Evaluation of antioxidant properties and quantification of key antioxidant phytochemicals in Hibiscus sabdariffa
Creators: name: Abdul Latif, Reem
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300699
subject: Food sciences not elsewhere classified
classificationCode: 321405
subject: Pharmaceutical sciences

description: This thesis reports research conducted to:

1. Determine antioxidant capacity of dry and fresh calyxes of Hibiscus sabdariffa.
2. Validate the HPLC method for analysis of the key antioxidant phytochemicals of Hibiscus sabdariffa.
3. Quantify the amounts of key antioxidants in the extracts of Hibiscus sabdariffa calyxes.

Hibiscus sabdariffa has gained researchers’ attention due to its high antioxidant content. The dry and, to a much less extent, fresh calyxes of Hibiscus sabdariffa are used in making of herbal tea beverages, jams, and other processed foods. Hibiscus sabdariffa plant has been used to treat common colds, coughs, tooth pain, infections of the urinary tract, hypertension and hangovers in traditional medicine. Dry Hibiscus sabdariffa extracts also have anti-inflammatory actions, weight loss effects, lipid lowering effects, antihypertensive effects, anti-diabetic effects, diuretic, antimicrobial, anticancer and immunomodulation effects.

Oxidation is defined as a chemical reaction that includes the transfer of an electron from a chemical entity to an electron lacking entity. Cell respiration of mitochondria is the oxidative phosphorylation reaction involving different metabolic processes occurring in the cells of living organisms. It involves the transfer of chemical energy from oxygen molecules and nutrients into energy used by the cell.

A free radical is defined as an atom or molecule that can receive one or more unpaired electrons. Free radicals present in the cell, derived from oxygen, are called reactive oxygen species. They render the cellular chemical reaction unstable and more reactive. They cause dysfunction of the mitochondria and / or mitochondrial DNA mutations, resulting in peroxidation of lipids and protein damage. Oxidative stress is defined as an equilibrium disorder between the reactive oxygen species production and antioxidant activity and can lead to tissue damage and inflammation.

An antioxidant is “a substance that opposes oxidation and inhibits reactions promoted by oxygen or peroxides”. Antioxidants are of two major types. The first type consists of the cell’s enzymes, for example superoxide dismutase, glutathione peroxide and catalase that target the reactive oxygen species. The second type are chain-breaking antioxidants found in the blood and extracellular fluids; such as vitamin E, these break the cascade of reactive oxygen species formation by electron donation.  The chemical structure of polyphenols shows that they act as hydrogen-donating radical scavenger compounds. These interact with the ions of metals, such as Cu, Zn, and Fe, as a chelating agent.
 
The use of different antioxidants in the treatment or prevention of cancer, respiratory illnesses, Parkinson’s / Alzheimer’s neurodegenerative diseases, cardiovascular diseases, diabetes mellitus and digestive diseases is well supported. The antioxidant capacity of natural products depends on different factors. These include extraction methods, composition of the extract and ecological factors, for example the type of weather, season of harvest, type of the soil and elevation from sea level. The physicochemical properties of the compounds such as the hydrophilicity and lipophilicity of the antioxidants also have important effects on the bioavailability of the antioxidant, affecting its clinical use.

Hibiscus sabdariffa extract contains mainly polyphenols, anthocyanins and flavonoids. Polyphenol flavonoids include quercetin, quercetin-3-rutinoside and kaempferol glycosides. Quercetin is one of the major antioxidant compounds of Hibiscus sabdariffa.
 
The anthocyanin antioxidant compounds are the key antioxidants of this plant including delphinidin 3-glucoside, delphinidin 3-sambubioside, cyanidin 3-sambubioside and cyanidin 3-glucoside.
 
In this study the key antioxidants in Hibiscus sabdariffa extracts will be evaluated. Quercetin, delphinidin 3-sambubioside and cyanidin 3-sambubioside were selected because of their high antioxidant potency as described in previous literature.  Quercetin (non - anthocyanin) belongs to the class of compounds called flavonoids while cyanidin 3-sambubioside and delphinidin 3-sambubioside are anthocyanins.

In this study, the extraction (in cold &amp; hot water, ethanol and methanol) was performed using ground dry Hibiscus sabdariffa and fresh Hibiscus sabdariffa calyxes. Fresh calyxes were freeze dried then put into the desiccator after grinding for further use in the experiments. The antioxidant capacity of dry and fresh Hibiscus sabdariffa calyxes was analyzed using two methods, α, α-DiPhenyl-β-PicrylHydrazyl (DPPH) and Ferric Reducing Antioxidant Properties (FRAP) assays.

To quantify the amounts of quercetin and anthocyanins, a reversed phase High Performance Liquid Chromatography (RP-HPLC) analytical method was used. Method validation was performed as per the International Council for Harmonization (ICH) guidelines.

The DPPH assay method was performed to define the effect of time (up to 7 days) and temperature (4˚C, -30˚C and -80˚C) on the stability of Hibiscus sabdariffa extracts. These conditions were chosen to determine extract stability conditions and to enable researchers to improve experimental efficacy.

The DPPH and FRAP assay of aqueous and organic extracts of dried calyxes of Hibiscus sabdariffa after 30, 60 and 90 minutes of the commencement of the reaction showed no statistically significant changes in the IC50. This indicated that the 30 minutes oxidant – antioxidant time period was sufficient for the DPPH reaction. 

There was a significant difference in IC50 values between the different solvent extracts (p ≤ 0.05). The methanol extract of dry and fresh Hibiscus sabdariffa had the greatest antioxidant capacity.

The validation of HPLC methods for quercetin and cyanidin-3-O-sambubioside showed reliable results in accordance with ICH and the guidelines of Center for Drug Evaluation and Research (CDER). Quercetin and cyanidin-3-O-sambubioside retention times were constant in all the samples tested and in the different concentrations used. The mean retention time of quercetin was 27.34 minutes and that of cyanidin-3-O-sambubioside was 11.20 minutes. The area under the curve calculated in both linearity tests showed directly proportional values against the concentration used. Quercetin and cyanidin-3-O-sambubioside test samples showed consistent results of precision (intermediate precision &amp; repeatability), specificity and accuracy. The system suitability tests of both quercetin and cyanidin-3-O-sambubioside showed the number of plates as more than 2000, tailing factors were &lt; 1.5, capacity factor of &gt; 2, and injection repeatability RSD of &lt; 1%. The LOD for quercetin was calculated to be 1.67 µg/mL while the LOQ was 5.06 µg/mL. The LOD and LOQ for cyanidin-3-O-sambubioside was 0.25 µg/mL and 0.76 µg/mL, respectively.

The HPLC protocol used to separate the major antioxidant compounds of Hibiscus sabdariffa extract showed a constant and reproducible reaction time and area under the curve. The amount of quercetin was 5.39 µg/mL while that of cyanidin 3- O- sambubioside was 174.86 µg/mL in dry Hibiscus sabdariffa extract. The standard utilized did not allow the researchers to accurately quantify the amount of delphinidin 3-sambubioside.

This research has concluded the following:

1. Hibiscus sabdariffa methanol extract showed the highest antioxidant capacity. Antioxidant capacity of fresh Hibiscus sabdariffa calyx methanol extract was significantly higher than that of dried calyx. 
2. Reverse Phase HPLC method is a reliable technique to determine the amount of antioxidant phytochemicals in Hibiscus sabdariffa extract. 
3. The antioxidants of Hibiscus sabdariffa are considerable and could be used in research to explore its benefits in the treatment of different oxidation–induced diseases.