Quality by design and microwave-assisted green silver nanoparticles loaded with Cassia tora extract for psoriasis management

Abdullah S. Alshetaili

doi:10.25259/AJC_79_2025

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Original Article

2025

:18;

792025

doi:

10.25259/AJC_79_2025

Quality by design and microwave-assisted green silver nanoparticles loaded with Cassia tora extract for psoriasis management

Abdullah S. Alshetaili^,*

Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173, Al-Kharj 11942, Saudi Arabia

* Corresponding author: E-mail address: a.alshetaili@psau.edu.sa (A. Alshetaili)

Received: 2025-1-25, Accepted: 2025-06-13, Epub ahead of print: 2025-09-22, Published: 2025-10-15

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Abstract

The aim of this study was to develop a Cassia tora (CT)-loaded silver nanoparticle (NP) gel to enhance dermal distribution at the target site. After Box-Behnken design optimisation, the produced formulation showed an NP size of 103.77 nm, polydispersity index (PDI) of 0.2672, zeta potential (ZP) of −24.38 mV, and entrapment efficiency (EE) of 82.06 ± 2.74%. The maximum in vitro drug release was 52.67 ± 3.71% at 6 h, and confocal laser scanning microscopy (CLSM) images of rat skin showed that rhodamine B-loaded silver NP (AgNP) gel permeated far deeper than the control rhodamine B hydroalcoholic solution. In addition, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay confirmed the antioxidant activity of the enhanced CT-AgNP formulation. In the characterisation study, the gel’s extrudability and spreadability were 16.05 ± 4.24 g and 31.96 ± 4.35 g.cm.s^-1, respectively. Additionally, the CT-AgNP formulation’s exceptional stability was clearly shown by the texture analysis results. The study confirms that the AgNP formulation serves as an effective drug delivery system specifically designed for dermal administration of CT in treating psoriasis.

Keywords

Antioxidant

Cassia tora

Psoriasis

QbD

Silver nanoparticles

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1. Introduction

Psoriasis, a skin condition affecting 2-5% of the global population, is characterized by persistent inflammation [1-3]. Psoriatic skin is identified histopathologically by hyperplasia of the epidermis with considerable differentiation of keratinocytes, increased angiogenesis, and massive inflammatory infiltrates [4,5]. Psoriasis can be treated in several ways; however, the three main types of treatment are local (topical therapy), external (phototherapy), and internal (systemic therapy) [6]. Topical therapy is often the first line of defence when treating mild conditions. However, severe cases may also require phototherapy or systemic therapy [7]. Current treatments for psoriasis can only alleviate the symptoms, not eliminate the condition [8].

Traditional topical medicine has many problems, such as inadequate drug penetration, increased dose frequency, severe side-effects, and worse patient compliance [9]. Hepatotoxicity, skin cancer, chronic kidney disease, and hypertension are some side effects of phototherapy and systemic therapy [9]. Therefore, these issues limit the utility of the standard psoriasis therapies available today [10].

The popularity of nanostructured metallic nanoparticles (NPs) has been observed due to the wide range of acceptance in medical applications [11-13]. As a safe and effective alternative therapy option for various diseases, silver NPs (AgNPs) fabricated using plant extracts are crucial for nanomedicine [14-16]. Moreover, experimental evidence suggests that AgNPs have anti-angiogenetic, anticancer, antiviral, antibacterial, wound healing, and anti-inflammatory properties [17]. The method of microwave synthesis was selected for the investigation because of its rapidity and capacity for consistent heating, which is a crucial factor when dealing with intricate biomolecules found in plant extracts.

Many plants have been identified for their potential to treat psoriasis, including Capsicum annum [2], Curcuma longa [18], Nigella sativa (thymoquinone) [19], Ulmus rubra [5], Smilax china [20], Rubia cordifolia, Scutellaria baicalensis [21], Indigo naturalis [22], Hypericum perforatum [23], Wrightia tinctoria [24], and Cassia bonduc [25]. In particular, the Cassia tora (CT) plant from the Fabaceae family has been identified as a valuable and promising resource for psoriasis treatment. Molecules such as luteolin, quercetin, and formononetin in CT reduce the thickness of the lower epidermal layer by inhibiting pro-inflammatory cytokines, including Interleukin 6 (IL-6), Interleukin 8 (IL-8), and Tumor necrosis factor (TNF-α) thus could be a promising agent in the amelioration of psoriasis. Moreover, administration of the methanolic extract of its leaves dramatically reduced epidermal thickness and spleen index in a study in which UV-B rays induced psoriasis in Wistar rats, suggesting a substantial anti-psoriatic effect [26]. However, sufficient scientific evidence to support the conclusion of its anti-psoriatic efficacy has still not been investigated [27-29].

Among different processes for AgNP synthesis, biosynthesis, which involves synthesis using plants, fungi, or bacteria and their enzymes, is commonly used because of its biocompatibility. Therefore, CT as a topical treatment for psoriasis appears promising, as it can mitigate the serious side effects associated with systemic treatment. Hence, we developed a CT-AgNP and optimized it using the Box-Behnken design, followed by evaluation using in vitro and ex vivo studies.

2. Materials and Methods

2.1. Materials

Fresh and original Cassia tora (CT) seeds were gathered from the global biotech market in July and August 2023 and confirmed by a botanist at Prince Sattam bin Abdulaziz University, Saudi Arabia. Silver nitrate in the form of solid crystals was obtained from Thomas Baker (Mumbai, India). Carbopol 934P and methanol LR grade were obtained from SD Fine Chemicals (Mumbai, India). Triethanolamine was obtained from Thermo Fisher Scientific (Mumbai, India). The chemicals and solvents used in the investigations were of analytical grade. All tests were conducted using high-performance liquid chromatography (HPLC) water.

2.2. Preparation of plant extract

CT seeds were processed into coarse powder. The powder was first defatted with petroleum ether, extracted with EtOH, and finally evaporated to dryness to produce an alcoholic extract. The extract was stored at 4°C and consumed within 2 weeks. This extract was used as a reducing and stabilizing agent.

