Identification of an Anthocyanin Compound from Strawberry Fruits then Using as An Indicator in Volumetric Analysis
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Diyar Salahuddin Ali
Salahaddin University - Science College - Chemistry Department, Erbil-Iraq

INTRODUCTION

Strawberry fruits (Fragaria ananassa) have been shown to possess high in vitro antioxidant activity that has been positively correlated with the content of polyphenolic compounds and, specifically, anthocyanin, the type of polyphenols quantitatively most important in strawberry¹.

The anthocyanin composition in strawberry has been the important matter of various studies, but is still not fully characterized regarding minor pigments. Strawberry anthocyanin derives from pelargonidin (Pg) and cyanidin (Cy) aglycones as shown in Fig. 1².

Fig. 1: Structures of main pigments which occur in Strawberry (Anthocyanin aglycones and with sugar attached)².

New HPLC conditions were developed for the separation of strawberry anthocyanins that provide a good resolution of peaks at low flow rate, compatible with the requirements of the mass spectrometry (MS) detector³.

The use of on-line photodiode array detection (DAD) coupled to HPLC has been shown to be a useful tool in assisting the characterization of anthocyanins. The UV-visible spectra of the anthocyanins yield information with regard to the nature of the aglycone and the sugar substitution pattern, while the retention characteristics on reversed-phase HPLC are closely related to their polarity and yield information on the nature of the sugar moieties, acylation and substitution of the anthocyanin B-ring using this technique⁴.
Various anthocyanins have been detected in strawberries^5,7. All researchers find that the cyanidin-3-glucoside, pelargonidin-3-glucoside, pelargonidin-3-rutinoside and pelargonidin-3-glucoside acylated with acetic acid are the major anthocyanins in strawberry.

According to the polarity property some scientists identified that the cyanidin-3-glycoside as the most polar anthocyanin in strawberry, which eluted followed by pelargonidin-3-glycoside and so on for other pelargonidin derivatives³.
Strawberry (Fragaria Ananassa.) fruits contain phenolic compounds that have antioxidant, anticancer, and anti-neurodegenerative properties⁸. Freeze-dried whole strawberry fruit powder and strawberry fruit extracts were analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) methods.
Phenolics were identified as ellagic acid (EA), EA-glycosides, ellagitannins, gallotannins,
anthocyanins, flavonols, flavanols and coumaroyl glycosides⁷.
Anthocyanins in strawberry were analyzed by HPLC; some identifications could only be achieved after semi-preparative HPLC, partial hydrolysis and analysis of the fragments obtained⁹.

The anthocyanins are naturally occurring pH indicators, and this property has been the subject of our article related to chemical education. It is through the work of Diyar et al.¹⁰, that our current understanding of the pH- induced color changes of the anthocyanins is based. Fig. 2 shows the results in an extended conjugation of double bonds through the three rings of the anthocyanin. The colored flavylium cation is in equilibrium with a colourless pseudo-base form, which begins to predominate as the pH of the solution is raised to values greater than three. The addition of the water molecule in the formation of the pseudo- base from the cation form of the molecule disrupts the conjugation of double bonds between the second and third ring and results in the absorption of photons in the ultraviolet region rather than in the visible ¹¹.

The aim of the present work is to update the knowledge about strawberry anthocyanins, for which the anthocyanin composition, qualitative and quantitative, has been analysed in strawberry fruits from five different cultivars, using HPLC coupled to diode array and MS detection.

Fig. 2: Chemical structures of the two forms of anthocyanin aglycones Cores responsible for the primary colour change.

