Gas chromatography flame ionization detection of alkaloids, phytochemicals, antimicrobial and antifungal potential of Ageratum conyzoides leaf extract in vitro and in silico studies

Introduction

Infection caused by fungi is a growing public health concern especially in immune-compromised patients. The incidence of fungal infections is steadily increasing year after year [1]. About three hundred fungals cause devastating fungal infections in humans [2]. Fungal infections are grouped into superficial mycoses, opportunistic infections, and systemic infections. The fungal infections implicated in many clinical conditions are the general candida, penicillium and Aspergillus [1]. About 1.5 million deaths and 13 million fungal infections are recorded per year, especially in compromised immune individuals [3]. A major clinical factor contributing to the increase of fungal infections in patients is drug resistance, also anti-fungal therapies like azoles that inhibit 14-α-lanosterol demethylase (fluconazole) and polyenes that inhibits ergosterol from membrane formation (Amphotericine) have encountered some limitations due to off-target effect and the development of resistance strains [4]. Also long term ventilation in critically ill patients has a higher rate of fungal infections in hospital setting [5]. Additionally, fungal pathogens can thrive in an environment altered by antibiotics, making them an opportunistic to establish infections [6]. Various factors that increased fungal infection rate are organ transplantation, the use of anti-cancer drugs, parenteral nutrition, treatment with corticosteroids, dialysis, prolonged use of catheters and  antibiotics [6].

Millions of individuals continue to die from bacterial infections every year despite the huge developmental strides in science and technology. Due to poverty and ignorance leading to overuse and misuse of over the counter antimicrobial drugs, this has really led to the emergence of antimicrobial resistance, resulting in millions of deaths in 2019 [7,8]. Apart from antimicrobial resistance due to mutation also sepsis a life threatening condition in which there is an uncontrolled response of the immune system to infection leading to the malfunctioning of vital organs [8]. In the United States, about 1.7 million people develop sepsis due to prolonged usage of antibiotics, leading to thousands of deaths [9]. The burden of antimicrobial resistance is forecasted to rise to 1.91 million deaths and 8.22 million deaths by 2050 [9]. Therefore, there is a drastic need for a reduction in anti-bacterial mortality now.

Ageratum conyzoides also known as goat weed, belongs to the plant family Asteraceae. It is found in Central America, Africa, Asia and South Pacific Islands [10]. It grows up to about 1 meter high, with small flowers, it can quickly dominate the ecology as an aggressive weed and it also possess aromatic properties [11]. The plant contains bioactives ranging from flavonoids, coumarins, alkaloids, terpenoids, essential oils and minerals [12-16]. Due to the presence of these bioactives, Ageratum conyzoides is used traditionally for the treatment of wounds, diabetes, fever, and many infectious and inflammatory diseases [15,17, 18]. Ageratum conyzoides has also been reported for antioxidant and free radical scavenging ability [19]. It also has antiflammtory, antibacterial and antihyperglycemic properties [15, 14. 20]. Therefore, this study aims to evaluate the phytochemicals and antimicrobial properties of Ageratum conyzoides in vitro and in silico.

Materials and method

Chemical/Reagents

Sodium hydroxide, FeCl₃, KI, iodine, lead acetate, NaNO₂, AlCl₃, Folin-Ciocalteu reagent, acetic acid, Sodium carbonate (Na₂CO₃), DMSO, methanol, ethanol, hydrochloric acid, normal saline, getamicin Injection, fluconazole, gallic acid, quercetin are all standard chemicals obtained from Fluka.

Plant collection and Identification

Fresh leaves of Ageratum conyzoides L. were collected from the Niger Delta University Botanical Garden. The plant was identified/authenticated and voucher specimen deposited at the Herbarium of the Department of Pharmacognosy and Herbal Medicine, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria, with Herbarium number NDUP/040.

