Determination of antioxidant and antibacterial activities of leaf extracts of Plumbago zeylanica (Amira)

Abstract

Traditionally, the leaves of Plumbago Zeylanica were used for the treatment of several diseases. Herein, we report phytochemical analysis, total flavonoid and phenolic content, antioxidant capacity, and antibacterial activities of the leaf extracts of the plant. The phytochemical analysis test of the extracts showed the presence of biologically important compounds such as phenolics, flavonoids, alkaloids, terpenoids, and glycosides. Moreover, the total flavonoid content of 69.566, 66.966, and 59.133 mgQE/100 g was obtained from methanol, ethyl acetate, and petroleum ether extracts respectively. And the total phenolic content of methanol, ethyl acetate, and petroleum ether extracts were 82.738, 69.784, and 51.630 mgGAE/g dry samples. The extracts have promising antioxidant and antibacterial activity against S.aureus (Staphylococcus aureus), S. pyogens (Streptococcus pyogens), E. coli (Escherichia coli), and K. peneumona (Klebsiella pneumonia). The high antibacterial and antioxidant activity was found for methanol extract of the leaf extract of P. Zeylanica is attributing to the presence of polar phytochemicals such as; flavonoids, phenolic, and glycosidic groups in the crude extract obtained from maceration of polar solvents.

Keywords:

• Plumbago Zeylanica
• Flavonoid content
• total phenolic content
• antioxidant activity
• antibacterial activity
• phytochemical analysis

PUBLIC INTEREST STATEMENT

An increase in the number of multi-drug resistant pathogens and cellular-based diseases is fast becoming a global concern. Thus, the discovery of novel active compounds against new targets is a matter of urgency. Medicinal plants have been utilized for thousands of years to treat various ailments and physical damages throughout the world, especially, in different parts of Ethiopia. In an attempt to modernize traditional way of using medicinal plants, researchers had paid much attention on isolating different kinds of bioactive compounds, which are highly potent from leaf, stem, stem-bark, root, seed, and fruit of herbal traditional plants. Therefore, qualitative phytochemical screening, quantitative phytochemical evaluation, structural elucidation, and determination of antioxidant and antibacterial activity of plant extracts obtained by different solvent systems. This helps us to introduce new potential drugs obtained from various parts of traditional herbs.

Competing interests

The authors declare that they have no competing interests.

1. Introduction

Medicinal plants have been utilized for thousands of years as major sources of cures for human beings throughout the world. Especially, in different parts of Ethiopia using traditional herbal medicine for addressing their primary health-care need and to treat various ailments and physical damages is a custom. And most of the people of Ethiopia are highly dependent on plant-based traditional medical practices. Plumbago Zeylanica belongs to the medicinal plants that were used traditionally. It is more commonly known as Doctorbush or Ceylon Leadwort and is a semi climbing subshrub that grows throughout Asia, Australia, Africa, and Ceylon and is widely used in ethnomedicine (Teshome et al., 2008). In Ethiopia, locally known as Amera (in Amharic), is a shrub widely distributed in the West and Northwest parts of Ethiopia at 1500–2200 m above sea level as can be seen in Figure 1 (Getahun et al., 2014). It is used in indigenous system of medicine, and commonly known as “Chitthra mulam” (in India). It is branched evergreen shrub growing up to 0.5–2 meters. The leaves are dark-green, ovate 2.2–3.8 cm long, and 1.2–2.5 cm wide. The flowers are white in thick racemes, individuals around 0.5 cm across, flowering throughout the year (Samar et al., 2015).

Figure 1. Fresh leaves of Plumbago zeylanica.

