World Applied Sciences Journal 29 (2): 247-251, 2014 ISSN 1818-4952 © IDOSI Publications, 2014 DOI: 10.5829/idosi.wasj.2014.29.02.1354

Anti Oxidant Activity and Anthocyanin Content in Flower of Mirabilis jalab L. Collected from Yemen

1Naji Ebrahim, 23 R.M.Kershi, Monica Butnariub, 4Nasser. A. Awadh. Ali, 55 Ahmed. A.T. A and M. Ramzy

1Department of Plant Production, Faculty of Agriculture and Veterinary Science, Ibb University, Ibb,Yemen 2Department of Physics, Faculty of Science, Ibb University, Ibb, Yemen 3Chemistry and Biochemistry Department, Banat's University of Agricultural Sciences and Veterinary Medicine, Romania 4Department of Pharmacy, Sana University, Sana'a, Yemen 5Aljazeerah University, Ibb, Yemen

Abstract: The aim of this study was to investigate and compare Mirabilis jalab L. extracts obtained in Ultrasonic condition with different water/methanol and water/ethanol extraction mixture acidified with 0.1% HCl. The extracts were analyzed for monomeric anthocyanins contents and antioxidant activities. The highest anthocyanins content (3197.8 mg/L) and the best antioxidant activity were obtained for the Mirabilis jalab extract with 100% ethanol. Also, there is a good correlations between antioxidant activity (R2 = 0.9332 and 0.9712) for water/ethanol and methanol series extracts respectively.

Key words: Mirabilis Anthocyanins Extraction Antioxidant Free radical Scavenging activity

INTRODUCTION oxymethylanthraquinone [1]. Furthermore, it has been reported that Mirabilis jalaba harbor antiviral activity Mirabilis jalapa is an erect herb that can grow up to against viruses [2] exhibit antifungal activity [3, 4] and 1 m tall. Leaves are simple, heart-shaped, 3-12 cm long, showing features as anticarcinogen chemotherapeutic opposite, tapering to a pointed end. Flowers are bisexual, potential. Moreover, in recent years, researches red, pink, yellow or white, with perianth distinctly determining the anthocyanin content, relation with constricted above and they bloom late in the afternoon. chlorophyll and regulation of pigments with genes Fruits are black and globose, 5-8 mm in diameter. The use [2, 5-8]. Anthocyanins are representative of plant of traditional medicine at the primary health care level is pigments widely distributed in colored fruits and flowers. widespread in Yemen; traditional healers use many local Because anthocyanins are widely consumed, finding out plants for the treatment of microbial disease. Mirabilis additional biological activities related to these compounds jalapa, a wild grown plant, is used to cure externally would be of great interest [9]. Anthocyanins are normally infected wounds. In Latin America, this plant is used in obtained by extraction from plants and the extraction traditional medicine because of the purgative or emetic methods currently employed are with the use methanol, cathartic properties contained in its roots [1], it is used ethanol, acetone, water or mixtures as solvents. In fact, to treat intestinal pains. In South Africa, the roots are the color stability of anthocyanins depends on a used as a purgative drug and it is reputed that the flowers combination of factors, such as the structure and of this plant at night exhale a strong odour which will concentration of the anthocyanin, pH, temperature and stupefy or drive away mosquitoes. The roots contain presence of complex agents such as phenols and metals

Corresponding Author: Naji Ebrahim, Department of Plant Production, Faculty of Agriculture and Veterinary Science, Ibb University, Ibb, Yemen.

