Sophoraflavanone G in Nano-niosomal Form: Implications for Bacterial Inhibition, Biofilm Disruption, and Cancer Suppression

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DOI: 10.18502/ajmb.v18i1.21049

• Teymouri, Manouchehr
  - Natural Products and Medicinal Plants Research Center, North Khorasan University of Medical Sciences, Bojnurd, Iran
  - Department of Medical Nanotechnology, School of Medicine, North Khorasan University of Sciences, Bojnurd, Iran

• Khayyer, Reyhaneh
  - Department of Advanced Sciences and Technologies, School of Medicine, North Khorasan University of Sciences, Bojnurd, Iran

• Iranshahy, Milad
  - Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

• Salarinia, Reza
  - Department of Medical Biotechnology, School of Medicine, North Khorasan University of Sciences, Bojnurd, Iran

• Zarghami Moghaddam, Parastoo
  - Natural Products and Medicinal Plants Research Center, North Khorasan University of Medical Sciences, Bojnurd, Iran

• Mohammdi, Ameneh
  - Natural Products and Medicinal Plants Research Center, North Khorasan University of Medical Sciences, Bojnurd, Iran

• Memariani, Toktam
  - School of Medicine, North Khorasan University of Medical Science, Bojnurd, Iran

• Mollazadeh , Samaneh Natural Products and Medicinal Plants Research Center, North Khorasan University of Medical Sciences, Bojnurd, Iran, Tel: +98 58 31513016; E-mail: samanehmollazadeh@yahoo.com
  

Sophoraflavanone G (5,7,2',4'-tetrahydroxy-8-lavandulylflavanone, SG) is a prenylated flavonoid compound extracted from the roots of Sophora species. This bioactive agent exhibits a multifaceted profile, including antimicrobial, radical scavenging, anti-inflammatory, and anticancer properties. The prenylation of SG has significantly improved its biological activity by enhancing lipophilicity and promoting affinity for the cellular membrane of both eukaryotic and prokaryotic cells, in addition to interaction with cellular components ¹. Consequently, SG accumulates in various tissues due to this modification ². Also, studies have suggested promising results of SG on Oral Squamous Cell Carcinoma (OSCC) and oral bacterial flora. However, this compound is highly hydrophobic and exhibits poor solubility in aqueous biological media, limiting its absorption and effective delivery in vivo.

To address this, a nanoformulated form of SG could improve its bioavailability, especially in the oral environment ^3,4. This limitation hinders the full therapeutic potential of SG. To overcome this issue, nanoparticle forms of SG are used to improve bioavailability, increase absorption, and reduce toxicity in non-tumor cells ⁵.

Among researchers, attention has turned to niosomes—a promising class of lipid-based nanocarriers ⁶. These nanocarriers have demonstrated remarkable efficiency in enhancing drug payload stability in both in vitro and in vivo settings. Specifically, niosomal formulations of bioactive agents have been associated with improved bioavailability, leading to enhanced antibacterial, antifungal, anticancer, and antibiofilm effects ⁷. Biodegradability, biocompatibility, non-toxicity, and encapsulation of a large amount of material in a relatively small volume are among the advantages of using niosomes as a suitable carrier for drug delivery ⁸. Niosomes are uniformly dispersed formulations containing high concentrations of nonionic detergents, such as polysorbates ⁹. Polysorbates, due to their large polar heads, can solubilize lipophilic compounds such as SG. For example, Tween 20 has a hydrophilic polar head due to the presence of hydroxyl (OH) groups, which can attract water molecules. In many cases, cholesterol and its derivatives are used to prepare niosomes. Cholesterol is uncharged and used to produce stable formulations. Cholesterol prevents the precipitation of the formulation and the sudden release of the drug. In the biological environment, i.e., cells, 40% of cholesterol is present, which provides stability to the cell membrane ⁷.

In light of SG’s high hydrophobicity and its low bioavailability, it is important to establish more applicable delivery systems like niosome which is an amphiphilic and flexible drug carrier. Accordingly, the current investigation focuses on developing a nano-niosomal formulation of SG. By addressing its poor aqueous solubility, it was attempted to improve SG’s dispersion in biological media, thereby improving its bioavailability and therapeutic efficacy ¹⁰.

