Antioxidant Activity of Phenolic and Alkaloid Fractions Accumulated in Artemisia Judaica and Artemisia Herba Alba

Eman Ramadan Elsharkawy1,2* and Mohamed H. Shiboob3 1Department of Ecophysiology, Plant Ecology and Range Management, Desert Research Center, Cairo, Egypt; elsharqawyeman2017@gmail.com 2Chemistry Department, Faculty of Science, Northern Borders University, Saudi Arabia 3Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia

Journal of Natural Remedies | ISSN: 2320-3358 www.informaticsjournals.com/index.php/jnr | Vol 17 (4) | October 2017 species (ROS) production. Cell injury results in cell viability compromise through apoptosis or necrosis. 2 Desert plants subject to water deficiency conditions produced high concentration concentrations of natural compound and secondary metabolite than same species grow under high water supply. Water deficiency induces drought stress-related metabolic responses, generating a large oxidative stress. As a consequence, metabolic processes are shifted towards biosynthetic activities that consume reduction equivalents. Accordingly, the synthesis of some products, terpene, phenols and alkaloids, are enhanced. Huda et al., 2015 and Esmaeili et al., 2011, 3,4 reported that secondary metabolites play key physiological roles in plant growth development, reproduction and disease resistance.

Aim of Study
Wild plant grow and survive in drastic conditions of water deficiency and high temperature, aridity try to adapt to these condition by accumulation some active compounds such as alkaloid, flavonoid and terpene, phonolic compound, in present study we discuss the accumulation of alkaloid and phenolic compound by which the plant Artemisia gudica and A. herba alba collected from two region, Tabuk, Saudi Arabia and North Sinai, Egypt adapt to oxidative stress.

Plant Material
Fresh aerial parts of two plants from genus Artemisia, namely Artemisia herba alba and Artemisia judaica were collected from the arid zone at Arish-North Sinai, Egypt, and Tabouk region, Saudi Arabia, during the spring seasons, 2016. Collected plants have been kindly verified and authenticated in the Desert Research Center, voucher specimens were deposited in the Herbraium of Desert Research Center.

Phytochemical Screening
The phytochemical analysis of methanol extracts were tests to identify different classes of bioactive chemical constituents (flavonoid-alkaloid-terpene and sterol) present in the plant using standard methods 11,18 .

Phenolic Compound Extraction
Phenolic compounds were extracted as described by 1 . Extraction was carried out in water bath. The solutions from all extractions were combined. After evaporating the organic solvent in a rotary evaporator at 45 0 C, the crude extracts were stored at -20 0 C until further analysis.

Extraction and Identification of Alkaloid
The fin powder aerial part of plant samples (5 g) were boiled in water bath with 20 ml of HCl in 50% methanol. The mixture was cooled and filtered. A portion was reserved. Another portion of the filtrate was put in 200 ml separating funnel then add drops of ammonia to alkaline the solution. Add equal volume of choloroform and shaken to allow the layer to separate. The chloroform layer was extracted with two quantities each of 5 ml of dilute hydrochloric acid 9 .

Analysis of Alkaloid Compound
The constituents of the alkaloid extract and methanol extract analyzed by GC-MS as reported by 12 . Compounds identified by comparison of their Retention Indices (RI), (C9 to C24 n-alkane mixture) and mass spectra with those reported in the literature 1,12 .

GC-MS Analysis
The compounds were analyzed using a Thermo GC-Trace ultra system (Thermo Co. USA), they were separated on 30m X0.25 mm X 0.25 µm Elite-5MS column (Thermo Scientific GC Column).

DPPH Radical Scavenging Capacity
In this study, 0.5 mL of each sample concentration was homogenized with the same volume of DPPH methanolic solution (0.04g/L). The mixture was shaken vigorously and allowed standing for 30 min in darkness at a temperature of 25°C; the absorbance of the resulting solution was measured at 517 nm with a spectrophotometer 13 . The percentage inhibition was calculated as: Ascorbic acid and quercetin were used as positive control, the concentration providing 50% inhibition (IC 50 ) was calculated from the graph of inhibition percentage plotted.

Results and Discussion
Methanol extracts of the aerial part of plants, (A.herba alba and A. judaica) Figure 1, grown in the arid zones in the Northern regions of Saudi Arabia and North Sinai in Egypt were subjected to primary phytochemical analysis which shown the presence of high amount of phenolic compounds, and flavonoids, absence of tannins in A.herba alba, alkaloid and sterols have moderate concentration as investigated in Table 1.

