Propolis örneklerinin kafeik asit fenetil ester içeriklerinin tayini ve anti-inflamatuvar özelliklerinin incelenmesi
Determination of the caffeic acid phenethyl ester (CAPE) content of the propolis samples and investigation of their anti-inflammatory properties
- Tez No: 559905
- Danışmanlar: PROF. DR. FATMA BEDİA BERKER
- Tez Türü: Yüksek Lisans
- Konular: Kimya, Chemistry
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2019
- Dil: Türkçe
- Üniversite: İstanbul Teknik Üniversitesi
- Enstitü: Fen Bilimleri Enstitüsü
- Ana Bilim Dalı: Kimya Ana Bilim Dalı
- Bilim Dalı: Kimya Bilim Dalı
- Sayfa Sayısı: 71
Özet
Propolis, son yıllarda en az bal kadar önemli hale gelen bir arı ürünüdür. Uzun yıllardan beri geleneksel tıpta en çok antibakteriyel, anti-inflamatuvar ve rejeneratif amaçlarla çeşitli hastalıkların tedavisinde kullanılmaktadır. Bu etkilerin çoğunun propolisin aktif bileşenlerinden biri olan kafeik asit fenetil estere (CAPE) bağlı olduğu düşünülmektedir. CAPE'nin insan sağlığı üzerine çok geniş spektrumlu etkileri bulunmakta olup, antiseptik, antibakteriyal, anti-inflamatuvar, antioksidan, antimutajenik aktiviteleri bilimsel çalışmalarda kanıtlanmıştır. Bu tez çalışmasının birincil amacı, CAPE'nin tayini için hızlı ve etkin bir analiz yöntemi geliştirilmesi ve Türkiye'nin farklı bölgelerinden toplanmış propolis örnekleri ile ticari propolis boğaz spreylerinin CAPE miktarlarının aydınlatılmasıdır. Propolisin aktif bileşenlerinden CAPE'nin antioksidan, anti-inflamatuvar, antikanserojen ve antibakteriyel özelliklerinin keşfedilmesiyle birlikte, CAPE son senelerde birçok yeni araştırmanın konusunu oluşturmuştur. Dünyanın farklı bölgelerindeki propolislerin CAPE'yi de içeren kimyasal bileşenlerini ve bunların miktarlarını veren çalışmalar bulunmaktadır. Bu çalışmalarda genellikle sıvı ve gaz kromatografisi tekniklerine başvurulmuştur. Kapiler elektroforez yöntemi yüksek ayırma etkinliğine sahip olması, kısa analiz süresi ve az miktarda örnek ve çözücü kullanılması bakımından diğer yöntemlerden üstündür. Bu tez çalışmasında CAPE'nin tayini için, bir misel elektrokinetik kapiler kromatografi (MEKC) yöntemi kullanılmıştır. Yapılan deneyler sonucunda optimum ayrıma ortamı 40 mM N-siklohekzil-3-aminopropansülfonik asit (CAPS), 50 mM sodyum dodesil sülfat (SDS) ve 10 mM 2-hidroksipropil-β-siklodekstrin (2-HP-β-CD) (pH: 10,4) olarak belirlenmiştir. Yöntem başarılı bir şekilde valide edilmiştir. Ticari propolis sprey örneklerinin (n=4) CAPE miktarları 16,88 – 80,45 mg CAPE/100 mL aralığında bulunmuştur. Sivas ve Zonguldak propolislerinin CAPE miktarları ise sırasıyla 330,8 – 441,7 mg CAPE/100 g propolis olarak bulunmuştur. Çalışmanın ikinci basamağında ise CAPE miktarları tayin edilen propolis örneklerinin anti-inflamatuvar aktiviteleri incelenmiştir. İnflamasyon, doku fonksiyonu kaybıyla sonuçlanan olayları zincirinin bütünüdür. İnflamasyon sürecinde inflamasyon aktivatörü olan tümör nekroz faktör-α (TNF-α) gibi bir çok pro-inflamatuvar sitokinlerin makrofajlar tarafından salımı gerçekleşir. Bu çalışmada propolis örneklerinin anti-inflamatuvar aktiviteleri, TNF-α enziminin in-vitro inhibitör aktivitesine dayanarak ölçülmüş, ticari propolis spreylerinin inhibisyonu %55,35 - %83,92 aralığında bulunmuştur. Sivas ve Zonguldak propolisleri ise TNFα'yı sırasıyla %68,75 ve %66,07 oranında inhibe etmiştir. Son olarak örneklerdeki CAPE miktarları ile örneklerin anti-inflamatuvar aktivitelerinin korelasyonu araştırılmış ve 0,944 olarak bulunmuştur. Bu yüksek korelasyon, propolisin anti-inflamatuvar etkisi üzerinde CAPE'nin önemli bir rolü olduğunun göstergesidir.
