Formulation & Characterization of Phytochemical Based Topical Analgesic gel in Management of Myogenous Temporomandibular Joint Pain.

Formulation & Characterization of Phytochemical Based Topical Analgesic gel in Management of Myogenous Temporomandibular Joint Pain. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Formulation & Characterization of Phytochemical Based Topical Analgesic gel in Management of Myogenous Temporomandibular Joint Pain. Ramya Suresh, Ramya Ramadoss, P Bargavi, Meenakshi Sundaram, Nitya Krishnasamy, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4832399/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Myogenous Temporomandibular joint disorder is characterised by pain & dysfunction in the masticatory muscles that are originated from pathologic and functional processes in the masticatory muscles. Most common symptoms are muscle discomfort, restricted range of motion, fatigue, stiffness, and subjective weakness. Boswellia serrata gum resin extracts have been widely employed years of years to address a wide range of chronic inflammatory conditions. These conditions include rheumatoid arthritis, osteoarthritis, asthma, inflammatory bowel disease, and other inflammatory disorders. Silver nanoparticles have a great potential for their mechanistic role. Green synthesized silver nanoparticles (AgNPs) have promising biomedical applications in healthcare. These nanoparticles are synthesized using plant-based compounds that act as moderators, resulting in AgNPs with high therapeutic potential. They offer an alternative approach to medicine utilizing the bioactive compounds of plants. The incorporation of these compounds enhances the biomedical properties of AgNPs, making them valuable for various therapeutic applications. Resinous component of Boswellia Serrata were used as a key ingredient and thymol, menthol camphor was added for topical gel formulation as it has analgesic properties which can be used for management of masticatory myalgia. Masticatory myalgia Orofacial Pain herbal gel Boswellia topical anti-inflammatory analgesic. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. INTRODUCTION Myogenous temporomandibular disorders (TMDs) are commonly referred to as masticatory myalgia which constitutes a diverse array of conditions characterized by pain and functional impairment arising from both pathological and functional abnormalities within the masticatory muscles ( 1 ). These disorders encompass various subtypes, including myofascial pain, myositis, muscle spasm, and muscle contracture, each presenting unique clinical manifestations and etiological factors. Myofascial pain is the most prevalent subtype that manifests as a chronic condition characterized by regional pain associated with tender areas known as trigger points (TrPs) within skeletal muscles, tendons or ligaments. Epidemiological studies indicate that approximately 50% of individuals with chronic head and neck pain experience symptoms attributable to myogenous TMDs. Moreover, these disorders affect the general population with the prevalence rates ranging from 20–50%, and approximately 6% exhibiting symptoms severe requiring therapeutic intervention ( 2 ). Understanding the etiology of myogenous temporomandibular disorders (TMDs) is challenging due to the absence of a specific underlying cause or identifiable pathology. One of the major causes is direct and indirect trauma that leads to muscle soreness and dysfunction. Events like a direct blow to the jaw or extended mouth opening during dental procedures or yawning may also contribute to pain ( 3 ). Furthermore, repetitive strain activities such as using chewing gum or participating in oral parafunctional habits like teeth clenching and jaw thrusting which places excessive strain on the masticatory muscles that can cause symptoms of discomfort and pain. Postural strain which are characterised by forward head posture or anomalies in cervical and lumbar lordosis can exacerbate the musculoskeletal burden may also major role in causing referred pain to masticatory muscles thus having a complex interplay between biomechanical variables and myogenous temporomandibular disorders (TMDs). Psychosocial stressors have a notable impact on the worsening of symptoms ( 4 ). Managing the myogenous TMDs possess significant challenges due to its complex and multifactorial nature. Treatment approaches primarily focus on symptom management and improving the patient's overall quality of life. Pharmacological interventions such as tricyclic antidepressants, muscle relaxants and NSAIDs, such as ibuprofen or piroxicam are suggested for myalgia, which can be used for short-term analgesic anti-inflammatory effects can be effective as a supplement to alleviate pain and overall management ( 5 ). However, it is essential to consider the potential side effects and limitations of this medications. Non pharmacological therapeutic intervention such as exercise, TrP injections, vapocoolant spray and stretch, topical analgesics, transcutaneous electrical nerve stimulation, Ultrasound therapy, biofeedback, posture correction also plays a vital role in the comprehensive management. Physical therapy, acupuncture, cognitive-behavioral therapy (CBT), and relaxation techniques may also be employed to address muscular tension, promote pain relief, and improve coping strategies ( 6 ). Boswellia serrata is also known as Indian frankincense, a tree native to India and other parts of Asia. Its gum resin has been used in traditional medicine for centuries due to its anti-inflammatory and analgesic properties ( 7 ). The active compounds found in Boswellia serrata, specifically the boswellic acids, have been extensively studied for their therapeutic potential in various inflammatory conditions. These compounds possess anti-inflammatory properties by inhibiting key enzymes and pro-inflammatory mediators involved in the inflammatory cascade. Furthermore, they exhibit analgesic properties by modulating pain perception and transmission. The use of Boswellia serrata extract in managing myalgia is gaining attention due to its potential to address both the inflammatory and pain components of the condition. By targeting the underlying inflammatory processes and providing analgesic effects, this herbal extract holds promise as a holistic and natural approach to pain management ( 8 ). Research studies evaluating the efficacy of Boswellia serrata extract in facial pain management are limited but encouraging ( 9 ). A study conducted by Sengupta et al. (2010) demonstrated significant pain reduction in patients with myogenous temporomandibular joint disorders, following the administration of Boswellia serrata extract. The findings suggested its potential as an adjunctive therapy in alleviating facial pain symptoms. The mechanisms through which Boswellia serrata extract exerts its therapeutic effects in masticatory myalgia management are not yet fully understood. However, it is believed that its anti-inflammatory actions contribute to reducing tissue inflammation thereby alleviating pain. Additionally, its analgesic properties may involve modulation of pain receptors and neurotransmitters involved in pain perception and transmission ( 10 ). While the use of Resinous part of Boswellia serrata extract appears promising it is important to acknowledge more studies are needed to establish its effectiveness and safety profile for pain management ( 11 ).Furthermore the optimal dosage, treatment duration and potential side effects require further investigation to ensure its appropriate use.The exploration of natural remedies such as Boswellia serrata extract for facial pain management aligns with the growing interest in complementary and alternative medicine approaches. Incorporating herbal extracts into conventional treatment regimens may offer a more holistic and personalized approach to managing facial pain considering the limitations and side effects associated with conventional pharmaceutical interventions. The utilization of Boswellia serrata extract in a topical gel formulation offers several advantages for facial pain management ( 12 ). Topical application allows for direct delivery of the active compounds to the affected area, facilitating targeted relief.Formulation of herbal gel also provides a convenient and non-invasive mode of administration enhancing patient compliance and minimizing systemic side effects associated with oral medications.Integration of silver nanoparticles to the herbal extract offers several advantages as silver nanoparticles possess unique physical and chemical properties such as a high surface-volume ratio and enhanced reactivity which contribute to their antimicrobial efficacy ( 13 ).It has the ability to modulate the inflammatory response reducing inflammation and promoting tissue regeneration. Incorporating silver nanoparticles into a herbal gel formulation offers several advantages. Gel base provides a suitable matrix for the dispersion and stabilization of silver nanoparticles ensuring their uniform distribution. This facilitates consistent and controlled delivery of the nanoparticles to the affected area maximizing their therapeutic potential. Moreover, the gel formulation allows for convenient topical application promoting patient compliance and localized treatment ( 14 ). In this study, herbal gel formulations were prepared using silver nanoparticle with synthesized boswellia, extracts of menthol, thymol, camphor and 1.5 percent of Carbopol which is the gelling agent, and they were evaluated for physical appearance, net content, primary skin irritation tests and pH ( 15 ). Obtained Extract was characterized using FTIR, Zeta Potential, Scanning Electron Microscope (SEM), EDS and UV. Stability test for the prepared topical herbal gel formulation was conducted as per the guidelines of ICH. Further, anti-Inflammatory & analgesic activity was also evaluated. The study aimed to formulate topical analgesic gel incorporated containing Boswellia serrata herbal extracts and silver nano particles for the management of Myogenous TMDs. 2. MATERIALS & METHODS 2.1. Materials: Gum Resin of Boswellia serrata extract, Thymol, menthol and camphor were obtained from K Ramaswamy Herbals Limited, Chennai. Carbopol (940) was purchased from Loba Chemie Private Ltd, Chennai. 2.2. Extract Preparation: This gum resin is broken into tiny pieces with a wooden mallet and all impurities including pieces of bark are removed manually during this process. Boswellia gum resin was extracted using a solvent extraction which is a widely employed method for extracting non-volatile compounds from plant materials ( 16 ). In this technique methanol were used to dissolve and extract the gum resin. Firstly, the tiny pieces gum resin was mixed finely with the methanol and was allowed to undergo agitation for a specific period of time. During this time the solvent interacted with the plant material extracting the target compounds into the solution. The solution is then separated from the solid plant material using filtration, the solvent was evaporated & concentrated extract is obtained. Subsequently Silver nano particles were integrated into the extract. 2.3. Synthesis of silver nanoparticles: A volume of 1 ml from the centrifuged filtrate was transferred into a larger volume of 40 ml aqueous silver nitrate solution with a concentration of 1 mM. Subsequently, the mixture was carefully monitored for any noticeable change in color ( 17 ). 