2.3. CT-AgNP production using the green synthesis method

In a 250 mL round-bottom flask equipped with magnetic stirring, a selected amount of CT was added to 100 mL of distilled water. The CT extract was then mixed with an aqueous solution of silver nitrate (AgNO₃). This mixture was exposed to microwaves using a Panasonic home microwave oven running at 800 W for a brief pulse of 30 s for fast microwave synthesis to facilitate the reduction of Ag⁺ ions to Ag⁰ NPs. Subsequently, the mixture was allowed to rest at room temperature before being used again. All reactions were conducted at room temperature in the dark. The shade dramatically changed from light yellow to dark brown, indicating AgNP formation. CT-AgNP production was largely reported by examining the color change from yellow to dark brown. Subsequently, UV spectroscopy confirmed that the λ_max was more than 400 nm. After centrifugation (Remi Centrifuge, Mumbai), the water-soluble components of AgNPs were washed off with water and acetone. CT-AgNPs were then lyophilized and stored for analysis.

2.4. Optimisation of CT-AgNPs

A Box-Behnken design with three factors at three distinct levels was used to optimize CT-AgNP production using Design Expert (version 13). We systematically investigated how the AgNO₃ concentration (X1), CT extract concentration (X2), and microwave power in Watts (X3) affect the size of AgNPs (Y1), polydispersity index (PDI) (Y2), and percentage entrapment efficiency (%EE) (Y3) (Table 1). These independent variables were evaluated at their lowest (−), medium (0), and highest (+) levels to identify the optimal composition. The experimental design encompassed 17 formulation runs, including three central points (Table 2). The effect of independent variables on dependent variables was assessed using polynomial equations and response surface plots. In addition, various models, including linear and quadratic models, were used to understand the influence of the independent variables on the dependent variables. The quadratic model was considered the most suitable because it accounted for the individual and combined effects of independent variables on dependent variables.

Table 1. Independent variables (X₁, X₂, and X₃) along with their levels and dependent variables (Y₁, Y₂, and Y₃) with their constraints in Box-Behnken design for CT-AgNP optimization.

Independent variables	Low (-1)	Medium (0)	High (+1)
X₁ = AgNO₃ concentration (mM)	2.5	5	7.5
X₂ = Cassia tora extract concentration (%)	5	7.5	10
X₃ = Power of the microwave in Watt	400	600	800
Dependent Variables
Particle size (Y₁)	Minimize
PDI (Y₂)	Minimize
EE%	Maximize

Table 2. Different CT-AgNP formulations obtained from Box-Behnken design.

RUNS	In-Dependent Variables
RUNS	X1	X2	X3
1.	2.5	7.5	400
2.	5	7.5	600
3.	5	10	800
4.	5	5	800
5.	7.5	10	600
6.	2.5	7.5	800
7.	5	7.5	600
8.	7.5	7.5	800
9.	7.5	5	600
10.	2.5	5	600
11.	5	5	400
12.	7.5	7.5	400
13.	5	7.5	600
14.	2.5	10	600
15.	5	7.5	600
16.	5	10	400
17.	5	7.5	600

2.5. Characterization of optimized CT-AgNP (Opt-CT-AgNP)

2.5.1. Particle size characterization

The synthesized Opt-CT-AgNPs were analyzed for particle size, PDI, and ZP using Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK). The surface charge of NPs is directly related to their electrostatic interactions or ZP. NPs with a ZP between −10 and +10 mV are considered neutral. The samples were analyzed by diluting them by a factor of 100 with double-distilled water and filtering them through membranes with a pore size of 0.45 µm.

2.5.2. Percentage entrapment efficiency (%EE)

%EE was measured using a published approach [30]. NPs were centrifuged at 14,000 rpm for 10 min to separate unentrapped CT. After centrifugation, the supernatant was collected and diluted, and UV-visible spectroscopy was used to calculate the quantity of free CT in the supernatant using Eq. (1).

(1)

% E E = \frac{(T o t a l d r u g a d d e d (m g) - F r e e D r u g i n s u p e r n a t a n t (m g))}{T o t a l d r u g a d d e d (m g)}

2.5.3. Transmission electron microscope (TEM) study

The size and shape of the NPs were determined under a transmission electron microscope (TEM-Tecnai, G20, Philips Scientific instrument) with an accelerating voltage of 100 kV. A drop of fluid containing AgNPs was placed on a carbon-coated copper grid. After drying, a diluted drop of the specimen was stained with phosphotungstic acid (1% w/v).

2.6. Preparation of Opt-CT-AgNP gel

A gel was formulated to prolong Opt-CT-AgNP retention on the skin. To make this gel, a gel dispersion was made by mixing Carbopol 934P (1% w/w) in double-distilled water. This dispersion was allowed to swell overnight. Subsequently, the following components were introduced: polyethylene glycol 400 (15% w/w) as a plasticizer, chlorocresol (0.1) for preservation, and triethanolamine to adjust the pH. The Opt-CT-AgNP formulation was slowly added drop-by-drop to the previously prepared gel while continuously stirring the mixture, resulting in a uniform formulation termed Opt-CT-AgNP gel.

2.7. Characterization of Opt-CT-AgNP gel

2.7.1. pH

The pH of the gel was measured using a calibrated pH meter (Mettler Toledo) to determine whether the gel could irritate the skin.

2.7.2. Spreadability

Gels that exhibit high consistency but minimal spreadability are the preferred choice. Spreadability of the gel was assessed by placing 2.5 g of Opt-CT-AgNP gel on a glass plate, covering it with another glass plate, and placing a 1 kg weight on the upper glass plate for 5 min. After removing the weight, the distance travelled by the gel was determined. Spreadability was evaluated using Eq. (2):

(2)

S = \frac{M L}{T}

“Where S = spreadability, M = weight (g) applied to the upper glass plate, L = length (cm) of the glass plate, T = time taken for the gel to spread the entire length”.

2.7.3. Extrudability

The percentage of gel expelled from a tube when subjected to significant weight was measured to assess the extrudability of the Opt-CT-AgNP gel. A higher amount of extruded gel indicates better extrudability. In a fresh, lacquered aluminum tube with a 5-mm tip, a one-ounce tube containing the Opt-CT-AgNP formulation was placed. To release the gel, 200 g was applied to the bottom of the tube. This experiment used the tip to determine the amount of gel extruded.

