Zeytin yaprağının önemli biyoaktif bileşeni oleuropeinin kapiler elektroforez yöntemiyle tayini
Determination of the major bioactive component oleuropein in olive leaf by capillary electrophoresis
Yazar: MERVE KOPAR
Danışman: PROF. DR. FATMA BEDİA BERKER
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı / Kimya Bilim Dalı
Derece: Yüksek Lisans, 2017
Konu: Kimya = Chemistry

Özet:
Son yıllarda gerek alınan gıdalardaki katkı maddeleri, gerek stres, gerekse çevresel faktörlerin olumsuzluğu başta metropollerde yaşayanlar olmak üzere insanların bağışıklık sistemini zayıflatmaktadır. Direnç düşmekte hastalık patojenlerine hassas hale gelinmektedir. Günlük yaşamda bazen farkına da varılamayan bu durumlarda insanlar, gıda takviyeleri veya çeşitli bitkisel çaylarla vücut dirençlerini muhafaza etme yoluna gitmektedirler. Buna bağlı olarak analitik kimyagerler doğal bitki bileşenlerini yoğun bir şekilde araştırmakta, alternatif tayin ve saflaştırma yöntemleri üzerinde durmaktadırlar.
Bu anlamda tarih boyunca farklı medeniyetlerin sıklıkla şifa amaçlı kullandığı zeytin yaprağı da bu bitkiler arasında çok büyük bir önem taşımaktadır. Nitekim zeytin dalı ve yaprağının çeşitli kültürlerde mistik anlamları da bulunmaktadır. Neredeyse tüm dünyada barışın da simgesi olarak bilinen zeytin dalı da farklı amaçlar için simya döneminden günümüze kadar kullanılmıştır.
Zeytin yaprağı pek çok fenolik bileşenler içermesiyle birlikte bunların içinde özellikle başlıca aktif bileşen olan oleuropein ve faydaları oldukça dikkat çekmektedir. Simya döneminden bu yana zeytin yaprağının sudaki özütlemesinden elde edilen çay şifa amaçlı tüketilmiş fakat dönemin koşulları itibariyle içeriğindeki bileşenlere vakıf olunamamıştır. Bugunkü şartlarda zeytin yaprağındaki bileşenler bilinmekte ve çeşitli rahatsızlıklar üzerindeki aktiviteleri incelenmektedir.
Bu bileşenlerden en önemlilerinden biri olan oleuropein polifenol grubu bir bileşik olup, önemli bir antioksidan aktiviteye de sahiptir. Bunun yanı sıra kanser çeşitleri üzerine oleuropein aktivitesi ile ilgili pek çok çalışma yapılmış olup, son derece olumlu veriler elde edildiği bildirilmiştir. Ayrıca Alzheimer hastalığı ve Tip-1 diyabet üzerine de etkisi literatürde araştırılmış ve tezde yer verilmiştir. Günlük yaşamda da sıklıkla edinilen viral ve bakteriyel patojenler üzerinde yapılan oleuropein ile ilgili çalışmalar bulunmakta olup bunların deteylarına ilerleyen bölümlerde yer verilmiştir.
Kapiler elektroforez (CE) gerek kısa analiz süresi ve yüksek ayırma etkinliği gerekse düşük miktarda numune ile çalışılması gibi pek çok yönden kolaylık sağlayan bir yöntem olup, az miktarda çözücü tüketimini sağlaması kolaylığı ile de nispeten çevre dostu bir yöntem olarak da düşünülmektedir.
Bu çalışmada zeytin yaprağındaki oleuropein bileşiğinin kapiler elektroforez ile tayini üzerine bir metot geliştirilmiştir. Kullanılan tampon karışımı; borat çözeltisi (pH=9,6), metanol ve 2-hidroksipropil beta siklodekstrin (2-HP-β-CD) çözeltisi ihtiva etmektedir. Ayrıca çalışmada pek çok ekstraksiyon yöntemi denenmiş ve optimum koşullar %80 metanol-%20 su ekstraktı ile sağlanmıştır. Sırasıyla Balıkesir, Bursa, İstanbul, İzmir, Manisa Muğla ve Yalova illerinden toplanan zeytin yaprağı numunelerinin yanında üç adet de ticari zeytin yaprağı çayı örnekleri incelenmiştir.
Örneklerin hepsi önceden kurutulmuş, akabinde öğütülmüş ve ekstraksiyon solventi içinde bir saat boyunca ultrasonik banyoda ekstrakte edilip süzülmüştür. Süzüntüler, geliştirilen analiz yöntemi şartlarında CE cihazına enjekte edilerek, zeytin yaprağı içerğindeki oleuropein aktif maddesinin kantitatif miktarları tayin edilmiştir.
