The antimicrobial activity of ethanolic extracts of Ziziphus spina- christi leafs were examined by using agar well diffusion method against five bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli and Staphylococcus epidermides) in addition to Candida albicans. Results of this present study indicated that the ethanol extract of Ziziphus spina- christi leafs was effective against tested gram positive and negative bacteria whereas the C. albicans was showed resistance to this extract. Results showed that the gram positive bacteria more sensitivity to ethanolic extract of Ziziphus spina- christi leafs at all concentration used (from 5% to 30%) than gram negative bacteria. The highest inhibition zone was by a high concentration of Ziziphus spina-christi ethanolic extract 30% against S. aureus with inhibition zone equal (19mm) while, the lower inhibition zone given by second concentration 20% against P. aeruginosa with inhibition zone equal to (9mm). The results of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity of ethanolic extract possess high antioxidant activity at different concentrations with IC₅₀ value equal to 176µg ml compared to the IC₅₀ value of control sample (L-Ascorbic acid) equal to 145.88µg/ml.

Keywords: antimicrobial activity, antioxidant activity, Ziziphus spina-christi, ethanolic extract, pathogenic microorganisms

Medicinal plant is defined as any plant which in one or more of its parts contains substance that can be used for therapeutic purpose or as precursors for the synthesis of useful drugs.¹ The medicinal plants have been used for traditional health remedies and considered the most popular for 80% of world population in Asia, Latin America and Africa and is reported to have minimal side effects.² In recent years, there is most interest in drugs derived from plants. This interest primarily stems from the belief that green medicine is safe and dependable, compared with costly commercial synthetic drugs that have adverse effects.^3,4

The family of Rhamnaceae included many genuses that have medicinal importance. One of these genuses is Ziziphus that comprises about 100 species of deciduous or evergreen trees and shrubs. distributed in the tropical and subtropical regions of the world. All parts of the plant Ziziphus has medicinal importance and used by the local Arab people to help maintain a healthy life style.⁵ Various species of Ziziphus are used frequently in traditional medicine in the middle-east, Africa and some Asian countries for acquiring good health and treating of many ailments; including headache, fever common cold, asthma, pulmonary ailment, malaria, wounds, burns, stomach discomfort and urinary infections.⁶

Ziziphus spina-hristi has been used in folk medicine as a depurative, demulcent, anodyne, stomach-ache, toothaches, emollient, astringents, antibacterial, antifungal and as a mouth wash.^7,8 Plant products are rich sources with different biological compounds, mainly phenolics, various flavonoids, saponins, tannins, betulinic, ceanothic acids, erols, and triterpenes. These phytochemicals have been found to possess different biological properties like antioxidant and antimicrobial effect.^9,10 The leafs are used to sores remedy, and the roots are used to cure and prevent skin diseases.⁵ The seeds are sedative and are taken sometimes with buttermilk to halt nausea, vomiting and abdominal pains associated with pregnancy.¹¹ Ziziphus spina-Christi (L.) has been reported to have activity against bacterial and fungal pathogens that are normally quite resistant to modern medications.¹²

The main objective of this study to investigate potential uses of Ziziphus spina- christi as antimicrobial and antioxidant against some foodborne pathogens and using ethanolic extract of Ziziphus spina- christi as alternative food preservative agent.

Preparation of sample

Sample collection

The useful plant leafs were collected from Yemen - Taize government - Bani Alhusam and identified according to guideline of the.^13,14 The collected plant material was dried in an open air protected from direct exposure to sun light. All dried plant sample was finely ground by using an electrical grinder to a fine powder and made ready for extraction.

Preparation of extract

The sample of Ziziphus spina- christi leafs was extracted by using 1:10 in ethanol 80% at room temperature for 48 hours with occasional shaking. Then filtered by using filter paper (Whatman No 1.5, whatman Ltd., England). The extract was evaporated to dryness in oven at 45ºC to yield dry crude extract. The obtained crude extract was filled to amber tight closed bottle glass in refrigerator until uses.

Antimicrobial assay

Microorganisms used

The microbial strains were get from faculty of science, Sana’a university, republic of Yemen in April-2018. These microorganisms include:

Pseudomonas aeruginosa MTCC2453 Mar.2001, Escherichia coli MTCC739 Mar.2001, Staphylococcus aureus (ATCC653-8), Staphylococcus epidermides (local isolate), Bacillus cereus (ATCC6633) and Candida albicans (ATCC2019).

