Assessment of antioxidant activities of three wild medicinal plants from Bahrain

Abdul Ameer Al-Laith; Jameel Alkhuzai; Afnan Freije

doi:10.1016/j.arabjc.2015.03.004

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ORIGINAL ARTICLE

12 (

); 2365-2371

doi:

10.1016/j.arabjc.2015.03.004

Assessment of antioxidant activities of three wild medicinal plants from Bahrain

Abdul Ameer Al-Laith^⁎, Jameel Alkhuzai, Afnan Freije

Department of Biology, College of Science, University of Bahrain, Sakhir Campus, P.O. Box 32038, Bahrain

⁎Corresponding author. Tel.: +973 17437890; fax: +973 17449158. biolaith@gmail.com (Abdul Ameer Al-Laith)

Received: 2014-10-18, Accepted: 2015-3-8, Published: 2015-12-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

This study reports the antioxidant properties of three wild medicinal plants from Bahrain, namely Aizoon canariense L., Asphodelus tenuifolius Cav., and Emex spinosus (L.) Campdera. Antioxidant and antiradical activities of dried materials of these plants were investigated using FRAP, DPPH and ABTS assays. Total phenolics, free phenolics and total flavonoids were also determined. E. spinosus was ranked by the assays as the plant possessing the highest antioxidant and antiradical activities with an average FRAP value of 1.84 mmol/g and IC₅₀ of 10.7 and 7.75 mg/ml for DPPH and ABTS assays, respectively. A. tenuifolius ranked second with a mean FRAP value of 0.69, IC₅₀ DPPH of 1.72 and ABTS of 0.36. A. canariense possessed the lowest activities with a mean FRAP value of 0.6, and averaged IC₅₀ of 103.8 and ABTS of 14.6. E. spinosus possessed the highest content of free phenolics (mg/100 g) (64.64) followed by A. tenuifolius (45.21) and A. canariense (32.23). E. spinosus also exhibited the highest total flavonoids with an average 82.71 mg/100 g followed by A. canariense (55.92) and A. tenuifolius (49.10). The studied medicinal plants possess considerable antioxidant activities and may contribute to the well-being of individuals who consume them.

Keywords

Aizoon canariense

Asphodelus tenuifolius

Emex spinosus

ABTS

DPPH

FRAP

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1 Introduction

Medicinal plants have been part of the human being therapy and well being since ancient times all over the world (Merlin, 2003). Globally, more than 75% of the world population, both in the developed and developing countries, relies with a varying degree on plant materials for health care and treatment of ailments, and the estimated number of plants which are used for medicinal purposes varies considerably between 21,000 and 70,000 plants (Rao and Arora, 2004). Herbal medicine is still widely practiced as therapeutical agents for various diseases in the Arab region including the Gulf states. Previous reports of works conducted at our institution included the study of various aspects of medicinal plants of Bahrain (Abbas et al., 1992; El-Oqlah and Abbas, 1994; Al-Saleh et al., 1997; Abbas and Al-Saleh, 2002; Alkhuzai et al., 2010a,b ). More recently, the potential of some medicinal plants of Bahrain as natural sources of important essential fatty acids has been explored (Freije et al., 2013).

Focus on the diverse and beneficial biological activities of medicinal plants has globally grown in recent years. Medicinal plants not only provide relatively affordable drugs with marginal side effects, but they are also sources of other beneficial substances including phytochemicals and phytoalexins (Iriti and Faoro, 2009). Both, phytochemicals and phytoalexins are made, among other things, of simple phenolics, polyphenolics and flavonoids, which are known as bioactive compounds responsible for the antioxidant and antiradical activities in plants, besides some vitamins (A, C and E). It is well-known that many biological activities of medicinal plants vary and are influenced, not only by the plant type and constituents, but also by many abiotic factors including the soil characteristics and geographical locations (Halvorsen et al., 2002).

Although more studies dealing with various aspects of indigenous medicinal plants from the Arab region are currently appearing, limited number of studies have been devoted to the antioxidant activity (Alali et al., 2007; Djeridane et al., 2007; Al-Jaber et al., 2011 ). On the other hand, antioxidant activity of many medicinal plants from different parts of the world has been extensively studied by many groups (Halvorsen et al., 2002; Dragland et al., 2003; Hu et al., 2011; Seth and Sharma, 2004; Surveswaran et al., 2007 ).

