*2.5. Release Kinetics*

Release kinetics of the optimized formulation F10 were investigated using zero-order, first-order, and Higuchi diffusion (Table 3 and Figure 4). Zero-order kinetics is defined as a constant amount of drug that is eliminated per unit of time, but the rate is independent of the concentration of the drug. First-order kinetics describes the constant proportion of the drug that is eliminated per unit of time. The rate of elimination is proportional to the amount of drug in the body. Higuchi's kinetics dominated drug release in the formulation and proves that the gel is a controlled drug delivery system. It also confirms a non-Fickian mechanism. Non-Fickian drug release refers to the drug being released from the gel through a diffusion mechanism as well as another method known as chain relaxation.

### *2.6. In Vitro Antioxidant Study*

The optimized formulation F10 was subjected to anti-oxidant analysis. The antioxidant activity of samples were investigated at various concentrations and standards (ascorbic acid). By scavenging DPPH (free radical) and converting it to DPPH, the samples demonstrated strong antioxidant activity (Figure 5). Nitric oxide (NO) is a quite unstable species that further, under aerobic conditions, reacts with O<sup>2</sup> to obtain uniform nitrate and nitrite products using NO2, N2O<sup>4</sup> and N3O<sup>4</sup> intermediates. In this study, the nitrite developed by the reaction mixture was limited.



**Table 2.** Physicochemical evaluation for various gel formulations.

**(6 months at room temperature)** 

**Drug content (mg /g)** 

0.17 ± 0.19

0.23 ± 0.01

0.30 ± 0.83

2.3.4. Drug Content Uniformity

*2.4. In Vitro Drug Diffusion Studies* 

2.3.3. Spreadability

2.3.5. Stability

formulation.

0.52 ± 0.06

high spreadability and extrudability (Table 2).

± SD, where *n* = 3 and SD stands for standard deviation.

0.55 ± 0.15

1.9 ± 0.34

After 1 min, the spreadability of the gels ranged from 10.73 ± 0.01 mm to 23.75 ± 0.03 mm. The gel formulations were prepared with Carbopol 934, and hence demonstrated

The percentage drug content of all formulations ranged from 0.2 ± 0.01% mg/g to

Physiological changes such as change in color, or segregation of fluid exudates of the gels were not observed after 6 months of stability studies. The pH of all the gels was unaffected and ranged from 6.5 ± 0.03 to 7.0 ± 0.06. After 6 months, and the drug content ranged from 0.17 ± 0.19 to 28.00 ± 0.004% mg/g (Table 2). The results are expressed as mean

Cumulative percent drug release ranged from 21.97 ± 0.02% for the open-ended cylinder and cumulative percent drug release ranged from 65.71 ± 0.04% for the diffusion cell apparatus across all formulations (Figure 3). As a result, F10 was chosen as the optimal

32.77 ± 0.20% mg/g (Table 2) The drug content was consistent across all gels.

5.34 ± 0.57

9.8 ± 0.93

15.34 ± 0.05

28.00 ± 0.004

**Figure 3.** Percent cumulative drug release from gel formulations. (**A**) Open−ended cylinder (F1 to F5), (**B**) diffusion cell apparatus (F1 to F5), (**C**) Open−ended cylinder (F6 to F10), (**D**) diffusion cell apparatus (F6 to F10), **Figure 3.** Percent cumulative drug release from gel formulations. (**A**) Open−ended cylinder (F1 to F5), (**B**) diffusion cell apparatus (F1 to F5), (**C**) Open−ended cylinder (F6 to F10), (**D**) diffusion cell apparatus (F6 to F10).


**Table 3.** Release kinetics for optimized formulation F10.

**R2** 0.9475 0.9475 0.9869 0.9448


portion of the drug that is eliminated per unit of time. The rate of elimination is propor-*2.7. Ex Vivo Permeation Study*

tion.

tional to the amount of drug in the body. Higuchi's kinetics dominated drug release in the formulation and proves that the gel is a controlled drug delivery system. It also confirms a non-Fickian mechanism. Non-Fickian drug release refers to the drug being released from The gel can permeate easily across the mucosal membrane. The permeation correlation coefficient range was 0.9586 and the percentage was found to be 21.97% in 3 h. The result of the study is shown in (Table 4 and Figure 6).

the gel through a diffusion mechanism as well as another method known as chain relaxa-

pendent of the concentration of the drug. First-order kinetics describes the constant pro-

 **Zero Order First Order Higuchi Diffusion Korsmeyer Peppas** 

**Figure 4.** Higuchi's kinetics drug release in the formulation. (**A**) Zero order, (**B**) first order, (**C**) Higuchi equation, (**D**) Korsmeyer Peppas. **Figure 4.** Higuchi's kinetics drug release in the formulation. (**A**) Zero order, (**B**) first order, (**C**) Higuchi equation, (**D**) Korsmeyer Peppas.