SG separation and purification

The roots (1,400 g) of the herbaceous plant Sophora pachycarpa (voucher number: 13275), preserved in the herbarium room of the School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran, were air-dried in darkness and finely ground using an electric grinder. The resulting herbal powder was subjected to maceration with 4 L of acetone solvent. The supernatant was then concentrated via vacuum evaporation using a rotary evaporator at ambient temperature to remove the solvent. This maceration and evaporation process was repeated three times. The final dry extract (35.56 g) was further purified by silica gel column chromatography (230-400 mesh, no. 377635, 5 cm width, Sigma-Aldrich) pre-equilibrated with petroleum ether. A gradient of petroleum ether/acetate was applied, starting from 1000 ml/0 ml and increasing to 500 ml/500 ml in 50 ml increments. Subsequently, a second gradient of acetate/methanol was used, ranging from 1000 ml/0 ml to 500 ml/500 ml with 250 ml increments.

The fractionated components were collected in separate 200 beakers. Finally, the solvents were evaporated, and the resulting powder was freeze-dried to eliminate residual solvent traces.

Subsequently, the fraction containing SG was identified using Thin-Layer Chromatography (TLC) against the standard SG (Sigma-Aldrich). The TLC process was carried out using the same solvent in which the fraction was initially collected, i.e., petroleum ether/ ethyl acetate 5.5/4.5 v/v. Various colored bands, including the SG, became visible under UV irradiation (256 nm) following the application of vanillin sulfuric acid reagent (100 g/dl in 95% ethanol) spray and heat treatment at 110°C.

Next, the identified fraction containing SG was further purified using preparative-TLC. A 20×20 cm PTLC silica gel plate was preheated at 110°C for 30 min. The fraction, in powdered form, was dissolved in a small amount of acetone and applied to the TLC plate. The mobile phase consisted of a petroleum ether/ethyl acetate mixture (4/6 v/v). The region corresponding to the SG band was then scraped off, removed, and dissolved in acetone to yield a purified SG solution.

SG chemical structure characterization

The chemical structure of SG was determined using one-dimensional ¹H-NMR spectroscopy with the aid of MestReNova (Mestrelab Mnova 15).

SG nanoformulation

The preparation of SG nano-niosomes was accomplished using the reverse-phase evaporation method, employing a solvent mixture of ethanol and deionized water. In brief, stock solutions of cholesterol, SG, and Tween 20 in ethanol were combined in a specific Molar ratio of 70:28:18 to form a final 25.91 mg/ml ethanol solution. This ethanol phase was then mixed with deionized water in a 1:9 volume ratio and subjected to 60 Hz sonication (Elmasonic ultrasound, Germany) at 80°C for 30 min to eliminate the ethanol phase. After sonication, the remaining ethanol and water were evaporated using the vacuum-equipped evaporator and subsequently lyophilized in a freeze-dryer. The resulting lyophilized powder was then reconstituted in a hot saline solution at an SG concentration of 8.5 mg/ml to yield lipid-based SG nano-niosome formulation.

Physicochemical characterization of SG niosomes

The liposome size distribution and Polydispersity Index (PDI) were measured using dynamic light scattering (DLS, Zetasizer Nano-ZS; Malvern, UK).

To quantify the amount of SG encapsulated within the niosomes, the niosomes were first dissolved in acetonitrile. This solution was then passed through a reverse-phase C18 HPLC column (250 mm×4.6 mm, either Alltech or Shiseido Capcell Pak) using the Lachrom Elite HPLC system from Shimadzu ¹¹. The mobile phase, a mixture of acetonitrile and water, was initially set at 30% acetonitrile and gradually increased to 100% over a period of 10 min. The flow rate was maintained at 0.1 ml/min at a temperature of 50°C. Following this, the area Under the Curve (AUC) of the resulting peaks was compared with the AUCs from the SG standard curve to determine the amount of encapsulated SG. The initial concentration of SG employed in the niosomal formulation, relative to the encapsulated SG following niosome formation, was quantified and reported as a percentage, representing the Encapsulation Efficiency (EE%) of the preparation.

