E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668
ORIGINAL RESEARCH ARTICLE
*1Umar, F. J., 1Adamu, A., 1Usman, A., 2Balarabe, F. T., and 1Musa, F. M.
1Department of Microbiology, Kaduna State University, Kaduna, Nigeria
2Scientific and Industrial Research, Departments, National Research Institute for Chemical Technology (NARICT), Zaria, Nigeria
Ziziphus jujuba is a common medicinal plant traditionally utilised in tropical and subtropical regions for its various biological functions, such as antibacterial, antifungal, and antidiarrheal properties. This study aimed to investigate the antibacterial activity of Ziziphus jujuba leaf extracts against clinical isolates of Streptococcus pneumonia and Salmonella species using the agar well diffusion method. Phytochemical screening, Minimum Inhibitory, and Minimum Bactericidal Concentration Assays (MIC/MBC) were also carried out using standard procedures. The results of phytochemical screening showed that tannins, phenols, saponins, quinines, terpenoids, and steroids were present in ethanolic extract. Similarly, Tannins, saponins, phenols, steroids, phytosteroids, and terpenoids were present in aqueous extract. Salmonella species were sensitive to the aqueous and ethanolic extracts at 500 and 125 mg/mL, respectively, compared to the 250 and 125mg/mL obtained against Streptococcus pneumoniae, respectively. The MIC values of the ethanolic extract were 62.5 and 125 mg/mL for Salmonella species and Streptococcus pneumoniae, while the aqueous extract exhibited MIC values of 62.5 and 125mg/mL for Streptococcus pneumoniae and Salmonella species, respectively. Regarding the MBC values, 125 and 62.5 mg/mL were obtained for the aqueous extract against Streptococcus pneumoniae and Salmonella species, compared to the 62.5 and 125 mg/mL for the aqueous extract. The antibacterial activity exerted by the extract against the test isolates suggests that Ziziphusjujuba leaf can be explored as a source of effective antibacterial medications targeting the test bacteria.
Keywords: Antibacterial activity, Phytochemical, Salmonella species, Streptococcus pneumonia, Ziziphus jujuba
The utilization of medicinal plants is unquestionably a skill that has been practiced since the beginning of humanity, employed as a remedy for illnesses in ancient times and early civilizations worldwide. Additionally, they have the potential to serve as sources of pharmacologically active substances that can be valuable candidates for drug development (Hosseini et al., 2020). Out of the roughly estimated 500,000 plant species, only around 10% are utilized as food by humans and other animals. Furthermore, a limited number of plants have undergone phytochemical investigation, and only a proportion have been exposed to biological or pharmacological screening (Chowdari et al., 2020).
Jujuba, scientifically known as Ziziphus jujuba (Magarya), is a species in the buckthorn family Rhamnaceae. It is also commonly referred to as red date, Chinese date, and Chinese jujuba. Ziziphus jujuba is alternatively known as Badari, Baer, Bogari, or Barihannu. The leaf of Ziziphus jujuba is extensively utilized in traditional medicine in Northern Nigeria (Abubakaret al., 2018). The jujuba fruit is a delectable fruit that serves as a potent herbal treatment, enhancing endurance and physical power and facilitating weight gain. It enhances liver function and boosts immune system resilience. It serves as an antidote, diuretic, emollient, and expectorant. The leaves are febrifuge, astringent, and claimed to enhance hair development. In the treatment of strangury, they are utilized to form a plaster. The dried fruits have sedative, anodyne, anticancer, refrigerant, styptic, pectoral, tonic, and stomachic properties. They aid in blood filtration and digestion (Venkatachalam et al., 2020). According to Al-Reza et al. (2010), the fruits from the Rhamnaceae family have high nutritional values and anti-infectious properties. Generally, Zizyphus species are widely used as medicinal plants in Asian countries, particularly in Taiwan and China, for the treatment of various liver diseases, urinary troubles, allergies, constipation, depression, chronic bronchitis, and insomnia (Afroz et al., 2014).