*2.6. In Vitro Antioxidant Study* **Table 4.** Ex vivo diffusion study of silymarin.

**Figure 5.** In vitro antioxidant study for optimized gel F10. (**A**) DPPH activity, which shows that it is dose-dependent, (**B**) nitric oxide, the scavenging activity. **Figure 5.** In vitro antioxidant study for optimized gel F10. (**A**) DPPH activity, which shows that it is dose-dependent, (**B**) nitric oxide, the scavenging activity. *Gels* **2023**, *9*, x FOR PEER REVIEW 9 of 15

8. 120 14.03 ± 0.09 9. 150 17.56 ± 0.04 **Figure 6.** Ex vivo permeation study for the optimized formulation F10. (**A**) Chicken buccal mucosa, (**B**) Cumulative drug release. **Figure 6.** Ex vivo permeation study for the optimized formulation F10. (**A**) Chicken buccal mucosa, (**B**) Cumulative drug release.

### 10. 180 21.97 ±0.18 **3. Discussion 3. Discussion**

Mucosal drug delivery systems have the superior advantage of intimate contact between the diseased mucosa and the drug, localizing the drug to the specific site and better patient compliance, compared to systemic drugs. Mucoadhesive forms also offer better plasma concentration and therapeutic efficacy [11]. The pre-formulation phase or study helps to lay down the foundation for transforming a new drug into a pharmaceutical formulation in such a way that it can be administered in the right manner and optimum dose. Moreover, pre-formulation studies provide better stability to the formulation by developing a proper design with adequate constituents, protecting the drug component from environmental conditions and evaluating the performance of the developed formulation to bring it to translational use in pharmacological practice [12]. Among various topical formulations such as ointments, creams, pastes, emulsions and gels, pastes and gels are the Mucosal drug delivery systems have the superior advantage of intimate contact between the diseased mucosa and the drug, localizing the drug to the specific site and better patient compliance, compared to systemic drugs. Mucoadhesive forms also offer better plasma concentration and therapeutic efficacy [11]. The pre-formulation phase or study helps to lay down the foundation for transforming a new drug into a pharmaceutical formulation in such a way that it can be administered in the right manner and optimum dose. Moreover, pre-formulation studies provide better stability to the formulation by developing a proper design with adequate constituents, protecting the drug component from environmental conditions and evaluating the performance of the developed formulation to bring it to translational use in pharmacological practice [12]. Among various topical formulations such as ointments, creams, pastes, emulsions and gels, pastes and gels are the

preferred topical formulations for the oral mucosa, because of their superior retentivity and mucoadhesive property. Gels, in particular, have higher water content and exhibit

Silymarin is a commonly used therapeutic drug with proven anti-inflammatory, antioxidant, anticancer and anti-fibrotic activities. Hence, the silymarin-based topical oral mucoadhesive gel could be a promising medication for inflammatory diseases of oral mucosa such as oral ulcers and oral mucositis, fibrotic diseases such as oral submucous fibrosis and radiation-induced fibrosis. To the best of our knowledge, silymarin-based oral mucoadhesive gel is not available for therapeutic use. However, a study done using silymarin cream for skin application is available in the literature, where its application in radiodermatitis of breast cancer patients, as a double-blinded placebo-controlled clinical trial, showed a significant delay in radiodermatitis development and progression in the silymarin group, with absence of notable side effects on the skin [8]. A study conducted using topical skin formulations of silymarin in melasma showed good results with minimal side effects on prolonged application for three months [13]. Silymarin also exhibited a gastroprotective effect in a study done by Sharma et al., in 2018, where silymarin-loaded beads of chitosan-MMT exhibited good mucoadhesion and efficient release of the drug, and were found to be a promising drug for the treatment of gastric ulcers [14]. Silymarin is known to be a safe drug with very minimal side effects. No major side effects or lifethreatening complications have been reported so far in the literature, especially on prolonged and high-dosage use [15,16]. In 2011, Becher-Schiebe et al., conducted an observational study on 101 women with breast cancer who had had breast-conserving surgery, where they used an alternate, open, nonrandomized schedule to assign patients to the silymarin and standard of care (SOC) groups. This trial exhibited silymarin-based cream preferred topical formulations for the oral mucosa, because of their superior retentivity and mucoadhesive property. Gels, in particular, have higher water content and exhibit lower friction and hence are better retained in the injured sites of the oral cavity for a prolonged period, thereby improving the drug's therapeutic efficacy [9].

