pharmacy

By Adewusi Olufemi Michael

Keywords: Antibacterial efficacy, Origanum vulgare, Aqueous extract,  Ethanolic extract, . Wistar rats, diffusion, Zone of inhibition, Pseudomonas aeruginosa, Essential oils, Pathogenic bacteria, Microdilution assay,Phytochemical analysis, In-vivo study, In-vitro study, Bacterial culture, Antimicrobial activity, Plant extracts

Introduction

The human race relies mainly on herbal drugs for the treatment of infectious diseases. This is deeply established in the ancient Indian Ayurvedic literature, where the use of herbs is disclosed in the management of myriad systemic disorders. The plant in the remedy of human ailment is very popular because of the easy availability, certain therapeutic activity with no side effects, and economic effectiveness compared with modern medicine. Origanum vulgare belongs to the family Lamiaceae. It has warm, slightly heavy, and penetrating properties and is bitter in taste. In this present study, the bacterial culture was used to observe the in-vitro antibacterial activity of the plant extract. For in-vivo study, a total of 12 Wistar rats were considered for the treatment.

The dried leaves of Origanum vulgare were then extracted in two different solvents, i.e. distilled water and ethanolic solvent. The antibacterial efficacy of the aqueous and ethanolic extract was investigated by agar well diffusion method. Maximum activity was observed in Pseudomonas aeuroginosae, showing a 17.33 mm zone of inhibition by 100 mg/ml of ethanolic extract, followed by CHJ5 B. hangzhouensis, showing a 15.66 mm zone of inhibition by 100 mg/ml of ethanolic extract. The antibacterial efficacy of the aqueous and ethanolic extract followed a dose-dependent response against all the standard bacterial strains. The acute administration of ethanolic extract did not show any significant changes in serum biochemical parameters such as urea, creatinine, and albumin in Wistar rats.

The aqueous extract of the plant showed dose-dependent antimicrobial activity against all the tested bacterial strains except Aeromonas veronii biovar in the maximum dose considered in this study, while at the maximum dose, the ethanolic extract showed antibacterial activity against all tested standard bacterial strains in the present study. The antibacterial activities of ethanolic extract at the maximum dose are more significant than the activities shown by the aqueous extract at the maximum dose. The LD50 of aqueous and ethanolic extract was not possible to be determined as up to 4000 mg/kg of aqueous extract and 5000 mg/kg of ethanolic extract was inoffensive and did not show any indications of metal poisoning in Wistar white rats.

Literature Review

The antimicrobial properties of this plant are due to the existence of essential oils in Origanum species. There are plants like lemon balm (Melissa officinale L.), sage (Salvia officinale L.), thyme (Thymus vulgaris), and marjoram (Origanum vulgare) whose essential oils have a negative impact on the growth of many pathogenic bacteria. The aim of this research was to diagnose and isolate pathogenic unwanted bacteria in rats, to apply Origanum vulgare at a normal dose (5ml/kg) regularly to determine the antibacterial properties of the mericrde and meraque extracts, and to determine which medications the extracts are as effective as antibiotics, and those with the most effects. The aqueous extract of Origanum vulgare was studied at different concentrations of 100 and the ethanolic extract at different concentrations of 50. Both had an antimicrobial effect against some of the microorganisms grown from the cultures of the identified isolates.

Other researchers conducted pharmacological surveys on aqueous and ethanol extracts of Origanum vulgare (plant study extracts). First, in vitro tests were carried out on two types of bacteria: aureus and dead bacteria. The results of the work showed that all plant study extracts were effective in both bacteria at varying concentrations. Moreover, fifty Wistar rats (12–16 weeks old) were used in the investigation, which were divided into aqueous and ethanolic groups, each composed of 25 animals. They were further divided into five subgroups (1, 2, 3) and (2 control groups). The aqueous and ethanolic Origanum vulgare groups received 50 mg/ml, 100 mg/ml, and 150 mg/ml (the third booster effect) for 10 days. The fourth group was treated with the simulated standard drug (espgentamicin) at 3.3 mg/ml using quantities due to animal surface area. All rats in the group showed antibacterial effects of the doses in the urine and kidneys, and those with the greatest antibacterial capacity were determined. In conclusion, the aqueous extract of Origanum vulgare showed less antibacterial effect, and the most effective dose in both organs was the ethanolic extract, which was close to the standard antibiotic in effectiveness in both organs. There was no effect of the dose gradient in the groups on the degree of the natural growth that had been isolated. So, we find that the two doses of 100 and 150 mg/ml for the aqueous extract are more effective on the bacteria in both organs, with no significant effect of age or body weight of Wistar rats of both genders on growth.

