Hibiscus Sabdariffa L. (Malvaceae): Phytochemical Study of Indigenously Grown and Biologically Authenticated Variety

Abstract

Herbal medicine has played a central role in health care since ancient times, serving both prophylactic and therapeutic purposes. People civilisations across have relied on ethnomedicinal knowledge and herbal remedies for the treatment and prevention of disease. In India, we see the government also support in continuing this legacy through supporting what is called AYUSH (Ayurveda, Yoga & Naturopathy, Unani, Siddha, and Homeopathy). A system working towards upholding the significance of traditional medicine, particularly Ayurveda, in promoting holistic well-being through herbal therapies. In the modern society, a growing integration of food-based medicine and nutraceuticals into daily life reflects a broader global shift toward preventive health strategies. Traditional ingredients of the likes of turmeric and olive oil that are well-documented for their cardioprotective properties are making a comeback into the everyday diet. A similar interest has emerged around Hibiscus sabdariffa (roselle), a versatile plant used as food, beverage, and therapeutic agent. A member of the Malvaceae family, H. sabdariffa is an annual or perennial herbaceous plant characterized by red stems, serrated leaves, and vibrant calyces. Its known by various aliases depending on its region of cultivation such as Indian sorrel, Jamaica sorrel, and Florida cranberry. In light of this shift to traditional ingredients for health benefits and the growing popularity of H. sabdariffa, this study focuses on the phytochemical evaluation the species cultivated in India. A particular emphasis has been placed on its anthocyanin and phenolic compound content—key bio actives associated with potent antioxidant, antidiabetic, antihypertensive, and diuretic effects. For this analysis, ethanolic extracts of the plant were analysed using UV-visible spectroscopy and high-performance liquid chromatography (HPLC) in a certified analytical laboratory. The findings of this study aim to contribute to the understanding of regional variations in phytochemical profiles and support the broader application of H. sabdariffa in evidence-based phytopharmacology.

Keywords

Introduction

A plan known for its rich phytochemical profile¹ ,Hibiscus sabdariffa (H. sabdariffa) is commonly found in tropical and subtropical regions such as Africa, India, Jordan, and Sudan. The flower contains a diverse array of bioactive constituents—including anthocyanins, flavonoids, and phenolic derivatives—each recognized for their therapeutic potential in the management and prevention of numerous diseases. ² Among these, phenolic compounds have attracted considerable attention due to their potent antioxidant properties, demonstrated in both in vitro and in vivo studies. A strong free radical scavenging activity has been exhibited in these compounds³, a feature that underpins many of their pharmacological benefits. The principal bioactive constituents of H. sabdariffa include organic acids (such as hydroxycitric acid and hibiscus acid), anthocyanins (notably delphinidin-3-sambubioside and cyanidin-3-sambubioside), and a range of flavonoids and phenolic acids (including gallic acid, quercetin, kaempferol, caffeic acid, chlorogenic acids, and galloyl esters). The biosynthesis and accumulation of these compounds is greatly influenced by genetic and environmental factors, such as rainfall, temperature, maturity stage, and cultivation practices.¹ Notably of these, the anthocyanin content is highly variable and dependent on regional climatic and soil conditions. Given this context, the present study aims to analyze the phytochemical composition of H.sabdariffa cultivated in India, with a specific focus on anthocyanins and phenolic compounds due to their diverse pharmacological activities. Ethanolic extracts of the plant were subjected to UV-visible spectroscopy and high-performance liquid chromatography (HPLC) analysis, performed by a certified standard laboratory. The findings are expected to highlight the plant's antioxidant potential and its associated antidiabetic, antihypertensive, and diuretic effects.

METHODS

To begin with, dried calyces of Hibiscus sabdariffa (15 g) were collected and authenticated⁹. The material was crushed into small pieces and immersed in 100 mL of absolute ethanol (99%) in a beaker. The mixture thus obtained was then covered with aluminium foil and left to macerate at room temperature for a duration of 72 hours. Post this, the ethanol was evaporated at 45?°C to yield approximately 15 mL of concentrated residue of this mixture. The extract obtained was further used for phytochemical analysis using ultraviolet-visible (UV-Vis) spectroscopy and high-performance liquid chromatography (HPLC) techniques.

