Phytochemical Compounds and the Antifungal Activity of Centaurium pulchellum Ethanol Extracts in Iraq

The current study included a variety of phytochemical substances that were extracted from Centaurium pulchellum and showed a wide range of medicinal properties from the plant's reproductive and vegetative parts against the pathogenic fungus Aspergillus flavus. The vegetative and reproductive components of Centaurium pulchellum were subjected to (GC-MS) analysis for phytochemical study. The data indicated that fungal activity was the highest. Four extract concentrations of 5, 10, 15, and 20 mg/ml were utilized in the investigation, and the diameter of the colonies measured at each concentration was 90.00, 36.00, 28.00, 18.00, and 0.00 mm, respectively.

Nine bioactive phytochemical compounds were found in Centaurium pulchellum's vegetative and reproductive portions, according to GC-MS analysis of the chemicals. Another study reported phytochemical substances that: 1-H-Imidazole-2-carboxaldehyde, 1-methyl-;Acetaminophen; n-Hexadecanoic acid; Mercaptoacetic acid, 2TMS derivative; 1.2,3-Dimethyl-5-(trifluoromethyl)-1,4-benzenediol #; Mercaptoethanol, 2TMS derivative-; Bis-(3,5,5-trimethylhexyl) phthalate Tetrakis(trimethylsilyl) orthosilicate #;- 1.1-Isopropoxy-3,3,3-trimethyl-1-[(trimethylsilyl)oxy]disiloxanyl tris(trimethylsilyl) orthosilicate #.

Centaurium pulchellum is a small flowering plant belonging to the genus Centaurium, which belongs to the Gentianaceae family, It is commonly known as the lesser centaury and is called (a dandelion weed) [1]. The plant under consideration is a perennial species known for its highly acerbic flavor, erect glabrous, stem 30 cm - 50 cm in height, 4-angled, leaves 2-4 pairs below the lowermost inflorescence branch, cyme branch spreading at a wide angle, calyx lobes 5, linear-subulate, 5 mm, corolla tube 10mm long, limb 5 rarely 4, lobes narrowly ovate-oblong, 4.5 mm, stamens inserted in throat, stigma distinctly 2- branched, capsule about as long as calyx [2,3]. It thrives in damp soil environments, rocky mountain clefts, and in proximity to pristine water sources, originating from the Mediterranean territories, it is indigenous to a vast expanse extending across most of Eurasia to the northerly regions, encompassing Scandinavia, and reaching as far as the Himalayas and China) [4-6].

This plant is a rich source of many active compounds that can be used in many fields, isolated as the secoiridoids and the xanthones found in the roots and stem of C. pulchellum [7-9]. Also, the plant extract acts as an antibacterial agent on brine shrimp, due to the two isocoumarin derivatives, one (erythricin) and the other (erythrocentaurin) [9]. Šiler, et al. [10,11] studied the main secondary metabolites: secoiridoid glycosides, monoterpenoid, and phenolics, and further investigated for antioxidant and antimicrobial(antibacterial and antifungal) activity for five centaury species one of them were C. pulchellum. The extract of centaur flowers contains (82 compounds) and six distinct groups have been identified: glycosides, iridoid glycosides, phenolic acid, aglycones, and their derivatives [12]. In the roots, the chemical constituent Ethyl N-docosanoylanthranilate, demonstrates efficacy in combating Aspergillus flavus fungal strains [13].

Aspergillus flavus represents a fungal species within the Aspergillus section Flavi and has served as a paradigmatic system for elucidating fungal development and toxin synthesis. The impact of A. flavus on human health is multifaceted, encompassing the generation of highly carcinogenic mycotoxins known as aflatoxins and the induction of aspergillosis in immunocompromised individuals. Furthermore, the infection of crops by A. flavus leads to economic repercussions due to diminished yields and aflatoxin contamination. A. flavus, classified as a saprophytic fungus, propagates in the environment predominantly through the production of asexual spores (conidia), which confer extended viability in adverse environmental conditions. Conidia exhibit a structural composition comprising the rodlet layer, cell wall, and melanin, originating from a specialized asexual structure termed the conidiophore [14].

Research subject and sample

"The fungi under study were identified in the agricultural college laboratory at Kerbala University. They were isolated from plants infected by Aspergillus in Kerbala fields."

Microscopic assessment

Samples were analyzed by the following procedure: The region was sterilized in 70% alcohol to obtain rid of saprophytes fungi and microbes, then a sample from the affected plant and put on a clean slide with a drop of 0% KOH and then covered was put on and heated on a flame then observed by a microscope looked after spores of Aspergillus flavus or hypha. The observed distinguished as in: [15-17].