Silymarin is a commonly used therapeutic drug with proven anti-inflammatory, antioxidant, anticancer and anti-fibrotic activities. Hence, the silymarin-based topical oral mucoadhesive gel could be a promising medication for inflammatory diseases of oral mucosa such as oral ulcers and oral mucositis, fibrotic diseases such as oral submucous fibrosis and radiation-induced fibrosis. To the best of our knowledge, silymarin-based oral mucoadhesive gel is not available for therapeutic use. However, a study done using silymarin cream for skin application is available in the literature, where its application in radiodermatitis of breast cancer patients, as a double-blinded placebo-controlled clinical trial, showed a significant delay in radiodermatitis development and progression in the silymarin group, with absence of notable side effects on the skin [8]. A study conducted using topical skin formulations of silymarin in melasma showed good results with minimal side effects on prolonged application for three months [13]. Silymarin also exhibited a gastroprotective effect in a study done by Sharma et al., in 2018, where silymarin-loaded beads of chitosan-MMT exhibited good mucoadhesion and efficient release of the drug, and were found to be a promising drug for the treatment of gastric ulcers [14]. Silymarin is known to be a safe drug with very minimal side effects. No major side effects or life-threatening complications have been reported so far in the literature, especially on prolonged and high-dosage use [15,16]. In 2011, Becher-Schiebe et al., conducted an observational study on 101 women with breast cancer who had had breast-conserving surgery, where they used an alternate, open, nonrandomized schedule to assign patients to the silymarin and standard of care (SOC) groups. This trial exhibited silymarin-based cream to be a promising candidate for a safe prophylactic treatment option of radiodermatitis [17].The absence of significant side effects, even upon long-term use makes it an ideal candidate for therapeutic use in oral inflammatory and fibrotic conditions, which warrants a longer duration of treatment and follow-up.

According to the literature survey, there is no silymarin-based mucoadhesive gel for oral topical application. The major hurdle is solubility, and the same is predicted to be overcome with formulation trials using a combination of newer polymers such as carbopol. Carbopol constitutes acrylic acid-poly alkenyl ether/divinyl glycol cross-linked primary polymer particles of about 0.2 to 6.0 micron average diameter with high molecular weight. Demonstrated to create a tenacious bond with the mucus membrane resulting in strong bio-adhesion, several commercial oral and topical products available today and under investigation have been formulated with carbopol polymers. Along with excellent adhesion forces, they lower the concentration of active ingredients and provide patient compliance with increased bioavailability of certain drugs. In the present study, silymarin gel was prepared using carbopol, propylene glycol, methylparaben, propylparaben, triethanolamine and peppermint oil. The stability, spreadability and release studies, *in vitro* antioxidant activity and *ex vivo* diffusion study using a chick embryo Franz diffusion model showed good promising results. These results are in concurrence with the previous study of silymarin gel for skin preparation, using the same ingredients, which demonstrated to have good viscosity, spreadability and antifungal properties with pH within the range of skin pH. The skin formulation also showed stability for up to two months at 40 ◦C, and had no skin irritation in human volunteers [7]. Among the three gel formulations prepared using pomegranate flowers by Aslan and his colleagues, one was preferred as a superior formulation because of its proper appearance and uniformity, acceptable viscosity, mucoadhesiveness and stability at different temperatures [9]. The present study of silymarin gel formulation trials resulted in an optimal gel formulation comparable to the gel obtained from the above-mentioned study with acceptable physicochemical parameters as shown in the results. Post six months of storage, no physiological alterations or changes of pH were observed. Non-fickian drug release and 22% drug permeation established its

oral mucosal membrane permeability. Some of the physical methods to improve drug permeation include nanoformulations, liposomes, ethosomes, microemulsion, hydrogels, etc, which can be taken up in future studies [18].