The bacterial strains were maintained on tryptone soya agar (TSA) and the fungal strains were maintained on Sabouraud dextrose agar (SDA).

Antimicrobial and Antibiotic assays

The Antimicrobial and Antibiotic assay was performed by using Agar Well Diffusion Method according to.^15,16

Antimicrobial assay

Four different concentrations of each extract of selected plants {300mg/ml (30%), 200mg/ml (20%), 100mg/ml (10%) and 50mg/ml (5%)} were dissolved in 10% {dimethylsulfoxide (DMSO) in Phosphate buffered saline} to be used in antimicrobial assay test. Extract solutions were prepared just before carrying out the test. Antimicrobial activity of the extracts was determined by agar well diffusion method as described by^15,16 on Tryptone soya Agar (TSA).

In each of these Plates four wells were cut out by using a standard cork borer (8 mm). About 60μl of each extract was added into different wells (triplicate each concentration). DMSO was used as a negative control. A positive control antibiotic disc was placed in the other plate. All the plates were incubated for 24h at 37°C. After incubation bioactivity was evaluated by measuring the zone of inhibition. All the experiment was performed in triplicates.

Antibiotic assays

The Antibiotic assay was performed by using antibiotic standard disc diffusion. The antibiotic standard disc was get from HiMedia and oxoid company which are Aztreonam (AT₃₀), Ceftazidin (CAZ₃₀), Co-Trimoxazole (COT₂₅), Gentamycin (Gen₁₀), Nystatin (100IU), Norfloxacin (NOR₅) and Vancomycin (VA₃₀)

Determination of DPPH radical scavenging activity:

The antioxidant activity of the ethanolic extract, on the basis of the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, with some modification according to.¹⁷ The antioxidant activity of the plant extract was estimated for a typical reaction, 1 ml of 0.2mM DPPH solution in methanol was mixed with 1 ml of (1, 0.5, 0.25 mg/ml ) of plant extract . The reaction mixture was incubated in the dark for 15 min. L-ascorbic acid was used as standard antioxidants and methanol was used as the control. An aliquot of 2 ml of a methanolic solution of DPPH (0.2mM DPPH) was added into the different concentration (1, 0.5, 0.25 mg/ml) of each extract and ascorbic acid as long as control sample (both extract and ascorbic acid were dissolved in methanol). All samples were incubated in the dark at room temperature for 15 min before absorbance values were read at 517 nm (PerkinElmer UV. visible spectrophotometer).The decrease in absorbance was calculated as an IC₅₀ and expressed as µg/ml, which is the concentration of sample required for 50% scavenging of DPPH radicals in the specified time period. The radical scavenging effect was calculated as follows:¹⁸

Radical scavenging effect (%) = Ac – As/Ac × 100

Ac = absorbance of control

As = absorbance of test sample.

Where control is the absorbance of the DPPH radical + methanol.

Antimicrobial activity of ethanol extract

The results showed in Table 1 explain the efficacy of the difference in the concentration as well as the type of tested microorganisms on the antimicrobial efficacy of the ethanol extract of the Ziziphus spina-christi which is directly proportional to the increase in concentration, that ranged between 5% and 30% where it was found that the ethanolic extract have antimicrobial activity against all tested microorganisms except C. albicans, which was resistance to this extract. Results also referred that the gram positive bacteria more sensitive than gram negative bacteria where the higher diameter of inhibition zone was 19 mm against S. aureus at the highest concentration 30%. While the lowest value of inhibition zone diameter was 9 millimeters against P. aeruginosa at the second concentration 20%.The results of sensitive and resistant of antibiotics (standards) was high with COT₂₅ (35mm) with gram-negative bacteria E. coli whereas the results showed resistance of P. aeruginosa to antibiotic (COT₂₅ and VA₃₀) respectively (Figure 1).

Figure 1 Inhibition zone diameters of Ziziphus spina- christi ethanolic extract against tested microorganisms.

DPPH radical scavenging activity

Results in table 2 revealed that the high antioxidants activity of Ziziphus spina- christi ethanol extract equal 92.32% at higher concentration (1 mg/ml) in comparison with those for sample control (L-ascorbic acid) equal 94.09% and with inhibitory concentration 50% (IC₅₀) value equal 176ug/ml compared to the IC₅₀ value of control sample (L-Ascorbic acid) that equal 145.88. The free radical scavenging efficacy was increasingly with the increased in concentration (Figure 2).