Phenolic Compound
The investigation of methanolic extract of plant samples of A.herba alba and A. judica by GC-MS exhibited different types of phenolic compounds, ferulic acid, 4-hydroxy-cinamic acid-ester, p-tert-Butyl-catechol, n-Propyl-cinnamate, Butyl-cinnamate, and ethyl-cinnamate and 2,4,6-Trimethyl-mandelic acid. Most compound found in ester form, compounds was shown in, The highest phenolics compound was shown in A. herba alba, collected from Saudi Arabia methyl cinnamate (8.6%) and the lowest for 1,3-Bis ( cinnamoyloxymethyl-adamantane (0.01%); also methyl-cinnamate are the highest (5.22%) phenolics compound in A. judica grown in Saudi Arabia. The results showed high concentration of some phenolic compounds in Saudi plant compared with the plant grow in Egypt. As well, the phenolic compounds have an adequate chemical structure capable of scavenging free radicals which produced as a result of oxidative stress.  Many studied reported that, drought stress inhibits the total synthesis of phenolic compounds in leaves and roots 19 , in earlier research on seedlings of V. vinifera exposed to chilling stress. Found he total content of tannins and phenolic acids in stress sample was lower than in control and author in 4 (Amarowicz et al., 2010) observed the same trend in leaves of V. vinifera subjected to low temperature stress. On the other hand (Chung et al., 2010), observed quite the opposite effect, the chilling stress and water deficit caused an increase in the total content of phenolic compounds, our result was agree with . Chung et al., where the plant which subject to more arid condition in Saudi Arabia accumulate higher amount of phenolic compounds. Many studied confirm that the production of phenolic compounds in plant tissues rises under a biotic stress condition 19,20 .

DPPH Radical Scavenging Activity
The antioxidant activity, DPPH free radicals scavenging activity reducing antioxidant assay of methanol extract of aerial parts of A.herba alba (0.122and 1.7 mg/mL) in Egypt and Saudi Arabia respectively, where A. judaica has an important antioxidant activity with an EC 50 of (0.56±0.04 and 0.67 mg/mL) as Table 3. These results is lower than that of ascorbic acid (EC 50 : 0.095±0.002) and quercetin (EC 50 : 0.019±0.003). The antioxidant activity of this extract is due to its chemical composition, which showed the presence of different phytochemical groups that have an antioxidant activity such as: alkaloids and phenolic compounds. Moreover, the chemical analysis using GC-MS allows identifying certain compounds which can be involved in the antioxidant mechanisms. Some studies were evaluated the efficacy ferulic acid and cinnamic acid as an antioxidant. Cinnamic acid derivatives 16 (Sharma, 2011), and Ferulic acid 10,17 .
The reduction of DPPH is accompanied by its passage from the violet color characteristic of DPPH solution to the yellow color measurable at 514-518 nm. In this study, the reduce 50% of the DPPH radical (IC 50 ) was measured and the results showed that Artemisia herba alba grown in Saudi Arabia present higher antioxidant capacity with an IC 50 of 0.122±0.001 mg/mL, followed by Artemisia judica grown Saudi with an IC 50 of (0.56±0.04 mg/ mL) However, the antioxidant capacity of ascorbic acid and quercetin were 0.091±0.001g/L and 0.026 ±0.002 g/L respectively (Table 3).

Total Alkaloid
The total alkaloid content of all the two plant varied from 0.24±0.2 to 1.26±0.3 mg/g of dried plant material. The Artemisia herba alba collected from Saudi Arabia show higher amount of alkaloid content. As in Table 1,this agree with (Mohammad Reza Amirjani 2013) 9 who reported that Seedlings of Catharanthus roseus was subjected to waterregimes, total alkaloid content significantly increased to maximum 178% compared to the control. Vincristine and vinblastine content of the seedlings grown water regimes increased to 175% and 171% compared to the control, respectively. Authors in 9 (Mohammad Reza Amirjani 2013) reported that the level of tropane alkaloid accumulation was

Alkaloid Compound
The GC-MS analysis of the Alkaloid fraction of Artemisia herba alba and A. judaica detected different types of alkaloid compounds, was shown in, Table 5. Pterin-6-carboxylic acid, Pseudosolasodine-diacetat, Dasycarpidan1methanol-acetate, Morphinan4,5epoxy3,6-diol, Aspidospermidin17ol and Dihydrovallesiachotamine in a different concentration of each compound according to plant and its region.    The concentration of some alkaloid compound in plant grow in Saudi Arabia was more than the plant grow in Egypt as (Dasycarpidan-1-methanol, acetate and Pterin-6-carboxylic acid) in two plant A. herba alba and A. judica this agree with 2 , Hansan et al., reported that, Gramine concentration in the leaf of Barley grown in Arimar was affected by temperature in the same way, gramine concentration remained low at suboptimal and optimal growth temperatures, increased sharply as temperature became moderately sup optimal, and began to fall when the heat stress was severe

Conclusion
The antioxidant activity of genus A. herba alba and A. judaica growing in Egypt and Saudi Arabia reflect the ability of plant to survive under drastic condition by the accumulation of some secondary metabolite, phenolic and alkaloid compounds, GC-MS-analysis of methanolic extract exposed numerous phenolic compound which have high antioxidant activity, cinammic acid, ferulic acid and nButyl-cinnamate, where alkaloid fraction analysis revealed the presence of Pseudosolasodine diacetate, Pterin-6-carboxylic acid and Aspidospermidin-17ol. These results proved the two plant species have high antioxidant activity espialy plant grow in Saudi Arabia which potent high antioxidant activity than plant grown in Egypt which reflexes the effect of environmental condition.