Antimicrobial activity of niosomal SG

The antimicrobial properties of the niosomal SG were evaluated using three methods: disk diffusion, well diffusion, and biofilm formation. The pathogens tested for antimicrobial activity included Streptococcus mutans (S. mutans) ATCC 25175, Staphylococcus aureus (S. aureus) ATCC 6538, and Candida albicans (C. albicans) ATCC 14053. In addition to niosomal SG (50 µl), the agents tested for their antimicrobial activity were non-niosomal SG (50 µl) suspended in DMSO, ciprofloxacin (50 µl, 0.3%), and nystatin (50 µl, 1×10⁵ units/ml).

For the disk-diffusion assay, 30 ml of freshly autoclaved, Tryptic Soy Agar, TSA (40.0 g/L) was poured into individual petri dishes under sterile conditions and allowed to cool at room temperature. A sample of the pathogens, with turbidity equivalent to 0.5 McFarland standards, was then collected from the microbial suspension using a sterile loop and streaked across the agar. Subsequently, 6.4 mm blank paper discs (Padtan Teb Co., Iran) were coated with the agents and left to dry for 30 min under sterile conditions. The discs were then flipped and placed on the petri dishes, which were incubated at 35°C for 48 hr to allow bacterial colonization. The inhibition zones (2r, mm) were finally measured using a ruler.

The well-diffusion test followed the same steps as the disk-diffusion assay, with the exception of creating a well in the agar using a Pasteur pipette (6 mm width) and filling it with 50 µl of each reagent.

Antibiofilm activity of niosomal SG

The antibiofilm efficacy of various agents, including niosomal SG, was evaluated using a microtiter assay ¹². In brief, a 96-well culture plate was prepared by dispensing 150 µl of TSA growth medium and microbial species, calibrated to a turbidity equivalent to 0.5 McFarland. Subsequently, 100 µl of the agents (niosomal SG, non-niosomal SG, PBS) were introduced into the wells. Control wells were established without either microbial species (negative control) or the agents (positive control). The plate was then incubated at 37°C for 24 hr.

Post-incubation, the medium and planktonic species were carefully removed and the wells were thoroughly rinsed with normal saline, three times. The now empty wells were treated with 200 µl methanol to stabilize the biofilm population. After 15 min, the contents of the wells were discarded and the plate was dried at room temperature. For staining, 200 μl of 2% crystal violet was added to each well for 5 min. Then, the excess dye was completely washed off with a gentle stream of water and the microplate was dried at room temperature. Lastly, contents of the well were dissolved by adding 200 µl of 33% v/v acetic acid, and the absorbance was quantified at 630 nm using a Multiskan Plus plate reader (Labsystems).

The cut-off value for Optical Density (ODc) ¹³ was determined based on the absorbance value of the negative control, calculated using the formula:

ODc=average OD of negative control+3×SD of negative control

The biofilm activity was then classified based on the absorbance as follows:

Absorbance ≤ODc indicates no biofilm activity

ODc <Absorbance ≤2×ODc indicates weak biofilm activity

2×ODc <Absorbance ≤4×ODc indicates medium biofilm activity

Absorbance >4×ODc indicates strong biofilm activity.

Antiproliferative activity of niosomal SG

The cytotoxic activity of the niosomal and non-niosomal SG was assessed in cancer KB cells (CRL-3596 ™, Pasteur Institute, Iran) and normal L929 cells (CCL-1 ™, Pasteur Institute, Iran) using the MTT assay. Briefly, 4×10⁴ viable KB cells and 1×0⁴ L929 cells were dispensed to a flat-bottomed 96-well plate and incubated at 37°C. After 24 hr, the cells were treated with niosomal and non-niosomal SG within the range of 0.015 mg/ml –1 mg/ml for 24- and 48-hr incubation periods at 37°C. For control, some wells were left either without cells (positive control) or without reagent treatment (negative control). Subsequently, the medium was replaced with fresh RPMI-1640 culture medium (Sigma-Aldrich) supplemented with 20 µl MTT solution (5 mg/ml) -3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide–and incubated for a further 4 hr. Finally, the medium was washed away extensively with normal saline, the formazan dye was dissolved in 0.2 ml/well DMSO, and the absorbance was measured at 550 nm using the ELISA reader.

