Sains Malaysiana 43(11)(2014): 1693–1697

Starvation Time and Successive Collection Effects on Leeches Saliva Collection Quantity and Proteins Quality and Quantity in Wet Season

(Kesan Masa Kebuluran dan Kejayaan Pengumpulan Kuantiti Air Liur Lintah serta Kualiti

dan Kuantiti Protein dalam Musim Hujan)

MOHAMED ALAAMA¹, ABM. HELALUDDIN¹*, ABBAS MOHAMMAD², AHMED MERZOUK³, ABDUALRAHMAN MOHAMMED ABDUALKADER¹

& MOHAMED BIN AWANG⁴

¹Department of Pharmaceutical Chemistry, Kulliyyah of Pharmacy, International Islamic University Malaysia (IIUM), 25200 Kuantan, Pahang, Malaysia

²Department of Basic Medical Science, Kulliyyah of Pharmacy, International Islamic University Malaysia (IIUM), 25200 Kuantan, Pahang, Malaysia

³Biopep Solutions Inc., Vancouver, BC Canada

⁴Faculty of Pharmacy, Cyberjaya University College of Medical Sciences (CUCMS), 63000 Cyberjaya, Selangor, Malaysia

Diserahkan: 12 Oktober 2012/Diterima: 17 Mac 2014

ABSTRACT

The salivary gland secretion of the haematophagous animals, leeches, has attracted the attention of therapists since the extreme old ages due to its wide range of medical properties. Thus, many researches have been done to develop and optimize new methods to collect leech saliva with high quality and quantity. In the present study, we aimed to evaluate the effects of starvation period and repeated collection on the quality and quantity of leech saliva extract LSE and its contents of proteins during the rainy season. Protein recovery in the LSE was also studied after first collection. It was found that leeches are able to produce protein-containing saliva whenever fed during the whole study period of 18 weeks with varied protein concentrations. The results showed that the highest protein concentrations (105-91 μg/mL) were produced after 12-15 weeks of starvation. The results of successive collection showed that leeches are able to produce proteins and peptides whenever they suck the solution after first collection with some varies in the concentrations. The concentrations varied between 0 and 72% of the initial concentration. Gel electrophoresis results showed absence for some bands when the concentrations are too low. Also the results showed that leeches are able to recover about 42% of their initial proteins concentration within four weeks of starvation after first feeding. The gel electrophoresis results showed the closeness between the first and second collections. To conclude, all test factors (starvation period, successive collection and recovery test) were shown to have an important impact on protein concentration of leech saliva and therefore its medicinal affectivity. The mentioned results are reported for the first time and they open the gate for further studies.

Keywords: Leeches; saliva; starvation; time; quality; quantity; wet season

ABSTRAK

Rembesan kelenjar air liur haiwan hematofaj, lintah, telah menarik perhatian ahli terapi sejak dulu kerana pelbagai sifat perubatan. Oleh itu, pelbagai kajian telah dijalankan untuk membangun dan mengoptimumkan kaedah baru untuk mengumpul air liur lintah dengan kualiti dan kuantiti yang tinggi. Dalam kajian ini, kami ingin menilai kesan tempoh kebuluran dan kutipan ulangan pada kualiti dan kuantiti air liur lintah ekstrak LSE dan kandungan proteinnya semasa musim hujan. Pemulihan protein di LSE itu juga dikaji selepas kutipan pertama. Didapati bahawa lintah boleh menghasilkan protein yang mengandungi air liur pada bila-bila masa setelah diberi makan sepanjang tempoh keseluruhan kajian selama 18 minggu dengan kepekatan protein yang berbeza-beza. Hasil kajian menunjukkan bahawa kepekatan protein tertinggi (105-91 μg/mL) telah dihasilkan selepas 12-15 minggu kebuluran. Hasil kejayaan kutipan menunjukkan bahawa lintah boleh menghasilkan protein dan peptida pada bila-bila masa mereka menghisap larutan selepas kutipan pertama dengan kepekatan berbeza. Kepekatan awal berbeza-beza antara 0 dan 72%. Hasil gel elektroforesis menunjukkan ketiadaan jalur apabila kepekatan terlalu rendah. Keputusan juga menunjukkan bahawa lintah mampu untuk memulihkan kira-kira 42% daripada kepekatan protein awal dalam tempoh empat minggu kebuluran selepas makan yang pertama. Keputusan gel elektroforesis menunjukkan kehampiran antara kutipan pertama dan kedua. Kesimpulannya, semua ujian faktor (tempoh kebuluran, kejayaan kutipan dan ujian pemulihan) telah terbukti mempunyai kesan penting ke atas kepekatan protein air liur lintah dan kesan perubatannya. Keputusan ini dilaporkan buat pertama kali dan membuka peluang untuk kajian lanjutan.

