Phytochemical and antibacterial properties of root extracts from Portulaca oleracea Linn . ( Purslane ) utilised in the management of diseases in Nigeria

The use of plants as medicine predates written human history. It is estimated that about 80% of people living in underdeveloped and developing countries rely on plant as a source of primary healthcare (Ajala, Olusola & Odeku 2020; Ojah, Moronkola & Osamudiamen 2020; Ojah & Kachi 2020; Rafiu, Sonibare & Adesanya 2019). Approximately half of medicines in the world are derived from natural products and more than a quarter of the prescriptions dispensed annually in the United States were initially derived from plants. It was also reported that 80% of the world’s population directly or indirectly utilise herbal medicine for the treatment or prevention of diseases (Newman, Cragg & Snader 2000). A wide variety of phyto-constituents perform essential biological, pharmacological, and physiological functions. Research has shown that at least 12 000 Background: Bacteria as etiological agents have been reported to cause many diseases and have increased the rate of mortality globally. Their resistance to conventional medicine has made medicinal plants a credible alternative in the management of diseases caused by bacterial infection. In the recent times many research efforts have been directed towards the exploration of phytoconstituents with antibacterial potentials. Medicinal plants are widely used as antibacterial agents because of their high therapeutic performance, low toxicity, and affordability.


Introduction
The use of plants as medicine predates written human history. It is estimated that about 80% of people living in underdeveloped and developing countries rely on plant as a source of primary healthcare (Ajala, Olusola & Odeku 2020;Ojah, Moronkola & Osamudiamen 2020;Ojah & Kachi 2020;Rafiu, Sonibare & Adesanya 2019). Approximately half of medicines in the world are derived from natural products and more than a quarter of the prescriptions dispensed annually in the United States were initially derived from plants. It was also reported that 80% of the world's population directly or indirectly utilise herbal medicine for the treatment or prevention of diseases (Newman, Cragg & Snader 2000). A wide variety of phyto-constituents perform essential biological, pharmacological, and physiological functions. Research has shown that at least 12 000 Background: Bacteria as etiological agents have been reported to cause many diseases and have increased the rate of mortality globally. Their resistance to conventional medicine has made medicinal plants a credible alternative in the management of diseases caused by bacterial infection. In the recent times many research efforts have been directed towards the exploration of phytoconstituents with antibacterial potentials. Medicinal plants are widely used as antibacterial agents because of their high therapeutic performance, low toxicity, and affordability. http://www.jomped.org Open Access bioactive compounds have been isolated in recent times (Dosumu et al. 2019;Motaleb 2010). Phytochemicals mediate their effect on the human body through processes similar to those understood in conventional drugs, thus plant medicines are not only as effective as orthodox medicines but also pose side effects. Plants parts such as roots, leaves, stem bark and seeds possess some active components that are of therapeutic value and hence useful in the treatment of diseases such as cancer, coronary heart disease, diabetes and infectious disease. Many of the herbal medicines that proved to be effective have been incorporated into modern medicine (Motaleb 2010).
Medicinal plants provide a wealth of antimicrobial agents, which can be used as an alternate source of antibiotics Walter et al. 2011;Prasannabalaji et al. 2012). Secondary metabolites in plants act as antibacterial agent that is utilised as therapy or prophylactics against several infections caused by bacteria (Nasrullah et al. 2012).
In the last few decades, most pathogenic bacteria developed resistance to many antibiotics and this is a major threat to human health. Medicinal plants are sources of diverse molecules, many of which display antimicrobial properties, which protect human body from pathogenic infections. Thus, it is important to characterise different medicinal plants for their antibacterial potential (Bajpai et al. 2005;Wojdylo, Oszmianski & Czemerys 2007). A large number of antibacterial agents derived from traditional medicinal plants are available for treating various diseases caused by microorganisms (Jain 1994). Plants generally, produce phytochemicals that have antibacterial activity. In the last few years, many bacterial organisms have continued to show increasing multidrug resistance to several antibacterial agents (Njenga & Mugo 2020). Although hundreds of plant species have been tested for antibacterial properties, the vast majority have not been adequately evaluated (Balandrin et al. 1985;Muthusamy et al. 2013).
Portulaca oleracea L. commonly known as purslane is a warm-climate herbaceous succulent annual plant with a cosmopolitan distribution belonging to Portulacaceae family. It is commonly known as purslane (United States and Australia), rigla (Egypt), pigweed (England), pourpier (France) and Ma-Chi-Xian (China) (Elkhayat, Ibrahim & Aziz 2008). It is distributed widely in the tropical and subtropical areas of the world, including many parts of the United States and is eaten extensively as a potherb and is added to soups and salads around the Mediterranean and tropical Asian countries (Palaniswamy, Bible & McAvoy 2002). This plant might have originated in Asia and is now ubiquitous in Africa and the Mediterranean region (Masoodi et al. 2011).
Portulaca oleracea also provides a source of nutritional benefits owing to its rich omega-3 fatty acids and antioxidant properties (Palaniswamy, McAvoy & Bible 2001). The plant contains many biologically active compounds, which are responsible for the wide application of the plant in medicine. The plant has been reported as a rich source of phytoconstituents, such as oxalic acids, alkaloids, omega-3 fatty acids, coumarins, flavonoids, cardiac glycosides and anthraquinone glycosides (Ezekwe et al. 1999). Crude extracts of P. oleracea have been found to possess potent wound-healing properties (Rashed, Afifi & Disi 2003). The plant possesses culinary property utilised in the preparation of salads, soups and pickles. It has been used in folk medicine in many countries as febrifuge, antiseptic and vermifuge (Lee et al. 2012.). It exhibits a wide range of pharmacological effects such as antiulcerogenic (Karimi, Hosseinzadeh & Ettehad 2004), anti-inflammatory (Chan, Islam & Kamil 2000), antioxidant (Rashed et al. 2003), and wound-healing (Xu, Yu & Chen 2006) properties. It is listed by the World Health Organization as one of the most used medicinal plants, and it has been given the term 'Global Panacea' (Chen, Wang & Wang 2009). The Chinese folklore described it as 'vegetable for long life' and it has been used for thousands of years in traditional Chinese medicine (Jin et al. 2013;Li, Wu & Chen 2013). It is cold in nature and sour in taste and is used to cool the blood, stanch bleeding, clear heat and resolve toxins. The dried aerial part of this plant is used for the treatment of fever, dysentery, diarrhoea, carbuncle, eczema, and hematochezia (Li et al. 2013;Zhao et al. 2014).
Although orthodox medicine has been accepted by some populations of the world, yet greater percentage still rely on natural remedies to diseases caused by bacteria. Hence, this study was designed to evaluate the antibacterial activity of phytoconstituents present in root extracts of Nigerian P. oleracea. L.

