Phytochemical analysis
In the present study, a preliminary qualitative phytochemical analysis was carried out to identify the major secondary metabolites such as tannin, phlobatannins, saponins, flavonoids, steroids, terpenoids and alkaloids.. The result of qualitative analysis was shown in Table
1. Alkaloids, saponins, steroids and tannins were detected in all leaf and bark guava extracts, but flavonoids and phlobatannins were not detected in the all extracts, and terpenoids in the Babile leaf and bark extracts. Qualitative phytochemical analysis was conducted by several researchers on guava extracts obtained by macerating leaf powder in ethanol. In one study, the presence of phenols, tannins, terpenoids, flavonoids and glycosides, and absences of saponins were revealed in the leaf collected from Fort Valley State University, USA [
27]. The presence of terpenoids, quinones, oil & fat, phenol, and the absence of alkaloids, flavonoids, sterols and anthocyanin were shown in the leaf collected from India [
32]. The presence of alkaloids, flavonoids, phenols, tannins, and the absences of saponins, steroids, terpenoids and cardiac glycosides were indicated in the leaf collected from Baringo Counties, Kenya [
33]. Leaves were collected from Botanical garden of Department of Biological Sciences, Bayero University, Kano, Nigeria and presence of alkaloid, glycosides, saponins and tannins detected in the leaf extract obtained by ethanol percolation at 30 °C, and the absence of flavonoid and steroids [
29]. The presence of phlobatannins, saponin, flavonoids, steroids, terpenoids, polyphenol and glycosides, and the absence of triterpenoids, alkaloid and anthraquinone were shown in the leaf collected from Thanjavur, Tamil Nadu, India [
11].
Similarly, qualitative phytochemical analysis was conducted on the guava bark collected from different regions. Barks were collected from Elele in Kelga Local Government Area of Rivers State, Nigeria and alkaloids, anthraquinone, cardiac glycosides, flavonoids, tannins and saponins were detected in the bark powder [
12]. Extraction was done by soaking powder of bark collected from Bodinga, Nigeria and the presence of tannin, saponin, flavonoid, alkaloid, steroid, terpenoids, and cardiac glycosides were revealed in the extract [
14]. Extraction was done by Soxhlet method from bark collected university of Maiduguri, Borno state Nigeria and presence of tannis, cardiac glycoside, flavonoids, terpenoid and saponins, and the absence of anthraquinone and alkaloid were shown in the extract [
15].
Quantitative determination of alkaloid, terpenoid, total phenolics and tannin contents were carried out and the result of quantities analysis was shown in Table
2.
Gursum guava extracts had generally higher contents of most quantified phytochemicals than Babile guava extracts even though most differences were insignificant (
p > 0.05). The alkaloid content of Babile bark extract (72.33 ± 3.93 mg/g) was lower than the alkaloid content of Gursum bark extract (82.33 ± 8.67 mg/g). The saponin content of Babile leaf extract (29.00 ± 4.93 mg/g) was lower than the saponin content of Gursum leaf extract (30.00 ± 5.77 mg/g). Babile bark extract showed significantly lower TPC (0.13 ± 0.00 mg/g as TAE) and TC (0.09 ± 0.01 mg/g as TAE) than that of Gursum bark extract (
p < 0.05). The results of phytochemical analysis revealed slight variation in type and content of the investigated phytochemicals between Babile and Gursum districts. This was supported by previously study [
30].
Quantitative phytochemical analysis of guava collected from different regions and extracted using ethanol was reported previously. In one study, total alkaloid extracted from leaves collected from Afaka, NDA permanent site, Kaduna, Nigeria and the obtained product was 20 mg/g of powder [
26]. Extraction was done by soaking leaf and bark collected from South Western part of Nigeria and the obtained extract showed concentrations of tannin (11.54 ± 0.14 mg/g), polyphenol (1.67 ± 0.24 mg/g), alkaloid (59.85 ± 0.34%) and saponin (1.86 ± 0.15%) in the leaf extract, whereas concentrations of tannin (3.85 ± 0.14 mg/g), polyphenol (0.45 ± 0.20 mg/g), alkaloid (6.65 ± 0.15% mg/g) and saponin (1.03 ± 0.15% in the bark extracts [
16]. Extraction was done by soxhlet apparatus and TPC of 7.30 ± 0.05 mg of catechol equivalent/g dry materials were obtained from the twig extract [
9].
The results of phytochemical analysis in present study and that of the above mentioned as previous studies revealed differences for the same plant species. it was reported that the results of phytochemical analysis may differ because of various factors such as biochemical reaction within species [
16], genes, environment and their interactions [
31], plant genotypes, developmental stages and geographical locations [
30], extraction methods [
8,
29]. Furthermore, variations in extraction methods are usually found in the length of the extraction period, pH, temperature, particle size, and the solvent-to-sample ratio [
45].