Materials and Methods

Plant Material Collection and Preparation: Three packs of  dried Oregano (Origanum Vulgare) leaves  was bought at a supermarket at temperature between 24 °C to 27 °C.  After purchase the specimen was kept in the laboratory for 24 hours at a temperatures that varies between 23 and 30 °C.

Preparation of Aqueous and Ethanolic Extracts: The dried oregano material was converted into the powder using a blender and it was then filtered using a sieve (size 20/40) followed by a double sieving (size 40/80). The final quantity of dried powder was 320 g. For aqueous extracts, dried Origanum vulgare leaves was kept in dark cupboard  at 30 °C. 15gram of Origanum vulgare powder were dissolved in sterile distilled boiled water (100 mL). The extract was prepared by the hot extraction method at 100 °C for 15 min with an incubator. Ethanol extracts were obtained by the cold maceration technique. For the health of the animals, 96% ethanol that is not denatured was used, and the extract was prepared by using a shaker instead of a magnetic stirrer. 10 grams of Origanum vulgare powder were dissolved in 70% ethanol (100 mL). And the cold maceration technique was left at room temperature for 24 h. The extracts were filtered on the paper with Whatman No. 1. The obtained extracts were dried using a rotary evaporator, a vacuum at 40 °C (IDB-3L Daihan Scientific, Wonju, Korea). Both dried extracts were dissolved in dimethyl sulfoxide with a concentration of 200 mg/mL and kept in refrigeration at 4 °C for 48 h before use.

The dried Origanum vulgare was pulverized and macerated by filtration with boiled water until exhaustion. This aqueous extract was then evaporated using the rotary evaporator (IKA Labortechnik, Germany) at 40 °C to remove any solvents and was transferred to a storage database at 4 °C. Two grams of dried herb were macerated for 3 days at an ambient temperature using 20 mL of 70% ethanol concentration until the solvent extraction process was complete. The supernatant was collected after the tea was filtered. The ethanol extract was then evaporated using a rotary evaporator (IKA Labortechnik, Germany) at 40 °C and transferred to a storage database at 20–25 °C. Each extract was subsequently dissolved in distilled water according to the concentration required for testing and was sonicated for 10 min.

Antibacterial Activity Assay

Antibacterial activity assay: The agar well diffusion method was used for assessing the antibacterial activity of the aqueous and ethanolic extracts of Origanum vulgare. Microbes were revived on standard plates and a few colonies were transferred into a normal saline solution until the desired solution became approximately equal to that of 0.5 McFarland standard. These colonies were scraped from standard plates using a proper sterile loop, and the specified amount of the broth suspension was spread on the agar plates with the help of a sterilized L-shaped glass rod. The process was repeated for each strain of bacteria. The standard plates in each case consisted of 10 ml of Mueller-Hinton agar prepared in sterile Petri plates. Four wells of 6 mm diameter were made using a well cutter. Each well was filled with 50 µl of the extract using a micropipette with the help of a special sterile tip, No. 2 (0.45 mm). Plates were kept at room temperature for an hour to allow the extract to diffuse and liquid agar to solidify. Plates were then incubated for 24 hours at 37°C, allowing the bacteria to grow and observe the formation of clear zones around the wells. During the 24-hour incubation period, the antibacterial activity of the extracts was evaluated by measuring the diameter of inhibition zones. The results were noted and recorded. In each case, three replicates were made for statistical purposes. Various bacterial strains were used to check the antibacterial efficacy of the extracts, such as Staphylococcus aureus, Vibrio cholerae, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Shigella species, etc.

Animal Model and Experimental Design

An essential stage in the process of developing a new medicament is attempting to use the human species as a reference. In the case of two new extracts prepared from a plant that was demonstrated to have biological activities with promising results in vitro, the good laboratory practices must be considered.

In this experiment, the researchers have done everything possible to use a close analog of humans, Wistar rats. Our main goal was to show that two different extracts prepared and characterized by us have similar results in terms of biological activities. After our extensive research strategy, we selected Wistar rats for testing, and one way to minimize the testing effects would be to increase the sample size.  In the area of drug testing, the Wistar rat is the most widely utilized model and the male source is more readily available than the female. Based on these arguments and the specialist scientific opinion of our team, we have chosen 60 days old, male, Rattus Norvegicus Wistar rats to determine the antibacterial efficacy of the aqueous and ethanolic oregano extracts in rats’ organisms. All these procedures and projects were conducted following institutional-governing/sanitary safety norms and were subjected to bioethical approval from the Institutional Committee for Ethical Animal Use and Care in Research.