2.4. Characterization of Boswellia serrata synthesized silver nanoparticles: The synthesis process of boswellia serrata silver nanoparticles was monitored by means of UV / Vis Spectrometer which is used to measure absorption spectra and the wavelength. Furthermore, to characterize the synthesized silver nanoparticles several characterization methods were employed ( 18 ). FTIR was employed to investigate the herbal synthesized silver nanoparticles, This analytical technique is used for understanding the molecular components and biomolecules involved in the biosynthesis of the silver nanoparticles.Infrared absorption spectra and functional groups present in the biomolecules responsible for the reduction and stabilization of silver ions during the nanoparticle synthesis process was analyzed.Scanning Electron Microscopy and Energy-Dispersive X-ray Spectroscopy was employed for the examination of the nanoparticles morphology and elemental composition, Nuclear Magnetic Resonance (NMR) spectroscopy provided in depth detail on the atomic level interactions and structural features of the herbal synthesized nanoparticles. Finally, Zeta Potential analysis was performed to determine the surface charge and stability of the herbal synthesized silver nanoparticles ( 19 ). 2.5. Gelation: Carbopol 940 was dissolved in a controlled manner by stirring it into 100 mL of distilled water over the course of 1 hour to prevent clumping or aggregation. Subsequently, separate solutions of Thymol, Menthol, and Camphor crystals were individually dissolved in 10 mL of distilled water and stirred for 10 minutes. These solutions were then combined with the carbopol solution. In addition, silver synthesized Boswellia serrata extract was introduced into the carbopol solution, followed by adjusting the pH to 7.4 through 10 minutes of stirring ( 20 ). Finally, the combined extract was left for stirring for another 10 minutes until a clear and homogeneous gel base was obtained. 2.6. Preparation of gel formulation in varied concentration A total of six topical gel formulations were developed using the Boswellia serrata extract and thymol, menthol, and camphor in accordance with the drug formulation manual. These formulations, designated as F1 to F6, employed a gel base consisting of 1.5 percent carbopol 940.Formulation compositions are recorded below in Table I. Table 1 Different formulation of topical gels using B. serrata extracts Ingredients F1 F2 F3 F4 F5 F6 Boswellia serrata 1.5gm 2gm 2gm 1.5gm 2.5gm 2.5gm Thymol 1 gm 1gm 1.5gm 1.5gm 1 gm - Menthol 1 gm 1.5gm 1 gm 1.5gm 1.5gm - Camphor 0.5gm 0.5gm 0.5gm 1gm 1gm - Carbopol 1.5gm 0.5gm 1 gm 1gm 0.5gm 0.5gm 2.7. Quantification the concentrations of active components in the formulated gel: To prepare each herbal formulation, one gram of the sample was placed in a 50 mL volumetric flask. Methanol was added to fill the flask and the mixture was vigorously agitated to dissolve the active constituents in the methanol. The resulting solution was then filtered through the Whatman filter paper ( 21 ). From the filtrate 0.1 mL was extracted using a pipette and diluted with methanol to obtain a final volume of 10 mL. The quantification of the active constituents was performed using spectrophotometric analysis at wavelength of 275 nm and a standard curve was constructed to determine their concentration. 2.8. PH measurement: The pH of the prepared herbal gel formulation was determined using a digitalized pH meter. The glass electrode of the pH meter was fully immersed in the gel system to ensure complete coverage of the electrode ( 22 ).This measurement was performed three times, and the average value of the 3 readings was recorded to ensure accuracy and reliability. 2.9. Appearance and Homogeneity of the Formulated herbal Gel: The homogeneity of the prepared gel was evaluated to ensure uniform distribution of the gel composition. Visual inspection was performed to assess color, transparency, absence of phase separation, clumping texture within the gel. 2.10. Viscosity of the Gel: The viscosity of the gel was determined using a Brookfield viscometer which is the instrument used for measuring the flow properties of fluids and gels. The viscosity measurement was conducted at a temperature of 25°C. The Viscometer was equipped with an appropriate spindle for gel viscosity measurements & a spindle speed of 12 rpm was selected for the rotational speed.This speed was chosen on the basis of the protocol followed for gelFormulated gels was loaded onto the viscometer. The gel samples were placed in the measuring chamber, viscometer was then activated & the spindle rotated at a constant speed of 12 rpm. During the rotational test the viscometer measured the resistance encountered by the spindle as it moved through the gel. This resistance which is called as torque was recorded by the viscometer ( 23 ). Based on the torque measurements the viscometer provided a viscosity reading for the prepared topical gel. 2.11. Spreadability of the Gel: Two sets of glass slides of standard dimensions were captured. On of the transparencies was covered with the herbal gel formulation. The second slide was placed on top of the gel, so that the gel was sandwiched between the two slides along a length of 7.50 cm. When applied or rubbed onto the skin's surface, the optimal topical gel must have a sufficient spreading coefficient. On a glass slide was placed approximately 1 g of the formulation for evaluation. A second glass slide of the same length was then set above it, and a mass of 500 g was placed on it so that the gel would be sandwiched between the two glass slides and spread over a specific distance ( 24 ). The time required for the gel to travel the distance from its position was recorded. 2.12. Anti-inflammatory assay: The assessment of the anti-inflammatory efficacy of formulated gels was conducted using BSA-mediated protein denaturation ( 25 ). The preparation and standardisation of formulated gels were conducted initially. Subsequently, Bovine Serum Albumin (BSA) underwent a denaturation procedure to simulate an inflammatory state. The structural integrity of BSA was evaluated after treatment using spectroscopic techniques. 2.13. Anti-microbial assay: The study assessed in vitro antimicrobial activity by determining minimum inhibitory concentrations (MIC) of prevalent microorganisms. To assess the inhibitory effects, an inhibition assay was performed using 96 well plates, following the methodology described by Liu et al. in 2015. In each well, 50 µL of the sample, 10 µL of bacterial culture suspension, and 100 µL of PBS were added. The plate was then incubated, and the optical density (O.D.) of the plate was measured at 600 nm using an ELISA plate reader the results indicate that all the tested samples, particularly those containing plant extracts in combination with gelling agents, exhibited notable activity against the bacterial strains used in the study. The inhibitory effects were evaluated through a standardized MIC test, which involved incubating the samples with bacterial cultures in 96 well plates and measuring the O.D. at 600 nm ( 26 ). 2.14. In-vivo Analysis- Behavioral test in zebrafish: The study of nociception and pain in zebrafish has primarily been conducted using behavioral analysis test. This test aim to break down complex locomotor patterns into simpler measures of locomotor activity. In zebrafish, behavioral tests have been widely used to assess nociception and pain ( 27 ). To study the effects of pain, low molecular weight substances are typically employed due to their stability and ease of administration. In the case of adult zebrafish, these substances are added to the water or injected at varying doses. Acetic acid is a well-established pain inducing algogen that is commonly administered in zebra fish experiments ( 28 ).In our study eight adult zebrafish referred to as group (A) and group (B) each group consisting of four zebra fish in which two male and two female in each tank were used for nociception assessment. A total of 0.1 mL of acetic acid was injected into both groups of zebrafish. Group (A) Zebrafish was placed in a separate tank, and behavior tests were conducted to evaluate its response to the pain stimulus. On the other hand, group (B) zebrafish was kept in a different tank, and a formulated herbal topical gel was exclusively applied to this fish. Subsequently, behavior analysis was performed to assess the effect of the herbal gel on pain response in zebrafish (B). 3. RESULTS AND DISCUSSION 3.1. UV-Visible spectroscopy analysis: UV-Visible spectral analysis is used to study the formation and completion of silver nanoparticles. The reduction of silver ions was observed by measuring the UV-Visible spectrum of the reaction medium, ranging from 200 to 800nm, using distilled water as a reference. The reduction of pure Ag + ions by Boswellia serrata was monitored after 5 hours using a UV-Vis spectrophotometer (UV-2450, Jasco). This reduction process was indicated by a color change from green to brown, signifying the formation of silver nanoparticles. These nanoparticles exhibit a Plasmon absorption band in the visible region, which is caused by the interaction between the free electrons in the nanoparticles and light waves. Specifically, silver nanoparticles are known to absorb UV-Visible light in the range of 380-420nm. Colloidal silver nanoparticles derived from Boswellia serrata exhibit a characteristic absorption band around 435nm, as observed in their UV-Vis spectra. The particle size decreases with lower AgNO3 concentration, and the silver nanoparticles have spherical shapes with diameters ranging from 22nm to 100nm. The synthesis of silver nanoparticles from plant extract shows an increase in absorbance at 430nm over time. When using Boswellia serrata, the final absorption intensities at 430nm can increase up to 1.5 Ǻ unit. In a study by Shakeel Ahmed et al., the UV-Visible spectrophotometer showed an absorbance peak in the range of 436-446nm for the silver nanoparticles ( 29 ). These nanoparticles also exhibited antibacterial activity against both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) microorganisms. Another study by Sinha Paul et al. reported that silver nanoparticles synthesized from Pithophora oedogonia extract showed an absorbance peak at 408nm, indicating their antimicrobial activity. The pure Boswellia serrata plant extract did not show any absorbance in the UV-Vis spectra range of 400-800nm, but upon exposure to AgNO3 solutions, a maximum absorbance was observed at 435nm due to the formation of nanoparticles. However, the formulation of a gel incorporated with silver nanoparticles and the characterization of these particles in Boswellia serrata are yet to be conducted. 3.2. NMR Spectroscopy analysis: During the NMR analysis of boswellic acids, the 1D-1H signals of the equatorial 17 H-3 and olefin H-12 are observed as multiplets with low resolution. These signals appear at 5.26 and 5.17 ppm, respectively. The multiplet of H-12 is consistent with the dihedral angles calculated using AM1 for H-1 and H-11, which are 48.0° and 68°, respectively. The proton signals from rings B to E tend to accumulate in the region between 2 and approximately 0.8 ppm. At a magnetic field strength of 500 MHz, these signals are generally not well resolved. The observed results also support the stereochemical structure of a possible boat conformation for ring A. The corresponding heat of formation for this conformation is calculated to be 226.1 kcal mol-1 using AM1 (1 kcal = 4.184 kJ). However, this conformation rapidly undergoes a change to the chair conformation, with both H-3 and C-23 in axial positions. The calculated heat of formation for this chair conformation is 235.3 kcal mol-1. In the COSY 45 experiment, the methyl groups 25, 26, 27, and 28 of the 3-acetyl of Boswellic serrata exhibit 4J coupling with their respective axial hydrogen neighbors, namely H-1, H-9, H-7, H-15, and H-16 ( 30 ). 