INTRODUCTION:

In the recent times, focus on the research has all over the world and a large body of evidence has been collected to show the immense potential of medicinal plants used in traditional system. Various medicinal plants have been studied using modern scientific approaches, and the results have revealed the potential of medicinal plants. ⁽¹⁾

Free radicals containing one or more unpaired electrons are highly unstable species, which can damage other molecules by getting electrons from them. In living organism, free radicals including hydroxyl radical, super oxide anion radical, peroxide radical are generated as the medium of energy supply, detoxification, chemical signal and immunity. But during the excessive metabolism, they cause extensive oxidative damage to cells leading to aging, cancer, neurodegenerative disorders, liver cirrhosis, atherosclerosis, diabetes, inflammation and other human diseases. More and more epidemiological reports associate diets on fruits and vegetables with reduced risk of heart disease and other chronic diseases. ⁽²⁾

The immediate family members of flavonoids include flavones, isoflavones and the 2, 3-dihydroderivatives of flavone, namely flavanones, which are interconvertible with the isomeric chalcones. Epidemiological evidence suggests an inverse relationship between dietary intake of flavonoids and the risk of coronary heart disease. It has been suggested that oxidative modification of LDL plays an important role in the development of human atherosclerosis. Thus, protecting LDL from oxidation by such compounds as flavonoids, may be an effective strategy to delay or prevent the progression of the disease. The leaves are diuretic, expectorant and stomachic. ⁽³⁾

The antioxidants have been widely used as food additives to provide protection against oxidative degradation of food. The most commonly used antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and ascorbic acid (ASA). However, (BHA) and (BHT) have been suspected of being responsible for liver damage and carcinogenesis. Therefore, a need for identifying alternative natural and safe sources of food additive antioxidants, especially of plant origin, has notably increased in recent years. ⁽⁴⁾

Our present study is designed to evaluate the antioxidant and scavenging potential of Hibiscus syriacus L. extract. For this reason, the major active compounds of its extract, which may be responsible for antioxidant activity, were determined through preliminary phytochemical screening. Total phenolic contents, total flavonoid contents and antioxidant properties were also assayed.

The genus Hibiscus is widely distributed over Korea, china, India, and Siberia. The dried root of Hibiscus syriacus L. are used as a fork medicine in the orient ⁽⁵⁾ for the cure of hematochezia, dysentery, obstruction due to wind phlegm, and vomiting of food. ⁽⁶⁾

Hibiscus syriacus L. (Rose-of-Sharon) is valued for large flowers produced in summer when few other shrubs bloom. It is useful as a garden accent due to its strict, upright habit. The open, loose branches and light green leaves make Rose-of-Sharon ideally suited to formal or informal plantings, and with a little pruning makes an attractive, small specimen tree. The plant grows in sun or partial shade and in any soil. Rose-of-Sharon grows 8 to 10 feet tall and spreads 4 to 10 feet. The growth rate ranges from slow to moderate, and transplanting is easy. Several roots are usually located just beneath the soil surface. The insects can be dislodged with high pressure water sprays from the garden hose or controlled by pinching off the part of the twig with the insects. Over-fertilizing increases aphid infestations. The single or double flowers are in shades of red, pink, white and purple, depending on the cultivar. Individual flowers stay open for one day and close at night. Since plants bloom on new growth, shaping or pruning can be done at any time. However, pruning is usually not required since the plant grows slowly and keeps a tight upright form. ⁽⁷⁾

MATERIALS AND METHODS:

Selection of plant

Plant was selected on the basis of literature survey.

Collection of plant materials

The leaves of Hibiscus syriacus L. was collected from the month of august-September from the Garden of Jawaharlal Institute of Technology and G. R. Y. Institute of Pharmacy Vidya Vihar Borawan District Khargone Western Nimar region of Madhya Pradesh,

Authentication of plant

The plant Hibiscus syriacus L. was identified and authenticated by Dr S.K. Mahajan, Botanist from Government College, Khargone Madhya Pradesh. The herbarium of the plant specimens were prepared and deposited in the Department of Pharmacognosy, G. R. Y. Institute of Pharmacy Vidya Vihar Borawan District Khargone Madhya Pradesh, India, under voucher no. G.R.Y.I.P. 43.

