Phytochemical characterization of Cleome droserifolia biomass and its application as in vitro antioxidant, anti-inflammatory, anti-diabetic, and anti-yeast agents

Abstract

This study investigated the anti-inflammatory, anti-diabetic, anti-yeast, and antioxidant properties of the ethanolic extract of Cleome droserifolia shoot (CDE). Thirteen phenolic compounds were shown in the CDE ethanolic extract using high-performance liquid chromatography (HPLC), with rutin and syringic acid being the predominant components. Rutin and syringic acid were found at elevated levels of 15,900 and 5320 µg//g of extract, respectively, in association with CDE. Additionally, gallic acid, chlorogenic acid, ellagic acid, and vanillin were quantified at 1150, 987, 2830, and 1425 µg/g of extract, respectively. The ethanolic extract of CDE exhibited detrimental impacts on the species of pathogenic yeast. The CDE demonstrated scavenging activity for 2,2-diphenyl-1-picrylhydrazyl to visualize an antioxidant action with IC₅₀ 8.95±1.023 µg/mL. CDE inhibited COX-1 and COX-2 to document its anti-inflammatory potential with IC₅₀ 12.91±0.5a µg/mL and 21.63±0.8 µg/mL, respectively. CDE inhibited amylase activity as a marker of diabetic management with IC_{50 of} 14.93±1.87µg/mL. The pathogenic yeasts including C. albicans, C. tropicalis, and C. glabrata were suppressed by CDE with inhibition zones 25±0.2, 20±0.1, and 25±0.1 mm, respectively. The findings suggest the utilization of CDE for the management of numerous health issues.

Full Article

Phytochemical Characterization of Cleome droserifolia Biomass and its Application as in vitro Antioxidant, Anti-Inflammatory, Anti-Diabetic, and Anti-Yeast Agents

Mohamed A. Amin  ,^a,* Mahmoud H. Hendy,^a Sulaiman A. Alsalamah  ,^b,* Salma Yousif Sidahmed Elsheikh, ^c Ahmed M. Abdel-Mageed,^c Shereen Ahmed Elwasefy,^d A. M. Elbasiony, ^e and Ismail M. A. M. Shahhat ^f

This study investigated the anti-inflammatory, anti-diabetic, anti-yeast, and antioxidant properties of the ethanolic extract of Cleome droserifolia shoot (CDE). Thirteen phenolic compounds were shown in the CDE ethanolic extract using high-performance liquid chromatography (HPLC), with rutin and syringic acid being the predominant components. Rutin and syringic acid were found at elevated levels of 15,900 and 5320 µg//g of extract, respectively, in association with CDE. Additionally, gallic acid, chlorogenic acid, ellagic acid, and vanillin were quantified at 1150, 987, 2830, and 1425 µg/g of extract, respectively. The ethanolic extract of CDE exhibited detrimental impacts on the species of pathogenic yeast. The CDE demonstrated scavenging activity for 2,2-diphenyl-1-picrylhydrazyl to visualize an antioxidant action with IC₅₀ 8.95±1.023 µg/mL. CDE inhibited COX-1 and COX-2 to document its anti-inflammatory potential with IC₅₀ 12.91±0.5a µg/mL and 21.63±0.8 µg/mL, respectively. CDE inhibited amylase activity as a marker of diabetic management with IC_{50 of} 14.93±1.87µg/mL. The pathogenic yeasts including C. albicans, C. tropicalis, and C. glabrata were suppressed by CDE with inhibition zones 25±0.2, 20±0.1, and 25±0.1 mm, respectively. The findings suggest the utilization of CDE for the management of numerous health issues.

DOI: 10.15376/biores.20.4.9020-9032

Keywords: Cleome droserifolia; Lipase; Amylase; Yeast; COX-1, COX-2

Contact information: a: Botany and Microbiology Department, Faculty of Science (Boys), Al-Azhar University, Cairo 11884, Egypt; b: Department of Biology, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh 11623, Saudi Arabia; c: Department of Biological Sciences, College of Science, Northern Border University, Arar, Saudi Arabia; d: Nursing Department, College of Applied Medical Sciences, Jouf University, Al Qurryat, Saudi Arabia, selwasifi@ju.edu.sa; e: Department of Chemistry, College of Science, Northern Border University (NBU), Arar, Saudi Arabia; f: Egyptian Drugs Authority, Department of Medicinal Plants and Natural Products, Cairo, Egypt, caironodcar@gmail.com;

* Corresponding author: mamin7780@azhar.edu.eg (M.A.A.) and SAAlsalamah@imamu.edu.sa (S.A.A.)