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[10]. The most common solvents used for anthocyanins MATERIAL AND METHODS extraction are aqueous mixtures of ethanol, methanol or acetone [11]. The adverse effects of oxidative stress Material: Fresh petals of the (Mirabilis jalaba) on human health have become a serious issue [12]. were collected randomly from the Ibb region during Under stress, our bodies produce more reactive oxygen June 2013 species (ROS) such as; superoxide anion radicals, hydroxyl radicals and hydrogen peroxide than enzymatic Extraction of Anthocyanins: The anthocyanins were antioxidants such as; superoxide dismutase [13]., extracted according to the methodology of [32]. Solvents glutathione peroxidase and catalase and non-enzymatic such as methanol and ethanol were used at antioxidants such as; ascorbic acids, glutathione, concentrations of 100, 75, 50, 25 and 0.0% in water, carotenoids and flavonoids. This imbalance leads to acidified with 0.1% hydrochloric acid (HCl) (Synth 37%) damage of biological structures such as proteins, Twenty five g of fresh petals of Mirabilis jalab were lipids and DNA and induce a variety of human diseases treated with 100 ml of different water/alcohol solutions [14-20]. Antioxidants from fruits and vegetables, acidified with 0.1% HCl (Merck, 37%) as extracting especially with an intense colouration, are considered an material (solid to solvent ratio 1:4 w/v). Each solution was important protection factor against oxidative stress and transferred to a 500 ml beaker, covered with parafilm and its deleterious consequences to human health [21, 22]. stored overnight at 4 C. The mixture was then filtered The antioxidative capacity, which is defined as the under vacuum using nº 1 Whatman paper and a Buchner capacity to inhibit or delay the oxidation of other funnel. Filtrate solution was taken and then 200 ml of molecules, anthocyanins and their aglycones solvent was added to complete the mixture. This mixture (anthocyanidins) and their free radical scavenging was later filtered and the residue washed with solvent activity have been revealed [23-25]. The antioxidant until obtained a total of 500 ml solution. A 5 ml aliquot activity of berries is directly proportional to the was removed from each extract, placed in a 50 ml anthocyanins content [26]. In response to the increased volumetric flask, the volume completed with two buffer popularity and greater demand for medicinal plants, a solutions: potassium chloride buffer 0.025 M (pH 1.0) and number of conservation groups are recommending that sodium acetate buffer 0.4 M (pH 4.5) and then the wild medicinal plants be brought into cultivation. Various absorbance was measured simultaneously at 516 nm and herbs and spices have been reported to exhibit 700 nm after 15 minutes of incubation at room temperature. anthocyanin content in their flowers including Mirabilis Absorbance readings were made at room temperature jalaba [10] and it is reported that,various herbs and against distilled water as blank was used for species have been reported to exhibit antioxidant measurements. activity, Ocimum sanctum, Piper cubeba, Allium sativum, Terminalia bellerica, Camellia sinensis and Zingiber Quantitative Determination of the Anthocyanins: officinale [27].Various analytical methods have been used to evaluate the antioxidant properties of phenolic Determination of total monomeric anthocyanins content compounds: the 1, 1-diphenyl- 2-picrylhydrazyl (DPPH) was quantified using a pH differential method described assay proves the capacity of the antioxidants to quench by [31]. The absorbance was measured simultaneously the PPH radical, whereas the ORAC method is based on at 516 nm and 700 nm after 15 minutes of incubation at the loss of fluorescence of the -phycoerythrin protein or room temperature. Absorbance readings were made at of fluorescein upon oxidation [28, 29]. Reactive oxygen room temperature against distilled water as blank. A - Jasco V 530 UV-Vis spectrophotometer was used for species (ROS), including super oxide anion (O2 ), hydroxyl measurements. The monomeric anthocyanin pigment radical (OH) and hydrogen peroxide (H22 O ), exist in living organisms [13]. The red colour of the wild Yemeni concentration was calculated according to the following Mirabilis jalaba is a consequence of its anthocyanin equation: contents that was not well scientifically investigated in Monomeric anthocyanin pigment (mg/L) = (A x MW extracts in Yemen. Therefore in the present study, x DF x 1000)/ ( x1) Where A= (A510-A700)pH 1.0 -(A516- Mirabilis jalaba flower plant was collected from Ibb A700)pH 4.5 , MW is the molecular weight (449.2) and is region in Yemen to study its anthocyanin content and the molar absorptivity, (26,900) and DF is the dilution antioxidant properties. factor.