Özet (Çeviri)
Propolis, a product collected by bees from diverse plants, protects bee habitats from insects and microorganisms, seal cracks and sterilizes their hives. The word“propolis”is of Greek origin,“pro”meaning“defense”and the“polis”meaning“city”. From here, the defense of the hive can be deduced. Propolis was discovered before the period of Christ. Aristotle who wanted to examine bees by using transparent hives realized that the transparency of the hive was closed by dark colored and viscous materials. These materials were thought to be propolis. It has been reported that propolis, traditionally used by the public since ancient times, has been used in the treatment of skin diseases, digestive system disorders and ulcers by Hippocrates. The most known feature of propolis is its effect against microorganisms. The beneficial properties of propolis have been utilized since its discovery and are still being used today. The biological activities of propolis, such as anti-inflammatory, antitumor, antiulcer, antimicrobial and antioxidant, have also become widespread in apitherapy, medicine, food and cosmetics. It is used in food and beverages because it increases body resistance and has antibiotic effects. It also has medicinal preparations against mouth and throat infections. Commercial propolis products such as tablets, lozenges, mouth spray, shampoo, toothpaste and creams are also available. In regards to the properties of propolis, the substances in the content of these products demonstrate differences in the construction of geographical regions and in the variety of bees. The studies are indicated that propolis contains more than 300 compounds. The chemical composition of propolis varies depending on the flora of the area it is collected in, the type of plants grown in that region, the climatic conditions of the region, the season and time of collection, the amount of resin in the bud, and the race of the bee and the content of the secreted material. Caffeic acid phenethyl ester (CAPE) is one of the most common substances among these compounds. CAPE has a great deal of pharmacological properties such as anti-inflammatory, anticarcinogenic, antimicrobial, antiseptic, and antioxidant properties. The research results of propolis and CAPE have shown that both properties have beneficial and extensive therapeutic effects. This discovery has increased the research of propolis and CAPE not only in Turkey, but around the world as well. Generally, chromatographic methods such as high performance liquid chromatography (HPLC), gas chromatography (GC) were used in these studies. Capillary electrophoresis (CE) is an analytical method which has high separation efficiency and short analysis time. It separates many different compounds from small ions to large molecules using small amounts of solvent and sample. Capillary electrophoresis consists of several different modes. In order to determine the CAPE in propolis content, micellar electrokinetic capillary chromatography (MEKC) method was applied. MEKC is used for the separation of neutral compounds with high efficiency and sensitivity. In the thesis study of CAPE content of the propolis samples, the working electrolyte composition was selected as 40 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 50 mM sodium dodecyl sulfate (SDS) and 10 mM 2-hydroxypropyl-βcyclodextrin (2HP-β-CD) at pH 10.4. The detection was performed by a UV-DAD detector at λ=230 nm. In this condition, the migration time was 6 minutes for CAPE. Validation studies of the applied methods were performed and successful results were obtained. The calibration equation was y = 3.478x – 11.17 with the correlation coefficient (R2) of 0.994. The precision of the method was performed in terms of inter-day and intra-day repeatability as RSD%. The standard CAPE solution, prepared at a concentration of 25 µg/mL, was injected into the capillary column seven times during the day. Repeatability was obtained by injecting 25 µg/mL CAPE solution seven times in the course of three