Plant extract extraction

The dried aerial parts were pulverized and extracted in a Soxhlet. The polar compounds were extracted with 1.5 liters of methanol, then filtered and evaporated. After that, the compounds were weighed, and their yield percentage, expressed as a percentage of their dry weight, was calculated. The yield of the dark green extract from methanol was 57.5 grams (0.095%), the plant extracts were placed in the dark and refrigerated at a temperature of 4 °C. Wahid & Jafar method [18].

Alcoholic extract from C. pulchellum plants is cultivated and used to study the growth of pathogenic fungi

Steps from El-Kady, et al. [19] were applied viz., "four concentrations (5, 10, 15, and 20 mg/ml) of the methanol extracted extracts of C. pulchellum were combined with (PDA) grown media (three tests for each concentration)." then hardening a medium and creating a hole in the middle of those dish with a cork borer that was pierced 5 mm. Placing a disk in the center of dishes that had a diameter of 5 mm, these dishes put fungal inoculum and allowed to grow for ten days. The plates were placed in the incubator at 25 °C, and after ten days, the developing colony's diameter was recorded. Results were noted, so the ratio of inhibition was measured. Then compute the inhibition ratio by [20]:

$Inhibitionratio=\frac{Average diameter of fungus in control dish\left(1\right)-Average diameter of fungus in tretment dish}{Average diameter of fungus in control dish(1)}\times 100$

Obtaining and preparing plant

The Centaurium pulchellum plant was collected from several parts of Iraq (Zakho, the mountain zones of Iraq, Mosul a hills zone, Baghdad, Karbala in the middle of Iraq a lowlands zone, Basra in the south of Iraq) in the period April-May 2023. Following that, the plants were cleaned and allowed to air dry. After being powdered, 40 g of plants were soaked in 200 ml of ethanol and filtered.

Participants extract identification using mass spectrometry and gas chromatography (GC/MS)

GC-MS analysis was used to identify C. pulchellum phytochemically in 'a (Scion Instruments Company. Model: SCiON 436) GC-MS Column: SCION-5MS {ID = 0.25 mm, length = 15 m, df = 0.25 μm} Carrier gas: Helium device controlled by a computer at 60 eV. Using a tiny syringe, approximately 1 μL of the ethanol extract was put into the GC-MS column, and the scanning process took 45 minutes. Column oven: initial temperature is 50 ℃ increase by 10 ℃ / min to 230 ℃, Sample Preparation: 100 μL of the sample is diluted with 5 ml of N-Hexane (HPLC-Grade) before injection. Gas flow ratio: 1 ml/min Pressure: 10 psi m/z Range: 1 – 2000, split ratio 1:50. (This method is innovative in Al-Zahraa Center for Medical and Pharmaceutical Research Sciences (ZCMPRS))[21].

Statistical analyses

Performed using STATGRAPHICS Centurion XV software, version 15.1.02 (StatPoint, Inc. 1982-2006, USA). The data analyzed of variance (ANOVA), and comparisons between the mean values of each of the concentrations investigated were made by the Least Significant Difference (LSD) test calculated at the 95% confidence level.

Antifungal activity

Aspergillus flavus was chosen for the current investigation to evaluate the ethanol extract's efficacy of C. pulchellum parts, the extract showed a high antifungal activity against Aspergillus flavus. Used 4 extract concentrations (5,10,15,20 mg/ml) showed results (36.00, 28.00, 18.00, 0.00 mm) diameter of colonies in Aspergillus flavus the results showed in Table 1. These results are in agreement with Šiler, et al. [10,11] and Todorovic, et al. 2022 [12] when they studied the activity of C. pulchellum against the Aspergillus flavus .while Janković, et al. Miana & Al-Hazimi [22,23] obtained some secondary metabolites that act as Antifungal and They discovered that fungi are inhibited by the ethanolic extract of C. pulchellum parts.

Evaluation of the biochemical substances of Centaurium pulchellum

Nine components are present in the GC-MS ethanolic extract of the vegetative and reproductive portions of C. pulchellum, as shown in Table 2. These substances have a variety of biological actions; a high level of phenols the examination of the acetaminophen compound revealed that 82% of the compounds were analgesic, antibiotic, antipyretic, and anti-inflammatory, the resulting in agreement with Šiler, et al. [10,11] study they mentioned that the most of compounds belong to phenols than the Organosilanes compounds which have antifungal activity (Table 2) (Figures 1-10) lists the remaining chemicals' antifungal and antioxidant properties.

In the current study, nine compounds from aerial parts of Centaurium pulchellum the plant collected from different places in Iraq, were identified by (GC-MS) analysis. this plant shows bioactive compounds useful for many herbal formulations especially antifungal activity against Aspergillus flavus because of the high levels of phenols and Organosilanes so it is important as a plant phytopharmaceutical. However, some studies are needed to undertake its chemotaxonomy properties, bioactivity, and profile of toxicity.

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