Preparation of Plant Extract          

The plant leaves were collected in large quantity, washed and left under shade at room temperature for two weeks to dry. Afterwards, they were ground into a fine powder. 500g of the powder was soaked in 1000ml of methanol and allowed to stand for 72 hours. The extract was filtered and centrifuged and the filtrate was evaporated in a water bath at 50 ⁰C. The paste formed was stored in the refrigerator for further use.

Qualitative phytochemicals

The presence of tannins, saponins, alkaloids, phenols and flavonoids were detected in Ageratum conyzoides extract according to the method of Harbone, [21].

Analysis of condensed tannins

The presence of condensed tannins in Ageratum conyzoides was based on the fact that in acid medium, tannins are transformed into anthocyanins when heated [22]. Two sets of tubes labelled series 1 and 2, in each tube 3ml of methanolic extract of Ageratum conyzoides were added, and 3ml of conc. HCl. One set of tubes were heated in a water bath at 100°C for 30 mins, followed by cooling to room temperature. The other set of tubes were placed at room temperature for 30mins. Later 0.5ml of ethanol was added to both sets of tubes and the absorbance was read at 550nm. The condensed tannins were detected through the following formular:

CT = (OD₁ – OD₂) X 19.33 (g/L)

Where:

CT = Condensed tannins

OD₁ = Optical density of tubes heated at 100°C

OD₂ = Optical density of tubes kept at room temperature

Flavonoid content

The total flavonoid content in Ageratum conyzoides was determined according to Zhishen et al., [23]. Exactly 0.5ml of distilled water was added to extract (1mg/ml) followed by the addition of 5% NaNO₂ (60 µL). This solution was kept at room temperature for 5 min. Thereafter 60 µl of 10% AlCl₃ was added and 400 µl of 1M NaOH and 450 µl of distilled water also added to the solution and was kept at room temperature for 30min. Later absorbance of the mixture was read at 510 nm. Quercetin served as reference compound and results were reported as quercetin equivalent per gram extract.

Phenol Content

The total content of phenol in Ageratum conyzoides was based on the Folin – Ciocalteu method of Singleton et al., [24] and Demiray et al., [25]. Ageratum conyzoides methanolic extracts (1ml) was mixed with 1ml of ten-fold diluted Folin – Ciocalteu reagent and was kept for 5 min. After that 1ml of sodium carbonate (0.7M) was added and the solution was kept at room temperature for 1hr with occasional shaking for the development of color. The absorbance was recorded at 765 nm, and gallic acid served as the standard. Total phenol was extrapolated and reported as gallic acid equivalent per gram extract.

Alkaloid Content

The total amount of alkaloids in Ageratum conyzoides was determined according to Unuofin et al., [26]. A weighed amount of 5g Ageratum conyzoides in triplicate was soaked in 100ml of 10 % acetic acid in ethanol. The solution was left standing for 4 hours at 25⁰ C. Later the solution was filtered. The filtrate was concentrated in a thermostat bath at 55⁰ C to a quarter of its original volume. Thereafter concentrated ammonium hydroxide was added in drops to precipitate all the alkaloids. The solution was filtered, and the alkaloids were weighed and calculated as percentage

Quantification of alkaloids by gas chromatography flame ionization detector

Exactly 0.2g of Ageratum conyzoides was added to a tube containing 15 ml ethanol and 10 ml 50% KOH. The tube was heated at 60°C in a water bath for 3hrs. Thereafter the extract was transported into a separating funnel and the extracted phytochemicals were solubilized in pyridine for alkaloid analysis. The GC-FID detection of alkaloid from Ageratum conyzoides was carried out using Agilent 6890 gas chromatography coupled with a flame ionization detector. Also a 15mm x 0.15mm MXT – 1 column was used. The injection temperature was 280°C and the carrier gas was helium with a flow speed of 40ml/min. Alkaloids were detected and expressed as ppm [27].

Antimicrobial activity of extract

Preparation of the extract concentrations: 100mg/ml, 50mg/ml and 25mg/ml concentrations were respectively prepared for each extract using 50% DMSO.

