Extraction yield and phytochemical screening

The yield of crude extracts from A. integrifolia leaf powder obtained via different solvents was determined to assess the effectiveness of different solvents in extracting specific components. Table 1 shows the mass and percentage yield of the samples dried with various solvents. The methanol leaf extract yielded the highest mass yield and percentage yield of secondary metabolites, whereas the water root extract had the lowest percentage yield in the following order: methanol > ethanol > water leaf extract. This trend is often observed in the extraction of bioactive substances and is believed to be due to the capacity of methanol to partially dissolve both polar and nonpolar compounds [28].

Table 1 Mass and percentage yield of A. Integrifolia leaf powder (10 g) extracted with different solvents

The yield of the methanol leaf extract was the highest (27.56%), followed by the ethanol extract at 16.985%. The ethanol and methanol extracts provided higher yields than did the water extract, probably because alcohol solvents contain both polar and nonpolar substances, enabling them to dissolve both polar and nonpolar compounds. Moreover, alcohol solvents are more effective at breaking down cell walls and extracting a greater number of polar molecules with intermediate and small polarities [29]. These results indicate that the secondary metabolite compounds found in A. integrifolia are mainly polar compounds and might be influenced by the nonpolar properties of the solvent [30].

Total phenolic content (TPC) and total flavonoid content (TFV)

The TPC and TFC of A. integrifolia leaf extracts varied in different solvent systems (Table 2). The results revealed that the extract was rich in phenolic and flavonoid compounds. A comparable TPC was reached at 18.74 ± 0.38 and 17.45 ± 0.38 mg GAE/g dried methanol and ethanol extracts, respectively. The water extract had the lowest value (6.67 ± 0.15 mg GAE/g dry extract). Similarly, the methanol and ethanol extracts had the highest TFC (38.47 ± 2.571 and 39.38 ± 2.314 mg CE/g dry extract, respectively). However, the water extract had the lowest value (6.67 ± 0.257 mg CE/g).

Table 2 TPC and TFC of A. integrifolia leaves extracted with various solvents

There were no significant differences (p < 0.05) in the TPC or TFC between the ethanol, and methanol, extracts. However, a significant difference was observed for the A. integrifolia water extract compared with both the methanol and ethanol extracts. A phytochemical screening test of A. remota leaf extracts using aqueous and 80% methanol revealed the presence of phenolic compounds and flavonoids [31]. Another study reported that flavonoids and phenolic compounds were identified in aqueous and 70% ethanol extracts of A. remota leaves [32]. The results of the previous total phenolic and flavonoid contents and the results of the present study support similar findings, indicating that the phenolic and flavonoid contents were relatively high in the leaf extract of A. integrifolia.

Antioxidant activityDPPH scavenging activity

The antioxidant activity potential is the degree of inhibition of free radical activity and was evaluated on the basis of its ability to scavenge stable free radicals by donating an electron or hydrogen [1]. The disappearance of the purple color of DPPH suggested the presence of antioxidant activity in the crude extract capable of releasing hydrogen. The antioxidant potential was monitored by measuring the absorbance following the production of stable free radicals via a spectrophotometer. A reduction in the absorbance value indicates that the extract has stronger antioxidant activity because it donates hydrogen atoms [33]. The methanol and ethanol extracts demonstrated a greater percentage of inhibition than did the water extract, indicating that phytochemicals are soluble in these solvents (methanol and ethanol) (Fig. 1).

Fig. 1

DPPH radical scavenging activity (%) of methanol, ethanol, and water leaf extracts of A. integrifolia and the control (ascorbic acid) at different concentrations. The values are expressed as the means ± standard deviations (n = 3)

At all the tested concentrations, the leaf extracts of A. integrifolia presented DPPH scavenging activity in the following order: ascorbic acid > methanol > ethanol > water. At a concentration of 1000 µg/mL, A. integrifolia leaf extract presented greater DPPH scavenging activity than did the other extracts, and there was no significant difference in DPPH scavenging activity between the methanol and ethanol extracts (94.15 ± 1.428% and 93.190 ± 0.275%, respectively), and results comparable with those of commercially available standard antioxidants (ascorbic acid) were obtained. In contrast, the water-containing leaf extract had 41.124 ± 1.363% activities and exhibited the lowest level of antioxidant activity (Fig. 1). These findings indicate that the dried leaf extract of A. integrifolia contains substances that are able to easily donate electrons/hydrogen and help neutralize unpaired electrons when extracted with methanol or ethanol. However, the utilization of water for extraction yields small electrons that can easily stabilize free radicals.

