- Research Article
- Open Access
Determination of antioxidant and antimicrobial activities of the extracts of aerial parts of Portulaca quadrifida
© The Author(s) 2018
- Received: 18 October 2018
- Accepted: 6 December 2018
- Published: 20 December 2018
The extracts from the aerial parts of Portulaca quadrifida have been reported to show the total flavonoid content, antioxidant and antibacterial activities.
Our results revealed that the total flavonoid content of methanol and chloroform extracts is 2.335 ± 0.0097 and 1.7312 ± 0.0082 mgQE/100 g respectively. The two extracts also showed good antioxidant activity and total phenolic content as well as weak to moderate antibacterial activity against some bacteria.
- Portulaca quadrifida
- Total flavonoid content
- Antioxidant activity
- Total phenolic content and antibacterial activities
Portulaca quadrifida, commonly known as “chicken weed”, a herb belongs to the Portulacaceae family, is endemic to Ethiopia, in most dry area of Benshangul Gumuz, konso, Ethio-Somalia, Oromia, Kaffa and Hararghe regions. Portulaca quadrifida (known as “Kimma” in Amharic and Kawa in shinasha) is a prostrate, mat-forming annual or short-lived perennial herb with much branched, spreading, articulated, fleshy stems up to 50 cm long or longer, rooting freely from the nodes, often flushed reddish; nodes with a dense whorl of whitish hairs . In Ethiopian traditional medicine, the aerial parts of Portulaca quadrifida used as food and used for the treatment of several diseases including gastric ulcer, ophthalmia, as an anti-microbial, anti-hyperglycemic, and for its antioxidant properties used in preventing different kinds of sickness and disorders . Several studies suggested that Portulaca quadrifida is a good natural antioxidant that can be used as health promoting agent for various disorders including diabetes mellitus and other kinds of diseases . Herein we reported the total flavonoid content, antioxidant activity and antibacterial activities of the extracts of the aerial parts of Portulaca quadrifida.
Determinations of total phonolic content
Total phenolic content of P. quadrifida extracts
TPC in mgGAE/g dry extracts
4.9029 ± 0.0087
4.5957 ± 0.0055
Petroleum ether extract
2.4914 ± 0.0028
Determination of total flavonoid content
Total flavonoid content of the extracts of P. quadrifida
TFC in mgQE/g dray extracts
2.335 ± 0.0097
1.7312 ± 0.0082
Petroleum ether extract
1.357 ± 0.0035
Determination of antioxidant capacity
The antioxidant activity of the extracts of the aerial parts of P. quadrifida was evaluated by using DPPH radical scavenging assay as shown in Table 3. The scavenging effect of different extracts of P. quadrifida on the DPPH radical decreases in the order of methanol extract, chloroform extract and petroleum ether extract. The highest percentage inhibition for methanol extract compared to two other solvents could be due to high polarity nature of solvent and highly polar miscible compounds found in the plant. As the concentration of phenolic compound increases, degree of hydroxylation of the phenolic compound also increases which resulted to increase the scavenging activity (% inhibition). The increase in DPPH scavenging activity directly related to antioxidant capacity of the P. quadrifida extracts. As a result methanolic extract of P. quadrifida which has highest DPPH radical scavenging activity showed fairly highest antioxidant activity in comparison to other extracts. The presence of phenolic acid in the extract of tested plant increases the probability of reaction with free radical, which lead to decrease the amount of free radical. In this study antioxidant assay of DPPH radical scavenging capacity (%), total phenolic, and total flavonoid contents were used to evaluate the antioxidant activity of P. quadrifida extracts. A good correlation between DPPH radical scavenging activity with TPC (R2 = 0.782) was observed and furthermore, a reasonable correlation between DPPH and TFC (R2 = 0.996) was observed. Results obtained from the experimental data showed that there was good correlation between total flavonoid and antioxidant activity of different extracts of P. quadrifida with correlation coefficient (R2) 0.996.
DPPH radical scavenging activity of P. quadrifida extracts at different concentrations
Concentration of extract (mg /L)
%DPPH scavenging activity
25.3541 ± 0.0272
23.1373 ± 0.00804
22.8602 ± 0.0020
30.9421 ± 0.0038
29.4874 ± 0.0100
26.4547 ± 0.0188
36.8042 ± 0.0056
34.1595 ± 0.0054
30.8344 ± 0.0047
38.2620 ± 0.0003
37.3599 ± 0.0062
33.0511 ± 0.0039
46.7595 ± 0.0069
42.4030 ± 0.0053
44.5428 ± 0.0174
Determination of antimicrobial activity of P. quadrifida
Different extracts from the aerial parts of P. quadrifida demonstrated antibacterial activities against both Gram-positive and Gram-negative bacteria strains. In this study 250 mg/L and 200 mg/L of methanol extract of Portulaca quadrifida recorded the most significant antimicrobial activity against all tested bacteria but chloroform extract showed good activity against fungi. Though methanol extract showed broad spectrum of activity against all tested bacteria, especially S. aurues and E. coli are the most susceptible pathogens. In addition, the methanol extracts of Portulaca quadrifida also showed significant antibacterial activity against Gram negative bacteria (Escherichia coli and Klebsiella pneumoniae) and Gram positive bacteria (Staphylococcus pyogenes and Staphylococcus aureus), respectively. However, petroleum ether extract showed least activity against all bacterial organisms. The significant and higher antibacterial activity might be due to the presence of flavonoids in the plant as described by Mutyala and Kishore . 0.3% w/v of Gentamycine drug reference and DMSO were used as a positive and negative control respectively. Gentamycine showed 27, 17, 29 and 19 mm zone of inhibition on E. coli, K. pneumoniae, S. aureus and S. pyogenes respectively while DMSO had no sensitivity on those four bacterial species.