Figure 2 Correlation between concentration of Ziziphus spina-christi ethanolic extract and antioxidant activity.

Antimicrobial Activity of ethanol extract

In ethanolic extract the highest antimicrobial activity was at 30% and 20% against all tested microorganisms except C. albicans that displayed resistance to all tested concentrations. The lower concentrations (10 and 5%) revealed antimicrobial effect only against gram positive bacteria (S. aureus, S. epidermides and B. cereus) While, gram-negative bacteria (P. aeruginosa and E. coli) resistant to the lower concentrations.

This Findings were in agreement with previous studies conducted by^8,19-24 who reported that there was antimicrobial activity against S.aureus. Results conducted by² that was found the alcohol extract of Ziziphus spina- christi leafs is high antimicrobial effect against bacterium B. cereus. As for the activity ethanol extract of the Ziziphus spina- christi plant against E. coli bacterium the results were confirmed with the previous studies^2,8,21,25,26 who mentioned that the ethanol extract of the Ziziphus spina- christi leafs have inhibitory efficacy against E. coli bacterium. Results conducted by^24,27 display the antimicrobial activity against P. aeruginosa this results agreed with the present study. On the regarding to effect of ethanol extract against E. coli the results disagree with study of.²⁴ On the other hand the results disagree with results of^20,2 which showed a significant effect of the ethanol extract against the C. albicans. Study by² concluded that the antimicrobial activity of Ziziphus spina-christi leafs extract on microorganisms increase with the increased in concentration. The activity of some extracts may be returned to presence of some effected compounds such as phytokumanins (a type of phenolic compound that has the ability to break down molecular biofilms). This efficacy was related to the high level of not only by the monoterpene hydrocarbons²⁸ but also by the tannins which bind cell walls of ruminal bacteria.²² The high activity of the alcoholic extract is due to the low value of acidity (pH) compared to some other extracts.²⁹ The same studies indicated to that Ziziphus spina-christi leafs contain several organic acids such as malic, citric, tannic, acetic and other acids that lead to a decrease in the acid function (pH) which increases the antimicrobial efficacy of the extract.

Antioxidant activity

The results of this study was appeared high antioxidant activity to ethanolic extract of Ziziphus spina- christi leafs comparing to results of standard (Ascorbic acid). The results was in agreement with the results of previous study by²³ about quantification of total phenolic, flavonoids and tannins in ethanol extract of other genus of Ziziphus (Ziziphus lotus and Ziziphus jujube) from many regions in Tunisia and their antimicrobial and antioxidants activity. The antioxidant activity of Ziziphus spina-christi attribute to its high quantity of phytochemical compounds (phenols, flavonoids, cardiac glycosides, polyphenols, saponins and tannins).^23,30 The phytochemicals activity was correlated with their chemical structures and degrees of polymerization.³¹ Sterols like ß-sitosterol, terpenoid, phytosterols, triterpenoids, alkaloids, saponins, flavonoids, glycosides and tannins have been reported to have antioxidant activity.^32,33 This antioxidant activity attribute to structure of phytochemicals content where flavonoids contain conjugated ring structures and hydroxyl groups that have the probable to function as antioxidants in vitro or cell free systems by scavenging lipid peroxyradicals, superoxide anion, singlet oxygen and stabilizing free radicals involved in oxidative processes through hydrogenation or complexing with oxidizing species.^34,35 Flavonoids can be act by various ways to prevent injury caused by free radicals. Through in vitro experiments, it has been found that flavonoids possess anti-inflammatory, antiviral, antiallergic and anticarcinogenic properties.³⁶ The flavonoids prevent synthesis of prostaglandins that suppress T-cells by forming prominent inhibitors of cycloxygenase and lipoxygenase. The immune cells communicate with chemical signals called cytokines, which are controlled by flavonoids.³⁷ Flavonoids directly act as antioxidant by reducing free radicals to produce a more stable and less reactive radical (oxidize the flavonoids).^38-41

The high antioxidant and antimicrobial clear efficiency to ethanolic extract of Ziziphus spina Christi leafs against many of microbial strains that causes different important diseases to human and animal confirming the traditional herbal medicines usages and provide a scientific basis for traditional medicine uses of Ziziphus spina Christi leafs ethanolic extract in primary health care.