3.3. Functional group analysis using FTIR: FTIR measurements were conducted to determine the biomolecules responsible for capping and stabilizing the synthesized metal nanoparticles. The IR spectrum of Boswellia serrata silver nanoparticles has the following interpretations. Figure 5 shows the FT-IR spectra of the crude Boswellia extract and the Boswellia-mediated nanoparticles. When the crude extract was converted into nano formulation, most of the peaks were retained, indicating that the constituents in the nano formulation are the same as those in the crude extract. In the crude boswellic extract, peaks were observed at 3448 cm-1 corresponding to the hydroxyl group, 2095 cm-1 for C = C & 1703 cm-1 for the carboxyl group. The spectrum of AgNPs reduced by the plant extract exhibited high similarities to the spectrum of the extract, suggesting a well-performed extraction process. Similar peaks were found in Boswellia nanoparticles as well. Comparing the FTIR spectrum of the extracted Boswellia serrata, the frequency range was between 2952 − 2852 cm-1, 1760 − 1460 cm-1 & 870 − 690 cm-1 with the wave numbers of 2957 cm-1, 1468 cm-1 & 732 cm-1 indicates the presence of the alkyl C-H stretch, aromatic C = C bending & aromatic C-H bending.The Aromatic C-H bending, N-H amide bending, C = O amide stretching & C-N stretching were observed at 1350–1470 cm-1, 1590–1650 cm-1, 1630–1695 cm-1, and 1000–1250 cm-1 in the FTIR spectra. Shima Edalat Fard et al. reported the presence of a C-O-C phenolic tensile vibration peak at 1271 cm-1 and tensile vibration peaks of C-O-C and C-O-H alcohols at 1070 cm-1 for evaluating apoptosis induction in the cancer cell line MCF-7 using silver nanoparticles biosynthesized from boswellia ( 31 ). Based on the analysis of FTIR spectra it was determined that the hydroxyl and carboxyl groups play a major role in reducing and stabilizing the silver nanoparticles while the phenolic group acts as a capping agent. The decrease in absorbance frequency observed in the stretching vibration of the carbonyl amide group indicated the binding of silver to this specific group. The presence of hydroxyl, amide & carbonyl groups in the plant extract is likely responsible for the biological reduction of silver ions to silver nanoparticles (AgNPs). 3.4. Morphological analysis Scanning electron microscope (SEM) analysis was conducted to examine the morphology of the synthesized silver nanoparticles (AgNPs) from Boswellia serrata. After the reaction was completed, the nanoparticles were placed on a carbon-coated copper grid, revealing both spherical and rod-shaped particles (Fig. 6 ). The size of the silver nanoparticles ranged approximately from 60 to 84 nm. In the study by Mojgan Goudarzi et al., highly agglomerated nanoparticles formed large aggregates with diameters ranging from 400 nm to 2.5 µm. Another study by Zhang et al. reported spherical AI-AgNPs with a particle size of 33 nm, consistent with the literature. Richa Sood et al. reported the optimized Ocimum Sanctum AgNPs, which exhibited colloidal shapes ranging from 45 nm to 1.5 µm.The elemental composition of the nanoparticles was investigated using energy-dispersive X-ray spectroscopy (EDX). The EDX pattern clearly indicated the crystalline nature of the silver nanoparticles, resulting from the reduction of silver ions using the extract. It showed a prominent peak in the silver region, confirming the formation of Ag nanoparticles. Silver nanocrystals exhibit a characteristic optical absorption peak at approximately 20.00 keV due to their surface plasmonic resonance. Other elemental peaks in the EDX spectrum could possibly be attributed to elements from enzymes or proteins present within the extract. 3.5. Surface charge analysis using Zeta Potential Zeta potential plays a significant role in determining the stability of nanoparticles, including drug-loaded nanoparticles. It is a measure of the electric charge present on the surface of particles dispersed in a liquid medium. The magnitude and polarity of the zeta potential provide information about the electrostatic forces between particles, which can influence their aggregation, dispersion, and stability maintaining stability is crucial to ensure the effectiveness and shelf life of the formulation. When nanoparticles have a higher zeta potential magnitude, either positive or negative, they tend to repel each other due to electrostatic forces. This electrostatic repulsion prevents their aggregation and keeps them dispersed in the medium. On the other hand, low zeta potential values can lead to particle aggregation and instability. For Boswellia serrata nanoparticles, the zeta potential measurement revealed a stability value of around − 39.8mV. This value is close to the previously reported zeta potential of -42.88mV, indicating a consistent stability pattern. A negative zeta potential suggests the presence of an excess of negatively charged species on the nanoparticle surface. In the case of low surface charges, this may be attributed to the Vanderwaals constant, which influences the stability of the nano material. Furthermore, the stability of integrated nanoparticles derived from Boswellia serrata, such as Boswellic acid nanoparticles, was also evaluated. The zeta potential values were found to be -45.1mV and − 50.0mV for integrated nanoparticles, respectively. These negative zeta potential values indicate a higher stability, as the particles possess a stronger repulsive force, hindering aggregation. A study conducted by Mehwish Andleeb et al. reported that the zeta potential value became increasingly negative with higher ethanol concentrations. This observation suggests that changes in the solvent composition can affect the surface charge and, subsequently, the stability of nanoparticles. The negative charge provided by a higher zeta potential is beneficial for material stability and the shelf life of a fabricated gel formulation. It helps prevent particle aggregation, maintaining a dispersed state. Moreover, negative zeta potential has been associated with enhanced permeation of nanoparticles, facilitating their penetration through barriers. Figure 9 displays the zeta potential values of different formulations and the negative zeta potential values observed indicate a stable gel formulation. This stability is crucial for ensuring the efficacy, bioavailability, and long-term storage of the gel. 3.6. Physical evaluation of the formulated Gel: The prepared gel was subjected to appearance, homogeneity, pH and viscosity and results are shown in Table 2 . All the gel formulations are homogeneous in nature without any gritty particles appearing from dark purple in F1, yellow color in F2 and F3, white color in F4 and F5 and transparent in F6. All these formulations have shown optimum viscosity. Table 2 – Color, pH, homogeneity and viscosity of the formulated AgNp-Boswellia gels. SI.NO PARAMETERS F1 F2 F3 F4 F5 F6 1. Color Dark purple Yellow Yellow White White Transparent 2. Homogeneity Smooth Smooth Smooth Smooth Smooth Smooth 3. pH 6.74 6.59 6.03 6.61 6.92 6.97 4. Net content (%) 99 98 99 97 96 98 5. Viscosity 0.387 0.375 0.382 0.367 0.395 0.386 The pH measurement of the gel was conducted using a digital pH meter. To ensure accurate readings, the glass electrode of the pH meter was completely immersed in the gel system, ensuring that the electrode was fully covered. This helps to minimize any potential interference or inaccuracies in the pH measurement. To ensure reliability and consistency the pH measurement was carried out in triplicate. This helps to account for any errors that can occur during the measurement process. The readings obtained from each measurement were then averaged to obtain a representative pH value for each formulation. When analyzing the different formulations, ranging from F1 to F6, a range of pH values was observed, ranging from 6.74 to 6.97.These values indicate the acidity or alkalinity of the gel formulations. The decrease in pH was observed in some formulations because of an increase in the Carbopol content within the gel. Therefore, the pH of the gel formulation needs to be within a suitable range to ensure it is compatible with the skin. pH variations in the gel formulations indicate differences in their surface reactivity and potential impact on the skin's pH balance. By closely monitoring and adjusting the pH of the gel formulation, it is possible to optimize its compatibility with the skin and enhance its effectiveness as a topical product. 3.7. Anti-inflammatory analysis: The anti-inflammatory activity of Boswellia extracts was demonstrated in BSA mediated protein denaturation. Previous research has already indicated that B. serrata extracts can induce inflammatory responses in various immune cells such as human and mouse macrophages, monocytes, and PBMCs upon exposure to LPS. However, in this particular study, the results highlighted the protective effect of B. serrata extracts against protein denaturation. Notably, the plant extract exhibited the highest efficacy in restoring and preserving protein structure. These findings suggest that the anti-inflammatory activity of Boswellia extracts is not solely reliant on the most extensively studied components ( 32 ). Other triterpenes, including incensole, have been recognized for their remarkable pharmacological properties in frankincense. Additionally, the gum resin contains polysaccharides, albeit in smaller quantities compared to other plant extracts. The variation in concentration could be attributed to the extraction medium used, which may influence the extraction of different components. Furthermore, the water-soluble fractions extracted from B. serrata, which contain galactose, arabinose, and D-glucuronic acid, were proposed to potentially enhance both humoral and cell-mediated immune responses ( 33 ). 3.8. Anti-bacterial analysis : The MIC analysis was conducted on topical gels formulated with different compositions, and the results demonstrated potent activity against oral pathogens including E. coli, S. aureus, C. albicans, S. mutans, and E. faecalis. The topical gels, which contained plant extracts in combination with other gelling agents, exhibited improved efficacy against all the tested strains. Interestingly, even the gel with a very low concentration of plant extract showed activity against all the bacterial strains employed in the study. To assess the inhibitory effects, an inhibition assay was performed using 96 well plates, following the methodology described by Liu et al. in 2015. In each well, 50 µL of the sample, 10 µL of bacterial culture suspension, and 100 µL of PBS were added. The plate was then incubated, and the optical density (O.D.) of the plate was measured at 600 nm using an ELISA plate reader the results indicate that all the tested samples, particularly those containing plant extracts in combination with gelling agents, exhibited notable activity against the bacterial strains used in the study. The inhibitory effects were evaluated through a standardized MIC test, which involved incubating the samples with bacterial cultures in 96 well plates and measuring the O.D. at 600 nm ( 34 ). 3.9. In-Vivo Analysis: Locomotion assay was done by evaluating the reflexive behavior of swimming activity can be evaluated in adult zebrafish using various parameters such as swimming velocity, distance traveled etc. This assay is used for distinguishing between emotional states specifically related to pain and other related reactions including stress, fear and anxiety. By utilizing objective measurements of swimming behavior, we can differentiate and analyze the distinct emotional responses exhibited by the zebrafish, thereby gaining valuable insights into their pain-related states. Increased erratic swimming and tail beating was seen initially in both group (A) & (B). After application of the formulated herbal topical gel, behavior tests were done the zebra fish Group (B) showing good response comparing Group (A) confirmed that formulated gel have a good effect in zebrafish. In a study Li F et al injected formalin into the lips of zebrafish to induce pain responses, locomotor activity was monitored which showed increased activity and face-rubbing behavior which was attenuated by analgesic compounds ( 35 ). In a study Yasmin et al developed a behavioral assay for assessing acute & chronic pain responses in zebrafish. He observed distinct locomotor responses to different intensities of thermal stimuli, indicating a dose-dependent response in zebra fish ( 36 ). 