Isolation, antimicrobial evaluation, and in silico docking of spinasterol from Impatiens rothii Hook. f. tuber extract

Debebe Birhanu; Avijit Mazumder; Mariamawit Y Yeshak; Kaleab Asres; Daniel Bisrat

doi:10.1186/s41182-026-00996-6

. 2026 Jun 11;54:143. doi: 10.1186/s41182-026-00996-6

Isolation, antimicrobial evaluation, and in silico docking of spinasterol from Impatiens rothii Hook. f. tuber extract

Debebe Birhanu ^1,³, Avijit Mazumder ², Mariamawit Y Yeshak ³, Kaleab Asres ³, Daniel Bisrat ^3,^✉

PMCID: PMC13488175 PMID: 42277980

Abstract

Background

The increasing prevalence of drug-resistant infections highlights the need for new antimicrobial agents. Ethiopia’s diverse plant resources remain largely underexplored, including Impatiens rothii tuber, an endemic species traditionally used to treat infections. This study therefore evaluated the antimicrobial activity of I. rothii tuber extract and its isolated compound, spinasterol.

Methods

Tuber extracts were prepared using 80% methanol and fractionated via silica gel chromatography to isolate spinasterol (1), identified by MS and NMR. Antimicrobial activity was tested against 26 bacterial and 4 fungal strains. Molecular docking assessed binding interactions with MRSA pyruvate kinase and sterol 14-alpha demethylase as antibacterial and antifungal targets, respectively.

Results

Both the extract and spinasterol (1) showed broad-spectrum antibacterial activity, with promising effects against Escherichia coli and Vibrio cholerae. Spinasterol (1) showed a minimum inhibitory concentration (MIC) of 25 µg/mL against non-resistant E. coli strains and V. cholerae, comparable to ciprofloxacin. Antifungal testing showed MIC values of 200 µg/mL against Candida albicans and 400 µg/mL against Aspergillus niger. Molecular docking revealed moderate binding of spinasterol (1) to MRSA pyruvate kinase (– 5.278 kcal/mol) and to sterol 14-alpha demethylase (– 8.523 kcal/mol), suggesting its antibacterial and antifungal mechanisms.

Conclusions

This study demonstrates the antimicrobial activity of I. rothii extract and its constituent spinasterol, supporting its ethnomedicinal use. Spinasterol demonstrates promise as a potential antimicrobial scaffold; however, further optimization is necessary to enhance its antimicrobial efficacy. Additional experimental studies, including enzyme inhibition assays, are also needed to validate these predictive molecular findings.

Supplementary Information

The online version contains supplementary material available at 10.1186/s41182-026-00996-6.

Keywords: Impatiens rothii, Spinasterol, Antimicrobial, Molecular docking, MRSA pyruvate kinase, Sterol 14-alpha demethylase, ADME

Background

Infectious diseases continue to emerge and reemerge due to factors such as microbial adaptation, host susceptibility changes, climate change, and socio-economic conditions [1–3]. Misuse of antimicrobials, including in veterinary practices, accelerates the rise of drug-resistant bacteria [4–6]. In traditional medicine, medicinal plants are used to meet the primary healthcare needs of about 80% of the global population, as highlighted by the World Health Organization (WHO) and are valuable sources of bioactive compounds [7]. Phytochemicals possess diverse pharmacological properties, notably antimicrobial activity, making them potential tools against infections and antibiotic resistance [8].

The genus Impatiens (family Balsaminaceae), comprising over 1000 species, is known for varied biological activities including antibacterial, antifungal, antioxidant, anti-inflammatory, antidiabetic, and anticancer effects [9–13]. Impatiens rothii, locally called Gesherit (Amharic), Buri (Oromifa), and Gurshit (Tigrigna), is an upright herbaceous perennial endemic to Ethiopia [14, 15]. Its wild-harvested tubers are traditionally used for cosmetic purposes, imparting a dark reddish color to skin, and for treating fungal infections such as athlete’s foot (tinea pedis) [16, 17]. Its restriction to Ethiopia reflects the country’s diverse topography, isolated highlands, and varied microclimates, which have promoted the evolution of locally adapted species. The Ethiopian Highlands are a major center of plant diversity and endemism, hosting narrowly distributed species such as I. rothii. However, endemic plants are highly vulnerable to habitat loss, deforestation, agricultural expansion, and climate change. Conserving Ethiopia’s endemic flora is therefore essential for protecting both national biodiversity and globally valuable genetic resources.

In our continued pursuit of plant-derived antimicrobial agents [18, 19], we evaluated the antibacterial and antifungal activities of I. rothii tuber extract along with its bioactive constituent. Although I. rothii has previously been reported to possess antifungal activity, including activity against athlete’s foot [17], the novelty of the present study lies in its comprehensive antimicrobial evaluation against 26 bacterial and 4 fungal strains, along with the isolation and antimicrobial assessment of spinasterol. In addition, the study integrates in vitro assays to evaluate antimicrobial efficacy and in silico analyses to predict molecular interactions, thereby providing a more comprehensive assessment of their potential as antimicrobial candidates.

Materials and methods

Plant material

I. rothii tubers were collected in October 2022 from Sela Dingay, North Showa (190 km from Addis Ababa, Amhara region; Fig. 1). The plant was identified and authenticated by botanist Melaku Wondafrash, Addis Ababa University, and a voucher specimen (collection number DB001) was deposited at the National Herbarium for future reference.

Chemicals, reagents and drugs

Analytical grade chemicals and reagents used included chloroform (Finkem Laboratory Reagent, India), ethyl acetate, methanol (Reagent Chemical Limited, UK), distilled water, ciprofloxacin, griseofulvin, nutrient agar, and Sabouraud dextrose medium.

Instruments

A rotary evaporator (Heidolph Instruments GmbH & Co., Germany), UV spectrophotometer (Shimadzu MultiSpec-1501, Japan), and NMR spectrometer (JNM-ECS400, JEOL Ltd., Akishima, Tokyo, Japan) were used.