None.

Authors declare that there is no conflict of interest.

1. Sofowora A, Eyitope O, Adedeji O. The role and place of medicinal plants in the strategies for disease prevention. Afr J Tradit Complement Altern Med. 2013;10(5):210‒229.
2. Abd El-Hameed AI. Using Natural Polyphenol Extracts of Sider (Ziziphus spina- christi L.) Leaves and Seeds as Anti-Microbial. Middle East J Appl Sci. 2017;7(2):262‒271.
3. Stuffness M, Douros J. Current status of the NCI plant and animal product program. J Nat Prod. 1982;45(1):1‒14.
4. Farombi EO. African indigenous plants with chemotherapeutic potentials and biotechnological approach to the production of bioactive prophylactic agents. African J Biotech. 2003;2(12):662‒671.
5. Adzu B, Amos S, Wambebe C, et al. Antinociceptive activity of Ziziphus spina christi root bark extract. Fitoterapia. 2001;72(4):334‒350.
6. Adzu SB, Amos S, Amizan MB, et al. Evaluation of the antidiarrhoeal effects of Ziziphus spina- christi stem bark in rats. Acta Trop. 2003;87(2):245‒250.
7. Mohammed GT, Yesuf HB, Abdulrahman FI, et al. Antimicrobial and toxicological screening of the aqueous stem-bark extract of Ziziphus spina-christi (Linnaeus Desf). J Microbial Biotech Res. 2012;2(2):337‒342.
8. Al-Mutairi MH, Sarah A, Salah MA, et al. Antibacterial activity of Sider (Ziziphus spina- christi) leaves extract against selected pathogenic bacteria. EJPMR. 2016;3(5):138‒144.
9. Ali SA, Hamed MA. Effect of Ailanthus altissima and Ziziphus spina- christi on Bilharzial infestation in mice: histological and histopathological studies. J Appl Sci. 2006;6(7):1437‒1446.
10. Kaneria M, Kanari B, Chanda S. Assessment of effect of hydroalcoholic and decoction methods on extraction of antioxidants from selected Indian medicinal plants. Asian Pac J Trop Biomed. 2012;2(3):195‒202.
11. Kaaria I. Seed production, dispersal and germination in Cryptostegia grandifolia and Ziziphus mauritiana, two invasive shrubs in tropical woodlands of Northern Australia. Australia J Ecology. 1998;21:324‒331.
12. Nazif NM. Phytoconstituents of Ziziphus spina- christi L. fruits and their antimicrobial activity. Food chemistry. 2002;76(1):77‒81.
13. Ministry of tourism and environment. The general consortium for environment protection, wild plants from Yemen. Ministry of tourism and environment, Yemen; 2002.
14. Al-Dubaie AS, Al-Khulaidi AA. Medical and Aromatic Plant in Yemen. Aubade center Sana'a–Republic of Yemen; 2005.
15. USDA. Chapter new, revised, MLG 34.03. Method number 34.6.3; 2007. 12-14 p.
16. Mahesh, B and Satish, S. Antimicrobial activity of some important medicinal plants against plant and human pathogens. World J Agric Sci. 2008;4:839‒843.
17. Ruchi GM, Majekodunmi OF, Al Mahrooqi Ramla, et al. Antioxidant capacity of some edible and wound healing plants in Oman. Food Chemistry. 2007;101(2):465‒470.
18. Al-Naqeb G. Antioxidant and Antibacterial Activities of Some Yemeni Medicinal Plants. International Journal of Herbal Medicine. 2015;3(3):06‒11.
19. Seyyednejad S, Maleki M, Damab S, et al. Antibacterial Activity of Prunus mahaleb and Parsley (Petroselinum crispum) Against Some Pathogen. Asian J Biological Sci. 2008;1(1):51–55.
20. Neama JD, Abu- Mejdad NMJ, Jaber AM. Evaluation the antimicrobial activity of aqueous and alcoholic extracts of leaves Ziziphus spina-christi (L) Desf. Basra Journal of Science (B). 2007;25(1):1‒16.
21. Altaf MT. Studies on two kinds of Ziziphus plants and their effects on some microorganisms, master brief. King Abdulaziz University-Jedda; 2008.