The plants of the flora of Bahrain comprise 323 species of which 25% of them are known to be used as medicinal plants (El-Oqlah and Abbas, 1992). The antioxidant activity of the medicinal plants native to Bahrain has not been previously reported. As part of our continuing efforts to explore indigenous Bahraini plants, three wild medicinal plants commonly found in Bahrain were selected for the study of their antioxidant activity. These plants included Aizoon canariense L., Asphodelus tenuifolius Cav., and Emex spinosus (L.) Campdera. The traditional medicinal applications of these plants have been recently described (Freije et al., 2013). In folk medicine, the A. canariense has been used to treat indigestion flatulence, and to reduce blood pressure. Chemical constituency of A. canariense includes alkaloids, coumarins, saponins, tannins, flavonoids, steroids, and triterpenes (Mossa et al., 1983; Al-Saleh et al., 1993). Medicinal usages of A. tenuifolius include treatment of constipation, measles, anemia, ulcers, inflammations, and as diuretic and laxative. Its chemical consistency includes anthraquinones, flavonoids, steroids, and triterpenes (Al-Yahya et al., 1987; Rizk and El-Ghazaly, 1995). The edible E. spinosus is used traditionally to treat constipation, indigestion, jaundice liver problems, male sexual disorders, and as diuretic and labor inducer. It contains alkaloids, anthraquinones, coumarins, flavonoids, saponins, sterols, and tannins (Mossa et al., 1983; Al-Saleh et al., 1993).

Aims of this study were to estimate the antioxidant and antiradical activities using several assays, and to estimate the concentration of several constituents with antioxidant activity. Findings of this study provide scientific basis for further exploitation that could bring selected medicinal plants, with known traditional and safe usage, as new potential natural antioxidants in pharmaceutical industries.

2 Materials and methods

2.1

2.1 Chemicals and equipments

2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ) and gallic acid were from Fluka Chemicals (Switzerland), 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and catechin from Sigma–Aldrich (Chemie, Germany); l-Ascorbic acid and Sodium acetate anhydrous from BDH (BDH Laboratory Supplies, England), Formaldehyde Solution from (Laboratory Rasayan, Boisar); Folin–Ciocalteu phenol reagent (FC) from Merck (KGaA, Germany). Other chemicals were of analytical grade. All spectrophotometric measurements were taken using Genesis Spectrophotometer (Novaspec, LKB Biochrom, UK). Centrifugation was performed using CS-6 ((Beckman Instruments Inc., Fullerton, CA, USA).

2.2

2.2 Plant materials and collection site

Three wild medicinal plants from Bahrain, namely A. canariense L., A. tenuifolius Cav., and E. spinosus (L.) Campdera were the subjects for the current study. Fresh samples were collected during February 2010. Site 1 (26°08′50.15″N; 50°27′29.38″E) was in an agricultural wasteland in a coastal area. Site 2 (26°02′43.79″N; 50°30′29.08″E) was in sandy habitat in the interior basin. Site 3 (26°02′42.20″N; 50°31′03.57″E) was in an agricultural wasteland but in a desert sandy habitat.

2.3

2.3 sample preparation

Fresh samples were transferred in polyethylene bags to laboratory in the same day of collection. They were identified, sorted, rinsed with de-ionized water, dried-plotted, weighed, and dried to a constant weight in an air-forced oven maintained at 25 °C. Dried samples were grounded using Moulinex coffee grinder, and stored until analysis in brown air-tight bottles. Analysis of each species was performed in triplicate using composite samples prepared from at least 30 individual plants.

2.4

2.4 Determination of antioxidant activities

2.4.1

2.4.1 Ferric Reducing/Antioxidants Power Assay (FRAP)

FRAP assay used was essentially as previously described (Benzie and Strain, 1996). Samples for FRAP assay were prepared by mixing 1 g of the ground materials with 9 ml of 75 mM of phosphate buffer (pH 6.7). The mixture was extracted by frequent shaking for 1 h, followed by centrifugation at 4000 g for 5 min. An aliquot (100 μl) of the appropriately diluted extract was added to 3 ml of the standard reaction solution and the absorbance was measured at 593 nm at 0 time and after 6 min standing at room temperature. The measurement was taken in triplicate. Ascorbic acid was used to generate the standard curve in the range of 50–1000 μM. The FRAP values for standard and samples were calculated and expressed as μM ascorbic acid equivalent (AAE/100 g of dried weight (dw) as: $FRAP value (μ M) = (A 593 sample / A 593 ascorbic acid) \times concentration of ascorbic acid \times dilution factor$

