ABSTRACT

This investigation looked at the effects of L. inermis extracts on vaginal isolates in terms of their antibacterial activity. Cold maceration was used to extract L. inermis leaf powder, and the resultant ethanol, aqueous, and chloroform fractions were separated. To produce residues that could be kept at 4°C, the resulting extracts were dried in an oven and then concentrated to a dry state. Saponin, tannin, alkaloids, terpenoids, and steroids were found in the residues after a phytochemical screening. Following accepted microbiological procedures, filthy HVS samples were randomly obtained from students, staff members, and inhabitants of hostels. They were then cultured. The samples showed the presence of Proteus spp., E. coli, Pseudomonas spp., Klebsiella spp., and Staph spp. The antibacterial activity that was concentration-dependent and varied with the organisms found was assessed using the agar diffusion technique on a broth culture, and it produced a result of 9.46%. For Proteus species and Staphylococcus species, the MIC of the crude extract and aqueous extract was 50 mg/ml, whereas it was 25 mg/ml for Klebsiella and Pseudomonas. For all of the isolated species, the aqueous extract showed a high MIC of 100 mg/ml. The chloroform extract (12.5 mg/ml) was shown to have the optimum minimal inhibitory concentration profile for Proteus species (8.0 + 1 mm), Staphylococcus species (12.0 + 0 mm), Klebsiella species (8.5 + 1.5 mm), and E. coli species (7.5 + 1.5 mm), as well as 6.25 mg/ml for Pseudomonas species (6.0 + 0). This study demonstrated that the leaves of Lawsomiainermis contain phytoconstituents that might cause an antibacterial response.

Key Words: Lawsomiainermis, Antimicrobial, Inhibition zone diameter, Phytoconstituents, MIC

INTRODUCTION

For ages, man has relied on nature to provide for his fundamental necessities, including food, clothes, shelter, and medicine [1, 2]. Many people consider plants to be a natural remedy for treating and preventing a variety of ailments [3, 4]. Plants have been employed in traditional herbal medicine for a long time [5, 6] because they are now recognized as natural sources of powerful and novel antibacterial agents [7, 8]. Because of the wide variety of medicines that are easily available, these plants are sources of natural compounds that are utilized as ethnomedicine in several nations across the world [9] and provide limitless prospects for developing novel pharmaceuticals [10, 11].

Traditional medicine is thought to be used by 80% of people in underdeveloped nations for their primary healthcare, and it is becoming more and more popular as a result of the high toxicity and negative side effects of orthodox medication [10, 12]. Herbal medicine has long been characterized as a course of care that entails a coordinated pharmacological intervention of several distinct substances interacting with numerous targets, as opposed to a single medicament engaging with a single target [13, 14]. Natural active compounds found in plants may be utilized to cure ailments and improve health, which is the basis for herbal treatments. Utilizing plants and plant extracts to treat a variety of illnesses [15].

Henna, mehndi, and mignonette trees are all common names for the tall shrub plant Lawsoniainermis, which is a member of the Kingdom. Plantae, Division: Angiospermae, Class: Dicotyledoneae, Order: Myrtales, Family: Lythraceae, Genus: Lawsonia, and Species: L. inermis L. A blooming plant with a height of 2–7 meters, henna is found in subtropical and tropical climates all throughout the world, including South Asia, Africa, Oasis in the Sahara Desert, and even northern Australia. The elliptic, obovate to oblanceolate, acute at the apex, sometimes mucronulate, constricted into a short petiole, pinnately nerved, and submembranous leaves of henna plants [16] have these characteristics. Lawsone (2-hydroxynaphthoquinone), mucilage, mannitol, gallic acid, and tannic acid make up the majority of henna's chemical components [17]. According to reports, henna has antibacterial [18], immunostimulant [17], and hypoglycemic effects. The goal of the current study is to ascertain if Lawsoniainermis leaves have any antibacterial properties against isolates of vaginal infections.

MATERIALS AND METHODS

Reagents and equipment

Ethanol (99.9%) (Loba  Chemie  PVT Ltd, Mumbai, India), Lawsomia inermis leaves, Petri dishes, test tubes, swap stick, cork-borer, spatula, precision balance, agar medium (Loba Chemie PVT Ltd, Mumbai, India), autoclave, flame, incubator, rotary evaporator, wireloop, meter rule, and agar broth. The different chemicals and tools needed for this project were acquired from the laboratories of the Pharmaceutical Microbiology Department of the Faculty of Pharmacy at Delta State University, Abraka (DELSU).

