Spiraea japonica var. fortunei bitkisinin fitokimyasal özelliklerinin spektroskopik ve kromatografik yöntemlerle belirlenmesi
Determination of photochemical properties of spiraea japonica var. fortunei plant by spectroscopic and chromatographic methods
Yazar: HAKA FAJRIANA FEBDA KURNIA
Danışman: DR. ÖĞR. ÜYESİ SEMRA YILMAZER KESKİN ; DOÇ. DR. AYŞE AVCI
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Biyokimya Ana Bilim Dalı
Derece: Yüksek Lisans, 2023
Konu: Biyokimya = Biochemistry

Özet:
Spiraea japonica var. fortunei, Rosaceae familyasına ait bir bitki olup yüze yakın spiraea bitkisi türü vardır. Çiçeklerinin rengi beyaz, kırmızı, pembe ve sarı olarak türlere göre farklılık göstermektedir ve 1,5 ile 3 m kadar uzayabilmektedir.  S. japonica var. fortunei, ağırlıklı olarak Asya ülkelerinde yetişen ve alkaloidleri ile tanınan bir çalı bitkisidir. Spiraea cinsi fenolik bileşikler açısından zengin olarak bilinmektedir. Fenolik bileşik sınıfına ait olan flavonoidler ayrıca antioksidan aktiviteye sahiptir. 
Bu çalışmanın amacı, S. japonica var. fortunei'nin toplam fenolik ve flavonoid içeriği ve FTIR analizi ile fenolikler ve flavonoidler ile ilişkili fonksiyonel gruplarını ortaya çıkarmaktadır. Bitkinin yaprakları ve çiçekleri, etanol veya metanol kullanılarak ayrı ayrı ekstrakte edilmiştir. Metanolün hem yaprak hem de çiçekler için etanole kıyasla ekstraksiyon için daha iyi bir çözücü olduğu belirlenmiş ve bununla daha yüksek ekstraksiyon verimleri, toplam fenolik ve flavonoid elde edilmiştir. Öte yandan, yaprakların fenoller ve flavonoidler açısından daha zengin olduğu ve çiçeklerden daha yüksek antioksidan aktiviteye sahip olduğu belirlenmiştir. Yaprakların etanol ve metanol ekstraktlarının toplam fenolik içerikleri sırasıyla 18,13±1,61 ve 25,64±0,32 mg GAE/g kuru madde iken çiçek ekstraktlarında 11,08±0,13 ve 19,33±1,24 mg GAE/g kuru madde olarak belirlenmiştir. En düşük flavonoid içeriği S. japonica var. fortunei çiçeklerinin etanol ekstraksiyolarında (16,98±3,07 mg QE/g kuru madde), en yüksek ise yaprakların etanol estraktında (46,46±2,96 mg QE/g kuru madde)  iken çiçeklerinin ve yapraklarının metanol ekstraktlarının toplam flavonoid içerikleri sırasıyla 22,68±2,63 ve 28,35±3,54 mg QE/g kuru madde olmuştur. Örneklerin antioksidan aktiviteleri de DPPH radikal süpürme hızı ölçülerek incelenmiş. En düşük konsantrasyonda (0,156 mg/mL) çiçeklerin ve yaprakların etanol ve metanol ekstraktlarının antioksidan aktiviteleri %12,30±0,79, 21,60±1,80, 29,40±4,30 ve 30,00±0,67 olarak belirlenirken en yüksek konsantrasyondaki (6,25 mg/mL) antioksidan aktiviteleri sırasıyla %66,43±0,11, 69,76±0,34, 65,71±0,90 ve 69,29±2,58 olarak tespit edilmiştir. Yaprakların antioksidan aktivitesi, 0,156 mg/mL konsantrasyonda çiçeklerden oldukça yüksektir. Öte yandan, tüm örneklerin antioksidan aktiviteleri 6,25 mg/mL'de hemen hemen aynıdır. Bu çalışmada da S. japonica var. fortunei çiçek ve yapraklarından elde edilen etanol ve metanol ekstraktlarının  IC50 değerleri sırasıyla 0,57±0,06, 0,48±0,01, 0,43±0,01, ve 0,45±0,00 olarak hesaplanmıştır. Ekstraktların UV-VIS spektrumları ve FTIR analizleri, fenoliklerin ve flavonoidlerin varlığını doğrulamıştır. Bu çalışmada ayrıca LC-MS analizine dayalı S. japonica var. fortunei bitkilerinin çiçek ve yapraklarının metanol ekstraktlarında 54 flavonoid bileşik ve 14 flavonoid olmayanlar bileşik tanımlanmıştır.

Abstract:
Oxidation reactions that occur constantly in our body trigger the formation of highly active free radicals that can damage cell structure and function. However, the reactivity of these free radicals can be inhibited by the antioxidant system that complements the immune system. By using antioxidants found in a plant called Spiraea japonica var. fortunei, oxidative stress can be reduced by increasing the body's immunity.
Spiraea japonica var. fortunei, also known as Japanese meadowsweet is a woody shrub of the rose family (Rosaceae), introduced as an ornamental plant which is spread from Asia to the Northeastern United States around 1870. There are close to a hundred species of spiraea plants. It can grow up to 1.5 to 3 meter. The color of the flowers varies according to the species, for example white, red, pink and yellow. Primary routes of propagation are wind, water and vegetative propagules; these features may make the Japanese meadowsweet a serious invasive plant in the future.
More than 80 alkaloids, six new flavonoid glycosides, one lignan, one neolignan glycoside, four monoterpene acylglycosides and one megastigman glycoside that are characteristic of the S. japonica complex have been found so far. Spiraea genus is known to be rich in phenolic compounds. Flavonoids, which belong to the class of phenolic compounds, also have antioxidant activity.
