Natural Products Chemistry & Research

Anthocyanin determination of Ethiopian Roselle (Hibiscus sabdariffa) genotypes

Bekri Melka Abdo^{* *}Correspondence: Bekri Melka Abdo, Wendo Genet Natural Product Research Laboratory, Ethiopian Institute of Agricultural Research, Ethiopia, Tel: +251911810857, Email:

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INTRODUCTION

Roselle (Hibiscus sabdariffa) belongs to the family Malvaceae, which originated from Africa and is native to tropical and sub-tropical regions of the world. The calyces of Roselle are used as a food colorant and are widely known as beverages called Bissap (Senegal), da Bilenni (Burkina Faso), Zobo (Nigeria), Sobolo (Ghana), Sorrel (Jamaica), Jamaica (Mexico)and Karkade tea (Sudan) [1-4]. Besides, it is used for the preparation of jelly, jam, marmalade, and cupcake. The consumption of Roselle calyces products has benefits for health such as inducing reduction of blood pressure, and reducing cardiovascular disease [5]. Further, it can be used as a source of nutrition [6] and increases the lactation at weaning time and increases the BMI (body mass index) of the pups [6,7]. Moreover, Roselle calyces extracts have antioxidant and antibacterial activities [8, 9].

The extracts of Roselle calyces have compounds of anthocyanin pigment (Figure 1) (delphinidin-3-sambubioside, cyanidin-3-sambubioside, delphinidin-3-glucoside and cyanidin-3-O-glucoside, malvidin-3-O-glucoside and petunidin-3-glucoside), which are responsible for its color. These compounds were characterized by using HPLC [10, 11]. The amount of anthocyanin present in the calyces can be measured using a UV-Visible spectrophotometer through the pH differential method [12].

Figure 1: Structure of common natural anthocyanins

In Ethiopia, there is no local beverage practice using Roselle calyces. Recently, the herbal tea of Roselle has emerged to be popularized as Karkade tea and soft drink. However, a good potential of Roselle plants is found in western Ethiopia from their natural habitat. Two varieties of Roselle (WG-H-Sudan and WG-H-Jamaica) were registered at Wendo Genet Agricultural Research Center and also have fourteen accessions of Roselle plant, which were collected from Benishangul Gumuz region. The anthocyanin content of the Ethiopian Roselle plants is not yet determined. This work had the aim of determining the total anthocyanin content of Ethiopian Roselle plant collections. Further, the superior anthocyanin yielded cultivar (WG-H-Sudan variety) was selected, and determined its nutritional profile and standardized the herbal tea preparation protocol.

Materials and Methods

Plant sample collection and preparation

The matured fruits of Roselle plants were collected from Wendo Genet Agricultural Research Center experimental field (A 1776m; N 07° 03' 19.1''; E 38° 30'08.4''). The calyces were striped and dried through in shade. The dried calyces were finely milled, and put in a plastic bag for analysis. The powdered calyx (0.1g) was extracted using 50mL of 5% HCl methanol solution for overnight shaking. The extraction was replicated three times for each accession sample. The extract was then analyzed its total monomeric anthocyanin content through a pH differential method. The analysis of variance and mean comparison of the total anthocyanin content between accessions were performed through statistical analysis software (SAS, version 9.1) with a completely randomized design sampling procedure.

Determination of total monomeric anthocyanin

The total anthocyanin content of the Roselle calyces was determined based on pH differential methods as described by Lee et al. [12]. Potassium chloride (1.86g) was added to a 980mL distilled water containing beaker, in order to prepare a 0.025 M solution. The crystals were dissolved well and adjusted the pH to 1.0 by slow dropping of HCl through pH meter reading.

