¹Department of Medicinal Chemistry, School of Pharmacy, College of Health and Medical Sciences, Haramaya University, Harar, Ethiopia

²Applied Chemistry Department, School of Applied Natural Sciences, Haramaya University, Harar, Ethiopia

³Department of Pharmacology, School of Pharmacy, Bisidimo Hospital, Harar, Ethiopia

Corresponding author: Terfo Yilma Mikre, Department of Medicinal Chemistry, School of Pharmacy, College of Health and Medical Sciences, Haramaya University, Harar, Ethiopia, Phone: 0976760242, e-mail: [email protected]

Received Date: February 12, 2026

Publication Date: April 16, 2026

Citation: Mikre TY, et al. (2026). Chemical Constituents of Ranunculus multifidus Aerial extract and Evaluation of Antimicrobial and Antioxidant Activities. Traditional Medicine. 7(1):36.

Copyright: Mikre TY, et al. © (2026).

ABSTRACT

Background: Infectious and inflammation diseases are still a growing public health concern, especially with the emerging challenge of drug resistance to the current drugs. Ranunculus multifidus, commonly known as Gubduu caaffee (in Oromiffa, Ethiopia), was a perennial herb traditionally used to treat malaria, asthma, wound healing, etc. Objective: The present study aimed to extract the bioactive metabolites from aerial part of R. multifidus and investigate the biological activity and isolation of the essential oils from solvent fractions using analytical grade solvents. Methods: In view of its medicinal uses, the powdered aerial part was extracted with methanol and successively fractionated with n-hexane, chloroform, ethyl acetate, n-butanol, and aqueous using solvent-solvent portioning technique. The fractions were evaluated in vitro using the disc diffusion method against S. pyrogens, S. aureus, E. coli, and P. aeruginosa. The antifungal activities of extracts were tested using disc diffusion methods against two fungal strains.  Radical scavenging power of extract and isolate was done using DPPH assay techniques. GC- MS analysis was used for isolation of solvent fraction essential oils. Results: The powdered aerial part was extracted with methanol and successively fractionated with n-hexane, chloroform, ethyl acetate, n-butanol, and aqueous using solvent-solvent portioning to afford 4.54, 10.87, 26.27, 15.15, and 38.08%, respectively. A phytochemical test revealed the presence of flavonoids, phenols, tannins, saponins, and steroids in both fractions of the plant. Strong inhibition observed against S. aureus (13.34 ±0.89 mm) and E. coli (11.55±1.1 mm) at 200 mg/ml of ethyl acetate fraction compared to ciprofloxacin as standard. Strong inhibition zone recorded for ethyl acetate fraction against C. albicans (14.5 ± 1.3 mm) and A. niger (16.93±1.11 mm) at 100 mg/ml compared to fluconazole (21 ±1.5 mm) and ketoconazole (23.43±0.65 mm) for strains respectively. The antiradical activities of each fraction were determined using the DPPH assay. The n-butanol and ethyl acetate fractions show (94.187 %) and (90.78%), respectively, and are comparable to ascorbic acid (98.59%) at 100 µg/mL. Using its spectacular microbial and radical scavenging power, the ethyl acetate fraction was selected for identification of compounds using GC-MS and afforded 45 compounds, including 9,12,15-Octadecatrienoic acid (Z, Z, Z) (24.09%), 9,12,15-Octadeca trienoic acid, methyl ester (23.46%), Methyl 9-cis,11-trans-octadecadienoate (7.07%), and hexadecanoic acid, methyl ester (12.69 %), as major compounds. Conclusion: The present finding of these pharmacologically important secondary metabolites from R. multifidus extracts brings the future attention of experts to the medicinal importance of the plant.