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Fig. 1: Concentration response curve for DPPH at 515 nm

Determination of Antioxidant Activity by the Dpph extraction system. This tendency also observed for Method: The 1, 1-diphenyl-2-picrylhydrazine (DPPH) water/ethanol extraction, where high values were obtained radical scavenging assay was first described by Blois in for 100 % ethanol extracting system. The amount of 1958 and was later modified slightly by numerous monomeric anthocyanins in Mirabilis jalab extracts researchers. It is one of the most extensively used ranged from 3197.8 mg/L to 55.7 mg/L for water/ethanol antioxidant assays for plant samples. DPPH is a stable free extraction and from 946 mg/L to 278.3 mg/L for radical that reacts with compounds that can donate a water/ethanol extraction. hydrogen atom. This method is based on the scavenging of DPPH through the addition of a radical species or Error! Not a Valid Link: Fig.1. Comparison of an antioxidant that decolourizes the DPPH solution. anthocyanins content from extracts obtained in The antioxidant activity is then measured by the decrease water/methanol and water/ethanol systems. in absorption at 515 nm. In this method, a 0.1mM solution Figure 2 shows the percentage of anti oxidant of DPPH in methanol is prepared (4 mg DPPH /100 ml activity after 2 hours of reaction between the methanol) and then stored at -20 and 2 ml of this solution extracts and DPPH radical for the two studied cases. are added to 0.5 ml of the sample solution in methanol. The lower this value, the higher is antiradical efficiency The mixture was left to stand at room temperature for 30 activity of Mirabilis jalab extracts increases with min in the dark before Absorbance measurement at 517 nm increasing the percentage of methanol and ethanol in the to assess the stability of the coloured reactive action,, A extraction system. Comparing antioxidant activities of the large decrease in the absorbance of the reaction mixture Mirabilis jalaba extracts in the two cases, it is observed indicates significant free radical scavenging activity of the similar antioxidant activities in the ethanol and methanol compound. The antioxidant activity of the extracts was series. estimated by the ability to scavenging the DPPH radical. The DPPH concentration in the reaction medium was Error! Not a Valid Link: Fig.2. Comparison of remaining calculated from the calibration curve (Figure 1) with DPPH of extracts obtained in water/methanol and the following equation determined by linear regression water/ethanol systems. (R2 = 0.988). The correlations between antioxidant activity and monomeric anthocyanins content for the two extraction

A515=11.368x -0.0437. systems are showed in Fig. 3. The following equations determined by linear regression regarding the relationship RESULTS AND DISCUSSIONS between antioxidant activity and anthocyanins content was obtained, Eq. (1) for ethanol series and Eq. (2) for The paper describes the extraction method of methanol series. There is good correlation between anthocyanins and antioxidant activity of Mirabilis jalab antioxidant activity and anthocyanins content for the selected from Yemen. The changes in total anthocyanins Mirabilis jalab extracts from methanol and ethanol series. content depending on the water/alcohol ratio are showed in Fig. 1. The monomeric anthocyanins content increases y = -0.0077x + 39.478 (R² = 0.9591) (1) with increasing the percentage of methanol in the y = -0.0682x + 79.824 (R² = 0.839) (2)

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Error! Not a Valid Link: Fig.3. Correlation between significant anti oxidant activity. The best results anthocyanin contents and anti oxidant activity. regarding monomeric anthocyanins content and Also, notice a good correlation between anti oxidant antioxidant activity were obtained at extraction with activity and extraction systems content for ethanol and 100% methanol and ethanol. However, for food methanol series extracts. The values of the determination industry, the extractions with ethanolic solution are coefficients are acceptable, indicating a high correlation more convenient. The correlations between anthocyanins by linear regressions between antioxidant activities and content and antioxidant activity depend on the extraction extraction systems Eq. (3) for ethanol series and Eq. (4) for solvent, the best determination coefficients was found for methanol series Mirabilis jalab extracts obtained in water/ethanol systems Fig. 2. y = -24.938x + 43.818 R² = 0.9332 (3) y = -51.636x + 68.801 (R² = 0.9712) (4) REFERENCES

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