Section II Extraction, Purification and Isolation of Anthocyanins

Transcription

1 Section II Extraction, Purification and Isolation of Anthocyanins Sweet potato germplasm field showing anthocaynic leaves 2.1 Introduction The study of natural colourants is an extensive and active area of investigation due to the growing interest of substituting synthetic colourants with toxic effects in humans (Chou et al., 2007). Carotenoids and anthocyanins are amongst the most utilized vegetable colourants in the food. Among the natmal colourants, anthocyanins are the largest group of water-soluble pigments in the plant kingdom. Another significant property of anthocyanins is their antioxidant activity, which plays a vital role in the prevention of neuronal

2 Section II. f :{fraction) Purification atufisofation ofjintliocyanins and cardiovascular illnesses, cancer and diabetes (Konczak and Zhang, 2004). A good extraction procedure should maximize anthocyanins recovery and minimize degradation or alteration of the native state. The presence of acids is an important requirement to stabilize anthocyanins in aqueous environment as acidic solutions are necessary for obtaining the flavylium cation form, which is the most stable form. The acids, however, may also change the native form of anthocyanins in the tissue during extraction. Hydrochloric acid is the most common mineral acid employed in anthocyanin extraction. High concentration of acids could cause the hydrolysis of anthocyanins. A concentration of 1% hydrochloric acid in methanol was found to cause partial hydrolysis of acyl moieties in acylated anthocyanins although it is helpful to increase the extraction rate of total monomeric anthocyanins (Revilla et al., 1998). Extraction with solvents containing Hel may cause pigment degradation during concentration. That is why acylations with aliphatic acids had been usually underestimated (Strack and Wray, 1989). To minimize the degradation of anthocyanins, weaker and volatile organic acids such as trifluoroacetic acid, about 0.5%-3%, are used in the solvents (Revilla et al., 1998). Many solvents can be used in extraction, such as methanol, acetone, ethanol and water, due to the polar character. Most anthocyanins occur naturally as glycosides. Anthocyanin glycosides have higher solubility in water than the corresponding aglycones. Giusti and Wrolstad have developed a method to extract anthocyanins, which involves powdering a sample with liquid nitrogen, extraction with aqueous acetone (30:70. v/v) followed by partition with chloroform (Giusti and Wrolstad, 1996). The young and emerging purple coloured leaves of sweet potato have been known as a potential source of anthocyanin (Jyothi et al., 2005). For a commercial production process, it is important to shorten the extraction time and to increase the extractability from natural sources. This section deals with 29

3 Section II. raction, Purification and Isofation of )fotfiocyanins the studies undertaken to optimize the conditions for the extraction of anthocyanin from the young and emerging purple coloured leaves of sweet potato using different solvent systems and also to purify and fractionate the pigments. The spectral properties of the pigments are also studied. 2.2 Materials and Methods Materials All the plant materials required for the anthocyanin estimation were collected from the experimental fields of CTCRI (Central Tuber Crops Research Institute, Thiruvananthapuram, India). From the germplasm field, genotypes having purple coloured leaves were selected for anthocyanin estimation. In majority of cases, only the young and emerging leaves at the apex of sweet potato vein ( 4-6 leaves from top) are pink coloured, but in the case of S-purple, almost all the leaves are purple coloured. Amberlite XAD-7 and Sephadex LH-20 were purchased from Sigma-Aldrich and all other organic solvents used were of analytical grade. Spectral analyses were done on a JASCO V-550 spectrophotometer. The HPLC equipment consisted of a liquid chromatograph system (Gilson, France) equipped with Gilson UV-Vis 156 detector attached to a Gilson 321 binary gradient pump. Extract concentration / was done in a Buchi R-200 (Buchi, Labortechnik, Switzerland) rotary evaporator. Remi Incubator (India), which operates in a temperature range, 0- -C, was used Experimental Methods Extraction of anthocyanin The solvent extraction has been the most common method for extraction of diverse compounds found in plants, including flavonoids. The phenolic compounds have been extracted by grinding, drying or lyophilizing the samples 30

4 Section II. P.x:J;raction} Purification andisofation offlntfwcyanins or only by soaking fresh. samples with subsequent solvent extraction (Merken and Beecher, 2000). The isolation, purification and structure determination of pure anthocyanins are time consuming processes and because most anthocyanins are prone to be unstable, these processes must be conducted with care. During the workup procedure, the anthocyanins may also become more fragile because of the removal of free sugars and other acylating groups which stabilize the pigment. Storage of anthocyanins in the dark, at low temperature and in the dry state, to reduce hydration and degradation, is therefore preferable. Typical procedures for isolation and characterization of pure anthocyanins consist of several steps: (1) extraction of the plant material, followed by a preliminary purification, (2) fractionation of the mixture followed by isolation of pure pigments and finally (3) characterization and identification of pure anthocyanins (Strack and Wray, 1989). Organic solvents efficiently extract anthocyanins by destroying cell membranes to dissolve the pigments from vacuoles. Traditionally, acidified organic solvents are used, with methanol being the most effective solvent. Solvents are typically acidified with weak organic acids such as formic, acetic or citric acid or low concentrations of strong acids such as hydrochloric or trifluoroacetic acid. Weak organic acids denature the cellular membranes to facilitate solubilization of the pigments, but addition of excess acid can result in hydrolysis of labile, acyl and sugar residues and may break down complexes with metals and co-pigments (Shahidi and Naczk, 2004). Furthermore, higher temperatures have been found to improve efficiency ofextraction, but can also increase the rate of anthocyanin degradation. Anthocyanins are polar molecules, thus the most commonly used solvents for the preparation ofplant extracts are methanol, ethanol and acetone, at a composition of70-80% in water (Kahkonen et al., 2001). 31

