Chemical profile and scavenging activity of four extracts from Hibiscus sabdariffa L. calyx CURRENT

Objective Hibiscus sabdariffa L., also known as roselle and karkadeh, is commonly used in traditional medicine and has many applications on food, pharmaceutical and cosmetic industries. More than 100 cultivars of H. sabdariffa are found. Despite the fact that Sudan is considered as one of the principal producer of H. sabdariffa there has been limited systematic study on the efficiency of different solvent extraction on its bioactive molecules. Therefore, this study was designed to investigate the chemical constituents of H. sabdariffa calyx using acetone, ethanol (EtOH), 50% EtOH and water as extraction solvents and their impact on the DPPH free radical scavenging activity. Results Results showed that the total polyphenol, flavonoids and vitamin C contents of the dried calyces were significantly (P < 0.05) affected by the solvent extraction used. HPLC analysis using 16 standards revealed the presence of 11–13 compounds in the four extracts with the 50% EtOH extract recovered the highest concentrations of identified compounds. Moreover, the 50% EtOH extract revealed highest DPPH free radical scavenging activity (IC 50 1.544 ± 0.3 µg/mL). Based on these results, maceration the calyx on 50% EtOH was therefore suggested for optimal extraction of bioactive compounds with high antiradical activity from H. sabdariffa calyx.


Introduction
Hibiscus sabdariffa L. (Family Malvaceae), with common names roselle and karkadeh, is believed to be domesticated by the people of western Sudan sometime before 4000 BC [1]. The calyx is widely used in Sudan to prepare cold or hot beverage, in addition to, a traditional fermented food known as Furundu is prepared from the seed [2]. H. sabdariffa is considered as important economic crop and a source of income for small farmers in western Sudan [3]. It is also well-thought-out as famine food because of its resistance to hard conditions of drought [4]. In traditional medicine, the calyces are mixed with fruits of Adansonia digitata and Tamarindus indica to cure malaria [5]. The calyx is also used to treat hypertension, flu, haemorrhoids [6], headache [7], fever, snake bite, scorpion sting [7; 8], as hypotensive and antispasmodic and for relaxation of uterine muscle [9]. A detailed review carried out by Da-Costa-Rocha [15] and Riaz and Chopra [16] summarized the chemical constituents and pharmacological properties of H. sabdariffa.
Biological activities of plants are associated with their phytochemical constituents [17], therefore it is important to determine the appropriate extraction method and extractive solvents used concerning a targeted biological activity. Despite the fact that Sudan is considered as one of the principal producer of H. sabdariffa and its different application on food, pharmaceutical and cosmetic industries there has been limited systematic study on the efficiency of different solvent extraction on bioactive molecules. In this context this study was designed to investigate the chemical constituents of H. sabdariffa calyx using acetone, ethanol and water as extraction solvents and their impact on antiradical property.

Materials And Methods Plant materials
Dried calyces of H. sabdariffa was purchase from the local market in Khartoum in June/2018.

Preparation of extracts
The dried calyces were cleaned from debris and were pulverized and subjected to extraction using four solvents; acetone, ethanol (EtOH), 50% ethanol/water (50% EtOH) and water. 10 g of sample was accurately measured and macerated in 50 mL of appropriate solvent for one hour. All extracts were filtered through Whatman No.1 filter paper. Then, acetone and EtOH extracts were concentrated using a rotary evaporator under reduced pressure while aqueous extracts were freeze-dried.

Determination of total polyphenol content
The total polyphenolic content was determined by using the method described by Wolfe et al. [18].
The total phenolic contents were expressed as gallic acid equivalents (mg/g on a dry weight basis).

Determination of total flavonoid content
The total flavonoid content was determined by adopting the method described by Ordonez et al. [19].
Total flavonoids content was expressed as quercetin (mg/g on a dry weight basis).

Determination of vitamin C content
The content of vitamin C was evaluated spectrophotometrically according to method PN-A-04019 [20].
Results are presented as mg vitamin C/100 g extract on a dry weight basis.
The column temperature was maintained at 35 °C. Compounds were identified and quantified (µg/g extracts) with comparison standards retention times and UV-Vis spectra.

Determination of antiradical activity
The antioxidant DPPH free radical scavenging activity was determined using the method described by Brand-Williams [21]. The IC 50 value was calculated from the linear regression of plots of concentration of the test sample against the mean percentage of the antioxidant activity.

Statistical analysis
All experiments were performed in triplicate and the obtained results were expressed as the mean ± standard deviation. One-way ANOVA was performed for determining significant differences between the four extracts and their antiradical activity.