2.4.2

2.4.2 The 2,2-diphenyl-1-picrylhydrazyl (DPPH^•) radical Scavenging assay

DPPH assay was carried out according to Molyneux (2004). Samples for DPPH assay were prepared by mixing the dried grounded materials (1 g) with 9 ml 80% methanol. Extraction was continued for 1 h, followed by centrifugation at 4000 rpm for 5 min. DPPH reaction mixture was prepared by mixing 0.3 ml DPPH^• radical solution (2 mM) with 3.7 ml of 80% methanol. To this 100 μl of extract was added at various dilutions. The absorbance was measured at 593 nm after 20 min standing at room temperature. The measurement was taken in triplicate. The positive standards trolox and vitamin C were used to calculate the Trolox equivalent antioxidant capacity (TEAC) and VCEAC, respectively. Percentage reduction (inhibition) of the DPPH^• radical scavenging activity was calculated as follows: % Inhibition = [(AB − AA)/AB] × 100, where AB – absorption of blank sample (t = 0 min); AA – absorption of tested sample solution (t = 20 min). IC₅₀, the amount of dried matters required to give 50% inhibition, was calculated by plotting the amount against the% inhibition.

2.4.3

2.4.3 ABTS^•+ radical cation assay

The ABTS method used was based on Re et al. (1999) with some modifications. Equal volumes of stock solutions of ABTS^•+ (7 mM) and potassium persulphate (2.45 mM) were mixed and allowed to react for 12–16 h in the dark at room temperature to generate the free radical. The formed ABTS^•+ solution was diluted with 60% methanol and the absorbance of the working solution was adjusted to 0.70 ± 0.02 at 734 nm. An aliquot (10–30 μl) of methanolic extract was mixed with 1 ml of the working ABTS^•+ solution and the absorbance was monitored at 734 nm for 6 min. The measurement was taken in triplicate. The ABTS^•+ radical scavenging activity was calculated as the percentage reduction (inhibition) of the ABTS^•+ as for DPPH. TEAC and VCEAC were calculated as described for the DPPH assay.

2.5

2.5 Determination of antioxidant constituents

2.5.1

2.5.1 sample extraction

For extraction of free phenolics and total flavonoids the method described by Vinson et al. (2001) was used employing 80% methanolic solution. For bound phenolics, samples were extracted using acidified methanolic solution.

2.5.2

2.5.2 Phenolics determination assay

Free, bound and total phenolics were essentially estimated using the FC reagent as described by Waterhouse (2009).

2.5.3

2.5.3 Extraction and determination of total flavonoids

Each sample (0.25 g) was extracted overnight with 5 ml 80% methanolic solution, followed by a brief sonication for 5 min. The mixture was centrifuged for 10 min at 4000 rpm. Total flavonoids was estimated using AlCl₃ precipitation method (Zhisen et al., 1999). A standard curve was generated using rutin and the results were expressed as mg rutin equivalents (RE) per gram dry weight of sample.

2.6

2.6 Statistical analysis

Excel (Microsoft Co, Redmond, WA, USA) and SPSS (version 19.0 statistical software, SPSS Inc., Chicago, USA) packages were used for statistical analysis. Results represented means ± s.d. of replicated determinations. Comparison and separation of the mean values was performed using Tukey’s multiple comparison test. Significant differences were established at the α level of 0.050. Pearson’s coefficient was used to evaluate the correlations between the assays and the assays with the antioxidant constituents.

3 Results

3.1

3.1 Antioxidant activities

3.1.1

3.1.1 FRAP assay

This assay measures the reducing power of a potential antioxidant which reduces the ferric ion (Fe³⁺) to the ferrous ion (Fe²⁺) leading to the formation of a deep blue complex (Fe²⁺/TPTZ). Leave samples of E. spinosus from both tested sites (S1 and S2) exhibited significantly (p 0.05) higher antioxidant activity than the two other medicinal plants, A. canariense, and A. tenuifolius. The highest FRAP value was reported in samples collected from S2 which was 30% greater than samples from site S1. Leave samples of E. spinosus from the two sites (S1 and S2) differed significantly in their FRAP values (p < 0.001). Similarly, leaves of A. tenuifolius from sites S2 and S3 exhibited significantly different FRAP values, whereas leaves samples of A. canariense from sites S1 and S3 showed no significant difference (p > 0.06). Furthermore, seeds of A. tenuifolius possessed lower antioxidant activity as compared to the leave sample of the same plant. All the tested plants showed lower FRAP values than the positive control, ascorbic acid (11.45 ± 0.001 mmol/g).

3.1.2

3.1.2 Free radical-scavenging ability by the DPPH assay

The radical scavenging ability measured by DPPH assay is given in Table 1 and expressed as IC₅₀ and as the VCEAC value which donates the amount in mg of vitamin C equivalent per gram of a dried sample. In this assay lower amount required to give IC₅₀ indicates higher free radical scavenging activity. Leaves samples of E. spinosus exhibited significantly higher free radical activities than A. canariense and A. tenuifolius. To cause IC₅₀ only about 1 mg/ml of dried leaves of E. spinosus was needed as compared to 1–4 and 6–13 mg/ml of A. canariense and A. tenuifolius, respectively. Leaves of E. spinosus from both sites (S1 and S2) had 7–15 times higher VCEAC value than A. canariense and about 2 times higher than A. tenuifolius.