2.7.4. Texture analysis

The texture was analysed by using a texture analyzer (Stable micro system, UK) in compression mode. In this procedure, 50 g of the optimized gel formulation was placed under the probe of the texture analyzer in a 100 mL beaker while ensuring that the gel’s surface remained smooth to prevent the trapping of air bubbles.

2.8. In vitro drug release study

Dialysis tubing of 25 mm (12 KD, Sigma Laboratories) was used for the release tests. CT-AgNP gel and CT-Conventional Formulation (CF) gel, respectively, were placed into the tubing before it was dipped in 50 mL of phosphate buffer (pH = 7.4). After placing the medium in the stirring apparatus, the temperature was increased to 37 ± 0.5°C. The stirring rate was set at 220 rpm for maximum efficiency. Subsequently, 0.6 mL samples were taken every hour and diluted with fresh buffer before being analysed using a UV absorbance spectrophotometer set to 260 nm. The error was kept to a minimum using a blank sample (containing AgNPs but no drug) to rule out polymer-induced absorbance. The percentage of drug released per hour was acquired from the calibration curve, and the release profiles were developed.

Furthermore, the release profiles of the Opt-CT-AgNP nanoformulation were fit using Eqs (3-5)

(3)

zero-order F (t) = k t \pm b,

(4)

first-order \ln (1 - \frac{M t}{M \infty}) = - k t and

(5)

Higuchi (\frac{M t}{M \infty}) = k t \frac{1}{2}

mathematical models, as well as the Korsmeyer-Peppas model [31]. Thus, the release and diffusion mechanisms were assessed effectively.

2.9. Ex vivo skin permeation study

To assess drug permeability through the skin, a 1 cm² section of the mice’s abdominal skin was excised using a scalpel, with hair removed, and the isolated skin washed 2–3 times with distilled water. Franz diffusion cells having a diffusion surface area of 1.5 cm² were utilized for the skin permeation study. The donor compartments were filled with CT-AgNP gel and CT-CF gel, respectively, while the receiver chamber contained 10 mL of phosphate-buffered saline (pH 7.4) maintained at 37 ± 1°C. A small magnetic bead, rotating at 600 revolutions per minute, provided continuous stirring. At specified time intervals (0, 0.25, 0.5, 1, 2, 4, 6, and 12 h), 1 ml of the sample was withdrawn and immediately replaced with an equal volume of fresh vehicle. The concentration of CT in each sample was analyzed using a UV spectrophotometer at a detection wavelength of 260 nm.

2.10. Confocal laser scanning microscopy

The confocal laser scanning microscopy (CLSM) was used to determine the penetration depth of AgNPs synthesized in this study [32]. To perform this assessment, we loaded the Opt-AgNP formulation with rhodamine B dye and examined its ability to penetrate rat skin using CLSM. This penetration depth was compared with that achieved by a control group using a rhodamine B hydroalcoholic solution. For the 6-h in vitro skin penetration analysis, we used Franz diffusion cells to investigate rhodamine B permeation of the Opt-AgNP formulation and rhodamine B hydroalcoholic solution (control) on rat skin. Glass slides of skin samples were prepared and observed under a CLS microscope (Leica TCS SPE, UK). The probe dye penetration depth was determined at a λ_max of 517 nm.

2.11. Dermatokinetic study

A dermatokinetic study was conducted to evaluate the drug concentration in the epidermis and dermis of mouse skin. An in vitro skin permeation study was performed using CT-AgNP gel and CT-CF gel on excised mouse skin. Skin samples mounted on Franz diffusion cells were collected at 0, 1, 2, 4, 6, and 8 h. The collected samples were rinsed with normal saline and heated at 60°C for 3 min. The skin layers were then separated, sliced into small pieces using forceps, and stored in MeOH for 24 h to ensure complete extraction of CT. The resulting methanolic extract was filtered and analyzed by UV spectrophotometry to determine CT concentration, and various dermatokinetic parameters were assessed.

2.12. Antioxidant activity using the 2,2-diphenylpicrylhydrazyl method

2,2-diphenylpicrylhydrazyl (DPPH) is a stable free radical at room temperature. However, it becomes a stable molecule upon accepting an electron or a hydrogen radical. Antioxidants convert the DPPH radical to its non-radical state in the DPPH assay. Thus, absorption decreases and the colour of the DPPH solution changes from purple to yellow. The percentage of free radical scavenging activity, along with the IC₅₀ values of several samples at concentrations ranging from 50 to 250 g/mL for AgNPs and the CT ethanolic extract, were determined using Eq. (6) [33]:

(6)

% scavenging = \frac{A b s_{b l a n k} - A b s_{s a m p l e}}{A b s_{b l a n k}} \times 100

2.13. Stability study

The stability experiments were performed on the Opt-CT-AgNP formulation and gel. Separate processes were used to prepare the Opt-CT-AgNP formulation and gel. After these processes were conducted, the formulation and gel were placed in a stability chamber with a temperature set at 25 ± 2°C and a relative humidity of 60 ± 5%. Additionally, monthly analyses of the gel and optimized formulation were conducted to find differences in PDI and particle size, loading efficiency, color, clarity, phase-separation, homogeneity, pH, and drug content. These analyses were performed to ensure consistent results throughout storage under different conditions.

3. Results and Discussion

3.1. Development of CT-AgNP and its characterization by UV spectroscopy

CT-loaded AgNPs were prepared using microwaves. The CT-AgNP solutions transitioned from clear to dark brown. AgNP production was initially identified by a visual shift in color (Figure 1a) and later verified by UV spectroscopy, which showed that the λ_max was greater than 400 nm (Figure 1b).

(a) Formation of CT loaded AgNP formulation and (b) UV-Spectroscopy of prepared CT loaded AgNP formulation.

