14 International Conference on Pure and Applied Sciences (ICPAS 2018)

In Vitro Evaluation of Antioxidant Activates for Parts of ( ) and Syrian Mesquite (Prosopis farcta)

Ismael H. Mohammed1, Esmail S. Kakey2 and Mahdi Moridi Farimani3

1Department of Medical Microbiology, Faculty of Health and Science, Koya University, Kurdistan - F.R. 3Department of Biology, Faculty of Health and Science, Koya University, Kurdistan - F.R. Iraq 2Department of Phytochemistry, Medicinal and Drugs Research Institute, Shahid Beheshti University, Tehran -

Abstract—Using antioxidant agents are the purpose of the present Its well-developed root system and can extend study. The antioxidant effects of n-hexane, ethyl acetate, and up to 15–20 m depth into the soil. Its stems are erect, and its methanol extracts of roots and stalks of Rhubarb (Rheum ribes) and slender branches have short prickles like roses. Leaves are roots, bark of roots, leaves, and fruits of Syrian mesquite (Prosopis bipinnately compound, with a 1.8–3.0 cm long rachis and farcta) on free radicals have been evaluated in this study. Different concentrations of extracts were subjected to evaluate total 9–13 pairs of small leaflets. Small yellow flowers appear from content, total phenolic content, and 2, 2-dipheny-l-picrylhydrazyl May to August. On each raceme, there are 1–2 oblong pods (DPPH) free radical scavenging activity. Total flavonoid content of which are dark brown when ripe. The mesocarp is pulpy [7,8]. the methanol extract for roots of P. farcta showed significantly higher P. farcta could be used as a folk remedy for the treatment content (331.62 µg rutin E/mg of extract) than other extracts. Ethyl of angina pectoris or to reduce cardiac or chest pain in Iran. acetate extract for leaves of P. farcta exhibited a significant dose- A possible explanation for this action might be extracted from dependent inhibition of DPPH activity with 50% of inhibition (half results of some researchers [9]. Furthermore, other research maximal inhibitory concentration) at concentration of 22.47 µg/ confirms the efficacy of P. farcta beans powder as a beneficial ml. Furthermore, it showed the highest value of phenolic content (143.7 µg Gallic acid equivalent /mg of the extract). Possessing agent to decrease low-density lipoprotein cholesterol and properties of antioxidant may qualifies these extracts to be used increase high-density lipoprotein cholesterol concentration [10]. as a medicines. Different species of P. farcta have antioxidant effects and are responsible for the collection and removing free radicals [11]. Index Terms—Alpha-glucosidase, Antioxidant, Prosopis farcta, Therefore, protective effects of the alcoholic extract are due Rhubarb, Syrian mesquite. to its antioxidant role [12]. P. farcta beans extract has some antioxidant property and may alleviate hepatotoxicity [13]. Rhubarb is a group of plants that belong to the genus I. Introduction Rheum in the family . It has many species The common name of the first in this study is Syrian and Rheum ribes [5] one of them which our study has been mesquite [1]. Synonyms of it are Lagonychium factum [2]; included. They are herbaceous perennial plants growing Prosopis stephaniana [3]; Mimosa farcta; and Mimosa from short, thick rhizomes. They have large leaves that are stephaniana [4]. It is belonged to Mimosaceae family and somewhat triangular, with long fleshy petioles. They have Prosopis genus [5]. Syrian mesquite (P. farcta) is a woody small flowers grouped in large compound leafy greenish- perennial dwarf legume shrub of 0.4–1 m in height, rarely white to rose-red . Although the leaves are up to 3 m. It can attain more than 2 m in height and grow as toxic because contain oxalic acid which is a nephrotoxic and tall as grapes and citrus trees in certain places where weed corrosive acid, various parts of the plants have medicinal and control is absent [6]. culinary uses [14]. The traditional Chinese pharmacopeia features rhubarb (as laxative). Rhubarb is commonly used Pure and Applied Science Conference | Koya University worldwide herb and often known as “the wondrous drug” Paper ID: ICPAS2018.MIM 41, 5 pages because of its extensive medicinal uses [15]. The genus DOI: 10.14500//icpas2018.mim41 Rheum consists of approximately 60 perennial species Received 27 February 2018; Accepted 31 March 2018 distribution around the world; several species are used in Conference paper: Published 01 August 2018 medicine [16]. It is a commonly used raw material for crude Conference track: Microbiology and Immunology (MIM) Corresponding author’s e-mail: [email protected] drugs in China, Japan, Europe, America, and Southeast Copyright © 2018 Ismael H. Mohammed, Esmail S. Kakey, Mahdi Asia [17]. Modern pharmacology has demonstrated that Moridi Farimani. This is an open access article distributed under the rhubarb can cure constipation, hyperlipidemia, hepatic Creative Commons Attribution License. injury, and diabetes [18-21]. Chemical analysis shows