Table 1 Mean and SD of FRAP value (expressed as mmol/g), IC₅₀ for DPPH and ABTS (expressed as the weight in mg of dw that gave 50% inhibition and as VCEAC value).

Plant	Part	Site	FRAP value (mmol/g) (±SD)	Amount of dried matter (mg/ml) required to scavenge 50% of the free radical (mg ± SD)		VCAEC (mg vitamin C equivalent per gram of dry weight
Plant	Part	Site	FRAP value (mmol/g) (±SD)	DPPH	ABTS	DPPH	ABTS
Aizoon canariense	L	S1	0.58 (0.016)^a	140.9 (2.9)^a	13.17^a (0.04)	0.033	0.36
Aizoon canariense	L	S3	0.61 (0.004)^a	66.56 (2.0)^b	16.06^b (0.04)	0.068	0.29
Asphodelus tenuifolius	L	S2	0.57 (0.006)^a	20.93 (0.09)^c	14.91^b (0.01)	0.22	0.24
Asphodelus tenuifolius	L	S3	0.81 (0.003)^b	18.37 (0.06)^c	11.72^c (0.02)	0.25	0.32
Asphodelus tenuifolius	S	S3	0.44 0.002)^c	37.24 (0.22)^d	19.78^d (0.01)	0.12	0.40
Emex spinosus	L	S1	1.46 (0.002)^d	10.22 (0.03)^e	9.70^e (0.01)	0.44	0.50
Emex spinosus	L	S2	2.21 (0.001)^e	11.13 (.05)^e	5.79^e (0.54)	0.40	0.83
Ascorbic acid	–	–	11.450 (0.001)	0.045 (0.00)	0.01125 (0.01)

Means followed with the same letter in each column are not significantly different at the level 5% level (α = 0.05).

3.1.3

3.1.3 Free radical-scavenging ability by the ABTS assay

The radical scavenging ability measured by ABTS assay is given in Table 1 and expressed as for DPPH assay. Percent inhibition of the ABTS radical activity followed similar pattern as for DPPH assay. On average, the amount required to cause the same effect as E. spinosus was 3 times and 8 times in the case of A. tenuifolius and A. canariense, respectively. Higher VCAEC values were found in leave samples from E. spinosus.

In both assays, DPPH and ABTS, the IC₅₀ of the pure ascorbic acid were lower than all tested plants, being 45 and 11.25 μg/ml, respectively. No significant difference was found between leave samples of E. spinosus from the two sites (S1 and S2) with regard to both the DPPH and the ABTS scavenging activities. However, A. canariense samples from sites S1 and S3 possessed significant differences on both of the free radical assays. All the three assays, FRAP, DPPH and ABTS, exhibited a dose–response relationship, with maximum attainable inhibition of the free radicals in DPPH and ABTS assays ranging between 80% and 90%.

3.2

3.2 Total, free, and bounded phenolics, and total flavonoids

3.2.1

3.2.1 Total phenolics

Levels of total, free, bounded phenolics and total flavonoids are given in Table 2. The highest level of total phenolics (TP) was found in leaves of A. tenuifolius at site S3, followed by leaves of E. spinosus at site S2. All other samples possessed comparable amount of total phenolics. Samples of A. tenuifolius from site S3 contained between 2 and 3 times as much as other samples, the exception was E. spinosus from site S2. The highest content of free phenolics (FP) was found in leaves of E. spinosus which was significantly different than the others (p < 0.001), whereas A. tenuifolius from site S3 was significantly different than A. canariense from site 8 (p = 0.014). With regard to bounded phenolics, A. tenuifolius from site S3 possessed the highest content, and was significantly different than others, whereas E. spinosus from site S2 possessed the second highest content of bound phenolics, and was also significantly different than the others.

Table 2 Mean (±SD) of total, free, and bound phenolics of the three medicinal plants (expressed as gallic acid equivalent (GAE) and total flavonoids (expressed as rutin equivalent (RE) mg/100 g dry matter.