The solution was transferred to a 1L volumetric flask and diluted to volume with distilled water. Sodium acetate (54.43g) was added to a 960mL distilled water containing beaker, in order to prepare a 0.4 M solution. The crystals were dissolved well and adjusted the pH to 4.5 by slow dropping of HCl. The solution was transferred to 1L volumetric flask and diluted to volume with distilled water. The extract solution of Roselle samples (2mL) was added to 8mL of pH1 and pH4.5 buffer solution independently. The test pH solutions were scanned on a UV- visible spectrophotometer at a wavelength of 200-800nm and selected the λ max value of 520nm. The absorbance of the dilute test solutions was measured at 520nm and 700nm wavelengths. Distilled water was used as a blank. The total anthocyanin pigment was expressed as in terms of Cyanidin-3-O-glucoside equivalents and determined according to the following formula:

Total anthocyanin content (%) = (A.V.DF.449.2) / (.W) X100% 1

Where; A ~absorbance = (A520 nm – A700 nm) pH1.0 – (A520 nm – A700 nm) pH 4.5;

Molecular weight of Cyanidin-3-O-glucoside = 449.2 g/mol; W ~Weight of sample; V ~Volume of sample; DF ~dilution factor; ~molar extinction coefficient = 26900 Lmol-1cm-1 for cyanidin-3-glucoside

Determination of coloring intensity

The coloring intensity of the Roselle calyces (WG-H-Sudan variety) was determined according to the method described by Herbal medicine compendium [13]. The powdered Roselle calyces’ sample (1g) was added into 25mL boiling water containing 50mL volumetric flask, and heated for 15 minutes in a water-bath with frequent shaking. The hot mixture was f iltered and added into a 50mL volumetric flask. The residue was rinsed and f iltered three times successively with 5 mL of warm water, and added into the 50mL flask. The filtrates were cooled and diluted to volume. The filtrate solution (5 mL) was taken and diluted to 50mL of water. The absorbance was measured by UV-VIS spectrophotometer at 520 nm wavelength using water as the blank.

Nutritional analysis

The proximate parameters of moisture, fat, protein, ash, crude fiber and carbohydrates of the powder of Roselle calyx were determined [14]. The dry ash method was used to prepare the tested sample for mineral analysis (Na, K, Ca, Mg, Fe, Cu, Zn and P) and governed by ICP-AES and AAS (inductively coupled plasma atomic emission spectrometer and atomic absorption spectrometer) [15].

Herbal tea preparation

The herbal tea was prepared from calyces of Roselle (WG-H-Sudan variety) through different concentrations (CC1, 0.5g powder/500mL water; CC2, 0.5g powder/250mL water; CC5, 0.5g powder/100mL water), temperature conditions of hot water (100°C) and cold water (25°C) and stirring time (1 and 3 minutes). The prepared tea was filtered through a muslin cloth and subjected to determine the Total Anthocyanin Content (TAC). The optimum anthocyanin extraction efficiency of the herbal tea preparation methods was optimized using response surface optimization method with a central composite design through Minitab software (version 19).

Results and Discussion

The anthocyanin content of the calyces of Roselle was determined by pH differential methods through UV-Visible spectrophotometer at λ max 0.266 0.001 0.000 0.702 0.444 0.420 0.899 value of 520nm (Figure 2). The total anthocyanin content was calculated in terms of Cyanidin-3-O-glucoside equivalents. Based on the ANOVA (F test), the total anthocyanin content was significantly varied between Roselle genotypes at probability level less than 0.0001 (Table 1). Thereby, acidic methanol solution extracts of calyces of Roselle genotypes yielded total anthocyanin content (TAC) in the range of 0.05-4.45% (Table 2). The WG-H-Sudan variety was superior in TAC (4.45%) and next to the code ETH/AsARC-29 genotype (3.37%). The WG-H-Jamaica variety had a TAC of 3.09%, which was comparable with some genotypes of Roselle found at accession level. Comparable results were reported by Beye et al. [10] with total anthocyanin content of 1.53% (Vimto/Sudan) and 0.69% (Koor) from Senegal cultivars, and 0.57% from Egyptian product [6]. Besides, 2.61% and 2.98% of the total anthocyanin content were reported from Burkina Faso cultivars of bright red and dark red Roselle, respectively [16].