Plant material
The roots of P. oleracea L. were collected from the Forest Research Institute of Nigeria (FRIN), Ibadan, Nigeria and authenticated in the Forest Research Herbarium, Ibadan, Nigeria where voucher samples were deposited with specimen voucher number FIH-112030.

Plant preparation and extraction
The roots of the plant were air-dried at a temperature below 40 °C and pulverised using a laboratory milling machine into fine powder after which a total of 500 g each of the ground powder were extracted successively in n-hexane, ethyl acetate, chloroform, and methanol by maceration using 5 L each of respective solvents (volume per volume [v/v]). The extract was concentrated using a rotary evaporator (Buchi model R210, Switzerland) and dried in a vacuum desiccator. The dried extract was reduced to powder using a laboratory mill and then sieved with a 250-μm mesh sieve.

Phytochemical screening
Phytochemical examinations were carried out for all the extracts using standard qualitative tests.

Liebermann-Burchard test
About 0.2 g of extract was dissolved in chloroform and few drops of acetic anhydride and concentrated sulphuric acid were added to the chloroform solution. Violet blue and finally green colour was formed indicating the presence of steroids (Harborne 1998;Talukdar et al. 2010).

Salkowski test
About 0.2 g of extract was dissolved in chloroform and a few drops of concentrated sulphuric acid were added to the solution. A reddish colour in the upper chloroform layer was observed indicating the presence of steroids (Kumar et al. 2007).

Test for alkaloids
Dragendroff's test

Mayer's test
To a few milliliters of filtrate, a few drops of Mayer's reagent were added by the side of the tube. A creamy white precipitate confirms the presence of alkaloids (Narasimhan et al. 2012).

Shinoda test
To 3 mL of 5 mg of methanolic extract, a piece of magnesium ribbon was added and 1 mL of concentrated hydrochloric acid. Pink-red or red colouration of the solution indicates the presence of flavonoids (Ajala et al. 2020).

NaOH test
About 0.5 g of extract was treated with 10% NaOH solution; formation of intense yellow colour indicates the presence of flavonoid (Sawant & Godghate et al. 2013).

Test for phenolic compounds
The extract (500 mg) was dissolved in 5 mL of distilled water. To this, a few drops of neutral 5% ferric chloride solution were added. A dark green colour indicated the presence of phenolic compounds (Mir, Sawheny & Jassal 2013).

Kellar-Kiliani test
A total of 2 mL of filtrate was added to 1 mL of glacial acetic acid, 1 mL ferric chloride and 1 mL concentrated sulphuric acid. Green-blue colouration of solution confirms the presence of glycosides (Chhetri et al. 2008;Parekh & Chanda 2007).

Test for tannins
To 0.5 mL of extract solution 1 mL of water and 1-2 drops of ferric chloride solution were added. Blue colour was observed for gallic tannins and green-black for catecholic tannins (Talukdar et al. 2010).

Frothing test or foam test
A total of 0.5 mL of filtrate was added to 5 mL of distilled water and shaken properly. Persistence of frothing on the solution confirmed the presence of saponins (Victor & Chidi 2009).