Moreover, the antioxidant capacity of the extracts was evaluated by determining the amount of sample capable of reducing the DPPH concentration to 50% (IC50) of the initial amount [34]. The IC50 values of A. integrifolia leaf extracts were calculated from the graph of percentage scavenging activity versus extract concentrations tested. An extract that has a lower IC50 value has greater antioxidant activity than Ismael et al. [19]. According to the results presented in Table 3, the IC50 values for the extracts ranging from 103 µg/mL for the water leaf extract presented the lowest DPPH scavenging activity to 48.01 ± 2.31 µg/mL for the methanol leaf extract, which presented the strongest DPPH scavenging activity.

Table 3 IC50 values of various solvents in A. Integrifolia leaf extracts

The results revealed that there was no significant difference in the IC50 values of the methanol and ethanol extracts. Moreover, the DPPH scavenging activities of the ethanol and methanol extracts were not significantly different from the DPPH scavenging activity of the standard (ascorbic acid). However, these values are greater than the IC50 values and indicate the lowest DPPH scavenging activity, with an IC50 value of 31.88 ± 0.20 µg/mL for the standard (ascorbic acid). However, the IC50 value of the water extract was significantly lower than that of both the methanol and ethanol extracts (p < 0.05). The water leaf extract had the lowest DPPH scavenging activity (IC50 > 103) of all the extracts. As a result, the IC50 values of the extracts decreased in the following order: ascorbic acid > ethanol > methanol > water leaf extracts. This is because A. integrifolia has high contents of phytochemicals, such as phenolics and flavonoids, which are responsible for its antioxidant properties, which are known for its free radical quenching potential [35]. Overall, these findings suggest that the leaves of A. integrifolia have a strong ability to scavenge free radicals. Hence, they have the capacity to react with free oxygen species to generate phenoxyl radicals, which are stable and responsible for stopping the chain reaction [1].

Ferric reducing antioxidant power

The reducing power of the extract was determined by the ferric reducing antioxidant power (FRAP) of the bioactive compounds, which is associated with the antioxidant activity of the natural molecules [1]. The ability of the compound to transfer electrons and reduce iron (III) suggests electron-donating activity, which is an important mechanism in phenolic antioxidant reactions [36]. The process of converting ferric iron (Fe3+) to its ferrous form (Fe2+) is initiated by antioxidants present in the sample that donate electrons. Thus, this activity is proportional to the antioxidant ability of the extracts. At a concentration of 1 mg/mL, the reducing capacity of the leaf extracts decreased in the following order: ethanol (141.224 ± 3.174 mg AAE/g) > methanol (85.714 ± 2.597 mg AAE/g) > water leaf extracts (27.244 ± 3.3 mg AAE/g) (Fig. 2). Compared with the methanol leaf extract and water leaf extract, the ethanol leaf extract of A. integrifolia presented the strongest FRAP (the weakest FRAP), with a significant difference (p < 0.05).

Fig. 2

Ferric reducing power (mg AAE/g) of water, ethanol, and methanol extracts from the air˗dried leaves of A. integrifolia. The data are the average of triplicate measurements (mean ± SD). Values with different letters in the histogram bar are significantly different (p < 0.05)

At 1 mg/mL, the ethanol extract had considerably greater ferric reducing power (p < 0.05) than did the methanol and water extracts (Fig. 2). This finding is consistent with the results of the present study, where the ethanol extract and water extract had the strongest and weakest total antioxidant activities, respectively, as determined by the DPPH scavenging activity. There was a significant difference in the antioxidant activity of the ethanol, methanol and water extracts (p < 0.05).

This study confirmed that the leaf extracts of A. integrifolia presented strong antioxidant properties. Keshebo et al. [37] and Mashwani ZUR et al. [38]. reported that the methanol leaf extract of A. integrifolia had strong antioxidant properties. Moreover, other recent studies by Fagbemi et al. [39] and Keshebo et al. [37] on bioactive compounds revealed that the extract has antioxidant activity, and the researchers attributed the presence of secondary metabolites, including phenolics, flavonoids, terpenoids, β-sitosterol, cis-vaccenic acid and other compounds, such as alkaloids and steroids, to these properties. This study and the previous findings therefore confirm the evidence that suggests the involvement of A. integrifolia in drug development to combat oxidative stress. As a result, this study validates the traditional use of this plant and its application in the pharmaceutical industry.