Extraction of lawsomia inermis leaves

The pulverized leaves of L. inermis were collected from a local outlet in Kaduna, Nigeria. The leaves were then divided into ethanol, aqueous, and chloroform fractions after being extracted using cold maceration. The fractions obtained were filtered and concentrated in an oven to 101.2g dryness, and the residues recovered were refrigerated at 4°C [19].

Phytochemical screening

L. inermis leaf extract was prepared for phytochemical analysis using the techniques described by Enwa et al. (2016) and Okafor et al. (2021), with a little modification.

Fractionation of lawsomia inermis extract

About 45.04g of the extract was weighed and transferred into a separating funnel. The aqueous phase was prepared with 100ml methanol and 75ml of distilled water. About 200ml of chloroform was measured and transferred into the separating funnel which was shaken vigorously for 5 minutes. The upper layer was decanted anda bottom layer containing the chloroform fraction was carefully transferred into a beaker. This process was repeated four times and the fractions were allowed to concentrate at room temperature for 72 hrs. Furthermore, the fractions were packaged and stored in separate beakers for further microbiological studies.

Identification of bacteria ısolate

Biochemical tests such as indole, catalase test, citrate utilization test, H2S test, urease, oxidase, MOT, coagulase, and gram staining were carried out. Glucose, lactose, and sucrose tests were carried out to determine the presence of these sugars [20].

Preparation of broth culture

15g of the nutritional broth powder were dissolved in 1 L of distilled water to create the nutritional broth. After thoroughly blending the solution for 5 minutes to achieve total dissolution, the mixture was placed into test tubes. The solution was then autoclaved for 15 minutes at 121°C. A sterile wire loop was used to inoculate the preparation with the microorganisms, and it was then left for 24 hours.

Evaluation of antimicrobial activity

Molten Mueller Hilton Agar (MHA) was allowed to solidify. Fifteen (15) test tubes were sterilized and labeled crude (CR 100 - 6.25 mg/ml), Aqueous (Aq 100 - 6.25 mg/ml), and Chloroform (Chl 100 - 6.25 mg/ml). Thereafter 0.4g of the extracts weredissolved in non-concentrated ethanol. Two-fold serial dilution was carried out for the crude, aqueous, and chloroform extracts inside the test tubes from a range of 100, 50, 25, 12.5, 6.25, and 3.125 mg/ml. The broth was streaked on the plates and a cork borer 6 mm diameter was used to punch the sides of the plate which was labeled according to the various concentrations used. The holes were sealed with molten agar to close up cracks. The various concentrations of the extract and the control (ciprofloxacin) were introduced into the holes and the plates were incubated for 24 hours. The zone of inhibition was read in duplicate and the average was taken. 

Evaluation of the minimum ınhibitory concentration (MIC)

Determination of minimum inhibitory concentration (MIC) was done using the method described by Enwa et al. 2023 with minimal adjustment.

Data analysis

In order to describe the data, graphs, frequency tables, means, and standard deviations were used. The data were examined using SPSS 22, the Statistical Package for Social Sciences. With a p-value of 0.05, an independent t-test was used for inferential statistics.

RESULTS AND DISCUSSION

Yield

The initial weight of the powdered leaves collected was 1100g. The weight obtained after concentrating was 104.04g. The yield was calculated using the formula below:

Phytochemical screening

The results of the phytochemical screening revealed that saponins, tannins, alkaloids, terpenoids, and steroids were present while flavonoids and reducing sugars were absent (Table 1).

Table 1. Phytochemical Screening

+ means Present

- means Absent

Identification of bacteria isolate

The bacteria isolate was identified using various biochemical tests. The result is presented in Table 2.  Klebsiellaspp, Proteus spp, Staphylococcus spp, and E. coli were mostly encountered.

Table 2. Bacteria isolate identified

KEY:C: Coursemates; H: Hosteller; W: University workers

Inhibition zone diameter of the extracts

Table 3 displays the diameter of the inhibition zone for each extract. The width of the inhibitory zone varies depending on the microorganism detected (Table 3).

Table 3. Inhibition Zone Diameter of the Extracts