In Ethiopian traditional medicine, the leaves of P. Zeylanica were used for the treatment of several diseases including intestinal warm, skin diseases and antitumor diseases, antimicrobial, and for its antioxidant properties used in preventing different kinds of disorders (Anubhuti & Pratibha, 2013; Olagunju et al., 1999; Oyedapo, 1996; Xu & Lu, 2010). Several studies suggested that, Plumbago Zeylanica is a good antimicrobial, antifungal, anti-inflammatory and anticancer, antihyperglycemic hypolipidaemic, and antiatherosclerotic (Devi & Thenmozhi, 2011; Madhava Chetty et al., 1998; Xu & Lu, 2010). Actually, antimicrobial and antioxidant activities of medicinal plants is directly linked to the presence of phytochemicals such as; alkaloids, phenolics, triterpenoids, flavonoids, gum, mucilage, protein, fatty acids, sapnonin, and different essential oils (Hamid et al., 2010; Kaushik et al., 2012; Lin et al., 2003; Rana, 2011; Subhash et al., 2013; Valko et al., 2007; Zhishen et al., 1999).

Different kinds of bioactive compounds have been previously isolated from the genus Plumbago. Studies revealed the presence of α-amyrin, stosterone, α-amyrin acetate, terpenoid, β-sitosterol, plumbagin and β-sitosterol-3β-glucoside, several naphthoquinones, binaphthoquinones, coumarins, di-phenyl sulfone, carboxylic acids and esters, meroterpenes, triterpenoids, amino acids, anthraquinones, steroids, steroid glucosides, and sugars in the genus Plumbago (Beigmohamadi et al., 2019; Singh et al., 2020). In addition, genus Plumbago has a lot of therapeutic applications such as antidiarroheal activities, antiallergic, insecticidal, antidiabetic, hepatoprotective, hypolipidaemic, anti-inflammatory, antitumor activity, antibacterial, antifungal, antimicrobial, and oral treatment for complaints related to infections of the urinary tract infections (Beigmohamadi et al., 2019).

The root of Plumbago zeylanica contains a numerous bioactive products such as two plumbagic acid glucosides (3ʹO-beta-glucopyranosyl plumbagic acid and 3ʹ-O-beta-glucopyranosyl plumbagic acid methylester) along with five naphthoquinones (plumbagin, chitranone, Maritinone, elliptinone, and isoshinanolone), and five coumarins (seselin, 5-methoxyseselin, suberosin, xanthyletin, and xanthoxyletin) respectively (Ganesan & Gani, 2013).

The leaves of P. zeylanica contain an alkaloid called plumbagin (2-methoxy-5hydroxy-1, 4-napthoquinone), which externally is a strong irritant but a powerful germicide. The following phytochemicals were isolated from Plumbago zeylanica root extract (Alam et al., 2013; Geng et al., 2012). 1-Plumbagin,2-(2, 4-Dihydroxy-phenyl)-3, 6, 8-trihydroxy-chromen-4-one, 3,3-diplumbagin, Chittanone, Isozeylenone, Maritinone, Elliptone, Droserone, Lupeol, and Stigmasterol. However, no detailed study was conducted on phytochemical screening, quantitative determination of phytochemicals, antioxidant, and antibacterial activity of the leaf extract of Plumbago zeylanica. In this paper we reported phytochemical analysis, total flavonoid and phenolic content, antioxidant, and antibacterial activities of P. Zeylanica leaf extracts obtained by different solvent extractions.

2. Results and discussion

2.1. Phytochemical screening

The presence of different bioactive compounds in different solvent extracts of the leaves of Plumbago Zeylanica was determined by using color change as a confirmatory test. Methanol extract was found to have a wide range of bioactive compounds because of its high polarity like glycosides, flavonoids, phenolic compounds, tannins, anthraquinone, terpenoids, protein, and alkaloids. The ethyl acetate extract was also positive for most qualitative tests except sapnonins and sterols. According to the study, petroleum ether extract has very limited phytochemicals as can be seen from Table 1. It confirms that methanol and ethyl acetate extracts have more phytochemical constituents than petroleum ether extract. However, Richa Tyagi and his co-workers reported that petroleum ether and ethyl acetate extracts of Plumbago Zeylanica are rich sources of phytochemicals than methanol extract (Tyagi & Menghani, 2014). But, petroleum ether is highly non-polar in nature, so it can’t extract polar phytochemicals such as anthraquinones, and alkaloids. The result of the test was summarized as follows in Table 1.