INTRODUCTION

The health of humans and animals can be enhanced by using nutraceuticals (food supplements) and medicinal plants as an alternative form of therapeutic medications (Alawlaqi et al. 2023; Al-Rajhi et al. 2025). In recent decades, there has been increased interest in looking at plants as potential sources of various medications, particularly antimicrobials, to combat bacteria that are resistant to numerous therapies (Hashem et al 2019; Almehayawi et al. 2024; Qanash et al. 2024). Cleome is one of the largest genera in the medicinal plant family Cleomaceae. Approximately 180 to 200 species of this genus can be found in arid and semi-arid regions like Egypt, Libya, Palestine, and Syria (Moustafa and Mahmoud 2023). They feature complexly branching stems, three-nerved leaves with enlarged glandular hairs, and are low, fragrant perennial shrubs that range in length from 25 to 60 cm. They also resemble cushions (El-Askary et al. 2019). Its remarkable array of naturally occurring phenolic compounds appears to be responsible for the strong antioxidant, antibacterial, and immunomodulatory properties of the aerial parts of the Cleome droserifolia shrub, which can enhance general health (Hashem and Shehata 2021). A substance known as 3-ethylsulfonyl-2,3-dimethoxypropyl was successfully isolated and identified from the methanolic extract of Cleome africana. Molecular docking studies indicate that this molecule exhibits promising activity and may serve as an effective antidiabetic agent by inhibiting glucosidase. Several extracts demonstrated hepato-protective and anti-inflammatory actions (Abdullah et al. 2021). Due to its antioxidative potential and antidiabetic properties, it can be concluded that C. droserifolia extract medication possesses a therapeutic protective role in diabetes by mitigating oxidative stress and damage to pancreatic β-cells (Helal et al. 2015). C. droserifolia, containing bioactive compounds, serves as a significant therapeutic plant capable of inducing apoptosis in human cancer cells (Panicker et al. 2020). This study focused on assessing the activity of C. droserifolia shoot extract against different species of pathogenic yeast, as well as the vital role of this extract versus COX-1 and COX-2 inhibitors. Also, the antioxidant and anti-amylase action of this extract was studied.

EXPERIMENTAL

Collecting and Preparing of Cleome droserifolia Shoot Extract (CDE)

In November 2023, C. droserifolia were collected from Saint Katrine in the South Sinai Governorate of Egypt. The plant shoots were cleaned under tap water, allowed to air dry at room temperature in the shade, and then ground into a finer consistency. For 72 h at 22 °C, a sealed bottle containing 25 g of pulverized C. droserifolia extract (CDE) was submerged in 0.25 L of 100% ethanol. After that, the extract was sonicated at 50 °C for 50 minutes. After filtration to create the crude CDE, the extracted materials were vacuum-concentrated in a rotary evaporator at 45 °C (Khowdiary et al. 2024).

HPLC Conditions

The phenolic and flavonoid profile was assessed by high-performance liquid chromatography using an Agilent 1260 series equipment. The separation process made use of a Zorbax Eclipse Plus C8 column that measured 4.6 mm by 250 mm i.d., and had a particle size of 5 μm. 0.9 mL/min of water (A) and 0.05% trifluoroacetic acid in acetonitrile (B) made up the mobile phase. The following describes the sequential programming of the mobile phase in an even gradient: 82% A for 0 min; 82% A for 0 to 1 minute; 75% A for 1 to 11 min; 60% A for 11 to 18 minutes; 82% A for 18 to 22 min; and 82% A for 22 to 24 min. At 280 nm, the multi-wavelength detector showed observation. Each specimen had an injection volume of 5 μL. The temperature of the column was maintained at 40 °C.

Antioxidant Activity

CDE’s capacity to scavenge DPPH was evaluated. Different double-fold dosages of the compounds under investigation, varying at different concentrations (1.95, 3.90, 7.81, 15.63, 31.25, 62.50, 125, 250, 500, and 1000 µg/mL), were combined with high-purity H₂O (Milli Q H₂O) while the DPPH was dissolution in methanol. In a test tube, 450 µL of Tris-HCl buffer (pH = 7.4), 1.0 mL of each concentration, and 1.0 mL of DPPH were mixed. After thoroughly mixing the contents of each tube, they were shaken (150 rpm) for 30 min at 37 °C in a dark environment. A test tube with all components except the tested compounds (ascorbic acid or extract) served as the negative control, while ascorbic acid served as the positive control at the same concentrations (Selim et al. 2025b). The scavenging activity was measured spectrophotometrically at 517 nm, and the percentage was computed as follows.

