Investigation of Anti-enzyme Activities of Primrose (Oenothera biennis) and Sweet Almond (Prunus dulcis Mill.) Oils

Abstract

In our study, the question of what might be the effects of Primrose (Oenothera biennis) and sweet almond (Prunus dulcis Mill.) oils on two different enzyme activities was asked. The first enzyme in our study is carbonic anhydrase I-II isoenzymes that play a very important role in many physiological events, by transforming catalysis of CO2 hydration and HCO3-dehydration in living things, by creating an intracellular bicarbonate buffer system. The second enzyme, the acetylcholinesterase enzyme, which was located in the synapse between the muscle cell and nerve, catalyzes the decomposition of the acetylcholine molecule. Inhibitors of both enzymes have the potential to be drugs. Therefore, the inhibition effect of the specified oils was investigated. For this purpose, the first carbonic anhydrase I-II isoenzyme was purified by the sepharose-4B-L tyrosine-sulfonamide affinity column. Then, by looking at enzyme activity in at least five different inhibitor concentrations, effects of evening primrose (Oenothera biennis) and sweet almond (Prunus dulcis Mill.) oils on enzyme activities were investigated. Finally, IC50 values of Primrose (Oenothera biennis) and sweet almond (Prunus dulcis Mill.) oils were determined by drawing activity%-[I] graph. Carbonic anhydrase I isoenzyme was purified from human erythrocytes 119 times in 20.12% yield, while carbonic anhydrase II isoenzyme was purified 535.72 times in 83.05% yield. IC50 values of the primrose oils (Oenothera biennis) on carbonic anhydrase I-II and acetylcholinesterase enzyme activity were 0,1950, 0,1406 and 0,1097 mg / mL, respectively. IC50 values of the sweet almond oil (Prunus dulcis Mill.) on carbonic anhydrase I-II and acetylcholine esterase enzyme activity were 0.0345, 0.0266 and 0.0394 mg/mL respectively. When both oils used in the study are compared, we see that sweet almond oil (Prunus dulcis Mill.) is more effective on both enzyme activities. Therefore, instead of synthetic drugs, sweet almond (Prunus dulcis Mill.) oil is thought may be used as a supplement in the treatment of diseases caused by the enzymes in question.

Keywords

• Acetylcholinesterase,carbonic anhydrase,in vitro

Çuha Çiçeği (Oenothera biennis) ve Tatlı Badem (Prunus dulcis Mill) Yağlarının Anti-enzim Aktivitelerinin Araştırılması

Abstract

Çalışmamızda iki farklı enzim aktivitesi üzerine Çuha çiçeği (Oenothera biennis) ile tatlı badem (Prunus dulcis Mill.) yağlarının etkileri ne olabilecek sorusu cevaplanmak istendi. Kullandığımız ilk enzim canlılarda CO2’in hidratasyonu ve HCO3-‘ın dehidratasyonunu dönüşümlü olarak katalizleyerek, hücre içi bikarbonat tampon sistemini oluşturarak, birçok fizyolojik olayda oldukça önemli rol alan karbonik anhidraz I-II izoenzimleridir. İkinci enzim ise asetilkolin molekülünün ayrışmasını katalizleyen, kas hücresi ve sinir arasındaki sinapsta yer alan asetilkolinesteraz enzimidir. Her iki enzimin inhibitörleri, ilaç olma potansiyeli taşımaktadır. Bu nedenle belirtilen yağların inhibisyon etkisi araştırıldı. Bu amaçla ilk olarak karbonik anhidraz I-II izoenzimi sefaroz-4B-L-tirosin-sülfanilamid afinite kolonu ile saflaştırıldı. Ardından en az beş farklı inhibitör konsantrasyonunda enzim aktivitesi bakılarak Çuha çiçeği (Oenothera biennis) ve tatlı badem (Prunus dulcis Mill.) yağlarının enzim aktiviteleri üzerindeki etkileri araştırıldı. Son olarak %aktivite-[I] grafiği çizilerek Çuha çiçeği (Oenothera biennis) ve tatlı badem (Prunus dulcis Mill.) yağlarının IC50 değerleri tespit edildi. İnsan eritrositlerinden karbonik anhidraz I izoenzimi %20,12 verimle 119 kat saflaştırılırken, karbonik anhidraz II izoenzimi %83,05 verimle 535,72 kat saflaştırıldı. Çuha çiçeği (Oenothera biennis) yağının karbonik anhidraz I-II ve asetilkolinesteraz enzim aktivitesi üzerindeki IC50 değerleri sırasıyla 0,1950, 0,1406 ve 0,1097 mg/mL olarak, tatlı badem (Prunus dulcis Mill.) yağının karbonik anhidraz I-II ve asetilkolin esteraz enzim aktivitesi üzerindeki IC50 değerleri ise sırasıyla 0,0345, 0,0266 ve 0,0394 mg/mL olarak tespit edildi. Çalışmada kullanılan her iki yağ karşılaştırıldığında, tatlı badem (Prunus dulcis Mill.) yağının her iki enzim aktivitesi üzerinde daha etkili olduğunu görülmektedir. Bu nedenle sentetik ilaçlar yerine tatlı badem (Prunus dulcis Mill.) yağının söz konusu enzimlerin sebep olduğu hastalıkların tedavisinde takviye ilaç olarak kullanılabileceği düşünülmektedir.