Preparation of Standard Microbial Inoculum: A standard inoculum of each of the isolate (Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Candida albican, Aspergilus niger and Mucor specie) was prepared by suspending few discrete colonies of the respective isolate into a sterile normal saline. The resultant turbidity for each suspension was matched/compared with that of 0.5M MacFarland standard.

Evaluation of Antimicrobial Activity of the Extracts against the Standardized Microbial Inocula: Antimicrobial activity of the extracts was assayed using the Agar Well Diffusion method with little modification. Mueller Hinton and Sabouraud Dextrose Agar media were prepared according to their manufacturer’s instruction and poured into different sterile petri dishes to a depth of about 4mm and were allowed to set. Thereafter, 0.2ml of each standardized inoculum was used to inoculate the appropriately labelled agar plate(s) surface(s) and spreading was done using the glass spreader. An 8mm sterile cork-borer was then used to make wells on the inoculated agar plates and the bottom of the wells were sealed with molten agar. Using a sterile glass pipette, a given volume of the prepared compounds’ concentration were respectively added into the various wells and allowed to diffuse for one hour prior to incubation. Fluconazole antibiotics discs and Gentamicin injection solution where used as the standard drugs against the fungi and bacteria isolates. The agar plates were then incubated at 37⁰C for 24hrs and the resulting zone diameters of inhibition were respectively measured around each of the extract concentration and recorded [28].

Molecular docking of Ageratum conyzoides bioactives

The molecular study of this work involves downloading the 3-D structures of the target proteins: Dihydrofolate reductase, PDB 1D, IAI9 and Pectin lyase, PDB 1D, 11DK from protein data bank (www.RCSBPDB.org). Also, the bioactives from Ageratum conyzoides methanolic extracts were camptothecin, jatrorrhizine, chelidonine, strychnine, leonurine and stachdrine were gotten from Pubchem. The downloaded proteins were prepared for docking studies by removing water and other group like the bound ligand, using the schrodinger suite. After this, ligands (Ageratum conyzoides bioactives) were docked into the active site of dihydrofolate reductase and pectin lyase. Best docking poses were selected via 2-D visualization [29].

ADMET evaluation of Ageratum conyzoides bioactives

Absorption, distribution, metabolism, excretion, and toxicity of camptothecin, jatrorrhizine, chelidonine, strychnine, leonurine and stachdrine were predicted by ADMET lab 2.0. The parameters predicted were very important such as X log P,  % human absorption and violation of Lipinski’s rule [30].

Statistical analysis

All results were processed utilizing GraphPad prism and the mean ± SD (n =3) were presented.

The table shows the antimicrobial activity of Ageratum conyzoides against three bacterial isolates: Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The effect is measured at different concentrations (100, 50 and 25 mg/ml), compared with standard antibiotics gentamicin respectively.

The table presents the antifungal activity of Ageratum conyzoides against three fungal isolates: Candida albicans, Aspergillus niger, and Mucor sp. The effectiveness is measured at different concentrations: 100, 50, and 25 mg/ml, compared with standard antifungal agent fluconazole at 25 mg/ml.

Discussion

Preliminary phytochemical analysis of the methanolic  extract of Ageratum conyzoides revealed the presence of tannins, saponins, alkaloids, phenols and flavonoid  as depicted  in table 1. The presence of these secondary metabolites are useful as antioxidant, anti-inflammatory, antimicrobial, immunomodulatory and metal ion chelating properties [31].