Plant	Part	Site	Total phenolics	Free phenolics	Bound phenolics	Total flavonoids
Plant	Part	Site	Mean (±SD)	Mean (±SD)	Mean (±SD)	Mean (±SD)
Aizoon canariense	L	S1	135.843^a (9.11)	28.096^a (2.50)	107.747^a (8.07)	37.882^ab (0.83)
Aizoon canariense	L	S3	155.210^a (14.32)	40.371^b (3.31)	114.839^a (12.50)	66.878^a (0.98)
Asphodelus tenuifolius	L	S2	157.119^a (2.84)	35.734^b (2.51)	121.386^a (4.12)	110.28^a (1.14)
Asphodelus tenuifolius	L	S3	442.444 ^b (13.23)	59.738^bc (6.55)	382.706^b (8.24)	47.05^ab (1.52)
Asphodelus tenuifolius	S	S3	139.662^a (4.73)	37.916^b (4.12)	101.746^a (1.89)	53.58^ab (1.09)
Emex spinosus	L	S1	167.485^a (18.25)	52.100^b (12.28)	115.385^a (6.55)	28.02^bc (1.66)
Emex spinosus	L	S2	343.315^c (36.39)	85.925^c (19.64)	257.229^c (22.10)	31.54^c (1.13)

Values having the same letter in a column are not significantly different at 5% level.

3.2.2

3.2.2 Total flavonoids

The highest level of total flavonoids was recorded in A. tenuifolius from site S2, followed by A. canariense from site S3, and the least was found in leaves of E. spinosus, site S1. Leave samples of E. spinosus from the two sites showed the lowest total flavonoids content.

4 Discussion

4.1

4.1 Antioxidant activities

This study investigates the antioxidant and antiradical activities of three medicinal plants known to be used in the folk medicine in Bahrain using three widely known methods; FRAP, DPPH and ABTS. Despite the fact that these methods have different reaction mechanisms and do not necessarily measure the same activity (Prior et al., 2005), the three methods clearly indicated that the studied plants possess variable but considerable antioxidant and antiradical activities. FRAP assay is based on electron transfer reaction, whereas DPPH and ABTS assays are based on electron and H atom transfer (Prior et al., 2005). Furthermore, the VCEAC method used to express the antioxidant activity provides a meaningful method for direct comparison with known potent antioxidant widely distributed in plant materials (Kim et al., 2002).

On weight-to-weight basis, the FRAP values of E. spinosus were about 20% of that of pure ascorbic acid. The results also showed that the three methods agreed on identifying the E. spinosus as the plant containing the most antioxidant and antiradical activities among the three studied plants. The three methods were also consistent to a certain degree on identifying A. canariense as the least potent in both types of activities of the three plants. It is not unusual that the three methods may generate inconsistent ranking results when used to test a particular plant (Halvorsen et al., 2002; Prior et al., 2005). However, the agreement between the three assays, in our study, probably indicate that these activities were mainly due to phenolics and flavonoids. It is known that vitamin C (ascorbic acid) and carotenoids are major source of discrepancy of antioxidant/antiradical activities in plant materials. Although these constituents were not investigated, their contributions toward antioxidant/antiradical activities of the three studied medicinal plants may be very minimal. It is well known that vitamin C is greatly affected by drying. Furthermore, the dried green leaves used in this study were low in total fat content, a parameter which is highly correlated with the total carotenoids content in many plant materials (Lu et al., 2009). Also, it is well known that the antioxidant effect of plant materials including medicinal plants can be attributed mainly to radical scavenging activity of phenolic compounds such as, simple phenolics, polyphenols, flavonoids, tannins, and phenolic terpenes.

Very few studies have been conducted to measure the antioxidant activity of the medicinal plants in the Arab region. Recently, Emam et al. (2010) reported that 20 μg/ml of aqueous extract of E. spinosus leaves produced IC₅₀ in the DPPH assay. This is a very small amount as compared to our result, (1 mg/ml produced IC₅₀). However, their sample preparation underwent several extraction, fractionation, and concentration steps, whereas our results were based on crude extract. Alali et al. (2007) reported an IC₅₀ value of 38.1 and 15.1 μmol Trolox Equivalent (TE) for aqueous and methanolic extracts of A. canariense.

FRAP values of some Chinese and Japanese medicinal herbs ranged between 0.3 and 120 mmol/g (Dragland et al., 2003). The three studied plants had FRAP values within the lower reported range. Leaves of E. spinosus possessed higher FRAP values than black tea (0.8 mmol/g) and comparable to green tea (2.5 mmol/g) (Blomhoff, 2005) two herbs widely promoted as good sources of antioxidants. The antioxidant activity of 70 medicinal plants obtained from local markets in Croatia was reported to have FRAP values ranging from 0.06 to 25 mM/L, or 0.004 to 1.67 mmol/g dried matters (Katalinic et al., 2006). Results from our study are within this range. Furthermore, and according to this study, E. spinosus can be grouped with medicinal plants possessing very high FRAP values, whereas A. canariense and A. tenuifolius with good and high categories.