Bacterial strains

The in vitro antibacterial assays were carried out against the following Gram-positive bacterial strains: Staphylococcus aureus ML 267, S. aureus MDR 1*, S. aureus MDR 2*, Bacillus pumilus 82 and B. subtilis ATCC 6633 and Gram-negative bacterial strains used were: Escherichia coli NCTC 5933, E. coli K88, E. coli NCTC 7360, E. coli LT37, E. coli 872, E. coli ROW 7/12, E. coli 3:37C, E. coli CD/99/1, E. coli HB101*, E. coli C600*, Pseudomonas aeruginosa MDR 1*, Salmonella typhi Ty2, S. enterica TD 01, Shigella boydii 8, S. dysentery D13629, S. flexneri Type 6, S. soneii 1, Vibrio cholerae NCTC 4693, V. cholerae NCTC5596, V. cholerae NCTC 10732, and V. cholerae NCTC 11501. All bacterial strains, including those marked with an asterisk (*) to indicate multidrug resistance, were obtained from the Department of Pharmaceutical Technology at Jadavpur University, the Central Drugs Laboratory in Kolkata, and the Institute of Microbial Technology in Chandigarh, India. Strain purity was confirmed by standard microbiological, cultural, and biochemical tests before sensitivity testing.

Fungal strains

Antifungal activity testing was carried out on the following fungal pathogens: Aspergillus niger ATCC 6275, Candida albicans ATCC 10231, Penicillium funiculosum and P. notatum ATCC 11625 NCTC 287. All the fungal strains were procured from Central Drugs Laboratory, Kolkata, India.

Preparation of extract

The tubers of I. rothii were selected for this study due to their traditional use in Ethiopia for treating infections. Fresh I. rothii tubers were cleaned, sliced, and shade-dried at room temperature for three weeks, then pulverized into coarse powder (400 g). The powder was cold-macerated with 80% methanol (2.5 L) for 72 h with occasional shaking, filtered (Whatman No. 1), and re-macerated for maximum yield. Combined filtrates were concentrated by rotary evaporation and oven-dried at 40 °C. The hydroalcoholic extract was weighed, yield calculated, and stored at 4 °C in an amber vial.

Solubility-based fractionation

Following a modified method of Kumar et al. [20] the 80% methanol extract (30 g) was sequentially fractionated by solubility into chloroform, ethyl acetate, methanol, and water fractions. The extract was sonicated twice with each solvent (100 mL), and organic fractions were concentrated by rotary evaporation under reduced pressure, while the aqueous fraction was oven-dried below 40 °C. All fractions were weighed, stored in amber vials, and refrigerated at 4 °C.

Compound isolation by column chromatography

The mobile phase for column chromatography was selected from preliminary TLC of the chloroform fraction, showing clear spots with diethyl ether/chloroform (3:1). A column was packed with a slurry of silica gel (80 g) in diethyl ether (100 mL). The chloroform fraction (1 g) was prepared by adsorption on silica gel (5 g) after dissolving in methanol and evaporating to dryness. Elution with diethyl ether–chloroform gradients yielded 159 fractions (10 mL each), monitored by TLC (silica gel 60 F₂₅₄) under UV light (254 and 360 nm). Fractions 15–21, each showing a single spot in multiple solvent systems, were combined to obtain a white powder (compound 1, 45 mg), which was stored in an amber vial at 4 °C.

Spinasterol (1): White amorphous powder; R_f = 0.57 (Silica gel-TLC using diethyl ether: chloroform (3:1)). + ve-ESI–MS (Supplementary Material 1): m/z = 395.5 [M + H-H₂O]⁺. ¹H-NMR (δ_H, ppm, CD₃OD, 400 MHz; Supplementary Material 2): δ 3.62 (1H, m, H-3), δ 5.05 (1H, dd, J = 8,16 Hz, H-23), δ 5.18 (1H, t, J = 12 Hz, H-7), δ 5.19 (1H, dd, J = 8,16 Hz, H-22); ¹³C-NMR (δ_C, ppm, CD₃OD, 100 MHz; Supplementary Material 3): δ 12.05 (C-18), δ 12.22 (C-29), δ 13.03 (C-19), δ 19.00 (C-26), δ 21.06 (C-21), δ 21.37 (C-27), δ 21.57 (C-11), δ 23.02 (C-15), δ 25.38 (C-28), δ 28.48 (C-16), δ 29.66 (C-6), δ 31.25 (C-2), δ 31.87 (C-25), δ 34.25 (C-10), δ 37.18 (C-1), δ 38.04 (C-4), δ 39.49 (C-12), δ 40.30 (C-5), δ 40.79 (C-20), δ 43.31 (C-13), δ 49.50 (C-9), δ 51.26 (C-24), δ 55.15 (C-14), δ 55.95 (C-17), δ 71.07 (C-3), δ 117.47 (C-7), δ 129.49 (C-23), δ 138.15 (C-22), δ 139.50 (C-8). Spinasterol (1) is in a good agreement with the NMR data reported for the same compound [21].

In vitro antibacterial and antifungal activity assay

Determination of minimum inhibitory concentrations (MICs) by broth dilution

The MICs of the tuber extract and the isolated compound were determined using the broth dilution method, as described by Nimse et al. [22]. The antibacterial activity was tested after preparing a stock solution of compounds in 1% dimethyl sulfoxide. A measured amount of the stock solution was dispensed in a series of Mc Cartney bottles previously containing calculated volumes of sterilized cooled molten Mueller–Hinton broth media (40–45 C) to prepare volumes of 30 mL each with dilutions of 5, 10, 25, 50, 100, 200, 400 and 800 µg/mL. These tubes were then kept in a refrigerator at 4 C for 24 h to allow uniform diffusion of the compounds throughout the media. Before spot inoculation, tubes were kept at 37 C for 2 h. Then one loopful (loop diameter: 3 mm) of an overnight grown culture of each microbes with 0.5 McFarland turbidity and optical density comparable to the density of a bacterial suspension with a 1.5 × 10^8 CFU/mL was inoculated in the tubes containing the compounds and media [22, 23]. The spot inoculated tubes were incubated at 37 C for 24 h for bacteria and minimum inhibitory concentration was determined.