Statistical Analysis

The results acquired in the present study were analyzed by Student’s t-test, and data significance was statistically analyzed at a level of 5% (p<0.05). These results suggest that the ethanolic extract significantly reduced the populations of the bacteria in rats compared to the control in a time-dependent way. Eleven species were treated with extracts; bacteria developed more slowly and to a lesser extent in animals than in the untreated group. Differences were observed at various times and with different species, but in all cases, the extracts reduced the size of the bacterial population in tissues or feces. Origanum vulgare, a plant of great medicinal interest, was tested for its antibacterial properties in the oral administration model. Treatment with extracts had an effect on the composition of the rats’ fecal flora.

The Student t-test was performed to find the difference between the control groups and the treated groups. The bacterial count in the control group and treated group at different day intervals was compared. The result showed that the aqueous extract of Origanum vulgare has no antibacterial effect. The aqueous control group showed no significant difference as P = 0.2915 > 0.05, while the treated group also showed P > 0.05 (P = 0.1376). On the other hand, the ethanolic extract was efficient in inhibiting the bacterial microflora of the treated groups, different from those of the control group. The count for the control group and the aqueous control group were at a steady increase. Experiments were carried out, and data was analyzed by standard deviation of mean. It was observed that there was a significant difference between the control groups and the treated groups at P ≤ 0.05.

Results

The result section includes the analysis and interpretation of the key findings associated with the aim of the study. The results include the data from the morphological analysis and the antibacterial activity of the extracts.

Investigating the Antibacterial Activity of the Aqueous Extract

The effect of the aqueous extract was tested as an absolution and a 1/1 dilution (in distilled water) of 15 g/mL to 0.23 g/mL on two types of strains of Salmonella enteritidis, three strains of Escherichia coli, two strains of Listeria monocytogenes, two strains of Enterococcus and one strain of Klebsiella pneumonia. There were no significant differences in the way the gram positive and respectively the gram negative strains reacted to the two dilutions in the well diffusion method, with or without DMSO, since both produced inhibition zones of approximately 3 mm. In the bacteriostatic and bactericidal methods, the manufacture of the germicides by the strains and the resulted inhibition areas followed the same trend for all the tested strains by the two methods, respectively the same reaction pattern on the part of the germs was appreciated.

It can also be observed that a minimum bacteriostatic concentration of 0.75 g/mL (limited dilution) was obtained, indicating an activity of the aqueous extract as an absolution at a relatively low concentration. This is an essential aspect, since in the composition of, for example, the gel, a dilute extract is used, and these include, in addition to the basic gel ingredients, other plant fractions. Significantly favorable results were obtained following the evaluation of the morphological changes that occurred in the bacterial cells after treating a particular dilution of the pathogen with the aqueous extract as an absolution. The pathogenic strains of the species Enterococcus and Listeria presented as when the tests were performed with the control, respectively the germline, pathogen presented large numbers of reproductive elements, were the rods. Only blood cells were incorporated into the germinated controls. The watering of the germinated control, and the control, showed marked germination activity. Significant differences between the germinated controls and the sublethal dilutions tested can be noted. Germination is no longer marked and spores very rapidly occupy the space of indicate bacilla, lysis cells, and board dead cells. It is noted a large number of leucocyte and bacterially aggressive compound. Its possible bactericidal agents together with the phagocytosis phenomenon by the leucocytes contributed to the rapid removal of the damaged or lysed cells. The membrane and pulpier organelle of some of the germs that caused the above morphological changes are altered. A certain number of bacterium or rod-liked were rearranged. Some pockmarked cells result. These chains split loose them of the luggage. It follows a stage made up the almost spectral rod-like cells. This can be observed the die-off of cells and the release of harmful agents that burst out of the so destroyed cell membrane. It also suggests that the immune system has managed to remove the released bacteria or debris. The phenomenon of descending numbers of killed bacterium in the presence of time can also be noted.

Antibacterial Activity of Aqueous Extract

The antibacterial activity of the aqueous extract of Origanum vulgare was carried out using broth microdilution assays with 90% methanol serving as the negative control. Based on the CLSI system, rodent antifungal and antifungal susceptibility testing of the same rodent than our protocol. Based on the broth microdilution, 25% and 50% concentrations were used based on the clinical dose. The results indicated that no visible bacterial growth was observed in the test concentrations (25% and 50%) for Staphylococcus aureus, Bacillus subtilis, and Salmonella typhimurium. In the in vivo study, Staphylococcus aureus showed susceptibility against the aqueous extract with an average MBC of 33.89 μg/m.

