Polygala major jacq. bitkisinin antioksidan ve antibakteriyel özelliklerinin araştırılması
Investigation of antioxidant and antibacterial properties of Polygala major jacq.
Yazar: LANA ARIF ALI ALI
Danışman: DR. ÖĞR. ÜYESİ SEMRA YILMAZER KESKİN
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı / Biyokimya Bilim Dalı
Derece: Yüksek Lisans, 2023
Konu: Biyokimya = Biochemistry

Özet:
Bitkiler sağlık açısından büyük önem taşımaktadır. Bitki ekstrelerinden elde edilen bileşenlerin kullanımı özellikle ilaç endüstrisinde önem kazanmaktadır. Bu çalışmada Türkiye'de yetişen ve tedavi amaçlı da kullanılabilen koca sütotu olarak bilinen Polygala major jacq. bitkisinin antioksidan ve antibakteriyel aktivitesi incelenmiştir. Polygala major jacq., bitkisi başlangıçta Avusturya'dan  tanımlanmış ve İtalya'dan batı Rusya'ya kadar bir dağılım aralığına sahiptir. Polygala cinsine  ait subtermofilik ve oligotrofik bir  biki türüdür. Çayırlarda ve çayırlıklarda sıkça karşılaşılır. Polygala cinsinin diğer türleri gibi acı maddeler  içermektedir. Dünyanın dört bir yanından 600'den fazla türden oluşur, bu türlerin 40'ı Çin'de mevcuttur. Türlerinin, enflamasyona, kardiyovasküler ve merkezi sinir sistemi bozukluklarına karşı çeşitli geleneksel kullanımları rapor edilmiştir. 
Polygala major jacq. bitkisi 2022 Mayıs ayında Esentepe/Sakarya'dan toplanmıştır. Çiçek ve yaprak kısımları oda sıcaklığında kurutulup, blender ile öğütüldükten sonra üç çözücü (aseton, etanol ve methanol) ile ekstrakte edilerek  toplam fenolik ve flavonoid madde içeriği, antioksidan ve antibakteriyel aktivitesi araştırılmıştır.
Her ekstrenin Folin Ciocalteu reaktifi (FCR) ile total fenolik madde içeriği, alüminyum klorür (AlCl3) metodu  ile total flavonoid madde içeriği ve 1,1-difenil-2-pikrilhidrazil (DPPH) kullanarak serbest radikal giderimi tayini UV–vis spektroskopisi ile yapılmıştır. Fourier Dönüşümlü Kızılötesi (FTIR) spektroskopisi gibi hızlı ve hassas bir yöntem kullanılarak bitkinin çiçek ve yaprak kısımlarında bulunan fonkesyönel gruplar incelenmiştir. Toplam fenolik madde tayininde en yüksek değer 48,51±1,35 mg GAE/g kuru madde yaprak etanol ultrasonik ekstraktında gösterirken, en düşük değer 29,04±2,93 mg GAE/g kuru madde çiçek aseton ultrasonik ekstraktında belirlenmiştir, toplam flavonoid tayininde değerler 4,17±39-16,56±0,72 mg QE/g kuru madde arasında değerlendirilmiştir. DPPH giderim aktivitesi ise en yüksek değer %50,40±0,25 yaprak metanol ultrasonik ekstraktında ve en düşük değer %39,90±1,25 yaprak aseton ultrasonik ekstraktında belirlenmiştir.
Koca sütotu (Polygala major jacq.) bitkisinin antibakteriyel aktivitesinin belirlenmesi için disk difüzyön yöntemi kullanılmıştır. Antibakteriyel aktivite ise Bacillus cereus, Escherichia coli O157:H7, Pseudomonas aeruginosa ve Staphylococcus aureus bakterileri gibi enfeksiyona neden olabilen farklı bakterilere karşı belirlenmiştir. Polygala major jacq. bitkisinin yaprak metanol ekstraktı Pseudomonas aeruginosa bakterisine karşı 10,5±0,6 mm çapında zon oluşturduğu gözlenmiştir. Diğer bakteri türlerine daha düşük etkisi olduğu belirlenirken Escherichia coli O157:H7 türüne etki etmediği gözlenmiştir.

Abstract:
Plants have been of great importance in terms of humen health from ancient times to the present day. Before the introduction of chemical medicines, man relied on the healing properties of medicinal plants. Some people value these plants due to the ancient belief which says plants are created to supply man with food, medical treatment, and other effects. The use of components obtained from plant extracts is increasing rapidly, especially in the pharmaceutical industry. Metabolites formed during the secondary metabolism of plants are used in the treatment of many diseases, but very few of them are used as antimicrobials.since plants are complex stractures containing different components, the biological effect of each is different. Accordingly, the degree of showing antimicrobial and antioxidant properties differs for each plant. 
Many medicinal plants have great antioxidant potential. Antioxidants reduce the oxidative stress in cells and are therefore useful in the treatment of many human diseases, including cancer, cardiovascular diseases and inflammatory diseases. The antioxidants are  extracted from the stems, roots, flowers, leaves, fruits and seeds of several important medicinal plants. In recent years, the emergence of antibiotic resistant bacteria and unwanted side effects in the synthetic drugs, which are used for the treatment of different inflamation diseases, has pioneered to produce antibiotics and drugs with the natural origin.
