Notes on antioxidant properties, volatile compounds, and sensory aroma profile of red and white Hibiscus sabdariffa (Sudanese Roselle)

Introduction

  • Roselle (Hibiscus sabdariffa L., family Malvaceae) is widely cultivated in tropical/subtropical regions; locally called “Karkade” in Sudan.
  • Four Sudanese roselle types analyzed: three red (Al-Rahad, Al-Fashir, Al-Gezira) and one white variety.
  • Antioxidant activity linked to phenolics, flavonoids, anthocyanins, and ascorbic acid; red varieties typically richer in anthocyanins.
  • Aroma profile and volatile compounds are important for product utilization and consumer acceptance.
  • Objectives: (1) determine antioxidant properties of Sudanese roselle flowers from different origins; (2) determine volatile profile, sensory descriptors, and the relationship between aroma/sensory data using partial least squares regression (PLSR).

Materials and Methods

  • Chemicals and samples
    • Reagents: Folin–Ciocalteu phenol reagent, gallic acid, ascorbic acid, ferric chloride hexahydrate, sodium hydroxide, sodium carbonate, aluminum chloride hexahydrate, trichloroacetic acid, ferric chloride, potassium ferricyanide, methanol, ethanol, hydrochloric acid, dipotassium phosphate, quercetin, BHT; DPPH.
    • 4 roselle samples from Sudan: red (Al-Rahad, Al-Fashir, Al-Gezira) and white type; collection sites: Al-Rahad (North Kurdufan), Al-Fashir (North Darfur), Al-Gezira (Al-Gezira), white from Khartoum.
    • Sample preparation: samples dried at 60 °C for 2 h, ground to powder, sieved through 500 μm mesh.
  • Preparing the roselle extracts
    • Aqueous extract: calyx powder to water ratio 1:50; sonicated 20 min at 25 °C; centrifuged 10 min at 3000 rpm; filtered through Whatman #1; stored at 4 °C; analyses within 24 h.
  • Total flavonoid content (TFC)
    • Method: mix 1 mL extract with 4 mL distilled water; add NaNO₂ (baseline), wait 5 min, add AlCl₃, wait 6 min, add NaOH; bring volume to 10 mL with water; vortex; measure absorbance at 510 nm.
    • Calibration: quercetin (0–100 μg/mL, R² = 0.98).
    • Expression: mg quercetin/100 g roselle dried powder (QE equivalents).
  • Total phenolic content (TPC)
    • Method: mix 1 mL diluted roselle (1:10 v/v) with 5 mL 0.2 N Folin Ciocalteu; vortex 10 min; add 2 mL sodium carbonate (75 g/L); incubate 2 h; measure at 760 nm.
    • Standard: gallic acid (0–200 μg/mL, R² = 0.99).
    • Expression: mg gallic acid/100 g dried sample (GAE equivalents).
  • Total anthocyanin content (TAC)
    • Method: 2 g roselle powder + 50 mL extraction solvent (95% EtOH: 1.5 N HCl, 85:15); stand overnight at 4 °C; filter; wash residue; pool filtrate to 100 mL; measure at 535 nm.
  • DPPH radical scavenging activity (RSA %)
    • Method: roselle extract (1:10 v/v) mixed with DPPH (0.02 mg/mL in methanol) in 2:1 ratio (sample:DPPH).
    • Incubate at room temp 15 min; measure absorbance at 517 nm; controls included (Ac = absorbance of control); negative and positive controls included (methanol; BHT 0.1 mg/mL).
    • Replicates: triplicate.
    • Inhibition% formula:
    • \text{Inhibition%} = \left(\frac{Abs{control} - Abs{sample}}{Abs_{control}}\right) \times 100.
  • Ferric reducing antioxidant power (FRAP)
    • Method: mix 2.5 mL diluted roselle extract (1:10) with 2.5 mL 0.2 M phosphate buffer (pH 6.6) and 2.5 mL 10% potassium ferricyanide; incubate 20 min at 50 °C; add 2.5 mL 10% trichloroacetic acid; centrifuge 10 min; take 2.5 mL supernatant, dilute with 2.5 mL distilled water and 0.5 mL 0.1% FeCl₃; measure absorbance at 700 nm.
    • Standard: ascorbic acid (0–10 μg/mL, R² = 0.99).
  • Gas chromatography–mass spectrometry (GC/MS) analysis of volatiles
    • Isolation: solid-phase microextraction (SPME) using a DVB/CAR/PDMS 50/30 μm coating fiber; preconditioned at 250 °C for 1 h.
    • Sample: 10 mL roselle solution (2% w/v) in a 15 mL vial; headspace extracted for 30 min at ~40 °C with stirring; desorbed in GC injector at 250 °C for 5 min.
    • Instrument: Agilent 6890 GC connected to Agilent 5973 MS; DB-WAX column (60 m × 0.25 mm × 0.25 μm).
    • Carrier gas: helium; split 1:10.
    • GC oven program: 40 °C (10 min) → 200 °C at 3 °C/min (3 min) → 250 °C at 10 °C/min (2 min).
    • Mass spectrometry: EI 70 eV; scan 33–500 m/z; source/interface temperatures 230 °C / 210 °C.
    • Compound identification: spectral matching to NIST05; Kovats retention indices (RI) confirmed where possible using C5–C25 n-alkanes; KI values provided in Table 2.
    • Quantification: relative amounts as percent of total ion current (TIC); values represent mean of triplicate analyses.
  • Descriptive sensory analysis
    • Panel: seven participants (4 female, 3 male, ages 23–43) from Jiangsu University.
    • Protocol: trained in descriptive sensory analysis; roselle infusion prepared as per cited method; served in coded form; aroma descriptors listed in Table 1.
    • Aroma descriptors (Table 1): Fruity; Floral; Burnt sugar/caramel; Grassy; Pant-like/green; Sweet; Total aroma intensity.
    • Scoring: 0–8 scale; 0–2 scarce; 2–4 light; 4–6 middle; 6–8 strong intensity.
  • Statistical analysis
    • Measurements: in triplicate.
    • Antioxidant data: analyzed by ANOVA; means compared by Tukey test (p < 0.05).
    • Multivariate: Partial least squares regression (PLSR) to relate sensory descriptors to SPME-GC/MS data; PLSR map generated with XLSTAT 2014.
    • Additional tools: radar graphs and ANOVA in Origin Pro 9.0.