5 Section II. (traction, Purification and Isofation of Jfotliocyanins l.l',1 I Sample Extraction with Methanol-acid Filtration I Solvent Extraction "ith Ethyl Acetate/Chloroform I Di scard the Ethyl Acetate /Chloroform layer +-- Freeze dryino/tlash i-, e, aporation I Purification -Amberliie :\.AD-7 chromatography I Fnwtio,rntion - SPphafl, I J-1-20 in f'".fh.' i(ln fhromatography Figure 2.1: Flow chart for the extraction and fractionation of anthocyanin from sweet potato leaves 32

6 Section II. p.:{!raction, CFurification atufisol4tion of)1.ntliocyanins Among the most common methods are those which use acidified methanol or ethanol as extractants (Amr and AI-Tamimi, 2007). After extraction, the extracts of anthocyanins are concentrated under vacuum, and purified using chromatographic techniques. A flow chart for the extraction and fractionation of anthocyanin from sweet potato leaves is given in Figure 2.1. The procedure must allow for recovery of the anthocyanins avoiding any chemical modification. The effect of various extraction solvent systems at various ph values and temperatures on anthocyanin extraction is discussed below. As a preliminary work, foliar anthocyanins present in various tuber crops were estimated. Fresh leaves were used for all the experiments and pigment was extracted using the following procedure. The leaves were chopped and weighed accurately (2.5 g). The sample was then homogenized with 50 ml of the extracting solvent, which was prepared by mixing 80 ml of methanol, O.lml concentrated HCI and 19.9 ml of water. The homogenized samples were kept overnight at 4 cc. The extract was then filtered through a Buchner funnel using Whatman No.1 filter paper and 8 ml of solvent was added to the residue; this procedure was repeated as many times as required until all colour had been extracted from the leaf tissues. Finally, the extracts' were combined and partitioned with chloroform to free it from chlorophyll and stored at -18 cc. Sweet potato germplasm varieties like S-402, S-406, S-347, S-480, S 491, and S-purple were screened. Pigments present in the leaves ofsweet potato variety, Sree Ratna, a released variety from CTCRI, were extracted using the above solvent. Anthocyanin present in young leaves ofyam, Orissa Elite and pink Xanthosoma were also extracted using methanolic HCI and were kept at 18 cc until analysis. 33

7 Section II. P.:{Jraction, <Purification an Isofation of )lntfiocyanins For comparative studies, anthocyanins present in commercial grape seed skins were also extracted and estimated spectrophotometrically. Flower samples collected from the sweet potato variety S-31 were also tested for plant pigments Extractability using different solvent systems A comparative study of extraction methods using acetone, methanol, and isopropanol was made. Six different combinations of solvents were prepared as follows ml acetone+ 0.1ml concentrated HCl ml water ml acetone + 2ml 20% trifluoroacetic acid + 18 ml water ml methanol+ 0.1ml concentrated HCl ml water ml methanol + 2ml 20% trifluoroacetic acid + 18 ml water ml isopropanol + 0.1ml concentrated HCl ml water ml isopropanol + 2ml 20% trifluoroacetic acid + 18 ml water Fresh leaves were used for all the experiments. The leaves were chopped and weighed accurately (2.5 g). The sample was then homogenized with 50 ml of the extracting solvent and extracted as detailed above Total monomeric anthocyanin The measurement of total anthocyanin pigment provides a basis for comparison and is useful in assessing the colour quality of many foods. Accurate quantification of anthocyanins and their degradation is of special interest when comparing fresh and processed fruits and vegetables and can also be a useful tool in assessing the colour quality of food colourants. The total anthocyanin content in crude extracts containing other phenolic materials has been determined by measuring absorptivity of the solution at a single 34

