Screening of Antioxidant and Radical Scavenging Activities of Some Omani Medicinal Plants

n-Butanol extracts of nine medicinal plants, Cressa cretica, Ziziphus spinachrist, Acacia tortilis, Tephrosia haussknechti, Aristolochiae bracteolata, Citrullus colocynthis, Teucrium mascatense, Rhazya stricta and Nerium oleander, found in Oman were screened for their antioxidant activity using phosphomolybdenum complex assays and their radical scavenging activity using DPPH assays. Ocimumi basilicum, a plant with well documented antioxidant activity, was used as a reference. A. tortilis, and T. haussknechti extracts possessed very high antioxidant activity (AOA) and high radical scavenging activity (RSA).


Introduction
pices have been used since ancient times to improve taste and aroma of different types of food and some of them are well known for their antioxidant properties (Madsen and Bertelsen, 1995).Antioxidants are widely used as additives in food, pharmaceutical, and cosmetic industries to provide protection against oxidative degradation.Numerous degenerative diseases such as brain dysfunction, cancer, heart diseases and immune system decline could be the result of cellular damage caused by reactive oxygen species and free radicals present in human diet and antioxidants may play an important role in the prevention such diseases (Aruoma, 1998).
A great number of aromatic, spicy, medicinal and non-medicinal plants contain chemical compounds exhibiting antioxidant properties.Several studies were carried out on plants, such as rosemary and sage have led to the development of natural antioxidant formulations for food, cosmetic, and other applications (Cuvelier et al., 1996).However, scientific studies on the antioxidant properties of several plants used in Omani traditional S medicine are still rather scarce.Therefore, the evaluation of such properties seems to be a useful task with a view to find new sources of natural antioxidants.
The present work deals with preliminary studies on the antioxidant and radical scavenging activities of the n-butanol extracts prepared from the following plants: Cressa cretica (Convolvulaceae), Ziziphus spina-christi (Rhamnaceae), Acacia tortilis (Leguminosae), Tephrosia haussknechtii (Leguminosae), Aristolochiae bracteolata (Aristolochiacae), Citrullus colocynthis (Cucurbitaceae), Teucrium mascatense (Labiatae), Rhazya stricta (Apocynaceae) and Nerium oleander (Apocynaceae) which are readily available plants growing in northern Oman.To the best of our knowledge, only little information on the antioxidant and radical scavenging properties of these plants is available in the literature.C. cretica is an annual plant that grows in saline soils and is used to treat minor wounds.The aerial parts of C. cretica are source of flavonoids (Shahat et al., 2004), and terpenoids (Ramachandran and Ali, 2003).Antioxidant properties of the plant have not been reported.Z. spinachristi is one of the five popular trees in Oman.The fruits are edible.The crushed leaves are used as a natural cleaning as well as a conditioner for the hair.Hair washed with Ziziphus leaves becomes particularly lustrous and soft (Miller and Morris, 1988).It has been reported that the volatile fraction of flowers and leaves of Z. spina-christi possesses antimicrobial activity (El-Hamouly and Mohamed, 2001) and the plant is rich in flavoniod glycosides and triterpenoid saponins.
A. tortilis is the most commonly seen tree in Oman (Miller and Morris, 1988).The plentiful flowers of A. tortilis attract honey bees and these flowers are known to produce the best honey in Oman.Even though the reports on flavonoid isolation from A. tortilis (Muhaisen et al., 2002) are available in the literature, the information on antioxidant properties of this plant are not found.
T. haussknechti is a bright-flowered shrub of rocky, lower altitude hill-sides which is the most common Tephrosia species in northern Oman (Mandaville, 1978).The leaves of T. haussknechti are used to treat earache, swollen joints (Ghazanfar, 1994) but no antioxidant properties are reported .A. bracteolata is a source of aristolochic acids (El Tahir, 1991), which is used to treat wounds, snakebites but no antioxidant properties are reported in the literature.
The seeds of C. colocynthis are used to treat tumors and the green flesh of the immature fruit is taken in small quantities as a purgative (Miller and Morris, 1988).The fruits of C. colocynthis contain flavones (Maatooq, et al., 1997), triterpenes glycosides (Hatam, 1989) but antioxidant properties of the plant have not been reported.
T. mascatense is an aromatic perennial, a native of Oman which is used in traditional medicine for fever, diabetes and stomach pain (Mandaville, 1978;Ghazanfar, 1994).The composition and antimicrobial activity of this plant was recently reported (Hisham et al, 2006) no studies on the antioxidant properties of this plant is available.
R. stricta, known as 'harmal' in Arabic is used in Arabian folk medicine as a remedy for respiratory ailments and fevers (Mandaville, 1978).Alkaloids have been isolated from the leaves of R. stricta (Atta-ur-Rahman and Habib-ur-Rahman, 1996) and antioxidant action of the leaves extract has been reported (Ali et al., 2000) In traditional medicine, various parts of N. oleander are used for treatment of cough, swellings, eyes and skin diseases.Methanolic extract of the fresh leaves of N. oleander showed central nervous system (CNS) depressant effect in mice (Begum et al., 1999).Flavonoids were present in all organs of the plant (Duret and Paris, 1977), but roots and the leaves of N. oleander contain triterpenoids (Begum et al., 1997) with no antioxidant properties reported in the literature.
It was noticed that the antioxidant activity of the above selected plants were either not investigated or poorly investigated and therefore testing of their antioxidant and antiradical properties is of interest, primarily to find new promising sources of natural antioxidants.