All chemicals and reagents were of analytical grade (BDH and Fluka) and deionized water used throughout.
Sample extraction. Strawberries, purchased from a local market, were disrupted in 0.1% HCl in methanol and later filtered through a Büchner funnel under vacuum. The solid residue was exhaustively washed with methanol and the filtrates obtained were centrifuged. After addition of water, the supernatant was concentrated under vacuum to total evaporation of the methanol.
UV-Visible double beam spectrophotometer Cecil 9000, was used with quartz cells (1cm).
Pye Unicam SP-3005 IR- spectrophotometer was used during this work, in the range of
600-4000 cm-1.
Bruker (600 MHz) instrument was used (from Bangor University-UK) to obtain 1HNMR
spectra.
A Bruker AMX500 13C-NMR was used (from Bangor University-UK) for identification.
Exeter analytical analyzer model CE-440 was used to find the percentage of C, H and O content.
Ultrasonic bath (Decon FS200) was used for degassing solutions in HPLC
HPLC analysis. The strawberry extract and the anthocyanins fractions were analysed
using a Hewlett-Packard 1000 Series liquid chromatograph, equipped with an AQUA
(USA) reversed-phase column (150¥ 4.60 mm, 5m, C18). The column temperature was
thermostatted at 35 °C using a column heater module (Waters, USA). Solvents were (A)
aqueous 0.1% trifluoracetic acid (TFA) and (B) 100% HPLC grade acetonitrile, which are used as a mobile phase, establishing the following gradient: isocratic 10% B for 5 min, from 10 to 15% B over 15 min, isocratic 15% B for 5 min, from 15 to18% B over 5 min, and from 18 to 35% B over 20 min, at a flow rate of 0.5 ml min-1. Detection was carried out in a photodiode spectrophotometer and 520 nm selected as preferred wavelength as a temporary selection before optimization process. HPLC solvents and samples were filtered through a 0.45 m Millipore filter. The extract was purified by using C18 column, the purification process was tested by PC and TLC with different solvent systems as mentioned in Table (1) and (2). All tests were done in Bangor University - UK
LC/MS analysis. MS was performed using a Finnigan LCQ (USA). The capillary
temperature 195°C. Spectra were recorded between m/z 150 and 1600. The mass
spectrometer was programmed to do a full mass, a zoom scan of the most abundant ion in
the first scan and MS-MS of the most abundant ion, using collision energy of 30. For the
analysis of anthocyanin fractions the same HPLC conditions already mentioned were used. All tests were done in Bangor University - UK⁷.
pH-meter. (Model Jenway 3305) (S. Korean). Furnace. (model Zorenta muffle furnace > 600 C) (USA).
Oven. (model KOTTERMANN < 300 C) (USA).
Press. (model Pye Unicam) (USA). Melting point apparatus. (Ger) (Electrothermal 9100). Electrical grinder. (model Thomas).

Determination of max
The absorbance of the extracted solution, which was used in volumetric analysis as
An indicator in acid-base titration was taken. Fig (3) shows that 517.3 nm was the max of the solution as an optimized maximum wavelength.

Fig. 3: The absorption spectrum of the pigment of strawberry

Chromatographic techniques for purification
All solvents which are used in TLC and PC were use for confirming the purification
process which was done by C18 column.
TLC: Table 1 shows different solvent mixtures in the present TLC procedure to test
the red solid material extracted on a glass plate coated by silica gel.
PC: Table (2) shows different solvent mixture used to test the red sample on the
sheets of Whatman No. 1 and 3 papers.
Extraction of malvidin-3-glycoside
The extracted pigment from strawberry sample which contains five compounds was
separated by PTLC technique. This method was applied for separation of the two
constituents in the pigment¹². Both constituents were tested by TLC plate the Rf values on the TLC plate compared with standards compounds as shown in Fig. 4.
The ready made plate was air-dried for 1 hour and activated in an oven at 115 C for 4 hours prior to use. 0.1 g of red pigment was dissolved in 10 ml of methanol, and then a capillary tube was used to make a small spot of the solution in more concentrated zone, 2 cm from the bottom of the plate. The TLC plate was then placed in the tank with different mobile phases for at least 3 hours.
The silica gel surrounding each spot was removed with a small spatula and dissolved in 25 ml methanol, followed by removing all silica gel from the solution by filtration.
Concentration of the mixture was done with vacuum rotary evaporator at 60 C. The residue was left in a watch glass for 2 hours to obtain the solid material¹³.

Fig. 4: Forestal chromatogram of the common flavonoid of plants [14]

Comparison between the extracted pigment and other common indicators

HCl (0.1M) was prepared by diluting 2.1 ml of the concentrated HCl to 250 ml with distilled water. Five drops of each indicator were added to 10 ml of 0.1M Na2CO3. The solution was titrated with 0.1M HCl solution.
A one-time massive titration interval of about 1 ml was used, then, the intervals decrease when the solution approached the endpoint. This process was repeated for a total of five times for each type. Indicators of universal indicator, methyl orange, methyl red, bromothymol blue, and extracted pigment were examined. The titration was considered complete when a permanent significant color change was achieved. Table (5) shows the results obtained.