8 Section II. P.:{j;raction, PurifUation atufisocation offlntliocyanins wavelength. This is possible because anthocyanins have a typical absorption band in the 490 to 550 run region of the visible spectra. This band is far from the absorption bands of other phenolics, which have spectral maxima in the UV range (Fuleki and Francis, 1968a). In many instances, however, this simple method is inappropriate because of interference from anthocyanin degradation products or melanoidins from browning reactions (Fuleki and Francis, 1968b). In those cases, the approach has been to use differential and/or subtractive methods to quantify anthocyanins and their degradation products. Francis (1989) suggested ph values of 1.0 and 4.5 when working with cranberries and these values serve as the basis for the ph-differential method (Wrolstad and Guisti, 2001; Francis, 1989). The ph-differential method is based on the reversible transformations that occur in anthocyanins with a change in ph. At ph 1.0, the flavylium cation is found in the coloured oxonium form, while at a ph 4.5, the colourless hemiketal form dominates. Methods that utilize spectroscopy allow for rapid and easy quantification of monomenc anthocyanins, even in the presence of polymerized degrading pigments and interfering compounds. Throughout this work, anthocyanins were quantified by the ph-differential method (Rodriguez-Saona et ai., 2001; Guisti and Wrolstad, 2001). Two dilutions ofthe sample were prepared, one with potassium chloride buffer (0.025M), ph 1.0, and the other with sodium acetate buffer (OAM), ph 4.5, and th.ese dilutions were allowed to equilibrate for 15 minutes. The absorbance of each dilution at the visible maximum (M max ) and at 700 run (to correct for haze A 700 ) were measured against a blank cell filled with distilled water. The absorbance ofthe diluted sample (A) can be calculated as follows: Monomeric anthocyanin pigment (mg/litre) = (A x MW x DF x 1000) -+- ( x 1) MW is the molecular weight; DF is the dilution factor, and the molar absorptivity. The MW used in this formula corresponds to the predominant 35

9 Section II. ~raction) Purification arufisocation ofjf.ntliocyanins anthocyanin in the sample. Ifthe molar absorptivity ofthe major pigment is not available or if the sample composition is unknown, pigment content is calculated as cyanidin-3-glucoside, where MW = and molar absorptivity = 26,900, the molar absorptivity reported in the literature for the anthocyanin pigment in acidic aqueous solvent. Quartz cuvettes of 1 cm path length (1) were used (Francis, 1989) Effect ofph on anthocyanin extraction This experiment was carried out in methanol-hci extracting system at different ph values (1, 2, 3, 4 and 5). The extracted anthocyanin concentration at different ph values was determined as explained above. Fresh emerging leaves of 8-406, and were used as anthocyanin sources. Three replications were done at each condition Effect of temperature on anthocyanin extraction Fresh young leaves of and were collected from field. A fixed weight (2.5 g) of the samples after homogenization with 50 ml of the extracting solvent (methanol-hci), were kept overnight at three different temperatures viz; 30, 35 and 40 C in an incubator. The extracted anthocyanin. concentration was determined spectrophotometrically. The measurements were done in triplicate Purification ofanthocyanins 1. Liquid-liquidpartition The anthocyanins extracted usmg methanolic HCI were purified by partition against chloroform to remove chlorophylls, stilbenoids, less polar flavonoids and other non polar compounds from the mixture. The extract was then concentrated by flash evaporation in rotary evaporator (Buchi R-200). 36

10 Section II. ~raction, (PUrification anaisocation of)f.ntfwcyanins 2. Amberlite XAD-7 adsorption chromatography The extracts obtained after the liquid-liquid partition step will also contain water soluble compounds other than anthocyanins, namely free sugars and aliphatic acids. These non-aromatic compounds were removed with the use of Arnberlite XAD-7 column chromatography (Andersen, 1988). Arnberlite XAD-7 adsorbs the aromatic compounds including anthocyanins and other flavonoids in aqueous solutions, whereas free sugars and other polar nonaromatic compounds were removed by washing with distilled water until the eluted water has a neutral ph. Then, the adsorbed anthocyanins were eluted using methanol containing 0.5% TFA as mobile phase (Andersen, 1988). The anthocyanins from sweet potato genotypes S-406 and S-purple were purified and for comparative studies, grape pomace anthocyanins also were extracted and purified Sample fractionation and isolation of pure pigments by gel filtration column chromatography In this work Sephadex LH-20 was used as column material. This material permits separation with respect to molecular size. The purified XAD-7 extracts were dissolved in a small amount of the initial mobile phase (MeOH: H 2 0: TFA; 20:80:0.5, v/v) (Andersen and Francis, 2004). Separation was achieved by isocratic or gradient elution using increasing amounts ofmethanol. Since the column functions on the size-exclusion principle, the anthocyanins may be mainly eluted in order of decreasing molecular mass. Anthocyanidin triglycosides are eluted prior to anthocyanidin di-glycosides followed by anthocyanidin monoglycosides. Acylated anthocyanins are usually more retarded than non-acylated anthocyanins because of increased adsorption (Jordheim et al., 2007). The anthocyanin containing fractions were collected on the basis ofobserved visual band separation and the spectra were recorded. 37