Antibacterial activity
In the present study, antibacterial activity tests were carried out using agar disc diffusion and broth dilution methods. Agar disc diffusion method was first used to identify sensitivity of the clinical isolates to the extracts. Once the extracts were found to have antibacterial activity through ZOI formation, broth dilution method was used to determine MIC. The ZOI of the tested clinical isolates was shown in Table
3. Babile and Gursum guava extracts at concentration of 2 mg/disc showed inhibitory effect on all tested clinical isolates. The standard antibiotics (ciprofloxacin) showed significantly higher ZOI against all the isolates than all guava extracts. Comparing antibacterial activity of guava extracts against each clinical isolates, Babile leaf extracts showed the highest ZOI against
S. Typhi (8.7 ± 1.01 mm),
S. boydii (13.0 ± 6.79 mm) and
E. faecalis (10.7 ± 1.59 mm) and Gursum bark extracts showed the highest ZOI against
S. aureus (10.0 ± 1.00 mm). This was happened may be due to the presence of a significant amount of alkaloid in Babile leaf extracts, the synergy of higher content of alkaloid and terpenoids in Gursum bark extracts and the degree of sensitivity of the isolates to the extracts. All the guava extracts showed the highest ZOI against
S. boydii and the lowest zone of inhibition against
S. Typhi. The antibacterial activity of Babile leaf and bark extracts was effective against the clinical isolates in order of
S. boydii >
E. faecalis >
S. aureus >
S. Typhi. The order of Gursum leaf and Gursum bark extracts were
S. boydii >
E. faecalis =
S. aureus >
S. Typhi and
S. boydii >
S. aureus >
E. faecalis >
S. Typhi, respectively.
MICs of the extracts were determined by preparing double serial dilutions. The ethanol extracts were dried and re-dissolved in mixture of ethanol and water in order to prepare the extracts’ double dilutions ranging from 20 mg/ml to 0.625 mg/ml. The MIC values of the guava extracts were shown in Table
4. All guava extracts showed different MIC against each clinical isolates. MIC of Babile leaf and Gursum bark extracts against
E. faecalis was significantly different (
p < 0.05). All the guava extracts showed the lowest MIC against
S. Typhi, except Babile leaf extracts that showed the lowest MIC against
S. boydii. The antibacterial activity of Babile leaf and bark extracts measured by MIC was effective against the clinical isolated in order of
S. boydii >
S. Typhi. >
E. faecalis >
S. aureus and
S. Typhi >
S. boydii >
S. aureus >
E. faecalis, respectively
. The order of Gursum leaf and Gursum bark extracts were
S. Typhi
> S. boydii >
S. aureus > E. faecalis and
S. Typhi >
S. aureus >
S. boydii >
E. faecalis, respectively. The difference in MIC among tested guava extracts is may be due to the difference in individual concentrations of constituents of phytochemicals that made up overall tested concentration of leaf and bark extracts and susceptibility of the isolates to the extracts’ constituent in a concentration dependent pattern as previously reported [
26].
The composition and quantity of alkaloids, saponins, tannins and terpenoids both in leaf and bark extracts of guava have medical implications that may be responsible for the observed antibacterial activities. Previous research report showed antibacterial activity of tannin [
46], alkaloid [
26], saponin [
47,
48] and terpenoid [
18,
49].
For guava collected from different regions, bark and leaf ethanolic extracts were shown to have in vitro antimicrobial activity against numerous bacteria in previous studies. Leaf was collected from Fort Bhaktapur, Kathmandu Valley, Nepal and the extract obtained from Soxhlet method showed 13, 20, 14 mm ZOI against
S. typhi, Salmonella paratyphi and
Shigella spp. [
1]. In another study, antimicrobial activity of leaf extract showed 11.0 ± 0.52 mm ZOI against
S. aureus [
27]. Antimicrobial activity of extract of leaf was carried out using well diffusion method and the results showed 3, 7, 6, 5 and 4 mm ZOI against
S. typhi,
Salmonella paratyphi A,
Salmonella paratyphi B,
Shigella sonnei,
Shigella dysentarie, respectively [
2]. Antimicrobial activity of extract of leaf was carried out using disc diffusion and broth dilution methods. The results showed that extract obtained by ethanol percolation at 30 °C inhibited
Salmonella spp. and
Staphylococcus spp. by 8 mm and the MIC of the extract were 0.25 mg/ml and
> 1 mg/ml against
Salmonella spp. and
Staphylococcus spp.
, respectively [
29]. Antimicrobial activity of extract of stem bark showed 12 ± 0.85 mm and 22 ± 0.96 mm ZOI against
Salmonella spp. and
S. aureusat 100 mg/ml, respectively, [
14].