DOI: http://dx.doi.org/10.14500/icpas2018 International Conference on Pure and Applied Sciences (ICPAS 2018) 15 that rhubarb contains anthraquinones, tannins, polyose, II. Materials and Methods phenylbutazones, and stilbenes [22-25]. Methanolic extracts A. Extraction from Rheum undulatum were found to show scavenging Fresh roots and stalks of rhubarb (R. ribes) and roots, bark activity for 2, 2-dipheny-l-picrylhydrazyl (DPPH) [26]. of roots, leaves, fruits of Syrian mesquite (P. farcta) were Rheum rhaponticum extract showed in vitro antioxidant properties against lipid peroxidation and free collected from Kurdistan of Iraq. They were dried and ground radical scavenging activities on human ultraviolet-stimulated into a fine powder. Extraction was carried out using n-hexane, melanocytes [27]. ethyl acetate, and methanol solvents at room temperature. Free radicals and oxidants play a dual role as both toxic The extract was obtained by extracting 10 grams of dried and beneficial compounds, since they can be either harmful plant powder in 50 ml of solvent on a magnetic stereo for 1 h or helpful to the body. They are produced either from normal then using the filtration and repeated that 3 times to obtain cell metabolisms in situ or from external sources (pollution, more extract. The extract was further concentrated using cigarette smoke, radiation, and medication). When an rotary vacuum evaporator at 45–50°C and stored at 0–4°C. overload of free radicals cannot gradually be destroyed, This extraction is done for selection the stronger plant part their accumulation in the body generates a phenomenon that does the best inhibition for 50% of alpha-glucosidase called oxidative stress. This process plays a major part in activity (half maximal inhibitory concentration [IC50]), the development of chronic and degenerative illness such as then goes to column chromatography. Determination of cancer, autoimmune disorders, aging, cataract, rheumatoid antioxidant activities which include: Total phenolic content, arthritis, cardiovascular, and neurodegenerative diseases. The total flavonoid content, and DPPH free radical scavenging human body has several mechanisms to counteract oxidative activity have been done for these extracts [33]. stress by producing antioxidants, which are either naturally produced in situ or externally supplied through foods and/ B. Determination of Total Phenolic Content or supplements [28]. Reactive oxygen species (ROS) is The concentration of total phenols was determined by the a collective term that describes free radicals derived from Folin–Ciocalteu method (Singleton and Rossi, 1965). The oxygen, such as superoxide anion, hydroxyl radical (HO•), procedure includes two steps; the first step was selecting the peroxyl radical and alkoxy radical, as well as hydrogen suitable concentration depending on the color (light green- peroxide, a non-radical species resulting from oxygen blue) and the second step was applying the method. metabolism. Mitochondria are the main source of ROS. The total phenolic content was expressed as Gallic acid About 1–2% of the oxygen consumed by this organelle equivalent (GAE) in microgram per milligram of extract. results in ROS production, mainly through the activity of Methanol (95–100%) was used for dissolving the extractor the electron transport chain [29]. Free radicals are generated can be used dimethyl sulfoxide (DMSO) for dissolving. from either endogenous or exogenous sources. Endogenous Sodium carbonate (Na2CO3) 7.5% was added to the multi- free radicals are generated from immune cell activation, plate wells. Folin reagent (Merck KGaA64271 Darmstadt, inflammation, mental stress, excessive exercise, ischemia, Germany) was used after diluting it 1:10 with D.W. Different infection, cancer, and aging. Exogenous free radicals result concentrations (10, 50, 100, 200, and 500 ppm) of Gallic from air and water pollution, cigarette smoking, alcohol, acid using methanol have been used as standards. Different heavy metals, certain drugs (cyclosporine and tacrolimus), concentrations of each extract sample (dissolving in DMSO industrial solvents, cooking, and radiation. After penetration 99%) have been used till get suitable concentration, and into the body, these exogenous compounds are decomposed after doing the assay, the calculation has been done based on into free radicals [30]. Oxidative stress results from an 1000 ppm. ELISA reader was used for reading of absorbance imbalance between formation and neutralization of free at wavelength 760 nm. The calculation was done by finding radicals. For example, HO• and peroxynitrite in excess can the formula of the y = ax-b using excel which can get it by damage cell membranes and lipoproteins by a process called insert the results of the average of standard absorbencies lipid peroxidation. This reaction leads to the formation of minus average of blank absorbencies which represent the y in malondialdehyde and conjugated diene compounds, which are cytotoxic and mutagenic. Lipid peroxidation occurs by a the formula and x represents the total phenol content. radical chain reaction, that is, once started; it spreads rapidly and affects a great number of lipid molecules [31,30]. Many C. Total Flavonoid Content diseases are caused by oxidative stress. Antioxidants has The total flavonoid content was determined by aluminum main role as defenses in the body against bad effects of chloride method using of ELISA reader at 510 nm. Quercetin ROS that cause oxidative damage. Several plants contain dissolving by the methanol (10, 50, 100, 200, and 500 ppm) components that have ability to reduce ROS-induced has been used as a standard. Methanol as a blank, D.W, oxidative damage. There are many assays in vitro used to NaNO2 (5%) Alcl3 (10%), and NaOH (4%), and different measure the antioxidant potential for the plants. Moreover, concentrations of extracts were used in this assay. in most of these assays they revealed potent antioxidant The procedure includes two steps; the first step is selecting activity [32]. the suitable concentration depending on the color (light As a follow-up to this, the aim of this study was to yellow) and the second step is applying the method. The unit evaluate the antioxidant activities of these two plants. was µg of rutin equivalent in 1 mg of extract [34].