Since these functions of plant are of great importance, a study on this subject has been preferred. In this study, the antioxidant and antibacterial activity of milkweed (polygala major jacq.) plant, was investigated. This plant is grown in Turkey and can also be used for therapeutic purposes. It is subthermophilic and oligotrophic species belonging to the genus polygala. İt is common in meadows. Like other species of the genus polygala,they are considered poisonous plants in grasslands. Polygala major jacq. was identified from Austria and has a distribution range from İtaly to western Russia.
Extraction is a critical method for isolating the effective component in plants. There are several parameters concerned in the extraction of antioxidative components from plant tissues. This includes the types of solvent, the ratio of solvent-to-sample, the extraction temperature and the extraction time. Ultrasonic probe extraction possesses greater solvent penetration into plant tissues due to the formation of cavitational bubble collapse, thus improving its solvent extraction ability.
Polygala major jacq. plant was collected from Esentepe/ Sakarya in May 2022. The flower and leaf parts were dried at room temperature, powdered with a blender, and extracted by using ultrasonic probe and normal extract method. Extract solutions are ethanol, methanol and acetone, antioxidant and antibacterial activity of P. major jacq. were determined by different methods.
In order to determine the antioxidant capacity of the plant,  the total phenolic content of each extract was determined with Folin Ciocalteu reagent, this method is developed by Singleton. It is used to measure the total phenolic compounds. The principle of FCR method is based on the transfer of electrons from phenolic compounds and other reducing compounds to molybdenum. The formation of blue complexes is evaluated spectrophotometrically at 750-765 nm. In this method we used gallic acid as standard curve. Phenolic compounds, with distinct and powerful antioxidant capabilities, can act as effective radical scavengers and diminish their devastating effects on body. Phenolic compounds are the major antioxidant contents of plant-based food products and have attracted increasing attention in scientific research, owing to their positive and restraining effects on the serious disorders caused by oxidative stress such as, inflammation, allergies and cancer.
The total amount of flavonoid substances was determined by the aluminum chloride method. In this method we used quercetin as standard curve. Flavonoids, as phenolic compounds, are also attracting great consideration due to their positive biological activities, comprising antioxidant, antiviral, antibacterial, anti-diabetic and anti- cancer activities. The total flavonoids content, as a significant index is usually used to check the quality and therapeutic value of traditional medicines. 
The DPPH free radical scavenging activity were determined by UV-vis spectroscopy. The 2,2-Diphenyl-1- picrylhydrazyl, DPPH method is rapid, simple and inexpensive assay for measuring the ability of different compounds to act as free radical scavengers and to evaluate the antioxidant activity of foods and plants.
Using a fast and sensitive method such as Fourier Transform Infrared (FTIR) spectroscopy, the functional groups in the flower and leaf parts of the plant were investigated. The presence of polyphenols and flavonoids were also confirmed by FTIR
The highest value in the determination of total phenolic substance 48.51±1.35 mg GAE/g dry matter in leaf ethanol ultrasonic extract, lowest value 29.04±2.93 mg GAE/gdry matter flower acetone ultrasonic extract, values in total flavonoid were ranged between 4.17±0.39 - 16.56±0.72 mg QE/g dry matter. DPPH removel activity was the highest value 50.40±0.25% in leaf methanol ultrasonic extract and the lowest value 39.90± 1.25% was determined in the ultrasonic extract of leaf acetone. 
The disk diffusion method  is classified as an agar diffusion method because the plant extract to be tested diffuses from its reservoir through the agar medium seeded with the test microorganism. Generally, the reservoir is a filter paper disk, which is placed on top of an agar surface. If tested plant extracts  are microbiologically active, an inhibition zone develops around the filter paper disk after incubation. The diameter of the inhibition zone properly describes the antimicrobial potency of plant extracts. Disk diffusion method was used to determine the antibacterial activity of the milkweed ( polygala major jacq) plant extracts.
The extracts were evaporated by using vakum evaporatore and then the antibacterial properties were determined against different microorganisms that can cause infection. Disk diffusion by the Kirby-Bauer method is a standardized technique for testing rapidly growing pathogens. Briefly, a standardized inoculum is swabbed onto the surface of MH agar. Because reproducibility depends on the log growth phase of organisms, fresh subcultures are used. Filter paper disks impregnated with a standardized concentration of a plant extract are placed on the surface, and the size of the zone of inhibition around the disk is measured after overnight incubation.  
The microorganisms are used Bacillus cereus, Escherichia coli O157,H7, Pseudomonas aeruginosa, Staphylococcus aureus bacteria. Polygala major jacq. it was observed that the leaf methanol extract of the plant formed a zone about 10.50± 0.6 mm in diameter against Pseudomonas aeruginosa bacteria, it was determined that it has a lower effect on other bacterial species, but it did not affect Escherichia coli O157,H7 species. İn the antibacterial tests of P. major jacq. plant with various bacteria, it is anderstood that the antibacterial active substance in the leaf part of the plant is best dissolved in methanol, one of the solvents we used. Today, in the face of increasing resistance to antimicrobial agents, P.major jacq. the active biological components in the plant can be evaluated as an important alternative source.