Active substances derived from higher plants have recently come to play a significant role in contemporary medicine. There are currently around 130 chemicals in use that have been isolated from higher plants and their synthetic or modified descendants. Several of these substances are used in conventional medicine and are thought to be effective antibacterial agents. However, a significant obstacle to treating infectious diseases in developing nations is the ongoing growth of bacteria resistant to traditional medications. This makes it necessary to look for novel, highly effective antibacterial compounds in locally grown plants (Abubakar et al., 2018).
Leaf samples were acquired from the Botanical Garden, Kaduna State University, Kaduna, Nigeria, and the plant was recognized and verified by a botanist at the Department of Biological Science of the same institution where 190/01 was assigned as herbarium/voucher number for the specimen. The Ziziphus jujuba leaves were carefully cleaned under running water and allowed to dry at room temperature for fourteen days. Using a mortar and pestle, it was ground into a coarse powder and kept in several airtight bottles (Periasamy et al., 2020).
Plant extraction was done using Periasamy et al.'s (2020) procedures. About 100 grams (100g) of powdered Ziziphus jujuba were suspended in 1000 milliliters of 70% ethanol in a conical flask, and another 100 grams of powdered Ziziphus jujuba were suspended in 1000 milliliters of distilled water in a different conical flask. This was combined and agitated briskly with a sterile glass rod before being stored at room temperature in tightly sealed containers. The mixture was continuously shaken with a rotary shaker for a full day. Whatman No. 1 sterile filter paper was used to filter the mixture and transfer it into a sanitized conical flask. After that, filters were placed in the rotary vacuum evaporator's sample container and left there for 24 hours at 28˚C. The resulting semi-solid extract was allowed to dry under a ceiling fan. Using the formula from Periasamy et al. (2020), the % yield of the ethanol and aqueous crude extract of Ziziphusjujuba was determined.
Percentage Yield of Plant Extract (%) ═ W2-W1/W0 × 100
Where
W1═ Weight of the container in grams
W2═ Weight of container + Extract
W0═ Weight of powdered leaf, root, or bark
Phytochemical components of the crude aqueous and ethanolic leaf extracts of Ziziphus jujuba were screened using the method described by Periasamy et al. (2020). Phytochemical components analyzed were Alkaloids, Steroids, Saponins, Phenol, Tannins, Glycosides, Terpenoids, Quinine, and Phytosteriod (Periasamy et al., 2020).
Salmonella species and Streptococcus pneumonia were the clinical isolates used. The isolates were obtained from the Department of Microbiology, Kaduna State University, labelled, and put in an icebox before being subjected to further analyses at the Laboratory of the Department.
Gram staining microscopy and biochemical analysis were used to reconfirm the clinical isolates used as described by Cheesbrough (2002).
For the antibacterial screening, Mueller Hinton agar was produced in accordance with the manufacturer's instructions. The antibacterial activity of the aqueous and ethanol extracts of Ziziphus jujuba against the clinical isolates of Streptococcus pneumonia and Salmonella species was determined using the agar well diffusion method, as described by Chowdari et al. (2020). A sterile cotton swab stick was used to evenly apply roughly 100μl of standardized inoculums of a bacterial solution across the entire surface of Mueller Hinton agar plates, which were inoculated using a micropipette. After giving the plates ten minutes, sterile cork borer wells of six mm were inserted into the agar. As a positive control, another well was filled with the antibiotic ciprofloxacin. After allowing the extract to diffuse into the agar for ten minutes at room temperature, the plates were incubated for twenty-four hours at 37˚C. Using a meter ruler, the diameter of each zone of growth inhibition for each tested bacterium was measured and recorded in millimeters.
