Antimicrobial and Phytochemical Evaluation of Ziziphus mauritiana var. spontanea Edgew. and Oenothera biennis L.

Ambrin1*, Ghulam Dastagir1, Jehan Bakht2 and Muhammad Adil1

1Department of Botany, University of Peshawar, Peshawar, KPK, Pakistan

2Institute of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar, KPK, Pakistan

ABSTRACT

The present study investigates antimicrobial activities of different solvent extracted samples of Ziziphus mauritiana var. spontanea and Oenothera biennis against different microbes. Ethyl acetate and ethanolic extracts of Z. mauritiana var. spontanea measured the highest zone of inhibition against Staphylococcus epidermidis, Escherichia coli and Salmonella typhi. The MIC values of ethyl acetate and ethanolic extracts of Z. mauritiana var. spontanea were the lowest for Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumonia, Shigella flexneri and Staphylococcus aureus. Highest zone of inhibition against Klebsiella pneumonia and Pseudomonas aeruginosa was shown by ethyl acetate and ethanolic extracts of O. biennis. Lowest MIC value was noted for ethyl acetate and ethanolic extracts of O. biennis against Staphylococcus epidermidis, Staphylococcus aureus, Salmonella typhi and Shigella flexneri. The ethyl acetate and ethanolic extracts of Z. mauritiana var. spontanea showed good antifungal activity against Aspergillus niger, Penicillium notatum. Minimum MIC value was shown by ethyl acetate and ethanolic extracts of Z. mauritiana against Aspergillus fumigatus and Aspergillus niger. The ethyl acetate and ethanolic extract of O. biennis revealed maximum antifungal activity against Aspergillus niger, Aspergillus flavus and Aspergillus fumigatus. The MIC value of ethyl acetate and ethanolic extracts of O. biennis was lowest for Fusarium solani and Penicillium notatum. Phytochemical screening of Z. mauritiana var. spontanea and O. biennis revealed the presence of amino acids, reducing sugar, proteins, triterpenoids, gums and mucilages, fats, steroids, phenols and phytosterol.

Article Information

Received 28 April 2019

Revised 22 June 2019

Accepted 21 October 2019

Available online 15 May 2020

Authors’ Contribution

Ambrin conducted the experiments and collected the data. GD designed the study and provided lab facilities.JB wrote the manuscript. MD did statistical analysis.

Key words

Z. mauritiana, O. biennis, Antibacterial, Antifungal, Phytochemical screening

DOI: https://dx.doi.org/10.17582/journal.pjz/20190428160433

* Corresponding author: Ambreenkhan727@gmail.com

0030-9923/2020/0005-1809 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

INTRODUCTION

Plants used in traditional medicine contain a wide variety of compounds which can be used to treat chronic and infectious diseases. It is estimated that about 35,000 to 70,000 plants species are used as medicinal plants worldwide (Bibi et al., 2011). Approximately 80% of the population in Pakistan use traditional medicines for several ailments (Haq, 1997; Munir et al., 2013). The antimicrobial potential of plant extracts may be due to the presence of secondary metabolites (Ganesh et al., 2014). Medicinal plants can be used to cure different microbial infections (Bakht et al., 2018; Bilal et al., 2018; Ayaz et al., 2017, 2018; Wajid et al., 2017). Contagious diseases are common in most of the developing countries, including Pakistan, therefore, it is important to search out and promote plant-based medication as natural medicines cost effective with fewer side effects as compared to the synthetic medicines. Synthetic antimicrobial agents are expensive and beyond the reach of common people.