Similar procedures were adopted to determine the minimum inhibitory concentration of fungi where we used Saborauds Dextrose Broth media containing test samples (50–2000 µg/mL) which were incubated at 25 °C for 3 days. The tests were done in triplicate.

Disc diffusion method

In vitro antibacterial activity was assessed using the disc diffusion method following the procedure of Mitchell and Carter [24]. The inhibition zones produced by the test samples were measured and compared with those of the standard drug, ciprofloxacin. Stock solutions of the tuber extract, spinasterol (1), and ciprofloxacin (1 mg/mL in 1% DMSO) were prepared. Sterile 6 mm discs were impregnated with 200 μg/ml of each sample; discs impregnated with 1% DMSO served as negative controls. Mueller–Hinton Agar plates (pH 7.2–7.4) were inoculated with standardized bacterial suspensions, allowed to dry, and the test discs were applied. Plates were incubated at 37 °C for 24 h, and the diameters of inhibition zones were measured in millimeters. All tests were performed in triplicate.

For antifungal activity, stock solutions of the tuber extract and spinasterol (1) (10 mg/mL in 1% DMSO) were prepared. Using the same disc diffusion procedure on Sabouraud Dextrose Agar, the tuber extract, spinasterol (1), and griseofulvin (1500 μg/mL) were tested. Plates were incubated at room temperature for 3 days before measuring inhibition zones. Negative controls contained 1% DMSO. Tests were performed in triplicate.

Determination of mode of action

Agar plugs were collected from the inhibition zones and re-inoculated into fresh culture media to determine whether the compounds exhibited bacteriostatic or fungistatic versus bactericidal or fungicidal activity. The cultures were incubated for 96 h at 37 °C for bacteria and at 25 °C for fungi, and monitored for microbial growth. The results were further confirmed by viable count assays performed at 24 h intervals, using compound concentrations of 200 μg/mL for bacteria and 2000 μg/mL for fungi [25, 26].

In silico study

Molecular docking

Molecular docking was performed to explore potential bacterial targets for spinasterol. Methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK) was selected as the primary target of spinasterol, based on preliminary results and prior studies [27]. Crystal structures of S. aureus pyruvate kinase (PDB ID: 3T07, antibacterial) [28] and C. albicans sterol 14-alpha demethylase (PDB ID: 5TZ1, chain A, HEC/HEM, antifungal) [29] with bound ligands were retrieved from the Protein Data Bank and prepared using Schrödinger Suite 2023–1 [30].

Protein preparation included adding hydrogens, correcting bond orders, adjusting ionization states with Epik, removing water molecules beyond 5 Å, and energy minimization using the OPLS3 force field (RMSD 0.3 Å). The structure of spinasterol was drawn in ChemDraw Ultra and prepared with Schrödinger’s LigPrep module, generating relevant ionization states and stereoisomers [31]. The receptor grid was centered on the co-crystal ligand binding site with a 6.0 Å radius. Van der Waals radii were scaled (0.8) with a partial charge cutoff of 0.15 to soften nonpolar interactions. Docking was performed using Glide in extra precision (XP) mode. The co-crystallized ligand was redocked as a control to validate binding process. Docking scores (kcal/mol) were used to assess binding affinity and pose.

Pharmacokinetics and drug-likeness properties

The physicochemical, pharmacokinetic, and toxicity profiles of spinasterol (1) were predicted using the QikProp module in Schrödinger Maestro (v13.5) [31, 32]. The structure of spinasterol was first drawn in ChemDraw 22.0.0, converted to SDF format, and then analyzed using the QikProp module.

Statistical analysis

SPSS (Statistical Package for the Social Sciences) software, version 26 was used to analyze antimicrobial data. ZOI are reported as mean ± standard error of the mean (SEM), including the 6 mm disc diameter. MIC values represent the mean of three independent experiments performed in triplicate. Statistical analysis was carried out using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc multiple comparison test, with p < 0.01 considered statistically significant.

Results

Extraction yield

In Ethiopian traditional medicine, fresh I. rothii tubers are typically pounded and boiled in water for wound treatment. In this study, however, 80% methanol was used for extraction due to its broader polarity range, which allows more effective recovery of plant constituents. The 80% methanol extract yielded 12% (w/w). Subsequent fractionation by solubility resulted in four fractions with yields of 23.3% (chloroform), 6.7% (ethyl acetate), 53.3% (methanol), and 13.3% (aqueous).

Structural elucidation of compound 1

Compound 1 was characterized using ¹H, ¹³C, DEPT-135 NMR and ESI–MS. The ¹³C-NMR spectrum (Supplementary Material 3) showed 29 carbon signals, with DEPT-135 (Supplementary Material 4) identifying six methyl, nine methylene, and eleven methine carbons, plus three quaternary carbons at δ 139.50, 34.25, and 43.31 ppm. A characteristic signal at δ 71.07 (C-3) suggested compound 1 is a sterol. Three olefinic methine carbons appeared at δ 117.47 (C-7), 129.49 (C-23), and 138.15 (C-22), along with a quaternary olefinic carbon at δ 139.50 (C-8). The ¹H-NMR spectrum (Supplementary Material 2) supported this, showing an oxymethine proton at 3.62 ppm (H-3) and three olefinic protons at δ 5.18 (H-7), 5.19 (H-22), and 5.05 ppm (H-23), consistent with two double bonds. In the positive ESI–MS spectrum (Supplementary Material 1), compound 1 exhibited a prominent dehydrated ion at m/z 395.5 ([M + H − H₂O]⁺), whereas the protonated pseudomolecular ion ([M + H]⁺) was not detected. This behavior is consistent with the ionization characteristics of sterols, which frequently display weak or absent [M + H]⁺ signals under ESI conditions.