3.2. Optimization of the CT-AgNP formulation

Response surface plots were generated to illustrate the effect of independent variables, namely AgNO₃ concentration (X1), CT extract concentration (X2) and microwave power (X3), on key parameters, including AgNP size (Y1), PDI (Y2) and %EE (Y3) (Table 3 and Figure 2). The plus and minus symbols in the equation indicate the corresponding effects on AgNP size and %EE. Various kinetic models, including linear and quadratic models, were fitted to evaluate the impact of independent factors. The quadratic effect was considered suitable for optimization because of the significant influence of the variables. Residual plots, derived from the Box-Behnken design, were used to correlate the actual and predicted values of AgNP size, PDI, and %EE (Figure 2).

Table 3. Arithmetic parameter summary for responses Y1, Y2, and Y3.

Responses	R²	Adjusted R²	Predicted R²	SD	%CV
NP size (nm)	0.9784	0.9506	0.7577	6.94	6.10
PDI	0.9761	0.9454	0.9121	0.0691	12.60
EE	0.9915	0.9806	0.9384	1.53	2.09

3D Contour plots and residual plots for responses; (a) particle size, (b) PDI, and (c) EE of CT-AgNP formulations.

3.2.1. Effect of independent variables on NP size (Y1)

The size of NPs in the developed formulation was within a certain range (i.e. 103.77 nm). According to the quadratic equation, the AgNO₃ concentration (in mm) positively affected particle size, indicating that an increase in AgNO₃ concentration led to larger NPs. Similarly, the CT extract concentration also positively affected the particle size. Conversely, microwave power negatively influenced NP size, which was responsible for its decrease. This decrease could be attributed to an increase in the ratio of surface area to volume, which ultimately enhanced the overall NP surface area. Consequently, this enhancement promoted microwave absorption. Higher microwave power resulted in greater absorption but a smaller NP size [28].

$\begin{matrix} Y 1 = + 107.40 + 27 . {88 X}_{1} + 2 0. {24 X}_{2} - 24 .0 8 X_{3} - 4.87 X_{1} X_{2} - 2.11 X_{1} X_{3} \\ + 8.28 X_{2} X_{3} + 3.45 X_{1}^{2} - 2.60 X_{2}^{2} + 12.48 X_{3}^{2} \end{matrix}$

A sequential increase in the AgNO₃ and CT extract concentrations led to a corresponding enlargement of particle size. This increase could be attributed to their incorporation into the NP layers, which caused the particles to expand. However, according to the equation, the interplay between microwave power, AgNO₃ concentration (AC), and CT extract concentration (BC) indicates that microwave power negatively influenced NP size.

3.2.2. Effect of independent variables on PDI (Y2)

According to the equation derived from this analysis, the AgNO₃ concentration negatively affected absorbance. Similarly, the CT extract concentration increased the absorbance value, as described in the equation. In addition, microwave power negatively influenced the absorbance value.

$\begin{matrix} Y 2 = + 0. 3152 - 0.0 {431X}_{1} - 0.0 50 {5 X}_{2} - 0.0 {429 X}_{3} + 0. {2513 X}_{1} X_{2} + 0. 1130 X_{1} X_{3} \\ - 0. {3518 X}_{2} X_{3} + 0. 170 {9 X}_{1}^{2} + 0.0 {336 X}_{2}^{2} + 0. 290 {9 X}_{3}^{2} \end{matrix}$

3.2.3. Effect of independent variables on %EE (Y3)

According to the equation derived from this analysis, the AgNO₃ concentration negatively affected absorbance, showing that the absorbance value dropped as the concentration rose. Conversely, an increase in the CT extract concentration was associated with an increase in the absorbance value, as outlined in the equation. Furthermore, microwave power exhibited a positive influence on the absorbance value.

$\begin{matrix} Y 3 = + 81.59 - 2 . {42 X}_{1} - 2 . {25 X}_{2} + 3 . {33 X}_{3} - 12 .0 {9 X}_{1} X_{2} + 11 . {97 X}_{1} X_{3} \\ + 4 . {24 X}_{2} X_{3} - 5 . {29 X}_{1}^{2} - 7 .0 {3 X}_{2}^{2} - 5 . {48 X}_{3}^{2} \end{matrix}$

3.3. Size and PDI

The particle size distribution revealed a PDI of 0.2672, indicating a low degree of size variation within the sample population (Figure 3). PDI values generally range from 0 to 1.0.

Optimized CT-AgNP formulation’s particle size distribution curve.

3.4. Zeta potential (ZP)

Opt-CT-AgNP exhibited a ZP of −24.38 mV, within the recommended range of −30 to +30 mV. This range is critical for ensuring that adequate repulsion forces are in place to prevent particle aggregation. Lower ZP values indicate the presence of an optimal distance between the charged particles within AgNPs loaded with the drug. This separation effectively prevents the coagulation, flocculation, and accumulation of particles (Figure 4).

Zeta potential of optimized CT-AgNP formulation.

3.5. Estimation of %EE

The %EE of the optimized preparation was 82.06 ± 2.74%. The enhancement in %EE was directly associated with the increased amounts of AgNO₃ and CT extract. The results align with the results reported previously [34].

3.6. TEM imaging

The TEM image of the CT-AgNP (Figure 5) displayed spherical NPs with dark cores, consistent with the size obtained from photon microscopy. This image demonstrated even distribution of particles within the formulation, with no signs of aggregation or crystal formation [35]. Furthermore, the TEM picture revealed well-defined sealed structures with a spherical shape and a homogeneous size distribution.

TEM images of CT-AgNP NPs depicting spherical-shaped NP.

3.7. Characterization of Opt-CT-AgNP gel

Table 4 displays the results of an analysis of the various distinguishing features of Opt-CT-AgNPs. The resulting CT-AgNP gel had a smooth, uniform appearance, and no abrasive particles were evident. Its pH was 7.04 ± 0.08, making it safe for topical application. It was experimentally determined that the Opt-CT-AgNP gel formulation had an extrudability of 16.05 ± 4.24 g and a spreadability of 31.96 ± 4.35 gcm/s. Figure 6 displays the results of a texture analysis performed on the Opt-CT-AgNP gel formulation, which had a firmness of 101.36 g, consistency of 659.41 g s, cohesiveness of −79.97 g, and a viscosity index of −502.80 g s.

Table 4. Evaluation of the optimized AgNP gel loaded with Cassia tora.