Keywords: Traditional Medicine, Ranunculus multifidus, Antimicrobial Activities, Antioxidant Effects, Phytochemical Screening

INTRODUCTION

The need for novel chemical compounds to treat human diseases is ever-increasing [1]. The rapid development of drug-resistant microbes, the discovery of new cases of life-threatening infections, and the constant recurrence of diseases have pushed for advances in the field of drug discovery [2]. Among the major strategies to alleviate the current situation is to search for new antibacterial, antifungal, and antioxidant agents from natural sources as an alternative to synthetic antibiotics. Plants are known to produce a variety of secondary metabolites to protect themselves against a variety of their own pathogens, and therefore, those with documented traditional use appear to be a rich source of new antibacterial and antioxidant drugs [3]. This prompts a revival in the search for antibacterial and antioxidant agents in natural products.

Medicinal plants have been used to treat a wide range of diseases throughout the history of human beings [4,5]. Today, it plays a role in primary health care as a therapeutic remedy in many developing countries. This is mainly because most of these herbals are available, affordable, and have little or no side effects [4,6]. In the 21^st century, studies have focused on a new group of bioactive components in medicinal plants that have protective effects against cell oxidation and alleviate certain degenerative diseases [7]. 

Ranunculus multifidus is one of the species growing in Ethiopia and is locally called Gubduu caaffee in Afan Oromo and Etse siol in Amharic. It is a perennial herb that is being used in Ethiopia for the treatment of a wide array of diseases, including cancer [8], malaria [9], eczema, toothache, anti-inflammatory [10], stomach ache, wounds, eye diseases, urinary tract infection [11], tuberculosis (TB) [12], shingles, sores and sexually transmitted disease (STD) [13] and others.  Some species in the genus Ranunculus were reported to contain secondary metabolites, including kaempferol-3-O-glucoside, tricin7-O-β-D-glucopyranoside, isoscopoletin, and Anemonin, β-sitosterol [14]. Protocatechuic acid, luteolin factors [15], ranuncoside [7], vanillic acid, and gallic acid  [16]. Other phytocompounds also reported from this species at different times from different parts of the plants using different solvents and extraction techniques such as bilobetin, sternbin, methyl paraben, 4-O-D-glucopyranosyl-p-coumaric acid, linocaffein, kaya flavone, podocarpus flavone A, isoginkgetin, amentoflavone, robusta flavone-4′-methyl-ether, and others [15].

Despite the enormous traditional use of this species against various diseases, there is no information on the chemistry or antimicrobial and antioxidant studies of the aerial extracts of R. multifidus. Hence, in this study, we report the isolation of 45 compounds from the ethyl fraction of aerial extracts of R. multifidus. The antimicrobial and antioxidant activities of different aerial fractions are reported herein for the first time.

MATERIALS AND METHODS

Plant Material

The fresh aerial parts as well as botanical specimens of Ranunculus multifidus were collected and packed in the plastic bag from Sultana village, Bekoji Woreda, Oromia, Ethiopia, which is 224 km from Addis Ababa, in November 2021. The plant material was authenticated by Melaku Wondafrash, and a voucher specimen (Voucher No. TY001) was deposited in the National Herbarium, Department of Botany, Addis Ababa University, for future references.

Figure 1: The image aerial parts of Ranunculus multifidus forks (Picture taken by Terfo Yilma in Bekoji wereda, Oromia region)

Extraction

The air-dried aerial materials (1kg) of R. multifidus were milled into powder and extracted with 6 L of methanol for 72 hours with occasional shaking at room temperature using an orbital mini shaker at 230 rpm. The extracts were filtered and concentrated under reduced pressure using a rotary evaporator at 40 °C, leaving behind the methanolic extract (120g). Using liquid-liquid partitioning techniques, the methanolic extracts were fractionated using different solvents, starting from non-polar to polar solvents, according to Khan [17] with slight modifications. In the process, the methanolic extract was suspended in water and partitioned successively into n-hexane, chloroform, ethyl acetate, and n-butanol. The whole procedure of liquid-liquid fractionation is depicted in (Figure 2). Each fraction of solvents concentrated under reduced pressure using rotavapor and aqueous phase concentrated using lyophilizes to dry after putting overnight in a deep freezer at -20 °C to afford 4.54, 10.87, 26.27, 15.15, and 38.08%, respectively.

The whole procedure of liquid-liquid fractionation was shown in (Figure 2). 