Table 1. Qualitative analysis of phytochemicals present in leaf extracts of Plumbago Zeylanica

Phytochemicals	Types of test or	Methanol	Ethyl	Petroleum
	Reagent		Acetate	Ether
Sapnonin	Foam test	+	-	-
Phenolic	Ferric chloride test	++	+	+
Flavonoids	With NH3	++	++	+
Alkaloids	Wagner’s reagent	+	++	-
Terpenoids	Chloroform	++	+	-
C.glycoside	Fecl3 + acetic acid	++	++	+
Sterols	Salkowski’s	++	-	-
Amino acid	Million’s reagent	++	++	-
Tannin	Ferric chloride test	++	+	-
Antherquinones	Hexane + NH3	++	+	-
Cholesterols	Chloroform +acetic anhydride +H2SO4	++	++	+
Quinones	With hcl	++	+	-

(++) highly present, (+) present, (−) not present; MeOH methanol extract, EtOAc ethyl acetate extract, PE petroleum ether extract.

2.2. Determination of total flavonoid content

The total flavonoid content of the extracts is determined by using quercetin standard calibration (Table 2 and Figure 2) and expressed as quercetin equivalent (mgQE) per dry sample. As it was observed from the table, methanol, ethyl acetate, and petroleum ether extracts of the leaves of Plumbago Zeylanica were contain flavonoids. The total flavonoid content of methanol, ethyl acetate, and petroleum ether extracts were 69.566, 66.966, 59.133 mgQE/g of dry sample mass respectively (Table 3). This means methanol extract contains more flavonoid compounds compared to ethyl acetate and petroleum ether extracts. This is due to the polar nature of methanol, so it can extract flavonoid compounds in greater extent than ethyl acetate and petroleum ether. Because flavonoid compounds are polyphenolic compounds thus have polar in nature.

Table 2. Absorbance of standard compound standard (quercetin) at λmax = 510 nm

Concentration of querecetin in (mg/L)	Absorbance of λmax at 510
10	0.0964
20	0.2462
30	0.3877
40	0.5332
50	0.7214

2.3. Determination of total phenolic content

The total phenolic content of the extracts is determined by using gallic acid standard calibration (Table 4 and Figure 3) and expressed as gallic acid equivalent (mgGAE)/g dry sample. All methanol, ethyl acetate and petroleum ether leaf extract of P. zeylanica contain high concentration of phenolic compound. The total phenolic content of methanol, ethyl acetate and petroleum ether extracts were 82.738, 69.784, and 51.630 mgGAE/g dry sample, which means methanol extract contains more phenolic compounds as compared to that of ethyl acetate and petroleum ether extracts (Table 5). Like flavonoid compounds phenolic compounds are polar in nature and they can easily extracted by methanol.

Table 3. Total favonoid content of leaf extracts of Plumbago Zeylanica

Extracts	Absorbance at 510 nm	Mg QE/g of sample
Methanol	0.1427	69.566
EtOAc	0.1349	66.966
Pet ether	0.1114	59.133

Table 4. Absorbance of standard compound (gallic acid) at λmax 765 nm

Gallic acid concentration in (mg/L)	Absorbance λmax at 765
20	0.106
40	0.358
60	0.584
80	0.857
100	1.206

Table 5. Total phenolic content of leaf extracts of P. Zeylanica

Extracts	Absorbance at 765 nm	mgGAE/g of sample
Methanol	0.3498	82.738
EtOAc	0.2656	69.784
Pet ether	0.1476	51.63

2.4. Antioxidant activity

The antioxidant activities of the extracts of the leaves of P.l zeylanica were evaluated by using FRAP and DPPH assays.

2.4.1. Ferric reducing antioxidant power (FRAP) assay

In this method, ascorbic acid was used as a standard to determine antioxidant activities of the extracts of the leaves of P. zeylanica (Table 6 and Figure 4). Table 7 and Figure 5 show that each extract has the highest reducing ability at higher concentration and methanol extract has higher mgAAE/100 g dry weight than ethyl acetate and petroleum ether extracts. This implies that methanol extract has higher reducing power than that of ethyl acetate and petroleum. And ethyl acetate extract has higher reducing power than petroleum ether extract within a corresponding concentration. The result of our study for each extract was supported by previous reported and this revealed that the extracts of more polar solvents exhibited better antioxidant activities than that of less polar solvents (Naz et al., 2014).

