Phytochemical analysis and biological activities of three wild Mesembryanthemum species growing in heterogeneous habitats

The objective of this study was to analyze the phytochemicals and to determine the antioxidant, antibacterial and allelopathic potential of three wild Mesembryanthemum species (M. crystallinum L., M. forsskaolii Hochst. Ex Boiss and M. nodiflorum L.). The phytochemical composition of the methanolic extract of studied species revealed the considerable quantities that might be responsible for their powerful antioxidant activity. The IC50 values were 386.51, 592.97, and 752.23μg/ml for M. nodiflorum, M. crystallinum and M. forsskaolii extracts respectively. The antibacterial activity index was calculated for each extract in comparison with the standard antibiotics. M. nodiflorum showed higher potency than ampicillin and penicillin G against against Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. The allelopathic potential showed that the studied Mesembryanthemum species expressed a significant phytotoxic activity against Chenopodium murale weed in a dose dependent manner. M. nodiflorum sample showed most phytotoxic effect among the studied species.


INTRODUCTION
There is a worldwide increasing interest in using the traditional foods and ingredients from natural resources (Muthukrishnan et al., 2018). The Egyptian desert is blessed with plenty of wild plants that significantly affect the daily life of Bedouins in urban areas (Zahran & El-Amier, 2013). The Egyptian flora plays a key role in maintaining the region's environmental sustainability (McClanahan, 1998;Zaki et al., 2018). In addition to their role in stabilizing slope and improving soils, plants are rich in chemical constituents and are used in insecticidal, herbicidal, folk medicine and other industrial applications (Hosseinzadeh et al., 2015;Naboulsi et al., 2018). The knowledge about the phytoconstituents of plants leads to a better understanding of their possible medicinal and agro-industrial uses (Yakhin et al., 2017). The biological activity of plants might be ascribed to the presence of secondary metabolites like polyphenols, flavonoids, lignins, alkaloids, terpenoids, carotenoids, vitamins, etc. (Vinson et al., 2005;Zaki et al., 2016;Zaki et al.,2017).
Family Aizoaceae is a widely distributed family in Africa that comprises around 127 genera and 1860 species distributed in tropical and sub-tropical areas. They are also cultivated as ornamental ground covers (Mabberley, 1997;Klak et al., 2004). In Egypt, it is represented by 9 species that are succulents ranged from pebble-like leaf succulents to small succulent shrubs. They are characterized by distinctive seed capsules. In Egyptian flora, three species of Mesembryanthemum (M. crystallinum L., M. forsskaolii Hochst. ex Bioss and M. nodiflorum L.) were recorded and is distributed in Mediterranean coastal strip, Desert and Oases (Boulos, 1999). Members of family Aizoaceae are known to have diverse biological activities including antihyperlipidemic, antipyretic, diuretic, antioxidant, anticancer, larvicidal, analgesic, anti-rheumatic, anticholera, emetic, laxative, anti-inflammatory and antimicrobial (Ibtissem et al., 2010;Mohammed et al., 2012;Ibtissem et al., 2012;Doudach et al., 2013;Moawad et al., 2016).
The biological activities of genus Mesembryanthemum like antioxidant, antimicrobial, anticarcinogenic and antiviral Phytochemical analysis and biological activities of three wild Mesembryanthemum species growing in heterogeneous habitats activity was reported (Rood, 1994;van Wyk, 2008;Falleh et al., 2011;Gawad et al., 2018). Moreover, this genus has recorded to possess several ethnomedicinal uses such as treatment of liver diseases, ocular infection, alexiteric, analgesic, laxative and diabetes (Mustafa et al., 1995;Bouftira et al., 2009;Al-Faris et al., 2010;Falleh et al., 2011). The phytochemical screening of Mesembryanthemum genus concluded the presence of carbohydrates, protein, antioxidant enzymes, tannins, triterpenes, alkaloids and flavonoids van (der Watt and Pretorius, 2001;Bouftira et al., 2008;Doudach et al., 2013). Therefore, our study aimed to determine the phytochemical constituents present in the studied Mesembryanthemum species collected from two different habitats in Egypt, to evaluate the antioxidant activity, the antimicrobial potential against several pathogenic bacterial strains and the allelopathic effect of the plants extracts against the noxious weed Chenopodium murale as potential green eco-friendly bioherbicide.