11 Section II. P.:(Jraction, (furi.fieation amiisofation of;4ntliocyanins High performance liquid chromatography High performance liquid chromatography (HPLC) has been the method of choice for the qualitative and quantitative analysis of anthocyanins. This is because of the capability of LC in preparation of samples on the gram scale using preparative-hplc and the purification of samples on the milligram scale with semi preparative-size LC columns and on the microgram scale with analytical-size column. HPLC is also used for identification of individual natural compounds in the plant matrices using coupled techniques such as HPLC with UV- Vis spectrophotometry detection (LC-UV), with mass spectrometry detection (LCMS), or with nuclear magnetic resonance detection (LC-NMR). There is not a single standard procedure for the analyses of anthocyanins using HPLC. Rather, there are a wide variety of columns and parameters that have been used in anthocyanin characterization from the same plant source resulting in equivalent separations. A high proportion of isolation methods for anthocyanins are generally run on reverse-phase columns, such as octadecyl silane (ODS), polystyrene or phenyl-bonded columns (da Costa et al., 2000). Also, HPLC methods tend to utilize gradient solvent systems of acetonitrile-water or methanol-water with a small amount of acid to lower- the solution ph and increase stability ofthe anthocyanins. These solvents are most popular due to their compatibility with gradient methods for isolation and the various detection methods coupled to the HPLC used for identification. To obtain reproducible results using this instrumentation, the ph of the mobile phase and temperature ofthe column must be controlled due to the instability of anthocyanin compounds in changing ph and temperature environments. For optimal results, acidic mobile phases lower than ph 2.0, are employed to ensure the anthocyanin remains in the more stable flavylium form and to reduce peak tailing of the chromatogram (da Costa et al., 2000). In reverse-phase chromatography, the retention time decreases with increasing polarity, which corresponds to increasing number of hydroxyl moieties on the flavylium ion 38

12 Section IL ~raction, (}!urification atufisofation of)f.ntliocyanins and the elution order ofdelphinidin, cyanidin, petunidin, pelargonidin, peonidin and malvidin. Due to reverse-phase elution, diglycosylated anthocyanins will have the lowest retention time, followed by monoglycosylated anthocyanins, aglycones and lastly the acylated anthocyanins (Mazza et al., 2004; da Costa et al., 2000). In the acidic media, the flavylium cation is red coloured and gives an absorption maximum around 520 nm, which avoids interference from other flavonoids that may be present in the plant extract. Because of these unique absorbance maxima, anthocyanins have been accurately identified and quantified from very crude plant extracts (da Costa et al., 2000). Nonnal phase systems or those using unmodified silica gel are not effective for the separation ofanthocyanins due to the polarity ofthese compounds (Andersen et al., 2006). The main drawback ofusing HPLC method for anthocyanin identification is the considerable difficulties in obtaining good quality standard samples of anthocyanin compounds, even from prominent chemical companies. In the absence of standards, many researchers identify anthocyanins by comparing the peaks with authentic standard peaks reported elsewhere. Identification of anthocyanin compositions by reverse phase HPLC was done by following the method ofislam et al. (2002). The HPLC analysis was carried out on a reversed-phase C I8 column, Kromasil 100 (5 ~m, 4.6 X 250 mm). The HPLC equipment consisted of a liquid chromatograph system (Gilson, France) equipped with Gilson UV-Vis 156 detector attached to a Gilson 321 binary gradient pump. The chromatographic data were analyzed with Gilson Unipoint software. A gradient solvent system was used for the separation of anthocyanins in a single HPLC run. The solvents used for separation were, (A) 1.5% phosphoric acid and (B) 1.5 % phosphoric acid, 20% acetic acid and 25 % acetonitrile. The elution profile was a linear gradient elution with 15-50% solvent B in solvent A for 90 minutes. In all experiments, 20 ~l ofthe leaf extract was injected and solvent 39

13 Section II. raction, Purification ana Isofation of }f.ntnocyanins flow rate was lml/minute and column pressure 3200 Psi. All solvents were of HPLC grade filtered through a 0.45-pm Durapore filter (Millipore, Bedford, MA). Identification of anthocyanins was done by comparing the retention time of peaks with those of the authentic standard of purple fleshed sweet potato anthocyanins (Islam et al., 2002; Terahara et al., 2004). The anthocyanin extracts from two sweet potato genotypes, S-406 and Orissa-3 were analyzed byhplc. 2.3 Results and Discussion Preliminary extraction A wide variability was found to exist in the anthocyanin content of sweet potato leaves, with values ranging from mg/loog fresh weight (FW) of the selected samples (Table 2.1 ). Among the crops selected for analysis, the highest value of anthocyanin content now observed was for the sweet potato variety, S-406 (165.0 mg/loog) followed by S-402, S-480, S-491 and S-purple. These results are in conformation with the results of Jyothi et al. (2005). These results show that sweet potato leaves contain considerable amount of anthocyanin and hence can be utilized as a cheap natural source of these pigments. Table 2.1: Anthocyanin contents in selected tuber crops Sample Anthocyanin content mg/100 g fresh weight S S S S S S-Purple Flower(S-31) Orissa Elite Sree Ratna