Antioxidant properties of Sudanese roselle (red vs white)

  • Overall finding: all four samples exhibited strong antioxidant activity; red varieties generally showed higher phenolic-related metrics than the white variety, though white roselle showed notable RSA (DPPH) and ascorbic-acid-related effects.
  • Anthocyanin content (TAC)
    • Al-Rahad: 32.96 ± 0.41 mg cyanidin/100 g.
    • Al-Fashir: 30.35 ± 0.59 mg cyanidin/100 g.
    • Al-Gezira: 29.51 ± 0.52 mg cyanidin/100 g.
    • White: 0.03 ± 0.00 mg cyanidin/100 g (negligible).
    • Interpretation: TAC correlates with red coloration; white roselle lacks anthocyanins, as expected.
  • Total flavonoid content (TFC)
    • Al-Rahad: 26.51 ± 0.81 mg QE/100 g.
    • Al-Fashir: 25.51 ± 0.80 mg QE/100 g.
    • Al-Gezira: 25.18 ± 0.61 mg QE/100 g.
    • White: 18.05 ± 1.09 mg QE/100 g.
    • Interpretation: red varieties richer in flavonoids than white.
  • Total phenolic content (TPC)
    • Al-Rahad: 148.17 ± 1.05 mg GAE/100 g.
    • Al-Fashir: 136.84 ± 0.84 mg GAE/100 g.
    • Al-Gezira: 137.17 ± 1.56 mg GAE/100 g.
    • White: 79.84 ± 2.01 mg GAE/100 g.
  • DPPH radical scavenging activity (RSA %)
    • White: 86.98 ± 1.43% (highest RSA among samples; note potential typographical inconsistency in table: “86.98d±1.43”).
    • Al-Rahad: 72.31 ± 1.80%.
    • Al-Gezira: 72.04 ± 0.49%.
    • Al-Fashir: 72.64? (text lists 2.64ac±0.41, which is likely a typographical error; intended value is around 72.6% by context).
    • Interpretation: white roselle shows unexpectedly high RSA despite negligible TAC, suggesting contributions from ascorbic acid and other phenolics.
  • Ferric reducing antioxidant power (FRAP)
    • Al-Rahad: 139.28 ± 1.34 mM Fe(II)/100 g.
    • Al-Gezira: 137.28 ± 1.21 mM Fe(II)/100 g.
    • Al-Fashir: 136.28 ± 1.22 mM Fe(II)/100 g.
    • White: 134.58 ± 3.35 mM Fe(II)/100 g.
    • ANOVA: differences among varieties were not significant at p > 0.05; all four samples exhibit strong reducing power.
  • Integrated interpretation
    • Red roselle varieties generally have higher TAC, TFC, and TPC, contributing to high FRAP; white roselle exhibits the highest RSA% perhaps due to ascorbic acid and other non-anthocyanin antioxidants.
    • The authors note that RSA% values exceed the FRAP values, indicating that multiple antioxidant mechanisms are at play and that relying on a single assay is insufficient.