Preparation of Plant Material
The aerial parts of M. crystallinum and M. nodiflorum were gathered during flowering stage from sand flats in north Nile Delta of Egypt (31°29'28.08"N 31°23'46.90"E), while M. forsskaolii was collected from Wadi Hagul, North Eastern Desert, Egypt (29°53'27.31"N 32°13'22.58"E). The studied species were identified according to Täckholm (1974) and Boulos (1999) (Table 1). The collected plants were dried in shade at room temperature for 21 days, grinded into fine powder and kept in a polyethylene container for further use.

Quantitative analysis
The total phenolics were measured according to the method adopted by Chlopicka et al. (2012), the total flavonoids were estimated according to Stankovic et al. (2011), the total tannins were measured according to van Buren and Robinson (1969) and the alkaloids were measured according to the assay adopted by Joshi et al. (2013).

DPPH radical scavenging capacity estimation
The radical scavenging activity of Mesembryanthemum species extracts was measured using 2,2-diphenyl-1-picryl-hydrazil (DPPH) radical according to the assay adopted by Miguel (2010). Briefly, 2 milliliters of 150 µM DPPH was added to 2 milliliters of plant extracts using concentrations from100 to 1000 mg/l and kept in dark at 40 ºC for 30 min. The absorbance was measured at 520 nm using Milton Roy Spectronic 21D UV-Visible Spectrophotometer (USA), all samples were analyzed in triplicate, and the IC 50 values were estimated using exponential curve.

Disc diffusion assay
Sterilized filter paper discs were immersed in the studied extracts then loaded over the plates seeded with the tested strains and incubated at 37 °C for 18-24 hours. The zone of growth inhibition was estimated and subtracted from the diameter of the filter paper discs (6 mm) (Cappuccino & Sherman, 2008). The Activity Index (AI) was used as a parameter for measuring the antibacterial potential of the studied extracts in comparison with standard antibiotics (Shekhawat & Vijayvergia, 2010).

Allelopathic Activity
Chenopodium murale seeds were gathered from cultivated fields in Mansoura city, Egypt. Uniform and ripened seeds were sterilized by immersing in NaOCl (0.3%) for three minutes then washed by bi-distilled sterilized water several times. The sterilized seeds were dried and kept for future use. The phytotoxic activity was measured using glass Petri dishes with bottom covered with Whatman No. 1 filter paper, on each of them 20 sterilized seeds were loaded. 4 ml of each extract was added in concentrations of 2.5, 5, 10, 20 and 40 g/l, then the dishes were incubated in growth chamber at 25-27 °C (Rice, 1972;Abd El-Gawad et al., 2015). Three replications for each treatment were measured. The percent of germination and the inhibition of germination of shoot and root growth was calculated as follows: Inhibition % = 100×(Length of control -Length of treatment)/Length of control.