Ziziphus mauritiana var. spontanea Edgew. commonly known as Beri, Jand, Mada bera, Ashar in Pakistan belongs to the family Rhamnaceae (Hussain et al., 2011; Ahmad et al., 2011; Ahmad et al., 2012). Members of this family are found in tropical and subtropical regions of the world including India, Sri Lanka, Pakistan, Iran, Greece and Afghanistan. Generally, Ziziphus reported to have anxiolytic, sedative, haemolytic, antidiabetic, analgesic, antiplasmodial, diuretic, anticonvulsant, hypoglycemic and anti-inflammatory activities (Goyal et al., 2012). It occurs commonly in Pakistan, China, Australia, India, Afghanistan and Ceylon. Several species of Ziziphus have shown medicinal value such as Z. oxyphylla is used in Pakistan for Obesity, urinary troubles, digestive disorders, skin infections, weakness, diabetes, diarrhea, liver complaints, fever and insomnia (Kaleem et al., 2014). Z. nummularia can be used as cooling, laxative, astringent, stomachic, cures mucous and for treating scabies. Z. spina-christi leaves are effective as anti-inflammatory, antiseptic and anti-fungal and used to treat obesity, digestive disorders, urinary troubles and can be used as anti-microbial agent. Fruit of Z. mauritiana var. spontanea is used as blood purifier and digestive stimulant. Stem bark is used for dysentery and diarrhea when mixed with honey or milk (Amjad et al., 2017). Oenothera biennis L. commonly known as evening primrose, evening star, or sun drop belongs to the family Onagraceae. It is found in eastern and central North America. Its seeds contain high quantities of Gamma Linolenic Acid (up to 20-25% GLA) and can be used for curing several ailments (Anwar et al., 1998). Oenothera biennis possesses antiviral, antibacterial, anti-inflammatory, anti-thrombolytic, anti-hyperlipidaemic, antioxidant and cytotoxic activities (Singh et al., 2012).

 

MATERIALS AND METHODS

Collection and identification of plant materials

The fresh plants of Ziziphus mauritiana var. spontanea Edgew. and Oenothera biennis L. were collected from Palosai area of district Peshawar KPK Pakistan and identified at the Department of Botany, University of Peshawar, KPK Pakistan. The shade dried plant materials were ground to get powder. Fifty grams of powdered plant materials were soaked in 250 ml each of ethanol and ethyl acetate in separate flat-bottomed flasks. The mixture was filtered by Whatman filter paper No. 1823 and the same procedures were followed three times. The pooled filtrates were concentrated by rotary evaporator (Heidolph Collegiate, LV28798826, New Jersey, USA) and stored at 4°C until used.

Antimicrobial activity

The antimicrobial activity of ethanolic and ethyl acetate extracts of Ziziphus mauritiana var. spontanea Edgew. and Oenothera biennis L. was studied using well diffusion agar method as described by Atta-ur-Rehman et al. (1991) against Staphylococcus aureus, Staphylococcus epidermidis and five Gram negative bacterial strains namely Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeraginosa, Shigella flexneri, Salmonella typhi, Aspergillus niger, Aspergillus flavus, Fusarium solani, Aspergillus fumigatus and Penicillium notatum.

Phytochemical screening

Steroids and reducing sugars were determined according to the methods of Edeoga et al. (2005) and Evans (2002) respectively. Protein was estimated by Kumar and Kiladi (2009). The method of Harborne (1998) was used for the determination of tri-terpenoids and phytosterol. Phenolic compounds, gum and mucilage was estimated by the procedures of Dahiru et al. (2006) and Ansari (2006), respectively.

Statistical analysis

Data are presented as mean values of three replications with standard deviation. MSTATC computer software was used for to carry out statistical analysis (Russel and Eisensmith, 1983).

 

RESULTS

Antibacterial activity

The antibacterial activity of ethyl acetate extract of Ziziphus mauritiana var spontanea showed the highest zone of inhibition (13 mm) against Staphylococcus epidermidis followed by Staphylococcus aureus, Shigella flexneri (10 mm) and the lowest zone of inhibition (6 mm) was shown by E. coli. Similarly, the ethanolic extract of Ziziphus mauritiana showed the highest antibacterial potential (14mm) against Escherichia coli and Salmonella typhi followed by Staphylococcus epidermidis and Staphylococcus aureus (10mm) and the lowest zone of inhibition (6 mm) was shown against Klebsiella pneumoniaee (Table I). The MIC values of ethyl acetate extract of Ziziphus mauritiana var. spontanea were the lowest (2µg/ml) for Staphylococcus aureus, Staphylococcus epidermidis and Klebsiella pneumoniaee (Table II). Likewise, the MIC values of ethanolic extract were the lowest (2 µg/ml) for Shigella flexneri and Staphylococcus aureus (Table II).

The antibacterial activity of ethyl acetate extract of Oenothera biennis showed the highest zone of inhibition (17mm) against Klebsiella pneumoniaee followed by E. coli, Pseudomonas aeruginosa and the lowest zone of inhibition (6 mm) was shown by Salmonella typhi. Similarly, the antibacterial activity of ethanolic extract of Oenothera biennis revealed the highest zone of inhibition (15mm) against Pseudomonas aeruginosa followed by Escherichia coli and Salmonella typhi (12mm) and the lowest zone of inhibition (8 mm) was shown by Klebsiella pneumoniaee (Table I). The MIC values of ethyl acetate extract of Oenothera biennis were the lowest (2 µg/ml) for Staphylococcus epidermidis, Staphylococcus aureus and Salmonella typhi (Table II). The MIC values of ethanolic extract were lowest (2 µg/ml) for Staphylococcus epidermidis and Shigella flexneri (Table II).