 (1)

The IC₅₀ value (the concentration of extract required to inhibit 50% of DPPH radicals) was determined from a dose–response curve plotted using the inhibition percentages.

In vitro COX-1 and COX-1 Inhibition Assay

Catalog number k548, Biovision, USA Kits were utilized to assess the test extract’s in vitro capacity for inhibiting COX-1 isoenzymes according to the manufacturer’s instructions. In an overall amount of 1 mL of the test, CDE were submerged in DMSO 1.0% and analyzed at doses ranging from 1000 to 0.5 μg/mL. Catalog number k547, Biovision, USA, kits were used to assess the efficiency of the extract to block the COX-2 isoenzyme. A final amount of 1 mL of the CDE was dissolved in DMSO 1.0% and analyzed at concentrations ranging from 1000 to 0.5 μg/mL. For the COX-1 and COX-2 inhibition test, celecoxib served as a positive control. The specimens were examined in duplicate at twelve distinct concentrations. Using GraphPad PRISM, a dose of the test CDE 50% inhibition (IC50) of COX-1 and COX-2 was determined from the concentration response curve (Selim et al. 2025a). The IC₅₀ values (concentration causing 50% inhibition) for COX-1 and COX-2 were calculated from the dose–response curves.

α-Amylase Inhibition Activity

Extract and acarbose, which had been prepared in various test tubes with 0.02 M phosphate buffer (pH=6.9), 500 µL of α-amylase (from Bacillus subtilis, Sigma-Aldrich, USA) was added, and the test components had been incubated for 10 min at 37 °C at various amounts (1000 to 1.95 µg/mL). After that, every tube received 500 µL of 1% a solution of starch, which was allowed to incubate for 10 min. To halt the reaction, 1 mL of 3,5-dinitrosalicylic acid was then added to each tube. The tubes were then incubated for 15 min at 60 °C in a water bath before being cooled and finished with 10 mL of dH₂O (Amin et al. 2025). The development color was measured at 540 nm, which indicates α-amylase % as follows,

 (2)

where Ac and At are the control and treatment absorbance, respectively.

The half-maximal inhibitory concentration (IC₅₀) of the extract was calculated based on dose-dependent inhibition. The IC₅₀ value, defined as the concentration of extract required to inhibit 50% of α-amylase activity, was determined using nonlinear regression analysis.

Anti-Yeast Activity

A well-plate agar diffusion experiment was used for assessing CDE’s yeast suppression versus three distinct kinds of Candida: C. glabrata (RCMB 027016), C. albicans (ATCC 10231), and C. tropicalis (ATCC 10243). Candida strains were collected from the culture collection of the microbiological lab at the Faculty of Science, Al-Azhar University, Egypt. Uncontaminated, melted Sabouraud dextrose medium was used to plant the plates containing yeasts. A sterilized cork borer was used to eliminate cups (6 mm radius) of the agar layer once the mixture had hardened. 100 μL from every CDE (20 μg/mL) was added to each cup using an automated microliter pipette. At 27 °C, the inoculation plates was incubated for 48 h. Fluconazole-containing wells were the control. After the incubation period, the inhibition zones were assessed (Abdelghany et al. 2021).

Minimum Concentration of CDE for Yeast Inhibition and as a Fungicidal Agent

The minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were determined using the twofold broth dilution method, as described by French (2006), with slight modifications. Sabouraud dextrose broth (SDB) was used as the culture medium. The extract was serially twofold diluted in SDB, starting from 125 μg/mL in a final volume of 2 mL. A negative control tube containing only sterile broth without extract was included. Yeast inocula were prepared by suspending 18-hour-old cultures in sterile saline and adjusting the turbidity to match the 0.5 McFarland standard (~1 × 10⁶ CFU/mL), using a densitometer. Each tube was inoculated with 0.5 mL of the standardized yeast suspension and mixed thoroughly. Tubes were incubated at 35 °C for 24 h. The MIC was defined as the lowest extract concentration with no visible turbidity (growth) observed by the unaided eye. To determine the MFC, 100 μL from tubes showing no visible growth were spread onto Sabouraud dextrose agar (SDA) plates and incubated at 35 °C for another 24 h. The MFC was the lowest concentration showing no colony growth.

Fig. 1. Biological functions and extraction of C. droserifolia

Statistical Analysis

Results were analyzed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). To calculate the ± standard deviation (SD) and mean values, tests were repeated three times.

