Ayçiçeği tohum kabuklarındaki antosiyaninlerin ekstraksiyonu ve karakterizasyonu
Ayçiçeği tohum kabuklarındaki antosiyaninlerin ekstraksiyonu ve karakterizasyonu
Dosyalar
Tarih
1997
Yazarlar
Günenç, Aynur
Süreli Yayın başlığı
Süreli Yayın ISSN
Cilt Başlığı
Yayınevi
Fen Bilimleri Enstitüsü
Özet
Bu araştırmada ayçiçeği tohum kabuklarından antosiyaninlerin ekstraksiyonu, saflaştırılması, yüksek basınçlı sıvı kromatografisi ve spektrofotometre ile karakterize edilmesi ve degradasyon mekanizmasının incelenmesi amaçlanmıştır. Dokuz farklı örnekle yapılan ön ekstraksiyon çalışmaları sonucunda yalnızca "Trakya Tarımsal Araştırma Enstitüsü Müdürlüğü" nden temin edilen Orta Anadolu'da yetiştirilen yerel çerezlik ayçiçeği türünün antosiyanin içerdiği tesbit edilmiş olup, daha sonraki analizlerde bu örnek kullanılmıştır. Gerek yüksek satış fiyatları gerek saflaştınlmış standardın bulunmasının çok zor olması nedeniyle antosiyanin bileşenlerinin standardlan (6 çeşit) satın alınamamış olup, "Lipton" firmasından temin edilen, ilgili literatürde kimyasal olarak ayçiçeği tohum kabuklan (ATK) antosiyaninlerine benzer olduğu belirtilen ve kompozisyonu kısmen bilinen "Üzüm Kabuğu Ekstraktı Tozu (ÜKE)" güvenilir örnek olarak kullanılmıştır. Hem ATK hem de ÜKE'de üç paralel olarak sabit sıcaklık ve pH'da, üç farklı çözgen ile yapılan ekstraksiyon çalışmalannda, toplam antosiyanin miktarlan iki farklı metoda göre hesaplanmıştır. Daha sonra, antosiyanin pigment ekstraktının bileşenlerinin saptanabilmesi amacıyla, C-18 mini kolondan geçirilerek antılan örneğin yüksek basınçlı sıvı kromatografisinde (YBSK) karakterizasyonu üzerine çalışılmıştır. Hem temizlenmiş ATK hem de ÜKE ekstraktında toplam onar adet pik gözlenmiştir. Bu piklerin standart kullanmaksızın teker teker tanımlanabilmesi amacıyla, her iki temizlenmiş pigment ekstraktına sırası ile alkali hidroliz, alkali borat tamponu ile seçici elüsyon ve asit hidrolizi olmak üzere 3 farklı yöntem uygulanmıştır. Sadece asit hidrolizi uygulanmış ve temizlenmiş örnek ekstraktı ile semi-preparatif olarak YBSK'de çalışılmış ve her bir pikin spektral özellikleri tesbit edilmeye çalışılmıştır. Bu analizlerden ve literatürden elde edilen verilerin birleştirilmesi sonucu ATK'da 6 tane açillenmiş, 4 tane açillenmemiş (3 tanesi o-dihidroksil grup içermeyen, diğeri içeren) pigment bileşeni bulunduğu tesbit edilmiştir. Degradasyon mekanizmasının belirlenmesi çalışmalannda örnek ekstraktlannda " degradasyon indeksi (Dİ) " ve " % alıkonulan antosiyanin miktan " cinsinden izlemeler yapılmış, sonuçta lineer olmayan bir degradasyon mekanizması gözlenmiş ve bu literatürde belirtilen benzer çalışmalarla desteklenmiştir.