RESULTS

Visual Characteristics of the Extract

The ethanolic extract of Hibiscus sabdariffa (HS-EE) exhibited a deep red coloration, suggestive of a high anthocyanin content. This qualitative observation supports existing reports that link the red hue of H. sabdariffa extracts to the presence of water- and alcohol-soluble anthocyanin pigments.

UV-Visible Spectrophotometric Analysis

UV-Vis analysis was performed to assess the absorbance spectra of the HS-EE. A prominent peak was observed at λ?=?535?nm, which falls within the characteristic absorption range of anthocyanins (typically 505–535?nm). This strong absorption at 535?nm confirms the presence of anthocyanins, further correlating with the intense red coloration of the extract obtained.

Additional bioactive compounds detected via UV analysis are presented in Table 1.

Table 1: UV Analysis of HS-EE

Extract	Wave length	Bio -Compound Shown
Raw HS-EE	252 nm(Peak 1)	Hydro benzoic acid and flavonols
Raw HS-EE	535 nm(Peak II)	Anthocyanin
Raw HS-EE	664 nm (Peak II)	Cyanidin -3-glucoside

UV-HPLC Analysis of Hibiscus sabdariffa Ethanolic extract for its Bio-active components evaluation:

In this study the authors have taken calyxes of H. Sabdariffa and made an ethanolic extract. We subject Hibiscus sabdariffa ethanolic extract (HS-EE) to UV and HPLC studies. The typical peaks shown at 535 nm⁵ in UV and Retention time RT 8.72. shown by HPLC shows emphatically presence of anthocyanin. The other peaks show presence of other bioactive components which are enlisted in Table-1 and Table 2. The deep red colour of HS -EE further confirms that anthocyanin is present in favourable amount. A UV peak at 535 nm ⁴ in Hibiscus sabdariffa is indicative of the presence of anthocyanins. Specifically, this wavelength is within the absorption range of anthocyanins, which are the pigments responsible for the red colour of Hibiscus sabdariffa . The absorption spectra of anthocyanin extracts from Hibiscus sabdariffa often show maximum absorption at wavelengths between 505-535 nm⁵. This is corellated in accordance to a study published by IOP Science.¹⁰ 

HPLC analysis of Hibiscus sabdariffa extracts is used in various research applications, identification of bioactive compounds, evaluation of antioxidant activity, and assessment of the quality and authenticity of different extracts. 

Table 2: HPLC Analysis “ISFAL Analytical Report”

HPLC Analysis

Specific Compounds: The following analysis of RT (Retention Time) has shown corresponding Bioactive compounds (Table 2) A Retention Time (RT) of 4.62 in our current study (Table 2) extract typically indicates the presence of chlorogenic in HPLC of a H.Sabdariffa ethanolic extract likely corresponds to delphinidin-3-O-sambubioside, a major anthocyanin compound known for its antioxidant and other biological activities. While this specific RT might vary slightly depending on the HPLC conditions, it's a common elution time for this particular compound in Hibiscus extracts.  A RT of 7.57 minutes in an HPLC analysis of a Hibiscus sabdariffa in our current study (Table 2) extract typically indicates the presence of chlorogenic acid. This compound is known to be present in hibiscus extracts, and its elution time in HPLC can be used to identify and quantify it.  The presence of a peak at 8.76, in our current study (Table 2) in conjunction with other peaks at different retention times, suggests the presence of multiple cinnamic derivatives or a related phenolic compound in the extract. The exact identity of the compound at 8.76 would require further analysis, such as mass spectrometry, to determine its specific structure and molecular weight. 

Elaboration:

Cinnamic Derivatives:

The peak at 8.76 could be indicative of a variety of cinnamic acid derivatives, including but not limited to caffeic acid, ferulic acid, or p-coumaric acid, which are commonly found in Hibiscus sabdariffa. 

Bioactivity:

Cinnamic derivatives are known for their various biological activities, including antioxidant, anti-inflammatory, and antimicrobial properties. Therefore, the presence of cinnamic derivatives in Hibiscus sabdariffa extract, including the one at 8.76, contributes to the overall bioactive potential of the extract. 

Research Applications:

HPLC analysis of Hibiscus sabdariffa extracts is used in various research applications, including the identification of bioactive compounds, evaluation of antioxidant activity, and assessment of the quality and authenticity of different extracts. 