Figure 2: UV-Visible spectrum of Roselle extract at pH 1 and pH 4.5 buffer solution

Table 1. Analysis of variance for total anthocyanin content in calyces of Roselle genotypes

Table 2. Mean comparison for total anthocyanin content (TAC) in calyces of Roselle genotypes

The Roselle of WG-H-Sudan variety was selected for further nutritional analysis and herbal tea standardization. Calyces of the WG-H-Sudan variety Roselle had a color intensity of 1.85, which indicated that the calyces had an intense red color. The proximate analysis of the calyces of the WG-H-Sudan variety Roselle revealed that it had moisture (10.2%), ash (6.9%), fiber (13.9%), protein (6.64%), fat (6.7%) and carbohydrate (69.56%). A total of 311.84Kcal energy was obtained from 100g of Roselle calyx. From the mineral analysis of 100g dried Roselle calyces, it was a source of K (20.42mg), Na (0.13mg), Fe (8.24mg), Zn (23.64mg), Cu (0.67mg), and Mn (18.49mg). A comparable result was reported by AbdelMoemin [6] from dried calyces of Roselle having moisture (9.5%), ash (5%), f iber (12%), protein (5.3%), and CHO (65%). Overall, the presented findings of color intensity, moisture and ash value were complying the herbal medicines compendium results of Roselle calyx with (>0.35), (<11%) and (<10%) respectively [13].

Means followed by the same letter under the same column are statistically nonsignificant at P<0.05 according to least significant difference (LSD) test.

The total anthocyanin content obtained from different modes of herbal tea preparations was optimized using response surface optimization method with a central composite design. The yield of active ingredients obtained in the prepared herbal tea was significantly influenced by the modes of herbal tea preparation. Rather than the interaction effect, the individual effect of the predictor was observed on the total anthocyanin content of the herbal tea. The predictors used in this particular experiment, such as the concentration and temperature condition of the water, have significantly influenced the yield of total anthocyanin content obtained from the prepared herbal tea at a probability level of less than 0.05 (Table 3). The model has a coefficient of determination (R2) 0.96 with the regression equation of

TAC(%) = 4.476 + 0.216 ST – 0.242 CC – 0.814 TC_ Cold + 0.814 Temperature_Hot – 0.0385 ST CC + 0.0692 ST TC_Cold – 0.0692 ST TC_Hot + 0.0062 CC TC_Cold – 0.0062 CC TC_Hot

Table 3. Influence of Roselle calyces (WG-H-Sudan variety) herbal tea preparation predictors on the mean square of total anthocyanin content from the analysis of variance

Where; TAC: total anthocyanin content; ST: stirring time; CC: concentration; TC: temperature condition

The hot water preparation of herbal tea maximized the content of the total anthocyanin by 0.814 times the normal value, and minimized that prepared in cold water. When the concentration of Roselle powder increased in a given solution, it decreased the total anthocyanin content by 0.242 times that of initial. The optimization plots of different modes of herbal tea preparation (Figure 3) showed that hot water (500mL) infusion of Roselle calyx powder (0.5g) for 3 minutes stirring acquired maximum total anthocyanin content of 5.37%, which is 26.85mg. Considering JECFA monographs [17] of the estimated acceptable daily intake of anthocyanin for man 0-2.5mg/kg bw, it can be set the amount of the herb taken by different age groups to utilize in the form of herbal tea and as application of colorants on beverage or local drinks.

Figure 3: Optimization plots of Roselle calyces' herbal tea towards maximum total anthocyanin content

Conclusion

A wide range of the value of total anthocyanin content had determined from Ethiopian Roselle plant collections. This indicates the presence of a huge potential Roselle plant in the country. It is a good opportunity to improve the Roselle plant with respect to responsible compounds of colorant trait. Concerning nutritional value, the calyces of the Roselle plant can be utilized as a good dietary source of energy and minerals. From the optimization of herbal tea preparation, hot water (500mL) infusion of 0.5g powder of Roselle calyx for 3 minutes stirring acquired the maximum total anthocyanin content of 26.85mg. This value is used to set the amount of Roselle calyx consumption in different products.

Acknowledgment

I am grateful to Mr. Henok Nahusenay and Mr. Nibret Mekonnen for their support on nutritional and mineral analysis of this work.

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