Surveswaran et al. (2007) screened 133 medicinal plants from India using several methods and reported values ranging from 0.16 to 124.05 umol TEAC/g in the FRAP assay, 0.00 to 679.69 and 0.16 to 500.70 mmol TEAC/100 g DW in the DPPH and ABTS assays, respectively. Our findings are comparable with the majority of the plants studied.

4.2

4.2 Antioxidant constituents

The fact that phenolic compounds are major antioxidant constituents in materials of plant origin including herbs and medicinal plants is well documented (Dorman et al., 2004; Velioglu et al., 1998). The three medicinal plants exhibited noticeable variations in the content of various phenolic compounds studied among the species, and for the same species from different locations. The percentage of free phenolics varied considerably, the lowest was found in A. tenuifolius, site 9 (13.5%) and highest in E. spinosus (31.1%). Free phenolics, but not bounded phenolics, are responsible for the antioxidant and antiradical activities under the experimental conditions employed in this study. The major types of phenolic compounds contributing to the antioxidant activity mainly include simple phenolic constituents, e.g., phenolic acids (hydroxycinnamic acids and hydroxybenzoic acids), polyphenolic compounds, e.g., tannins, flavonoids, curcuminoids, coumarins among others (Dorman et al., 2004; Velioglu et al., 1998). It should be noted that Folin-Ciocalteu assay not only measures the total phenolic compounds, but is prone to various interfering compounds of plant origins including ascorbic acid, some nitrogen-containing and thiol compounds (Everette et al., 2010).

Katalinic et al. (2006) reported that total phenolic compounds of 70 medicinal plants ranged between 9 and 2218 mg CE/L which, if converted to 100 g dried matters rather than a liter of infusion, is equivalent to 60–14,786 mg/g dw. On the other hand, Surveswaran et al. (2007) reported that the total phenolics of the 133 Indian medicinal plants mentioned above ranged from 0.06 to 41.47 g of gallic acid equivalents (GAE)/100 g (DW) with a mean of 2.44 g GAE/100 g DW. Our findings were consistent with these reported values.

4.3

4.3 Folk usage of E. spinosus

E. spinosus which is an annual herb grow throughout Bahrain in stony or gravelly soils. Though its roots are edible, the whole plant can be used as a remedy for stomach disorders including the relief of dyspepsia and colic, as laxative, diuretic, purgative (Abbas and Al-Saleh, 2002). It can also be used an appetizer. Previous studies already demonstrated an association between acute abdominal pain and oxidative stress as measured by total antioxidant capacity (Chi et al., 2002). Our results show a considerably high antioxidant capacity of E. spinosus, which may indicate the relevance of this capacity to its folk usage. Furthermore, the results also indicate that E. spinosus has a potential for further studies to establish the causal relationship between these health benefits and nutritive attributes.

4.4

4.4 Correlation

Table 3 shows the correlation between the three assays and between the assays and the antioxidant constituency of the medicinal plants under investigation. FRAP was significantly and negatively correlated with DPPH and ABTS. Negative correlation, in the cases of DPPH and ABTS, indicates lower amount of plant materials required to produce higher% inhibition of the radicals. DPPH was strongly correlated with ABTS. FRAP, DPPH and ABTS were all strongly correlated with free phenolics. FRAP and DPPH, but not ABTS, were significantly correlated with total phenolics. Only FRAP was strongly correlated with total flavonoids. Bounded phenolic compounds apparently were not statistically correlated with any of the methods used. Floegel et al. (2011) recently reported a strong correlation between DPPH and ABTS methods, and between these methods and phenolics and flavonoids.

Table 3 Pearson’s correlation coefficients between the three assays and between the assays and the phenolic constituents.

	DPPH	ABTS	Total phenolics	Free phenolics	Bound phenolics	Total flavonoids
FRAP	−0.484^⁎	−0.465^⁎	0.451^⁎	0.823^⁎⁎	0.348	0.835^⁎⁎
DPPH		0.943^⁎⁎	−0.464^⁎	−0.576^⁎⁎	−0.413	−0.181
ABTS			−0.403	−0.569^⁎⁎	−0.344	−0.147

⁎ Correlation is significant at the 0.05 level.

⁎⁎ Correlation is significant at the 0.01 level.

5 Conclusions

The three medicinal plants collected from various sites from the main island of the Kingdom of Bahrain exhibited variable but considerable antioxidant and antiradical activities. Among the three studied plants, E. spinosus possessed the highest activities in the three methods used, FRAP, DPPH and ABTS. This plant was characterized by relatively higher content of free phenolics and total flavonoids. The strongest correlation coefficients were found between the three assays and the free phenolics.