Antibacterial activity

Table 4 shows the antibacterial activity of A. integrifolia leaf extracts in various solvents. The antibacterial effect of Tween 20 (considered a negative control) did not hinder the growth of the tested bacterial strains. All extracts at the tested concentrations presented lower antibacterial activity than did the antibiotics used (positive control). In this study, ethanol and methanol leaf extracts of A. integrifolia presented increased antibacterial activity against the gram-positive bacteria S. aureus, followed by antibacterial activity against S. pyrogens, E. coli, and P. aeruginosa. However, the results showed that the water extract is resistant to gram-positive bacteria (S. pyrogens). According to the results of this study, among the bacteria tested, gram-negative bacteria were more resistant to A. integrifolia water leaf extract.

Table 4 Antibacterial activity of A. Integrifolia leaf extracts in various solvents at various concentrations

In this study, the Gram˗positive bacterium S. aureus was more susceptible to ethanol and methanol leaf extracts of A. integrifolia at all the tested concentrations. This is due to the antimicrobial activity of polyphenols and flavonoid compounds [40, 41]. Table 4 shows that at a concentration of 100 mg/mL, the methanol extracts had the strongest bacterial inhibitory effect (9.5 ± 0.5 mm) on S. aureus, which was not significantly different from that of the ethanol extract. However, the results indicated that the extracts are resistant to S. pyogenes, E. coli, and P. aeruginosa. The water leaf extract of A. integrifolia had the lowest antibacterial efficacy against the gram-positive bacteria S. pyogenes, while the gram-negative bacteria E. coli and P. aeruginosa were more resistant to the water leaf extract of A. integrifolia. Many studies have suggested that the antibacterial effect of plant extracts is due to the interaction of the extract with the cell proteins of bacterial strains, leading to cell death [19, 42]. The synergetic effect of the high content of major flavonoids and their interaction due to their structure may lead to the high biological activities of the extracts.

Minimum inhibitory concentration (MIC)

Table 5 summarizes the minimum inhibitory concentrations (MICs) of the A. integrifolia leaf extracts. The MIC values for the investigated bacterial strains ranged from 3.125 mg/mL to 100 mg/mL. The ethanol and methanol extracts displayed the highest activity against S. aureus, with an MIC of 3.125 mg/mL, followed by moderate activity against E. coli, S. pyogens, and P. aeruginosa, with MIC values of 12.5 mg/mL for the ethanol extract and 50 mg/mL for the methanol extract. The MIC value for the water extract was 50 mg/mL against Gram˗positive bacteria (S. aureus and S. pyogens) and 100 mg/mL against Gram˗positive bacteria (E. coli and P. aeruginosa). The differences in the MICs obtained among the different solvent extracts were probably due to their ability to extract secondary metabolites [14]. Furthermore, S. aureus was the most susceptible bacterium to ethanol and methanol extracts, with an MIC value of 3.125 mg/mL. However, S. pyogenes, E. coli, and P. aeruginosa were more resistant to the water extract. Certainly, Gram˗negative bacteria are more resistant to the extracts; this could be due to their impermeable external membrane, which limits the amount of antibiotics that may enter the cell [43].

Table 5 Minimum inhibitory concentrations (MICs) of A. Integrifolia leaf extracts in various solvents

A comparison of these findings with those of other studies confirmed that A. integrifolia has antibacterial activity. Keshebo et al. [37] reported that methanol extracts showed antibacterial activity against Escherichia coli, Shigella, Staphylococcus aureus, and Bacillus subtilis. Another study by Mashwani et al. [38] on A. integrifolia leaf extract revealed strong antibacterial activity. Moreover, Nagarkoti et al. [44] reported the antibacterial activity of Ajuga species (Ajuga brachystemon, Ajuga integrifolia, Ajuga macrosperma and Ajuga parviflora) belonging to the Lamiaceae family. Similarly, more recent studies by Fagbemi et al. [39] and Keshebo et al. [37] on bioactive compounds revealed that the extract had antibacterial activity on some tested bacterial strains, and the results were attributed to the presence of secondary metabolites, including phenolics, flavonoids, terpenoids, β-sitosterol, cis-vaccenic acid and other compounds, such as alkaloids and steroids, as reported by other researchers. The present study and the previous findings therefore confirm the evidence suggesting the involvement of A. integrifolia in drug development against pathogenic bacteria. Thus, the results of this study validate the traditional application of this plant and suggest its application in the pharmaceutical industry for the development of drugs from it.

The findings of this study have many important advantages, such as highlighting the potential of this plant for biological application. It can also be used for scientific communities by paving the way and promoting community awareness in conserving plants for medical assets.