RESULTS AND DISCUSSION

Assessment of Phenolic and Flavonoid Compounds of Cleome droserifolia Shoot Extract (CDE) by HPLC

The Cleome droserifolia shoot was extracted with ethanol, and HPLC was used to track the phytoconstituents, antioxidants, anti-yeast, anti-inflammatory, and anti-diabetic properties (Fig. 1). The wide range of secondary compounds present in plants of the Cleome genus is linked to the biological consequences. Shrubs belonging to the Cleome species have yielded a variety of flavonoids, alkaloids, and polyphenols (Moustafa and Mahmoud 2023). The examination of phenols and flavonoids in the shoot extract of Cleome droserifolia (CDE) is shown in Table 1 and the HPLC chromatogram (Fig. 2). Elevated levels of rutin and syringic acid (15854.59 and 5319.18 µg/g, respectively) were linked to CDE. There are numerous medicinal uses for syringic acid (SA), including the prevention of diabetes, cancer, and cerebral ischemia. It also has antioxidant, antibacterial, and anti-inflammatory properties. It reduces oxidative stress indicators and is an efficient free radical scavenger. Methoxy groups attached to the aromatic ring at positions 3 and 5 are responsible for SA’s medicinal properties. SA’s potent antioxidant activity might be the source of its positive health effects (Srinivasulu et al. 2018).

Also, gallic acid, chlorogenic acid, ellagic acid, and vanillin were identified at quantities of 1150, 987, 2830, and 1420 µg/g, respectively. Conversely, a minimal concentration of coumaric acid (19.4 µg/g), succeeded by rosmarinic acid (21.0 µg/g), methyl gallate (22.1 µg/g), and ferulic acid (125 µg/g) was identified in CDE. Other chemicals were observed in moderate concentrations, such as caffeic acid, catechin, and naringenin (Table 1).

Table 1. HPLC Analysis of Cleome droserifolia Shoot Extract (which Area [mAU*s]: milli-absorbance unit and RT: retention time)

Fig. 2. HPLC diagram for detection of phenolic compounds in Cleome droserifolia shoot extract (A) and HPLC chromatograms of the standard for the phenolic and flavonoids compounds (B)

Gallic acid has been shown to have a number of positive effects, such as anti-inflammatory, antioxidant, and anti-cancer effects. It has been reported that this chemical has therapeutic properties for metabolic and cardiovascular diseases (Kahkeshani et al. 2019). Also, chlorogenic acid has many benefits, including anti-oxidant, anti-bacterial, anti-tumor, lipid and glucose metabolism management, anti-inflammatory, nervous system protection, and blood vessel activity.

Phenolic and flavonoid components in C. droserifolia extract play a crucial role in its medicinal effects. These chemicals enhance their antibacterial, anti-inflammatory, and antioxidant properties (Joshi et al. 2023, Amin et al. 2024, and Khowdiary et al. 2024). The principal constituents in the methanolic extract of C. droserifolia shoot extract are benzoic acid, rutin, ellagic acid, naringenin, and o-coumaric acid (Hashem and Shehata 2021). Figure 2B shows an HPLC chromatogram of the standard for the phenolic and flavonoids compounds.

Antioxidant Activity of CDE

Many therapeutic chemicals that can be used safely to treat a variety of human and animal ailments can be found naturally in medicinal plants (Abdel Ghany and Hakamy 2014; Abdelghany et al. 2019; Al-Rajhi et al. 2023). Clarifying plants as a significant source of many medications has received more attention in recent decades (Bakri et al. 2024; Bazaid et al. 2025). CDE exhibited antioxidant activity, which increased with its concentration increase from 1.95 to 1000 µg/mL. Compared with the standard ascorbic acid, the DPPH scavenging % by CDE differed significantly at concentrations from 1.95 to 250 µg/mL, while the difference was not significant at 500 and 1000 µg/mL. The obtained IC₅₀ dose of CDE (8.95±1.023 µg/mL) indicated its potential as an antioxidant agent if compared with the ascorbic acid IC₅₀ dose (3.07±1.08 µg/mL) (Table 2). In another investigation, the IC₅₀ methanolic CDE was 470.27 ± 2.24 μg/mL compared to IC₅₀ 14.03 ± 0.67 μg/mL of ascorbic acid, respectively, via DPPH (Hashem et al. 2021). Elevated level of rutin (15854.59 µg/g) in C. droserifolia extract, which acts as an antioxidant agent by enhancing catalase and superoxidase dismutase enzymes, which also possesses anti-inflammatory properties. Rutin can also dramatically reduce lipid peroxidation in a variety of tissues, including the liver and brain (Bakhtiari et al. 2017).