Figure 2: Schematic representation of liquid-liquid partitioning of aerial part of R. multifidus

Phytochemical Screening of Fractions

The word "Phyto" is the Greek word for plant. Phytochemicals are not only nutritive plant chemicals; they also have protective or preventive properties and also protect humans from host diseases [18]. Qualitative screening of various extracts of the plant was performed for the identification of various classes of chemical constituents in the plant based on the standard procedures with slight modifications as required [18–23].

Isolation of Compounds

Analytical TLC was applied to the detection and monitoring of compounds through a separation process and was used to optimize solvent systems for column chromatography. The band separation on the TLC describing the separation of compounds was detected under UV absorbance at 254 nm (absorption) and 365 nm (fluorescence), followed by spraying the TLC plates with vanillin-H₂SO₄ reagent and subsequent heating at 80 °C. The ethyl acetate extract (12 g) was adsorbed and fractionated over silica gel (200 g) by column chromatography with increasing polarity of EtOAc in n-hexane to afford 100 mL of each of the 52 major fractions. The first eight fractions were collected with 100 % n-hexane. Fr8-14, Fr15, Fr16-18, Fr19-21, Fr21, Fr22-27, Fr28-32, Fr33-36, F37-40, Fr41-44, Fr45-48, and Fr49-50 were eluted with 9:1, 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, and 100% n-hexane: EtOAc, respectively, and the last 9:1 and 4:1 fraction were eluted using ethyl acetate to methanol. Fractions 15 (785 mg) after silica gel Column chromatography with 4:1 n-hexane: EtOAc as an eluent furnished a bright green, oily, non-separable spot with (30 mg. Fractions 28 _32 (453 mg) showed three spots on TLC, which were combined and subjected to column chromatography on silica gel with 85:15 EtAcO: MeOH as an eluent is still inseparable and has a yellow colour and oily nature (36 mg). Both fractions were submitted to GC-MS analysis to furnish 45 compounds together with some major.

Antimicrobial Investigation

Previously different scholars reported as this plant have different bioactivities, especially antibacterial and anti-malarial effects. According to Dilebo et al. [10] the leaf extracts of the plant show strong anti-inflammatory effects compared to indomethacin as standard  anti-inflammatory. In the same scenario the anemonin isolated from hydroalcoholic extracts from leaf extracts of this plant show anti-malaria effects [9]. Having this in mind the present work tried to extracts the aerial arts of this plant and investigated its antibacterial, antifungal and antioxidant effects of different solvent extracts using standard methods.

Antibacterial Activity Test

Antimicrobial effects of aerial fractions of R. multifidus were investigated in vitro using the Kirby Bauer Disc diffusion technique against test bacterial and fungal strains [24]. The agar-well diffusion method is used to determine whether a bacterium is resistant or susceptible to fractions and pure compounds. This method was selected due to its simplicity, capacity to analyze multiple samples simultaneously, and ability to work well with defined inhibitors [25]. The activity of fractions was tested against four American Type Culture Collection (ATCC) bacterial strains. In the process, two Gram-positive bacteria, Streptococcus pyrogens (ATCC 12696) and Staphylococcus aureus (ATCC 25923), and two Gram-negative bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853), were used to evaluate the antibacterial activities. The medium was prepared using Muller-Hinton agar (MHA). The autoclaved medium um was poured into sterile plates (35–40 mL/plate), and the plates were allowed to solidify under sterile conditions at room temperature. After solidification, the plates were seeded with overnight-grown cultures of approximately 1.5 X 10⁸ CFU/mL of bacterial strain and 10⁴ CFU/mL of fungal strain by swabbing evenly onto the surface of the medium with a sterile cotton swab. Ciprofloxacin was used as a positive control, while DMSO was used as a negative control. The four bacterial strains were tested with 50, 100, and 200 mg/mL fraction concentrations using the paper disc-diffusion method [26] with slight modifications. For each concentration, 6 mm-diameter Whatman filter paper discs were soaked with a 100 µL solution of the above concentration for each fraction, and then these saturated paper discs were inoculated at the center of each petri dish having a bacterial lawn with the help of a sterile Cork borer [14]. The plates were incubated at 37 °C for 48 hours, and the inhibition zone that appeared around the paper disc in each plate was determined by measuring the diameter of the inhibition using a caliper.