The extracted pigment from strawberry gave five spots using the different chromatographic techniques. The extracted compounds were red solid material, noncrystalline.
The compound decomposed at 195-198 C. Table (3) shows some physical and chemical properties for the extracted pigments. Total anthocyanin content ranged between 25-125 mg kg-1. This range was considered as the good yield for use as an indicator.
New HPLC conditions have been developed for the separation of strawberry anthocyanins. The use of a C18 column end capped with a hydrophilic (polar) reagent and 0.1% TFA as aqueous solvent effected a good chromatographic resolution of anthocyanins using a low flow rate (0.5 ml min-1), compatible with the requirements of the MS detector.
Figure (5) shows the HPLC chromatogram recorded at 517 nm corresponding to the complete strawberry anthocyanin profile.

Fig. 5: HPLC chromatogram (517 nm) showing the anthocyanin profile of strawberry extract

Two peaks were assigned respectively to the main two anthocyanin compounds cyaniding-3-glucoside and malvidin-3-glucoside based on their relative elution order, which was mentioned in the absorption spectra obtained with the diode array detector. Our results compared with the data mentioned according by others^3,4,12. Table (4) shows the results, which were obtained from ESI-MS analysis.
Pigment which was isolated from strawberry then used as indicator showed a positive molecular ion [M+] at m/z 510 (malvidin-3-glycoside).
The percentage of pigment extracted (malvidin-3-glycoside) from strawberry was found to be 4.63%. From our results it seems that the best conditions for these extractions are 70 C, pH= 3.0 and 3 hours time for strawberry extraction. However, the extracted pigment from strawberry gave five spots using the different chromatographic techniques.
The extracted compound was red solid material, non-crystalline. The compound decomposed at 195-198 C.
Generally, this work has a big economical value in the world, and especially in our region. All common indicators have a high cost, but our work obtained a natural indicator in a good precision and accuracy by lower cost.

Comparison of red pigment with common indicators
Table 5 shows the results of standardization of 10 ml 0.1N HCl with 10 ml, 0.1N Na2CO3 solution using different common indicators and extracted pigment from strawberry. The results were obtained using different common indicators with our pigment, and indicates that all common indicators give titration error (0-0.2 ml) while the extracted pigment gave a titration error (0.23 ml). The precision (strawberry extract) for five titrations (n=5) was found to be 0.0162 as standard deviation and 0.158% as relative standard deviation. These values indicate a good precision.

The use of optimized HPLC conditions coupled to diode array and mass detection allowed us to detect up to five different anthocyanin pigments in strawberry fruits in the north of Iraq.
Most anthocyanin showed Pelargonidin and Malvidin as an aglycone and with sugar attached, although some cyanidin was also present. Fructose was the most usual substituting sugar. In different strawberry samples collected, pelargonidin derivatives are always predominant anthocyanins; usually these compounds represented more than 70% of total anthocyanin content in strawberry.

Acknowledgments
Thanks are due to Salahaddin University (Iraq) for financial support (project number 7/54/1022). Many thanks to school of chemistry in Bangor University - UK for sample analysis.

* Universal indicator: Universal indicators were prepared by dissolving 0.05 g methyl orange, 0.15 g methyl red, 0.30 g bromothymol blue and 0.35 g phenolphthalein in 1L ethanol/ H2O mixture made up of two parts ethanol to one part water (by volume), solutions stirred overnight to dissolve the more insoluble compounds¹⁵.

Solvent mixture Ratio
n-Hexane: Diethyl ether: Acetic acid 8:2:0.1
n-Butanol: Acetic acid: Water 4:1:5
Conc. HCl: Formic acid: Water 2:5:3
Solvent mixture (Mobile phase) Ratio
Conc. HCl: Acetic acid: Water 3:30:10
Conc. HCl: Formic acid : Water 2:5:3
n-Butanol: Acetic acid: Water 4:1:5
Tests Characteristic of extracted pigment from strawberry
Physical state Bloody red
Solubility 100 % soluble in water, ethanol
Molisch test (+) ve
Moisture (pigment) 2.3%
Water content (fruit sample) 73%

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