Phytochemical Analysis of Aqueous Flower Extract of Hibiscus sadariffa (Zobo Flower)

¹Obidoa, Onyechi*, ¹Joshua, Parker Elijah, ¹Egemole, John C. and ²Ikeyi Adachukwu

¹Department of Biochemistry, University of Nigeria, Nsukka, Enugu, Nigeria

²Department of Science Laboratory Technology (Biochemistry Option), Institute of Management & Technology, Enugu, Enugu State, Nigeria.

ABSTRACT:

The phytochemical analyses of Hibiscus sabdariffa was extensively carried out using Harborne method. The qualitative test of the phytochemical analyses revealed the presence of some secondary metabolites such as flavonoids, glycosides, tannins, acidic compounds and steroids. Macronutrients such as proteins, carbohydrates and reducing sugars were also found to be present. The results of the analyses also revealed the differences in solubility of the phytochemicals and macronutrients in polar solvent (water). In this study, flavonoids, tannins and carbohydrates were found to be present in higher concentration. On the other hand, bioactive compounds such as glycosides, steroids, acidic compounds, proteins and reducing sugars were found to be present in moderate concentration while alkaloids, saponins, terpenoids and fats and oil were absent in the zobo flower.

KEYWORDS: Hibiscus sabdariffa; Phytochemicals; Macronutrients; Zobo.

INTRODUCTION:

The plant Hibiscus sabdariffa is commonly called “Roselle” and “Zobo” in Nigeria which belongs to the family Malvaceae. It is a popular vegetable in Indonesia, India, West Africa and tropical regions (Adanlawo and Ajibade, 2006). In Nigeria, two botanical varieties are recognized. The calyces (Hibiscus sabdariffa) of the red variety are used for the preparation of “Zobo” drink and soup while calyces of gree variety are used to cook soup, stew and sauces. The calyx of roselle (green) is rich in vitamin C and riboflavin with some major minerals present (Babalola, 2000).

Hibiscus sabdariffa plant has been used in many ways both for industrial purposes and medicinal uses. It has been incorporated in the treatment of many ailments such as hypertension, arterioclerosis, neurosis, cancer, etc (Tseng et al., 1996; Duh and Yed, 1997; Tsai et al., 2002). Hibiscus sabdariffa has been used for many industrial products. They are used fresh for making wine, juice, jam, jelly, syrup, gelatin, pudding, cakes, ice cream and flavors and also dried and brewed into tea, spice and used for butter, pies, sauces and the juice preparation of roselle involves boiling it for some time and sieve the flowers out to obtain the juice, sweetening it with sugar and adding ginger. This makes a wonderful juice (Wikipedia, 2008).

The plant has been used in the treatment of many ailments and used in many folk medicines. The leaves and flowers are used as a tonic tea for digestive and kidney function (Yadong et al., 2003). The leaves and flowers contains antioxidant including flavonoids (gossypetine), glycosides, etc and also it is said to have diuretic effects, to help lower fever and is antiscorbutic. The flower decreases the viscosity of the blood, reducing blood pressure and stimulating intestinal peristalsis. It is used in the folk remedy in the treatment of abscesses, bilious conditions, cancer, cough, debility, dyspepsia, dysuria, fever, hangover, heart ailment, hypertension, neurosis, scurvy and strangurg. It also has value in the treatment of arterioclerosis (Ali et al., 2005).

Hibiscus sabdariffa is commonly called Roselle and the red Roselle contains calcium, iron, protein (seed) (Morton, 1975). It is also known that the plant sample (Hibiscus sabdariffa) is rich in citric acid, pectin and so useful for making jams, jellies, etc. The fresh plant is also rich in riboflavin, ascorbic acid which is major in vitamin, niacin, carotene, calcium and iron that are of nutrition important. The seeds are high in protein; it can be roasted and ground into powder then used in soup and sauces (Yadong et al., 2003).

Phytochemicals are plant or fruit derived chemical compounds that can be used as therapeutic agents. They reduce the risk of cancer due to dietary fibres, polyphenol antioxidants and anti-inflammatory effects (Kindersley, 2006). The phytochemicals are produced via secondary metabolism in relatively small amounts (Hasler, 1998).

This work is aimed at determining the presence of secondary metabolites (phytochemicals) in Hibiscus sabdariffa (Zobo flower).

MATERIALS AND METHODS:

Plant Materials:

The flowers of Hibiscus sabdariffa (Zobo Flowers) were bought from a Nsukka Main Market, Enugu State of Nigria. The identification was made by Mr. A. Ozioko, a taxonomist of Bio-resources Development and Conservation Programme center, Nsukka (BDCP).