Keywords

• Asetilkolinesteraz,karbonik anhidraz,in vitro

References

1. Adem, S., Akkemik, E., Aksit, H., Guller, P., Tüfekci, A.R., Demirtas, İ., Ciftci, M. (2019). Activation and inhibition effects of some natural products on human cytosolic CAI and CAII. Medicinal Chemistry Research, 28:711–722.
2. Aggarwal, M., Kondeti, B., McKenna, R. (2013). Insights Towards Sulfonamide Drug Specificity in Α-Carbonıc Anhydrases. Bioorganic & Medicinal Chemistry, 21(6): 1526-1533.
3. Ahmad, Z. (2010). The uses and properties of almond oil. Complementary Therapies in Clinical Practice, 16(1), 10-12 4.
4. Akalın, Ş. (1952). Büyük bitkiler kılavuzu. Cilt 1, Güzel Sanatlar Matbaası, 628s Ankara.
5. Akıncıoğlu A, Akıncıoğlu H, Gül.in I, Durdağı, S., Supuran, C.T., Göksu, S. (2015). Discovery of potent carbonic anhydrase and acetylcholine esterase inhibitors: novel sulfamoylcarbamates and sulfamides derived from acetophenones. Bioorg Med Chem 23(13):3592-602
6. Akıncıoğlu, A., Topal, M., Gülçin, I., Göksu, S., (2014). Novel Sulphamides and Sulphonamides Incorporating the Tetralin Scaffold as Carbonic Anhydrase and Acetylcholine Esterase Inhibitors. Arch Pharm 347:1, 68-76.
7. Akkemik, E., Aybek, A., Felek, I. (2019). Effects of Cefan Melon (Cucumıs Melo L.) Seed Extracts on Human Erythrocyte Carbonıc Anhydrase I-II Enzymes. Applıed Ecology And Envıronmental Research, 17(6):14699-14713.
8. Akkemik, E., Cicek, B., Camadan, Y., Calisir, U., Onbasioglu, Z. (2018). The determination of the carbonic anhydrase’s activators in vitro effect of mixed donor crown ethers. Journal of Biochemical and Molecular Toxicology, 32(3): e22032. Akkemik, E., Çalışır, Ü., Çiçek, B. (2017). İnsan karbonik anhidraz I,II izoenzim aktiviteleri üzerine bazı tiyocrown eterlerin etkisi. J. BAUN Inst. Sci. Technol, 19(2), 192-199.