Volatile compositions and aroma profile (GC/MS/SPME)

  • Number and categories of volatiles
    • 19 compounds detected across roselle extracts (A1–A19).
    • Major groups and approximate ranges across varieties:
    • Aldehydes: 18.14%–19.35%
    • Alcohols: 5.63%–9.73%
    • Ketones: 8.26%–10.08%
    • Esters: 0.92%–6.67%
    • Phenols: 0.21%–1.70%
  • Key aldehydes and notes
    • Furfural (A8) present in high amounts across varieties; often associated with drying and aroma notes (bread, almond, sweet).
    • Hexanal (A4) associated with green/fresh/grass notes; white roselle showed the highest hexanal content among varieties.
    • Other aldehydes include benzaldehyde (A10) with almond/burnt sugar notes.
  • Esters and fruity/sweet notes
    • Ethyl acetate (A2), 2-methylpropyl acetate (A3), 1-butanol, 3-methyl-, acetate (A5) contribute fruity/sweet aromas; Al-Rahad exhibited the highest ester content, followed by Al-Fashir and Al-Gezira; white showed the lowest ester content.
  • Alcohols and floral/rose notes
    • 1-hexanol (A7) and phenylethyl alcohol (A19) contribute floral/rose-like aromas; present across varieties with varying intensities; white showed higher 1-hexanol, 2-ethyl- and 1-nonanol (A14) contributing fat/green notes.
  • Ketones and sweet/aromatic notes
    • 5-hepten-2-one, 6-methyl- (A6) and geranyl acetone (A15) contribute sweet, fruity, and green/hay notes; these ketones may arise from carotenoid degradation pathways and contribute to Al-Rahad and Al-Fashir/Al-Gezira aroma profiles.
  • Phenol and antioxidant-related aroma
    • Phenol, 2,4-di-tert-butyl (A16) detected in trace amounts; a phenolic compound with antioxidant properties that could contribute to aroma and health benefits, particularly noted in white roselle.
  • Notable individual compounds and varietal patterns (Table 2)
    • A1 Methacrolein (KI 775): floral note; present in all varieties with varying intensities.
    • A2 Ethyl acetate (KI 877): fruity/aromatic notes; higher in red varieties.
    • A3 2-Methylpropyl acetate (KI 1009): banana/fruit notes; higher in red types.
    • A4 Hexanal (KI 1077): green/fresh notes; higher in white variety (A4 values: Al-Rahad 4.23, Al-Fashir 2.61, Al-Gezira 2.72, White 7.69).
    • A5 1-Butanol, 3-methyl-, acetate (KI 1121): apple/banana/pearl notes; moderate across varieties.
    • A6 5-Hepten-2-one, 6-methyl- (KI 1338): sweet/fruit/green; higher in red types.
    • A7 1-Hexanol (KI 1455): banana/flower/grass; present across varieties.
    • A8 Furfural (KI 1460): bread/almond/sweet; high across varieties (notably high in some red variants).
    • A9 1-Hexanol, 2-ethyl- (KI 1488): rose/green; varied among varieties.
    • A10 Benzaldehyde (KI 1515): almond/burnt sugar; present across varieties.
    • A11 2-Furancarboxaldehyde, 5-methyl- (KI 1575): almond/caramel/burnt; moderate across varieties.
    • A12 Safranal (KI 1664): herb/sweet notes.
    • A13 Ethanone,1-(2-methyl-1-cyclopenten-1-yl): (KI 1600): present in some varieties.
    • A14 1-Nonanol (KI 1661): fat/floral/green notes; higher in white.
    • A15 Geranyl acetone (KI 1761): green/hay/magnolia notes; notable in Al-Fashir/Al-Gezira.
    • A16 Phenol, 2,4-di-tert-butyl (KI 1910): toasted cereal notes; antioxidant-associated aroma.
    • A17 Nonanal (KI 1400): fat/floral/green/lemon; present variably.
    • A18 2-Phenyl-2-butenal (KI 1953): cocoa/roast/rum/sweet notes.
    • A19 Phenylethyl alcohol (KI 1932): rose/honey/spice notes.
  • Overall volatile profile patterns by variety (Fig. 2 interpretation)
    • Aldehydes dominated (18–19%), followed by ketones, alcohols, esters, phenols.
    • White roselle showed a relatively higher hexanal level and lower ester/aldehyde balance compared with red types, contributing to a greener aroma.
    • Al-Rahad typically showed higher ester content, associated with fruity/floral aromas; Al-Fashir and Al-Gezira showed intermediate profiles; white had distinctive compounds such as higher hexanal and some phenols.
  • Sensory relevance (Table 1 descriptors)
    • Fruity and Floral descriptors were associated with pleasant aroma notes; Green/Grassy was linked to less desirable notes in this context.