Preparation of extract

Extraction of organic component

The leaves were initially separated from the main plants body and rinsed with distilled water, dry in shade then homogenized into fine powder and finally stored in air tight bottles. It was then passed through the 40 mesh sieve. A dried and powered plant material was defatted firstly to remove fatty material. For this purpose 1000 gm of weighed powered plant of Hibiscus syriacus L. was packed in Soxhlet apparatus and extracted with petroleum ether at 60-80°C for 36 hrs and completion of extraction was confirmed by discoloration of the solvent. The marc was removed and dried then it was subjected to continuous hot extraction with another organic solvent like benzene, chloroform, and methanol in soxhlet apparatus for 36 hrs and completion of extraction was confirmed by discoloration of the solvent. After complete extraction of plant materials the solvent was evaporated and concentrated the extracts. The petroleum ether, benzene, chloroform, and methanolic leaves extract of Hibiscus syriacus L. yielded greenish brown, green, brown and deep blue semi solid residue, then it were filtered with the help of muslin cloth. The supernatant was collected and the solvent was evaporated by solvent distillation apparatus and concentrate the extract in reduce pressure. ⁽⁷⁾

Extraction of aqueous component

The marc was removed and dried then it was subjected to boil with 2 liter of water for 24 hrs, and then filtered through muslin cloth, and extract was evaporated by solvent distillation apparatus.⁽⁷⁾

Phytochemical analysis of extract

The methods described by Harborne (1978) with slight modifications were used to test for the presence of the active ingredients in the test sample.

Test for steroids

A 10 ml extract of the test plant leaves were evaporated to a dry mass and the mass dissolved in 0.5 ml of chloroform. Acetic anhydride (0.5 ml) and 2 ml of concentrated sulphuric acid were added. A blue or green color or a mixture of these two shades was regarded as positive for the presence of steroidal compounds.

Test for terpenoids

The presence of terpenoids was determined as described for steroids except that red, pink or violet colour indicates the presence of terpenoids.

Test for tannins

i) 1 ml of freshly prepared 10% KOH was added to 1 ml of the extract. A dirty white precipitate indicated the presence of tannins.

ii) Powdered leaves extract of the test plant (1.0 g) were weighed into a beaker and 10 ml of distilled water added. The mixture was boiled for five minutes. Two drops of 5% FeCl3 were then added. Production of greenish precipitate indicated the presence of tannins.

Test for flavonoids

A small piece of magnesium ribbon was added to ethanolic extract of the plant material, this was followed by the drop wise addition of concentrated hydrochloric acid. Colors varying from orange to red indicated flavones, red to crimson indicated flavonols, crimson to magenta indicated flavonones.

Test for alkaloids

The extract of the plant leaves sample (0.5 g) was stirred with 5 ml of 1% HCl on a steam bath. The solution obtained was filtered and 1 ml of the filtrate was treated with two drops of Mayer’s reagent. The two solutions were mixed and made up to 100 ml with distilled water. Turbidity of the extract filtrate on addition of Mayer’s reagent was regarded as evidence for the presence of alkaloids in the extract.

Test for saponins

0.5 g of the powdered leaves extract was introduced into a tube containing 5.0 ml of distilled water, the mixture was vigorously shaken for 2 min, and formation of froth indicated the presence of saponins.

Test for glycosides

Powdered leaves extract (1 g) were added into two separate beakers. To one of the beakers was added 5 ml of dilute sulphuric acid while 5 ml of water was added to the other beaker. The two beakers were heated for 3 – 5 min and the contents filtered into labeled test tubes. The filtrate was made alkaline with 5% sodium hydroxide and heated with Fehling’s solution for 3 min. The presence of reddish precipitate in the acid filtrate and the absence of such precipitate in the aqueous filtrate were regarded as positive for glycosides. ⁽⁸⁾

Determination of Total Flavonoid Contents

TFC was determined by a colorimetric method with some modification. A 0.5 ml extract or rutin standard solution was added to a 10 mL volumetric flask. Then 60% (V/V) ethanol was added up to the volume of 5 ml and mixed with 0.3 ml of NaNO2 (5%, W/V). After 6 min, 0.3 ml of AlCl₃ (10%, W/V) was added. The mixture was mixed and kept for another 6 min at room temperature, and then 4.0 ml NaOH (1.0 mol/l) was added. The reaction mixture was diluted with 0.4 ml double distilled water and kept for 15 min. The increase in absorbance was read at 510 nm against the blank (without extract or rutin). The flavonoid content was expressed as rutin equivalents in milligrams per gram of dry basis, using a standard curve generated with rutin. ⁽⁹⁾