Figure 2. Calibration curve of querecetin standard

Figure 3. Calibration curve of gallic acid to determine the total phenolic contents

Figure 4. Calibration curve of AA for FRAP assay

Figure 5. Ferric reducing power of standard ascorbic acid and methanol, ethyl acetate and petroleum ether extracts

Table 6. % Reducing power and absorbance of AA at different concentrations

Concentration of	Absorbance at 700 nm	% reducing power
ascorbic acid in (mg/L)		of ascorbic acid
50	0.778	85.218
100	1.341	91.424
150	1.693	93.207
200	2.141	94.628

Table 7. FRAP values of % reducing power and absorbance for leaf extracts of P. zeylanica at different concentration

Concentration	Pet ether extract		EtOAc extract		Methanol extract
(mg/L)	Abs	%RP	Abs	%PR	Abs	%RP
50	0.375	66.666	0.393	70.737	0.445	74.151
100	0.445	73.563	0.441	72.819	0.513	77.582
150	0.483	76.239	0.525	78.695	0.768	84.915
200	0.517	77.752	0.542	78.782	0.909	87.348

2.4.2. DPPH radical scavenging activity

The DPPH-free radical scavenging ability of the extracts of the leaves of P. zeylanica was determined by using ascorbic acid standard calibration (Table 8 and Figure 6) and expressed using mgAAE/100g of dry sample. The DPPH-free radical scavenging ability of methanol, ethyl acetate, and petroleum ether extracts were evaluated by using color change as the reagent was added and recorded the absorbance of each extract at different concentrations. The change of a color from pink to yellow in each extract as well as standard solution confirmed that they have DPPH radical scavenging capacity. The faster the disappearance of the color revealed that the extract has higher DPPH-free radical scavenging activity. According to our study, methanol extract has greater DPPH radical scavenging power compared to that of ethyl acetate and petroleum ether extracts. But, such extract has lower DPPH radical scavenging activity than the standard antioxidant ascorbic acid. In addition, the lower IC₅₀ value of methanol extract reflects a better protective action than ethyl acetate and petroleum ether extract. As the concentration increases, the scavenging activity of the extracts were also increased as shown in Table 9 and Figure 7. Similarly, Yohannes Weldemariam Getahun and his co-workers also examined the radical scavenging activity of chloroform and ethanolic crude extracts of P. Zeylanica. The ethanolic extracts showed high percent of radical scavenging activity than chloroform crude extracts and comparable with that of standard ascorbic acid at higher concentration (Getahun et al., 2014).

Figure 6. Calibration curve of AA for DPPH assay

Figure 7. % inhibition versus concentration of standard of ascorbic acid and plant extracts that shows DPPH scavenging capacity

Table 8. % Inhibition activity and absorbance of AA at various concentrations

Concentration (mg/L)	Absorbance of AA	DPPH scavenging value (mg AAE/100g of dry weight)
	at 517 nm
25	0.177	63.195
50	0.139	71.263
75	0.104	78.464
100	0.072	85.062
125	0.036	92.501

Table 9. DPPH radical scavenging value of leaf extracts of P. zeylanica at different concentrations

Plant extract	Concentration	% Inhibition	regression	IC50 value
	in µg/ml	of activity	equation of sample	in (µg/ml)
Methanol	25	31.951
	50	49.378
	75	65.767	y = 0.5369x + 21.515	53.143 (µg/ml)
	100	76.141
	125	85.684
Ethyl acetate	25	23.651
	50	41.286	y = 0.3793x + 18.892	82.541(µg/ml)
	75	50.207
	100	57.676
	125	62.863
Petroleum	25	3.464
Ether	50	16.804	y = 0.3075x − 1.7049	172.103(µg/ml)
	75	21.182
	100	30.081
	125	35.269

2.5. Antibacterial activity

Antibacterial activity of the extracts of the leaf of P. zeylanica was evaluated by using Agar well diffusion method. Four bacteria in which two of them were gram-negative bacteria (E. coli and K. pneumoniae) whereas the remaining two were gram-positive bacteria (S.aureus and S. pyogens) were used for the determination. As shown in Table 10, methanol and ethyl acetate extracts showed relatively significant antibacterial activity whereas petroleum ether extract showed lower activity in all four bacteria.