4. CONCLUSION Metallic nanoparticles particularly silver nanoparticles have gained significant acceptance and utility in the field of biomedicine. In this study the anti-inflammatory potential of a gel formulation containing silver nanoparticles synthesized using boswellia gum resin extract and thymol, menthol and camphor extract was investigated. The formulated gels exhibited homogeneity and stability.The study demonstrated that Formulations 2 and 6 exerted a robust rubefacient effect, showcasing their potential in the treatment of facial pain.In vivo analysis was conducted in zebrafish, formulation 6 topical herbal gel was applied in group (B) Zebra fishes which showed better response comparing the group (A) demonstrating the formulated topical herbal gel has good analgesic properties which can be further used in Atypical facial pain. Declarations Data Availability The data presented in the study are available on request from the corresponding author. Conflicts of Interest The authors of this study declare no conflict of interest. Ethical Approval: The study has been ethically approved by Saveetha dental college, Saveetha Institute of Medical and Technical Sciences ethical committee, BRULAC/SDC/SIMATS/IAET/12-2023/09. Funding: The project is supported by the research grant from TMJ Foundation, TMJ Consultancy Services, Bhopal (Madhya Pradesh), India and DARSN Academy for Maxillofacial Education and Research, DAMER, India. Author Contribution Study concepts: Dr. Ramya Ramadoss, Dr. Ramya SureshStudy design: Dr. Ramya Ramadoss, Dr. Ramya SureshData acquisition: Dr. Ramya Ramadoss, Dr. Ramya Suresh, Dr. Bargavi PQuality control of data and algorithms: Dr. Ramya Ramadoss, Dr. Ramya Suresh, Dr. Bargavi PData analysis and interpretation: Dr. Ramya Ramadoss, Dr. Bargavi P, Dr.Meenakshi SundaramManuscript preparation: Dr. Ramya Ramadoss, Dr. Ramya Suresh Manuscript editing: Dr. Ramya Ramadoss, Dr. Ramya Suresh, Dr. Bargavi P Manuscript review: Dr. Ramya Ramadoss, Dr. Radha G, Dr.Meenakshi Sundaram References Elbarbary, M., Oren, A., Goldberg, M., Freeman, B. V., Mock, D., Tenenbaum, H. C., & Azarpazhooh, A. (2022). Masticatory Myofascial Pain Syndrome: Implications for Endodontists. Journal Of Endodontics , 48 (1), 55–69. 10.1016/j.joen.2021.10.004 Epub 2021 Oct 25. PMID: 34710470. Macrì, M., Rotelli, C., Pegreffi, F., & Festa, F. (2023). Non-Pharmacological Pain Treatment of Patients with Myofascial Pain Syndrome of the Masticatory Muscles—Case Series. Biomedicines , 11 (10), 2799. https://doi.org/10.3390/biomedicines11102799 Chan, N. H. Y., Ip, C. K., Li, D. T. S., & Leung, Y. Y. (2022). Diagnosis and Treatment of Myogenous Temporomandibular Disorders: A Clinical Update. Diagnostics (Basel Switzerland) , 12 (12), 2914. https://doi.org/10.3390/diagnostics12122914 Ahmed, J., Nath, M., Sujir, N., Shenoy, N., Ongole, R., & Binnal, A. (2021). Effect of Myogenous Temporomandibular Joint Disorders on Cervical Range of Motion: A Prospective Study. Journal of Orofacial Sciences 13(1):p 33–38, Jan–Jun. | 10.4103/jofs.jofs_158_19 Kalamir, A., Graham, P. L., Vitiello, A. L., et al. (2013). Intra-oral myofascial therapy versus education and self-care in the treatment of chronic, myogenous temporomandibular disorder: a randomised, clinical trial. Chiropr Man Therap , 21 , 17. https://doi.org/10.1186/2045-709X-21-17 Quek, S. Y. P., Kalladka, V., & Subramanian (2018). Gayathri. A new adjunctive tool to aid in the diagnosis of myogenous temporomandibular disorder pain originating from the masseter and temporalis muscles: Twin-block technique. The Journal of Indian Prosthodontic Society 18(2):p 181–185, Apr–Jun | 10.4103/jips.jips_293_17 Siddiqui, M. Z. (2011). Boswellia serrata, a potential anti-inflammatory agent: an overview. Indian J Pharm Sci. ;73(3):255–61. http://dx.doi.org/10.4103/0250-474X.93507 Xu, L., Wang, Y. Y., Huang, J., et al. (2020). Silver nanoparticles: synthesis, medical applications and biosafety. Theranostics , 10 , 8996–9031. Shaik, M. R., Khan, M., Kuniyil, M., et al. (2018). Plant-extract-assisted green synthesis of silver nanoparticles using Origanum vulgare L. extract and their microbicidal activities. Sustain , 10 , 1–14. https://doi.org/10.3390/su10040913 Shin, M. R., Kim, H. Y., Choi, H. Y., Park, K. S., Choi, H. J., & Roh, S. S. Extract, 5-Loxin®, Prevents Joint Pain and Cartilage Degeneration in a Rat Model of Osteoarthritis through Inhibition of Inflammatory Responses and Restoration of Matrix Homeostasis. Evid Based Complement Alternat Med. Choi, J. H., Hyun, K. Y., & Lee, M. K. (2018). Orofacial Pain Anti-Inflammatory Activity of Extracts of Boswellia Serrata in Rats. Biomed Sci Letters 2018 Sep 30 [;24(3):239–44. https://www.bslonline.org/journal/view.html?doi=10.15616/BSL .24.3.239. Harsha, K., & Shreya, K. K. (2018). Topical Nanoemmigel Formulation of Boswellia serrata. Indian J Pharm Sci .; 80(2). Henrotin, Y., Dierckxsens, Y., Delisse, G., Maes, N., & Albert, A. (2022). Curcuma longa and Boswellia serrata extract combination for hand osteoarthritis: an open-label pre-post trial. Pharmaceutical Biology , 60 (1), 2295–2299. https://doi.org/10.1080/13880209.2022.2147550 Simon, S., Sibuyi, N. R. S., Fadaka, A. O., Meyer, S., Josephs, J., Onani, M. O., Meyer, M., & Madiehe, A. M. (2022). Biomedical Applications of Plant Extract-Synthesized Silver Nanoparticles. Biomedicines , 10 (11), 2792. https://doi.org/10.3390/biomedicines10112792 Aiyalu, R., Govindarjan, A., & Ramasamy, A. (2016). Formulation and evaluation of topical herbal gel for the treatment of arthritis in animal model. Braz J Pharm Sci , 52 (3), 493–507. Shi, Z., Pan, S., Wang, L., & Li, S. (2021). Topical gel-based nanoparticles for the controlled release of oleanolic acid: design and in vivo characterization of a cubic liquid crystalline anti-inflammatory drug. BMC Complementary Medicine and Therapies , 21 (1), 1–13. Chatur, V. M., Walode, S. G., Awate, S. A., Gandhi, M. U., & Thorat, V. S. (2021). Formulation and physical characterization of herbal face gel toner. World Journal of Advanced Research and Reviews , 11 (1), 138–145. Rahman, N., & Varshney, P. (2020). Assessment of ampicillin removal efficiency from aqueous solution by polydopamine/zirconium(iv) iodate: optimization by response surface methodology. RSC Advances , 10 , 20322–20337. https://doi.org/10.1039/d0ra02061c Andleeb, M., Shoaib Khan, H. M., & Daniyal, M. (2021). Development, Characterization and Stability Evaluation of Topical Gel Loaded with Ethosomes Containing Achillea millefolium L. Extract. Frontiers In Pharmacology , 12 , 603227. 10.3389/fphar.2021.603227 PMID: 33912036; PMCID: PMC8074965. Shukla, R., & Kashaw, V. (2019 Mar). Development, characterization and evaluation of poly-herbal ointment and Gel formulation containing Nerium Indicum Mill, Artocarpus Heterophyllus Lam, Murraya Koenigii Linn, Punica Granatum Linn. Journal of Drug Delivery and Therapeutics , 15 (2), 64–69. Chellathurai, B. J., Anburose, R., Alyami, M. H., Sellappan, M., Bayan, M. F., Chandrasekaran, B., Chidambaram, K., & Rahamathulla, M. (2023). Development of a Polyherbal Topical Gel for the Treatment of Acne. Gels , 9 (2), 163. 10.3390/gels9020163 PMID: 36826332; PMCID: PMC9956052. Ohnesorge, N., Heinl, C., & Lewejohann, L. (2021). Current Methods to Investigate Nociception and Pain in Zebrafish. Front Neurosci , 15 , 632634. doi.org/10.3389/fnins.2021.632634 Antinociceptive activity of standardized extract (of Bacopa monnieri in different pain models of zebrafish. J Ethnopharmacol 2022). ;282:114546. dx. doi.org/10.1016/j.jep.2021.114546 Bertocchi, M., Isani, G., Medici, F., Andreani, G., Tubon Usca, I., Roncada, P. Anti-Inflammatory Activity of Boswellia serrata Extracts: An In Vitro Study on Porcine Aortic Endothelial Cells. Oxid Med Cell Longev.2018 Jun 25 [cited 2023 Jul 6];2018. from: https://doi.org/10.1155/2018/2504305 Azmi, S. N. H., Al-Jassasi, B. M. H., Al-Sawafi, H. M. S., et al. (2021). Optimization for synthesis of silver nanoparticles through response surface methodology using leaf extract of Boswellia sacra and its application in antimicrobial activity. Environmental Monitoring And Assessment , 193 , 497. https://doi.org/10.1007/s10661-021-09301-w Yousaf, H., Mehmood, A., Ahmad, K. S., & Raffi, M. (2020). Green synthesis of silver nanoparticles and their applications as an alternative antibacterial and antioxidant agents. Materials Science and Engineering C , 112 , 110901. https://doi.org/10.1016/j.msec.2020.110901 Bhinge, S. D., Bhutkar, M. A., Randive, D. S., Wadkar, G. H., Todkar, S. S., Kakade, P., & Kadam, P. (2017). Formulation development and evaluation of antimicrobial polyherbal gel. Annales pharmaceutiques francaises , 75 5 , 349–358. Anti inflammatory activity of Boswellia. In. (2021). Inflammation and Natural Products (pp. 147–159). Academic. http://dx.doi.org/10.1016/B978-0-12-819218-4.00010-9 Zanfirescu, A., Nitulescu, G., Stancov, G., Radulescu, D., Trif, C., Nitulescu, G. M., et al. (2020). Evaluation of Topical Anti-Inflammatory Effects of a Gel Formulation with Plantago Lanceolata, Achillea Millefolium, Aesculus Hippocastanum and Taxodium Distichum. Scientia Pharmaceutica , 88 (2), 26. Jurca, T., Józsa, L., Suciu, R., Pallag, A., Marian, E., Bácskay, I., Mureșan, M., Stan, R. L., Cevei, M., Cioară, F., Vicaș, L., & Fehér, P. (2020). Formulation of Topical Dosage Forms Containing Synthetic and Natural Anti-Inflammatory Agents for the Treatment of Rheumatoid Arthritis. Molecules (Basel Switzerland) , 26 (1), 24. https://doi.org/10.3390/molecules26010024 Malafoglia, V., Bryant, B., Raffaeli, W., Giordano, A., & Bellipanni, G. (2013). The zebrafish as a model for nociception studies. J Cell Physiol. Oct 1 [6];228(10):1956–66. https://onlinelibrary.wiley.com/doi/abs/10.1002/jcp.24379 Li, F., Lin, J., Liu, X., Li, W., Ding, Y., Zhang, Y., et al. (2018). Characterization of the locomotor activities of zebrafish larvae under the influence of various neuroactive drugs . Annals of Translational Medicine. Sison, M., Cawker, J., Buske, C., et al. (2006). Fishing for genes influencing vertebrate behavior: zebrafish making headway. Lab Anim (NY) , 35 , 33–39. 10.1038/laban0506-33 Anderson, K. V., & Ingham, P. W. (2003). The transformation of the model organism: a decade of developmental genetics. Nature Genetics , 33 . 10.1038/ng1105 . Suppl:285 – 93. Xi, Y., Ryan, J., Noble, S., et al. (2010). Impaired dopaminergic neuron development and locomotor function in zebrafish with loss of pink1 function. European Journal Of Neuroscience , 31 , 623–633. 10.1111/j.1460-9568.2010.07091.x Brockerhoff, S. E., Hurley, J. B., Janssen-Bienhold, U., et al. (1995). A behavioral screen for isolating zebrafish mutants with visual system defects. Proc Natl Acad Sci U S A , 92 , 10545–10549. 10.1073/pnas.92.23.10545 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4832399","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":346418144,"identity":"c60c4000-dfb5-4e0f-8d6f-29fa7f5d90ab","order_by":0,"name":"Ramya Suresh","email":"","orcid":"","institution":"Saveetha Dental College and Hospitals, Saveetha University","correspondingAuthor":false,"prefix":"","firstName":"Ramya","middleName":"","lastName":"Suresh","suffix":""},{"id":346418145,"identity":"aeaa01d6-8b5c-4ac2-8409-393a66ce39fb","order_by":1,"name":"Ramya 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14:53:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":605352,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation of different formulation of topical gels using B. serrata extracts.