14 Section II. ~raction, Purification adisocation of;4.ntfwcyanins Extractability of different solvent systems Figure 2.2 shows the comparison of extractability of anthocyanins from S-406, S-408 and S- 480 when different solvent systems were used. In all cases, 1.0g of fresh, homogenized leaf samples were taken and extractions were carried out in triplicate, and it was found that methanol-hci and isopropanol HCI solvent gave good quantitative extraction when compared to the other four solvents. Methanol-HCI system gave a slightly better extraction than isopropanol-hci system, this could be due to higher polarity of methanol and the ability to diffuse into cell membranes is better for methanol compared to isopropano1.,-....c tl!l 'a:; ~ 200 ~ 150 a.>..:: tl!l c:> c:> ~ E ':: 100 =.2:l = Q<.l = 'c~ ~ 50 Q.c-= ~ o solvent system used Figure 2.2: Comparison of anthocyanin extractability of different solvent systems for sweet potato genotypes, S-406, S-408 and S (1 Acetone and HCI, 2-Acetone and TFA, 3- Methanol and HCI, 4 Methanol and TFA, 5- Isopropanol and HCI, 6- Isopropanol and TFA) 41

15 Section II. ~raction, CPu:ri.fication atufisofiltion of;f.ntfwcyanins The effect of ph on anthocyanin extraction The results obtained after extracting at different ph levels are shown in a Table 2.2. There was no marked change in the amount of anthocyanin extracted when ph of the medium was increased from 1 to 2; however, a noticeable decrease in the pigment extraction was observed when ph was further increased from 2 to 5. The colour and stability of an anthocyanin in. solution is highly dependent on ph. Anthocyanins are most stable and most highly coloured at low ph values and gradually lose colour as the ph increases, becoming almost colourless between ph 4.0 and 5.0 (Ozela et al., 2007). Therefore, an increase in ph may lead to a reduction of pigment stability and results in a decrease in the extraction yield. This is clear from the low extractability at ph 5.0. Additionally, a low ph may Increase the cell membrane permeability leading to higher diffusion coefficient values for the extracting solvent. Table2.2: Anthocyanin extracted at different ph values Source ofleaf Anthocyanin extracted, mg/100g* (fresh weight) at ph sample S ± ± ±.11 98±.28 69±.21 S ± ± ± ±.22 71±.15 S ± ± ±.13 79±.24 49±.19 * - values with standard deviation obtained from three replications The effect oftemperature on anthocyanin extraction The results obtained after extracting anthocyanins at different temperatures are shown in Table 2.3. There is obviously an increase in extraction efficiency as temperature is increased. In addition to the effect of temperature, denaturation ofthe cell membranes might also occur at higher 42

16 Section II. P.xJraction, lrification anaisofation ofjintliocyanins temperatures, affecting the extraction process. High temperature renders the plant cell wall permeable and increases solubility and diffusion coefficient of compounds and decrease the viscosity of solvents, thereby a resulting improvement of the efficiency of the extraction. However, anthocyanins are sensitive to heat and easily convert to the colourless chalcone form during heating (Wrolstad et al., 2002). Hence, it can be inferred that temperature ofthe extracting medium cannot be increased indiscriminately to maximize the process. Temperature and ph ofthe extraction medium were found to affect the anthocyanin extraction. Therefore, shortening the extraction time by employing a low ph (below 2.0) and a high temperature (40 C) may be considered to be better suited and more feasible for an industrial process. Table 2.3: The effect oftemperature on anthocyanin extraction Sample Anthocyanin extracted, mg/l00g* 30 C 35 C 40 C S ± ± ±.18 S ± ± ±.49 * - values with standard deviation obtained from three replications UV-Visible absorption spectra of purified and fractionated anthocyanins Figure 2.3 (A and B) shows the UV-Visible absorption spectra of purified anthocyanins from S-406 and S-purple. The absorption maxima in the visible region are mainly dependent of the nature of the aglycone, the position of sugar substituents on the aglycone and the presence of aromatic acyl groups. Secondly, spectral measurements are of value in determining the position of attachment of the sugars in the anthocyanin molecular framework. The most common sugar moieties are glucose, galactose, rhamnose, xylose, arabinose, as mono-, di-, and tri-glycosides. The sugars may be acylated with aromatic acids, 43