Antifungal activity

The ethyl acetate extract of Ziziphus mauritiana var. spontanea showed good antifungal activity (16mm) against Aspergillus niger followed by Fusarium solani (12 mm) and lowest antifungal activity against Aspergillus flavus (8mm). Similarly, the ethanolic extract of Ziziphus mauritiana var. spontanea measured the highest antifungal activity (14 mm) against Penicillium notatum followed by Aspergillus fumigatus (12mm) and the lowest antifungal activity (6mm) against Fusarium solani at 40 µg/ml extract concentration (Table III). The MIC value of ethyl acetate extract of Z. mauritiana was lowest for Aspergillus fumigatus whereas the MIC value of ethanolic extract of Z. mauritiana was lowest for Aspergillus niger (Table IV).

 

Table IV. MIC values of ethyl acetate and ethanolic extract of Ziziphus mauritiana var. spontanea Edgew. and Oenothera biennis L.

Ziziphus mauritiana. var. spontanea Edgew.			Oenothera biennis L.
MIC values (µg/ml)
Fungi	Ethyl acetate extract	Ethanol extract	Ethyl acetate extract	Ethanol extract
Aspergillus flavus	7 ±0.56	8 ±0.52	7 ±0.49	6 ±0.15
Aspergillus fumigatus	3 ±0.37	6 ±0.58	6 ±0.30	5 ±0.15
Fusarium solani	5 ±0.76	6 ±0.76	2 ±0.23	6 ±0.30
Aspergillus niger	7 ±0.26	2 ±0.15	4 ±0.65	4 ±0.55
Penicillium notatum	5 ±0.26	5 ±0.45	8 ±0.35	2 ±0.60

 

Table V. Phytochemical analysis of Ziziphus mauritiana var. spontanea Edgew. and Oenothera biennis L.

	Ziziphus mauritiana var. Spontanea Edgew.		Oenothera biennis L.
Chemical constituents	Ethyl acetate extract	Ethanol extract	Ethyl acetate extract	Ethanol extract
Amino acids	+	+	+	+
Reducing sugar	+	+	+	-
Gum and mucilage	-	+	+	+
Phenols	+	+	-	+
Phytosterol	+	-	-	+
Proteins	+	+	+	+
fats	-	+	+	+
Triterpenoids	+	+	+	-
Steriods	+	-	+	+

 

The ethyl acetate extract of Oenothra biennis revealed maximum antifungal activity (15mm) against Aspergillus niger followed by Fusarium solani (12 mm) and minimum (2mm) against Penicillium notatum. Similarly, the ethanolic extract of Oenothera biennis showed highest antifungal activity (16 mm) against Aspergillus flavus, Aspergillus fumigatus followed by Penicillium notatum (12mm) and the lowest antifungal activity (8mm) against Fusarium solani at the maximum 40 µg/ml extract concentration (Table III). The MIC value of ethyl acetate extract of O. biennis was lowest for Fusarium solani whereas the MIC value of ethanolic extract of O. biennis was lowest for Penicillium notatum (Table IV).

Phytochemical analysis of different solvent extracted samples of Ziziphus mauritiana var. spontanea revealed the presence of amino acids, reducing sugar, phenols, phytosterol, proteins, steroids, terpenoids and gum and mucilages. Similarly, phytochemical screening of whole plant of Oenothera biennis indicated the presence of amino acids, fats, proteins, reducing sugar, steroids, phenols, phytosterol, gums and muclilages and terpenoids and (Table V).

 

