Cestrum- nocturnum is a woody, evergreen shrub that can reach a height of 4 metres (13 feet) with thin branches. Simple, slender, shiny, smooth leaves with a full border [1]. The greenish-white blooms unfold around dusk. It releases a strong, pleasant scent at night [2-4] (Figure 1-3) (Table 1,2).

Figure1: Cestrum Nocturnum Flowers

Plant Collection

Cestrum nocturnum leaves were gathered from a nursery in Baghdad.  The plant material was gathered in January and allowed to air dry in the shade at room temperature. followed by weighing and powder grinding [5].

Figure 2: Cestrum Nocturnum Fruit

Figure 3: Cestrum Nocturnum Leaves

Table 1: Phyto Constituents of Cestrum Nocturnum

Table 2: Pharmacological Activity of Cestrum Nocturnum

Preliminary Phytochemical Study Of Cestrum Nocturnum Leaves

In order to conduct a qualitative analysis of the major classes of secondary metabolites, the plant extract underwent phyto-chemical screening [6].

Test- for Saponins (Foam Test)

Ethanolic extract of the flowers (1mL) was mixed with distilled water (5mL) in a test tube and then shaken until persistent foam appear.

Test- for Terpenoids (Salkowski Test)

Plant extract (5 mL) was combined with 2 mL of chloroform, and 3 mL of pure sulfuric acid was added very carefully. When terpenoids are present, an interface with a reddish-brown colour forms.

Test- for Flavonoids

To 1 mL of ethanolic Cestrum nocturnum extract, 2 mL of ethanolic KOH was applied. The development of a yellow hue signifies the existence of flavonoids.

Test- for Tannins

In a test tube, a 0.5 g powdered plant sample is cooked with 20 ml of distilled water. After filtering, the filtered material was mixed with 1% ferric chloride. The presence of tannins is indicated by the colour, which is either brownish-green or blue-black.

Preparation of Extract

Method of extraction (Hot extraction): 50 grammes of ground-up plant material from plant leaves that had been shade-dried and ground up were placed inside a soxhlet apparatus thimble and extracted for eighteen hours using 500 millilitres of hexane. To obtain a dry brown extract, the extract was filtered and the solvent was removed using a rotary evaporator. After being repacked in the soxhlet apparatus thimble, the residue was extracted for 16 hours using 500 mL of ethanol.  To obtain a dry extract, the extract was filtered and the solvent was removed using a rotary evaporator.

Microorganisms under test included one species of gram-negative bacteria (Escherichia coli) and one species of gram-positive bacteria (Staphylococcus aureus). The isolates were from Al-Esraa University College's Department of Pharmacy and Microbiological Laboratory.

Extract of ethanol Agar well diffusion bioassay: The agar well diffusion technique was used to evaluate the antibacterial activity of ethanol extract using pure cultures of all kinds of bacteria. The test microorganisms were grown in stock cultures for 22 hours at 37˚C in Muller Hinton Broth (MHB, Merck, Germany) medium. The final cell concentrations were calibrated to 1.5 108 CFU/ML using the McFarland turbid metre as a reference (10, 11). After allowing the media to settle, wells were made in the seeded agar plates using a cup borer (6 mm) into agar. The bacteria under test were then placed into four wells in each agar plate, and (100µl) of extract dilutions (2 grams/mL, 1 gram/ml, and 500 mg/mL) were added [6,7].

Figure 4: The GC Mass Analysis was Carried Out at the Ibn AL-Betar Research Centre/Ministry of Industry/Baghdad

Using ethanol as a negative control was done. The antibacterial impact was estimated using the diameter of the inhibitory zone after the plates were incubated for 24 hours at 37 °C. The antibacterial action was assessed based on the diameter of the inhibitory zone that formed across the well.

Gas Chromatography (GC) Analysis of Hexane Extract

GC analysis was carried out to detect the presence non-polar active constituents in the plant extract. Helium was employed as the GC carrier gas, and it was injected in a volume of 1L with a splitting ratio of 2.0. The injection temperature was 250°C. The temperature of the column was gradually increased from 80°C to 310°C at a rate of 10°C per minute (Figure 4).