consecutive days (n = 7x3). The intraday and inter-day repeatability values were obtained by calculating the relative standard deviation (%) of corrected peak area and the migration time. The accuracy of the method was examined by recovery analysis. Standard CAPE was added to the two propolis samples in three different concentrations. These concentrations were added as half of the amount, its own amount, and twice of the amount of CAPE detected in the propolis samples. The limit of detection (LOD) was calculated as 1.48 µg/mL and the limit of quantification (LOQ) was 4.93 µg/mL. The developed MEKC technique was applied for two propolis samples collected from Sivas and Zonguldak and four commercial propolis throat spray samples. Solid propolis samples were homogenized after collection. The sample of Sivas propolis was weighed at 10.14 g and extracted in a mixture of 140 mL of 98% ethanol - 2% water (v/v) (72.429 mg/L). 3.18 g of Zonguldak propolis was precisely weighed and extracted in a mixture of 63 mL of 98% ethanol - 2% water (v/v) (50.476 mg/L). The samples were filtered through 0.45 µm diameter microfilter. Propolis samples collected from Sivas and Zonguldak were diluted 25 times with methanol and the commercial propolis spray samples were diluted 50 times. They were then injected into the capillary column. CAPE amounts of the commercial propolis spray samples were between in the range of 16.88 – 80.45 mg CAPE/100 mL. CAPE levels of Sivas and Zonguldak propolis samples were found to be 330.8 mg and 441.7 mg CAPE/100g propolis, respectively. In the second part of this thesis study, anti-inflammatory activities of propolis samples were investigated. Inflammation is a chain of events that results in loss of tissue function. In the inflammation process, many pro-inflammatory cytokines, such as inflammatory activator tumor necrosis factor-α (TNF-α), are released by macrophages. In this study, anti-inflammatory activities of propolis samples were measured based on the in-vitro inhibitory activity of TNF-α enzyme. For this purpose, Human TNF alpha ELISA (Enzyme Linked-Immuno-Sorbent Assay) kit (Thermo Fisher Scientific, Waltham, MA) was used. Enzyme Linked-ImmunoSorbent Assay (ELISA) is an easy, fast and frequently used biochemical technique which detects the presence of antigen or antibody in a sample. It has four different types; sandwich (non-competitive), competitive, direct and indirect. The human TNF alpha ELISA assay performed in this thesis is based on the sandwich ELISA technique. In the study, antigen concentration in unknown samples is determined. The kit contains a 96-well microplate with a broken and detachable lane coated with a monoclonal antibody. After the materials in the kit are prepared in accordance with the procedure given by the manufacturer, they are pipetted into this microplate together with the samples of propolis. At the last step of the analysis, the absorbance of the wells was read at 450 nm. Absorbance measurements were performed using the BioTek Power Wave XS (Winooski, VT, USA) branded microplate reader and Gen5 Data Analysis software. Inhibition values of commercial propolis sprays were found between 55.35% and 83.92%. The propolis samples collected from Sivas and Zonguldak inhibited TNF-α by 68.75% and 66.07%, respectively. Finally, the correlation between the amount of CAPE in the samples and the anti-inflammatory activities of the samples was investigated and found to be 0.944. This high correlation indicates that CAPE has an important role on the anti-inflammatory effects of propolis. In the literature, there are not any studies that reveal the CAPE amount in Turkish propolis. In this thesis, Turkish propolis was first evaluated in regards to their CAPE amounts. Afterwards, the propolis' anti-inflammatory activities were compared and lastly, their correlation with CAPE contents were examined. In addition, this study was the first to analyze CAPE by using the capillary electrophoresis method.
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