DISCUSSION

The selection of the plants reported in this study was based on the medicinal uses reported by various workers in literature review. The antibacterial activity revealed that E. coli and Salmonella typhi were the most sensitive bacteria to ethanolic extract of Ziziphus mauritiana var. spontanea. These results agree with Okwulehie et al. (2013) who reported similar results for Ficus experata and Camellia sinensis. Staphylococcus epidermidis was the most sensitive to the ethyl acetate extract of Ziziphus mauritiana var. spontanea. These results are in line with Prabhakaran et al. (2014) and Mills-Robertson et al. (2012) who reported similar findings for Careya arborea and Cryptolepis sanguinolenta. The MIC values revealed that Staphylococcus aureus, Staphylococcus epidermidis and Klebsiella pneumoniae were the most susceptible bacteria to the ethyl acetate extract of Ziziphus mauritiana var. spontanea whereas Shigella flexneri and Staphylococcus aureus were the most susceptible bacteria to ethanolic extract of Ziziphus mauritiana var. spontanea. Pseudomonas aeruginosa was the most sensitive bacteria to ethanolic extract of Oenothera biennis. These results agree with Abalaka et al. (2017) and Awah et al. (2017) who reported similar antibacterial potential of ethanolic extract of Garcinia kola and Carica papaya. Klebsiella pneumoniae was the most sensitive bacteria to ethyl acetate extract of Oenothera biennis. Idris et al. (2016) reported similar antibacterial effect of the ethyl acetate extract of Peperomia pellucida and Bridelia ferruginea. The MIC values exhibited that Staphylococcus epidermidis, Staphylococcus aureus and Salmonella typhi were the most susceptible bacteria to the ethyl acetate extract of Oenothera biennis whereas Staphylococcus epidermidis and Shigella flexneri were the most susceptible bacteria to ethanolic extract of Oenothera biennis. These results are in line with Bajpai et al. (2009) and Leela and Satirapipathkul (2011) who reported that susceptibility of Gram positive bacteria as compared to the Gram negative bacteria due to a mesh-like peptidoglycan layer which is more accessible to permeation by the extracts (Biswas et al., 2013). Interaction of phenols, terpenoids and steroids with membrane proteins of bacteria can modify membrane permeability resulting in cell destruction. They can also penetrate into bacterial cells and coagulate cell content (Sengul et al., 2009; Shihabudeen et al., 2010).

The ethyl acetate extract of Ziziphus mauritiana var. spontanea revealed maximum antifungal activity against Aspergillus niger. These results agree with Rashid et al. (2017) who reported significant antifungal activity against Aspergillus niger by the ethyl acetate extract of Seidenfia rheedii and Hartmannia rosea. Similarly, the ethanolic extract of Ziziphus mauritiana var. spontanea measured the highest antifungal activity against Penicilium notatum. Kumar et al. (2010) reported highest inhibition of Penicilium notatum due to ethanolic extract of Murraya koenigii and Rhizophora mucronata. The MIC value showed that A. fumigatus was susceptible to the ethyl acetate extract of Z. mauritiana and A. niger was susceptible to the ethanolic extract of Z. mauritiana.

The ethyl acetate extract of Oenothera biennis indicated good antifungal activity against Aspergillus niger. These results are in line with Bharathi et al. (2014) who reported highest antifungal against Aspergillus niger by the ethyl acetate extract of Azadirachta indica. Similarly, the ethanolic extract of Oenothera biennis revealed highest antifungal activity against Aspergillus flavus, Aspergillus fumigatus. Abbas et al. (2017) reported highest antifungal activity against Aspergillus flavus and Aspergillus fumigatus by the extracts of Heterostemma tanjorense and Melia azedarach. The MIC value showed that F. solani was susceptible to the ethyl acetate extract of O. biennis and P. notatum was susceptible to the ethanolic extract of O. biennis).

Phytochemical analysis showed the occurrence of various groups of compounds in both plants. Both plants revealed the presence of amino acids, reducing sugar, phenols, phytosterol, proteins, steroids, terpenoids and gum and mucilages (Khamis and Aly, 2017; Tikadar et al., 2017). Manchu et al. (2015) reported that phenols, terpenoids, steroids and phytosterols possess antimicrobial properties. The secondary metabolites present in plants reveal biological activities including antimicrobial activities (Yadav et al., 2014). These results showed that both plants are rich sources of secondary metabolites that can be used for the synthesis of new drugs for curing various diseases.

 

CONCLUSION

These results revealed the antimicrobial potentiality of ethyl acetate and ethanolic extracts of Ziziphus mauritiana and Oenothera biennis. The ethanolic extract of Z. mauritiana caused greater inhibition of bacteria as compared to ethyl acetate extract. Whereas ethyl acetate extract of O. biennis was more potent against different strains of bacteria as compared to ethanolic extract. Similarly, the ethyl acetate extract of Z. mauritiana showed highest antifungal activity and in case of O. biennis the ethanolic extract showed greater antifungal activity. Phytochemical screening revealed the the presence of phenols, terpenoids, amino acids, gums and mucilages, phytosterols, reducing sugar, steroids, proteins in the extracts of both plants.

 

Statement of conflict of interest

The authors have declared no conflict of interest.

 

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