Anadolu adaçayı türlerinin vitamin-b2 içeriklerinin kapiler elektroforez lazer indüklenmiş florasans dedektör ile saptanması
Determination of ri̇boflavin content in anatolian sage samples using capillary electrophoresis laser induced fluorescence detector
Yazar: GÖRKEM GEZEK
Danışman: PROF. DR. FATMA BEDİA ERİM BERKER
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı
Derece: Yüksek Lisans, 2016
Konu: Kimya = Chemistry

Özet:
Lamiaceae familyasının bir cinsi olan Salvia L. ya da ülkemizde kullanılan ismiyle adaçayıdünya genelinde yaygın olarak 900 civarında çeşide sahiptir. Eski çağlarda geleneksel tıpta geniş kullanım alanları olmasının yanısıra süs ve tat verici amaçla da kullanılmıştır. Adaçayı bitkisinin içeriklerinin çok sayıda biyoaktif özelliği olduğu bilimsel çalışmalarla gösterilmiştir. Bitkininen güçlü antioksidan içerikleri karnasol, karnosik asit ve rosmarinik asittir. Ayrıca adaçayı türleri çok sayıda bioaktif flavonoid ve terpenoidler içermektedir. Bitkinin farklı bileşenleri antioksidan, anti tümörijenik, anti-enflamatuar, antimiktobiyel özelliktedir. Adaçayının yetiştiği bölgelere göre kimyasal içeriklerinin biyojenik aktivitesi çeşitlenebilir. 
Riboflavin, vitamin B2 olarak bilinen ışığa karşı duyarlı ve suda çözünebilen bir vitamindir. Riboflavin insan metabolizmasında önemli bir yere sahiptir. Flavoenzimlerin yapısında kullanılan öncü bir bileşendir aynı zamanda proteinlerin ve yağ asitlerinin enzimatik proseslerinde de son derece önemli bir yere sahiptir. Ayrıca önemli koenzimler olan flavin mononükleotid (FMN) ve flavin adenin dinükleotid (FAD) yapılarının oluşturulmasında başlıca ihtiyaç duyulan parametredir. Metabolizmada önemli roller üstlenmesine rağmen riboflavin insan vücudunda sentezlenemez ve dışardan alınan besinler aracılığı ile vücuda sağlanması gerekir ancak vitamin B2 kaynağı olan birçok gıda ürününün vitamin içeriği düşüktür. 
Son yıllardaliteratürde kapiler elektroforezin tıbbi bitkilerin analizlerine uygulandığıgörülmektedir. Kapiler elektroforetik yöntemlerin klasik kromatografik yöntemleregöre avantajı reaktif tüketiminde, örnek hazırlama ve analiz süresinde belirgin azalmadır. Tüketilen örnek hacmi her injeksiyonda birkaç nanolitre olduğu için son derece azdır. CE yüksek ayırma gücü nedeniyle özellikle kompleks yapıdakidoğal matriksler içindeki biyoaktif bileşenlerin analizi için uygun bir yöntemdir.
Bu çalışmada 10'u endemik olan, 13 farklı adaçayı türünün riboflavin içeriği kapiler elektroforez yöntemi ve lazer indüklenmiş florasans dedektör (CE-LIF) yardımıyla ilk kez tespit edilmiştir. Geliştirilen yöntem için optimumayırma koşulları, 20 mM borat tamponu pH:9,5 ve 30kV voltaj, optimum enjeksiyon şartı ise 50 mbar da 6sn sürelihidrodinamik enjeksiyon olarak belirlenmiştir. 
Riboflavinin doğrusal kalibrasyon aralığı, 0,998 regrasyon ile 0,05-1,00 µg/mL arasındadır. Yöntemin en düşük dedeksiyon sınırı (LOD)8,00 ng/mL olarak tespit edilmiştir.
Yapılan literatür araştırmaları sonucu adaçayı örneklerinde bugüne kadar riboflavin tayini yapılmadığı görülmüştür ve böylece literatüre önemli bir katkı sağlanmıştır. Validasyonu yapılan yöntem, 13 farklı adaçayının riboflavin miktarı tayinine başarılı bir şekilde uygulanmıştır.

Abstract:
Salvia is the genus of Lamiaceae family, which is commonly called as Sage. In Turkey, sage is known as 'adaçayı' and is commonly consumed as teas, which are also used in Anatolian folk medicine. Sage genus subtends 900 species widespread around the world and from ancient centuries various species of sage have been used for ornamental and flavouring purposes besides their wide use in folk medicine. Essential oil of sage leaves also have gained reputation for several health beneficial effects, including antitumorigenic activity, antioxidant capacity, antimicrobial and anti-inflammatory properties. As the main antioxidant ingredients, carnosol, carnosic acid and rosmarinic acid were considered as the responsible agents for healing properties of sage plant. Sage also contains many bioactive flavonoids and terpenoids. It is important to know that how various environmental conditions may diversify the chemical composition and biological activity of salvia species.