9. Aksu, K., Özgeriş, B., Taslimi, P., Naderi, A., Gülçin, I., Göksu, S. (2016). Antioxidant Activity, Acetylcholinesterase, and Carbonic Anhydrase Inhibitory Properties of Novel Ureas Derived from Phenethylamines.Arch Pharm 349(12):944-954.
10. Ammar, N.M., Soroor, K.H.A., Mohammed, D.A. (2000). Impact of natural oils supplements on disease activity and antioxidant state of Egyptian patients with rheumatoid arthritis. MJIAS; 13: 161-171.
11. Anonim, (2020). https://www.pfizer.com.tr/arge/yeni-ila%C3%A7-geli%C5%9Ftirme-s%C3%BCreci, 14.02.2020, 18:36
12. Balta, M.F. (2013). Fatty acid profiles for almond [Prunus amydalus Batsch] genotypes with different kernel taste and formation. Iğdır Univ. J. Inst. Sci. Tech, 3(1), 17-24. 3.
13. Bangou, J. M., Kiendrebeogo, M., Compaore, M., Coulibaly, A. Y., Roland Meda, N. T., Abarka Almaraz, N., Zeba, B., Millogo-Rasolodimby, J., Nacoulma, O.G. (2011). Enzyme inhibiting effect and polphenolic content of medicinal plant extracts from Burkina Faso. Journal of Biological Sciences, 11(1): 31-38.
14. Bayrak, S., Yılmaz, Ö. (2009). Ceviz- Badem yetiştiriciliği. Rekmay Reklam ve Tanıtım Ltd. Şti., 321s Ankara.
15. Berchtold, N. C., and Cotman. C.W. (1998). Evolution in the conceptualization of dementia and Alzheimer’s disease: Greco-Roman period to the 1960s. Neurobıol Agıng 19(3) 173–189,
16. Birch, A.E., Fenner, G.P., Watkins, R., Boyd, L.C. (2001). Antioxidant properties of evening primrose seed extracts. J Agric Food Chem, 49: 4502-s4507.
17. Boztaş, M., Çetinkaya, Y., Topal, M., Gülçin, I., Menzek, A., Şahin, E., Tanc, M., Supuran, C.T. (2015). Synthesis and carbonic anhydrase isoenzymes I, II, IX, and XII inhibitory effects of dimethoxybromophenol derivatives incorporating cyclopropane moieties. J Med Chem 58 (2), 640-650.
18. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-54.
19. Chen, C.Y., Lapsley, K., Blumberg, J. (2006). A nutrition and health perspective on almond. J. Sci. Food Agric. 86, 2245–2250.
20. Costa, L.G. (2006). Current issues in organophosphate toxicology. Clinica Chimica Acta, 366, 1–13.
21. Darvesh, S., Darvesh, K.V., McDonald, R.S., Mataija, D., Walsh, R., Mothana, S., Lockridge, O., Martin, E. (2008). Carbamates with differential mechanism of inhibition toward acetylcholinesterase and butyrylcholinesterase. J Med Chem, 51(14):4200-12.
22. Drachman, D.A., Leavitt, J. (1974). Human Memory and the Cholinergic System A Relationship to Aging? Arch Neurol. 30(2):113-121
23. Davis, P.A., Iwahashi, C.K. (2001). Whole almonds and almond fractions reduce aberrant crypt foci in a rat model of colon carcinogenesis. Cancer. Lett, 165(1):27–33
24. El‐Hadary, A.E., Ramadan, M.F. (2019). Phenolic profiles, antihyperglycemic, antihyperlipidemic, and antioxidant properties of pomegranate (Punica granatum) peel extract. Journal of Food Biochemistry, e12803.
25. Ellman, G.L., Courtney, K.D., Andres, V., Featherston, R.M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem Pharmacol 7:88-95.
26. Favati, F., King, J.W., Mazzati, M. (1991). Supercritical carbon dioxide extraction of evening primrose oil. JAOCS, 68: 422-427. Fulton, M.H., and Key, P.B. (2001). Acetylcholinesterase Inhiıbition In Estuarine Fish and Invertebrates As an Indicator of Organophosphorus Insecticide Exposure and Effects, Environmental Toxicology And Chemistry, 20:1, Pp. 37–45,
27. Glenner, G.G., Wong, C.W. (1984a). Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun, May 16;120(3):885-90.
28. Glenner, G.G., Wong, C.W. (1984b). Alzheimer's disease and Down's syndrome: sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res Commun. Aug 16;122(3):1131-5.
