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Biyoaktif maddelerden berberin, ursolik asit ve oleanolik asidin bitkilerde tayini için yeni kromatografik yöntemler

Novel chromatographic methods for the determination of the bioactive materials in plants: Berberin, ursolic acid, and oleanolic acid

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  1. Tez No: 485326
  2. Yazar: SESİL ERKAN
  3. Danışmanlar: PROF. DR. FATMA BEDİA ERİM BERKER
  4. Tez Türü: Doktora
  5. Konular: Kimya, Chemistry
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 2017
  8. Dil: Türkçe
  9. Üniversite: İstanbul Teknik Üniversitesi
  10. Enstitü: Fen Bilimleri Enstitüsü
  11. Ana Bilim Dalı: Kimya Ana Bilim Dalı
  12. Bilim Dalı: Kimya Bilim Dalı
  13. Sayfa Sayısı: 70

Özet

Bitkiler yapısal olarak birbirinden farklı çok sayıda kimyasal bileşik içerirler. Tıbbi bitkiler, biyoaktif bileşenlerinden dolayı, potansiyel yeni ilaçların keşfinde önemli kaynaklardır. Birçok yeni biyoaktif bileşiğin yapısı bitkilerden izole edilerek aydınlatılmıştır. Farklı bitkilerden yeni bileşenlerin izole ve teşhis edilmesine ait rapor edilen çalışmalara kıyasla, biyoaktif maddelerin bitkilerdeki kantitatif miktarlarının aydınlatılması konusundaki çalışmalar oldukça azdır. Bitki ekstraktlarının matriksleri oldukça karmaşıktır ve spesifik bir maddenin bu matriks içinde tayini güçtür. Biyoaktif maddelerin, farklı bitkilerdeki ve bitkinin kök, yaprak, çiçek gibi farklı bölgelerindeki miktarları geniş bir spektruma yayılır. Bitkilerdeki biyoaktif maddelerin yararlı ve toksik etkilerinin kontrolü için, kantitatif miktarlarının bilinmesi ve bunun için de hassas analitik yöntemler gerekmektedir. Bu çalışmanın amacı, biyoaktif özellikleri ispatlanmış üç önemli bitki aktif maddesi olan berberin, oleonolik asit ve ursolik asidin, bulundukları bitki grubu içindeki miktarlarını aydınlatacak analitik ayırma ve analiz yöntemleri geliştirilmesi ve biyoaktif maddelerin miktarlarının raporlanmasıdır. Tez çalışmasının ilk bölümünde, berberin tayini için kapiler elektroforetik yeni bir analiz yöntemi geliştirilmiştir. Berberinin doğal floresans özelliğinden yararlanılmış ve floresans şiddetini arttıracak yönde çalışmalar yapılmıştır. Kapiler elektroforez (CE) yöntemi ile ayrılan berberin, CE cihazına adapte edilen lazer indüklenmiş floresans dedektörü ile hassas olarak tayin edilmiştir. Geliştirilen yöntem, berberin içerdiği bilinen ve bu amaçla ticari olarak satılan bir grup bitki ekstraktına uygulanmıştır. Yöntemin düşük tayin sınırı sayesinde, berberin içerikleri oldukça düşük olan bitkilerde kantitatif tayin gerçekleştirilmiştir. Bu çalışma Journal of Chromatography A dergisinde, (vol. 1338, 184–187, 2014) yayınlanmıştır. Tez çalışmasının ikinci bölümünde oleanolik asit ve ursolik asit izomerlerini bir arada tayin edecek yeni bir HPLC yöntemi geliştirilmiştir. Literatürde bu iki asidin bazı Salvia türlerinden izole edildiği konusunda çalışmalar mevcuttur. Adaçayı (Salvia), ballıbabagiller (Lamiaceae) ailesinden Salvia cinsini oluşturan kokulu bitkilere verilen addır. Yaklaşık olarak bilinen 900 türü ile ballıbabagiller (Lamiaceae) familyasının en geniş cinslerinden biridir. Ballıbabagiller ülkemizde 45 cins ve 546 tür ile temsil edilmektedir. Bu aileden 28 cins Türkiye'de yaygın olarak bulunmaktadır ve bunların 240 türü Türkiye için endemiktir. Adaçayı türleri dünyanın her yerinde tıbbi bitki olarak kullanılmaktadır. Geliştirilen HPLC yöntemi ile 14 adet Anadolu adaçayı türünün oleanolik asit ve ursolik asit içerikleri aydınlatılmıştır. Her iki asidin, önemli sayıda Adaçayı türündeki miktarlarının bir arada raporlandığı ilk çalışmadır. Çalışmanın sonuçları yayına hazırlanmaktadır.