The MIC of the plant extracts was ascertained by the broth dilution method, as described previously (Bashir et al., 2022). Muller-Hinton broth in six tubes, each holding five milliliters, was made. In tubes 1-4, one (1) milliliter of the crude extract from (500, 250, 125, and 62.5 mg/mL) was added, and it was well mixed. After that, four tubes each received 0.5 mL of the test organisms' broth cultures, with the final tube acting as the broth control for each. The inoculation tubes were incubated for twenty-four hours at 37°C. 500 mg/mL of ciprofloxacin was employed as the positive control. MIC is defined as the lowest concentration at which no growth was observed.
Mueller-Hinton broth that had been produced was poured into sterile test tubes in five-milliliter increments, equal to the number of tubes that had no discernible growth from the MIC. Then, tubes holding the 5 mL Mueller Hinton broth were filled with 0.1 mL of the broth culture. The tubes were stored on a test-tube rack and labeled. A sterile Petri plate was filled with prepared Mueller-Hinton agar, which was then left to harden. A sterile pipette was used to transfer 0.1 mL from each tube onto the agar's surface. A sterile, smooth glass rod that was bent was used to distribute the inoculum. For twenty-four hours, tubes and plates were stored in an incubator at 37°C. It was noted whether the broth culture was turbid or cloudy, as well as whether bacterial colonies were growing on solid agar media. The MBC was determined by using the lowest concentration of the MIC culture that did not exhibit any apparent bacterial growth (Bashir et al., 2022).
The findings of the phytochemical screening of Ziziphus jujuba leaf extracts in ethanol and aqueous form are shown in Table 1. The findings demonstrated that whereas alkaloids, glycosides, and phytosteroids were absent from the ethanolic extract, tannins, phenol, saponin, quinines, terpenoids, and steroids were present. Additionally, the aqueous extract included tannin, saponin, phenols, steroids, phytosteroids, and terpenoids but not alkaloids, glycosides, or quinines.
| S/N | Constituents | Aqueous Extract | Ethanol Extract |
|---|---|---|---|
| 1. | Tannin | + | + |
| 2. | Saponin | + | + |
| 3. | Alkaloid | - | - |
| 4. | Glycoside | - | - |
| 5. | Quinines | - | + |
| 6. | Phenols | + | + |
| 7. | Terpenoids | + | + |
| 8. | Steroid | + | + |
| 9. | Phytosteroid | + | - |
Key: + = Detected
- = Not detected
The antibacterial activity of an aqueous and ethanol extract of Ziziphus jujuba leaf was tested against Salmonella species and Streptococcus pneumonia, as shown in Tables 2 and 3. Compared to an aqueous extract, the results of the ethanol extract demonstrated more antibacterial activity.
Table 2: Antibacterial susceptibility of the aqueous leaf extract of Ziziphus jujuba against the test isolates solution.
| Organism | 500mg/ml | 250mg/ml | 125mg/ml | 62.5mg/ml | Positive Control Ciprofloxacin mg/mL |
|---|---|---|---|---|---|
| Streptococcus pneumonia | 13mm | 10mm | R | R | 9mm |
| Salmonella species | 14mm | R | R | R | 8mm |
Key: R= resistant
Table 3: Antibacterial susceptibility of the Ethanolic extract of Ziziphus jujuba leaf against the test isolates
| Organism | 500mg/ml | 250mg/ml | 125mg/ml | 62.5mg/ml | Positive Control Ciprofloxacin mg/mL |
|---|---|---|---|---|---|
| Streptococcus | 15mm | 14mm | 12mm | 6mm | 9mm |
| Salmonella | 16mm | 14mm | 12mm | 6mm | 8mm |
The results of the Minimum Inhibitory Concentration of the leaf's ethanolic and aqueous extract on the clinical isolates of bacteria are displayed in Table 4.
Table 4: Minimum Inhibitory Concentration Result of Ziziphus jujuba against the test Isolates
| Organism | Aqueous | Ethanolic |
|---|---|---|
| Streptococcus pneumonia | 250 mg/mL | 125 mg/mL |
| Salmonella species | 500 mg/mL | 125 mg/mL |
The results of the MBC of the leaf's ethanol and aqueous extract on the clinical isolates of bacteria are displayed in Table 5.