Phytochemical Constituents
Nowadays, plants are considered as important sources of bioactive molecules that are be beneficial in various fields and could be used as drugs, food supplements, antimicrobials, allelochemicals, etc. The phytochemical screening of the dried aerial parts of the studied Mesembryanthemum species revealed the presence of all of the estimated phytoconstituents except anthraquinones in M. crystallinum and M. nodiflorum species while M. forsskaolii showed absence of anthraquinone and glycosides and presence of the other phytoconstituents in traces as recorded in Table 2. The ethanolic extract obtained from each of the studied plants was quantitatively assayed for phenolics, flavonoids, tannins and alkaloids. Ethanol was chosen due to its efficiency of extraction for biologically active compounds like phenolics (Raks et al., 2018;Krakowska-Sieprawska et al., 2020).
In the present study, there were variable levels of alkaloids, phenolics, flavonoids and tannins in Mesembryanthemum species. M. nodiflorum expressed the highest content of tannins, alkaloids and phenolics (36.14, 9.22 and 21.36 mg/g dried plant, respectively) followed by M. crystallinum (31.64, 6.21 and 19.55 mg/g dried plant, respectively) while the lowest content was in M. forsskaolii (22.87, 3.88 and 19.22 mg/g dried plant, respectively). The highest content of flavonoids recorded in M. crystallinum (11.41 mg/g dried plant) as illustrated in Figure 1. Our data were comparable with earlier studies on common desert plants (Hariprasad & Ramakrishna, 2011;Alzuaibr et al., 2020;El-Amier & Al-hadithy, 2020), but were not in agreement of Bohnert and Cushman (2000) who depicted that M. crystallinum tissues contain negligible amounts of phenolic and flavonoid compounds.

Antioxidant Activity
Halophytic plants have the ability to overcome and scavenge harmful reactive oxygen and reactive nitrogen species produced under stress of increased salinity, since they possess a powerful antioxidant system (Qiu-Fang et al., 2005;Kapoor et al., 2019).
The antioxidant properties of Mesembryanthemum species have been evaluated by measuring their DPPH radical scavenging activity using the ethanolic extracts of the studied species. All organic extracts of Mesembryanthemum species exhibited an antioxidant activity in a dose dependent manner, which was comparable with ascorbic acid as reference standard (Table 3). IC 50 values were calculated and recorded in Table 3. The IC 50 value of M. nodiflorum, M. crystallinum and M. forsskaolii extracts were 386.51, 592.97, and 752.23 µg ml -1 , respectively and that of ascorbic acid was112.31 µg ml -1 . These results suggest  Values are means ± standard error (n=3). IC 50 : the antioxidant concentration capable of diminishing 50% of the used DPPH radical that ethanol extracts of Mesembryanthemum species has an obvious effect on scavenging of DPPH radical. M. nodiflorum expressed the highest antioxidant activity followed by M. crystallinum while M. forsskaolii was the lowest. These results could be attributed to the levels of phytoconstituents that are responsible for the antioxidant activity. Antioxidant enzymes and compounds like ascorbic acid, phenolics, flavonoids and glutathione provide protection for living organisms against free radical damage (Rice-Evans et al.,1996;Chen et al., 2020).

Antibacterial Activity
The antibacterial activity of crude extracts of Mesembryanthemum species was estimated in vitro using 8 different pathogenic bacterial strains. The studied extracts exhibited broad antibacterial spectrum (Table 4).
The results in Figure 2 showed that the ethanolic extract of The phytochemical constituents like tannins, triterpenes, alkaloids, phenolics and flavonoids might be the motive of the activity against the examined bacterial strains (van der Watt & Pretorius, 2001;Bouftira et al., 2008;Doudach et al., 2013) and this agrees with the results obtained as M. nodiflorum content of these chemicals was the highest among the three studied Mesembryanthemum species, meanwhile it expressed the broadest antibacterial spectrum with significant inhibitory activity.
It is obvious from the results that that gram-negative bacteria are more sensitive to three plant extracts than gram-positive bacteria and these results agrees with the previously reported by Kaneria et al. (2009)  Streptococcus epidermis n.a n.a n.a n.a n.a n.  the tested microbes (Cowan, 1999;Kaneria et al., 2009;Kumar et al., 2016;Manandhar et al., 2019).
Moreover, the results revealed that the antibacterial performance of ethanolic extracts of M. nodiflorum and M. crystallinum was better than the antibiotics ampicillin and penicillin as presented in Table 4, thus it could be suggested that ethanolic extracts of these species could be used as good alternative for antibiotics especially for antibiotic resistant bacteria (Table 4).

