Farklı bitkilerin antioksidan aktivitelerinin belirlenmesi
Determination of antioxidant activities of different plants
Yazar: DUYGU ELİF BİLGE
Danışman: PROF. DR. GÜLNUR ARABACI
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı / Biyokimya Bilim Dalı
Derece: Yüksek Lisans, 2023
Konu: Kimya = Chemistry

Özet:
Antioksidanlar, sağlığa olan faydaları ile pek çok kişi tarafından bilinen ve gıda takviyeleri, kozmetik, işlenmiş tahıl gevrekleri gibi ürünler de çoğunlukla yer alan bileşenlerdir.
Sağlık üzerindeki faydaları saymakla bitmeyen ve besinlerin içerisinde değişen oranlarda yer alan antioksidanlar, serbest halde bulunan radikaller nedeniyle oluşan hücresel tahribatın önlenmesinde veya yavaşlatılmasında katkı sağlayan maddelerdir ve vücuda besinler ya da takviye edici gıdalar (özellikle bitkiler) ile alınabilmektedir.
Aynı zamanda antioksidan maddeleri vücudun kendi içinde, serbest halde bulunan radikallere karşı savunma amacıyla üretilir. Antioksidan kapasitesi,varlığı keşfedilmiş birçok farklı madde vardır.
Canlı metabolizmasında olağan şartlar altında her zaman serbest radikaller meydana gelir. Serbest radikaller; belirlenmiş bir seviyenin üstünde olunca kararsız kimyasal yapıları sebebiyle sağlıklı olan hücreleri tahribata uğratarak kanser, diyabet, Alzheimer, kalp hastalıkları gibi pek çok ciddi hastalığa yakalanma olasılığını arttırmış olur. Eğer vücut serbest radikalleri etkili olarak ortadan kaldıramazsa oksidatif stres adı verilen bir durum meydana gelebilir.
Metabolizmadaki oksidatif stres, tütün ve alkol tüketimi, ağır metallerle etkileşim, çevre kirliliği, radyasyon, sık geçirilen enfeksiyonlar, antioksidan azlığı sonucuna bağlı olarak daha da artabilir. Geniş bir zaman boyunca var olan yüksek oksidatif stres, hücrelerin genetik bileşeni olan DNA'da tahribatlara neden olarak kanser ve kronik hastalıklara, hatta hücre ölümlerine dahi başlangıç oluşturabilir. Tüm bunlara ek olarak serbest radikallerin metabolizmada pozitif yönde faaliyetleride bulunmaktadır. Örneğin immün sistem, metabolizmaya katılan enfeksiyon ajanları ile savaşırken serbest radikalleri kullanır. Sağlığımızın stabil kalabilmesi için serbest radikaller ve antioksidanlar metabolizmada belirli bir denge içerisinde bulunmalıdır. Lakin sağlıksız ve dengesiz diyete bağlı olarak antioksidanlar, besinlerle birlikte gerektiği düzeyde metabolizmaya alınmaz ise bu denge bozulur ve vücut, hastalıklara karşı savunmasız bir yapıda olmuş olur. Bu nedenle gerektiği kadar ve belirli periyotlarla antioksidan kullanımına dikkat etmek oldukça önemlidir.
Canlı metabolizmaların toksik maddelerle mücadele etmek amacıyla ürettiği antioksidanlar; katalaz, glutatyon peroksidaz, askorbat peroksidaz ve SOD (superoksit dismutaz) gibi enzimlerdir. Antioksidanların pek çok çeşitleri bulunur. Bunlardan E Vitamini (alfa tokoferol) içinde alfa, beta, gama ve delta tokoferolleri bulundurur. Bunlardan en önemli antioksidan olanı alfa tokoferoldir. Diğerleri; Askorbik Asit (C Vitamini), beta caroten ve flavonoid gibi antioksidanlardır. Tüm bunların dışında var
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olduğu kanıtlanmış onlarca farklı antioksidan çeşidi bulunmaktadır. Bitkisel kökenli pek çok türden besinin yapı taşında farklı tür ve miktarlarda bulunan antioksidanlar beslenme yoluyla vücuda alınır.