29. Goedert, M., Spillantini, M.G., Jakes, R., Rutherford, D., Crowther, R.A. (1989). Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease. Neuron. Oct;3(4):519-26.
30. Gokcen, T., Topal, M., Gulcin, I., Ozturk, T., Goren, A.C. (2017). Synthesis of some natural sulphonamide derivatives as carbonic anhydrase inhibitors. Org Commun, 10:15–23
31. Göksu, H., Topal, M., Keskin, A., Gültekin, M.S., Çelik, M., Gülçin, I., Tanc, M., Supuran, C.T. (2016). 9,10-Dibromo-N-aryl-9,10-dihydro-9,10-[3,4]epipyrroloanthracene-12,14-diones: Synthesis and Investigation of Their Effects on Carbonic Anhydrase Isozymes I, II, IX, and XII. Arch Pharm, 349(6):466-74.
32. Grutzendler, J., Morris, J.C. (2001). Cholinesterase inhibitors for Alzheimer’s disease. Drugs, 61(1):41-52.
33. Gul, H.I., Demirtas, A., Ucar, G., Taslimi, P., Gülçin, I. (2017a). Synthesis and biological evaluation of aminomethyl and alkoxymethyl derivatives as carbonic anhydrase, acetylcholinesterase and butyrylcholinesterase inhibitors, Lett Drug Des Discov, 32(1): 1174–1182.
34. Gul, H.I., Mete, E., Taslimi, P., Gulcin, I., Supuran, C.T. (2017b). Synthesis, carbonic anhydrase I and II inhibition studies of the 1,3,5-trisubstituted-pyrazolines, J Enzym Inhib Med Chem, 32:1, 189-192.
35. Gülçin, I., Scozzafava, A., Supuran, C.T, Akıncıoğlu, A., Köksal, Z., Türkan, F., Alwasel, S. (2016). Rosmarinic acid inhibits some metabolic enzymes including glutathione S-transferase, lactoperoxidase, acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase isoenzymes. Journal of Enzyme Inhibition and Medicinal Chemistry, 31: 1698-1702
36. Habtemariam, S. (2019). Natural Products in Alzheimer’s Disease Therapy: Would Old Therapeutic Approaches Fix the Broken Promise of Modern Medicines?. Molecules, 24, 1519;
37. Hederos, C.A., Berg, A. (1996). Epogam evening primrose oil treatment in atopic dermatitis and asthma. Arch Dis Child, 75: 494-497.
38. Hostettmann, K., Borloz, A., Urbain, A., and Marston, A. (2006). Natural Product Inhibitors of Acetylcholinesterase, Current Organic Chemistry, 10, 825-847
39. Hyson, D.A., Schneeman, B.O., Davis, P.A. (2002). Almonds and almond oil have similar effects on plasma lipids and LDL oxidation in healthy men and women. J Nutr, 132(4):703–7. 8.
40. Ismail, M.F., EL-Maraghy, S.A., Sadik, N.A.H. (2008). Study of the immunomodulatory and anti- inflammatory effects of evening primrose oil in adjuvant arthritis. AJBR, 2: 74- 80.
41. Jenkins, D.J., Kendall, C.W., Josse, A.R., Salvatore, S., Brighenti, F., Augustin, L.S. (2006). Almonds decrease postprandial glycemia, insulinemia, and oxidative damage in healthy individuals. J. Nutr, 136, 2987–2992.
42. Jenkins, D.J., Kendall, C.W., Marchie, A., Parker, T.L., Connelly, P.W., Qian, W., et al. (2002). Dose response of almonds on coronary heart disease risk factors: blood lipids, oxidized low-density lipoproteins, lipoprotein a, homocysteine, and pulmonary nitric oxide: a randomized, controlled, crossover trial. Circulation, 106(11):1327–32. 9.
43. Jennifer, M., Moody, W., Heywood, J.S. (2001). Pollination limitation to reproductive success in the missouri evening primrose, Oenothera Macrocarpa (Onagraceae). Am J Bot; 88: 1615-1622.
44. Kaya, Z. (2010). Arsenı̇kle Lı̇pı̇d Peroksı̇dasyon Oluşturulan Ratlarda Çuha Çı̇çeğı̇ Yağının Etkı̇lerı̇ Farmakoloji-Toksikoloji Anabilim Dalı, Yüksek Lisans Tezi, Kayserı̇ Ercı̇yes Ünı̇versı̇tesı̇, Sağlık Bı̇lı̇mlerı̇ Enstı̇tüsü, 45p, Kayseri
45. Kocyigit, U.M., Budak Y., Gürdere, M.G.,·Dürü, N., Taslimi, P.,· Gülçin, İ., Ceylan, M. (2019). Synthesis and investigation of anticancer, antibacterial activities and carbonic anhydrase, acetylcholinesterase inhibition profiles of novel ( 3a R, 4S ,7 R ,7a S )‑2 ‑[4 ‑[1 ‑ac ety l‑5 ‑(a ryl /he ter oar yl) ‑4, 5‑ d ihy dro ‑1 H‑ pyr azo l‑3 ‑yl ]ph eny l]‑ 3a, 4,7 ,7a ‑te tra hyd ro‑ 1H ‑4,7‑ methanoisoindole‑1,3(2H)‑diones, Monatsh. Chem. 150:721-731.