Preparation of Plant Material:

The fresh flowers of Hibiscus sabdariffa were collected, dried and milled to coarse coarse powder using laboratory mill for the analyses.

Extraction of Plant Material:

A small quantity (150 g) of the milled Hibiscus sabdariffa flower was extracted in cold distilled water and used for the analyses.

Phytochemical Analyses of Hibiscus sabdariffa:

The phytochemical tests below were carried out on the semi-solid extract of Hibiscus sabdariffa to determine the active constituents according to the procedures and methods outlined in Trease and Evans (1989) and Harborne (1998). These phytochemical tests were done to detect the presence of secondary metabolites, such as alkaloids, tannins, saponins, resins, flavonoids, steroid, glycosides and terpenoids in the plant under investigation.

Test for Alkaloids:

A quantity (0.2g) of the sample was boiled with 5ml of 2% HCl on a steam bath. The mixture was filtered and 1ml portion of the filtrate was measured into four test tubes. The 1ml filtrate was treated with 2 drops of the following reagents.

A. Dragendorff’s Reagent: A red precipitate indicates the presence of alkaloids.

B. Mayer’s Reagent: A creamy-white colored precipitate indicates the presence of alkaloids.

C. Wagner’s Reagent: A reddish-brown precipitate indicates the presence of alkaloids.

D. Picric Acid (1%): A yellow precipitate indicates the presence of alkaloids.

Test for Flavonoids:

A quantity (0.2g) of the extract was heated with 10ml of ethylacetate in boiling water for 3 minutes. The mixture was filtered differently and the filtrates used for the following tests:

A. Ammonium Test: A quantity (4ml) of the filtrates was shaken with 1ml of dilute ammonia solution (1%). The layers were allowed to separate. A yellow coloration was observed at the ammonia layer, which indicates the presence of flavonoids.

B. Aluminum Chloride Test: A quantity (4ml) of the filtrates was shaken with 1ml of 1% aluminum chloride solution and observed for light yellow coloration. A yellow precipitate indicates the presence of flavonoids.

Test for Glycosides:

Dilute sulphuric acid (5ml) was added to 0.1g of the extract in a test tube and boiled for 15 minutes in a water bath. It was then cooled and neutralized with 20% potassium hydroxide solution. A mixture, 10ml of equal parts of Fehling’s solution A and B was added and boiled for 5 minutes. A more dense red precipitate indicates the presence of glycoside.

Test for Steroids and Terpenoids:

A quantity (9ml) of ethanol was added to 1g of the extract and refluxed for a few minute and filtered. The filtrate was concentrated to 2.5ml in a boiling water bath. Distilled water, 5ml was added to the concentrated solution, each of the mixtures was allowed to stand for 1 hour and the waxy matter was filtered off. The filtrate was extracted with 2.5ml of chloroform using a separating funnel. To 0.5ml of the extract in a test tube was carefully added 1ml of concentrated sulphuric acid to form a lower layer. A reddish-brown interface shows the presence of steroids.

To another 0.5ml of the extract was evaporated to dryness on a water bath and heated with 3ml of concentrated sulphuric acid for 10 minutes on a water bath. A grey colour indicates the presence of terpenoids.

Test for Saponins:

A quantity (0.1g) of the extract was boiled with 5ml of distilled water for 5 minutes. The mixture was filtered while still hot and the filtrate used for the following tests:

A. Emulsion Test

A quantity (1ml) of the filtrate was added drops of olive oil. The mixture was added to another two drops of olive. The mixture was shaken and observed for the formation of emulsion.

B. Frothing Test

A quantity (1ml) of the filtrate was diluted with 4ml of distilled water. The mixture was shaken vigorously and then observed on standing for a stable froth.

Test for Tannins:

A quantity (2g) each, of the extract was boiled with 5ml of 45% ethanol for 5 minutes. Each of the mixtures was cooled and filtered. The different filtrates were subjected to the following tests.

A. Lead Sub-acetate Test

To 1ml of the different filtrate was added 3 drops of lead sub-acetate solution. A cream gelatinous precipitate indicates the presence of tannins.

B. Ferric Chloride Test

A quantity (1ml) of the filtrate was diluted with distilled water and added 2 drops of ferric chloride. A transient greenish to black color indicates the presence of tannins.

Test for Acidic Compounds:

A quantity (0.1g) of the extract was placed in a clear dry test tube and sufficient water added. These were warmed differently in a hot water bath and cooled. A piece of water-wetted litmus paper was dipped into the different filtrates and observed for color change. Acidic compounds turn blue litmus paper red.

