene of antitumor ativity of euphorbia tirualli (4). A study on latex revealed that it enhaned the growth of EBV whih led to Burkitts lymphoma (7). Literature searh did not reveal many artiles related to the antitumor ativity of this herb. Most of the information provided in literature laked sientifi basis. No artiles were deteted in literature dealing with the effets of aveloz on breast aner ells tested in this work or any other ell lines. The antibaterial and antifungal properties of latex were not investigated in literature. The only artile found indiated that extrats from the stem and leaves of Euphorbia tirualli have anti herbes simplex virus -2 effets (8). As indiated in the results obtained in this projet, the aveloz latex gave some antifungal ativity. The numbers of the fungal isolates tested were not enough to draw major onlusion. Extrats from the leaves and stems of this herb demonstrated antibaterial ativity. Most of the gram positive and gram negative baterial isolates were suseptible to this extrat to a reasonable dilution (1:4). Exeptions were seen in multidrug resistant A. baumanii, Pseudomonas aeruginosa, and Salmonella and Shigella speies. The drawbaks of the antimirobial effets of this herb are the unavailability of literature disussing this issue. Therefore we are unable to ompare our results with others. V. Conlusion Aveloz may be a promised medial plant to produe mediinal preparations for treatment different baterial, fungal and tumor diseases, so the results of this researh give informational supports to work on this plant by pharmaeutial ompanies researhes. Conflit of Interest Statement The authors delare that there is no onflit of interest with any finanial organization or orporation or individual that an inappropriately influene this work. 62 Page

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