46. Krasavin, M., Korsakov, M., Zvonaryova, Z., Semyonychev, E., Tuccinardi, T., Kalinin, S., Tanç, M., Supuran, C.T. (2017). Human carbonic anhydrase inhibitory profile of mono- and bis-sulfonamides synthesized via a direct sulfochlorination of 3- and 4-(hetero)arylisoxazol-5-amine scaffold Bioorg Med Chem 25(6):1914-1925
47. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–5. Lin, G., Tsai, Y.C., Liu, H.C., Liao, W.C., Chang, C.H. (1998). Chang Enantiomeric inhibitors of cholesterol esterase and acetylcholinesterase, Biochimica et Biophysica Acta, 1388 161-174
48. Lee, S., Lee, D., Baek, J Jung, E.B., Baekb, J.Y., Leec, I.K., Jangd, T.S., Kangb, K.S., Kima K.H. (2017). In Vitro Assessment Of Selected Korean Plants For Antioxidant And Antiacetylcholinesterase Activities, Pharmaceutical Biology, VOL. 55, NO. 1, 2205–2210
49. Mills, C., Cleary, B. V., Walsh, J.J., Gilmer, J.F. (2004). Inhibition of acetylcholinesterase by Tea Tree oil. Journal of Pharmacy and Pharmacology, 56:3, 375–379
50. Miyazawa, M., Yoshio, K., Ishikawa, Y., Kameoka, H. (1998). Insecticidal Alkaloids against Drosophila melanogaster from Nuphar japonicum DC. J. Agric. Food Chem. 46, 1059−1063.
51. NTP. (2009). İnformation review document for evening primrose oil (Oenotherabiensis L.), CAS No: 90028-66-3, 1-37. Oluba, O., Adeyemi, O., Ojieh, G., Isiosio, I. (2007). Fatty acid composition of Citrullus lanatus (Egusi melon) oil and its effect on serum lipids and some serum enzymes. The Internet Journal of Cardiovascular Research, 5(2): 1-7.
52. Perry, E.K., Gibson, P.H., Blessed, G., Perry, R.H., Tomlinson, B.E. (1977). Neurotransmitter enzyme abnormalities in senile dementia: Choline acetyltransferase and glutamic acid decarboxylase activities in necropsy brain tissue, Journal of the Neurological Sciences, 34:2, 247-265.
53. Perry, N., Court, G., Bıdet, N., Court, J., Perry, E. (1996). European Herbs with Cholinergic Activities: Potential in Dementia Therapy, International Journal of Geriatric Psychiatry, 11: 1063-1069
54. Perry, N.S.L., Houghton, P.J., Theobald, A., Jenner, P., Perry, E.K. (2000). In-Vitro Inhibition of Human Erythrocyte Acetylcholinesterase Bysalvia Lavandulaef oli essential Oil and Constituent Terpenes. Journal of Pharmacy and Pharmacology, 52(7), 895–902.
55. Polat, K.L., Gülçin, I., Gören, A.C., Namiesnik, J., Martinez-Ayala, A.L., Gorinstein, S. (2015). LC–MS/MS analysis, antioxidant and anticholinergic properties of galanga (Alpinia officinarum Hance) rhizomes. Ind Crops Prod, 74: 712-721
56. Quinn, D.M. (1987). Acetylcholinesterase: Enzyme Structure, Reaction Dynamics, and Virtual Transition States, Ct”. Rev. 87. 955-979.
57. Rahman, A., Choudhary, M.I. (2001). Bioactive natural products as a potential source of new pharmacophores. A theory of memory, Pure Appl. Chem., 73:3, pp. 555–560,
58. Riaz, A., Khan, R.A., Ahmed, S.P. (2009). Assessment of anticoagulant effect of evening primrose oil. Pak J Pharm Sci, 22: 355-359.
59. Ronceroa, J.M., Álvarez-Ortí, M., Pardo-Giménez, A., Gómez, R., Rabadán, A., Pardo, J.E., (2016). Virgin almond oil: Extraction methods and composition. Grasas Aceites, 67:(3): e143.
60. Sacan, O., Yildiz, E. (2014). Turhan, lipoxygenase inhibitory activities of some plant extracts and chemical compounds. The Journal of Biological Chemistry, 73(2): 47-52.
61. Santos, T.C., Gomes, T.M., Pinto, B.A.S., Camara, A.L., Andrade Paes, A.M. (2018). Naturally Occurring Acetylcholinesterase Inhibitors and Their Potential Use for Alzheimer’s Disease Therapy, Frontiers in Pharmacology, 9, 1192