Compound 1 was conclusively identified as spinasterol (stigmasta-7,22-dien-3-ol) by comparing its NMR data with literature reports from I. balsamina [21]. To our knowledge, this is the first report of spinasterol in I. rothii tubers. Its structure is shown in Fig. 2.

Fig. 2 — Chemical structure of spinasterol (1)

Antimicrobial activity of tuber extract and spinasterol

Antibacterial activity of tuber extract and spinasterol

In this study, the tuber extract and spinasterol (1) were evaluated against 26 bacterial strains and demonstrated significant antibacterial activity, with inhibition zones of 8–17 mm at 200 µg/mL and MICs values of 25–400 µg/mL (Table 1). According to previous reports [33], plant extracts that produce inhibition zones greater than 10 mm at 500 µg/mL are considered to exhibit strong antibacterial activity; therefore, the activities observed in this study can be regarded as strong.

Table 1.

Zones of inhibition and minimum inhibitory concentrations (MICs) of the tuber extract and spinasterol isolated from I. rothii against bacterial strains

Bacteria	Zone of inhibition in mm (200 µg/mL)ⁱ			MIC (µg/mL)ⁱⁱ
	Extract	Spinasterol	Ciprofloxacin	Extract	Spinasterol	Ciprofloxacin
Bacillus pumilus 82	8.5 ± 0.06^a	8.0 ± 0.09^a	19.0 ± 0.12^b	200	NA	100
Bacillus subtilis ATCC 6633	8.0 ± 0.06^a	8.0 ± 0.06^a	18.0 ± 0.12^b	200	NA	100
Escherichia coli 3:37C	13.0 ± 0.09^a	14.0 ± 0.17^b	16.5 ± 0.00^c	50	25	25
Escherichia coli 872	13.0 ± 0.12^a	14.0 ± 0.11^b	16.0 ± 0.14^c	50	25	25
Escherichia coli C600*	12.5 ± 0.03^a	12.5 ± 0.03^a	13.5 ± 0.12^b	100	100	25
Escherichia coli CD/99/1	13.5 ± 0.12^a	15.5 ± 0.11^b	17.0 ± 0.09^c	50	25	25
Escherichia coli HB101*	14.0 ± 0.11^a	13.0 ± 0.03^b	14.0 ± 0.15^a	100	100	25
Escherichia coli K88	14.0 ± 0.09^a	15.1 ± 0.07^b	17.1 ± 0.13^c	50	25	25
Escherichia coli LT37	13.0 ± 0.15^a	14.5 ± 0.15^b	16.0 ± 0.02^c	50	25	25
Escherichia coli NCTC 5933	13.5 ± 0.03^a	14.5 ± 0.14^b	16.0 ± 0.15^c	50	25	25
Escherichia coli NCTC 7360	14.5 ± 0.14^a	14.5 ± 0.15^a	17.0 ± 0.09^b	50	25	25
Escherichia coli ROW 7/12	13.5 ± 0.03^a	14.5 ± 0.15^b	16.5 ± 0.01^c	50	25	25
Pseudomonas aeruginosa MDR 1*	11.5 ± 0.14^a	11.5 ± 0.14^a	12.5 ± 0.04^b	100	400	50
Salmonella enterica TD 01	15.1 ± 0.07^a	15.1 ± 0.12^a	19.0 ± 0.04^b	50	100	150
Salmonella typhi Ty2	14.0 ± 0.09^a	14.0 ± 0.15^a	16.0 ± 0.09^b	50	100	200
Shigella boydii D13629	13.5 ± 0.06^a	13.5 ± 0.06^a	20.0 ± 0.00^b	200	100	150
Shigella dysentery 8	14.5 ± 0.03^a	14.5 ± 0.15^a	20.0 ± 0.05^b	100	100	150
Shigella flexneri Type 6	13.5 ± 0.00^a	13.5 ± 0.12^a	20.5 ± 0.00^b	200	100	150
Shigella soneii 1	14.0 ± 0.12^a	14.0 ± 0.12^a	19.5 ± 0.00^b	100	100	150
Staphylococcus aureus MDR 1*	10.5 ± 0.15^a	10.0 ± 0.20^b	11.5 ± 0.14^c	100	400	200
Staphylococcus aureus MDR 2*	11.1 ± 0.07^a	10.5 ± 0.15^b	12.1 ± 0.07^c	100	400	200
Staphylococcus aureus ML 267	17.0 ± 0.03^a	17.0 ± 0.03^a	18.0 ± 0.12^b	25	50	100
Vibrio cholerae NCTC 4693	13.0 ± 0.03^a	13.0 ± 0.15^a	17.5 ± 0.07^b	50	25	25
Vibrio cholerae NCTC 5596	13.0 ± 0.15^a	12.9 ± 0.18^a	18.5 ± 0.03^b	50	25	25
Vibrio cholerae NCTC 10732	13.0 ± 0.15^a	13.0 ± 0.06^a	19.0 ± 0.12^b	50	25	25
Vibrio cholerae NCTC 11501	13.5 ± 0.12^a	13.5 ± 0.12^a	18.5 ± 0.09^b	50	25	25

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ⁱZones of inhibition are expressed as mean ± SEM, including the 6 mm disc diameter. Different superscript letters within the same row indicate statistically significant differences (p < 0.01) among extract, spinasterol and ciprofloxacin; ⁱⁱMIC values represent the mean of three independent experiments performed in triplicate; NA– No activity; * = multidrug resistance

Both the tuber extract and spinasterol displayed antibacterial activity against a wide range of bacterial strains. The strongest effects were observed against clinically important Gram-negative pathogens. Particularly, Gram-negative bacteria are typically less susceptible to antibiotics because their outer membrane limits permeability. However, in our study, several Gram-negative bacteria were sensitive to both the tuber extract and spinasterol.