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4832399/v1/4ccb031bd5a5ad746c27d7aa.png"},{"id":63699162,"identity":"03bbfa23-a68d-446b-b1d5-61bc5ef93c46","added_by":"auto","created_at":"2024-08-31 14:45:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41459,"visible":true,"origin":"","legend":"\u003cp\u003eUV spectrometer of the synthesized aqueous leaf extract AgNp-Boswellia\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4832399/v1/64067c8114e779e90b93438e.png"},{"id":63698828,"identity":"1a2bfdd0-cb10-4801-b332-0b6b3fffa74a","added_by":"auto","created_at":"2024-08-31 14:37:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44974,"visible":true,"origin":"","legend":"\u003cp\u003eNMR analysis of the synthesized AgNp-Boswellia\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4832399/v1/bae1d1419fb3a70eaeec91be.png"},{"id":63699383,"identity":"05c5b3f4-8908-478e-9eb3-68fc8b2d2c8b","added_by":"auto","created_at":"2024-08-31 14:53:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":40937,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectrometer of the synthesized AgNp-Boswellia\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4832399/v1/4661a01e3d4526aea6b69e85.png"},{"id":63698832,"identity":"eef73324-b962-4d13-a98d-0f3922fd3dff","added_by":"auto","created_at":"2024-08-31 14:37:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":462381,"visible":true,"origin":"","legend":"\u003cp\u003eSEM/EDS analysis of the synthesized AgNp-Boswellia\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4832399/v1/342db57afcbde05003c523f5.png"},{"id":63699161,"identity":"caa4a496-8173-4d83-b4a1-fa68556f1f2f","added_by":"auto","created_at":"2024-08-31 14:45:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":74135,"visible":true,"origin":"","legend":"\u003cp\u003eZeta Potential of the synthesized AgNp-Boswellia\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4832399/v1/1132bec0ca05327877afa784.png"},{"id":63699164,"identity":"b4ee9909-7366-4759-9ef4-b589709c6417","added_by":"auto","created_at":"2024-08-31 14:45:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":18085,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of inhibition - anti-inflammatory activity of topical gel.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4832399/v1/f2920f10d233ffaa301f8e96.png"},{"id":63698830,"identity":"9d527822-9ff7-4056-a28d-54489b6fb710","added_by":"auto","created_at":"2024-08-31 14:37:39","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":113911,"visible":true,"origin":"","legend":"\u003cp\u003eMinimum inhibitory concentration analysis for topical gel.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4832399/v1/d590810406b2681b0f022517.png"},{"id":63698825,"identity":"a9f67d84-7bc2-4803-af7d-fc90bb37aaf5","added_by":"auto","created_at":"2024-08-31 14:37:39","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":136506,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of locomotor activity of zebrafish in Group A \u0026amp; Group B.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4832399/v1/cfb8a9ecc209a34be2ab7475.png"},{"id":64024599,"identity":"9e39f7af-1a25-4074-905f-c5205af88b4e","added_by":"auto","created_at":"2024-09-05 07:26:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2553140,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4832399/v1/38da1274-0aab-4168-809c-b6089b6fa8e6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Formulation \u0026 Characterization of Phytochemical Based Topical Analgesic gel in Management of Myogenous Temporomandibular Joint Pain.","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eMyogenous temporomandibular disorders (TMDs) are commonly referred to as masticatory myalgia which constitutes a diverse array of conditions characterized by pain and functional impairment arising from both pathological and functional abnormalities within the masticatory muscles (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). These disorders encompass various subtypes, including myofascial pain, myositis, muscle spasm, and muscle contracture, each presenting unique clinical manifestations and etiological factors. Myofascial pain is the most prevalent subtype that manifests as a chronic condition characterized by regional pain associated with tender areas known as trigger points (TrPs) within skeletal muscles, tendons or ligaments. Epidemiological studies indicate that approximately 50% of individuals with chronic head and neck pain experience symptoms attributable to myogenous TMDs. Moreover, these disorders affect the general population with the prevalence rates ranging from 20\u0026ndash;50%, and approximately 6% exhibiting symptoms severe requiring therapeutic intervention (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnderstanding the etiology of myogenous temporomandibular disorders (TMDs) is challenging due to the absence of a specific underlying cause or identifiable pathology. One of the major causes is direct and indirect trauma that leads to muscle soreness and dysfunction. Events like a direct blow to the jaw or extended mouth opening during dental procedures or yawning may also contribute to pain (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Furthermore, repetitive strain activities such as using chewing gum or participating in oral parafunctional habits like teeth clenching and jaw thrusting which places excessive strain on the masticatory muscles that can cause symptoms of discomfort and pain. Postural strain which are characterised by forward head posture or anomalies in cervical and lumbar lordosis can exacerbate the musculoskeletal burden may also major role in causing referred pain to masticatory muscles thus having a complex interplay between biomechanical variables and myogenous temporomandibular disorders (TMDs). Psychosocial stressors have a notable impact on the worsening of symptoms (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eManaging the myogenous TMDs possess significant challenges due to its complex and multifactorial nature. Treatment approaches primarily focus on symptom management and improving the patient's overall quality of life. Pharmacological interventions such as tricyclic antidepressants, muscle relaxants and NSAIDs, such as ibuprofen or piroxicam are suggested for myalgia, which can be used for short-term analgesic anti-inflammatory effects can be effective as a supplement to alleviate pain and overall management (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, it is essential to consider the potential side effects and limitations of this medications. Non pharmacological therapeutic intervention such as exercise, TrP injections, vapocoolant spray and stretch, topical analgesics, transcutaneous electrical nerve stimulation, Ultrasound therapy, biofeedback, posture correction also plays a vital role in the comprehensive management. Physical therapy, acupuncture, cognitive-behavioral therapy (CBT), and relaxation techniques may also be employed to address muscular tension, promote pain relief, and improve coping strategies (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBoswellia serrata is also known as Indian frankincense, a tree native to India and other parts of Asia. Its gum resin has been used in traditional medicine for centuries due to its anti-inflammatory and analgesic properties (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The active compounds found in Boswellia serrata, specifically the boswellic acids, have been extensively studied for their therapeutic potential in various inflammatory conditions. These compounds possess anti-inflammatory properties by inhibiting key enzymes and pro-inflammatory mediators involved in the inflammatory cascade. Furthermore, they exhibit analgesic properties by modulating pain perception and transmission. The use of Boswellia serrata extract in managing myalgia is gaining attention due to its potential to address both the inflammatory and pain components of the condition. By targeting the underlying inflammatory processes and providing analgesic effects, this herbal extract holds promise as a holistic and natural approach to pain management (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearch studies evaluating the efficacy of Boswellia serrata extract in facial pain management are limited but encouraging (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). A study conducted by Sengupta et al. (2010) demonstrated significant pain reduction in patients with myogenous temporomandibular joint disorders, following the administration of Boswellia serrata extract. The findings suggested its potential as an adjunctive therapy in alleviating facial pain symptoms. The mechanisms through which Boswellia serrata extract exerts its therapeutic effects in masticatory myalgia management are not yet fully understood. However, it is believed that its anti-inflammatory actions contribute to reducing tissue inflammation thereby alleviating pain. Additionally, its analgesic properties may involve modulation of pain receptors and neurotransmitters involved in pain perception and transmission (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile the use of Resinous part of Boswellia serrata extract appears promising it is important to acknowledge more studies are needed to establish its effectiveness and safety profile for pain management (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).Furthermore the optimal dosage, treatment duration and potential side effects require further investigation to ensure its appropriate use.The exploration of natural remedies such as Boswellia serrata extract for facial pain management aligns with the growing interest in complementary and alternative medicine approaches. Incorporating herbal extracts into conventional treatment regimens may offer a more holistic and personalized approach to managing facial pain considering the limitations and side effects associated with conventional pharmaceutical interventions. The utilization of Boswellia serrata extract in a topical gel formulation offers several advantages for facial pain management (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Topical application allows for direct delivery of the active compounds to the affected area, facilitating targeted relief.Formulation of herbal gel also provides a convenient and non-invasive mode of administration enhancing patient compliance and minimizing systemic side effects associated with oral medications.Integration of silver nanoparticles to the herbal extract offers several advantages as silver nanoparticles possess unique physical and chemical properties such as a high surface-volume ratio and enhanced reactivity which contribute to their antimicrobial efficacy (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).It has the ability to modulate the inflammatory response reducing inflammation and promoting tissue regeneration. Incorporating silver nanoparticles into a herbal gel formulation offers several advantages. Gel base provides a suitable matrix for the dispersion and stabilization of silver nanoparticles ensuring their uniform distribution. This facilitates consistent and controlled delivery of the nanoparticles to the affected area maximizing their therapeutic potential. Moreover, the gel formulation allows for convenient topical application promoting patient compliance and localized treatment (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, herbal gel formulations were prepared using silver nanoparticle with synthesized boswellia, extracts of menthol, thymol, camphor and 1.5 percent of Carbopol which is the gelling agent, and they were evaluated for physical appearance, net content, primary skin irritation tests and pH (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Obtained Extract was characterized using FTIR, Zeta Potential, Scanning Electron Microscope (SEM), EDS and UV. Stability test for the prepared topical herbal gel formulation was conducted as per the guidelines of ICH. Further, anti-Inflammatory \u0026amp; analgesic activity was also evaluated. The study aimed to formulate topical analgesic gel incorporated containing Boswellia serrata herbal extracts and silver nano particles for the management of Myogenous TMDs.