Antifungal Activity Test

The anti-fungal activity was conducted against two common human pathogenic fungal strains, Candida albicans (ATCC 26555) and Aspergillus niger (ATCC 10864), which were incubated on Potato dextrose agar (PDA) for three days at 30 °C [27,28]. The anti-fungal activity was carried out by the agar-well diffusion method. Different dilutions of aerial fractions (10, 50, and 100 mg/mL) in DMSO were tested, while fluconazole and ketoconazole (50 µL, 1 mg/mL in DMSO) were used as the standard drugs. The zone of inhibition was recorded in the same fashion as above for each bacterial strain using a caliper. The results were expressed as the mean ± standard deviation. The bacterial and fungal strains were collected from Harar Health Science Medical Laboratory and Haramaya University Plant Pathology Department, respectively, and the work was done in collaboration with the medical microbiology laboratory and biology department at Haramaya University.

Antioxidant Activity Test

Evaluation of the DPPH radical scavenging assay (RSA)

Antioxidants play an important role as health protection factors. Scientific evidence suggests that antioxidants reduce the risk of chronic diseases, including cancer and heart disease [3]. The DPPH assay was used to assess the free radical scavenging activity of fractions. With this method, the radical scavenging power of an antioxidant compound was measured with the principle of a decrease in absorbance due to the donation of hydrogen from the sample to the DPPH radical to produce stable DPPH-H, and the colour change from purple to yellow observed as shown in (Figure 3) below.

Figure 3: The structure of the DPPH radical and the scavenged radical

The radical-scavenging activities of the fractions were determined using the DPPH method [29].  In the process, the n-hexane, chloroform, ethyl acetate, n-butanol, and aqueous fractions of aerial extracts were dissolved in methanol to afford 1mg/mL. It was serially diluted in methanol to give a concentration of 500, 250, 125, and 62.5 μg/mL. To 1 mL of each concentration, 4 mL of DPPH (0.04% DPPH in MeOH) was added to make 100, 50, 25, and 12.5 μg/mL solutions. All samples were incubated in an oven at 37 °C for 30 min, and then absorbance was recorded at 517 nm using a UV-Vis spectrophotometer (Jenwey 6858, England). A reaction solution without DPPH was used as a blank, a DPPH solution as a control, and L-ascorbic acid as a standard. The percentage inhibition was calculated using the formula:

% Inhibition (RSA)= [A control –A test] / A control × 100…………………… (1)

RESULTS AND DISCUSSION

Phytochemical screening: Phytochemical screening tests were done to determine the classes of compounds present in each fraction following standard experimental protocols. The preliminary phytochemical screenings of different solvent fractions of aerial R. multifidus were tested. The results suggested that flavonoids, saponins, phenols, tannins, terpenoids, steroids, phytosterols, and glycosides are present within the fractions in different ranges (Table 1). The results obtained in this study suggest the identified phytochemicals may be the bioactive constituents of this plant, suggesting the plant is a valuable reservoir of bioactive compounds of substantial medicinal importance.

Table 1: Phytochemical screening of different fractions of aerial extracts of R. multifidus

Phytochemical screening	Test type	n-hexane fraction	EtOAc fraction	CHCl3 fraction	n-BuOH fraction	Aqueous Fraction
Flavonoids	Alkaline test	-	+	+	+	+
Saponins	Foam test	+	-	+	-	+
Phenols	Alkaline test	+	+	+	+	+
Tannins	Ferric chloride test	-	+	+	+	-
Terpenoids	Salkowski test	-	+	-	-	+
Steroids	-	-	+	-	+	+
Phytostero	Salkowski test	-	+		-	+
Cardiac glycosides	Keller-Killiani test	-	+	+	+	+
Alkaloids	Wagner’s test	-	-	+	+	-
Anthocyanin	-	-	+	-	-	+
Quinones	-	+	-	+	-	+
Reducing sugar	Fehling’s test	-	+	+	+	+
Anthraquinone	-	-	+	+	+	+