Kata kunci: Air liur; kebuluran; kualiti; kuantiti; lintah; masa; musim hujan

RUJUKAN

Abdualkader, A., Ghawi, A., Alaama, M., Awang, M. & Merzouk, A. 2013a. Leech therapeutic applications. Indian Journal of Pharmaceutical Sciences 75(2): 127-137.

Abdualkader, A., Ghawi, A., Mohamed, A., Mohamed, A. & Ahmed, M. 2013b. In vivo anti-hyperglycemic activity of saliva extract from the tropical leech Hirudinaria manillensis. Chinese Journal of Natural Medicines 11(5): 488-493.

Abdualkader, A., Merzouk, A. & Ghawi, A.M. 2011. Some biological activities of Malaysian leech saliva extract. IIUM Engineering Journal Special Issue on Biotechnology 12(4): 1-9.

Alaama, M., AlNajjar, M., Abdualkader, A., Mohammad, A. & Merzouk, A. 2011. Isolation and analytical characterization of local Malaysian leech saliva. IIUM Engineering Journal Special Issue on Biotechnology 12(4): 51-59.

Bagdy, D., Barabás, E. & Gráf, L. 1973. Large scale preparation of hirudin. Thrombosis Research 2(3): 229-238.

Baskova, I.P., Zavalova, L.L., Basanova, A.V., Moshkovskii, S.A. & Zgoda, V.G. 2004. Protein profiling of the medicinal leech salivary gland secretion by proteomic analytical methods. Biochemistry (Mosc) 69(7): 770-775.

Baskova, I.P., Nikonov, G.I. & Cherkesova, O.U. 1984. Antithrombin, antitrypsin and antichymotrypsin activities of the salivary gland secretion and intestinal chyme of medicinal leeches. Antichymotrypsin activity of partially purified preparations of hirudin and pseudohirudin. Folia Haematologica 111(6): 831-837.

Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254.

Chopin, V., Salzet, M., Baert, J., Vandenbulcke, F., Sautiere, P.E., Kerckaert, J.P. & Malecha, J. 2000. Therostasin, a novel clotting factor Xa inhibitor from the Rhynchobdellid Leech, Theromyzon Tessulatum. The Journal of Biological Chemistry 275(42): 32701-32707.

Ghawi, A.M., Merzouk, A., Abdualkader, A. & Alaama, M. 2013. Treating cancer with a whole, leech saliva extract. U.S. Patent No. 8,501,241. 6 Aug. 2013.

Götz Nowak, K.S. 2007. Hirudin - the long and stony way from an anticoagulant peptide in the saliva of medicinal leech to a recombinant drug and beyond. Thromb Haemost 98: 116-119.

Merzouk, A., Ghawi, A.M., Abdualkader, A., Abdullahi, A.D. & Alaama, M. 2012. Anticancer effects of medical Malaysian leech saliva extract (LSE). Pharm. Anal. Acta. S15. pp. 1-5.

Rigbi, M., Levy, H., Iraqi, F., Teitelbaum, M., Orevi, M., Alajoutsijarvi, A., Horovitz, A., Galun, R. 1987. The saliva of the medicinal leech Hirudo medicinalis--I. Biochemical characterization of the high molecular weight fraction. Comp. Biochem. Physiol. B 87(3): 567-573.

Salzet, M., Chopin, V., Baert, J., Matias, I. & Malecha, J. 2000. Theromin, a novel leech thrombin inhibitor. The Journal of Biological Chemistry 275(40): 30774-30780.

Sawyer, R.T. 1986b. Leech Biology and Behaviour: Volume II: Feeding Biology, Ecology, and Systematics. Oxford: Oxford University Press.

Schägger, H. & von Jagow, G. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochemistry 166(2): 368-379.

*Pengarang untuk surat-menyurat; email: mohdhelal@hotmail.com