Table 5: Minimum Bactericidal Concentration result of Ziziphus jujuba against the test Isolates
| Organism | Aqueous | Ethanolic |
|---|---|---|
| Streptococcus pneumonia | 250 mg/mL | 125 mg/mL |
| Salmonella species | 500 mg/mL | 125 mg/mL |
Aqueous and ethanol leaf extracts of Ziziphus jujuba were subjected to phytochemical screening, and the results indicated the presence of several phytochemical compounds, including tannins, phenols, saponins, quinines, terpenoids, steroids, and phenols, which have been reported to have antibiotic properties (Deshpande et al., 2019; Elshahir et al., 2020; Soni et al., 2021).
According to the study by Duan et al. (2023), the susceptibilities of test bacteria to the active fractions of Ziziphus jujuba leaf were likewise shown to be concentration-dependent, meaning that the higher the concentration, the higher the activity.
Ziziphus jujuba extract has strong antibacterial action,
while ethanol plant extract demonstrated broad-spectrum efficacy. The
study also demonstrates that Streptococcus pneumonia and
Salmonella species were both susceptible to the extract, with
the highest zones of inhibition for both species at 500 mg/mL (16 mm for
Salmonella species and 15 mm for S. pneumonia on the
ethanolic extract), while the zones of inhibition for both species on
the aqueous extract were (13 mm) and (14 mm) for S. pneumoniae
and Salmonella species, respectively. The results of the
investigation corroborate those of Babatunde et
al. (2023), who found that the zone of inhibition increased
with concentration. The results showed that the bacterial isolates'
susceptibility to Ziziphus jujuba leaf extracts was similar to
that of ciprofloxacin (positive control). This suggests that
Ziziphus jujuba leaf extracts could be used as an additional or
alternative antibacterial agent to reduce the risk of bacterial vaginal
infection in women who are antibiotic-resistant.
The results of a previous study by Medini et
al. (2014), where the selected bacterial isolates used in the
study showed good susceptibility to aqueous and ethanolic extracts of
Ziziphus jujuba at varied concentrations, may explain why the
ethanol extract demonstrated more antibacterial activities than the
aqueous extract. Ethanol is an organic solvent and can extract more
phyto-constituents than water. The differences in the cell walls of the
bacteria may be the cause of the isolates' varying susceptibilities to
different concentrations. While Gram-negative bacteria have
lipopolysaccharides, which may have prevented the active components of
extracts from penetrating, Gram-positive bacteria tend to allow
diffusion of the active components. This was also consistent with
research by Yahia et al. (2020), which
found that Gram-positive bacteria had a larger zone of inhibition than
Gram-negative bacteria. However, it was at odds with research by Abubakar et al. (2018), which found that
at 150 mg/ml concentration, Gram-negative bacteria were more susceptible
than Gram-positive bacteria. All of the extract's actions, however, were
dose-dependent, which is consistent with studies by Sakha et al. (2018) that found that the
extract's antibacterial activity increased at higher concentrations.
According to earlier studies (Elaloui et al., 2017; Abubakar et al., 2018; Yahia et al., 2020), Ziziphus jujuba has excellent antibacterial activity against both Gram-positive and Gram-negative bacterial isolates. The MIC and MBC of the study's results are comparable to these studies' findings. The plant's ability to suppress the isolates with low MIC and MBC values was ascribed to the potency of the plant extracts. This is in contrast to the Dubey et al., (2010) findings, which had greater MIC and MBC.
This study revealed the presence of tannins, saponins, terpenoids, alkaloids, glycosides, phenol, steroids, quinines, and phytosteroids as the phytochemical constituents of Ziziphus jujuba leaf extracts, which underscore its antibacterial action against the test isolate.
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