Plant active part: Roots belonging to S. costus had been procured from indigenous markets, and thereafter recognized based on their characteristics [12], The roots of this plant were cleansed, desiccated, and stored under [13], (Fig: 1).

The crude alkaloid components had been extracted according to the method reported in [14].

The crude flavonoid components had been extracted according to the method reported in [15].

The Crude Terpenoid components had been extracted according to the method reported in [16]. A 100 mg/ml stock solution of Alkaloid, Flavonoid, and Terpenoid was made in a 10% Dimethyl Sulfoxide (DMSO) solution. The solution was then sterilized using a Millipore filter with a pore size of 0.22µm and stored at -20°C pending it was ready for use [17].

Rice Seed collection: rice seeds were gathered from various parts of indigenous shops in the area of Babil, namely in Hillah-Iraq, 2022.

Isolation and diagnosis: To separate the Aspergillus fungi, a total of one-hundred seeds were haphazardly selected from each sample. The sterilization process involved treating it with a 2% solution of sodium chlorate for 2 minutes. Subsequently, it was rinsed twice with sterile distilled water to eliminate any remaining sterile substances. Finally, it was dried using sterile filter paper. The specimen was carefully transferred using sterilized forceps onto 9cm Petri dishes that contained 20 ml of preprepared Dextrose Agar of Potato (PDA) supplemented with 50mg/l of chloramphenicol to inhibit bacterial growing [18], each dish was seeded with 5 seeds, and each sample was replicated three times. The dishes had been incubated at 25°C for 5 to 7days. The fungi that were present in the seeds were subsequently isolated using secondary cultures in order to be identified. The isolated were then identified using the keys in [19, 20]. The isolates were stored in aseptic glass containers filled with Nutrient Agar media. The containers' glass had been kept at 25°C for a duration of 7days, thereafter deposited in a fridge-freezer 4°C pending they were utilized.

Antifungal assay: The PDA medium had been prepared and sterilized. Then, a volume of (1, 2, 3ml) of each plant extract had been placed in the center of the Petri dishes. The volume had been completed to 20ml with PDA medium to achieve the desired final volume concentrations (5, 10, and 15 mg/ml) of the secondary metabolites under study. Later the medium hardened entirely, a disc about of 5mm in diameter of a 7-day-old of the test fungus had been placed in the center of the Petri plates under sterile conditions. The plates were then incubated at a temperature of 25±2C˚ for 7days. Simultaneously, 0. 02ml of an antibiotic solution had been added to each assay plate to check for bacterial contamination, as recommended by the suggested protocol [21]. The positive control utilized in the experiment was Carbendazim, a fungicide with a concentration of 500g/l. The negative control, on the other hand, was dimethyl sulfoxide with a concentration of 10%. Data was documented on the 7^th day. The measurement of the colony's diameter had been expressed in millimeters. A control had been used as a medium without extract. Three replicates had been maintained for each treatment. The toxicity of the f extracts had been determined by calculating the percentage of inhibition in mycelial growth, by means of the specified formulation [22].

Percent Inhibition% = [dc – dt / dc] × 10²

dc=The mean increment in mycelial diameter colony under normal conditions.

dt=The mean increment in mycelial diameter colony within the experimental treatment.

Statistical analysis: The data for treatments had been obtained from 3 replicates. The data underwent an analysis of variance by means of the SPSS, 20.0 version. A design of completely randomized had been employed, the least significant difference (L.S.D.), test had been conducted at a significance level of Probability ≤ 0.05.

Alkaloid antifungal crude compounds extracted from S. costus roots in contradiction of Aspergillus spp. isolated from seeds of Rice had been assessed in Table, 1. The findings demonstrated that Alkaloid secondary products derived from S. costus roots exhibited a significant decline at P≤ 0.05 of Aspergillus spp. growth and the same effect of the positive control. Antifungal action was applied at (5.0, 10.0, and 15.0) mg/ml. mycelial reduction fluctuating from 66.0% in 5.0 mg/ml, 71.6% in 10.0 mg/ ml, and 100% in 15.0 mg/ml, (Figure 2: A, B), compared with negative control treatment represented by 10% DMSO and positive fungicide Carbendazim 500g/l control (Figure 2: C) where redaction proportion was 0.00% for control treatment and 100% for fungicide Carbendazim 500g/l  positive control treatment, (Figure 2: D).

Within the same context, the crude Flavonoid compounds exhibited a 62.3% suppression of mycelial growth at a concentration of 5.0 mg/ml, and complete inhibition at concentrations of 10.0 mg/ml and 15.0 mg/ml, as indicated in table 2. Therefore, it exhibited substantial differences in comparison to the control treatment, as well as producing a similar effect to the positive control, (Figure 2: E, F).

The crude Terpenoid compounds exhibited noteworthy efficacy at three doses (5.0, 10.0, and 15.0 mg/ml), in comparison to the negative control. Additionally, they demonstrated a similar impact as the positive control at concentrations of 10.0 and 15.0 mg/ml, (table 3). The inhibitory action exhibited a percentage of 34.1% at a concentration of 5.0 mg/ml, while reaching 100% at concentrations of 10.0 and 15.0 mg/ml, respectively, (Figure 2: G, H). Finally, Secondary metabolite products, including Alkaloid products, Flavonoid products, and Terpenoid products, isolated from S. Costus roots exhibit similar effects to the positive fungicide Carbendazim 500g/l control.