Acknowledgment

The authors are very grateful for the financial support by the Deanship of Scientific Research, the University of Bahrain, Project # 16/2008.

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Lu Q.-Y., Zhang Y., Wang Y., Wang D., Lee R.-P., Gao K., Byrns R., Heber D., . California hass avocado: profiling of carotenoids, tocopherol, fatty acid, and fat content during maturation and from different growing areas. J. Agri. Food Chem.. 2009;57:10408-10413.
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Show Sections

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[8] Al-Saleh F.S., Ali H.H., Mirza M., . Chemical constituents of some medicinal plants growing in Bahrain. Fitoterapia LXIV. 1993;3:251-256.
[Google Scholar]

[9] Al-Yahya M.A., Mossa J.S., Al-Meshal I.A., . Medicinal Plants of Saudi Arabia. Vol vol. VI. Saudi Arabia: King Saud University Library; 1987.

[10] Benzie I.F., Strain J.J., . The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem.. 1996;239:70-76.
[Google Scholar]

[11] Blomhoff R., . Dietary antioxidants and cardiovascular disease. Curr. Opin. Lipidol.. 2005;16(2005):47-54.
[Google Scholar]

[12] Chi C.H., Shiesh S.C., Lin X.Z., . Total antioxidant capacity and malondialdehyde in acute abdominal pain. Am. J. Emerg. Med.. 2002;20:79-82.
[Google Scholar]

[13] Djeridane A., Yousfi B., Nadjemi B., Vidal N., Lesgards J.F., Stocker P., . Screening of some Algerian medicinal plants for the phenolic compounds and their antioxidant activity. Eur. J. Food Res. Tech.. 2007;224:801-809.
[Google Scholar]

[14] Dorman H.J., Bachmayer M., Kosar O.M., Hiltunen R., . Antioxidant properties of aqueous extracts from selected lamiaceae species grown in Turkey. J. Agr. Food Chem.. 2004;52:762-770.
[Google Scholar]

[15] Dragland S., Senoo H., Wake K., Holte K., Blomhoff R., . Several culinary and medicinal herbs are important sources of dietary antioxidants. J. Nutr.. 2003;133:1286-1290.
[Google Scholar]

[16] El-Oqlah A.A., Abbas J.A., . Preliminary studies on the natural flora and vegetation of Bahrain. Dirasat. 1992;19:123-137.
[Google Scholar]

[17] El-Oqlah A.A., Abbas J.A., . A checklist of vascular plants of Bahrain. Dirasat. 1994;21:95-118.
[Google Scholar]

[18] Emam A.M., Mohamed M.A., Diab Y.M., Megally N.Y., . Isolation and structure elucidation of antioxidant compounds from leaves of Laurus nobilis and Emex spinosa. Drug Discov. Therapeu.. 2010;4:202-220.
[Google Scholar]

[19] Everette J.D., Bryant Q.M., Green A.M., Abbey Y.A., Wangila G.W., Walker R.B., . Thorough study of reactivity of various compound classes toward the Folin-Ciocalteu reagent. J. Agric. Food Chem.. 2010;58:8139-8144.
[Google Scholar]

[20] Floegel A., Kim D.-O., Chung S.-J., Koo S.I., Chun O.K., . Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich US food. J. Food Compos. Anal.. 2011;24:1043-1048.
[Google Scholar]

[21] Freije A., Alkhuzai J., Al-Laith A.A., . Fatty acid composition of three medicinal plants from Bahrain: new potential sources of gamma-linolenic acid and dihomo-gamma-linolenic. Indust. Crops Prod.. 2013;43:218-224.
[Google Scholar]

[22] Halvorsen B.L., Holte K., Myhrstad M.C., Barikmo I., Hvattum E., Remberg S.F., Wold A.B., Haffner K., Baugerod H., Andersen L.F., Moskaug Ǿ., Jacobs D.R. Jr., Blomhoff R., . A systematic screening of total antioxidants in dietary plants. J. Nutr.. 2002;132:461-471.
[Google Scholar]

[23] Hu, J., Zhang, J., Zhao, W., Zhang, Y., Zhang, L., Shang, H. 2011. Cochrane Systematic Reviews of Chinese Herbal Medicines: An Overview.