Color has an important role in human life, both by enriching the world and by enchancing the visual appeal of foods. As long as there is continuous consumer interest in natural ingredients, there will be a market for isolated natural pigments (Meggos, 1984; Mazza and Miniati, 1993). Among the natural pigments, anthocyanins are the largest water soluble group responsible for the attractive colors ranging from salmon and pink, through scarlet, violet to purple and blue of most fruits, juices, flower petals and leaves. These ubiquitous compounds are fascinating in that it is now known that they can exist in many structural forms, both simple and complex, governed by certain physico- chemical phenemona which have profound effects on their colors and stabilities. Studies on anthocyanins cover many aspects of chemistry, biochemistry, biology, botany and medicine (Timberlake, 1980; Mazza and Miniati, 1993). Anthocyanins are glycosides of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium or flavilyum salts (figure 1). Differences between individual anthocyanins are the number of hydroxyl groups in the molecule, the degree of methylation of these hydroxyl groups, the nature and number of sugars attached to the molecule and the position of the attachment, and the nature and number of aliphatic or aromatic acids attached to the sugars in the molecule. The known naturally occuring anthocyanidins or aglycones are listed in Table 1 (Chapter 2). Since each anthocyanidin may be glycosylated and acylated by different sugars and acids, at different positions, the number of anthocyanins is 15 to 20 times greater than the number of possible anthocyanidins (Harborae and Mabry, 1982). Figure 1. Chemical structure of anthocyanins (Mazza and Miniati, 1993). In aqueous media, most of the natural anthocyanins behave like pH indicators, being red at low pH, bluish at intermediate pH, and colorless at high pH. The nature of the chemical structures which these anthocyanins can adapt upon changing the pH has already been clarified. Sunflower (Helianthus ammus, L) is one of the world's leading oilseed crops, second only to soybean in world oil production. A sunflower cultivar with red pigment in the corolla and in other part of the plant was first reported in the early 1900's; the first studies on color inheritance were also conducted during this period (Mazza and Miniati, 1993). Extracts from the purple hulls of these cultivars of sunflower have been proposed for use as natural red food colorants. Their appeal as natural ingredients also carries over to non-food uses, such as textiles and cosmetics, even though the extracts may be more expensive to produce and less stable than artificial red colorants. However, use of artificial red colorants in foods have the disadvantage of being perceived unfavorably by consumers. The purplish-red pigments in purple sunflower hulls are classified as anthocyanins. Thus, they are chemically similar to pigments in grape skin, red cabbage and some other sources of natural red colorants, and they have similar applications. Purple sunflower hull extract is not yet commercially available or approved as food colorant, but it has several desirable features. The pigment is highly stable within the hulls, and it can be extracted under mild conditions. Through conventional plant breeding, the purple-hulled trait could be bred into high-oil sunflower types. Thus, an abundant, inexpensive supply of raw material could become available as a by-product of the sunflower oil industry (Wiesenborn and et al. 1993 and 1995). The objectives of this study are the extraction of natural red pigments from the hulls of native sunflower seeds, purification, identification of their chemical structure, and evaluation of its stability. The work included the extraction of pigments by different solvents at the same temperature and pH conditions, purification of the pigments from other interfering materials and the determination of total anthocyanin content. The components of extracted pigments have been analyzed by using HPLC, and spectrophotometry techniques were used to characterize their structure. The stability of the pigments has also been evaluated by the relationship between the pigment quantity and the percent of anthocyanins remaining in the solution with respect to time. There is a general scarcity of anthocyanin standards. There are, however a number of commonly available natural materials for which the anthocyanins have been throughly characterized. These materials can often be used as "reliable references". The methods of quantitative determination, chromatographic and spectrofotometric identification, and stabilization characterization of anthocyanins in sunflower seed- hulls were also applied to the GSE (Grape Skin Extract ) which was used as reliable refrence for this study. Anthocyanins have previously been separated by a number of techniques including paper, thin-layer, and column chomatographic methods. Of these methods, paper chromatography has been the most widely used method for separation of the individual anthocyanins (Harborne, 1975; Pifferi and Vaccari, 1981; Thomas, 1984). A number of workers have reported high-performance liquid chromatographic (HPLC) methods for separation of both anthocyanins and their aglycons (Rommel, A., and et al., 1992; Bakker and et al., 1994;). One of the problems