Determination of Total phenolic Contents

TPC was estimated by the Folin-Ciocalteu method with a little modification. 1.0 ml extract (0.5 mg/ml) was mixed with 0.5 ml Folin-Ciocalteau reagent and 2.5 ml Na2CO3 (7.5%, W/V) (added 2 min after the Folin-Ciocalteau reagent) in a 10 ml volumetric flask. The absorbance was determined at 760 nm against the blank (without extract) after incubation for 2 h at 25°C. TPC of the extracts was expressed as milligrams per gram of gallic acid equivalents of dry basis, using a standard curve generated with gallic acid. ⁽⁹⁾

Determination of antioxidant activity

DPPH free radical scavenging assay ^{(9, 10, 11, 12)}

The scavenging activity of the extract on the DPPH radical was assayed by the method described in the literatures with a minor modification. A 2×10-4mol/L of DPPH ethanol solution was prepared. A 1.0 ml different concentration of sample was added to 2.0 ml of DPPH solution. The mixture was incubated for 60 min at 25°C in the dark. Then the discolorations were measured at 517 nm against ethanol. The ascorbic acid was used as the positive control compound. Percent of sample on DPPH was calculated as the following equation.

Where, Sa is the scavenging activity of the tested sample (%); A_sample is the absorbance of 1.0 ml sample and 2.0 ml DPPH; A_self is the absorbance of 1.0 ml sample and 2.0 ml ethanol; A_control is the absorbance of 1.0 ml double distilled water and 2.0 ml DPPH.

Statistical analysis

All the experiments were carried out three times and the three repeated samples were employed in colorimetric assays. Data were shown in means values ± standard deviations.

RESULTS AND DISCUSSION:

Preliminary phytochemical screening

Preliminary phytochemical screening for Hibiscus syriacus L. leaves (table 1) revealed the presence of carbohydrate, glycosides, steroids, proteins, flavonoids, tannins and alkaloids.

Total flavonoid contents

Many reports indicated that an antioxidant activity was in accordance with TFC.Total flavonoids were expressed in terms of rutin equivalents per gram. The plant leaves extract was rich with flavonoids. TFC of extracts were estimated to be 23.45mg rutin equivalents/g for petroleum ether extract, 25.13mg rutin equivalents/g for benzene extract, 26.89mg rutin equivalents/g for chloroform extract, 36.54mg rutin equivalents/g for methanol extract and 35.05mg rutin equivalents/g for aqueous extract. This also explains why the extract had strong antioxidant activity.

Table 1 Phytochemical screening of leaves extract of Hibiscus syriacus L.

S. No.	Phytochemical constituents	Pet. Ether extract	Benzene extract	Chloroform extract	Methanol extract	Water extract
1	Carbohydrates	-	+	+	+	+
2	glycosides	-	-	-	+	+
3	Steroids	+	+	+	-	-
4	Proteins	-	-	+	+	-
5	Flavonoids	-	-	-	+	+
6	Tannins	+	+	+	+	+
7	Alkaloids	-	-	-	-	+

Fig.1. Standard graph of rutin

Fig.2. Standard graph of Gallic acid

Table 2. Absorbance of Rutin Std/ Various extract of Hibiscus syriacus L.

S.No.	Concentration (mg/ml)	Absorbance of Rutin Std/ various extract
1.	10	0.1675
2.	20	0.2365
3.	40	0.3524
4.	60	0.4744
5.	80	0.6124
6.	100	0.8024
7.	Petroleum ether Extract	0.5820
8.	Benzene Extract	0.3854
9.	Chloroform Extract	0.5814
10.	Methanol Extract	0.7421
11.	Water Extract	0.6182

Total phenolic contents

Phenolic compounds are responsible for the antioxidant activity of plant materials due to their redox properties and the phenolic hydroxyl group helps them to work as reducing agents, hydrogen donors and singlet oxygen quenchers ⁽¹³⁾. Many reports indicated that antioxidant activities were in accordance with TPC. ⁽¹⁴⁾ Total phenols were expressed in terms of gallic acid equivalents per gram. The leaves extract of Hibiscus syriacus L. was rich in phenols. TPC of leaves extract of Hibiscus syriacus L. estimated to be 69.10 mg gallic acid equivalents/g for petroleum ether extract, 41.23 mg gallic acid equivalents/g for benzene extract, 69.31 mg gallic acid equivalents/g for chloroform extract, 92.15 mg gallic acid equivalents/g for methanol extract, 74.26 mg gallic acid equivalents/g for aqueous extract. This also explains why the leaves extract of Hibiscus syriacus L. have strong antioxidant activity.