Subsequently, the antiproliferative activity of the niosomal and non-niosomal SG was determined for each SG concentration using the following formula:

Antiproliferative activity=(OD550 nm of negative control–OD550 nm of reagent)×100/(OD550 nm of negative control–OD550 nm of positive control).

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 6 (GraphPad Software, San Diego, CA) at a significance level of 0.05. One-way ANOVA was employed followed by the Bonferroni’s multiple comparisons test to determine significant differences between the treatment groups.

SG chemical structure characteristics

The ¹H NMR spectrum (A) along with the chemical structure (B) of SG were characterized by ¹H NMR (Supplementary 1).  Based on ¹H NMR spectrum the following hydrogen shifts were observed: Hydrogen at Carbon 2 (H2) that appeared as a doublet of doublets (dd) at 5.67 ppm with coupling constants of 13.2 Hz and 7.2 Hz; central and equatorial Hydrogen at Carbon 3 (H3) that were observed at 3.0 ppm and 2.79 ppm, respectively, with coupling constants of 13.2 Hz, 2.17 Hz, and 2.7 Hz. Methyl groups attached to carbons 9", 10", and 4" exhibit chemical shifts of 1.50 ppm, 1.58 ppm, and 1.68 ppm, respectively. The hydrogen attached to the hydroxyl group at position 5 forms a hydrogen bond with the carbonyl group. Its sharp singlet peak appears weak due to its position between several electronegative groups. The peak is observed at 12.2 ppm. Hydrogen at Carbon 6 (H6) appeared as a sharp singlet at 6.05 ppm. Methylene Hydrogens (H5") showed two peaks, corresponding to the methylene hydrogens, appeared as a doublet of doublets at 4.56 ppm and 4.64 ppm. Ring B Hydrogens (H3", H5‎", and H6'‎) exhibited chemical shifts of H3" 6.5 ppm; H5‎" 6.46 ppm; H6" 7.4 ppm. For a more detailed overview, refer to the complete data in supplementary 2.

Physicochemical characteristics of niosomal SG

Figure 1 depicts the size distribution of niosomal SG with an average size of 115 nm. The first quartile of the particles exhibited a size distribution of <90 nm and third quartile displayed a size distribution of <164 nm. Overall, the majority (95%) of the particles displayed a size distribution of <209 nm. The PDI of the particles varied between 0.18-0.22 (of three independent niosomal preparations) with zeta potential, ranging from -10 to -20 mV. Regarding SG loading, the SG contained 4.75 mg SG/ml niosomal formulation, corresponding to EE of 56% of the initial amount of SG used.

Antimicrobial properties of niosomal SG

Figure 2 presents the results of the inhibition zones around the discs and wells treated with various reagents, while Figure 3 illustrates biofilm formation in the treated wells. It was observed that non-niosomal SG exhibited superior antimicrobial activity compared to niosomal SG.

The most substantial inhibition zones were associated with the reference antibiotics, specifically ciprofloxacin for bacteria and nystatin for C. albicans (Figures 2A and B). The inhibition zone exceeded 50 mm for S. aureus, while for S. mutans and C. albicans, it was approximately 25 mm around the discs. In contrast, the inhibition zone for non-niosomal SG was less than 20 mm for the bacteria and was negligible (6 mm) for C. albicans (Figures 2A and B) when compared to PBS. A similar pattern was observed around the wells. Niosomal SG did not exhibit any significant antibacterial or antifungal activity compared to PBS. Similar results could be seen in the disc-diffusion plats for S. aureus and S. mutans (Figures 2C and D).

The diminished antimicrobial efficacy of niosomal SG was evident in its limited capacity to inhibit biofilm formation. Wells treated with niosomal SG exhibited strong biofilm activity, indicating insufficient suppression of microbial colonization. In contrast, non-niosomal SG demonstrated moderate biofilm activity against S. aureus and C. albicans, suggesting a partial inhibitory effects on biofilm development (Figure 3).