with the use of HPLC for analysis is that absolute peak retention times can very from worker to worker even under similar analytical conditions. This is made more complex by the fact that there is a general lack of availability of pure anthocyanin standards. The combination of a lack of standards and the problems with specification of absolute peak retention times are two factors contributing to the difficulty of using HPLC for analysis of anthocyanins (Hong and Wrolstad, 1990a and 1990b). The total anthocyanin pigment content was determined by using two methods: the single pH and pH differential methods. The extraction was realized with three different solvents (for sunflower seed-hulls and GSE) at the same pH and temperatures. The results are given in Table 1. Table 1. Total anthocyanin pigment contents for sunflower seed-hulls and GSE. For HPLC analysis, the anthocyanin isolate was obtained by passing it through C-18 reversed-phase-cartridge to remove other interfering phenolics. The chromatogram of the cleaned anthocyanin extract is given in Figure 2a. Auxiliary sample treatment techniques useful for pigment characterization were also applied. The first one of these was alkaline hydrolysis of the anthocyanins for determination of acylation. The second one was alkaline-borate buffer elution for separation of anthociyanins not containing an o-diphenolic system. The last one was acid hydrolysis of the anthocyanins for determination of the aglicone moiety. All the related chromatograms are given in Figure 2 b, c, d. 0-">0 J 00 2 ÛO 3.OO Figure 2a HPLC chromatogram of cleaned anthocyanin extract. 0 > 1 a '¦ /V-» O Alt' A \ - 1 - 1 0<) :>.<>. 3.0) Figure 2b HPLC chromatogram of alkali-hydrolyzed anthocyanin extract. I )l>..'. Hü I 0 BO > 0 r| * '.SO II M O.I I ^_/^V_J 0 SO 40 0. r,ı i o u< Figure 2d HPLC chromatogram of acid-hydrolyzed anthocyanin extract. The combined interpretation of these results together with relevant literature data have given idea for identification of the sunflower seed-hull anthocyanins. The anthocyanin identification scheme used is summerized in Table 2a. Tablo 2a. Identification scheme used for characterizing sunflower seed-hull anthocyanins (SSHA). (+) : Present in chromatogram (-) : Absent in chromatogram * : The peaks are numbered according to their retention times on SSHA chromatogram. Tablo 2b. Identification scheme used for characterizing grape-skin extract anthocyanins (GSE). (+) : Present in chromatogram (-) : Absent in chromatogram * : The peaks are numbered according to their retention times on GSE chromatogram.
Color has an important role in human life, both by enriching the world and by enchancing the visual appeal of foods. As long as there is continuous consumer interest in natural ingredients, there will be a market for isolated natural pigments (Meggos, 1984; Mazza and Miniati, 1993). Among the natural pigments, anthocyanins are the largest water soluble group responsible for the attractive colors ranging from salmon and pink, through scarlet, violet to purple and blue of most fruits, juices, flower petals and leaves. These ubiquitous compounds are fascinating in that it is now known that they can exist in many structural forms, both simple and complex, governed by certain physico- chemical phenemona which have profound effects on their colors and stabilities. Studies on anthocyanins cover many aspects of chemistry, biochemistry, biology, botany and medicine (Timberlake, 1980; Mazza and Miniati, 1993). Anthocyanins are glycosides of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium or flavilyum salts (figure 1). Differences between individual anthocyanins are the number of hydroxyl groups in the molecule, the degree of methylation of these hydroxyl groups, the nature and number of sugars attached to the molecule and the position of the attachment, and the nature and number of aliphatic or aromatic acids attached to the sugars in the molecule. The known naturally occuring anthocyanidins or aglycones are listed in Table 1 (Chapter 2). Since each anthocyanidin may be glycosylated and acylated by different sugars and acids, at different positions, the number of anthocyanins is 15 to 20 times greater than the number of possible anthocyanidins (Harborae and Mabry, 1982). Figure 1. Chemical structure of anthocyanins (Mazza and Miniati, 1993). In aqueous media, most of the natural anthocyanins behave like pH indicators, being red at low pH, bluish at intermediate pH, and colorless at high pH. The nature of the chemical structures which these anthocyanins can adapt upon changing the pH has already been clarified. Sunflower (Helianthus ammus, L) is one of the world's leading oilseed crops, second only to soybean in world oil production. A sunflower cultivar with red pigment in the corolla and in other part of the plant was first reported in the early 1900's; the first studies on color inheritance were also conducted during this period (Mazza and Miniati, 1993). Extracts from the purple hulls of these cultivars of sunflower have been proposed for use as natural red food colorants. Their appeal as natural ingredients also carries over to non-food uses, such as textiles and cosmetics, even though the extracts may be more expensive to produce and less stable than artificial red colorants. However, use of artificial red colorants in foods have the disadvantage