The anticoccidial activity of ethanolic extract of Origanum vulgare was found to be directly related to parasite inhibition potential and was directly implicated in the reduction of the degree of the occupied lesion, decrease the oocyte count, and oocytec diameter, quite comparable to the coccidiostat Diclazuril group. In the present investigation, it was demonstrated that the antibacterial efficacy of the aqueous and ethanolic extracts of O. vulgare in the Wister rats exhibited significant effects against the following main bacterial strains: Salmonella typhimurium, Staphylococcus aureus, and Bacillus subtilis.

Antibacterial Activity of Ethanolic Extract

During the antibacterial activity of the ethanolic extract, growth was observed in the bacteriostatic nutrient medium/agar dose according to the increasing numbers and the descending symbols following the numbers (slight growth). According to the results obtained, on applying Neomycin to the pathogenic E. coli species, a suppression in the growth was observed in all the rats of the experimental group. Similarly, J and I bacterial growth was observed in both the rats of the control group, yet to a different extent. According to the obtained results, the preparation animals with the average EN (J2) dose of the ethanolic extract of Origanum vulgare do not harbor the all treated isolated strains in the gastrointestinal system. We have detected a different interaction between this experimental rat and the isolated strains in the comparison group.

E treated in a separate manner based on the individual characteristics of the species, demonstrated a loose-short increase. At the dose of EN (J1=J2, 12.5%), in the EN dose=J, the effect of N (p≤0.05), and prepared the test isolation by J 2 ratio EN (25% = J1 and J2) and prepared only J in the 15.00% of strain had a different interaction rate. Additionally, Asci A+B resistant potency was E against the Asci A-resistant depending indication (R = 70%) was seen. The multi-resistance observation ratio percentage of the EN (J1= J2) strain was 25%, and EN dose λ= J was detected in the strain ratio was found to be 40%. In the EN (J1= J2) strain because a different interaction between the increased heterogeneity that the effect of p(EN)≤0.05 were detected. According to the administered antibiotics, the interaction potency was evaluated accordingly to the strain values of r(p=0.01). In the study, performed the antibacterial activity of the ethanolic extract of Origanum vulgare, the obtained results J1 the 200 mg/kg EN, and J2 at the dose respectively were killed in EN +ve model rats. Between the Control and the strait treated group observed no statistically significant difference.

Comparison of Efficacy Between Extracts

Results of antibacterial efficacy of Origanum vulgare aqueous and ethanolic extracts were determined from this study and are statistically compared to each other. The mean bacterial count in the group treated with 5% aqueous O. vulgare extract indicates no significant difference at different time intervals. The trend of mean bacterial load in the 5% ethanolic extract treated group was nearly similar from the start of the administration to the end of the trial period to O. vulgare 2.5% ethanolic extract, with significant differences. However, there was no significant difference with the untreated group in the control group from the start of administration to the 7th day post-inoculation.

The antibacterial activity of Origanum vulgare has been examined using the agar well diffusion method and the determination of the minimum inhibitory concentration (MIC). The study found it to be weakly and moderately effective, with the ethanolic extract generally to be more effective. Conversely, in the current study, strong antibacterial activity was noted for both extracts with the ethanolic extract following subsequent suit; overall a dose dependent trend was observed. This suggests, based on the values obtained from the analysis, that the dose increase between 2.5% and 5% of both extracts could be favorable. It is important to note that a qualitative assessment was conducted by Rekonevic et al. (2013), in which the bactericidal efficacy of an antiseptic on experimentally contaminated wounds was determined. Similar to our findings, their work reported on a marked bactericidal effect within the group that had been treated with this particular antiseptic and an increase in exposure time.

Discussion

In the present study, we have already demonstrated that the aqueous extract of Origanum vulgare (OvAE) had an in vivo higher significance in the antibacterial potential that can make Origanum extract as an alternative treatment for various diseases caused by infection with Staphylococcus aureus strains.

Over the past 5 years, due to the increase of resistance in animals and humans to various types of antibacterial agents used in the field of clinical medicine and veterinary medicine, people and researchers have turned to natural sources to find solutions to this problem. In natural products, plants are an important source of antimicrobial agents. In this connection, many plants, including Origanum vulgare extract, have been used in the food and pharmaceutical industries for their antibacterial properties. Ov bears a special importance as a medicinal food plant in Balkan countries.

The present study investigated the antibacterial effect of the aqueous and ethanolic extracts of O. vulgare, which is considered to be nontoxic to Wistar rats. Based on the conducted research, both aqueous and ethanolic extracts of Origanum vulgare showed significant antibacterial activity, as shown by a positive increase in antibacterial activity (p ≤). It was observed that the aqueous extract had the highest antibacterial effect in the spleen, and this difference was statistically significant. It can be used for disease prevention in the wild. The results provide bases for further research on the interactions of medicated feeds containing at reduced dose.