DPPH radical-scavenging activities
The n-BuOH extracts of the plants were prepared as described in section 3 and their free radical scavenging activity was determined by DPPH free radical method (Brand-Williams et al., 1995).The n-BuOH extract of Ocimumi basilicum (Labiatae), a plant well known for its antioxidant and radical scavenging properties, was used as a reference.
The 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical is a stable radical with a maximum absorption at 517 nm.When DPPH • reacts with an antioxidant compound, which can donate hydrogen, it is reduced and the color changes from deep-violet to light-yellow.The results of DPPH radical-scavenging activity of n-butanol extracts are shown in Figure 1.

Antioxidant activity evaluation by the phosphomolybdenum method
The antioxidant activity of the extracts was evaluated by phosphomolybdenum method, which is based on the reduction of Mo(VI) to Mo(V) by the antioxidant compound(s) and the formation of a green Plant phosphate/Mo(V) complex with a maximal absorption at 695 nm.The results of antioxidant activity exhibited by different plant extracts are summarized in Table 1.The extract of A. tortilis exhibited higher antioxidant activity than O. basilicum, while extracts of Z. spinachristi, T. hausknechti and N. oleander showed antioxidant activities, similar to the reference extract.The extracts of A. bracteolate, T. mascatense and C. colocynthis showed moderate antioxidant activities, whereas C. cretica and R. stricta exhibited only weak activities compared to the reference plant extract.

Plant material
Ocimum basilicum (leaves and stems), Cressa cretica (leaves and stems), Ziziphus spina-christ (leaves), Acacia tortilis (leaves), Tephrosia haussknechti (leaves and stems), Aristolochiae bracteolata (leaves and stems), Citrullus colocynthis (fruits), Teucrium mascatense (leaves and stems), Rhazya stricta (leaves and stems) and Nerium oleander (leaves) were collected from Northern Oman during 2003 and were authenticated by Department of Biology, Sultan Qaboos University.The freshly cut plants were dried in the drying room with ventilation at ambient temperature prior to use for investigation.

Extraction
The dried, powdered plant materials were extracted with methanol (500 mL)at room temperature and the methanol extracts were concentrated under reduced pressure to get greenish viscous materials which were first partitioned with hexane/H 2 O, then with EtOAc/H 2 O and finally with n-Butanol/H 2 O to give hexane extracts, ethyl acetate extracts and n-Butanol extracts.The concentrated n-Butanol extracts were used for the evaluation of the antioxidant activities.

DPPH radical-scavenging activity
The free radical scavenging activity of the plant extracts was determined by DPPH free radical method (Brand-Williams et al., 1995).A 2.0 ml methanolic solution of DPPH (0.1 mM) was mixed with 0.1 ml of extract solution (0.1 mg/ml) in methanol and, after 60 min standing; the absorbance of the mixture was measured at 517 nm against methanol as the blank on a UV/visible light spectrophotometer (Shimadzu UV 1650 PC, Japan).Triplicate measurements were made and the radical scavenging activity was calculated by the percentage of DPPH that was scavenged using the following formula: % Reduction = [(A B -A A )/A B ] x 100 Where: A B -absorption of blank sample; A A -absorption of tested extract solution.

Evaluation of antioxidant activity
The antioxidant activity of plant extracts was evaluated by the phosphomolybdenum method according to the procedure of Prieto et al., (1999).An aliquot of 0.3 ml of sample solution (1 mM in methanol) was combined in a 4-ml vial with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate).The vials were capped and incubated in a water bath at 95 °C for 90 min.After the samples had cooled to room temperature, the absorbance of the mixture was measured at 695 nm against a blank.The antioxidant activity was expressed relative to that of Ocimumi basilicum (Labiatae).

Conclusions
The n-BuOH extracts A. tortilis, T. mascatense and T. hausknechti were found to be the strongest radical scavengers in DPPH radical assay and the strongest antioxidants in phosphomolybdenum assay among the plants screened.They are promising plants for further investigation of antioxidant properties particularly A. tortilis of which antiradical and antioxidant activities were higher than that of O. basilicum.In a previous work, we showed that aristolochic acid and aristolochic acid-D isolated from A. bracteolata, is as effective as vitamin C in antioxidant activity on a molar basis (Al-Busafi et al., 2004).The results in Table-1 suggests that the plants A. tortilis, T. hausknechtii and Z. spina-christi may have better antioxidants than vitamin C.

Acknowledgements
We acknowledge, with thanks, financial support from the Sultan Qaboos University.

Table 1 .
Antioxidant activities of n-BuOH extracts a A w , activity relative to Oscimum basilicum on a weight basis