Activity Index (AI)
The significance of using the prepared extracts in comparison with the standard antibiotics (ampicillin, penicillin, gentamicin and chloramphenicol) against the tested pathogenic bacterial strains was estimated using the activity index ( Table 5). The activity index varied in gram positive from 0.09 to 3.61 and in gram-negative bacteria from 0.2 -2.20. The maximum activity index values were observed against Pseudomonas aeruginosa and Escherichia coli (3.61 and 2.17, respectively) while the lowest activity index value was for Escherichia coli (0.09). Ampicillin and penicillin expressed moderate and low effects against the tested pathogenic strains. Activity index values above one expressed higher potential of herbal extracts while those below one expressed higher antibiotics potential against tested pathogenic strain (Shekhawat & Vijayvergia, 2010). The obtained results affirmed the strength of using the studied extracts in comparison with the tested antibiotics except chloramphenicol.
M. nodiflorum showed higher potency than ampicillin and penicillin G against K. pneumoniae, E. coli, P. aeruginosa, S. aureus and B. subtilis. It was also more potent than gentamicin and chloramphenicol against P. aeruginosa and B. subtilis. On the other hand, M. monocytogenes showed higher potency against L. monocytoyenes, P. aeruginosa, S. aureus and B. subtilis than ampicillin and penicillin G. These results indicated the probability of using such these potent extracts against antibiotic resistance bacteria like P. aeruginosa, S. aureus and K. pneumoniae.

Allelopathic Activity
The ethanolic extracts of Mesembryanthemum species significantly exhibited allelopathic effect against C. murale weed in a concentration dependent manner (Figure 3). M. nodiflorum showed most phytotoxic effect among the three Mesembryanthemum samples. At the highest concentration (40 g mL -1 ), the ethanolic extracts of M. nodiflorum, M. crystallinum and M. forsskaolii inhibited the germination of  From these results, the allelopathic effect of Mesembryanthemum species could be attributed to the bioactive components that act in a synergistic manner or to compounds which regulate one another such as flavonoids, phenolics, tannins and alkaloids. The allelopathic activity of the coastal (M. nodiflorum and M. crystallinum) may be due to the high content of carbohydrates, protein, antioxidants enzymes, tannins, triterpenes, alkaloids and flavonoids (van der Watt & Pretorius, 2001;Bouftira et al., 2008;Doudach et al., 2013). Phenolic compounds are a class of the most important and common plant allelochemicals in the agro-ecosystem that could diffuse into the rhizosphere soil and inhibit germination and growth of the plants (Inderjit, 1996;Li et al., 2010).
In reality, weeds (Chenopodium murale, C. album, Phalaris minor, Amaranthus hybridus, etc.) are more than just a nuisance; weeds have severe economic impacts and threaten the global food and natural ecosystem. Weeds compete with crops for moisture, nutrients, sunlight, and space (Capinera, 2014). Chenopodium murale is recorded as nuisance weed that compete with various crops such as barley (Al-Johani et al., 2012), wheat (Majeed et al., 2012), and chickpea (Batish et al., 2007). In this context, the present results revealed that the ethanol extract from costal samples showed higher phytotoxic activity against tested weed C. murale. Therefore, ethanol extract still considered as new promising resource and eco-friendly bioherbicide against harmful weeds like C. murale.

CONCLUSION
In conclusion, the studied Mesembryanthemum species could be used as sustainable sources of antioxidants and antimicrobials that could be added to foods for preservation, to fodders for raising the immunity and for protection of feeded animals against diseases. These results indicated the probability of using such these potent extracts against antibiotic resistance bacteria like P. aeruginosa, S. aureus and K. pneumoniae. They could be used as source of phytotoxic extracts that possess a significant allelopathic activity against C. murale weed that impact threats on valuable economic crops. El-Amier, et al.