Also quantitative evaluation of phytochemicals in methanolic extract of Ageratum conyzoides shows the presence of tannins 10.76 ± 1.52 g/l, alkaloids 3.67 ± 1.07 %, phenol 39.42 ± 3.6 GAE/g and flavonoids 24.34 ± 2.06 mg QE/g. Tannins are polyphenolics existing as hydrolysable and condensed the presence of the above secondary metabolites have various effects against many diseases related free radicals and possess neuroactive, astringent, anti-inflammatory and antimicrobial properties. Our results are similar to the findings of Habu and Ibeh [32] who also reported the presence of secondary metabolites in Newbouldia laevis

The antimicrobial and antifungal activity of Ageratum conyzoides was tested against many isolates including C. albicans, A. niger, Mucor sp., S. aureus, K. pneumoniae and P. aeruginosa. The results showed in table 3 and 4 revealed that Ageratum conyzoides methanolic extract at concentrations of 100, 50 and 25 mg/ml inhibited S. aureus, K. pneumoniae and P. aeruginosa with a zone of inhibition of 11mm for S. aureus at 100 mg/ml, 8mm and 7mm for K. pneumonia and P. aeruginosa  respectively. Also gentamicin at 25mg/ml inhibited all three isolates that is S. aureus, K. pneumoniae and P. aeruginosa at 26, 22, and 16mm respectively. The extract inhibition at 25 and 50mg/ml concentrations were in-significant. This report is in line with the report of Eboh et al.,  and Mir et al., [33, 34]

The antifungal ability of fluconazole (standard) shows no inhibition against C. albicans,  A. niger and Mucor sp at 25mg/ml. However methanolic extract of Ageratum conyzoides at 50mg/ml and 100mg/ml inhibited only Mucor sp with a zone of inhibition of 11mm and 13mm respectively. Therefore the antimicrobial and antifungal activities of Ageratum conyzoides could be partly due to the presence of a wide range of secondary metabolites detected in the extract qualitatively and quantitatively [35].   

The docking results of the selected compounds against dihydrofolate reductase and pectin lyase were depicted in table 5. Camptothecin, Jatrorrhizine, chelidonine and strychnine were docked against dihydrofolate reductase (PDB: 1AI9) and the result revealed jatrorrhizine as having the highest docking score of -6.81, and least score was strychnine -5.04 kcal/mol. The scores of camptothecin and chelidonine were -6.40 and – 6-34 kcal/mol respectively. Jatrorrhizine made good interactions with the amino acids of dihydrofolate reductase for example H-bonding between ALA-115, GLU-32 and functional groups of jatrorrhizine like -OH and phenolic groups. Also there were hydrophobic Interactions between ILE-19, MET-25, VAL-10 and ILE-33 and the hydrophobic regions of jatrorrhizine (benzene rings). Polar interactions also strengthen the binding affinity between Jatrorrhizine and PDB:1AI9 due to the polar interactions of THR-147, SER-61 and THR-58. Therefore, camptothecin, Jatrorrhizine, chelidonine and Strychnine made interactions with dihydrofolate reductase ranging from H-bond,hydrophobic and polar interactions hence the higher docking scores Also selected phytocompounds docked against pectin lyase were leonurine,starchdrine,Jatrorrhizine and chelidonine. The result revealed leonurine has the highest docking score of -6.13 kcal/mol and the least was jatrorrhizine -4.74 kcal/mol. Leonurine made H-bonds:ARG-236, ASP-242 and ASP-217, hydrophobic interactions of TYR-215, TRP-212, TRP-151 and TRP-81 and also a polar bonding of GLN-177 and GLN-241 and the functional groups of leonurine such as -OH groups. Also stachdrine, jatrorrhizine and chelidonine made favorable interactions too [34, 36, 37]. Therefore the compounds to a greater extend inhibited the activity of these microbial and fungal enzymes. Also the ADMET parameters revealed that leonurine, stachdrine, camptothecin, Jatrorrhizine, chelidonine and strychnine do not violate lipinski rule of 5. Also the percentage human oral absorption was higher in stachdrine and strychnine. The number of primary metabolites of these phytocompounds were also in the standard range of 1-8 metabolites [34,36].

Conclusion

This study shows that Ageratum conyzoides has antimicrobial and antifungal activity against pathogenic isolates. Molecular binding interaction shows that leonurine, camptothecin, jatrorrhizine, strychnine, chelidonine and stachdrine, have binding potential against  dihydrofolate reductase and pectin lyase. Therefore these compounds can be exploited as antimicrobial.

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