Kalibrasyon eğrisinin doğrusal konsantrasyon aralığı 50 ppm ile 1000 ppm arasında çizilmiş ve doğrunun regrasyon değeri 0.996 olarak saptanmıştır. Yöntemin gün içi ve günler arası alan tekrarlanırlığı düzeltilmiş alana göre sırasıyla % 2,11 ve % 5,23 RSD olarak saptanmıştır. Metodun zeytin yaprağı ekstraktlarında oleuropein için geri kazanım değerleri %106 ile 117 arasında bulunmuştur.
Bu tez çalışmasında, kapiler elektroforezle zeytin yaprağından oleuropein tayini için geliştirilen yöntem farklı zeytin yaprağı örneklerine başarıyla uygulanmış olup; kısa analiz süresi, iyi bir tekrarlanırlık ve yüksek geri kazanım elde edilmiştir

Abstract:
In recent years, additive foods, stress and environmental factors have weakened the immune system of humans, particularly those living in metropolitan areas. Resistance is becoming more susceptible to pathogenic diseases. In these situations where everyday life is sometimes unrecognizable, people are trying to maintain their body resistance with food supplements or various herbal teas. Accordingly, analytical chemists are intensively investigating natural plant components, focusing on alternative detection and purification methods.
In this sense, the olive leaf used by different civilizations throughout the history for healing purposes is of great importance among these plants. As a matter of fact, olive branch and leaf have mystical meanings in various cultures. The olive branch, known as the symbol of peace in almost the entire world, has also been used until today from alchemy epoch for different purposes.
Although olive leaf contains many phenolic compounds, the major active compound oleuropein and its beneficial effects are particularly noteworthy. Since the time of alchemy, the tea obtained from the extract of olive leaf in water has been consumed for healing purposes, but the components in its have not been found in terms of the period. Nowadays components of olive leaf are known and their activities on various diseases are examined.
The olive tree (Olea Europaea) is known as one of the most durable trees in the world. The chemical composition of olive leaves may vary depending on the different parameters. These parameters are, the type of the tree, the climate that tree grows, the locality and the ratio of branches to tree.
Olive leaf generally contains essential oils, tannins, resins and organic acids. Olive leaf is very rich in terms of phenolic compounds. Some of the phenolic compounds found in the olive leaf are tyrosol, hydroxytyrosol, verbascosite, lutein-7-glucoside and oleuropein. Oleuropein and hydroxytyrosol are phenolic compounds naturally found in olive and also oleuropein is found in abundant amounts in untreated olive and leaves while hydroxytyrosol is abundant in processed olive and olive oil.
Oleuropein is found on the highest level of olive leaf, although it is present in all parts of the olive fruit. This compound is responsible for the bitterness of immature and unprocessed olives. As it known, olives cannot be consumed without being subjected to any treatments after the hazel because of its bitter taste. In order to remove this taste oleuropein from the medium, processes such as brine, alkali treatment, enzymatic method or microorganisms treatment are applied.
Oleuropein has many pharmacological properties researched to date. These are antioxidative, antimicrobial, antiviral, anti-inflammatory, anticancer, neuroprotective cardioprotective and hypolipidemic properties.

The anticancer studies of oleuropein have been based on cancer types such as breast cancer, colon cancer, liver cancer, prostate cancer, bladder cancer and the activity on various cancer cells has been reported.
When we examined the effects of oleuropein in literature on cardiovascular system it has been reported that studies have shown that the compound is a protective effect against acute antibiotic cardiotoxicity. It has also been reported that oleuropein is an antiatherosclerotic effect in studies conducted.
Alzheimer's is an irreversible and progressive disease. It is thought that the pathogenesis of Alzheimer's disease is caused by the production and accumulation of β-amyloid peptide (Aβ). In one study, oleuropein was reported to reduce or even prevenets Aβ aggregation which is inherent to Alzheimer's disease.
When we search the literature, we see that different methods of analysis oleuropein have been done. We know that there is some work done with HPLC for the determination of oleuropein from olive leaf. In addition various electrochemical methods have been developed for oleuropein determination. When we look at literatures about oleuropein determination by capillary electrophoresis, the analysis made from olive oil are often striking. As a result we could not come across a developed method for oleuropein determination from olive leaf by capillary electrophoresis.