Colour	Appearance	Washability	Phase separation	Odor
Slightly brownish	Translucent	Washable	No	No
Homogeneity	Drug content	pH	Extrudability	Spreadability
Good/excellent	96.25 ± 3.16s	7.04 ± 0.08	16.05 ± 4.24 g	31.96 ± 4.35 g cm/s
Texture analysis	Cohesiveness	Firmness	Consistency	Viscosity index
Texture analysis	-79.97 g	101.36 g	659.41 g s	-502.80 g s

Texture analysis diagram of optimized CT-AgNP gel formulation denoting consistency, firmness, cohesiveness, and work of cohesion.

3.8. In vitro drug release study

The drug release study indicated that the maximum drug release was 96.61 ± 3.12% at 6 h from CT suspension, whereas drug release from CT-AgNP was 52.67 ± 3.71% at 6 h, which indicated drug distribution in a controlled manner. However, the maximum release of CT-AgNP was observed to be 67.63 ± 2.87% at 12 h. Drug release was significantly increased in Opt-CT-AgNP compared with the pure suspension. This pattern of release is optimal for maximising the therapeutic benefits. The therapeutic concentration required to show beneficial effects can be achieved by an initial rapid release, and this therapeutic effect can be enhanced by a slower release over a longer period. We observed a statistically significant difference (p < 0.05) in the first 2 h (Figure 7a-c). The Higuchi equation (R² = 0.918) provided the best explanation for the permeation investigations, followed by first-order kinetics (R² = 0.882) and zero-order kinetics (R² = 0.746) (Figure 7). The Korsmeyer–Peppas model predicted a non-Fickian release form at n = 0.510 (Figure 7d) [36].

Figure 7.

Release kinetics and in vitro release profile of CT-AgNP formulation: (a) The Higuchi model, (b) First order release model, (c) zero order release model and (d) Korsmeyer–Peppas model.

3.9. Ex vivo permeation

Permeation studies using conventional and Opt-CT-AgNP gel formulations on rat skin revealed that the Opt-CT-AgNP gel formulation had superior permeability compared to the conventional formulation. The permeability optimized gel was measured with permeation of 75.08 ± 3.14%, whereas the conventional formulation had a permeation of 40.98 ± 1.97% (Figure 8) [37,38]. Therefore, compared with the conventional formulation, the Opt-CT-AgNP gel formulation had superior skin permeability because of its nano-size particles; hence, CT-AgNPs infiltrate the inner layers of the skin and cause greater absorption of the formulation.

Ex vivo permeation studies of CT-AgNP gel and CT-CF gel.

3.10. Confocal laser scanning microscopy (CLSM)

A comparative CLSM study was conducted to assess the penetration depth of the examined samples. The CLSM analysis results showed that the rhodamine B-loaded Opt-CT-AgNP gel formulation had a significantly higher fluorescence intensity by penetrating deeper into the skin (up to 35 µm) compared with rhodamine B hydroalcoholic solution (up to 10 µm) [39]. Thus, CT-AgNPs permeated more deeply and uniformly throughout the tissue because of their nano-size (Figure 9).

CLSM images in optical cross section perpendicular to rat’s skin surface “(a) treated with hydroalcoholic solution of Rhodamine B and (b) treated with Rhodamine B-loaded opt-AgNP formulation (Containing Cassia tora)”.

3.11. Dermatokinetic study

Figure 10 displays the drug distribution of the generated formulation in the rat skin’s dermis and epidermis. CT-AgNP significantly increased drug delivery in the epidermal layer compared with the dermal layer (p < 0.05). The skin disposal rate (Ke), AUC0–8h, C_max skin, and T_max skin numerical values are displayed in Table 5. In addition, the AUC of CT-CF was substantially higher in the epidermis and dermis (p < 0.001 for each). By penetrating the deep layers of the skin and enhancing drug retention in both skin layers, the CT-AgNP gel demonstrated enhanced efficacy in treating psoriasis.

CT concentration on after topical application of (a) CT-AgNP and (b) CT-CF on excised rat skin.

Table 5. Dermatokinetic parameters of CT-AgNP and CT-CF (Mean ± SD).

Dermatokinetic parameters	CT-AgNP		CT-CF
Dermatokinetic parameters	Epidermis	Dermis	Epidermis	Dermis
T_{skin max} (h)	2 ± 0.1	2 ± 0.2	2 ± 0.2	2 ± 0.1
C_{skin max (}μg/cm²)	203.01 ± 3.29	172.989 ± 8.32	104.02 ± 12.25	91.8496 ± 7.38
AUC_{0-8 (}μg/cm²h)	810.21 ± 16.28	694.79 ± 14.32	469.02 ± 18.29	398.63 ± 12.38
Ke (h^-1)	0.13128 ± 0.02	0.13102 ± 0.05	0.11798 ± 0.03	0.13169 ± 0.02

3.12. DPPH assay

Bioactive chemicals found in plants are largely responsible for their biological effects and antioxidant properties. Because of the potential for excipient interaction with antioxidants, assessing antioxidant activity after formulation preparation is important. We determined whether the CT-AgNP formulation possessed antioxidant properties, and its efficacy was compared with that of ascorbic acid. The antioxidant activity was 93.13 ± 3.23%, 76.38 ± 2.52%, and 81.84% for ascorbic acid, CT extract, and CT-AgNP, respectively. The enhanced antioxidant value confirmed the increased efficacy of the CT-AgNP. The antioxidant activity between ascorbic acid and CT-AgNP was not significantly different.

3.13. Stability

Short-term stability studies were performed over 6 months. Particle size, ZP, drug content, reconstitution time, colour, phase separation, clarity, homogeneity, pH, taste, and drug content remained consistent throughout the study (Tables 6 and 7). During lyophilization, the cryoprotectant mannitol vitrified and formed a hard glassy matrix, aiding NP resistance to aggregation. The glassy matrix dissolved during reconstitution, enabling efficient particle redistribution.

Table 6. Accelerated stability assessment of CT-loaded i-optimized AgNP formulation in the short term.