Using the disk diffusion method, spinasterol showed significantly higher activity (p < 0.01) than the tuber extract, particularly against most non-MDR E. coli strains. However, no significant difference (p > 0.01)was observed between spinasterol and the tuber extract against the MDR E. coli C600 variant, whereas the tuber extract exhibited significantly higher activity (p < 0.01) than spinasterol against the MDR E. coli HB101 variant. In the broth dilution assay, spinasterol exhibited antibacterial activity against multiple E. coli strains (MIC = 25 µg/mL, excluding MDR variants), V. cholerae (MIC = 25 µg/mL), and all tested Salmonella and Shigella species (MIC = 100 µg/mL each). This degree of susceptibility is interesting considering the well-established resistance mechanisms of Gram-negative bacteria.

According to González-Pastor et al. [34], plant extracts with MIC values between 100 and 1000 µg/mL are classified as antimicrobials, while values below 400 µg/mL indicate strong antimicrobial activity [35]. In our study, both the tuber extract of I. rothii and spinasterol (1) fell within this range, with MIC values from 25 to 400 µg/mL across most tested strains (Table 1). The tuber extract showed significantly higher activity (p < 0.01) than spinasterol against Gram-positive MDR S. aureus strains (MDR-1 and MDR-2) using the disk diffusion method. Among all tested bacterial strains, Gram-positive B. pumilus 82 and B. subtilis ATCC 6633 exhibited significantly lower susceptibility (p < 0.01) to both the tuber extract and spinasterol compared to the positive control, ciprofloxacin. Previous work on whole-plant extracts of Impatiens balsamina reported relatively high MICs (2500–10,000 µg/mL) against S. aureus, S. epidermidis, S. pyogenes, S. pneumoniae, P. aeruginosa, and E. coli using the broth dilution method [36], suggesting that I. rothii may contain more potent antibacterial constituents.

In certain cases, the tuber extract was even more effective than spinasterol, particularly against MDR strains. For example, against P. aeruginosa MDR-1, the extract achieved an MIC of 100 µg/mL compared to 400 µg/mL for spinasterol. These differences may reflect may be due to possible synergistic or additive effects among multiple phytochemicals present in I. rothii, which likely enhance its overall antibacterial potency. This explanation is consistent with earlier reports [12, 37], which proposed that the antibacterial activity within the genus Impatiens often results from interactions among multiple compounds, rather than the action of a single constituent alone.

Incubation of each bacterial strain with the extract or spinasterol, following the method of Miles et al. [38], did not result in any colony-forming units, indicating that both the extract and spinasterol are bactericidal rather than bacteriostatic in their mode of action.

Antifungal activity of tuber extract and spinasterol

The antifungal evaluation of the tuber extract and spinasterol from I. rothii demonstrated significant activity against four fungi strains. Using the disk diffusion method, the tuber extract exhibited inhibition zones ranging from 11.0 to 13.5 mm, which were comparable to those of the standard antifungal agent griseofulvin (13.0–15.2 mm) (Table 2). These antifungal results suggest that the tuber extract has significant antifungal potential, in line with previous reports showing that phytochemicals from Impatiens species exhibit activity against diverse microbial strains [39].

Table 2.

Zones of inhibition (ZoI) and minimum inhibitory concentrations (MICs) of the tuber extract and spinasterol isolated from I. rothii against fungal strains

Fungi	Zone of inhibition in mm (1500 µg/mL)ⁱ			MIC (µg/mL)ⁱⁱ
Fungi	Extract	Spinasterol	Griseofulvin	Extract	Spinasterol	Griseofulvin
Aspergillus niger ATCC 6275	11.0 ± 0.15^a	12.0 ± 0.09^b	14.5 ± 0.23^c	800	400	400
Candida albicans ATCC 10231	12.4 ± 0.13^a	13.0 ± 0.09^a	15.2 ± 0.17^b	800	200	500
Penicillium funiculosum NCTC 287	13.5 ± 0.03^a	12.0 ± 0.15^b	11.0 ± 0.12^c	1000	1000	500
Penicillium notatum ATCC 11625	10.0 ± 0.12^a	10.5 ± 0.03^a,b	12.0 ± 0.43^b	800	800	500

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ⁱZones of inhibition are expressed as mean ± SEM, including the 6 mm disc diameter. Different superscript letters within the same row indicate statistically significant differences (p < 0.01) among extract, spinasterol, and griseofulvin; ⁱⁱMIC values represent the mean of three independent experiments performed in triplicate

The MIC values obtained via broth dilution further confirmed the antifungal potency of the extract. The lowest MICs (800 μg/mL) were observed against C. albicans, A. niger, and P. notatum, suggesting a moderate inhibitory effect. In contrast, P. funiculosum was less susceptible to the extract, indicating potential species-specific variability in response to phytochemicals present in the tuber.

Spinasterol exhibited higher antifungal activity than the tuber extract. It was most potent against C. albicans (MIC = 200 μg/mL) and A. niger (MIC = 400 μg/mL), while P. funiculosum and P. notatum showed lower susceptibility (Table 2). The enhanced activity of spinasterol against C. albicans is particularly significant, as this fungus is a common human pathogen responsible for opportunistic infections [40].

In our study, the tuber extract demonstrated promising antimicrobial activity, supporting the traditional medicinal use of I. rothii. Traditionally, decoctions and infusions prepared from the leaves, stems, and tubers of I. rothii have been used to treat both localized and systemic bacterial and fungal infections. Our findings support these ethnomedicinal practices and confirm that I. rothii is a valuable source of extracts with potent antimicrobial properties.

Incubation of plugs taken from the zones of inhibition for 96 h did not yield any growth of any of the tested fungal strains, suggesting that both the extract and spinasterol are fungicidal rather than fungistatic.

In Silico analysis

Molecular docking analysis

Molecular docking is a powerful tool in drug discovery, as it provides valuable insights into the interactions between ligands and their protein targets. To explore the antibacterial mechanism of spinasterol, docking studies were performed against the active site of MRSA pyruvate kinase (PK, PDB ID: 3T07) using the Schrödinger 2023–1 Suite [28]. Figure 3 depicts both the 2D and 3D binding modes of spinasterol within the active site. The docking analysis yielded a moderate binding score of ‒ 5.278 kcal/mol (Table 3), suggesting that while spinasterol binds favorably, and its affinity is limited. This may be attributed to the absence of strong hydrogen bonding.