Extraction yield
Calyx of H. sabdariffa was macerated in four solvents; acetone, EtOH, 50% EtOH and water to evaluate the effect of type of solvent on the chemical composition and consequently the antiradical activity of the plant. Results of extraction yield (percentage of mass of extract/mass of dry matter) are depicted in Table 1. The four extracts yield were ranked in a descending order as follows: EtOH (77.2%) > 50% EtOH (58.4%) > water (6.6%) > acetone (3.1%). (138.11 µg/g) extracts and absent or in low amount in the water and acetone extracts. Vitamin C content Vitamin C content in the four extracts of H. sabdariffa calyx was also determined and results are presented in Table 1. The highest content in vitamin C was obtained from the acetone (420 ± 9.7 mg/100 g) and EtOH (230 ± 7.5 mg/100 g) extracts respectively while the water (100 ± 1.2 mg/100 g) and 50% EtOH (80 ± 0.7 mg/100 g) extracts had the least content.
DPPH free radical scavenging activity

Discussion
Active metabolites usually exist in low concentration in plants [22,23]. Therefore, it is necessary to select the appropriate solvent to be able to obtain extracts with high yield and best properties for the targeted biological activity. Extraction yield was used as an indicator of the effects of the extraction conditions. In this study the four solvents used to extract metabolites from H. sabdariffa calyx varied in their extraction yield with best amount obtained from the EtOH (77.2%) and 50% EtOH (58.4%) solvents respectively. This results were in agreement with finding of Grigonisa et al. [24] who reported that the highest extract yields were obtained with polar alcohol-based solvents.
The results of the present study demonstrated that the total content of polyphenol, flavonoid and Vitamin C in calyces of H. sabdariffa significantly (p ≤ 0.05) influenced by type of solvent used for extraction. EtOH and 50% EtOH extracted highest content of polyphenols while the water and acetone extracts showed the highest flavonoid and vitamin C contents respectively. Previous study by Koffi et al [25] showed that alcohol (ethanol and methanol) was better in extracting phenols than acetone.
However, it would also be important to consider that the high contents of phenolics in alcoholic extracts are more likely associated with biomolecules including proteins, carbohydrates (glycosides), terpenes, chlorophyll, lipids and inorganic compounds, which could be also extracted by these solvents and consequently interfere in determination of total polyphenol by Folin-Ciocalteu reagent [26].
Total flavonoid content in the present study were best extracted by the water extract and was in contradiction to the results of Koffi et al [25] who showed that flavonoids in H. sabdariffa calyx were better extracted from the acetone extract. This difference in results could be attributed to nature of flavonoid molecules present in H. sabdariffa sample from Sudan which could be affected by many biotic and abiotic factors such as differences of cultivars, soil of cultivation, climatic conditions and storage condition [27,28].
Furthermore, the chemical profiling of the four extracts revealed that out of the 15 phenolic standards used 12 compounds were present in H. sabdariffa with chlorogenic acid, naringenin rutin and gallic acid at noteworthy concentrations in most extracts. Generally, the highest amount of the identified compounds was obtained from the 50% EtOH extract. Previous studies [15,16] reported the presence of most of these compounds in H. sabdariffa calyx, but to the best of our knowledge this is the first reports on comparing the effect of the four used solvents on the recovery of these compounds.
Result of the antioxidant DPPH free radical scavenging activity showed that 50% EtOH extract followed by EtOH extract exhibited the best anti-DPPH radical activity while acetone extract revealed the least activity. Previous study showed that the alcoholic (methanol) extract gave the highest DPPH value in comparison with ethyl acetate and acetone [28,29]. As noted in the phenolic profile of these two extracts, many compounds are well known for their antiradical activity such as rutin, qurecetin [30], gallic acid [31], ellagic acid [32], chlorogenic acid, coumaric acid [33], caffeic acid [34] vanillin and catechin [35]. Although the water extract contained also considerable amount of these compounds, its antiradical activity was lower than that observed in the 50% EtOH and EtOH extracts.
This might be to antagonistic effect and steric hindrances of other components which are also present in the crude extract [36]. Furthermore, the antiradical activity of the acetone extract was probably associated to its high content in vitamin C. Soobrattee et al. [37] reported that although the antiradical potential of vitamin C was found to be weaker than that of quercetin and similar to that of trolox, it can still contribute to the antiradical activity of berries and fruits.
From the Pearson's correlation coefficient of total polyphenols, flavonoids, vitamin C with antioxidant activity, it was suggested that the antioxidant DPPH free radical scavenging activity of H. sabdariffa calyx was mainly associated to polyphenolic content (R² = 0.9836) rather than the total flavonoid content (R² = 0.0625) or vitamin C (R² = 0.3962).

Conclusion
The present study provide a better insight into the influence of type of solvent extraction on the chemical composition of metabolites from H. sabdariffa calyx and their amount. The obtained results suggested that 50% EtOH could extract more phenolic compounds with highest concentration which was reflected on highest DPPH free radical scavenging activity.

Limitation
Types of anthocyanins and organic acids should be determined.
Other antioxidant assays should be performed.