DOI: http://dx.doi.org/10.14500/icpas2018 16 International Conference on Pure and Applied Sciences (ICPAS 2018)

D. DPPH Free Radical Scavenging Activity bioactivity of appears to be mediated through a DPPH (Sigma-Aldrich – Germany) was evaluated by variety of mechanisms, particular attention has been focused on dissolving 7.886 mg in 100 ml methanol to get 200 µM. their direct and indirect antioxidant potentials. The antioxidant 50 µL of extract samples in different concentration was added properties are conferred on flavonoids by the phenolic hydroxyl to multi-plate. Adding 200 µL of DPPH with concentration groups attached to ring structures and act as free radical to wells of the multi plate from line A to D and adding 200 scavengers, reducing agents, and metal chelators [42]. µL of methanol from E to H of the multi plate. Control has Expressed as µg of Quercetin Equivalent Per 1 mg of been done by adding 50 µL DMSO in column 12 from A to Extract (Has Been Calculated at 1000 Ppm) H and adding 200 µL DPPH in this column (12) from A to D and adding 200 µL methanol from E to H. The absorbance C. DPPH Free Radical Scavenging Assay was measured at 517 nm against methanol as a blank using Ethyl acetate extract for leaves of P. farcta exhibited ELISA reader. a significant dose-dependent inhibition of DPPH activity The scavenging effect of DPPH radicals was calculated with 50% of inhibition (IC50) at concentration of using the following equation: 22.47 µg/ml, followed by methanol extract for leaves of DPPH Scavenging effect (%) = A – B/A * 100 P. farcta at concentration of 49 µg/ml, methanol extract Where A is the absorbance of the control, B is the for root barks of P. farcta at concentration of 76.32 µg/ml, absorbance of the sample. ethyl acetate extract for fruits of P. farcta at concentration of 83.61 µg/ml, methanol extract for roots of Rhubarb at E. Statistical Analysis concentration of 96.70 µg/ml, and other extracts showed IC50 Determination of assays was conducted in triplicates. The more than 100 µg/ml (Table III). value for each sample was calculated as the mean ± standard DPPH radical has been used extensively as a stable free deviation. The results were evaluated using the SPSS radical to determine the reducing substances or antioxidant and one-way ANOVA. Differences were considered to be activity of plant extracts [43,44]. The antioxidant activity of statistically significant if P < 0.05. plant extract containing polyphenol compound is due to their capacity to be donors of hydrogen atoms or electrons and to capture the free radicals [45,46]. The purple-colored DPPH III. Results and Discussion will reduce to yellow-colored complex. This DPPH assay A. Total Phenolic Content is considered the most valuable procedure for the in vitro According to results in Table I, ethyl acetate extract for evaluation of antioxidant activity [47]. leaves of P. farcta showed the highest value of phenolic content (143.7 µg GAE/mg of the extract). The second TABLE I highest value was methanol extract for roots of P. farcta Total Phenolic Content of Extracts for Syrian Mesquite (P. farcta) (116.22 µg GAE/mg of the extract) and followed by methanol