Fig. 3 — A 3D representation of spinasterol docked within the active site of Methicillin-resistant *Staphylococcus aureus* (MRSA) pyruvate kinase (PK, PDB ID: 3T07); B The zoomed 3D view of the ligand–protein interactions; C 2D interaction map of spinasterol with key residues in the MRSA pyruvate kinase active site

Table 3.

Docking scores of spinasterol and native ligand with in the active site of 3T07

Ligand	Glide Score (kcal/mol) within 3T07	Interaction with amino acid residues
		H-bonds	Non-H-bonds
Spinasterol (1)	‒5.278	‒	ALA358; ASN369; HIS365; ILE361; LEU370; SER362, THR348; THR353; THR366
Native ligand	‒8.877	HIE365; SER362	ALA358; ASN369; HIS365; ILE361; LEU370; MET467; THR348; THR353; THR366

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Native ligand = bis-indole alkaloid ((3S,5R)-3,5-bis(6-bromo-1H-indol-3-yl)piperazin-2-one)

Spinasterol, being a predominantly non-polar compound, interacts mainly with non-polar amino acid residues located in the hydrophobic pocket of the protein. As illustrated in Fig. 3, its steroidal core is stabilized by hydrophobic contacts with residues such as ALA358, ILE361, and LEU370, while additional polar interactions are formed with ASN369, HIS365, SER362, THR348, THR353, and THR366. Collectively, these interactions indicate that spinasterol achieves stabilization within the pocket through a balance of hydrophobic and polar contacts, although the lack of hydrogen bonding may explain its moderate docking score. The docking process was validated using a redocking approach with the native ligand, a bis-indole alkaloid [(3S,5R)-3,5-bis(6-bromo-1H-indol-3-yl)piperazin-2-one], which yielded a docking score of ‒ 8.877 kcal/mol (Supplementary Material 5). Inhibition of MRSA pyruvate kinase by spinasterol could potentially disrupt glycolytic ATP generation and carbon flux, impairing essential biosynthetic pathways and compromising bacterial survival. Nevertheless, experimental enzyme assays are required to validate this predicted mechanism.

The molecular docking results revealed that spinasterol exhibited a more favorable binding affinity (Glide score: ‒ 8.523 kcal/mol) toward the active site of C. albicans sterol 14-α demethylase (CYP51, PDB ID: 5TZ1; chain A, HEC/HEM, antifungal [29]. Validation of the docking procedure was performed by re-docking the native ligand VT-1161 into its respective binding site, which produced a Glide score of ‒ 7.089 kcal/mol (Supplementary Material 6). Since a lower docking score indicates a stronger and more stable interaction, these findings suggest that spinasterol may inhibit CYP51 more effectively than the native ligand. Analysis of the docking poses showed that spinasterol did not form hydrogen bonds within the binding site of CYP51. However, it established multiple stabilizing interactions, including hydrophobic contacts (ILE379, LEU87, LEU88, LEU121, LEU376, MET508, PHE228, PHE233, PHE380, PRO230, TYR64, TYR118, TYR132, TYR505, VAL509), polar interactions (HIE377, SER378, SER506, SER507, THR122, THR311), and a metal coordination bond with HEM601, within the heme-binding pocket of CYP51 (Table 4).

Table 4.

Docking scores of spinasterol and native ligand with in the active site of 5TZ1

Ligand	Glide Score (kcal/mol) within 5TZ1	Interaction with amino acid residues
Ligand	Glide Score (kcal/mol) within 5TZ1	H-bonds	Non-H-bonds
Spinasterol (1)	‒ 8.523	‒	HEM601; HIE377; ILE379; LEU87; LEU88; LEU121; LEU376; MET508; PHE228; PHE233; PHE380; PRO230; SER378; SER506; SER507; THR122; THR311; TYR64; TYR118; TYR132, TYR505, VAL509
Native ligand	‒ 7.089	TYR132	HEM601; HIE377; ILE131; ILE304; ILE379; LEU87; LEU121; LEU139; LEU376; MET508; PHE126; PHE228; PHE380; PHE233; SER378; SER507; TYR64; TYR118; THR122; THR311

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Native ligand = VT-1161 ((2S,4S)-2-(4-Fluorophenyl)-4-(4-fluorophenyl)-1,3-thiazolidine-2,4-diol)

These findings highlight the ability of spinasterol to fit tightly within the enzyme’s active site (Fig. 4), thereby potentially blocking the ergosterol biosynthesis pathway, which is crucial for fungal cell membrane integrity. Although docking provides valuable insights, these predictions require further validation through molecular dynamics simulations, enzyme inhibition assays, and antifungal susceptibility testing. Overall, the strong binding profile of spinasterol underscores its promise as a candidate for further investigation.

Pharmacokinetics and drug-likeness properties of spinasterol

In drug discovery, predicting the physicochemical properties and drug-likeness compatibility of compounds is essential. Early evaluation of these parameters, for instance through Lipinski’s rule of five, increases the likelihood of compounds advancing successfully to lead optimization. In this study, QikProp (Schrödinger 2023) [32] was employed to analyze the physicochemical characteristics and drug-likeness profile of spinasterol (Table 5).

Table 5.

Physicochemical properties, lipophilicity, and ADME descriptors of spinasterol

Compound	mol MW (g/mol)	accptHB	donorHB	#rotor	PSA (Å²)	QPlogS	QPlogPo/w	QPlogKhsa	LR-5
Spinasterol (1)	412.698	1.700	1.000	6	22.414	‒ 8.278	7.426	2.039	1
Recommended values³¹	130–725	2–20	0–6	≤ 10	≤ 140	− 6.5 to 0.5	− 2.0 to 6.5	− 0.5 to + 0.9

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mol MW= molecular weight; accptHB= number of hydrogen bond acceptors; donorHB= number of hydrogen bond donors; #rotor = number of rotatable bonds; PSA=polar surface area; QPlogS = predicted aqueous solubility; QPlogPo/w = Predicted octanol/water partition coefficient at pH=7.4; QPlogKhsa = Prediction of binding to human serum albumin (plasma protein binding); LR-5=Lipinski rule of 5.