Figures & Tables

Discussion

Medical plants are regarded as a crucial resource for the treatment and prevention of numerous types of diseases [23]. Every plant has numerous essential components that have potential use in the field of medicine and can contribute to the creation of various pharmaceuticals [24]. The current investigation demonstrated that chemical secondary compounds, including Alkaloids, Flavonoids, and Terpenoids, derived from S. costus roots exhibited anti-fungal properties counter to Aspergillus spp. obtained from rice seeds collected from several local markets of Hillah city located in Iraq. Chemical secondary compounds derived from several active parts of different medicinal plants such as Lactuca serriola leaves, Lepidium sativum leaves, Myrtus Communis leaves, Cassia senna leaves, Ricinus communis leaves, Cassia didymobotrya leaves, Melia azedarach leaves, Dianthus caryophyllus flower buds, and Salvia hispanica seeds, exhibit antibacterial properties capable of suppressing various pathogenic microorganisms obtained from diverse clinical samples [25-33]. Hussein et. al. [34] it has been shown that phytochemical substances isolated from the unicellular primitive plant Chlorella vulgaris have the capacity to counteract harmful bacteria by acting as antibacterial agents. Kamal et. al. [35] Used Hibiscus sabdarifa extracts for controlling pathogenic microorganisms such as E. coli and Proteus sp. Kamal et. al. [36] Employed Ficus carica for inhibiting Pseudomonas aeruginosa and Escherichia coli pathogenic bacteria. AL-Masoodi et. al.  [37] Utilized substances derived from Curcuma longa and Boswellia carteri to manage Fusarium spp. obtained from corn seeds. Referring to[38, 39] Employed terpenoids derived from seeds of Carthamin tinctorius and flavonoids obtained from M. Communis leaves to combat Aspergillus spp. identified from preserved seeds of medicinal plant. Secondary compounds found in M. Communis leaves, including alkaloids and flavonoid compounds, are considered valuable for their ability to inhibit pathogenic microorganisms that are isolated from hemodialysis fluid specimens [40]. Radhi et. al. [41] Employed extracts from Callistemon viminalis leaves to manage isolates of Urinary Tract Infections. Allium sativum is considered a reliable means of suppressing some bacterial species that have been isolated from people infected with the Corona Virus [42]. The flower buds of D. caryophyllus contain secondary metabolite chemicals, specifically terpenoids and flavonoids, which have potent antifungal properties against Candida species [43]. S. costus was regarded as a therapeutically significant plant, the numerous compounds extracted from it have remedial properties [12]. The roots of S. costus contain alkaloids and terpenoids that exhibit potent antifungal properties against Candida species [44]. Several chemicals were extracted from the roots of S. costus and evaluated against four species of Aspergillus such as A. niger, A. ochraceus, A. versicolor, A. flavus, and two species  of Penicillium such as P. ochrochloron, P. funiculosum, and other species such as   Cladosporium cladosporioides, Alternaria, and Trichoderma viride. The chemical exhibited anti-fungal properties ranging from temperate to strong  [45]. The polyphenol content of S. costus was found to be highest in the ethanol and ethyl acetate extracts, n-butanol extract came next, while n-hexane extract came last, these compounds exhibit antibacterial and antifungal properties against a wide range of microorganisms [46]. Saussurea lappa had the most potent antifungal action against A. flavus, with Trapa natans and Mangifera indica showing lesser efficacy [47]. The methanolic extract of S. costus roots is abundant in many components including flavonoids, terpenoids, alkaloids, phenols, polyphenols, tannins, resins, coumarins, quinines, steroids,  and cardiac glycosides exhibited antifungal properties against A. niger strain (ATCC6275), [48]. Conversely, the mechanism by which Alkaloids exert their antifungal effects is typically pleiotropic, involving inhibition of protein synthesis and intercalation into fungal DNA, or by improving the production of fungal inhibitors. [49]. Terpenoids decreased the number of mitochondria, hence altering the levels of production of ATP and reactive oxygen species. Additionally, it has been found that triterpenoids have greater antifungal action in comparison to tetraterpenoids [50]. Secondary products such as Terpenoid and flavonoid exert their own effects by destabilizing microbial membrane components [51]. Flavonoids commonly hinder the growth of fungi by diverse ways, such as disrupting the plasma membrane, causing dysfunction in the mitochondria, and inhibiting processes including cell wall production, cell division, RNA, and protein synthesis, as well as the efflux-mediated pumping system. [52]. Therapeutic herbs exhibit anti-fungal properties through numerous methods. They disrupt the cell membrane, leading to an injury of membrane integrity. They also hinder DNA transcription and decrease the number of fungal cells. Additionally, they block the action of fungal antioxidant enzymes and prevent the creation of fungal biofilms [53, 54].  Finally, the anti-fungal activity of S. costus roots may be attributed to alkaloids, flavonoids, and terpenoids. These compounds could affect proteins and DNA synthesis, as well as impair membrane permeability and disturb metabolic activity.

The roots of S. costus include alkaloids, flavonoids, and terpenoids that exhibit potent antifungal properties against Aspergillus spp. that exist in rice seeds.

Conflict of Interest

The authors declare that there is no conflict of interest.

Author Contributions

Fatima S. Al-Nafie: study design, Hussein J. Hussein: Refinement of study design and supervision, Abeer Fawzi Al-Rubaye: Refinement of study design and supervision.

References