Test for Resins:

Two tests were carried out to detect the presence of resins in the plant under investigation.

A. Precipitate Test

A quantity (0.2g) each of the extract was treated with 15ml of 96% ethanol. The alcoholic extract was then poured into 20ml of distilled water in a beaker. A precipitate occurring indicates the presence of resins.

B. Color Test

A quantity (0.12g) each of the extract was treated with chloroform and the extract concentrated to dryness. The residues were re-dissolved in 3ml of acetone and 3ml of concentrated hydrochloric acid added. The mixtures were now heated differently in a water bath for 30 minutes. Pink color, which changes to magenta-red, indicates the presence of resins.

MACRONUTRIENT Analyses of Hibiscus sabdariffa:

The tests below were carried out to determine the presence of macronutrients in the flower extract of Hibiscus sabdariffa.

Test for Proteins:

A quantity (5ml) of distilled water was added to 0.1g of the extracts. This was left to stand for 3 hours and then filtered. To 2ml portion of the filtrate was added 0.1ml Million’s reagent. It was shaken and kept for observation. A yellow precipitate indicates the presence of proteins.

Burette Test:

A quantity (2ml) of the extract was put in a test-tube and 5 drops of 1% hydrated copper sulphate was added. A quantity, 2ml of 40% sodium hydroxide was also added and the test-tube shaken vigorously to mix the contents. A purple coloration shows the presence of proteins (presence of two or more peptide bonds).

Test for Carbohydrate:

A quantity of 0.1g each of the extract was shaken vigorously with water and then filtered. To the aqueous filtrate was added few drops of Molisch reagent, followed by vigorous shaking again. Concentrated sulphuric acid, 1ml was carefully added to form a layer below the aqueous solution. A brown ring at the interface indicates the presence of carbohydrate.

Test for Reducing Sugar:

A quantity of 0.1g of the extract was shaken vigorously with 5ml of distilled water and filtered. To the filtrate was added equal volumes of Fehling solutions A and B and shaken vigorously. A brick red precipitate indicates the presence of reducing sugars.

Test for Fats and Oil:

A quantity of 0.1g of the extract was pressed between filter paper and the paper observed. A control was also prepared by placing 2 drops of olive oil on filter paper. Translucency of the filter paper indicates the presence of fats and oil.

RESULTS:

Percentage Yield of Extract:

The percentage yield of the aqueous flower extract of Hibiscus sabdariffa was found to be 10.47%.

Phytochemical Compositions of Hibiscus sabdariffa Flower Extract:

The result of the phytochemical analyses carried out on the sample of Hibiscus sabdariffa flower (Zobo) using polar solvent extract was shown in Table 1. The result revealed the relative high concentration of flavonoids under the ammonium and aluminium chloride tests with yellow colour and yellow precipitate observations respectively. Bioactive compound that was found in relative abundance was tannins using lead sub acetate and ferric chloride test.

Table 1 shows moderate presence of glycosides (using Fehling’s solution and Sulphuric acid tests), steroids (using concentrated tetraoxosulphate (iv) acid test) and acidic compounds (using litmus paper test).

However, bioactive compounds such as alkaloids, saponins, resins and terpenoids were not detected in the sample.

No phytochemical was found in low concentration as shown in Table 1.

Table 1: Phytochemical constituents of Aqueous Flower Extract of Hibiscus sabdariffa

Test

Observation

Inference

Alkaloids

i. Dragendorff’s Reagent

ii. Mayer’s Reagent

iii. Wagner’s Reagent

iv. Picric Acid Solution (1%)

No Reaction

”

–

Flavonoids

i. Ammonium Test

ii. Aluminium Chloride Test

Yellow colour.

Yellow precipitate.

+++

Glycosides

i. Fehling’s Solution Test

ii. Sulphuric Acid Test

Brick red precipitate.

”

Saponins

i. Emulsion Test

ii. Frothing Test

No Emulsion.

Stable froth not formed.

–

Tannins

i. Lead Sub Acetate Test

ii. Ferric Chloride Test

Cream gelatinous precipitate.

A transient greenish to black colour change.