1. Antioxidant Activity of Leaf Extracts from Different Hibiscus sabdariffa

DISCUSSION

The present study investigated the phytochemical composition of  HS-EE, emphasizing its anthocyanin, phenolic acid, and cinnamic acid derivative content. The analytical profiling was conducted through UV-visible spectrophotometry and high-performance liquid chromatography (HPLC), techniques widely recognized for their precision in bioactive compound analysis (Snyder et al., 2011)?¹¹?. HPLC, in particular, is a robust method for separating and identifying chemical constituents within complex plant matrices. In the context of H. sabdariffa, it plays a vital role in detecting phenolics such as flavonoids and cinnamic acid derivatives, which are often responsible for the plant's therapeutic effects (Chen et al., 2003; Obouayeba et al., 2014)?¹²,¹³?. Our study, conducted at ISFL Laboratory, Moga, India (ISFL, 2025)???, yielded significant findings: a RT of 4.62?min identified delphinidin-3-O-sambubioside, 7.57?min indicated chlorogenic acid, and 8.76?min pointed to potential cinnamic derivatives such as caffeic and ferulic acids (Iqbal et al., 2020)?¹³?. UV-visible spectroscopy revealed a strong absorbance peak at 535?nm, a spectral hallmark of anthocyanins (Yang & Zhang, 2001; Iqbal et al., 2020)?¹³,¹??. The anthocyanins not only confer the characteristic red pigmentation to H. sabdariffabut also act as potent antioxidants, mitigating oxidative damage through free radical scavenging (Duenas et al., 2005)?¹⁵?. The authenticated sample (ISFL, 2025)??? confirmed the presence of delphinidin-3-O-sambubioside, chlorogenic acid, cinnamic derivatives, and various polyphenols. These were confirmed using both UV and HPLC methods (Lin et al., 2007; Mullen et al., 2007)?¹⁶,¹⁷?. As outlined in Tables 1 and 2, these constituents were observed across multiple peaks, graphically substantiating their presence. Polyphenols, including anthocyanins and related compounds, are known for their broad spectrum of biological activities. The phytochemical constituents detected in HS-EE are linked to antioxidant, antidiabetic, antihypertensive, diuretic, and antihyperlipidemic effects (Yang & Zhang, 2001; Lin et al., 2007)?¹⁸,¹⁹?. This multifunctionality enhances the therapeutic promise of H. sabdariffa, aligning with prior ethnobotanical and pharmacological reports (Obouayeba et al., 2014)?²⁰?. Furthermore, the extraction protocol—employing absolute ethanol, fine particle size, ambient temperature, and a pH of 5.76 for 72 hours—ensured optimal yield and phytochemical stability, consistent with literature indicating these conditions favour anthocyanin recovery (Mullen et al., 2007)?²¹?. In summary, the phytochemical fingerprint established by this study reinforces the medicinal significance of H. sabdariffa, particularly regarding its polyphenol-rich profile. Future research using advanced techniques such as LC-MS and NMR could provide deeper structural validation of minor constituents and illuminate their pharmacodynamic mechanisms.

CONCLUSION

Based on the findings outlined in the results, the HS-EE demonstrates a rich phytochemical composition, notably characterized by the presence of anthocyanins, chlorogenic acid, and cinnamic acid derivatives. The extract’s intense red colour and UV absorption at 535?nm strongly support the abundance of anthocyanins, with HPLC analysis further confirming the presence of delphinidin-3-O-sambubioside, a potent antioxidant compound. Additional RT peaks indicate the presence of chlorogenic acid and bioactive cinnamic derivatives such as caffeic and ferulic acid, which collectively contribute to the extract’s therapeutic potential. The extraction protocol—employing absolute ethanol, ambient temperature, optimal pH, and prolonged exposure time—proved effective in maximizing the yield and stability of these constituents. These bioactive compounds are associated with a spectrum of pharmacological properties, including antioxidant, antidiabetic, antihypertensive, diuretic, and antihyperlipidemic effects. Altogether, the study reinforces H. sabdariffa's value as a phytochemical-rich herb with promising applications in traditional medicine, functional foods, and nutraceutical development.

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