and Rhubarb (R. ribes) extract for root barks of P. farcta (101.22 µg GAE/mg of the Extracts of plant parts n‑hexane Ethyl acetate Methanol extract). Other extracts showed fewer values. Root barks of P. farcta 28.00±0.16a 76.47±0.35f 101.22±0.45k Many plants contain phenolic compounds which have been Roots of P. farcta 9.50±0.09b 74.78±0.35f 116.22±0.51 c h j showed important activities including a potent antioxidant Fruits of P. farcta 5.13±0.07 52.59±0.26 40.50±0.21 e w i activity [35,36]. Phenolic compounds have an important Leaves of P. farcta 69.93±0.33 143.7±0.62 88.53±0.40 Roots of Rhubarb 20.65±0.13d 88.28±0.40i 82.84±0.38m role in stabilizing lipid oxidation and are associated with the Stalks of Rhubarb 11.72±0.10b 21.91±0.14d 37.84±0.20j antioxidant activity. The mechanism of phenolic compounds Each value represents the average ± SD (µg GAE/mg of extract). Averages in a for doing antioxidant belongs to their radical scavenging ability column followed by the same letter are not significantly different and different letters mediated by hydroxyl groups [37]. This mechanism comes are significantly different. SD: Standard deviation, GAE: Gallic acid equivalent, from their redox characteristics, through attenuating high- P. farcta: Prosopis farcta, R. ribes: Rheum ribes energy form of oxygen (singlet) and triplet oxygen, neutralizing and absorbing free radicals or decomposing peroxides [38]. TABLE II Total Flavonoid Content of Extracts for Syrian Mesquite (P. farcta) and Rhubarb (R. ribes), Expressed as µg of Quercetin Equivalent Per 1 B. Total Flavonoid Content mg of Extract (Has Been Calculated at 1000 Ppm) As shown in Table II, total flavonoid content of the Extracts of plant parts n‑hexane Ethyl acetate Methanol methanol extract for roots of P. farcta showed significantly Root barks of P. farcta 65.9±3.00a 199.50±5.24g 292.70±6.99k higher content (331.62 µg q E/mg of extract) than the other Roots of P. farcta 50.98±2.03b 141.11±4.76h 331.62±12.99l solvent extracts, and this is followed by methanol extract Fruits of P. farcta 1.16±0.36c 5.62±0.36i 23.75±2.88m for root barks of P. farcta (292.70 µg q E/mg of extract), Leaves of P. farcta 34.48±2.75d 187.26±5.57j 217.83±4.34n methanol extract for roots of Rhubarb (278.19 µg qE/mg of Roots of Rhubarb 15.90±1.80e 200.00±6.46g 278.19±5.76o extract), and other extracts showed less contents. Stalks of Rhubarb 10.23±1.58f 19.03±1.07e 8.317±0.76f Group of plant polyphenols includes flavonoids which Each value represents the average ± SD (µg quercetin E/mg of extract). Averages in a column followed by the same letters are not significantly different and different have antidiabetic, anti-inflammatory, antibacterial, antiviral, letters are significantly different. SD: Standard deviation,P. farcta: Prosopis farcta, anticancer, and antiallergic activities [39-41]. Although the R. ribes: Rheum ribes

DOI: http://dx.doi.org/10.14500/icpas2018 International Conference on Pure and Applied Sciences (ICPAS 2018) 17

TABLE III [14] Available: http://www.flipper.diff.org/app/items/info/4591. Rhubarb and

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DOI: http://dx.doi.org/10.14500/icpas2018