The name of riboflavin is used for vitamin B2 due to its ribose and flavin moieties. Riboflavin (vitamin B2) is a photo sensitive and water soluble micronutrient with native fluorescent. It is considered as one of the most crucial substances for flavoenzymes in the human body and also has an important role in proteins and fatty acids enzymatic processes. Moreover, riboflavin is the exclusive source of flavin adenine dinucleotide (FAD) and flavin mononucleotides (FMN) coenzymes in cell. Although riboflavin has a crucial role in human metabolism but human body doesn't have the ability to synthesize it and must provide its riboflavin need by taking from dietary sources like eggs, liver, cheese, cereals and plants. Considering the importance of riboflavin source foods for human body, determination of riboflavin in different types of nutrients is valuable. Generally, foods with riboflavin content have a complex matrix with trace amounts of riboflavin and this brings the necessity of utilizing very accurate and sensitive methods of determination.
There are considerable amounts of studies in literature, on determination of riboflavin contents of different kinds of food stuffs. Various determination techniques especially including chromatographic techniques have been used for selective separation and determination of riboflavin in different matrices. Capillary electrophoresis as one of the recently introduced separation techniques, has gained a great popularity in analysis of medicinal plants in last decades. Capillary electrophoresis has considerable superiorities on previous chromatographic techniques including low cost, low sample and reagent consumption, high resolution, ease of automation and less analysing time. Capillary electrophoresis with strong ability of separation comes as one of the first candidatesfor analysis in complex matrices.
Capillary electrophoresis is a chromatographic separation techniques which was introduced as the simple, rapid, inexpensive and compatible with green chemistry for separation of wide spectrum of components including smallest organic anions to biggest molecules like DNA under high electrical field which was held by applying voltage between to electrode. Analytes in the form of ions migrate through electrolyte solutions in electrical field andcan be separated according to ionic mobility and/or partitioning into an alternate phase via non-covalent interactions.Capillary electrophoresis (CE) has a high separation power. Thus, it is especially suitable for complex matrices. Several modes of capillary electrophoresis are existing which according to the type of matrix and analytes, researchers decide to use one of them. Capillary zone electrophoresis (CZE) is one of the most useful modes of CE. In CZE, the separation is based on differences in electrophoretic mobilities of the separated ions. In capillary zone electrophoresis all ions are introduced to the column from anodic side. Analytes or ions are carried to the cathode electrode where the detector was placed near it, regardless of their charge by strong electroosmotic flow (EOF). In this study we used capillary zone electrophoresis as separation mode for riboflavin determination.
Our study includes very simple, rapid and inexpensive method for determining riboflavin amounts in 13 salvia species from various heights of Turkey which 10 of them are endemic for Turkey. Capillary zone electrophoresis coupled with laser induced fluorescence used for sensitive determination of riboflavin in salvia species. Dried samples of salvia were separated and cleaned from unwanted stalks and wooden parts and then well grinded with mortar and sieved through 500 µm mesh size. Samples are stored at cold and dry place. 100 mg of sample was extracted into 10 ml of boiling water and incubated for 5 minutes, then extract was passed through a 45 µm pore sized microfilters and after appropriate dilution with water injected to the CE-LIF system.
Agilent 1600 capillary electrophoresis system (Waldbronn, Germany) equipped with a ZETALIF 2000 laser-induced fluorescence detector (Picometrics, Montlaur, France) was used for the determination of riboflavin in salvia samples. The excitation was made by a 488 Ar-ion laser and emission at 520 nm.
The fused silica capillary with effective length of 50 cm and 75 µm I.D. with the total length of 65cm was used for separation. 
Optimum condition for separation was obtained first by choosing background electrolyte. Considering the basic pKa= 9,6 for riboflavin, at borate buffer, riboflavin turns to its negative form and can migrate easily and fast through anode. Considering the aforementioned information, we tested borate buffer as the first candidate for background electrolyte and found that in borate natural pH value there is not any interfering compound signal for riboflavin.  Borate buffer with its natural pH= 9,5 value was used as back ground electrolyte. Different concentrations of borate were tested at 5, 10, 15 and 20 mM and and desirable signals in terms of symmetry and height was obtained at 20 mM borate as background electrolyte. Sample solutions were injected hydrodynamically at 50 mbar for 6 s and voltage was set at 30 kV. Temperature was 25 Celsius.
The linearity of the method for the riboflavin standards were between 0,05-1 µg/mL. The repeatability was calculated for standards of riboflavin by consecutive injections in the same day (n = 7) and in three different non-consecutive days (n = 21). The intraday RSD of riboflavin according to corrected peak areas was 2,19% and the inter-day RSD was 3.04%. The limit of detection (LOD) as three times of the average noise taken for three different baseline areas was 8 ng/mL. Consequently, 10 times the average signal to noise ratio was taken as the limit of quantification (LOQ), and it was 27 ng/mL. Riboflavin amount for various species of salvia samples from different heights of Turkey were between 1,65-11,90 µg/g. The lowest riboflavin content was belonging to the Salvia aucheri var. aucheri and Salvia hypergia with 1,65 and 1,85 µg/g respectively. Moreover, highest concentration of riboflavin was detected in Salvia limbata and Salvia sclarea with 11,87 and 11,90 µg/g respectively. Our results show that salvia can be considered as one of the rich sources for riboflavin.