QikProp analysis of spinasterol revealed favorable oral absorption characteristics, with high predicted Caco-2 (3397 nm/sec) and MDCK (1855 nm/sec) permeability values, and 100% human oral absorption. The compound’s low polar surface area (22.4 Å²) and moderate brain–blood partition coefficient (QPlogBB = –0.27) indicate excellent membrane permeability and potential CNS penetration. However, its very high lipophilicity (QPlogPo/w = 7.43) and poor aqueous solubility (QPlogS = –8.28) suggest limited bioavailability and formulation challenges. Strong serum albumin binding (QPlogKhsa = 2.04) and six predicted metabolic sites point to reduced free plasma concentrations and possible metabolic instability. Furthermore, the hERG liability (QPlogHERG = –4.31) raises some concern for cardiotoxicity (Table 6). Collectively, these findings highlight spinasterol (1) as a compound with excellent absorption but limited solubility and suboptimal safety profiles, requiring structural optimization to improve its drug-likeness.

Table 6.

Pharmacokinetics and toxicity profile prediction of spinasterol

Compound	QPPCaco (nm/sec)	%HIA	QPlogBB	QPlogHERG	QPPMDCK (nm/sec)	QPlogKp	#metab
Spinasterol (1)	3397.080	100%	‒ 0.268	‒ 4.311	1855.293	− 1.783	6
Recommended values³¹	> 500	> 80%	− 3.0 to + 1.2	> − 5	> 500	− 8.0 to − 1.0	1–8

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QPPCaco = Predicted apparent Caco-2 cell permeability in nm/sec; %HIA = %Human Oral Absorption; QPlogBB= Predicted brain/blood partition coefficient; QPlogHERG= Predicted IC50 for blockage of HERG K+ channels; QPPMDCK= Predicted Apparent Permeability across a MDCK cell monolayer; QPlogKp= predicted skin permeability coefficient; #metab= ; Predicted metabolism that may be prone to metabolic instability

Discussion

AMR poses a persistent challenge to global public health, and the search for new bioactive scaffolds from natural sources remains a priority. Natural products currently contribute to about 35% of antimicrobial drugs [41], and phytosterols such as spinasterol represent an important class of bioactive compounds.

In this study, spinasterol, a phytosterol previously reported in several plant species, including Cucumis dipsaceus [27], Melia azedarach [42], Securidaca inappendiculata [43], Aster pseudoglehnii [44] and Acacia auriculiformis [45] was isolated and characterized for the first time from I. rothii. Both the tuber extract and spinasterol demonstrated antibacterial activity against diverse bacterial strains, particularly against Gram-negative bacteria such as E. coli and V. cholerae. Despite the usual challenge to treat Gram-negative bacteria due to their outer membrane of lipopolysaccharides [46], our findings showed positive results against Gram-negative bacteria. Gram-negative bacteria's higher fat content may contribute to this unexpected outcome. Spinasterol showed MIC values as low as 25 µg/mL, which is promising given the intrinsic resistance barriers in Gram-negative species. This finding is consistent with previous studies on spinasterol isolated from different plant species, which also highlight its antimicrobial potential. For example, Assefa et al. [27] reported that spinasterol extracted from the fruits of C. dipsaceus exhibited antibacterial activity against E. coli, P. aeruginosa, and S. pyogenes at a concentration of 100 µg/mL. Likewise, Fufa et al. [42] demonstrated the in vitro antibacterial activity of spinasterol obtained from the leaves of M. azedarach and the stem bark of Albizia schimperiana against B. subtilis, S. aureus, P. aeruginosa, and E. coli. Interestingly, both the extract and spinasterol demonstrated bactericidal rather than bacteriostatic activity. Bactericidal agents are typically preferred because they can rapidly eradicate bacteria regardless of host immunity and may lower the risk of resistance development [47, 48].

Likewise, the I. rothii tuber extract and its isolated compound, spinasterol, exhibited moderate antifungal activity, with spinasterol showing particularly promising inhibitory effects against C. albicans. The higher activity of spinasterol may be due to its greater purity, concentration, and specific antifungal mechanism compared to the tuber extract. Furthermore, the fungicidal activity exhibited by the I. rothii extract and spinasterol provides important therapeutic advantages, particularly in cases of severe infection and among immunocompromised patients. Unlike fungistatic agents, which only inhibit fungal growth and rely on host immune mechanisms for pathogen clearance, fungicidal compounds directly kill fungal cells, leading to rapid elimination of the infection. This property not only reduces the risk of relapse but may also limit the development of resistant strains [49–51]. The present study underscores the potential implications of these findings for the development of broad-spectrum antifungal agents, suggesting that spinasterol may represent a promising lead compound for future antifungal drug discovery efforts.

The in silico analyses provided further insight into the molecular basis of these antimicrobial effects. Molecular docking revealed that spinasterol binds moderately to MRSA pyruvate kinase (‒ 5.278 kcal/mol), potentially interfering with glycolysis and energy production, while showing stronger affinity for C. albicans sterol 14-α demethylase (‒ 8.523 kcal/mol), a key enzyme in ergosterol biosynthesis. This strong binding affinity, surpassing that of the native ligand VT-1161, suggests that spinasterol may act as an inhibitor of fungal membrane biosynthesis, in agreement with the antifungal assay results. Despite not forming hydrogen bonds, spinasterol shows stronger docking affinity due to strong hydrophobic interactions that dominate its stabilization within the active site, consistent with its lipophilic nature. However, these molecular docking findings remain predictive, and additional experimental validation, such as enzyme inhibition assays, is necessary to confirm them.