Determination of anti-fungal activity of the extracts of P. quadrifida
Antibacterial sensitivity of Portulaca quadrifida extracts
Concentration in (mg/L)
Mean zone of inhibition ± SD (mm)
7.1 ± 1.6
8.0 ± 2.0
11.88 ± 1.0
5.3 ± 1.3
4.0 ± 0.7
9.7 ± 0.8
1.2 ± 0.8
1.8 ± 2.6
6.4 ± 1.3
3.8 ± 1.7
5.3 ± 1.1
5.78 ± 0.5
10.3 ± 1.3
2.5 ± 0.6
3.1 ± 2.1
5.2 ± 0.6
1.5 ± 1.4
10.3 ± 2.2
10.7 ± 3.0
17.3 ± 2.0
6.2 ± 2.1
6.8 ± 2.5
10.2 ± 1.75
1.2 ± 1.0
1.5 ± 0.7
5.3 ± 0.6
1.2 ± 0.5
2.35 ± 0.5
8.7 ± 1.7
9.3 ± 2.1
10.8 ± 2.5
5.0 ± 1.3
6.8 ± 1.2
7.3 ± 1.8
2.9 ± 1.8
1.4 ± 2.3
3.0 ± 2.5
Inhibition zone of P. quadrifida extracts against standard fungus
Concentration in (mg/L)
Mean zone of inhibition ± SD (mm)
8.4 ± 0.6
11.8 ± 1.6
10.2 ± 2.1
4.3 ± 0.7
6.0 ± 2.2
7.2 ± 2.0
2.4 ± 2.5
3.0 ± 1.3
1.2 ± 1.0
12.5 ± 2.1
15.2 ± 1.5
10.5 ± 1.6
8.3 ± 1.3
7.7 ± 1.7
9.7 ± 1.5
3.3 ± 0.8
3.0 ± 0.7
4.3 ± 2.1
1.2 ± 1.7
Estimation of acute toxicity
Experimental weight of animals that were recorded within fixed time interval
Animal weight (g)
Mice 1 in gram
Mice 2 in gram
Mice 3 in gram
Mice 4 in gram
The fresh aerial parts of Portulaca quadrifida were collected in September 2017 from Benishangul Gumuz Regional State, Bullen district, Western Ethiopia, which is 310 km away from Bahir Dar and 680 km from Addis Ababa. The plant species was identified and authenticated by Dr Ali Seid at Biology department, Bahir Dar University.
Chemicals and reagents
The analytical grade chemicals and reagents used for this study were petroleum ether (Blulux, india), chloroform (Lobachemie, India), methanol (Lobachemie, India), dimethyl sulphoxide (DMSO), potassium iodide (Lobachemie, India), Wagner’s reagent (Iodine in potassium iodide), aluminum chloride (Blulux, India), iodine (Blulux, India), sodium nitrite, hydrochloric acid (LOBAChemie, India), sulfuric acid (Lobachemie, India), sodium hydroxide (Mumbai-400002, India), nitric acid, sodium carbonate, disodium hydrophosphate (Blulux, India), phosphoric acid (Blulux, India), sodium molybdate, sodium tungstate, iron chloride (Alpha Chemica, India), bromine, ascorbic acid (Blulux, INDIA-121005), Gallic acid, DPPH (himedia, India), quercitine (Alpha chemika, India), lithium sulphate, Muller Hinton agar and ammonia solution. All chemicals used for laboratory analysis were analytical grade that is greater than 97% in purity.
Extraction and isolation
The air-dried and ground aerial parts of P. quadrifida (200 g) were extracted by soaking successively in petroleum ether, chloroform (CHCl3) and methanol (MeOH) each for 48 h (two times with each solvent) and removal of the solvent under reduced pressure using a BUCHI flash evaporator to afford extracts of 2.70 g (for petroleum ether), 7.30 g (for chloroform) and 8.26 g (for MeOH).
Determination of total phenolic content (TPC)
Total phenolic content of the aerial parts of P. quadrifida extracts was determined according to the Folin-ciocalteu method as described before by different researchers [7, 8]. Each extract was dissolved in methanol (100 g/L) and 1 mL of extract, 5 mL of Folin-Ciocalteu reagent (diluted tenfold) and 4 mL (75 g/L) of sodium carbonate (Na2CO3) solution were added together. After the reagents mixed with the extracts, the flasks were filled with distilled water up to the mark and the mixtures left for 30 min in dark area and absorbances were measured at 765 nm.
