Stabilization of Hibiscus sabdariffa anthocyanins using polyphenols

Natural colorants are widely used in food and beverage products due to increasing consumer demand for natural ingredients (Katz & Williams 2011). Anthocyanin is one of the most commonly utilized water-soluble natural colorants and a subclass of flavonoids (Mercadante & Bobbio 2007). They are typically extracted from the red and blue parts of certain plants, including fruits, vegetables, flowers, and leaves. The color stability of anthocyanin is strongly dependent on the pH of the surrounding aqueous phase and they are more stable in acidic rather than in neutral or alkaline solutions. The rate of degradation is affected by many factors including pH, light, temperature, oxygen, enzymes, and ingredient interactions (Malien-Aubert et al. 2001). Besides, anthocyanin also has anti-oxidant and bioactive properties linked to certain health benefits e.g. anti-diabetic, anti-inflammatory, and anti-cancer effects (Tzulker et al. 2007, Olaleye 2007). The stability of anthocyanin color can be enhanced by co-pigmentation phenomenon where the anthocyanin molecule reacts with other natural plant substances through weak interactions forming an enhanced and stabilized color (Gordillo et al. 2012). Co-pigmentation is a solution phenomenon in which pigments and co-pigment molecules form molecular complexes. These cause the pigments to exhibit high color intensity than would be expected from their original. This interaction prevents water attack on the flavylium cation. The most common co-pigments are flavonoids, polyphenolic compounds, alkaloids, amino acids and organic acids (Mazza & Brouillard 1990). Hibiscus sabdariffa is a tropical plant, which belongs to the Malvaceae fam. It is rich in anthocyanins, minerals, pectin. The major reported anthocyanins are delphinidin-3-glucoside, delphinidin-3-sambubioside, and cyanidin-3-sambubioside chiefly responsible for their color and antioxidant properties (Pau-Ling et al. 2002, Jadhav & Bhujbal 2019). Phytochemicals, these natural components, may be able to inhibit the chemical degradation of the anthocyanins and to prolong their color stability. For this reason, many investigations have been carried out to improve the stability of anthocyanins. Therefore, they can be used more widely in food products (Eiro & Heinonen. 2002, Talcott et al. 2003, Clemente & GallI 2011, Jadhav & Bhujbal 2019). The present study is carried out to extract anthocyanins from Hibiscus saddariffa and to improve its stability by copigmentation by four polyphenols (green tea extract, rosemary extract, rose extract, sage extract and in the presence of ascorbic acid). The color stability of anthocyanins is determined by evaluation of the amount anthocyanins, destructive index and total polyphenol during storage at elevated temperature in the presence of light and ascorbic acid in order to accelerate the degradation of anthocyanin pigments.

In order to extract Hibiscus saddariffaas, a rich source of anthocyanins, the sepals of this plant were used. First, a weighed amount (6 g) of Hibiscussepal was dissolved in Methanol and water in a ratio of 0.5 to 1.5 as a solvent system. Then, it was kept in an orbital shaker at 40 ℃ for 4 hours to complete anthocyanin pigment extraction. The extract was filtered using whatman No.1 filter paper and concentrated by rotary evaporator at 60℃ to reach 12 brix. In order to prepare other phytochemical extracts (green tea, rosemary, sage and rose), a weighed amount of (260 g) of each phytochemical was dissolved in deionized water and stirred until fully hydrated. Next, 30% w/w of each extract were added to the hibiscus extraction (as a source of anthocyanins) as well as 0.05% w/w ascorbic acid. The pH of the solutions was then adjusted to pH2 using 1 M citric acid. Then, the amount of anthocyanin compounds was determined using pH change method. Besides, destructive index of anthocyanins was evaluated by using absorbance at 420 nm divided by the absorbance at 520 nm. The total amount of polyphenolic compounds of treatments by the folin-ciocalteu reagents was investigated. For this aim, all experiments were analyzed in triplicate in a completely randomized design using the Minitab 16 software using one-way analysis of variance Duncan's test.

The color stability of anthocyanin in the sample reduced in the presence of ascorbic acid. In the absence of ascorbic acid, the anthocyanin color was stable over seven days. The color fading caused by the presence of ascorbic acid had been proposed to occur through two main mechanisms: (a) a condensation reaction between anthocyanin and ascorbic acid and/or (b) autoxidation of ascorbic acid generating free radicals (e.g., hydrogen peroxide) that cleave the flavylium core of the anthocyanins (Mercadante & Bobbio 2007). In addition, the polyphenols extracts in the presence of ascorbic acid increased the anthocyanin content compared to the control and treatment sample (T2) and also increased the stability of anthocyanin content during storage. Among the co-pigments used (polyphenols extracts), T3 treatment, which contained 30% w/w green tea, had the highest amount of anthocyanin compounds (260/49 mg / l), which could be due to the phenomenon of co-pigmentation. In other words, the higher anthocyanin stability upon green tea addition was due to co-pigmentation, inhibited the ability of water molecules to attack the flavylium ion and caused color loss (Kopjar et al. 2014). According to the obtained results of Degradation index, treatment (T3) with addition of ascorbic acid and green tea extract had the lowest, while the sample (T2) with the addition of ascorbic acid only had the highest degradation index. Those results were in correspondence with the results for anthocyanin content. After preparation, samples with green tea extract addition had the highest anthocyanin content and the lowest degradation index, which means that anthocyanins were more stable. During storage, degradation of anthocyanin occurred and the destructive index increased (Piližota et al. 2012). In terms of total phenolic content, under the defined conditions, T3 treatment containing of green tea extract (30% w/w) had more phenolic compounds than other treatments (5.78 mg/l) and T2 treatment containing ascorbic acid had the lowest polyphenol content (2.85 mg/l). Whereas T3 treatment had more polyphenolic compounds than other samples, anthocyanins and polyphenol structures could be more exposed to each other and more effective co-pigmentation occurred.

The results revealed that by using plant extract/polyphenol, solutions could significantly increase the stability of anthocyanins. Among these extracts, green tea extract was the most effective extract in inhibiting the destruction of anthocyanins.