\u003c/p\u003e"},{"header":"2. MATERIALS \u0026 METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials:\u003c/h2\u003e \u003cp\u003eGum Resin of Boswellia serrata extract, Thymol, menthol and camphor were obtained from K Ramaswamy Herbals Limited, Chennai. Carbopol (940) was purchased from Loba Chemie Private Ltd, Chennai.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Extract Preparation:\u003c/h2\u003e \u003cp\u003eThis gum resin is broken into tiny pieces with a wooden mallet and all impurities including pieces of bark are removed manually during this process. Boswellia gum resin was extracted using a solvent extraction which is a widely employed method for extracting non-volatile compounds from plant materials (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In this technique methanol were used to dissolve and extract the gum resin. Firstly, the tiny pieces gum resin was mixed finely with the methanol and was allowed to undergo agitation for a specific period of time. During this time the solvent interacted with the plant material extracting the target compounds into the solution. The solution is then separated from the solid plant material using filtration, the solvent was evaporated \u0026amp; concentrated extract is obtained. Subsequently Silver nano particles were integrated into the extract.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Synthesis of silver nanoparticles:\u003c/h2\u003e \u003cp\u003eA volume of 1 ml from the centrifuged filtrate was transferred into a larger volume of 40 ml aqueous silver nitrate solution with a concentration of 1 mM. Subsequently, the mixture was carefully monitored for any noticeable change in color (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Characterization of Boswellia serrata synthesized silver nanoparticles:\u003c/h2\u003e \u003cp\u003eThe synthesis process of boswellia serrata silver nanoparticles was monitored by means of UV / Vis Spectrometer which is used to measure absorption spectra and the wavelength. Furthermore, to characterize the synthesized silver nanoparticles several characterization methods were employed (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). FTIR was employed to investigate the herbal synthesized silver nanoparticles, This analytical technique is used for understanding the molecular components and biomolecules involved in the biosynthesis of the silver nanoparticles.Infrared absorption spectra and functional groups present in the biomolecules responsible for the reduction and stabilization of silver ions during the nanoparticle synthesis process was analyzed.Scanning Electron Microscopy and Energy-Dispersive X-ray Spectroscopy was employed for the examination of the nanoparticles morphology and elemental composition, Nuclear Magnetic Resonance (NMR) spectroscopy provided in depth detail on the atomic level interactions and structural features of the herbal synthesized nanoparticles. Finally, Zeta Potential analysis was performed to determine the surface charge and stability of the herbal synthesized silver nanoparticles (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Gelation:\u003c/h2\u003e \u003cp\u003eCarbopol 940 was dissolved in a controlled manner by stirring it into 100 mL of distilled water over the course of 1 hour to prevent clumping or aggregation. Subsequently, separate solutions of Thymol, Menthol, and Camphor crystals were individually dissolved in 10 mL of distilled water and stirred for 10 minutes. These solutions were then combined with the carbopol solution. In addition, silver synthesized Boswellia serrata extract was introduced into the carbopol solution, followed by adjusting the pH to 7.4 through 10 minutes of stirring (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Finally, the combined extract was left for stirring for another 10 minutes until a clear and homogeneous gel base was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Preparation of gel formulation in varied concentration\u003c/h2\u003e \u003cp\u003eA total of six topical gel formulations were developed using the Boswellia serrata extract and thymol, menthol, and camphor in accordance with the drug formulation manual. These formulations, designated as F1 to F6, employed a gel base consisting of 1.5 percent carbopol 940.Formulation compositions are recorded below in Table I.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDifferent formulation of topical gels using B. serrata extracts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF6\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBoswellia serrata\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.5gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThymol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMenthol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCamphor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbopol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Quantification the concentrations of active components in the formulated gel:\u003c/h2\u003e \u003cp\u003eTo prepare each herbal formulation, one gram of the sample was placed in a 50 mL volumetric flask. Methanol was added to fill the flask and the mixture was vigorously agitated to dissolve the active constituents in the methanol. The resulting solution was then filtered through the Whatman filter paper (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). From the filtrate 0.1 mL was extracted using a pipette and diluted with methanol to obtain a final volume of 10 mL. The quantification of the active constituents was performed using spectrophotometric analysis at wavelength of 275 nm and a standard curve was constructed to determine their concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. PH measurement:\u003c/h2\u003e \u003cp\u003eThe pH of the prepared herbal gel formulation was determined using a digitalized pH meter. The glass electrode of the pH meter was fully immersed in the gel system to ensure complete coverage of the electrode (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).This measurement was performed three times, and the average value of the 3 readings was recorded to ensure accuracy and reliability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Appearance and Homogeneity of the Formulated herbal Gel:\u003c/h2\u003e \u003cp\u003eThe homogeneity of the prepared gel was evaluated to ensure uniform distribution of the gel composition. Visual inspection was performed to assess color, transparency, absence of phase separation, clumping texture within the gel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Viscosity of the Gel:\u003c/h2\u003e \u003cp\u003eThe viscosity of the gel was determined using a Brookfield viscometer which is the instrument used for measuring the flow properties of fluids and gels. The viscosity measurement was conducted at a temperature of 25\u0026deg;C. The Viscometer was equipped with an appropriate spindle for gel viscosity measurements \u0026amp; a spindle speed of 12 rpm was selected for the rotational speed.This speed was chosen on the basis of the protocol followed for gelFormulated gels was loaded onto the viscometer. The gel samples were placed in the measuring chamber, viscometer was then activated \u0026amp; the spindle rotated at a constant speed of 12 rpm. During the rotational test the viscometer measured the resistance encountered by the spindle as it moved through the gel. This resistance which is called as torque was recorded by the viscometer (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Based on the torque measurements the viscometer provided a viscosity reading for the prepared topical gel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Spreadability of the Gel:\u003c/h2\u003e \u003cp\u003eTwo sets of glass slides of standard dimensions were captured. On of the transparencies was covered with the herbal gel formulation. The second slide was placed on top of the gel, so that the gel was sandwiched between the two slides along a length of 7.50 cm. When applied or rubbed onto the skin's surface, the optimal topical gel must have a sufficient spreading coefficient. On a glass slide was placed approximately 1 g of the formulation for evaluation. A second glass slide of the same length was then set above it, and a mass of 500 g was placed on it so that the gel would be sandwiched between the two glass slides and spread over a specific distance (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The time required for the gel to travel the distance from its position was recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Anti-inflammatory assay:\u003c/h2\u003e \u003cp\u003eThe assessment of the anti-inflammatory efficacy of formulated gels was conducted using BSA-mediated protein denaturation (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The preparation and standardisation of formulated gels were conducted initially. Subsequently, Bovine Serum Albumin (BSA) underwent a denaturation procedure to simulate an inflammatory state. The structural integrity of BSA was evaluated after treatment using spectroscopic techniques.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Anti-microbial assay:\u003c/h2\u003e \u003cp\u003eThe study assessed in vitro antimicrobial activity by determining minimum inhibitory concentrations (MIC) of prevalent microorganisms. To assess the inhibitory effects, an inhibition assay was performed using 96 well plates, following the methodology described by Liu et al. in 2015. In each well, 50 \u0026micro;L of the sample, 10 \u0026micro;L of bacterial culture suspension, and 100 \u0026micro;L of PBS were added. The plate was then incubated, and the optical density (O.D.) of the plate was measured at 600 nm using an ELISA plate reader the results indicate that all the tested samples, particularly those containing plant extracts in combination with gelling agents, exhibited notable activity against the bacterial strains used in the study. The inhibitory effects were evaluated through a standardized MIC test, which involved incubating the samples with bacterial cultures in 96 well plates and measuring the O.D. at 600 nm (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. In-vivo Analysis- Behavioral test in zebrafish:\u003c/h2\u003e \u003cp\u003eThe study of nociception and pain in zebrafish has primarily been conducted using behavioral analysis test. This test aim to break down complex locomotor patterns into simpler measures of locomotor activity. In zebrafish, behavioral tests have been widely used to assess nociception and pain (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). To study the effects of pain, low molecular weight substances are typically employed due to their stability and ease of administration. In the case of adult zebrafish, these substances are added to the water or injected at varying doses. Acetic acid is a well-established pain inducing algogen that is commonly administered in zebra fish experiments (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).In our study eight adult zebrafish referred to as group (A) and group (B) each group consisting of four zebra fish in which two male and two female in each tank were used for nociception assessment. A total of 0.1 mL of acetic acid was injected into both groups of zebrafish. Group (A) Zebrafish was placed in a separate tank, and behavior tests were conducted to evaluate its response to the pain stimulus. On the other hand, group (B) zebrafish was kept in a different tank, and a formulated herbal topical gel was exclusively applied to this fish. Subsequently, behavior analysis was performed to assess the effect of the herbal gel on pain response in zebrafish (B).