Bu tez kapsamında Sakarya Üniversitesi'nin Biyokimya laboratuvarında farklı bitkilerin farklı kısımlarının etanol ekstraktlarının antioksidan aktivite değerleri DPPH serbest radikali yakalama testi, Demir (II) iyonları şelatlama aktivitesi ve indirgenme kapasitesini içeren üç farklı yöntem ile belirlenmiştir. DPPH (1,1-Difenil-2– pikrilhidrazil radikali; C18H12N5O6) antioksidan analizi, farklı bileşiklerin antioksidan kapasitesini değerlendirmek için kullanılan hızlı, yaygın ve maliyetsiz bir yöntemdir. Metal iyonları şelatlama testi serbest halde bulunan ağır metallerin bozucu etkilerini baskılamak amacıyla kullanılan bir test yöntemidir.
Çalışma için, Bursa'nın Mudanya ilçesinden ; acı kavun otu ((Ecballium elaterium), dul avrat otu (Arctium lapa ), sazlık otu (Phragmites australis) ve mürver otu (Sambucus nigra) gibi dört farklı bitki yaprak ve meyveleri ile beraber toplanarak antioksidan aktiviteleri belirlenmiştir. Bu dört bitki halk arasında hastalıklara tedavi amacıyla veya cilt bakımı gibi pek çok alan için yaygın olarak tüketilmektedir. Mürver otu dünya üzerinde astorpikal bölgelerde çoğunluktadır ve koyu mavi ya da siyah renkte bulunur. Canlı metabolizmasında bir antioksidan ve anti-inflamatuar ajan olarak görev alır.
Toplanan dört bitki türünün yaprak ve meyve kısımları etanol ile ekstraksiyon işlemine tabi tutulmuştur. Bu işlem için uygun kimyasallar kullanılarak kurutma işlemi gerçekleştirilmiştir. Tüm bitkilerin hem yaprak hem de meyve kısımlarından antioksidan aktivite taraması yukarıda bahsedilen üç yöntem ile yapılmıştır.
DPPH serbest radikal süpürme testinde karanlıkta inkübasyon yapılarak 517 nm de değerler okunmuştur. Demir (II) iyonları şelatlama testinde deiyonize su ile FeCl2 oda sıcaklığında inkübasyona bırakılmıştır ve 562 nm de değerler okunmuştur.
İndirgenme kapasitesi testi için ise fosfat tamponu ve kompleks bir tuz ile su banyosunda inkübasyonu yapılmıştır. 700 nm de değerler okunmuştur. DPPH serbest radikal bağlama aktivitelerine bakıldığında; derişim oranları arttıkça aktivite oranlarının da arttığı gözlemlenmiştir. Demir (II) iyonlarının şelatlama işleminde de konsantrasyon değerleri arttıkça aktivite oranının arttığı görülmüştür.
Analizi yapılan çalışmanın sonucunda en yüksek DPPH aktivitesi, etanolde mürver bitkisinin meyve kısmında % 82,12 olarak belirlenmiş olup yaprak kısmında %72,12 olarak ölçülmüştür. İndirgenme kapasitesi tayininde en yüksek değer etanol ile ekstrakte edilen hem kabuk hem de meyve kısmı için yine mürver otunda ölçülmüştür. Demir (II) iyonlarını şelatlama tayininde de tekrardan etanol ile ekstraktesi yapılan hem yaprak ve meyve kısımları için mürver otu yüksek değere sahiptir. Yapılan üç yöntemde de en düşük orana sahip olan ot ise sazlık otu olmuştur.
Alternatif tıpta daha çok tercih edilen mürver otunun sinüzit rahatsızlığı gibi pek çok rahatsızlığı gidermesi, sazlık otunun ise sadece süs eşyaları yapımında kullanılması, insan vücuduna bir faydasının görülmemesi bu çalışmayı desteklemektedir. Bu çalışmaya göre uygulanan yöntemlerden en iyisi DPPH yönetemi demek kesin sonucu oluşturmayacaktır.

Abstract:
Antioxidants are components that are known to many people for their health benefits and are often included in products such as food supplements, cosmetic products, enriched cereals.
In this study, the antioxidant activities of bitter melon grass (Citrullus colocynthis ), dul avrat grass (arctium lapa ), reed grass (phragmites ) and elderberry grass (Sambucus Nigra) of four different plants collected in Mudanya district of Bursa were determined. These plants are widely consumed among the population Decently.
The benefits on health do not end with counting and are contained in varying proportions in nutrients antioxidants are substances that contribute to the prevention or slowing of cellular destruction caused by free radicals and can be taken into the body with foods or supplements (especially plants).
At the same time, antioxidant substances are produced in the body itself for the purpose of defense against free radicals.