22. Bukar AM, Kyari MZ, Gwaski M, et al. Evaluation of phytochemical and potential antibacterial activity of Ziziphus spina-christi L. against some medically important pathogenic bacteria obtained from University of Maiduguri Teaching Hospital, Maiduguri, Borno State-Nigeria. J Pharmacogn Phytochem. 2015;3(5):98‒101.
23. Elaloui M, Amel E, Hanene G, et al. Quantification of total phenols, flavonoides and tannins from Ziziphus jujuba (mill.) and Ziziphus lotus (l.) (Desf). Leaf extracts and their effects on antioxidant and antibacterial activities. Int J SecMetabolite. 2017;4(1):18‒26.
24. Temerk HA, Salem WM, Sayed WF, et al. Antibacterial Effect of Phytochemial Extracts from Ziziphus-spina christi against Some Pathogenic Bacteria. Egypt J Bot. 2017;57(3):595–604.
25. Doughari JH. Antimicrobial activity of Tamarindus indica Linn. Trop J Pharm Res. 2006;5(2):597‒603.
26. Motamedi H, Safary A, Maleki S, et al. Ziziphus spina- christi, A native plant from khozestan, Iran, as apotential sours for discovery new antimicrobial agents. Asian Journal of Plant Sciences. 2009;8(2):187‒190.
27. Motamedi H, Seyyed MS, Zahra H, et al. Comparative Study on the Effects of Ziziphus spina- christi Alcoholic Extracts on Growth and Structural Integrity of Bacterial Pathogens. Iranian Journal of Pharmaceutical Sciences. 2014;10(2):1‒10.
28. Amri I, Hamrouni L, Hanana M, et al. Chemical composition, physicochemical properties, antifungal and herbicidal activities of Pinus halepensis Miller essential oils. Biol Agric Hortic. 2013;29(2):1‒16.
29. Rajakarune N, Harris CS, Towers GHN. Antimicrobial activity of plants collected from Serpentine outcrops in Srilanka. Pharmaceutical Biology J. 2002;40(3):235‒244.
30. Abalaka ME, Mann A, Adeyemo SO. Studies on in-vitro antioxidant and free radical Scavenging potential and phytochemical screening of leaves of Ziziphus mauritiana L. and Ziziphus spina-christi L. compared with Ascorbic acid. Journal of Medical Genetics and Genomics. 2011;3(2):28‒34.
31. Lu Y, Foo LY. Antioxidant and radical scavenging activities of polyphenols apple pomace. Food Chemistry. 2000;68:81–85.
32. Dragland SM, Senoo H, Wake K, et al. Antioxidant activity of Terpenoids. J Nutr. 2003;133:1286‒1290.
33. Cai YZ, Luo Q, Sun M, et al. Antioxidant activity of Sterols. (B-sitosterol) Life Sciences. 2004;74:2157‒2184.
34. Duthie SJ, Dobson VL. Dietary flavonoids protect human colonocyte DNA from oxidative attack in vitro. Eur J Nutr. 1999;38(1):28–34.
35. Birt DF, Hendrich S, Wang W. Dietary agents in cancer prevention: Flavonoids and isoflavonoids. Pharmacol Therapeut. 2001;90:157–177.
36. Middleton EJ. Effect of plant flavonoids on immune and inflammatory cell function. Adv Exp Med Biol. 1998;439:175‒182.
37. Pal RS, Ariharasivakumar G, Girhepunjhe K, et al. In vitro antioxidative activity of phenolic and flavonoids compounds extracted from seeds of Abrus precatorius. Int J Pharm Pharm Sci. 2009;1(2):136‒140.
38. Korkina LG, Afanas’ev IB. Antioxidant and chelating properties of flavonoids. Adv Pharmacol. 1997;38:151‒163.
39. Hossein M, Seyyed MS, Zahra H, et al. Comparative Study on the Effects of Ziziphus spina- christi Alcoholic Extracts on Growth and Structural Integrity of Bacterial Pathogens. Iranian Journal of Pharmaceutical Sciences. 2014;10(2):1‒10.
40. Hussein HM, El-Sayed EM, Said AA. Antihyperglycemic, antihyperlipidemic and antioxidant effects of Ziziphus spina Christi and Ziziphus jujube in Alloxan diabetic rats. Int J Pharmacol. 2006;2(5):563‒570.
41. O’Shea R, Moser HE. Physicochemical properties of antibacterial compounds: implications for drug discovery. J Med Chem. 2007;51(10):2871‒2878.