17 Section II. ~raction, CFuri.fication atufisofation of;fntfiocyanins such as p-coumaric, caffeic, ferulic, sinapic, gallic or p-hydroxybenzoic acids or aliphatic acids, such as malonic, acetic, malic, succinic or oxalic acids (Robbins, 2003). Confirmation of the presence (or absence) of a hydroxy aromatic acid as an acyl component can be made by spectral means, as has already been indicated (Harbome, 1958; Hong and Wrolstad, 1990). Acylated anthocyanins indicate a low sensitivity to ph changes and possess higher colour stability in neutral and alkaline solutions. This increased stability is due to the fact that acylated anthocyanins are more resistant to hydration of the flavylium ion leading to the equilibrium pushed more towards quinonoidal base forms (Torskangerpoll and Andersen, 2005). In effect, the acylating groups encourage the production ofthe bluer pigmentation that glucosidic moieties bring forth but also counteract the instability attributed to the sugars. A mechanism called intramolecular/ intermolecular copigmentation was introduced to illustrate the increased stability of acylated anthocyanins (Mazza and Miniati, 1993; Harbome and Williams, 2000). The charged C ring in anthocyanidins can react with nucleophiles as an electrophilic center. The acyl groups not only affect the colour but also protect the chromophores from the nucleophilic attack through an intermolecular stacking of chromophore in a planar or a sandwich spatial structure for anthocyanins containing two or more aromatic acyl groups (Giusti and Wrolstad, 2003). The spatial proximity between the hydrogens from aglycone and acyl groups in radish was observed to be very close to each other in two-dimensional NMR spectroscopy analyses (Giusti et al., 1998). The spectra of anthocyanins acylated with p-coumaric acid show two peaks in the ultraviolet at 289 and 310 nm, due to the superimposition of the absorption of the cinnamic acid upon that of the pigment absorption. The spectra of simple anthocyanins exhibit a simple peak at about 270 nm and only very weak absorption at 310 nm (Harbome, 1958). Figure 2. 3 (A) shows both S-406 and S-purple show strong peaks around 310 nm showing a high degree 44

18 Section II. ~ractionl Cl'urification atufisofation offlntfwcyanins ofacylation and greater stability compared to other anthocyanins. Glycosylation at the 3 and 5 positions ofthe anthocyanidin molecule has an important impact on the spectral characteristics, and extensive research has been published in that area (Harborne, 1967; Giusti and Wrolstad, 1996). The most important spectral parameters derived from the UV-Vis absorption spectra of anthocyanins are; AVis-max, the absorption (A) at A = 440 nm compared to A at AVis -max (A 4401A Vismax), and A at AUv-max compared to A at AVis-max (A Uv- max 1 AVis-max)' For anthocyanin 3-0-glycosides having a free 5-hydroxyl, the value of A 4401 A Vismax is in the range of Harborne found that, when the 5-position in the flavanoid structure is glycosylated, the value of A AVis-max is in the range of (Harborne, 1958). From Table 2.4 it can be inferred that for anthocyanins of S-purple and S-406, the value ofa 440 1AVis-max is in the range of showing that the 5-position is glycosylated. For grape pomace anthocyanins the value of A Avis-max is 0.36, indicating that the anthocyanin 3-0-glycosides having a free 5-hydroxyl group. The presence of aromatic acylation can be determined using the ratio Auv-maxl AVis-max. Typical monoaromatic acylation of anthocyanins may give an Auv-max 1AVis-max ratio of ~ A higher ratio may indicate several aromatic acyl residues (Ando, et ai., 1999). From the values presented in Table 2.4, it can be concluded that for anthocyanins of S-purple and S-406, the value ofa uv -max 1 AVis-max is a higher ratio (more than 10) indicating that several aromatic acyl residues are present in the sweet potato leaf anthocyanin molecule. S-Purple anthocyanins have a greater degree of acylation compared to the anthocyanins of S-406 and hence more stable and this is confirmed by the stability studies carried out in our laboratory (Rajeswari et ai., 201 Oa). The value of Auv-max 1 AVis-max obtained for grape pomace anthocyanins is very low (1.0) as compared to those extracted from sweet potato leaves. Hence it can be 45

19 Section II. P.tf;raction, <Purification anti IsoCation of )f.ntli.o cy anins (A) --S-purple --S grape pomace 2.5 ctl.c.c ctl wavelength(nm) (B) --S31 -- OrissaElite -- Xanthosoma 3.0 Cl) 2.5 ctl.c 2.0.c ctl wavelength Figure 2.3: The UV-visible absorption spectra of XAD-7 purified anthocyanins from S-purple, S-406 and grape pomace (A)and Orissa elite, S-31 and Xanthosoma(B) sources 46