Antioxidant capacity,there are many different substances whose existence has been discovered. Under normal conditions in living metabolism, free radicals occur all the time. Free radicals; When they are above a certain level, they destroy healthy cells due to their unstable chemical structure, increasing the likelihood of developing many serious diseases such as cancer, diabetes, Alzheimer's, heart diseases.Oxidative stress in the body may increase due to conditions such as smoking and alcohol consumption, environmental pollution, exposure to heavy metals, radiation, frequent infections, antioxidant deficiency.
Oxidative stress in metabolism, tobacco and alcohol consumption, environmental pollution, exposure to heavy metals, radiation, frequent infections, may increase even more depending on the result of antioxidant deficiency. High oxidative stress, which has existed for a long time, can cause damage to the DNA, which is the genetic component of cells, causing the onset of cancer and chronic diseases, even cell death. In addition to all these, free radicals also have positive activities in metabolism. October 19, 2016. For example, the immune system uses free radicals when fighting infectious agents involved in metabolism. In order for our health to remain stable, free radicals and antioxidants must be present in a certain balance in metabolism. However, if antioxidants are not metabolized at the required level along with nutrients due to an unhealthy and unbalanced diet, this balance is disrupted and the body becomes vulnerable to diseases. For this reason, it is very important to pay attention to the use of antioxidants as much as necessary and with certain periods.
Antioxidants produced by living metabolisms in order to combat toxic substances; catalase, glutathione peroxidase, ascorbate peroxidase and SOD (superoxide dismutase). There are many types of antioxidants. Alpha tocopherol (Vitamin E) contains alpha, beta, gamma, and delta tocopherols. Among them, alpha tocopherol is an important antioxidant. Others; Antioxidants such as Ascorbic Acid (Vitamin C), beta carotene and flavonoids. Apart from all these, there are dozens of different types of antioxidants that have been proven to exist. Antioxidants, which are found in different types and amounts in the building blocks of many types of plant-based foods, are taken into the body through nutrition.
A large number of methods have been developed to date to measure antioxidant capacity. Methods that allow measuring the total antioxidant capacity can be divided into two, namely methods based on hydrogen atom transfer (HAT) reactions and methods based on electron transfer (ET). Many of the LINE-based methods use competitive kinetic reactions based on the competition of antioxidant and substrate for peroxyl radicals formed by the degradation of azo compounds. MEAT-based methods, on the other hand, measure the antioxidant's ability to reduce oxidants by color change. The degree of color change is related to the antioxidant concentration of the samples.
ORAC; This method, which examines the effect of free radicals and uses the area under curve (AUC) technique in determining the amount, is a method that can express both the percentage of antioxidants inhibiting free radicals and the inhibition time as a single value. The net AUC is proportional to the amount of antioxidants.
In the TRAP method, the delay time of the reaction is measured and the total amount of antioxidants is calculated in terms of trolox by comparing the results found with trolox. The disadvantage of this method is that the endpoint of the oxygen electrode is not precisely detected, and the stability of the oxygen electrode cannot be maintained within the required time frame.
This method, which is called DCFH-DA and is based on the TRAP method, the total antioxidant capacity is calculated in two stages. At the first stage, the capacity of the antioxidants in the sample is calculated in terms of delay time, and then trolox solution, the amount of which is known, is added to the same sample. After the Trolox solution is consumed by free radicals, the second delay time is calculated. The total antioxidant capacity in trolox is calculated by taking advantage of the difference between these two Deceleration times.
The luminol method is used by oxidizing the luminol substance with hydrogen peroxide or perborate. In order for the reaction to occur faster, HRP (horse radish peroxidase) was used as a catalyst and the light propagation was faster. Under normal conditions, this reaction occurs as a low intensity light emission that decreases rapidly. When p-iodophenol is added to the reaction medium, the light emission becomes more intense, long-lasting and stable. In order for light to be emitted by luminol radicals, all antioxidants in the environment must be consumed. Therefore, this method is sensitive to antioxidant interference. The most significant disadvantage of this method is that the antioxidants studied reduce not only the radicals formed from AAPH, but also the luminol radicals.In the crocin bleaching method, the degree of bleaching of crocin, a carotenoid, is measured by peroxyl radicals formed as a result of thermal degradation of the azo initiator. Fr chromogenic oxidant in the cuprac method(II)-reagent using neokuproin, plasma antioxidants, flavonoids, polyphenols food, vitamin C and vitamin E for a simple, widely applicable, an antioxidant capacity determination method has been developed. This reagent is a stable, inexpensive, easily accessible reagent that can respond to hydrophilic and lipophilic antioxidants. This method used in the determination of total antioxidant capacity has been introduced into the world literature under the name CUPRAC (copper(II) ion reduction antioxidant capacity).