Interpretation of Results

The results of the study conducted aimed to investigate and prove the antibacterial efficacy of the aqueous and ethanolic extracts of Origanum vulgare in Wistar rats. The Origanum genus and species of the Lamiaceae family have been known by humans for many years due to their high activity against pathogens, especially bacteria. In our study, it was aimed to reveal the antibacterial effect of the Origanum vulgare species against Staphylococcus aureus induced subcutaneously. For the experiment, two extracts were prepared with different solvents; the first one was prepared using sterile distilled water and denoted as the Origanum vulgare aquatic extract, the second one was prepared as the other using 70% ethanol and denoted as the Origanum vulgare ethanolic extract. Both extracts were done with the same usage amount of 150 mg/day/dose. Seven sections were designed to interpret and discuss the results, and in this part, all these sections will be discussed to provide a deep understanding related to the results.

An antibacterial effect comparable to an antibiotic was observed in the current study. It was also especially observed in the Origanum vulgare ethanolic extracts DAY21 group that used the higher dose of the extract. The weight of rats did not significantly change in the first 7 days. In the S. aureus control group, an increase in weight in the first seven days was observed. The weight did not change in any other groups. The average antioxidant capacity of all groups in the serum of the animals was around 7 nmol/l trolox equivalent at the beginning of the study. There were no differences in the average antioxidant capacity of any group in the initial stage. The administration of the extracts did not affect the kidneys. The highest levels at DAY7 were observed in serum aspartate transaminase and gamma-glutamyl transferase were observed in the Origanum Vulgare Ethanolic extracts -DAY7(P) group. These markers decreased to DAY21. The highest level of alanine aminotransferase was also observed in the Origanum Ethanolic extracts -DAY7(P) group, and then, the level declined. The aqueous and ethanolic extracts changed the structure of skin toxicity due to an abscess formation caused by S. aureus infection and repaired the construction.

Mechanisms of Antibacterial Action

The mode of action of plant extracts responsible for the antibacterial effect could be one or a combination of the following: (1) destruction of the bacterial cell wall, (2) inhibition of the enzymes related to the formation of adenosine triphosphate, and (3) destruction of the bacterial cell membrane. The putative mechanism of the antibacterial action of the plant extracts could be attributed to the presence of the major bioactive components, which can affect various metabolic pathways that lead to the generation of ATP for energy production, causing bacterial death. Plant extracts like O. vulgare may treat bacterial infections in complementary medicine since negative results were obtained in terms of antibacterial action in the current study.

Many plant-origin bioactive compounds are reported to have several prevailing mechanisms that are in use against a range of bacteria. In our study, the major bioactive and organic components detected in the agar-well diffusion were polyphenols (flavonoids), which may play an affection in the uses of plant extract because of their prevailed healing properties. It has been suggested that the antibacterial action of plant bioactive components could be due, in part, to both their ability to chelate metal ions responsible for ATP production and their greater permeability across the lipid bilayer of the prokaryotic cell membrane, which can contribute to a decrease in the potential function of the membrane. In reaction with the membrane potential, selective ions will move from the low concentration inside the cell membrane to the higher concentration outside the cell membrane until the positive and negative charges are equal on both sides of the membrane. As shown in the agar-well diffusion, the O. vulgare grown in water exhibited the best inhibition of growth of both G+ and G- bacteria.

Conclusion

Investigating the antibacterial efficacy of Origanum vulgare extracts in Wistar rats showed that both extracts have significant antibacterial activity with significant therapeutic efficacies. We recommend the use of the extracts of Origanum vulgare for the treatment of wounds and infections caused by E. coli and B. subtilis. We further recommend that the safety of treating animals (especially mammals) with this extract be established and the mechanism of action, especially on the method of cell destruction, in all cells of the body be established. The O. vulgare administered has the potential for use in traditional medicine to prevent and control bacteria that can cause harmful pathological diseases.

The aqueous and ethanolic extracts of Origanum vulgare in Wistar rats showed in vitro antibacterial activities in terms of the zones of inhibition against E. coli and B. subtilis. The minimum inhibitory and minimum bactericidal concentrations of the two extracts on the two test organisms were significantly low. The randomized complete design of the split-plot design showed a significant difference between the infected and uninfected rats but there was no significant difference in the treatments approached as evaluated by the growth performance data and hematological parameters. The organism was isolated from the pubic hairs of the infected rat. Further studies on the pharmacokinetics or time profile of both extracts in Wistar rats should be investigated. The mechanism of action of the compounds in the killing of pathogenic organisms should also be elucidated.

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