Table 2. Cleome droserifolia Shoot Extract’s Antioxidant Activity (µg/mL) as Determined by the DPPH Free Radical Scavenging Assay

Anti-Inflammatory Activity of CDE

The anti-inflammatory effects of CDE were represented by the inhibition of COX-1 and COX-2 (Table 3). From the results, COX-1 was more sensitive to CDE than COX-2 at all tested concentrations, where their inhibitions were 47.6±0.4 and 37.2±1.1 % at 7.8 µg/mL: 60.1±1.0 and 54±0.4 at 31.25 µg/mL, respectively. Inhibition of COX-1 and COX-2 was compared to Celecoxib as standard drug which reflected high inhibition levels against both enzymes if compared to CDE, however the IC₅₀ dose of CDE is considered good against COX-1 (12.91±0.5a µg/mL) and COX-2 (21.63 ±0.8 µg/mL) compared celecoxib against COX-1 (6.41 ± 0.7 µg/mL) and COX-2 (3.44 ±0.8 µg/mL). in vivo study, the inflammatory markers were decreased by CDE (Alqahtani et al. 2024).

Table 3. Anti-inflammatory of CDE via Inhibition of COX-1 and COX-2 Compared to Celecoxib

Anti-amylase Activity of CDE

CDE exhibited anti-diabetic activity via inhibition of amylase compared to acarbose as a standard drug. Amylase inhibition by CDE and acarbose was 50.9±4.32 and 64.3±2.32 % at 15.62 µg/mL and reached to 93.9±2.36 µg/mL and 97.4±2.36 µg/mL at1000 µg/mL, respectively (Fig. 3).

Fig. 3. Anti-amylase activity of Cleome droserifolia shoot extract, with Acarbose as positive control

The obtained IC₅₀ dose of CDE (14.93±1.87µg/mL) indicated its potential as an anti-diabetic agent, which documented with the aid of acarbose acid IC₅₀ dose (4.2±2.32µg/mL). The present results were agreement with in vivo study (El Naggar et al. 2005) which indicated that CDE possess anti-diabetic potential due to the presence of quercetin, kaempferol, and isorhamnetin quercetin, kaempferol, ferulic acid, 4-coumaric acid, isorhamnetin and syringic acid, these active compound may change the dynamics of proteins, enzymes, and other transcription factors linked to cancer, inflammation, and diabetes (Srinivasulu et al. 2018).

In another investigation, Helal et al. (2015) found that CDE reflected therapeutic protective against diabetes via declining oxidative stress as well as pancreatic β-cells’ injury which is due to its anti-diabetic and antioxidative properties.

Anti-yeast Activity of CDE

The recorded and apparent inhibition zone indicated the efficacy of CDE against tested yeasts C. albicans (25±0.2mm), C. tropicalis (20±0.1mm), and C. glabrata (25±0.1mm) (Table 4 and Fig. 4). The standard antifungal agent showed better inhibition zones against C. albicans and C. tropicalis but lower against C. glabrata than CDE. Moreover, promising values of MIC and MFC were attributed to CDE against the examined yeasts. Various bacteria and Candida albicans were previously shown to be inhibited by CDE (Hashem et al. 2021). The antifungal activity of was documented by El-Alem et al. (2024), where the growth of the fungus C. albicans was suppressed with zone of inhibition 10 mm.

Overall, these findings recommend the possibility of utilizing CDE as a therapeutic agent. Prospective experiments are required to separate the bioactive constituents of the CDE and their specific activities in vitro and in vivo.

Table 4. Anti-yeast Activity of Cleome droserifolia Shoot Extract, with Fluconazole as a Positive Control

Fig. 4. Anti-yeast Activity of Cleome droserifolia Shoot Extract (E), with Fluconazole as a Positive Control (S), and ethanol as negative control (NC)

CONCLUSIONS

1. The findings of the current investigation point to the remarkable range of flavonoids and phenols ingredients of the CDE with several biological functions.
2. The CDE demonstrated antioxidant, anti-inflammatory, anti-diabetic, and anti-yeast activities in vitro, suggesting its potential—pending further modification such as purification or fractionation, stability enhancement, and targeted delivery—for evaluation in in vivo studies.

ACKNOWLEDGMENT

The authors extend their appreciation to the Deanship of Scientific Research at Northern Border University, Arar, KSA for funding this research work through the project number “NBU-FFR-2025-2585-01”.

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Article submitted: May 13, 2025; Peer review completed: June 30, 2025; Revised version received and accepted: July 31, 2025; Published: August 25, 2025.

DOI: 10.15376/biores.20.4.9020-9032