The TEAC/ABTS method, expressed as Trolox equivalent antioxidant capacity, is a method based on the inhibition of the absorbance of the 2,2' azinobis(3- ethylbenzothiazoline-6-sulfonate) chromogenic radical cation by hydrogen-donating antioxidants. By taking advantage of the decrease in absorbance, the total antioxidant capacity is given in trolox.
In the original FRAP method, the Fe3+-tripridyltriazine (Fe3+-TPTZ) complex is reduced to the Fe2+-tripridyltriazine (Fe2+-TPTZ) complex by an antioxidant substance with reducing properties in an acidic pH environment (pH=3.6).The Fe2+-TPTZ complex gives a violent blue color. The absorbance of this Fe2+-TPTZ complex formed at 593 nm is measured and the reducing power (or capacity) of electron-donating antioxidants is measured in total.
The DPPH method is a method based on the measurement of the scavenging effects of antioxidants on a stable free radical, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical. This radical is reduced to hydrazine when hydrogen interacts with donors. The red-colored DPPH radical provides maximum absorption at 517 nm. With the addition of antioxidants to ethanol or methanol DPPH solution, there is a decrease in absorption and the color of the radical turns from red to yellow with the presence of antioxidants. This method is known as an easy and valid method for evaluating the radical scavenging abilities of antioxidants.
In this study, DPPH free radical capture test, Iron (II) ion chelating activity, reduction capacity were studied by three different methods in the Biochemistry laboratory of Sakarya University. DPPH (1,1-Diphenyl-2-picrylhydrazil radical; C18H12N5O6) antioxidant analysis is a fast, common, efficient, and inexpensive method used to evaluate the antioxidant capacity of different compounds. Metal ion chelation test is a test method used to suppress the destructive effects of free heavy metals.
In this study, antioxidant effects of elderberry (Sambucus Nigra) were determined. Elderberry is concentrated in the astorpical regions of the world and is available in dark blue or black. It functions as an antioxidant and anti-inflammatory agent in the human body. Elderberry herb was first subjected to the extraction process. Drying was carried out using suitable chemicals for this process. Antioxidant activity screening was done from both leaf and fruit parts of elderberry.
For the study, four different plants such as bitter melon grass (Ecballium elaterium), widow avrat grass (Arctium lappa), reed grass (Phragmites australis) and elderberry grass (Sambucus nigra) from Mudanya district of Bursa were collected together with their leaves and fruits and their antioxidant activities were determined. These four
plants are widely consumed among the people for the treatment of diseases or for many areas such as skin care. Dec. Elderberry grass is mostly found in astorpical regions around the world and is found in dark blue or black color. It acts as an antioxidant and anti-inflammatory agent in live metabolism.
The leaf and fruit parts of the four plant species collected were subjected to ethanol extraction process. The drying process was carried out by using suitable chemicals for this process. The screening of antioxidant activity from both leaf and fruit parts of all plants was carried out by the three methods mentioned above.
In the DPPH free radical scavenging test, values were read at 517 nm by incubating in the dark. In the chelating test of iron (II) ions, deionized water and FeCl2 were incubated at room temperature and values were read at 562 nm.
For the reducing capacity test, it was incubated in a water bath with phosphate buffer and a complex salt. Values were read at 700 nm. Considering the DPPH free radical binding activities; It was observed that as the concentration ratios increased, the activity ratios also increased.
As a result of the analyzed study, the highest DPPH activity was determined as 82.12% in the fruit part of the elderberry plant in ethanol and was measured as 72.12% in the leaf part. The highest value in determining the reduction capacity was again measured in elderberry grass for both the peel and the fruit part extracted with ethanol. In the determination of chelating iron (II) ions, elderberry grass has a high value for both leaf and fruit parts, which are extracted with ethanol again. The grass with the lowest rate in all three methods was reed grass.
In fact, when we look at daily life, elderberry herb relieves many ailments such as sinusitis discomfort, while reed grass is only used in the production of ornaments, no benefit to the human body is seen supports this study.
According to this study, DPPH management is the best method, which will not create a definitive result.