of being perceived unfavorably by consumers. The purplish-red pigments in purple sunflower hulls are classified as anthocyanins. Thus, they are chemically similar to pigments in grape skin, red cabbage and some other sources of natural red colorants, and they have similar applications. Purple sunflower hull extract is not yet commercially available or approved as food colorant, but it has several desirable features. The pigment is highly stable within the hulls, and it can be extracted under mild conditions. Through conventional plant breeding, the purple-hulled trait could be bred into high-oil sunflower types. Thus, an abundant, inexpensive supply of raw material could become available as a by-product of the sunflower oil industry (Wiesenborn and et al. 1993 and 1995). The objectives of this study are the extraction of natural red pigments from the hulls of native sunflower seeds, purification, identification of their chemical structure, and evaluation of its stability. The work included the extraction of pigments by different solvents at the same temperature and pH conditions, purification of the pigments from other interfering materials and the determination of total anthocyanin content. The components of extracted pigments have been analyzed by using HPLC, and spectrophotometry techniques were used to characterize their structure. The stability of the pigments has also been evaluated by the relationship between the pigment quantity and the percent of anthocyanins remaining in the solution with respect to time. There is a general scarcity of anthocyanin standards. There are, however a number of commonly available natural materials for which the anthocyanins have been throughly characterized. These materials can often be used as "reliable references". The methods of quantitative determination, chromatographic and spectrofotometric identification, and stabilization characterization of anthocyanins in sunflower seed- hulls were also applied to the GSE (Grape Skin Extract ) which was used as reliable refrence for this study. Anthocyanins have previously been separated by a number of techniques including paper, thin-layer, and column chomatographic methods. Of these methods, paper chromatography has been the most widely used method for separation of the individual anthocyanins (Harborne, 1975; Pifferi and Vaccari, 1981; Thomas, 1984). A number of workers have reported high-performance liquid chromatographic (HPLC) methods for separation of both anthocyanins and their aglycons (Rommel, A., and et al., 1992; Bakker and et al., 1994;). One of the problems with the use of HPLC for analysis is that absolute peak retention times can very from worker to worker even under similar analytical conditions. This is made more complex by the fact that there is a general lack of availability of pure anthocyanin standards. The combination of a lack of standards and the problems with specification of absolute peak retention times are two factors contributing to the difficulty of using HPLC for analysis of anthocyanins (Hong and Wrolstad, 1990a and 1990b). The total anthocyanin pigment content was determined by using two methods: the single pH and pH differential methods. The extraction was realized with three different solvents (for sunflower seed-hulls and GSE) at the same pH and temperatures. The results are given in Table 1. Table 1. Total anthocyanin pigment contents for sunflower seed-hulls and GSE. For HPLC analysis, the anthocyanin isolate was obtained by passing it through C-18 reversed-phase-cartridge to remove other interfering phenolics. The chromatogram of the cleaned anthocyanin extract is given in Figure 2a. Auxiliary sample treatment techniques useful for pigment characterization were also applied. The first one of these was alkaline hydrolysis of the anthocyanins for determination of acylation. The second one was alkaline-borate buffer elution for separation of anthociyanins not containing an o-diphenolic system. The last one was acid hydrolysis of the anthocyanins for determination of the aglicone moiety. All the related chromatograms are given in Figure 2 b, c, d. 0-">0 J 00 2 ÛO 3.OO Figure 2a HPLC chromatogram of cleaned anthocyanin extract. 0 > 1 a '¦ /V-» O Alt' A \ - 1 - 1 0<) :>.<>. 3.0) Figure 2b HPLC chromatogram of alkali-hydrolyzed anthocyanin extract. I )l>..'. Hü I 0 BO > 0 r| * '.SO II M O.I I ^_/^V_J 0 SO 40 0. r,ı i o u< Figure 2d HPLC chromatogram of acid-hydrolyzed anthocyanin extract. The combined interpretation of these results together with relevant literature data have given idea for identification of the sunflower seed-hull anthocyanins. The anthocyanin identification scheme used is summerized in Table 2a. Tablo 2a. Identification scheme used for characterizing sunflower seed-hull anthocyanins (SSHA). (+) : Present in chromatogram (-) : Absent in chromatogram * : The peaks are numbered according to their retention times on SSHA chromatogram. Tablo 2b. Identification scheme used for characterizing grape-skin extract anthocyanins (GSE). (+) : Present in chromatogram (-) : Absent in chromatogram * : The peaks are numbered according to their retention times on GSE chromatogram.
Açıklama
Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Sosyal Bilimler Enstitüsü, 1997
Anahtar kelimeler
Antosiyaninler,
Ayçiçeği,
Tohumlar,
Özütleme,
Anthocyanins,
Sun flower,
Seeds,
Extractation