Table 10. Antibacterial activity and comparison of MZI among leaf extracts of P. zeylanica

Extract and	Concentration	Zone of inhibition (mm) for bacteria
	in (mg/L)	Gram positive		Gram negative
		S. aureus	S.pyogens	E. coli	K. peneumona
Methanol	25	0	0	0	0
Extract
	50	11 ± 0.29	11 ± 0.25	11 ± 0.09	12 ± 0.21
	75	12 ± 0.35	15 ± 0.36	13 ± 0.33	14 ± 0.29
	100	17 ± 0.3	17 ± 0.33	16 ± 0.23	16 ± 0.43
	125	19 ± 0.4	20 ± 0.26	21 ± 0.16	16 ± 0.43
Ethyl	25	0	0	0	0
Acetate
Extract	50	10 ± 0.13	10 ± 0.17	10 ± 0.14	10 ± 0.24
	75	13 ± 0.37	13 ± 0.17	18 ± 0.24	11 ± 0.34
	100	15 ± 0.14	16 ± 0.29	20 ± 0.10	15 ± 0.26
	125	18 ± 0.11	22 ± 0.18	21 ± 0.14	17 ± 0.41
Petroleum	25	0	0	0	0
Ether
Extract	50	0	0	0	0
	75	7 ± 0.11	0	7 ± 0.18	7 ± 0.16
	100	9 ± 0.21	8 ± 0.15	9 ± 0.19	9 ± 0.17
	125	10 ± 0.17	12 ± 0.16	11 ± 0.24	11 ± 0.11
Gentamycine	200	32 ± 0.12	31 ± 0.08	28 ± 0.07	29 ± 037

S. aureus = Staphylococcus aureus, S. pyogens = Streptococcus pyogens, E. coli = Escherichia coli, K.pneumonia = Klebsiella pneumonia, Gen = Gentamycin.

For instance, methanol extract showed a good antibacterial result against S. aureus, S. pyogens, E. coli and K. peneumona with minimum zone of inhibition 19 ± 0.4, 20 ± 0.26, 21 ± 0.16, and 16 ± 0.43 respectively. However, the highest minimum inhibition zone was recorded in ethyl acetate extract against S. pyogenes and E. coli with minimum zone of inhibition 22 ± 0.18 and 21 ± 0.14 respectively. For the extracts with higher antibacterial activities, the IC50 value is calculated. In this regard the IC50 of methanol extract toward gram-negative E. coli is 292 mg/L whereas for ethyl acetate extract toward gram-positive S. pyogens is 294 mg/L. But for petroleum ether extract the IC50 value is determined to be 747. Generally methanol and ethyl acetate extracts P. zeylanica showed a remarkable antibacterial activity than petroleum ether extract. According to the result of qualitative and quantitative phytochemical investigation, methanol and ethyl acetate extracts have more phytochemical constituents than petroleum ether extract. So, the presences of more polar phytochemical constituents lead to higher antibacterial activity.

3. Experimental section

3.1. Chemicals and reagents

Chloroform, acetone, iodine powder, potassium iodide, Muller Hinton agar, ethyl acetate, methanol, petroleum ether, ferric chloride (FeCl₃), Wagner’s reagent (Iodine in potassium iodide), hydrochloric acid, sulfuric acid (H₂SO₄), sodium hydroxide (NaOH), nitric acid (HNO₃), hydrated aluminum chloride (AlCl₃.6H₂O), sodium nitrite (NaNO₂), sodium carbonate (NaCO₃), monosodium hydrogen phosphate (NaH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), trichloroacetic acid, potassium hexacyano-ferrate (II) (K₂[Fe(CN)₆]), Ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, gallic acid, ammonia solution, distilled water, and deionized water were some of the chemicals and reagents that were used for the experimental work during our study.