20 Section II. ~raction, (]!Urifieation atufisocation of)f.ntfwcyanins concluded that stability of foliar anthocyanins of sweet potato are higher than those of pomace pigments. Since aliphatic groups do not have significant UV Vis absorption, their presence can not be directly detected by UV-visible spectroscopy. Similarly, from Figure 2.3 (B) it can be estimated that Auv-max 1 AVis-max value of anthocyanins from pink Xanthosoma, Orissa Elite and S-31 is considerably high compared to that of grape pomace anthocyanin, indicating the higher stabilityofthe corresponding pigments. Table2. 4: Spectral details ofanthocyanins extracted from S-purple, S-406 and grape pomace Absorbance S-purple S-406 Grape pomace A 440nm A Vis-max A Uv-max A 440lA Vis-max A Uv-maxlA Vis-max The fractionation of anthocyanins by Sephadex LH-20 The absorption spectra of fractionated anthocyanins using Sephadex LH-20 size exclusion chromatography are shown in Figure 2.4. Three fractions were obtained for S -406, having AVis-max at 527 nm, 528 nm and 529 nm respectively and for S -purple also three fractions were obtained having AVis-max at 524 nm, 525nm and 526 nm respectively. The value of A 4401A Vis-max calculated for the first, second and third fractions of the pigments of S-406 are 0.19, 0.18 and 0.17 respectively, indicating that the 5 position is glycosylated. The value ofa Uv-max I AVis-max for the first, second and third fractions of S-406 anthocyanin are 2.2, 2.5, 5.6 respectively, showing that the extent ofacylation is more for the third fraction when compared to the other two fractions. Similarly for S-purple also, the third fraction is more acylated and in all the three fractions, the 5-position is glycosylated. 47

21 Section II. f t!;raction, CJluri.fication atufisofation ofjfntliocyanins 5 4 (A) --fraction 1 --fraction 2 --fraction 3 Ql g 3 I'll.Q... o III.g 2 1 o+-----r :;::=:::;:=::::::;:=~~-~...,._---r'-_,..._----, wavelength (A) --fraction 1 --fraction 2 --fraction 3 Ql g 3 I'll.Q... o III.Q 2 I'll 1 o+---,-~:::::::=~::::;:~~!!t-~---,-...,----r------, wavelength Figure 2.4: The absorption spectra ofsephadex fractionated anthocyanins extracted from S-purple (A) and S-406 extract (B) 48

22 Section II. P.J(f;raction, (}!uri.fieation anaisofation ofjfntftocyanins The HPLC chromatographic separation and characterization of anthocyanins HPLC chromatographic pattern ofthe anthocyanins of S-purple leaves is shown in Figure 2.5. Among the peaks in the chromatogram, nine peaks could be identified as due to known anthocyanins, by comparing with the retention times with those of authentic standard peaks of purple fleshed sweet potato anthocyanins (Islam et al., 2002; Terahara et al., 2004) and their chemical names are shown in Table 2.5. The major peaks, 8, 14 and 17 were identified as caffeoylated peonidin 3-sophoroside-5-glucoside, peonidin 3-(6,6'-caffeoylferuloylsophoroside)-5 glucoside and cyanidin 3-0-(2-0-(6-0-(E)-p-coumaroyl-~-D-glucopyranosyl) 6-0-(E)-p-coumaroyl-~-D-glucopyranoside)-5-0-~-glucopyranosiderespectively by comparing the peaks with standard peaks (Islam et al., 2002; Terahara et al., 2004). In the leaves of S-purple, the major pigments are peonidin type and are acylated with aromatic acids. Sample SP 2.5 <t..,. t--..,. (Il ~ E 0.0 o Minutes Figure 2.5: HPLC chromatogram of the pigment of the purple sweet potato leaves (Ipomoea batatas L.), S-purple 49

23 Section IL ~raction, fpurification atufisolation of)f.ntfiocyanins The reversed phase-hplc-chromatogram ofthe anthocyanins of Orissa 3 is shown in Figure 2.6. Nine peaks were identified which include the major peaks and their chemical names are shown in Table 2.5. The single, major anthocyanin present in Orissa-3 leaves is caffeoylated (cyanidin 3-sophoroside-5-glucoside), peak No.2. The second largest peak in terms ofpeak area is peak No.7, which corresponds to feruloylated (cyanidin 3 sophoroside-5-glucoside). Thus, we now confirm that the major pigments present in foliar anthocyanins of the sweet potato genotype Orissa-3 are of cyanidin based and are acylated with caffeic and ferulic acids. Table 2.5: Chemical names corresponding to the anthocyanin peaks in the HPLC chromatograms of foliar anthocyanins of S-purple and Orissa-3 Peak No. S-purple Peak No. Orissa Anthocyanin identified (S-purple) Cyanidin 3-sophoroside-5-glucoside Peonidin 3-sophoroside-5-glucoside p-hydroxybenzoylated (cyanidin 3-sophoroside-5-glucoside) p-hydroxybenzoylated (peonidin 3-sophoroside-5-glucoside) Caffeoylated (peonidin 3-sophoroside-5-glucoside) Feruloylated (cyanidin 3-sophoroside-5-glucoside) Cyanidin 3-(6,6'-caffeoylferuloylsophoroside)-5-glucoside Peonidin 3-(6,6'-caffeoylferuloylsophoroside)-5-glucoside Cyanidin 3-0-(2-0-(6-0-(E)-p-coumaroyl-~-D-glucopyranosyl) 6-0-(E)-p-coumaroyl-~-D-glucopyranoside)- 5-0-~-D glucopyranoside(e- p-coumaryl) Anthocyanin identified (Orissa-3) Caffeoylated (cyanidin 3-sophoroside-5-glucoside) p-hydroxybenzoylated (peonidin 3-sophoroside-5-glucoside) Caffeoylated (peonidin 3-sophoroside-5-glucoside) Feruloylated (cyanidin 3-sophoroside-5-glucoside) Cyanidin 3-(6,6'-caffeoyl-p-hydroxybenzoylsophoroside)-5 glucoside Cyanidin 3-(6-caffeoylsophoroside)-5-glucoside Cyanidin 3-(6,6'-caffeoylferuloylsophoroside)-5-glucoside Peonidin 3-(6,6'-dicaffeoylsophoroside)-5-glucoside Peonidin 3-(6,6'-caffeoylferuloylsophoroside)-5-glucoside 50