Antibacterial Assay by Agar Well Diffusion Method

The antibacterial activity of hexane and ethanolic extracts was tested using pure cultures of all bacterium species and the agar well diffusion method. The bacteria were grown in stock cultures for 22 hours at 37 degrees Celsius in Muller Hinton Broth (Merck, Germany) medium. The McFarland turbidity metre was used to adjust the final cell concentrations to 1.5 108 CFU/mL.  The medium was allowed to solidify, then cup borer (6 mm) holes were made in the seeded agar plates. The hexane and ethanolic extracts were divided into three wells, and 100 µl dilutions of each extract, ranging from 2 grammes per millilitre to 500 millilitres per millilitre, were added to the wells on the plate as stock solutions. The negative controllers were ethanol and hexane. The diameter of the inhibition zone was used to determine the antibacterial effect of the plates after they were stored for 24 hours at 37°C. The inhibitory zone diameter around the well was measured in order to assess the antibacterial activity.

Preliminary phytochemical analysis of Cestrum nocturnum leaf ethanolic extract. According to Table 3, Figure 5, the ethanolic extract of Cestrum nocturnum leaves underwent preliminary phytochemical testing that revealed the presence of terpenoids, terpenes, flavonoids, and tannins [8-10].

Anti-Bacterial Assay of Cestrum Nocturnum Leaves

Anti-Bacterial Assay of Hexane Extract of Cestrum Nocturnum Leave: Agar well diffusion method was used to screen the hexane extract of Cestrum nocturnum leaves for antibacterial- activity. Hexane was used as a negative- control against gramme positive (S. aureus) and gramme negative (E. coli) bacteria at concentrations of 2000 mg/mL, 1000 mg/mL, and 500 mg/mL of hexane extract, as indicated in Table 4 [11,12].

Figure 5: Oleic Acid Chemical- Structure

Figure 6: Results of Antibacterial Activity of Cestrum Nocturnum Leaves Ethanol Extract Against S. Aureus by Agar Well Diffusion Method

Figure 7: Results of Antibacterial Activity of Cestrum Nocturnum Leaves Ethanol Extract Against E.coli by Agar Well Diffusion Method

Table 3: Qualitative Profile of the Phytochemicals Found in The Leaves of the Studied Plant

Table 4: GC Mass Information of Oleic- Acid

Table 5: Using the Agar Well Diffusion Method, the Antibacterial Activity of Cestrum Nocturnum Leaves Hexane Extract Was Determined in Millimetres with Respect to Various Bacterial Species

Table 6: Using the Agar Well Diffusion Method, The Antibacterial Activity of an Ethanol Extract Made from The Leaves of Cestrum Nocturnum Was Evaluated in Millimetres Against Various Bacterial Species

At varying concentrations, the hexane extract has antibacterial action against S. aureus but not against E. coli.

Anti-Bacterial Assay of Ethanol Extract of Cestrum Nocturnum Leave

Agar well diffusion method was used to screen the ethanol extract of Cestrum nocturnum leaves for antibacterial activity. Ethanol was used as a negative control against gramme positive (S. aureus) and gramme negative (E. coli) bacteria at concentrations of 2000 mg/mL, 1000 mg, and 500 mg/mL of hexane extract, as indicated in Table 5. At varying concentrations, the ethanol extract has antibacterial action against S. aureus but not against E. coli [13-16]. 

The preference of Gram-positive bacteria for extracts (hexane and ethanol) over Gram-negative bacteria was studied in this study. The study concluded that Gram-positive bacteria are preferred because they lack the hydrophilic polysaccharide chain that Gram-negative bacteria use as a barrier. Because of this distinction, Gramme negative bacteria are less susceptible to the Cestrum nocturnum extract (Table 6) [17-20].

Because of the antibacterial characteristics of the flavonoids and glycosides found in ethanol extracts, the ethanolic extract outperforms the hexane extract in antibacterial activity against S. aureus (Figure 6,7).

• A phytochemical analysis of Cestrum nocturnum grown in Iraq found significant amounts of terpenoids and flavonoids, two types of beneficial compounds

• The antibacterial qualities of ethanol extract are potent against pathogens that are Gramme positive (Staphylococcus aureus) and Gramme negative (Escherichia coli)

• This study examined the preference of Gram-positive bacteria for ethanol extract over Gram-negative bacteria. The study concluded that Gram-positive bacteria are preferred because they lack the hydrophilic polysaccharide chain that acts as a barrier for Gram-negative bacteria. This difference means that gramme negative bacteria are less susceptible to the Cestrum nocturnum extract

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