3.2. Plant materials

Fresh leaves of P. zeylanica were collected from Mentawuha, which is located in Awi zone and 212 km away from Bahir Dar, Amhara regional state, Ethiopia in March 2019. The plant material was identifed and authenticated by Dr. Ali Seid, botanist in biology department, Bahir Dar University.

3.3. Extraction of samples

The air-dried and ground leaves of P. zeylanica were extracted by maceration process. Leave powder (100 g) was soaked successively in petroleum ether, ethyl acetate (EtOAc) and methanol (MeOH) each for 24 h (two times with each solvent) and removal of the solvent under reduced pressure using a rotatory evaporator to afford extracts of 4.32 g (for pet ether), 7.33 g (for EtOAc), and 10.52 g (for MeOH).

3.4. Phytochemical analysis

The phytochemical analysis of methanol, ethyl acetate, and petroleum ether extracts of the leaves of P. zeylanica were studied by slight modifications based on standard procedures described on different literatures (Edeoga et al., 2005; Okwu, 2001; Parekh & Chanda, 2007; Pranoothi et al., 2014).

3.5. Determination of total phenolic content

Total phenolic content was analyzed by using standard method with slight modifications (Pranoothi et al., 2014). Five milligrams of the methanol, ethyl acetate, and petroleum ether extracts of the sample were weighed and dissolved in 10 mL of distilled water. 1 mL of this solution was transferred to separate test tube and mixed with 5 mL distilled water, then 0.5 mL of Folin-Ciocalteu reagent was added and vortexes. After 5 min, 1.5 mL 20% of Na₂CO₃ solution was added and ultimately the volume was made up to 8 mL with distilled water and finally incubated for 60 min at room temperature. After incubation, the absorbance against the reagent blank was determined at 765 nm. A reagent blank was prepared using distilled water instead of the plant extract. These data were used to estimate the total phenolic content using a standard calibration curve obtained from various diluted concentrations of gallic acid (20, 40, 60, 80, and 100 μg/mL). The total phenolic content of the plant was expressed as gallic acid equivalent mgGAQ/g dry Samples were analyzed in triplicates.

$\mathrm{F}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{C}:\mathrm{M}\mathrm{o}(\mathrm{V}\mathrm{I}),(\mathrm{y}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{o}\mathrm{w})+{\mathrm{e}}^{-}(\mathrm{F}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t})----\to \mathrm{M}\mathrm{o}(\mathrm{V}),(\mathrm{B}\mathrm{l}\mathrm{u}\mathrm{e})$

3.6. Measurement of total flavonoid content

Total flavonoid content was measured with aluminum chloride colorimetric assay as described by different researchers with minor modifications (Abdel-Sattar et al., 2008; Bhagwat et al., 2005). In brief, 1 mL of methanol, ethyl acetate, and pet ether extracts and 1 mL of standard quercetin solutions (10, 20, 30, 40, 50 μg/mL) were positioned into test tubes and 4 mL of distilled water and 0.3 mL of 5% sodium nitrite solution were added into each solutions. After 5 min, 0.3 mL of 10% aluminum chloride was added. At 6th min, 2 mL of 1 M sodium hydroxide was added and orange yellowish color was developed (Nagda et al.,). The absorbance was measured at 510 nm by using UV-visible spectrophotometer. The blank was performed using distilled water. Quercetin was used as standard. The calibration curve was plotted using standard quercetin. The data of the total flavonoid contents was expressed as mg of quercetin equivalents/100g of dry mass. pneumoniae, Klebsiella Pneumoniae; Gen, Gentamycin.

3.7. Measurement of free radical scavenging activity ferric reducing antioxidant power (FRAP) assay

The reducing power of methanol, ethyl acetate, and petroleum ether extracts was determined according to the method described in Kriengsak et al. (2006). with slight modification. In brief, 2.5 mL of different concentration of methanol, ethyl acetate and petroleum ether extracts were mixed with 2.5 mL of phosphate buffer solution (PH = 6.6, 0.2 M) and 2.5 mL of potassium hexacyanoferrate ([K₃Fe(CN)₆]) (1%). The mixture was incubated at 50°C for 20 min in water bath. Then 2.5 mL of Trichloroacetic acid (10%) was added to the mixture to terminate the reaction. 5 mL of the upper layer of the solution was mixed with 5 mL of distilled water and 0.5 mL of FeCl₃ solution (0.1%). The reaction mixture was left for 10 min at room temperature and the absorbance developed bluish-green color was measured at 700 nm by using UV-spectrophotometer against a blank solution. Distilled water was used instead of extracts or standard to prepare a blank solution.