Sensory analysis and aroma profiling

  • Descriptive sensory results (Table 1 and Fig. 3, radar plot)
    • White roselle: highest overall aroma intensity but with the strongest unpleasant descriptor “Green/Grassy”; lower scores for pleasant descriptors “Fruity” and “Floral.”
    • Al-Rahad: highest positive descriptors “Fruity” and “Floral”; low “Green/Grassy,” indicating superior aroma quality.
    • Al-Fashir and Al-Gezira: high scores for “Sweet”; moderate scores for “Floral,” “Fruity,” and “Green/Grassy”; overall intermediate aroma profiles.
    • Aroma-based grading (from radar): high grade (Al-Rahad), middle grade (Al-Fashir, Al-Gezira), low grade (White).
  • Link to market value and potential applications
    • Aroma profile suggests Al-Rahad as the preferred cultivar for aroma quality; white roselle offers high antioxidant potential but less favorable aroma.

Relationship between aroma compounds and sensory data (PLSR)

  • Multivariate approach
    • 19 volatile compounds (A1–A19) as independent variables; five sensory attributes (fruity, floral, sweet, caramel, green/grassy) as dependent variables.
    • PLSR explained 94.63% of total variance with the first two components, indicating a strong relationship between GC/MS data and sensory descriptors.
  • Key correlations (Figure 4 interpretation)
    • Positive correlations with fruity/floral/caramel attributes to: methacrolein (A1), ethyl acetate (A2), 2-methylpropyl acetate (A3), 1-butanol, 3-methyl-, acetate (A5), 1-hexanol (A7), benzaldehyde (A10), 2-furancarboxaldehyde,5-methyl- (A11), phenylethyl alcohol (A19).
    • Positive correlations with sweet descriptor to: furfural (A8), 2,6,6-trimethylcyclohexa-1,3-dienyl methanol (A12), geranyl acetone (A15), 2-phenyl-2-butenal (A18).
    • Green/grassy descriptor positively correlated with: hexanal (A4) and 5-hepten-2-one,6-methyl- (A6).
    • Some aroma attributes could not be fully explained by the measured volatiles, suggesting contributions from other odorants or interactions not captured in this dataset.
  • Practical interpretation
    • The PLSR results support a robust link between GC/MS-detected volatiles and sensory perception, validating GC/MS as a predictive tool for aroma quality in roselle products.