Table 3. Absorbance of Gallic acid Std/ Various extract of Hibiscus syriacus L.

S.No.	Concentration (mg/ml)	Absorbance of Gallic acid Std/ various extract
1.	10	0.0578
2.	20	0.1671
3.	30	0.3132
4.	40	0.4471
5.	50	0.6012
6.	60	0.7611
7.	Petroleum ether Extract	0.2328
8.	Benzene Extract	0.2460
9.	Chloroform Extract	0.2489
10.	Methanol Extract	0.4221
11.	Water Extract	0.3995

DPPH radical scavenging activity

DPPH radical is a stable nitrogen-centered on free radical with an unpaired electron. Its solution appears deep violet and shows a strong absorbance at 517 nm. Substances, which make its color lighter and absorbance descendent, can be considered as antioxidants and therefore radical scavengers. ⁽¹⁵⁾ Now, it is well accepted that DPPH radical is used to analyze the scavenging potential of compounds. ⁽¹⁶⁾As shown in Figure 3, 4, 5, 6 and 7 all the extract of Hibiscus syriacus L. showed appreciable free radical scavenging activity, which was weaker than ascorbic acid in the same concentrations. There was a dose-effect relationship with the increasing concentration. The IC50 value of petroleum ether extract of Hibiscus syriacus L. estimated to DPPH radical were to 175 ug/ml, IC50 value of benzene extract were to 312 ug/ml, IC50 value of chloroform extract were to 167 ug/ml, IC50 value of methanol extract were to 152 ug/ml, IC50 value of aqueous extract were to 170 ug/ml, also it were lower than the ascorbic acid (IC50=113 ug/mL). Methanolic extract contain higher amount of antioxidant activity compare all other leaves extract of Hibiscus syriacus L.

Table 4 Absorbance of various extract of Hibiscus syriacus L.

S.No.	Conc. mg/ml	Abs of Ascorbic acid	Abs of Pet. ether	Abs of Benzene	Abs of Chloroform	Abs of Methanol	Abs of Water
1	0.1	0.2650	0.2933	0.3555	0.3511	0.2825	0.2715
2	0.2	0.1475	0.2126	0.3009	0.1918	0.2217	0.2611
3	0.4	0.0797	0.1700	0.2300	0.1818	0.1536	0.2261
4	0.6	0.0734	0.1362	0.2080	0.1746	0.1162	0.1823
5	0.8	0.0261	0.1188	0.2042	0.1136	0.1025	0.1514
6	1.0	0.0115	0.1024	0.1952	0.1050	0.0901	0.1265

Table 5 Percentage scavenging activity of various extract of Hibiscus syriacus L.

S.No.	Conc. mg/ml	% scavenging of Ascorbic acid	% scavenging of Pet. ether	% scavenging of Benzene	% scavenging of Chloroform	% scavenging of Methanol	% scavenging of Water
1	0.1	49.52	44.13	32.28	32.22	45.23	48.28
2	0.2	72.45	59.50	42.68	60.66	57.77	50.26
3	0.4	84.81	67.61	56.19	65.37	70.74	56.93
4	0.6	86.01	74.05	60.38	66.74	77.86	65.27
5	0.8	95.02	77.37	61.10	78.36	80.47	71.16
6	1.0	98.35	80.49	62.81	80.00	82.85	75.90

Fig.3 Antioxidant activity of petroleum ether extract compare with Ascorbic acid

Fig.4 Antioxidant activity of Benzene extract compare with Ascorbic acid

Fig.5 Antioxidant activity of Chloroform extract compare with Ascorbic acid

Fig.6 Antioxidant activity of Methanolic extract compare with Ascorbic acid

Fig.7 Antioxidant activity of water extract compare with Ascorbic acid

CONCLUSION:

In present study, antioxidant activities of various leaves extract of Hibiscus syriacus L. was determine by DPPH. The extracts were found to possess radical scavenging and antioxidant activities. Generally, IC₅₀values of lower than 10 mg/ml indicated that the extracts were effective in antioxidant properties. In the present study it is found that methanol extract of Hibiscus syriacus L. contains substantial amount of phenolics and flavonoids responsible for antioxidant activity. Thus, it can be concluded that methanol extract of Hibiscus syriacus L. can be used as an accessible source of natural antioxidants with consequent health benefits.

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Received on 21.11.2014 Modified on 05.12.2014

Res. J. Pharmacognosy & Phytochem. 7(1): Jan.-Mar. 2015; Page 18-24

DOI: 10.5958/0975-4385.2015.00005.9