Molisch's test
Few drops of Molisch's reagent were added to each of the portion dissolved in distilled water, this was then followed by addition of 1 mL of concentrated H 2 SO 4 by the side of the test tube. The mixture was then allowed to stand for 2 min and then diluted with 5 mL of distilled water. Formation of a red or dull violet colour at the interphase of the two layers was a positive test (Sofowora 1993).

Fehling's test
About 0.5 g each of the extract was dissolved in distilled water and filtered. The filtrate was heated with 5 mL of equal volumes of Fehling's solution A and B. Formation of a red precipitate of cuprous oxide was an indication of the presence of reducing sugars (Sofowora 1993).

Test organisms
The antibacterial activities of the extract were determined using the agar well diffusion method of Balouiri, Sadiki and Ibnsouda (2016

Antibacterial screening procedure
The antibacterial activities of the extracts were tested against the selected strains using agar well diffusion method as described by . An amount of 20 mL of sterilised nutrient agar medium was poured into each sterile Petri dish and allowed to solidify. The test bacteria cultures were standardised to 0.5% McFarland standard (NCCLS 1993) and evenly spread over the appropriate media with the aid of a swab stick.
Then wells of 6 mm were made in the medium using a sterile cork borer (Bhargav et al. 2016). Concentrations of sample solutions were prepared followed by appropriate dilutions to the required concentration (10 mg/mL). These concentrations (at 0.1 mL) were transferred into separate wells, followed by the incubation of the plates at 35 °C for 24 h. After the incubation period, the zones of growth inhibition (ZI) were observed and measured using transparent ruler . Each test was repeated three times to ensure reproducibility. The mean of the triplicate tests ± their standard error of mean (

Statistical analysis
The experiments were conducted three times and all determinations were performed in triplicates (n = 3) and results were expressed as mean ± s.d. Statistical analysis was performed by one-way analysis of variance (ANOVA) with GraphPad Prism statistical software package, version 8. Duncan's new multiple range test were applied to the result at 0.05 level of significance (p < 0.05).

Ethical consideration
This article followed all ethical standards for research without direct contact with human or animal subjects.

Results and discussion
The present study identified secondary metabolites present in root hexane, ethyl acetate, chloroform and methanol extracts of P. oleracea using standard methods (Trease & Evans 2002). Phytochemical screening on root extracts showed the presence of carbohydrates, steroids, triterpenes, cardiac glycosides and saponins (Table 1). The presence of these useful phytochemicals could be responsible for the observed antibacterial activities and can be seen as a potential source of antibiotic drugs. In general, the accumulation and concentration of secondary metabolites are responsible for the antibacterial activity of a plant (Tim-Cushnie & Andrew 2005). Flavonoids possess antibacterial, antifungal and antiviral activity (Cowan 1999). Tannins are known for their astringent property and antimicrobial activity. Alkaloids are good antibacterial drugs whilst saponins possess antibacterial and anticandidal activity as reported in literature (Maatalah et al. 2012;Ramanathan et al. 2013;Tim-Cushnie, Benjamart & Andrew 2014).
Antibacterial screening on root hexane, ethyl acetate, chloroform and methanol extracts of P. oleracea exhibited good activity against the tested organisms from the zones of inhibition obtained ( Table 2). The inhibitory effect was compared with standard antibiotic drugs ciprofloxacin at 10 mg/mL. The significant activity of methanol extract was maximum against E. cloacae (24 ± 0.3 mm) followed by B. subtilis (23 ± 0.6 mm). Staphylococcus aureus, E. coli had 22 ± 0.4 mm as zone of inhibition. Streptococcus agalactiae had zone of inhibition of 21 ± 0.4 mm when exposed to the extracts. Salmonella typhi and S. dysenteriae had the least zones of inhibition (20 ± 0.7 mm). Enterobacter cloacae had the highest zone of inhibition in the ethyl acetate extract (31 ± 0.4 mm) whilst the least activity was observed in S. dysenteriae (22 ± 0.4 mm). Enterobacter cloacae had the highest zone of inhibition in the chloroform extract (31 ± 0.4 mm) whilst the least activity was observed in S. dysenteriae (22 ± 0.4 mm). Streptococcus agalactiae had the highest inhibition in the hexane extract (28 ± 0.5 mm) whilst S. dysenteriae had the least activity (23 ± 0.2 mm).  The MIC and MBC of extracts were determined in mg/mL as presented in Tables 3 and 4,

Conclusion
The root of P. oleracea L. was collected to investigate its phytoconstituents and antibacterial potentials, with the goal of establishing the presence of bioactive constituents responsible for the medicinal applications of the plant.
The study revealed antibacterial phytochemicals present in root extracts of P. oleracea L., which support its vast utilisation in ethno-medicine. Our study suggests that P. oleracea L. could be a potential source for antibacterial drug discovery.