Parameters	0 month	4±2ᵒC (months)			25±2ᵒC/60±5% RH (months)
Parameters	0 month	1	3	6	1	3	6
Appearance	+++	+++	++	++	+++	++	++
Phase separation	−	−	−	−	−	−	−
Shape	Spherical	Spherical	Spherical	Spherical	Spherical	Spherical	Spherical
PDI	0.197	0.197	0.252	0.265	0.210	0.260	0.265
Size (nm)	78.48	78.65	87.01	91.34	80.58	88.93	92.36
%EE	78.65	78.24	76.12	74.97	75.96	73.19	70.43
Reconstitution (second)	8±2	9±2	9±3	13±2	14±4	15±3	21±3

“+ Satisfactory, ++ Good, +++ Excellent”

Table 7. Short-term accelerated stability evaluation of CT-loaded optimized AgNP gel formulation.

Parameters	0 month	4±2ᵒC (months)			25±2ᵒC/60±5% RH (months)
Parameters	0 month	1	3	6	1	3	6
Color	Pale brownish yellow	Pale brownish yellow	Pale brownish yellow	Pale brownish yellow	Pale brownish yellow	Pale brownish yellow	Pale brownish yellow
Appearance	Transparent	Transparent	Transparent	Transparent	Transparent	Transparent	Transparent
Phase separation	−	−	−	−	−	−	−
Clarity	clear	clear	clear	clear	clear	clear	clear
pH	7.04	7.04	7.02	7.12	7.08	6.80	7.26
Homogeneity	***	***	***	***	**	**	*
Washability	washable	washable	washable	washable	washable	washable	washable
Odor	−	−	−	−	−	−	−
Taste	Salty bittersweet	Salty bittersweet	Salty bittersweet	Salty bittersweet	Salty bittersweet	Salty bittersweet	Salty bittersweet

* Satisfactory, ** Good, ** Excellent

3.14. Discussion

The study provides valuable insights into the efficacy of quality by design (QbD) and microwave-assisted green AgNPs loaded with CT extract for the management of psoriasis. We investigated the preparation and optimization of AgNPs and conducted in vitro drug release, CLSM, dermatokinetic, antioxidant, skin permeation, and stability studies of the formulation.

The Box-Behnken design was used to optimize the AgNP formulation. It was observed that an increase in the AgNO₃ and CT extract concentrations increased the NP size, which decreased when the microwave power increased. An increase in AgNO₃ and CT extract concentrations decreased PDI, and microwave power showed a negative effect on the absorbance value. Furthermore, an increase in the quantity of AgNO₃ and CT extract decreased %EE, and microwave power showed a positive effect on the absorbance value.

The NP size was 103.77 nm, and ZP was −24.38 mV, which are suitable for skin delivery. In the CT-AgNP formulation, NPs were present without crystal formation or aggregation as well-defined sealed structures with a spherical shape and uniform size distribution. The in vitro drug release showed that the maximum drug release was 96.61 ± 3.12% at 6 h from CT suspension, whereas drug release from CT-AgNP was demonstrated to be 52.67 ± 3.71% at 6 h, thereby indicating drug distribution in a controlled manner. However, the maximum release of CT-AgNP was observed to be 67.63 ± 2.87% at 12 h. The antioxidant activity of ascorbic acid was 93.13%, whereas that of the Opt-CT-AgNP formulation was 81.84% and that of the CT extract was 76.38%. Stability testing was performed at 1 month, 3 months, and 6 months. No changes in appearance, phase separation, shape, PDI, particle size, %EE, and reconstitution of the formulation were noted. Gel characterization studies revealed an extrudability of 16.05 ± 4.24 g and a spreadability of 31.96 ± 4.35 g cm/s for the produced gel formulation. Furthermore, texture analysis results showed that the Opt-CT-AgNP gel formulation had a firmness of 101.36 g, consistency of 659.41 g s, cohesiveness of −79.97 g, and viscosity index of −502.80 g s, affirming the stability of the prepared gel. In the skin permeation study, CT-AgNP showed a 1.8-times increase in drug permeation. In the CLSM study, CT-AgNP demonstrated 3.5 times deeper penetration compared with rhodamine B hydroalcoholic solution.

4. Conclusions

The Box-Behnken design ensures the production of consistent, safe, and effective spherical CT-AgNPs with a size of 103.77 nm, PDI of 0.2672, and ZP of −24.38 mV. The Opt-CT-AgNP formulation resulted in a %EE of 82.06% and in vitro drug release of 52.67%. The CLSM analysis results showed that the Opt-CT-AgNP formulation loaded with rhodamine B permeated rat skin more effectively than the control. Furthermore, the dermatokinetic analysis demonstrated enhanced permeability of the CT-AgNP gel compared with the CT-CF formulation. The DPPH assay was used to verify the antioxidant activity of the Opt-CT-AgNP formulation. According to the results of the gel characterization analysis, the gel formulation exhibited an extrudability of 16.05 ± 4.24 g and a spreadability of 31.96 ± 4.35 g cm/s. The findings from the texture analysis indicate that the Opt-CT-AgNP gel formulation exhibited a firmness of 101.36 g, consistency of 659.41 g s, cohesiveness of −79.97 g, and viscosity index of −502.80 g s. These results provide additional evidence of the stability of the prepared gel. The outcomes of the ex vivo investigation demonstrated that the CT-AgNP gel formulation exhibited a greater rate of permeation compared with the control in both dermis and epidermis. The results of the short-term stability experiments validated the assumption that NPs remain stable during storage. The results of the present study confirm that the Opt-CT-AgNP gel formulation is an effective drug delivery system for the dermal delivery of Cassia tora in the treatment of psoriasis. Although the obtained results are seminal in concluding the effectiveness of CT-AgNP in psoriasis treatment.

Acknowledgment

The authors extend their appreciation to Prince Sattam bin Abdulaziz University for funding this research work through the project number (PSAU/2024/03/31903).

CRediT authorship contribution statement

Abdullah S. Alshetaili: Methodology, Investigation, Data curation. Abuzer Ali: Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition, Methodology, Resources.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.