+++

Resins

i. Precipitate Test

ii. Colour Test

No precipitate

No colour change

–

Steroids

i. Conc. H₂SO₄ Test

Reddish brown interface

Terpenoids

i. Conc. H₂SO₄ Test

Grey colour not observed

–

Acid Compounds

i. Litmus Paper Test

Blue litmus turned red

Key:-

+++ = Relative abundance of compound: ++ = Moderate abundance of compound: + = Relative low presence of compound: ND = Not detected

Macronutrient Compositions of Hibiscus sabdariffa Flower Extract

Table 2 shows relative moderate abundance of macronutrient such as carbohydrates using Molisch test with the observation of brown colouration at the interface. Bioactive compounds such as protein and reducing sugars were found to be in relative moderate abundance while fats and oil were not detected in the sample as shown in Table 2 using translucency test.

Table 2: Macronutrient constituents of Aqueous Flower Extract of Hibiscus sabdariffa

Test

Observation

Inference

Protein

i. Millon’s Test

ii. Biuret Test

White precipitate

Purple colour

Carbohydrates

i. Molisch Test

A brown colouration observed at the interface.

+++

Reducing sugar

i. Fehling’s Solution Test

Brick red precipitate.

Fats and Oil

i. Translucency Test

Translucency not observed.

–

Key:-

+++ = Relative abundance of compound: ++ = Moderate abundance of compound

+ = Relative low presence of compound: ND = Not detected

DISCUSSION:

The phytochemical analyses of Hibiscus sabdariffa (Zobo flower) showed the presence of some phytonutrients and some nutrients. The phytonurtient otherwise known as phytochemical present on the plant sample are flavonoids, glycosides, phytosterols and tannis while the macronutrients are proteins, fats and oil and reducing sugar and carbohydrates. The role of Hibiscus sabdariffa in the folk medicine has been attributed to the treatment of abscesses, bilious conditions, cancer, cough, debility, dyspepsia, fever, heart ailments, more especially in cardiovascular diseases, cancer, allegic reactions etc. flavonoids are most commonly known for their antioxidant activity, however it is now know that the health benefit they provide against cancer and heart diseases are the result of other mechanism (Lotito and Frei, 2006). Flavonoids have been referred to as nature’s biological response modifiers because of strong experiment evidence showing their inherent ability to modify the body’s reaction to allegens, viruses and carcinogens. They also show anti-allergic, anti-inflammatory, anti-microbial and anti-cancer activities, these effects have also been attributed to the action of zobo flower (Kushi et al., 2006).

Flavonoids like gossypetin and anthocyanin have effects on decreasing the viscocity of blood, reducing blood pressure and stiulating intestinal peristalsis (Perry, 1980). Studies have showed that the specific flavonoids contained in Hibiscus sabdariffa F. are gossypetin and anthocyanin which make the plant very useful in the treatment of some ailments (Wikipedia, 2006). From results of this study the presence of the flavonoids agreed that of previous studies. This agreement concern the fact that flavonoids are poorly absorbed by human body (less than 5%) and most of what is absorbed is quickly metabolized and excreted, the action of excreting the flavonoids from the body induces the phase II enzymes that gets rid of carcinogens and mutagens that causes diseases like cancer, arteriosclerosis and other cardio vascular diseases (Slavin, 2003).

The anti-microbial activity of Hibiscus sabdariffa could also be attributed to the presence of tannins found on the analyses carried. Tannins have anti-microbial activity by precipitating protein content of the outer wall of the microbes, thereby forming complex with the proteins and stop their activities. Also tannins have an antioxidants activity which helps in mopping up the free radicals that causes oxidative damages of the cell which results in many known diseases. The study also revealed the presence of phytosterols, which play major role in inhibiting the intestinal absorbption of cholesterol. Studies showed that zobo F. contains β-sitosterol as one of the phytosterols. Β-sitosterol has been used in lowering serum cholesterol levels in hypercholesterolemic individuals (Hicks and Moreau, 2001). This can attributed to use of Hibiscus sabdariffa F. in the treatment of arteriosclerosis which occurs as a result of increase in cholesterol levels.

CONCLUSION:

The study reaveled the presence of the most important polyphenolic compounds (flavonids and tannins) and phytosterols which have many biological activities have justified the use of Hibiscus sabdariffa F. in the treatment of many ailments in folk medicines. Also previous studies showed that the Hibiscus sabdariffa F. contains many minerals like calcium, phosphours and iron, the presence of macronutrients like proteins, reducing sugars and carbohydrates in the study are essential for good health.

Further research on this work can include proximate analyses of Hibiscus sabdariffa, to determine the quality of macronutreients present and also to the extent of determining its dose respose data and to evaluate the pharmacological and toxicological effects.

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Received on 10.05.2011

Accepted on 02.06.2011

Research Journal of Pharmacognosy and Phytochemistry. 3(4): July- August 2011, 169-173