The pH adjustment in the F10 was optimized maximally to 6.4, with further adjustment bearing on its viscosity. The normal salivary pH is 6.2–7.6, varying to 6–7 in various oral pathologies. A study performed by Foglio-Bonda et al., in 2017, reported that the salivary pH of 88 patients with oral lesions was 6.7 and the mean pH of 80 patients without oral mucosal lesions was 6.95 [19]. Oral disease severity, due to smoking and poor oral hygiene habits, is also strongly correlated with low salivary pH values (6.25 in stage IV periodontal disease) [20]. Hence, the pH of the oral mucosa tends to vary with oral disease presentations and habits such as tobacco.

A study employing ultrasonication (for better drug permeation) for the preparation of a silymarin-loaded nanostructured lipid carrier (NLC) gel showed antiproliferative, antioxidant, anti-inflammatory and antitumor activity in ex vivo and in vivo studies of skin cancer models [21]. The establishment of clinical efficacy of the gel shall be the impetus for further expansion of its therapeutic index, possibly as nano gel. Plans of preparation of a nano-based silymarin gel could probably increase the release percentage and offer better drug availability for improved efficacy. This formulation has set the pace for the development of the needed therapeutic option for oral diseases such as oral ulcers (aphthous ulcers and traumatic ulcers), oral mucositis and potentially malignant disorders such as oral submucous fibrosis, in the form of optimally bioavailable silymarin, thus potentializing its antifibrotic, anti-oxidant, anti-inflammatory and cytoprotective properties. Hence, multi-centric clinical trials on the topical application of silymarin mucoadhesive gel for different oral diseases can pave the way for identifying its therapeutic efficacy in different oral diseases.

### **4. Conclusions**

Silymarin mucoadhesive gel was prepared using a carbopol base and was found to show optimum results with pre-formulation studies. The prepared silymarin oral mucoadhesive gels, formulation F10, had improved mucoadhesive properties, drug delivery and bioavailability to the oral mucosa, according to the physicochemical evaluation results. Our *ex vivo* analysis confirms formulation permeation and investigated the potential for oral mucosal delivery. The next step in the research process is to develop a nano gel using poly(lactic-co-glycolic acid) nanoparticles and conduct clinical trials in different oral pathologies.

### **5. Materials and Methods**

The Serum Institute of India Pvt. Ltd. Pune, India supplied silymarin. Carbopol 934, methyl paraben and propyl paraben were purchased from Loba Chemie Pvt. Ltd. Mumbai, India. Propylene glycol and triethanolamine were obtained from SRL Chemicals Pvt. Ltd. Chennai, India.

### *5.1. Preparation of Silymarin-Based Mucoadhesive Gel*

Silymarin, which is the bioflavanoid present in *Silybum marianum*, has poor water solubility. Because of its low bioavailability, mucoadhesive gels with optimized polymer combinations were planned as an improved silymarin formulation for potential use in oral mucosa as a topical preparation.

The gel was prepared by dispersing carbopol into distilled water to which propylene glycol had previously been added. Methylparaben and propylparaben were added to this precisely weighed quantity. Then, silymarin, which was previously solubilized in propylene glycol, was slowly added with gentle stirring in the carbopol gel base. Triethanolamine was added to neutralize the mixture and peppermint oil was added as a flavoring agent.

### *5.2. Pre-Formulation Studies*

The color, odor, taste, and melting point of silymarin were determined. According to the certificate of analysis on the label, the purity was found to be 70% total silymarin. The standard stock solution was prepared in distilled water, and 100 mg of silymarin was accurately weighed and transferred to a 100 mL standard volumetric flask, which was then filled with distilled water to a volume of 100 mL. A sufficient aliquot of the standard stock solution (10 mL) was transferred to a 100 mL standard volumetric flask; the volume was then made up to 100 mL with distilled water. 1 mL, 2 mL, 4 mL and 5 mL was pipetted out, and the final volume was made up to 100 mL with distilled water to achieve concentrations of 1–5 µg/mL solutions. The absorbance was measured against distilled water at 287 nm (λ max silymarin) and the standard curve of concentration versus absorbance was plotted. The Fourier-transform infrared spectroscopy (FT-IR) spectra matching system was used to identify any potential chemical reactions between the drugs and polymers. It was scanned between 4000 and 600 cm−<sup>1</sup> .

### *5.3. Evaluation Parameters*

### 5.3.1. Viscosity

The measurement of the viscosity of the formulated gel was determined by a Brookfield Viscometer.

### 5.3.2. pH

The pH of the gel formulation was determined by using a digital pH meter [22].