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1. Introduction

2. Materials and Methods

2.1. Materials
2.2. Preparation of plant extract
2.3. CT-AgNP production using the green synthesis method
2.4. Optimisation of CT-AgNPs
2.5. Characterization of optimized CT-AgNP (Opt-CT-AgNP)
2.6. Preparation of Opt-CT-AgNP gel
2.7. Characterization of Opt-CT-AgNP gel
2.8. In vitro drug release study
2.9. Ex vivo skin permeation study
2.10. Confocal laser scanning microscopy
2.11. Dermatokinetic study
2.12. Antioxidant activity using the 2,2-diphenylpicrylhydrazyl method
2.13. Stability study

3. Results and Discussion

3.1. Development of CT-AgNP and its characterization by UV spectroscopy
3.2. Optimization of the CT-AgNP formulation
3.3. Size and PDI
3.4. Zeta potential (ZP)
3.5. Estimation of %EE
3.6. TEM imaging
3.7. Characterization of Opt-CT-AgNP gel
3.8. In vitro drug release study
3.9. Ex vivo permeation
3.10. Confocal laser scanning microscopy (CLSM)
3.11. Dermatokinetic study
3.12. DPPH assay
3.13. Stability
3.14. Discussion

4. Conclusions

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[1] Khan R., Mirza M.A., Aqil M., Alex T.S., Raj N., Manzoor N., Naseef P.P., Saheer Kuruniyan M., Iqbal Z.. In vitro and in vivo investigation of a dual-targeted nanoemulsion gel for the amelioration of psoriasis. Gels (Basel, Switzerland). 2023;9:112. https://doi.org/10.3390/gels9020112
[Google Scholar]

[2] Dabholkar N., Rapalli V.K., Singhvi G.. Potential herbal constituents for psoriasis treatment as protective and effective therapy. Phytotherapy Research. 2021;35:2429-2444. https://doi.org/10.1002/ptr.6973
[Google Scholar]

[3] Jyothi S.L., Krishna K.L., Ameena Shirin V.K., Sankar R., Pramod K., Gangadharappa H.V.. Drug delivery systems for the treatment of psoriasis: Current status and prospects. Journal of Drug Delivery Science and Technology. 2021;62:102364. https://doi.org/10.1016/j.jddst.2021.102364
[Google Scholar]

[4] Sondhi S., Singh N., Jindal S.. Natural remedies used in the treatment of psoriasis: A short review. Asian Journal of Pharmaceutical Research. 2021;11:43-45. https://doi.org/10.5958/2231-5691.2021.00009.5
[Google Scholar]

[5] Daniyal M., Akram M., Zainab R., Munir N., Shah S.M.A., Liu B., Wang W., Riaz M., Jabeen F.. Progress and prospects in the management of psoriasis and developments in phyto-therapeutic modalities. Dermatologic Therapy. 2019;32:e12866. https://doi.org/10.1111/dth.12866
[Google Scholar]

[6] Herman A., Herman A.P.. Topically used herbal products for the treatment of psoriasis - mechanism of action, drug delivery, clinical studies. Planta Medica. 2016;82:1447-1455. https://doi.org/10.1055/s-0042-115177
[Google Scholar]

[7] Meng S., Lin Z., Wang Y., Wang Z., Li P., Zheng Y.. Psoriasis therapy by Chinese medicine and modern agents. Chinese Medicine. 2018;13:16. https://doi.org/10.1186/s13020-018-0174-0
[Google Scholar]

[8] Svendsen M.T., Jeyabalan J., Andersen K.E., Andersen F., Johannessen H.. Worldwide utilization of topical remedies in treatment of psoriasis: A systematic review. The Journal of Dermatological Treatment. 2017;28:374-383. https://doi.org/10.1080/09546634.2016.1254331
[Google Scholar]

[9] Gisondi P., Del Giglio M., Girolomoni G.. Treatment approaches to moderate to severe psoriasis. International Journal of Molecular Sciences. 2017;18:2427. https://doi.org/10.3390/ijms18112427
[Google Scholar]

[10] Gaikwad R.G., Shinde A.J., Hajare A.A.. Herbal treatment for management of psoriasis: An overview. Research Journal of Pharmacy and Technology 2022:1385-1392. https://doi.org/10.52711/0974-360x.2022.00231
[Google Scholar]

[11] Pradhan M., Alexander A., Singh M.R., Singh D., Saraf S., Saraf S., Ajazuddin N.. Understanding the prospective of nano-formulations towards the treatment of psoriasis. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie. 2018;107:447-463. https://doi.org/10.1016/j.biopha.2018.07.156
[Google Scholar]

[12] Dawadi S., Katuwal S., Gupta A., Lamichhane U., Thapa R., Jaisi S., Lamichhane G., Bhattarai D.P., Parajuli N.. Current research on silver nanoparticles: Synthesis, characterization, and applications. Journal of Nanomaterials 2021:1-23. https://doi.org/10.1155/2021/6687290
[Google Scholar]

[13] Yadav N., Aggarwal R., Targhotra M., Sahoo P.K., Chauhan M.K.. Natural and nanotechnology based treatment: An alternative approach to psoriasis. Current Nanomedicine. 2021;11:21-39. https://doi.org/10.2174/2468187310999201022192318
[Google Scholar]

[14] Ashij M.A., Al-Shmgani H.S., Sulaiman G.M., Mohammed H.A., Abdalrazaq E.A., Albukhaty S.. Investigation of Antibacterial activity and wound healing promotion properties induced by bromelain-loaded silver nanoparticles. Plasmonics. 2024;19:1903-1916. https://doi.org/10.1007/s11468-023-02127-x
[Google Scholar]

[15] Hussein N.N., Al-Azawi K., Sulaiman G.M., Albukhaty S., Al-Majeed R.M., Jabir M., Al-Dulimi A.G., Mohammed H.A., Akhtar N., Alawaji R., A Alshammari A.A., Khan R.A.. Silver-cored Ziziphus spina-christi extract-loaded antimicrobial nanosuspension: Overcoming multidrug resistance. Nanomedicine (London, England). 2023;18:1839-1854. https://doi.org/10.2217/nnm-2023-0185
[Google Scholar]

[16] Perveen R., Shujaat S., Naz M., Qureshi M.Z., Nawaz S., Shahzad K., Ikram M.. Green synthesis of antimicrobial silver nanoparticles with Brassicaceae seeds. Materials Research Express. 2021;8:055007. https://doi.org/10.1088/2053-1591/ac006b
[Google Scholar]