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. UV-Visible spectroscopy analysis:\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUV-Visible spectral analysis is used to study the formation and completion of silver nanoparticles. The reduction of silver ions was observed by measuring the UV-Visible spectrum of the reaction medium, ranging from 200 to 800nm, using distilled water as a reference. The reduction of pure Ag\u0026thinsp;+\u0026thinsp;ions by Boswellia serrata was monitored after 5 hours using a UV-Vis spectrophotometer (UV-2450, Jasco). This reduction process was indicated by a color change from green to brown, signifying the formation of silver nanoparticles. These nanoparticles exhibit a Plasmon absorption band in the visible region, which is caused by the interaction between the free electrons in the nanoparticles and light waves. Specifically, silver nanoparticles are known to absorb UV-Visible light in the range of 380-420nm. Colloidal silver nanoparticles derived from Boswellia serrata exhibit a characteristic absorption band around 435nm, as observed in their UV-Vis spectra. The particle size decreases with lower AgNO3 concentration, and the silver nanoparticles have spherical shapes with diameters ranging from 22nm to 100nm. The synthesis of silver nanoparticles from plant extract shows an increase in absorbance at 430nm over time. When using Boswellia serrata, the final absorption intensities at 430nm can increase up to 1.5 Ǻ unit.\u003c/p\u003e \u003cp\u003eIn a study by Shakeel Ahmed et al., the UV-Visible spectrophotometer showed an absorbance peak in the range of 436-446nm for the silver nanoparticles (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). These nanoparticles also exhibited antibacterial activity against both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) microorganisms. Another study by Sinha Paul et al. reported that silver nanoparticles synthesized from Pithophora oedogonia extract showed an absorbance peak at 408nm, indicating their antimicrobial activity. The pure Boswellia serrata plant extract did not show any absorbance in the UV-Vis spectra range of 400-800nm, but upon exposure to AgNO3 solutions, a maximum absorbance was observed at 435nm due to the formation of nanoparticles. However, the formulation of a gel incorporated with silver nanoparticles and the characterization of these particles in Boswellia serrata are yet to be conducted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. NMR Spectroscopy analysis:\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring the NMR analysis of boswellic acids, the 1D-1H signals of the equatorial 17 H-3 and olefin H-12 are observed as multiplets with low resolution. These signals appear at 5.26 and 5.17 ppm, respectively. The multiplet of H-12 is consistent with the dihedral angles calculated using AM1 for H-1 and H-11, which are 48.0\u0026deg; and 68\u0026deg;, respectively. The proton signals from rings B to E tend to accumulate in the region between 2 and approximately 0.8 ppm. At a magnetic field strength of 500 MHz, these signals are generally not well resolved. The observed results also support the stereochemical structure of a possible boat conformation for ring A. The corresponding heat of formation for this conformation is calculated to be 226.1 kcal mol-1 using AM1 (1 kcal\u0026thinsp;=\u0026thinsp;4.184 kJ). However, this conformation rapidly undergoes a change to the chair conformation, with both H-3 and C-23 in axial positions. The calculated heat of formation for this chair conformation is 235.3 kcal mol-1. In the COSY 45 experiment, the methyl groups 25, 26, 27, and 28 of the 3-acetyl of Boswellic serrata exhibit 4J coupling with their respective axial hydrogen neighbors, namely H-1, H-9, H-7, H-15, and H-16 (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Functional group analysis using FTIR:\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFTIR measurements were conducted to determine the biomolecules responsible for capping and stabilizing the synthesized metal nanoparticles. The IR spectrum of Boswellia serrata silver nanoparticles has the following interpretations. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the FT-IR spectra of the crude Boswellia extract and the Boswellia-mediated nanoparticles. When the crude extract was converted into nano formulation, most of the peaks were retained, indicating that the constituents in the nano formulation are the same as those in the crude extract. In the crude boswellic extract, peaks were observed at 3448 cm-1 corresponding to the hydroxyl group, 2095 cm-1 for C\u0026thinsp;=\u0026thinsp;C \u0026amp; 1703 cm-1 for the carboxyl group. The spectrum of AgNPs reduced by the plant extract exhibited high similarities to the spectrum of the extract, suggesting a well-performed extraction process. Similar peaks were found in Boswellia nanoparticles as well. Comparing the FTIR spectrum of the extracted Boswellia serrata, the frequency range was between 2952\u0026thinsp;\u0026minus;\u0026thinsp;2852 cm-1, 1760\u0026thinsp;\u0026minus;\u0026thinsp;1460 cm-1 \u0026amp; 870\u0026thinsp;\u0026minus;\u0026thinsp;690 cm-1 with the wave numbers of 2957 cm-1, 1468 cm-1 \u0026amp; 732 cm-1 indicates the presence of the alkyl C-H stretch, aromatic C\u0026thinsp;=\u0026thinsp;C bending \u0026amp; aromatic C-H bending.The Aromatic C-H bending, N-H amide bending, C\u0026thinsp;=\u0026thinsp;O amide stretching \u0026amp; C-N stretching were observed at 1350\u0026ndash;1470 cm-1, 1590\u0026ndash;1650 cm-1, 1630\u0026ndash;1695 cm-1, and 1000\u0026ndash;1250 cm-1 in the FTIR spectra. Shima Edalat Fard et al. reported the presence of a C-O-C phenolic tensile vibration peak at 1271 cm-1 and tensile vibration peaks of C-O-C and C-O-H alcohols at 1070 cm-1 for evaluating apoptosis induction in the cancer cell line MCF-7 using silver nanoparticles biosynthesized from boswellia (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on the analysis of FTIR spectra it was determined that the hydroxyl and carboxyl groups play a major role in reducing and stabilizing the silver nanoparticles while the phenolic group acts as a capping agent. The decrease in absorbance frequency observed in the stretching vibration of the carbonyl amide group indicated the binding of silver to this specific group. The presence of hydroxyl, amide \u0026amp; carbonyl groups in the plant extract is likely responsible for the biological reduction of silver ions to silver nanoparticles (AgNPs).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Morphological analysis\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eScanning electron microscope (SEM) analysis was conducted to examine the morphology of the synthesized silver nanoparticles (AgNPs) from Boswellia serrata. After the reaction was completed, the nanoparticles were placed on a carbon-coated copper grid, revealing both spherical and rod-shaped particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The size of the silver nanoparticles ranged approximately from 60 to 84 nm. In the study by Mojgan Goudarzi et al., highly agglomerated nanoparticles formed large aggregates with diameters ranging from 400 nm to 2.5 \u0026micro;m. Another study by Zhang et al. reported spherical AI-AgNPs with a particle size of 33 nm, consistent with the literature. Richa Sood et al. reported the optimized Ocimum Sanctum AgNPs, which exhibited colloidal shapes ranging from 45 nm to 1.5 \u0026micro;m.The elemental composition of the nanoparticles was investigated using energy-dispersive X-ray spectroscopy (EDX). The EDX pattern clearly indicated the crystalline nature of the silver nanoparticles, resulting from the reduction of silver ions using the extract. It showed a prominent peak in the silver region, confirming the formation of Ag nanoparticles. Silver nanocrystals exhibit a characteristic optical absorption peak at approximately 20.00 keV due to their surface plasmonic resonance. Other elemental peaks in the EDX spectrum could possibly be attributed to elements from enzymes or proteins present within the extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Surface charge analysis using Zeta Potential\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eZeta potential plays a significant role in determining the stability of nanoparticles, including drug-loaded nanoparticles. It is a measure of the electric charge present on the surface of particles dispersed in a liquid medium. The magnitude and polarity of the zeta potential provide information about the electrostatic forces between particles, which can influence their aggregation, dispersion, and stability maintaining stability is crucial to ensure the effectiveness and shelf life of the formulation. When nanoparticles have a higher zeta potential magnitude, either positive or negative, they tend to repel each other due to electrostatic forces. This electrostatic repulsion prevents their aggregation and keeps them dispersed in the medium. On the other hand, low zeta potential values can lead to particle aggregation and instability.\u003c/p\u003e \u003cp\u003eFor Boswellia serrata nanoparticles, the zeta potential measurement revealed a stability value of around \u0026minus;\u0026thinsp;39.8mV. This value is close to the previously reported zeta potential of -42.88mV, indicating a consistent stability pattern. A negative zeta potential suggests the presence of an excess of negatively charged species on the nanoparticle surface. In the case of low surface charges, this may be attributed to the Vanderwaals constant, which influences the stability of the nano material. Furthermore, the stability of integrated nanoparticles derived from Boswellia serrata, such as Boswellic acid nanoparticles, was also evaluated. The zeta potential values were found to be -45.1mV and \u0026minus;\u0026thinsp;50.0mV for integrated nanoparticles, respectively. These negative zeta potential values indicate a higher stability, as the particles possess a stronger repulsive force, hindering aggregation. A study conducted by Mehwish Andleeb et al. reported that the zeta potential value became increasingly negative with higher ethanol concentrations. This observation suggests that changes in the solvent composition can affect the surface charge and, subsequently, the stability of nanoparticles. The negative charge provided by a higher zeta potential is beneficial for material stability and the shelf life of a fabricated gel formulation. It helps prevent particle aggregation, maintaining a dispersed state. Moreover, negative zeta potential has been associated with enhanced permeation of nanoparticles, facilitating their penetration through barriers. Figure\u0026nbsp;9 displays the zeta potential values of different formulations and the negative zeta potential values observed indicate a stable gel formulation. This stability is crucial for ensuring the efficacy, bioavailability, and long-term storage of the gel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Physical evaluation of the formulated Gel:\u003c/h2\u003e \u003cp\u003eThe prepared gel was subjected to appearance, homogeneity, pH and viscosity and results are shown in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. All the gel formulations are homogeneous in nature without any gritty particles appearing from dark purple in F1, yellow color in F2 and F3, white color in F4 and F5 and transparent in F6. All these formulations have shown optimum viscosity.