In this thesis, a method was developed by capillary electrophoresis for the determination of oleuropein in olive leaf. The method was optimized for the appropriate extraction method after buffer optimization. Chemicals used in the study are ethanol, methanol, 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD), sodium tetraborate decahydrate and sodium hydroxide. In all solutions, pure water obtained with Elga Purelab Option brand purified water device was used. In the study olive leaf samples from Balıkesir, Bursa, Istanbul, Izmir, Manisa, Mugla and Yalova regions were used and three different brand commercial olive leaf tea samples were examined.
Olive leaf samples were dried and then grinded. 0.1 g were taken from samples and added to 10 mL 80% methanol (20% water). In vortex device, the samples were mixed for 2 min. at 2000 rpm and then extracted for 1 hour. After extraction, the filration was carried out using Sartorious RC 0.45 μm filter and samples were made ready.
Agilent 1600 capillary electrophoresis device and diode array detector were used in study. The detections were made at 240 nm and were operated with a voltage of 28 kV. The injection time is set to 6 seconds and the injection pressure to 50 mbar. The temperature is set to 25 ºC. Silica capillary columns are used and the column inner diameter is 50 μm. The silica capillary column was washed with 1M NaOH solution for 30 minutes and then 10 minutes with pure water for the first time. At the beginning of the day, the column was washed with a 1M NaOH solution for 10 minutes, followed by 5 minutes with pure water and 5 minutes with buffer solution respectively. In addition intermediate washes were performed prior to injection of each sample.
100mM stock solution was prepared from sodium tetraborate decahydrate as the buffer solution to be worked up. The optimum concentration of borate buffer was determined by diluting the prepared solution. The optimal solution in the prepared buffers of 20, 25, 30 and 40 mM respectively was determined as 30 mM borate buffer. Buffer additives were made to separate more effectively from the peaks next to the oleuropein. For this, 5 mM β-CD solution was added to the buffer but the desired separation was not achieved. Subsequently, a study was made with 2-HP-β-CD. For this, 2-HP-β-CD solutions at 20, 25, 30 mM concentrations were added respectively to the borate buffer at a concentration of 30 mM. Among them 2-HP-β-CD at 25 mM concentration was chosen as the most suitable solution. Finally 10% methanol solution added to the buffer mixture for a better separation of peaks. The buffer to be worked up was identified as a mixture of 30 mM borate (pH=9.6), 25 mM 2-HP-β-CD and 10% methanol.
In this study, methanol, ethanol solutions at different concentrations and distilled water used to select the most suitable extraction method. Olive leaf samples from Mugla province weighing 0.1 g each time were taken in 10 mL of 100% ethanol, 100% methanol, 80% ethanol, 80% methanol solutions respectively then mixed with vortex for 2 minutes at 2000 rpm and extracted in ultrasonic bath for 1hour. Furthermore, olive leaf samples of Mugla province weighing 0.1 g were taken to 10 mL pure water and mixed in vortex for 2 minutes at 2000 rpm and extracted for 1 and 2 hours respectively. After all extractions all samples were filtered and subsequently injected. Finally, 80% methanol solution was chosen as the most suitable extraction condition.
1000 ppm standard solution prepared with methanol was diluted and injected into the device by the selected buffer medium. Oleuropein peak was obtained before 6 minutes. Then the olive leaf extracts injected into the device respectively. It was seen that the oleuropein peak good separated on the electropherogram.
The calibration curve was prepared by diluting the preared 1000 ppm oleuropein standard solution. The oluropein standards taken at 1000, 500, 400, 250, 125, 100 and 50 ppm respectively, were injected into the device and the calibration curve was drawn. Accordingly, the calibration equation was found as y=0.00035849x-0.0040164 and the correlation value (r2) was determined as 0.996.
In order to determine the precision of the method, 500 ppm oleuropein standard solution injected into the device five times a day. For day-to-day repeatability, 500 ppm oleuropein standard solution injected into the device in three consecutive days, five times per day. The repeatability values were calculated according to corrected peak areas and arrival times of peaks. Intra-day and inter-day repeatability values were found as RSD 2,11 % and 5,23 % respectively according to the corrected peak areas. LOD (limit of detection) and LOQ (limit of quantification) values were calculated as 13,4 ppm and 44,8 ppm respectively.
In this thesis, a method was developed for the determination of oleuropein, the major bioactive component of olive leaf using capillary electrophoresis and using a small amount of sample and buffer with high separation efficiency. As mentioned before, oleuropein amounts were studied by taking samples from different trees growing in different climatic and soil conditions since the chemical composition of the olive leaf is dependent on many parameters. The developed method was successfully applied for the determination of oleuropein in olive leaf.