24 Section II. ~raction, (purification arufisocation of;f.ntfiocyanins 5.0 Sample FS 2.5 o Minutes Figure 2.6: HPLC chromatogram of the pigment of the purple sweet potato leaves, Orissa-3 From the above results, structures ofthe major foliar anthocyanins ofthe sweet potato genotypes S-purple and Orissa-3 are presented in Figure 2.7. Depending on the nature of R I, R 2 and R 3, there are different types of anthocyanins. Based on the aglycon present in the structure, anthocyanins can be classified as peonidin type and cyanidin type. In S-purple, the predominant anthocyanins are ofpeonidin type and in S-406; they belong to the cyanidin group. Terahara et al. studied the stability of anthocyanins having various degrees of acylation and found that diacylated anthocyanins were more stable than monoacylated and nonacylated ones (Terahara et al., 2004). The effect of the substituents on the stabilities of the anthocyanins suggested that the two aromatic acids protect the aglycon nucleus by a sandwich-type hydrophobic stacking mechanism and inhibit the attack of a water molecule that leads to a loss of colour. The studies conducted in our laboratory on the storage stability of foliar anthocyanins of sweet potato also show similar results (Rajeswari et al., 2010a). In addition to this, Terahara et al., (2004) reported that cyanidin 51

25 Section II. P.:(f:raction, <Pu,rification ana Isolation of )1.ntfwcyanins HO Pigment R1 R2 R3 Cyanidin 3-0-(2-0-( 6-0-(E)-p-coumaroy I -P-D-glucopyranosy 1)- 6-0-(E)-p-coumaroy I H p-coumaric p-coumaric -P-D-glucopyranoside)-5-0-P-D-glucopyranoside Caffeoy lated ( cyanidin 3-sophoroside-5-glucoside) H Caffeic H Peonidin 3-( 6,6' -caffeoy lferuloy lsophoroside) -5-glucoside CH3 Ferulic Caffeic Figure 2.7: Structures of predominant anthocyanins present in sweet potato genotypes S-purple and Orissa-3 based pigments exhibited higher free radical scavenging activity than peonidin based ones and the acylated anthocyanins showed higher antioxidant ability compared to nonacylated pigments. Both the anthocyanin extracts studied here, isolated from S-406 and Orissa-3, are acylated and hence showed extra stability as a food colourant and as a nutritional additive. The pigment profile of anthocyanin-rich extracts has been suggested to impact biological activity: purified cyanidin and delphinidin exhibited superior anticancer activity (Wang et al., 1997) to other types of anthocyanins such as peonidin. The major 52

26 Section II. f :{tractiot; CJ!urification atufisofation of}f.ntliocyanins pigments in Orissa-3 are cyanidin based and hence these may prove to be good chemo preventive agents Conclusion The results of the present study show that anthocyanins ofsweet potato leaves can be easily extracted with methanolic HCI, preferably at ph levels less than 2.0 and at an extraction temperature of 40 C. The foliar anthocyanins of the sweet potato genotypes and 8-purple are more stable compared to the anthocyanins of grape pomace as evidenced by their UV-visible spectra. Among the two sweet potato anthocyanins studied, 8-purple anthocyanins are. more acylated and hence more stable than that of The foliar anthocyanin content of8-406 and 8-purple lies in the range mg/loo g fresh weight, which is higher than the reported value ofabout mg anthocyanins/g fresh weight for purple-fleshed sweet potatoes developed in Japan (Furuta et al., 1998). HPLC studies on the pigments of sweet potato genotypes Orissa-3 and 8-purple indicate that the predominant pigments are cyanidin and peonidin based anthocyanins acylated with caffeic, ferulic and coumaric acids. This acylation with aromatic acids imparts greater stability to the pigment. The results ofour study indicate that th~ sweet potato leaves can be used as a cheap source of anthocyanin pigments and these findings may be useful for further optimization studies to design a commercial production of anthocyanins from sweet/potato leaves. 53