Conclusions and implications

  • This is the first study to address antioxidant properties, volatile aroma profile, and descriptive sensory analysis of Sudanese Hibiscus sabdariffa L. together.
  • Red roselle (Al-Rahad, Al-Fashir, Al-Gezira) generally exhibit stronger antioxidant-related metrics (TAC, TFC, TPC, FRAP) than the white variety, while white roselle showed the highest RSA% (likely due to ascorbic acid and other non-anthocyanin antioxidants).
  • Volatile analysis revealed 19 compounds with distinct varietal profiles: aldehydes dominated, followed by alcohols, ketones, esters, and phenols; white roselle had a higher hexanal content, contributing grassy notes, whereas red varieties carried more fruity/floral esters contributing pleasant aromas.
  • Sensory analysis aligned with instrumental data: Al-Rahad possessed the most favorable aroma descriptors (fruity, floral, caramel); white roselle had a more green/grass-oriented aroma profile; Al-Fashir and Al-Gezira showed intermediate profiles.
  • PLSR demonstrated a strong relationship between volatile composition and sensory attributes (R² ≈ 0.9463 for the first two components), indicating that GC/MS data can predict sensory descriptors to a significant extent.
  • Overall, Al-Rahad stands out as having a superior combination of antioxidant properties and an aroma profile deemed most favorable by sensory judges; white roselle offers remarkably high RSA but different aroma characteristics; selection of cultivar could be guided by the intended application (functional antioxidant beverage vs. aroma-driven product).

Connections to broader concepts and applications

  • Link between phytochemicals and health benefits: anthocyanins, flavonoids, phenolics, and ascorbic acid contribute to antioxidant capacity; variability can arise from genotype, geography, and processing conditions.
  • Aroma science principles: volatile compounds arise via multiple biosynthetic pathways (lipid oxidation, carotenoid degradation, amino acid catabolism); aldehydes and esters commonly shape fruit/floral notes, while green/grass notes relate to aldehydes like hexanal and certain alcohols.
  • Methodological integration: combining chemical analytics (SPME-GC/MS) with sensory science (descriptive analysis) and chemometrics (PLSR) provides a holistic view of product quality and potential market value.
  • Practical implications for product development: cultivar selection (Al-Rahad vs white) can be tailored to target antioxidant benefits, flavor/aroma profiles, or a balance of both depending on the product (beverage, tea, extract).
  • Ethical/real-world relevance: work supports agricultural and industrial decisions for Roselle production in Sudan and similar regions, potentially impacting local livelihoods and food systems by validating varieties with desirable health and sensory attributes.

Key data at a glance (selected values)