[24] Iriti M., Faoro F., . Chemical diversity and defence metabolism: how plants cope with pathogens and ozone pollution, a review. Int J. Mol Sci.. 2009;10:3371-3399.
[Google Scholar]

[25] Katalinic V.M., Milos M., Kulisic T., Jukic M., . Screening of 70 medicinal plant extracts for antioxidant capacity and total phenols. Food Chem.. 2006;94:550-557.
[Google Scholar]

[26] Kim D.O., Lee K.W., Lee H.J., Lee C.Y., . Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. J. Agric. Food Chem.. 2002;50:3713-3717.
[Google Scholar]

[27] Lu Q.-Y., Zhang Y., Wang Y., Wang D., Lee R.-P., Gao K., Byrns R., Heber D., . California hass avocado: profiling of carotenoids, tocopherol, fatty acid, and fat content during maturation and from different growing areas. J. Agri. Food Chem.. 2009;57:10408-10413.
[Google Scholar]

[28] Merlin M.D., . Archaeological evidence for the tradition of psychoactive plant use in the old world. Econ. Bot.. 2003;57:295-323.
[Google Scholar]

[29] Molyneux P., . The use of the stable free radical diphenylpicryl-hydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. Sci. Technol.. 2004;26:211-219.
[Google Scholar]

[30] Mossa J.S., Al-Yahya M.A., Al-Meshal I.A., . Phytochemical and biological screening of Saudi medicinal plants. Fitoterapia LXIV. 1983;4:147-158.
[Google Scholar]

[31] Prior R.L., Wu X.L., Schaich K., . Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agr. Food Chem.. 2005;53:4290-4302.
[Google Scholar]

[32] Rao, V.R., Arora, R.K., 2004. Rationale for conservation of medicinal plants. In: Medicinal Plants Research in Asia, vol. 1: The Framework and Project Workplants. International Plant Genetic Resources Institute, Regional Office for Asia, the Pacific and Oceania (IPGRI-APO). Malaysia, pp. 9.

[33] Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evan C., . Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med.. 1999;26:1231-1237.
[Google Scholar]

[34] Rizk A.M., El-Ghazaly G.A., eds. Medicinal and Poisonous Plants of Qatar. Qatar: Doha Modern Printing Press Ltd.; 1995.

[35] Seth S.D., Sharma D., . Medicinal plants of India. Indian J. Med. Res.. 2004;120:9-11.
[Google Scholar]

[36] Surveswaran S., Cai Y.-Z., Corke H., Sun M., . Systematic evaluation of natural phenolic antioxidants from 133 Indian medicinal plants. Food Chem.. 2007;102:938-995.
[Google Scholar]

[37] Velioglu Y.S., Mazza G., Gao L., Oomah B.D., . Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J. Agr. Food Chem.. 1998;46:4113-4117.
[Google Scholar]

[38] Vinson J.A., Su X., Zubik L., Bose P., . Phenol antioxidant quantity and quality in foods: fruits. J. Agr. Food Chem.. 2001;49:5315-5321.
[Google Scholar]

[39] Waterhouse, A., 2009. Folin–Ciocalteau Micro Method for Total Phenol in Wine. <http://www.waterhouse.ucdavis.edu/phenol/folinmicro.htm>.

[40] Zhisen J., Mencheng T., Jiamming W., . Determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem.. 1999;64:555-559.
[Google Scholar]

Assessment of antioxidant activities of three wild medicinal plants from Bahrain

Abstract

Keywords

Aizoon canariense

Asphodelus tenuifolius

Emex spinosus

ABTS

DPPH

FRAP

1 Introduction

2 Materials and methods

2.1 Chemicals and equipments

2.2 Plant materials and collection site

2.3 sample preparation

2.4 Determination of antioxidant activities

2.4.1 Ferric Reducing/Antioxidants Power Assay (FRAP)

2.4.2 The 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical Scavenging assay

2.4.3 ABTS•+ radical cation assay

2.5 Determination of antioxidant constituents

2.5.1 sample extraction

2.5.2 Phenolics determination assay

2.5.3 Extraction and determination of total flavonoids

2.6 Statistical analysis

3 Results

3.1 Antioxidant activities

3.1.1 FRAP assay

3.1.2 Free radical-scavenging ability by the DPPH assay

3.1.3 Free radical-scavenging ability by the ABTS assay

3.2 Total, free, and bounded phenolics, and total flavonoids

3.2.1 Total phenolics

3.2.2 Total flavonoids

4 Discussion

4.1 Antioxidant activities

4.2 Antioxidant constituents

4.3 Folk usage of E. spinosus

4.4 Correlation

5 Conclusions

Acknowledgment

References

Suggested read for related articles:

2.4.2 The 2,2-diphenyl-1-picrylhydrazyl (DPPH^•) radical Scavenging assay

2.4.3 ABTS^•+ radical cation assay