62. Schumock, G.T. (1998). Economic considerations in the treatment and management of Alzheimer’s disease. Am J Health-Syst Pharm. 55(suppl 2):S17-21.
63. Scozzafava, A., Mastrolorenzo, A., Supuran, C. T. (2006). Carbonic anhydrase inhibitors and activators and their use in therapy. – Expert Opinion on Therapeutic Patents 16(12): 1627-1664.
64. Selkoe, D.J. (1992). Aging brain, aging mind. Sci Am. 267(3):134-42.
65. Sevim, O. (2018): Determination of some macro and micro element contents of multi-medical plants growed in agri and the effects of these elements on metabolic enzyme. Ağrı İbrahim Çeçen Üniversitesi, Fen Bilimleri Enstitüsü, Kimya Anabilim Dalı, Yüksek Lisans Tezi, Ağrı, Türkiye.
66. Sharma, K. (2019). Cholinesterase inhibitors as Alzheimer's therapeutics (Review), Molecular Medicine REPORTS 20: 1479-1487. Smirnovienė, J., Smirnovas, V., Matulis, D. (2017). Picomolar inhibitors of carbonic anhydrase: Importance of inhibition and binding assays. Anal Biochem, 522:61-72.
67. Spiller, G.A., Jenkins, D.A., Bosello, O., Gates, J.E., Cragen, L., Bruce, B. (1998). Nuts and plasma lipids: an almond-based diet lowers LDL-C while preserving HDL-C. J. Am. Coll Nutr. 17, 285–290.
68. Sultana, Y., Kohli, K., Athar, M., Khar, R.K., Aqil, M. (2007). Effect of pre‐treatment of almond oil on ultraviolet B–induced cutaneous photoaging in mice. Journal of cosmetic dermatology, 6(1), 14-19. 10.
69. Supuran, C.T. (2007). Curr Top Med Chem 7:825
70. Supuran, C.T. (2008). Carbonic anhydrases as drug targets. Current Pharmaceutical Design, 14(7): 601-2.
71. Tarawneh, R., Holtzman, D. M. (2012). The clinical problem of symptomatic Alzheimer disease and mild cognitive impairment. Cold SpringHarbor Perspect. Med. 2:a006148.
72. Taslimi, P., Gülçin, I., Özgeriş, B., Göksu, S., Tümer, F., Alwasel, S.H., Supuran, C.T. (2016b). The human carbonic anhydrase isoenzymes I and II (hCA I and II) inhibition effects of trimethoxyindane derivatives, J Enzym Inhib Med Chem, 31:4, 152-157.
73. Taslimi, P., Gülçin, I., Öztaşkın, N., Çetinkaya, Y., Göksu, S., Alwasel, S.H., Supuran, C.T. (2016a). The effects of some bromophenols on human carbonic anhydrase isoenzymes, J Enzym Inhib Med Chem, 31:4- 603-607.
74. Topal, F., Gulcin, I., Dastan, A., Guney, M. (2017). Novel eugenol derivatives: Potent acetylcholinesterase and carbonic anhydrase inhibitors. Int J Biol Macromol, 94, 845-851.
75. Verpoorte, J., Mehta, S., Edsall, J.T. (1967). Esterase activities of human carbonic anhydrases B and C. The Journal of Biological Chemistry, 242, 4221–4229,
76. Vullo, D., Del Prete, S., Nocentini, A., Osman, S.M., Al Othman, Z., Capasso, C., Bozdag, M., Carta, F., Gratteri, P., Supuran, C.T. (2017). Dithiocarbamates effectively inhibit the β-carbonic anhydrase from the dandruff-producing fungus Malassezia globosa, Bioorg Med Chem 25:3; 1260-1265
77. Whitehouse, P.J., Price, D.L., Struble, R.G., Clark, A.W., Coyle, J.T., Delon, M.R. (1982). Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain, Science 215 (4537), 1237-1239
78. Wiśniewski, H.M., Narang, H.K., Terry, R.D. (1976). Neurofibrillary tangles of paired helical filaments. J Neurol Sci., 27(2):173-81.
79. Yurtvermez, B. (2016). Isolation of bioactive secondary metabolites from tarragon (Artemisia dracunculus L.) and identification of their chemical structures. Ağrı İbrahim Çeçen Üniversitesi, Fen Bilimleri Enstitüsü, Kimya Anabilim Dalı, Yüksek Lisans Tezi, Ağrı, Türkiye.
80. Zarotsky, V., Sramek, J.J., Cutler, N.R. (2003). Galantamine hydrobromide: An agent for Alzheimer’s disease, Am J Health-Syst Pharm-Vol 60:1,
81. Zohary, D., Hopf, M. (2001). Domestication of plants in the old world. 3rd ed. London: Oxford University Press; 2000. p. 186. 6.