Antiproliferative properties of niosomal SG

Figure 4 presents the dose-response graph for both non-niosomal and niosomal SG in the cancerous KB (A) and normal L929 cell lines (B). While niosomal SG showed only a marginal increase in the inhibition of cell proliferation in normal L929 cells, it exhibited a significant difference in the KB cells when compared to non-niosomal SG.

This study delved into the potential of niosomal formulation to enhance the efficacy of the prenylated flavonoid, SG. Given the high hydrophobicity and poor water solubility of SG, it was finely dispersed using either DMSO supplementation or niosomal formulation for its biological activity assessment.

Numerous studies have underscored the role of niosomal formulations in unlocking and reintroducing the beneficial properties of highly hydrophobic herbal compounds. These compounds would otherwise precipitate in biological media, resulting in significantly low bioavailability. For instance, Agarwal et al ¹⁴ utilized a niosomal system comprising non-ionic surfactant span 60 and cholesterol to create a well-dispersed suspension of Morusin, another prenylated flavonoid. Similarly, Murugesan et al ¹⁵ used a comparable niosomal system for the dietary polyphenols quercetin and resveratrol. Without the niosome, the water solubility of these compounds is considerably lower than their effective biologically-active concentrations–the solubility of quercetin and resveratrol is 0.01 mg/ml and 0.05 mg/ml, respectively ^16,17.

Through the niosomal formulation, we attained a high loading concentration of 4.75 mg/ml, adequate to trigger antimicrobial activity. However, the niosomal SG did not exhibit any antibacterial activity or fungal growth inhibition (Figure 2), and only marginal antibiofilm activity was observed for S. mutans (Figure 3).

In contrast, non-niosomal SG demonstrated some antibacterial (Figure 2) and antibiofilm activity (Figure 3). The absence of a DMSO control group in this study limits the interpretation of these results. The observed effects could be attributed to either the synergistic antimicrobial activity of DMSO or its enhanced penetration and availability. Previous studies have highlighted the antimicrobial activity of DMSO across a broad spectrum of bacteria and fungi ^18,19, and its ability to penetrate deeply into tissues affected by dental infections ²⁰. Thus, it appears that niosomal SG may not be suitable for dental hygiene in terms of controlling bacterial and fungal growth.

Turning to the anticancer potential of niosomal SG, intriguing and promising findings were uncovered. Niosomal SG slightly enhanced its ability to inhibit cell growth in the KB cell line and showed strong protection against cell damage in normal L929 cells (Figure 4). KB is a human epithelial cell originally derived from a patient with epidermal carcinoma of the mouth and L929 is a fibroblast cell; therefore, it appears that niosomal formulation enhanced cancer-specific cytotoxicity for oral malignancies. 

The anticancer activity of SG aligns with previous literature. Huang et al ²¹ reported the antiproliferative effect of SG on human leukemia HL-60 cells and the triple-negative breast cancer MDA-MB-231 cells. In the present study, both niosomal and non-niosomal SG exhibited antiproliferative activity against cancer KB cells, while only the niosomal SG exhibited no toxicity against the normal L929 cells, suggesting that niosomal SG delivers SG at non-toxic concentrations to normal cell lines.

This study evaluated the biological activity of SG encapsulated in a niosomal system, comprising cholesterol and tween 20. SG is a biologically active compound but unstable in the biological environment, which causes to precipitate rapidly in the aqueous environment and become unavailable. Encapsulation of SG in niosomes resulted in the formation of a stable SG suspension in the aqueous media at nanoscale, mostly in the range of 100 to 200 nm. It appeared that the niosomal system somewhat obscured the antimicrobial activity of SG when compared to that of non-niosomal SG dissolved in DMSO. It is plausible that a niosomal formulation with elevated concentrations of tween 20 or substitution with alternative ionic detergents could enhance the antimicrobial activity of SG, a prospect worth exploring. Despite this, the niosomal formulation provided valuable insights, indicating SG might be effectively utilized as a highly selective formulation in the treatment of gingival cancers, while SG may not be a potent antimicrobial agent. The use of a nano-niosomal form of SG has the potential for targeted elimination of cancer cells in clinics, while reducing the toxicity of SG. This could position niosomal SG as a suitable therapeutic candidate, especially in the cancer treatment.