[17] Messaoudi O., Benamar I., Azizi A., Albukhaty S., Khane Y., Sulaiman G.M., Salem-Bekhit M.M., Hamdi K., Ghoummid S., Zoukel A., Messahli I., Kerchich Y., Benaceur F., Salem M.M., Bendahou M.. Characterization of silver carbonate nanoparticles biosynthesized using marine actinobacteria and exploring of their antimicrobial and antibiofilm activity. Marine Drugs. 2023;21:536. https://doi.org/10.3390/md21100536
[Google Scholar]

[18] Campora S., Ghersi G.. Recent developments and applications of smart nanoparticles in biomedicine. Nanotechnology Reviews. 2022;11:2595-2631. https://doi.org/10.1515/ntrev-2022-0148
[Google Scholar]

[19] Nardo V.D., Gianfaldoni S., Tchernev G., Wollina U., Barygina V., Lotti J., Daaboul F., Lotti T.. Use of curcumin in psoriasis. Open Access Macedonian Journal of Medical Sciences. 2018;6:218-220. https://doi.org/10.3889/oamjms.2018.055
[Google Scholar]

[20] Negi P., Sharma I., Hemrajani C., Rathore C., Bisht A., Raza K., Katare O.P.. Thymoquinone-loaded lipid vesicles: A promising nanomedicine for psoriasis. BMC Complementary and Alternative Medicine. 2019;19:334. https://doi.org/10.1186/s12906-019-2675-5
[Google Scholar]

[21] Qadir A., Aqil M., Ali A., Warsi M.H., Mujeeb M., Ahmad F.J., Ahmad S., Beg S.. Nanostructured lipidic carriers for dual drug delivery in the management of psoriasis: Systematic optimization, dermatokinetic and preclinical evaluation. Journal of Drug Delivery Science and Technology. 2020;57:101775. https://doi.org/10.1016/j.jddst.2020.101775
[Google Scholar]

[22] Zhang Q., Xie J., Li G., Wang F., Lin J., Yang M., Du A., Zhang D., Han L.. Psoriasis treatment using Indigo naturalis: Progress and strategy. Journal of Ethnopharmacology. 2022;297:115522. https://doi.org/10.1016/j.jep.2022.115522
[Google Scholar]

[23] Gendrisch F., Haarhaus B., Krieger N., Quirin K.W., Schempp C.M., Wölfle U.. The effect of herbal medicinal products on psoriasis-like keratinocytes. Biomolecules. 2021;11:371. https://doi.org/10.3390/biom11030371
[Google Scholar]

[24] Sundarrajan S., Lulu S., Arumugam M.. Deciphering the mechanism of action of Wrightia tinctoria for psoriasis based on systems pharmacology approach. Journal of Alternative and Complementary Medicine (New York, N.Y.). 2017;23:866-878. https://doi.org/10.1089/acm.2016.0248
[Google Scholar]

[25] Juvatkar P., Jadhav A.. Caesalpinia bonducella: A medicinal potential value. Journal of Pharmacognosy and Phytochemistry. 2021;10:206-214.
[Google Scholar]

[26] Geetha M.. Anti-psoriatic activity of flavonoids from Cassia tora leaves using the rat ultraviolet B ray photodermatitis model. Brazilian Journal of Pharmacognosy. 2014;24:322-329. https://doi.org/10.1016/j.bjp.2014.07.010
[Google Scholar]

[27] Spoorthi, Ashoka Shenoy M.. Exploring the Therapeutic Potential of Cassia Tora Linn: An in-depth Review of its Phytochemistry, Traditional Uses and Pharmacology. International Journal of Pharmaceutical Sciences. 2025;3:4818-4826. https://doi.org/10.5281/zenodo.15547185
[Google Scholar]

[28] Anheyer M., Cramer H., Ostermann T., Längler A.. Herbal medicine for treating psoriasis: A systematic review. Complementary Therapies in Medicine. 2025;90:103173. https://doi.org/10.1016/j.ctim.2025.103173
[Google Scholar]

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Quality by design and microwave-assisted green silver nanoparticles loaded with Cassia tora extract for psoriasis management

Abstract

Keywords

Antioxidant

Cassia tora

Psoriasis

QbD

Silver nanoparticles

1. Introduction

2. Materials and Methods

2.1. Materials

2.2. Preparation of plant extract

2.3. CT-AgNP production using the green synthesis method

2.4. Optimisation of CT-AgNPs

2.5. Characterization of optimized CT-AgNP (Opt-CT-AgNP)

2.5.1. Particle size characterization

2.5.2. Percentage entrapment efficiency (%EE)

2.5.3. Transmission electron microscope (TEM) study

2.6. Preparation of Opt-CT-AgNP gel

2.7. Characterization of Opt-CT-AgNP gel

2.7.1. pH

2.7.2. Spreadability

2.7.3. Extrudability

2.7.4. Texture analysis

2.8. In vitro drug release study

2.9. Ex vivo skin permeation study

2.10. Confocal laser scanning microscopy

2.11. Dermatokinetic study

2.12. Antioxidant activity using the 2,2-diphenylpicrylhydrazyl method

2.13. Stability study

3. Results and Discussion

3.1. Development of CT-AgNP and its characterization by UV spectroscopy

3.2. Optimization of the CT-AgNP formulation

3.2.1. Effect of independent variables on NP size (Y1)

3.2.2. Effect of independent variables on PDI (Y2)

3.2.3. Effect of independent variables on %EE (Y3)

3.3. Size and PDI

3.4. Zeta potential (ZP)

3.5. Estimation of %EE

3.6. TEM imaging

3.7. Characterization of Opt-CT-AgNP gel

3.8. In vitro drug release study

3.9. Ex vivo permeation

3.10. Confocal laser scanning microscopy (CLSM)

3.11. Dermatokinetic study

3.12. DPPH assay

3.13. Stability

3.14. Discussion

4. Conclusions

Acknowledgment

CRediT authorship contribution statement

Declaration of competing interest

Declaration of generative AI and AI-assisted technologies in the writing process

References

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