Özet (Çeviri)

The plants contain a large number of chemical compounds that are structurally different from each other. Medical plants are important sources for the discovery of potential new drugs because of their bioactive components. The structure of many new bioactive compounds has been identified which are isolated from plants. The studies on the quantification of bioactive substances in plants are scarce compared to the reported studies on the isolation and identification of new compounds from different plants. The matrix of plant extracts is very complex, and it is difficult to identify the specific substance in this matrix. The contents of bioactive compounds of plants are spread over a wide spectrum due to their collected regional areas and also their parts like roots, leaves and flowers. For the control of the beneficial and toxic effects of bioactive substances in plants, it is necessary to know quantities of them. Therefore, scientists need sensitive analytical methods. The aim of this study was to develop analytical separation methods to quantify the amounts of berberine, oleonic acid and ursolic acid, three important plant active substances with proven bioactive properties. Various medicinal plants contain depending amounts of berberine which is an isoquinoline alkaloid. In recent years there has been growing interest on berberine due to its pharmaceutical activities. It is proven that berberine is effective on inflammation, infectious diseases, cardiovascular disorders, type 2 diabets, dyslipidemia and cancer. In parallel to the studies of pharmaceutical activities of berberine, interest has increased in medicinal plants and supplements containing berberine. Also, analytical studies on berberine sources, that shows wide spectrum due to plant species and plant regions, come into question. In recent years, HPLC and CE methods which are two main analytical methods, used to determine berberine amounts of plants. Berberine is a basic drug so it shows peak tailing disadvantage in HPLC analysis. But sharp peaks can be obtained with CE analysis when an acidic medium is used. However, the sensitivity of CE may not enough for low berberine contents in some plants. To overcome this difficulty, sample stacking methods have been developed for CE and MEKC analysis. However, sample stacking methods are complex and it is hard to work both in aqueous and non-aqueous media. The combination of capillary electrophoresis with laser induced fluorescent detector (CE-LIF) is very suitable for fluorescent substances or for substances which are derivatized with fluorescent dyes. In this way very low detection limits can be reached and analysis of plants with low berberine amounts can be carried out. Despite berberine is naturally fluorescent molecule, its fluorescence intensity is too low in aqueous media. In the first part of present study, a sensitive method for the analysis of berberine was developed using capillary electrophoresis coupled with LIF detector. To overcome the determination hurdle of berberine in aqueous medium, the fluorescence intensity of berberine molecule is enhanced by the addition of 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) to the separation electrolyte. The optimum separation electrolyte composition was selected as 20 mM acetic acid, 35 mM 2-HP-β-CD and 20% methanol at pH 5. According to the comparison of the detection limits of the molecule in the 2-HP-β-CD-free and 2-HP-β-CD containing separation medium, 162-fold enhancements in sensitivity were obtained by the use of 2-HP-β-CD in the buffer. The limit of detection of the method for berberine was 15.7 ng/mL. The intra and interday repeatabilities of corrected peak areas as RDSs were 1.83% and 3.75%, respectively. The developed method was successfully applied for the determination of berberine contents in eight Chinese medicinal plants and herbal supplemental tablets. The berberine contents of plants range over wide limits from 55.9 µg/g to 46.8 mg/g. The found berberine hydrochloride equivalents per tablet were compatible with the label. This study was published in Journal of Chromatography A, (1338, 184–187, 2014). Salvia is the name given to the scented plants in the Lamiaceae family. The genus is distributed throughout Mediterranean and Asia, with about 900 known species. Lamiaceae family is represented by 45 genera and 546 species in our country. There are 28 species from this family in Turkey and 240 species are endemic for Turkey. Traditionally, Salvia has been used in digestive problems, colds, ulcer pain and heart disease. Today's modern studies indicate that the Sage species have antioxidant, antidiabetic and antitumor effects. These properties can be attributed to the various active components which plants contain. Salvia species contain phenolic acids, essential oils, flavonoids, monoterpenes, diterpenes and triterpenes. Ursolic acid and oleanolic acid are pentacyclic triterpenes which exist in Salvia species. Recently, both acids gained huge attention due to their pharmaceutical effects. Pharmacological investigations have shown that ursolic acid and oleanolic acid have anti-inflammatory, antitumor and anti-microbial effects. These isomeric compounds protect the liver and have analgesic activity. It is also known that ursolic acid plays an important role in the treatment of ulcer and diabetes mellitus. Therefore, considering the side effects of synthetic drugs, the use of ursolic acid is an important alternative in the treatment of these diseases. Due to its pharmacological effects, in recent years, many scientists have been interested in developing sensitive, efficient and simple analytical techniques for the separation and identification of ursolic acid and oleanolic acid in herbs. Ursolic acid and oleanolic acid have hydrophobic structure due to pentacyclic hydrocarbon skeleton and their solubility in aqueous media is very low. These two molecules with identical molecular weights are the isomers of each other, and the position of only one methyl group in the chemical structure is different. Therefore, the structural similarity of ursolic acid and oleanolic acid caused some difficulties in the simultaneous identification of these triterpenic acids. The quantitative analysis of ursolic acid and oleanolic acid are present in previous studies by capillary electrophoresis, high performance liquid chromatography and gas chromatography. But the combination of oleanolic acid and ursolic acid isomers at the same time is still missing. In the second part of present study, a new HPLC method was developed to determine the combination of oleanolic acid and ursolic acid isomers in 14 Turkish Salvia species which 12 of them are endemic. The optimal mobile phase was selected as 85% acetonitrile solution. The limit of detection of the method for ursolic acid and oleanolic acid were 14 ng/L and 13 ng/L, respectively. The method showed good precision and accuracy with intra-day and inter-day variation of 0.54% and 7.33% for ursolic acid, intra-day and inter-day variation of 0.51% and 5.26% for oleanolic acid and overall recoveries of 97.8% - 98.5 for ursolic acid and oleanolic acid, respectively. Oleanolic and ursolic acid contents of 14 Salvia samples were analyzed by HPLC. All Salvia species contain both oleanolic and ursolic acid. Ursolic acid concentrations of Salvia extracts changed between 0.21 and 15.5 mg/g. Oleanolic acid concentrations of Salvia extracts changed between 0.05 and 12.7 mg/g. This study was ready to be published.

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