QikProp-based ADME predictions indicated that spinasterol has excellent intestinal absorption and membrane permeability, suggesting favorable bioavailability. However, it also exhibited poor aqueous solubility, strong plasma protein binding, high lipophilicity, and potential hERG liability, which may negatively affect its pharmacokinetic and safety profile. High lipophilicity is often associated with poor solubility, tissue accumulation, rapid metabolic clearance, and increased risk of off-target toxicity, while poor solubility can reduce drug dissolution and absorption. In addition, potential hERG inhibition raises concerns regarding cardiotoxicity, including QT interval prolongation and arrhythmias. Therefore, although spinasterol demonstrates promising drug-like properties, structural optimization or advanced formulation strategies may be required to improve its solubility, safety, and overall therapeutic potential.

The present findings demonstrate the dual antibacterial and antifungal potential of spinasterol. Interestingly, the extract of I. rothii showed greater activity than spinasterol against some multidrug-resistant strains, suggesting possible synergistic or additive effects among its phytochemicals. Such synergism is consistent with previous reports from Impatiens species [36, 37]. However, these interactions were not experimentally quantified in this study, and not all constituents were identified. Thus, future work involving fractionation and synergy testing has been suggested. Other key limitations include the absence of in vivo validation, cytotoxicity assessment, and experimental mechanistic studies such as enzyme inhibition assays. Therefore, the observed antimicrobial effects and predicted molecular interactions require further confirmation. Overall, this finding supports the plant’s long-standing ethnomedicinal use in Ethiopia for the treatment of infectious diseases [52–54].

Conclusions

In this study, spinasterol was successfully isolated for the first time from the tubers of I. rothii and evaluated for antimicrobial potential. Both the hydroalcoholic extract and spinasterol displayed antibacterial and antifungal activity against a wide range of microbes, particularly against E. coli, V. cholerae, and C. albicans. The tuber extract outperformed spinasterol against some multidrug-resistant strains, suggesting synergistic or additive effects among its phytochemicals. Molecular docking analyses revealed that spinasterol interacts moderately with MRSA pyruvate kinase and strongly with C. albicans sterol 14-α demethylase, supporting its antibacterial and antifungal mechanisms. In silico ADME predictions showed excellent absorption and permeability but highlighted issues of poor solubility, high protein binding, and potential cardiotoxicity, underscoring the need for optimization. Overall, the results may support the ethnomedicinal use of I. rothii, and spinasterol highlights its potential as a source of antimicrobial scaffolds, warranting further structural, synergistic, mechanistic, and in vivo investigations.

Supplementary Information

41182_2026_996_MOESM1_ESM.docx^{(2MB, docx)}

Supplementary material 1. Positive-mode electrospray ionization mass spectrum (+-mode ESI-MS) of spinasterol (1). ¹H-NMR spectrum of spinasterol (1). ¹³C-NMR spectrum of spinasterol (1). DEPT-135 NMR spectrum of spinasterol (1). Interaction between native ligand and Methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK, PDB ID: 3T07): (A): 2D and (B): 3D. Interaction between native ligand and sterol 14-alpha demethylase from Candida albicans (PDB ID: 5TZ1); (A): 2D and (B): 3D.

Acknowledgements

The authors are indebted to Melaku Wondafrash, National Herbarium, College of Natural and Computational Sciences, Addis Ababa University, for the identification of plant. DeB would like to thank Addis Ababa University and Hawassa University for sponsoring the study.

Author contributions

DeB initiated the research agenda, collected and prepared the plant material, conducted the laboratory works, and prepared the draft manuscript. AM oversee the antimicrobial activity. MYY assisted in the laboratory with compound isolation and provided guidance during the research work. KA and DaB supervised the study, involved in the analyses, conducted interpretation of the experimental results, and edited the final manuscript. All authors read and approved the final version of the manuscript.

Funding

The present study was financially supported by the International Science Program (ISP), Uppsala University through ETH:02 project.

Data availability

All data generated or analyzed during this study are included in this article.

Declarations

Ethics approval and consent to participate

Ethical approval is not applicable for this article.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

41182_2026_996_MOESM1_ESM.docx^{(2MB, docx)}

Data Availability Statement

All data generated or analyzed during this study are included in this article.

[CR1] 1.Gebreyes WA, Dupouy-Camet J, Newport MJ, et al. The global One Health paradigm: challenges and opportunities for tackling infectious diseases at the human, animal, and environment interface in low-resource settings. PLoS Negl Trop Dis. 2014;8(11):e3257. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR2] 2.Weiss RA, McMichael AJ. Social and environmental risk factors in the emergence of infectious diseases. Nat Med. 2004;10(12):70–6. 10.1038/nm1150. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR3] 3.Institute of Medicine. Microbial threats to health in the United States. In: Lederberg J, Shope RE, Oaks SC Jr., eds. Emerging Infections. 2nd ed. Washington, DC: National Academy Press; 1992:chap 2. [PubMed]

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PERMALINK

Isolation, antimicrobial evaluation, and in silico docking of spinasterol from Impatiens rothii Hook. f. tuber extract

Debebe Birhanu

Avijit Mazumder

Mariamawit Y Yeshak

Kaleab Asres

Daniel Bisrat

Abstract

Background

Methods

Results

Conclusions

Supplementary Information

Background

Materials and methods

Plant material

Fig. 1.

Chemicals, reagents and drugs

Instruments

Bacterial strains

Fungal strains

Preparation of extract

Solubility-based fractionation

Compound isolation by column chromatography

In vitro antibacterial and antifungal activity assay

Determination of minimum inhibitory concentrations (MICs) by broth dilution

Disc diffusion method

Determination of mode of action

In silico study

Molecular docking

Pharmacokinetics and drug-likeness properties

Statistical analysis

Results

Extraction yield

Structural elucidation of compound 1

Fig. 2.

Antimicrobial activity of tuber extract and spinasterol

Antibacterial activity of tuber extract and spinasterol

Table 1.

Antifungal activity of tuber extract and spinasterol

Table 2.

In Silico analysis

Molecular docking analysis

Fig. 3.

Table 3.

Table 4.

Fig. 4.

Pharmacokinetics and drug-likeness properties of spinasterol

Table 5.

Table 6.

Discussion

Conclusions

Supplementary Information

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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