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026ndash; Color, pH, homogeneity and viscosity of the formulated AgNp-Boswellia gels.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSI.NO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePARAMETERS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eF6\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eColor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDark purple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWhite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHomogeneity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNet content (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eViscosity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.387\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.382\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.395\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.386\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe pH measurement of the gel was conducted using a digital pH meter. To ensure accurate readings, the glass electrode of the pH meter was completely immersed in the gel system, ensuring that the electrode was fully covered. This helps to minimize any potential interference or inaccuracies in the pH measurement. To ensure reliability and consistency the pH measurement was carried out in triplicate. This helps to account for any errors that can occur during the measurement process. The readings obtained from each measurement were then averaged to obtain a representative pH value for each formulation. When analyzing the different formulations, ranging from F1 to F6, a range of pH values was observed, ranging from 6.74 to 6.97.These values indicate the acidity or alkalinity of the gel formulations.\u003c/p\u003e \u003cp\u003eThe decrease in pH was observed in some formulations because of an increase in the Carbopol content within the gel. Therefore, the pH of the gel formulation needs to be within a suitable range to ensure it is compatible with the skin. pH variations in the gel formulations indicate differences in their surface reactivity and potential impact on the skin's pH balance. By closely monitoring and adjusting the pH of the gel formulation, it is possible to optimize its compatibility with the skin and enhance its effectiveness as a topical product.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Anti-inflammatory analysis:\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe anti-inflammatory activity of Boswellia extracts was demonstrated in BSA mediated protein denaturation. Previous research has already indicated that B. serrata extracts can induce inflammatory responses in various immune cells such as human and mouse macrophages, monocytes, and PBMCs upon exposure to LPS. However, in this particular study, the results highlighted the protective effect of B. serrata extracts against protein denaturation. Notably, the plant extract exhibited the highest efficacy in restoring and preserving protein structure. These findings suggest that the anti-inflammatory activity of Boswellia extracts is not solely reliant on the most extensively studied components (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Other triterpenes, including incensole, have been recognized for their remarkable pharmacological properties in frankincense. Additionally, the gum resin contains polysaccharides, albeit in smaller quantities compared to other plant extracts. The variation in concentration could be attributed to the extraction medium used, which may influence the extraction of different components. Furthermore, the water-soluble fractions extracted from B. serrata, which contain galactose, arabinose, and D-glucuronic acid, were proposed to potentially enhance both humoral and cell-mediated immune responses (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.8. Anti-bacterial analysis\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThe MIC analysis was conducted on topical gels formulated with different compositions, and the results demonstrated potent activity against oral pathogens including E. coli, S. aureus, C. albicans, S. mutans, and E. faecalis. The topical gels, which contained plant extracts in combination with other gelling agents, exhibited improved efficacy against all the tested strains. Interestingly, even the gel with a very low concentration of plant extract showed activity against all the bacterial strains employed in the study. To assess the inhibitory effects, an inhibition assay was performed using 96 well plates, following the methodology described by Liu et al. in 2015. In each well, 50 \u0026micro;L of the sample, 10 \u0026micro;L of bacterial culture suspension, and 100 \u0026micro;L of PBS were added. The plate was then incubated, and the optical density (O.D.) of the plate was measured at 600 nm using an ELISA plate reader the results indicate that all the tested samples, particularly those containing plant extracts in combination with gelling agents, exhibited notable activity against the bacterial strains used in the study. The inhibitory effects were evaluated through a standardized MIC test, which involved incubating the samples with bacterial cultures in 96 well plates and measuring the O.D. at 600 nm (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.9. In-Vivo Analysis:\u003c/h2\u003e \u003cp\u003eLocomotion assay was done by evaluating the reflexive behavior of swimming activity can be evaluated in adult zebrafish using various parameters such as swimming velocity, distance traveled etc. This assay is used for distinguishing between emotional states specifically related to pain and other related reactions including stress, fear and anxiety. By utilizing objective measurements of swimming behavior, we can differentiate and analyze the distinct emotional responses exhibited by the zebrafish, thereby gaining valuable insights into their pain-related states. Increased erratic swimming and tail beating was seen initially in both group (A) \u0026amp; (B). After application of the formulated herbal topical gel, behavior tests were done the zebra fish Group (B) showing good response comparing Group (A) confirmed that formulated gel have a good effect in zebrafish. In a study Li F et al injected formalin into the lips of zebrafish to induce pain responses, locomotor activity was monitored which showed increased activity and face-rubbing behavior which was attenuated by analgesic compounds (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In a study Yasmin et al developed a behavioral assay for assessing acute \u0026amp; chronic pain responses in zebrafish. He observed distinct locomotor responses to different intensities of thermal stimuli, indicating a dose-dependent response in zebra fish (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eMetallic nanoparticles particularly silver nanoparticles have gained significant acceptance and utility in the field of biomedicine. In this study the anti-inflammatory potential of a gel formulation containing silver nanoparticles synthesized using boswellia gum resin extract and thymol, menthol and camphor extract was investigated. The formulated gels exhibited homogeneity and stability.The study demonstrated that Formulations 2 and 6 exerted a robust rubefacient effect, showcasing their potential in the treatment of facial pain.In vivo analysis was conducted in zebrafish, formulation 6 topical herbal gel was applied in group (B) Zebra fishes which showed better response comparing the group (A) demonstrating the formulated topical herbal gel has good analgesic properties which can be further used in Atypical facial pain.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in the study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors of this study declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study has been ethically approved by Saveetha dental college,\u0026nbsp;Saveetha Institute of Medical and Technical Sciences\u0026nbsp;ethical committee, BRULAC/SDC/SIMATS/IAET/12-2023/09.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project is supported by the research grant from TMJ Foundation, TMJ Consultancy Services, Bhopal (Madhya Pradesh), India and DARSN Academy for Maxillofacial Education and Research, DAMER, India.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eStudy concepts: Dr. Ramya Ramadoss, Dr. Ramya SureshStudy design: Dr. Ramya Ramadoss, Dr. Ramya SureshData acquisition: Dr. Ramya Ramadoss, Dr. Ramya Suresh, Dr. Bargavi PQuality control of data and algorithms: Dr. Ramya Ramadoss, Dr. Ramya Suresh, Dr. Bargavi PData analysis and interpretation: Dr. Ramya Ramadoss, Dr. Bargavi P, Dr.Meenakshi SundaramManuscript preparation: Dr. Ramya Ramadoss, Dr. Ramya Suresh Manuscript editing: Dr. Ramya Ramadoss, Dr. Ramya Suresh, Dr. Bargavi P Manuscript review: Dr. Ramya Ramadoss, Dr. Radha G, Dr.Meenakshi Sundaram\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eElbarbary, M., Oren, A., Goldberg, M., Freeman, B. 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A behavioral screen for isolating zebrafish mutants with visual system defects. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e, \u003cem\u003e92\u003c/em\u003e, 10545\u0026ndash;10549. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.92.23.10545\u003c/span\u003e\u003cspan address=\"10.1073/pnas.92.23.10545\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Masticatory myalgia, Orofacial Pain, herbal gel, Boswellia, topical, anti-inflammatory, analgesic.","lastPublishedDoi":"10.21203/rs.3.rs-4832399/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4832399/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMyogenous Temporomandibular joint disorder is characterised by pain \u0026amp; dysfunction in the masticatory muscles that are originated from pathologic and functional processes in the masticatory muscles. Most common symptoms are muscle discomfort, restricted range of motion, fatigue, stiffness, and subjective weakness. Boswellia serrata gum resin extracts have been widely employed years of years to address a wide range of chronic inflammatory conditions. These conditions include rheumatoid arthritis, osteoarthritis, asthma, inflammatory bowel disease, and other inflammatory disorders. Silver nanoparticles have a great potential for their mechanistic role. Green synthesized silver nanoparticles (AgNPs) have promising biomedical applications in healthcare. These nanoparticles are synthesized using plant-based compounds that act as moderators, resulting in AgNPs with high therapeutic potential. They offer an alternative approach to medicine utilizing the bioactive compounds of plants. The incorporation of these compounds enhances the biomedical properties of AgNPs, making them valuable for various therapeutic applications. Resinous component of Boswellia Serrata were used as a key ingredient and thymol, menthol camphor was added for topical gel formulation as it has analgesic properties which can be used for management of masticatory myalgia.\u003c/p\u003e","manuscriptTitle":"Formulation \u0026amp; Characterization of Phytochemical Based Topical Analgesic gel in Management of Myogenous Temporomandibular Joint Pain.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-31 14:37:34","doi":"10.21203/rs.3.rs-4832399/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09d2baf2-329c-426f-a254-3983efb90f13","owner":[],"postedDate":"August 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-05T07:18:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-31 14:37:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4832399","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4832399","identity":"rs-4832399","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}