  • Anthocyanin content (TAC, mg cyanidin/100 g)
    • Al-Rahad: 32.96 ± 0.41
    • Al-Fashir: 30.35 ± 0.59
    • Al-Gezira: 29.51 ± 0.52
    • White: 0.03 ± 0.00
  • Total flavonoid content (TFC, mg QE/100 g)
    • Al-Rahad: 26.51 ± 0.81
    • Al-Fashir: 25.51 ± 0.80
    • Al-Gezira: 25.18 ± 0.61
    • White: 18.05 ± 1.09
  • Total phenolic content (TPC, mg GAE/100 g)
    • Al-Rahad: 148.17 ± 1.05
    • Al-Fashir: 136.84 ± 0.84
    • Al-Gezira: 137.17 ± 1.56
    • White: 79.84 ± 2.01
  • DPPH RSA (% inhibition)
    • White: 86.98 ± 1.43% (highest RSA)
    • Al-Rahad: 72.31 ± 1.80%
    • Al-Gezira: 72.04 ± 0.49%
    • Al-Fashir: 72.64? (text shows 2.64; likely a typographical error; intended around 72.6%)
  • FRAP (mM Fe(II)/100 g)
    • Al-Rahad: 139.28 ± 1.34
    • Al-Fashir: 136.28 ± 1.22
    • Al-Gezira: 137.28 ± 1.21
    • White: 134.58 ± 3.35
  • Volatile compounds identified (19; KI values shown in Table 2)
    • A1 Methacrolein, KI 775; floral aroma; present in all varieties (quantitative values per variety in Table 2)
    • A2 Ethyl acetate, KI 877; fruity/aromatic; high in red varieties
    • A3 2-Methylpropyl acetate, KI 1009; banana/fruit notes
    • A4 Hexanal, KI 1077; green/apple; highest in White
    • A5 1-Butanol, 3-methyl-, acetate, KI 1121; apple/banana/pearl notes
    • A6 5-Hepten-2-one, 6-methyl-, KI 1338; sweet/fruit/green
    • A7 1-Hexanol, KI 1455; banana/flower/grass
    • A8 Furfural, KI 1460; bread/almond/sweet
    • A9 1-Hexanol, 2-ethyl-, KI 1488; rose/green
    • A10 Benzaldehyde, KI 1515; almond/burnt sugar
    • A11 2-Furancarboxaldehyde, 5-methyl-, KI 1575; almond/caramel/burnt
    • A12 Safranal, KI 1664; herb/sweet
    • A13 Ethanone,1-(2-methyl-1-cyclopenten-1-yl), KI 1600
    • A14 1-Nonanol, KI 1661; fat/floral/green
    • A15 Geranyl acetone, KI 1761; green/hay/magnolia
    • A16 Phenol, 2,4-di-tert-butyl, KI 1910; toasted cereal
    • A17 Nonanal, KI 1400; fat/floral/green/lemon
    • A18 2-Phenyl-2-butenal, KI 1953; cocoa/roast/rum/sweet
    • A19 Phenylethyl alcohol, KI 1932; rose/honey/spice
  • PLSR findings (relationship between GC/MS data and sensory data)
    • First two components explain 94.63% of total variance.
    • Fruity, Floral, Caramel positively associated with A1, A2, A3, A5, A7, A10, A11, A19.
    • Sweet associated with A8, A12, A15, A18.
    • Green/Grassy associated with A4 and A6 (negative relation to Fruity/Floral/Caramel).
    • Indicates aroma perception results from a balance and interaction among multiple volatiles, not just single compounds.

Summary of key takeaways

  • Sudanese red roselle varieties (Al-Rahad, Al-Fashir, Al-Gezira) show superior antioxidant profiles (TAC, TFC, TPC, FRAP) compared with the white variety, while white roselle exhibits the highest RSA% potentially due to ascorbic acid and non-anthocyanin antioxidants.
  • White roselle has distinct volatile composition with higher hexanal and different esters/alcohols, contributing to greener/aroma profile that is less fruity/floral than red types.
  • Descriptive sensory analysis aligns with instrumental data: Al-Rahad provides the most desirable aroma profile (high Fruity/Floral), while White is characterized by greener notes and lower Fruity/Floral scores.
  • PLSR demonstrates a strong link between GC/MS volatile data and sensory descriptors, supporting the use of instrumental aroma profiling to predict sensory impressions in roselle products.
  • Practical implication: Al-Rahad is recommended for aroma-rich products with strong antioxidant properties; white roselle could be explored for antioxidant applications, with consideration of its distinct aroma profile.
  • Limitations and future work: the RSA value for Al-Fashir in the reported table may contain a typographical error; future work should confirm RSA values with replicated measurements and expand the volatile profile to include odor-active compounds not captured in this study.