Experiments are made for the total flavonoid content using the colorimetric AlCl3 method, the total phenolic content using the Folin-Ciocalteu method, and the antioxidant content using the DPPH method. Flavonoids and phenolics can be extracted optimally if appropriate extraction solvent is used.
S. japonica var. fortunei was collected from Sakarya University Engineering Faculty backyard. The flowers and leaves of the plant were dried separately in the shade at room temperature. Next, the dried flowers and leaves were ground in a Waring blender and then sieved to obtain a fine powder. Samples were stored in plastic bags at room temperature. Chemicals used include methanol, ethanol, sodium carbonate, gallic acid, aluminum chloride, sodium nitrite, sodium hydroxide and Folin-Ciocalteu reagent purchased from Merck (Darmstadt, Germany), 1,1-diphenyl-2-picrylhydrazil (DPPH) Sigma -Obtained from Aldrich (St., Louis, USA).
Then, both methanol and ethanol extracts of leaves and flowers were prepared. For this, 0.25 g of powder sample was weighed and 10 mL of 80% (v/v) was added. Samples for extraction were moderately shaken on a magnetic stirrer for 24 h at room temperature. The solution was then centrifuged at 9000 rpm for 10 minutes and the pellet was discarded while the supernatant was collected for further analysis. The extracts were stored at -30 °C until analysis.
To calculate the extraction yield, the clear filtrate was dried under vacuum at 40 °C using a rotary evaporator (IKA KS 400, Switzerland). The resulting dry crude extract was weighed to calculate the extraction yield.
For determining total phenolic contents of S. japonica extracts using the Folin Ciocalteau method, 75 µL of a suitable solvent (80% methanol or ethanol) was mixed with 25 µL of extract, 200 µL of Folin-Ciaocalteu reagent and 2 mL of distilled water. After three minutes, 1 mL of Na2CO3  solution (20% w/v) was added and the mixture was left in a dark drawer for one hour. The absorbance of the extract solution was read with a UV-Vis spectrophotometer (Agilent Model 8453 Diode Array Spectrophotometer) at a wavelength of 765 nm. A standard curve was created using gallic acid. Total phenolic content was expressed as mg Gallic Acid Equivalent (GAE) in g dry matter of the sample. Unless otherwise stated, all analyzes were performed in triplicate and results are expressed as mean ± standard deviation.
To determine the total flavonoid content using aluminum chloride colorimetric assay, 100 µL of the extract was mixed with 300 µL of distilled water and 30 µL of NaNO2 (5%, w/v) and left at room temperature for 5 minutes. Then, 30 µL of AlCl3 (10%, w/v) was added to the mixture. After 5 minutes, 200 µL of NaOH (1 mM) was added. Finally, the volume was made up to 1 mL with distilled water and vortexed. The intensity of the orange-yellow color produced by the flavonoids was read with a UV-Vis spectrophotometer at a wavelength of 510 nm. Quercetin standard (10-800 ppm) was prepared under conditions to calculate the number of flavonoids in the sample. The results are expressed as mg (QE)/g dry matter of the sample.
For determining the antioxidant activity by using DPPH radical scavenging activity, various amounts of extract (2.5, 5, 10, 50, and 100 µL) were added to the test tube and the volumes were made up to 200 µL with 70% methanol (v/v). The samples were reacted with 3 mL of DPPH (0.051 mmol; 70% in methanol) solution for 30 minutes at room temperature. Then, the decrease in absorbance compared to the control was observed at 517 nm. The control sample contained 200 µL of 70% methanol instead of the sample. Antioxidant activity was calculated and the results are given as %DPPH scavenging activity.
Leaf and flower extracts obtained with different solvents were subjected to UV-VIS scanning analysis. They were diluted 30 times and absorbance spectra were recorded between 200 and 800 nm wavelengths.
By using Fourier Transform Infrared (FTIR) spectrometry (Shimadzu IR, Prestige 21, Nakagyo-ku, Japan), functional groups associated with phenolic compounds were identified. The scanning range is between 4000 and 400 cm-1.
The goal of this study was to determine the total phenol, and flavonoid contents of S. japonica var. fortunei and to reveal the functional groups associated with the phenolics, and flavonoids by FTIR analysis. The leaves and the flowers of the plant were analyzed separately by using either ethanol or methanol as extraction solvents. Methanol was determined to be a better solvent for extraction compared to ethanol for both leaves and flowers with which higher extraction yields, total phenolic, and flavonoid contents were observed. On the other hand, leaves are richer in phenols and flavonoids and have higher antioxidant activity than flowers. The total phenolic contents of ethanol and methanol extracts of the leaves were 18,13±1,61 and 25,64±0,32 mg GAE/g dry matter, respectively, while they were 11,08±0,13 and 19,33±1,24 mg GAE/g dry matter in the extracts of flower, respectively. In S. japonica var. fortunei flavonoid determination, the lowest flavonoid content was in the ethanol extraction of the flowers (16.98±3.07 mg QE/g dry matter) and the highest was in the ethanol extract of the leaves (46.46±2.96 mg QE/g dry matter) while the total flavonoid contents of the methanol extracts of the flowers and leaves were 22.68±2.63 and 28.35±3.54 mg QE/g dry matter, respectively.Antioxidant activities were also investigated by measuring DPPH radical scavenging rate and they ranged from 65,71±0,90 to 69,76±0,34 % and IC50 values were calculated to be between 0.48±0.01 and 0.57±0.06 mg/mL. UV-VIS spectra and FTIR analyses of the extracts confirmed the presence of phenolics, and flavonoids. In this study, the methanol extracts of flowers and leaves of S. japonica var. fortunei plant based on LC-MS analysis indetified 54 flavonoid compounds and 14 phenolic compounds.