Determination of the total flavonoid content (TFC)
The total flavonoid content of P. quadrifida crude extracts were determined by aluminum chloride assay as described before by different researchers [9, 10]. 0.25 mL of the extract was mixed with 1.25 mL of distilled water in 50 mL volumetric flask, followed by an immediate addition of 0.075 mL of 5% NaNO2 and 5 min later, 0.15 mL of 10% AlCl3 solution was added. After 6 min 0.5 mL of 1 M NaOH solution was added followed by 0.275 mL of distilled water and immediately the absorption at 510 nm was recorded by using UV-Vis spectrophotometer.
Free radical scavenging activity
Free radical scavenging activity of the extracts was determined by using the DPPH assay. DPPH assay is popular in natural product antioxidant studies and the antioxidant activity of P. quadrifida extracts was also evaluated on the bases of the radical scavenging effect of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) by using those methods described previously by different researchers . The methanol, chloroform, and petroleum ether extracts of P. quadrifida with different concentrations (20%, 40%, 60%, 80% and 100% (v/v)) were prepared by methanol to determine DPPH scavenging activity. An aliquot of 2 mL of 0.004% of DPPH solution was mixed with 1 mL of each extracts and the solutions were kept in dark for 30 min and the absorbance of the combination was measured at 517 nm using UV–Vis spectrophotometer.
Antimicrobial activity test
Antimicrobial activities of the plant extracts P. quadrifida were done in microbiology laboratory, department of Biology at Bahir Dar University by using agar well diffusion method. Muller Hinton agar media was prepared for culturing selected Gram negative and positive bacteria by using standard methods. Four bacteria were selected, two Gram positive (S. aureus and S. pyogenes) and two Gram negative (E. coli, and K. pneumoniae). A series of plant extracts with concentrations (50, 100, 150, 200 and 250 mg/L) and standard antibiotics (Gentamycin) were added to the incubated plate by using filter paper. Then it was incubated for 24 h at 37 °c and the experiment was repeated thrice, and the average values of zone of inhibition was recorded in mm for antimicrobial activity [12–14].
Estimation of cytotoxicity
The toxicity of the plant was done at Ethiopian food, medicine and health care administration and control authority. Experiments were performed using healthy young adult mice, non-pregnant and weighing 35–40 g. The experimental animals were divided into control and test groups containing four animals each. The animals were grouped in to their order of age and feed the plant for experimental animals and pellet for control group. Young rats were chosen because of their greater sensitivity to treatment. All the rats were observed individually at least once during the first 30 min, periodically during the first 24 h with special attention given during the first 4 h, and then daily for a total of 14 days. All the rats were observed at least twice daily with the purpose of recording any symptoms of ill-health or behavioral changes .
Methods of data analysis
In this study, the antioxidant activity, total phenolic content and total flavonoid content of P. quadrifida extracts were calculated and reported in terms of ascorbic acid (AA), gallic acid (GA), and querecetin (QT) equivalent per gram of extract. The equation stated below used for calculation from Y = BR + C linear equation.
Total phenolic content, total flavonoid content, antioxidant activities in DPPH assay and zone of inhibition in antimicrobial activities were measured in triplicates to take the mean ± SD value. The calibration curves and graphs were constructed by using Microsoft excel 2007. Statistical analysis was also undertaken by analysis of variance (one way ANOVA) with Least Significant Difference (LSD) to compare result between extracted plants by different solvents at the same concentrations using SPSS statistics version 20. Result was considered statistically significant at P-value < 0.05.
The extracts of the aerial parts of the Portulaca quadrifida were subjected to qualitative and quantitative analysis. The result of the study clearly indicated that nearly all investigated extracts showed antioxidant activity against DPPH radical scavenging activity. In antioxidant activity measurement, methanol extract, showed the highest percentage of DPPH radical scavenging activity. In addition to this, the study also showed that P. quadrifida extracts were found to contain measureable amount of total phenolic and flavonoid content which play major role in inhibiting oxidative stress in the body. Moreover, the results can also provide the effectiveness of P. quadrifida extracts for antimicrobial activity. All extracts of the plant also showed antimicrobial activity against different bacteria and fungi species. The acute toxicity result also revealed that the plant has no observable side effects. Generally the result of the study showed that the plant contains significant secondary metabolites and can be used as easily accessible source of food, natural antioxidant and antimicrobial activity.
ZYD was supervised the whole work as well as organized the manuscript as a whole and DAC did the experiment. All authors contributed to manuscript finalization. All authors read and approved the final manuscript.
We would like to thank Bahir Dar University for financial support to do this research. DAC also thanks Agricultural and Nutritional Research Laboratory, Ethiopian Institute of Agricultural Research (EIAR) for study leave as well as financial support for his study.
The authors declare that they have no competing interests.
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