3.8. DPPH radical scavenging assay

The antioxidant activity of methanol, ethyl acetate, and petroleum ether extracts was measured on the basis of the scavenging activity of the stable 1,1-diphenyl-2-picrylhyorazyl (DPPH) free radical according to the method described with slight modifcations (Pranoothi et al., 2014). In brief, 1 mL of DPPH solution was added to 4 mL of various concentrations of methanol, ethyl acetate, and petroleum ether extracts and ascorbic acid to be tested. After 30 min, absorbance was measured at 517 nm. Ascorbic acid with a series of concentration was used as a reference.

3.9. Antibacterial activity

Antibacterial activities were performed in microbiology laboratory, department of Biology, Bahir Dar University by using agar well-diffusion method. Muller Hinton agar media was prepared for culturing selected gram-positive and gram-negative bacteria by using standard methods. Four bacteria [2-g positive (S. aureus and S. pyogens) and 2-g negative (E. coli and K. pneumoniae) were tested. A series of P. zeylanica leaf extract concentrations (25, 50, 75, 100, and 125 μg/mL) and standard antibiotics (Gentamycin) were added to the incubated agar by using discs. Then it was incubated for 24 h at 37°C and the experiment was repeated three times, and average values of zone of inhibition were recorded in mm for antimicrobial activity as described before (Bull et al., 1968; Sarker & Nahar, 2007).

3.10. Data analysis

Most results were reported as mean±standard deviation (SD). The calibration were constructed by using Microsoft excel window 10 and origin 8 pro.

4. Conclusion

The result of the study clearly indicated that, the leaf extracts of P. zeylanica have bioactive phytochemicals those are sapnonin, phenolic compound, flavonoid, terpenoids, tannins, steroids, sterols, quinone, anthraquinone, glycosides, alkaloid, cholesterol, and protein. And it showed good total flavonoid content, remarkable total phenolic content, good DPPH radical scavenging activity, and weak to moderate antibacterial activity. Among those extracts, methanol and ethyl acetate extracts have showed significant activities compared to petroleum ether extract. So, the polar and medium-polar solvent extracts of Plumbago zeylanica leaf show better antioxidant and antibacterial activities than non-polar solvent extract due to their higher phytochemical constituents. Generally, this study showed that leaf extracts of P. zeylanica had good antioxidant and antibacterial activities. Therefore, further investigations will be needed to isolate individual compounds from this plant which have higher antioxidant and antibacterial activities.

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Cover image

Source: Author.

Acknowledgments

We would like to thank Bahir Dar University and Ministry of Education, Ethiopia for financial support to do this research.

Additional information

Funding

The authors have no funding to report.

Notes on contributors

Belete B. Beyene

Belete B. Beyene (Ph.D.) is a full-time associate professor of organic chemistry at the department of chemistry, Bahir Dar University, Ethiopia. He received B.Ed. in chemistry from Bahir Dar University, M.Sc. in organic chemistry from Addis Ababa University in Ethiopia and Ph.D. in Chemistry from National Tsing Hua University in Tiwan (ROC). His research group is composed of some staff members of Bahir Dar University and postgraduate students. He also has research collaboration with professor from institute of chemistry Academia Sinica, Taiwan. Belete’s research group works in the development of metal complexes of organic ligands (mainly porphyrins) as efficient catalysts and potential antimicrobials. Furthermore, the group is working in the synthesis of organic ligands for environmental applications, such as ion sensing as well as phytochemical investigation and antimicrobial activity study of medicinal plants. Dr. Belete B. Beyene has authored/coauthored more than 26 peer-reviewed articles encompassing original research papers, a review article, and a book review.