Int J App Pharm, Vol 14, Issue 3, 2022, 110-116Original Article

OPTIMIZATION OF DIMETHYL SULFOXIDE AS AN ENHANCER ON EX VIVO PENETRATION OF SESEWANUA (CLERODENDRUM FRAGRANS WILD) LEAF EXTRACTS EMULGEL

PAULUS PANGALO1, ZULFIAYU SAPIUN2* , ARLAN K. IMRAN2, MUINDAR MUINDAR 3, SABARUDDIN SABARUDDIN 3, PRISCA SAFRIANI WICITA2, YSRAFIL, YSRAFIL4 , FIHRINA MOHAMAD2

1Department of Nursing, Gorontalo Polytechnic of Health of Ministry of Health, Indonesia, 2Department of Pharmacy, Gorontalo Polytechnic of Health of Ministry of Health, Indonesia, 3Laboratory of Traditional Drug and Cosmetics, National Agency of Drug and Food Control in Gorontalo, Indonesia, 4Faculty of Medicine, Universitas Palangka Raya, Palangka Raya, Indonesia
Email: zulfiayu@poltekkesgorontalo.ac.id

Received: 20 Dec 2021, Revised and Accepted: 12 Feb 2022

ABSTRACT

Objective: The present study aims to investigate the effect of using DMSO as an enhancer on the ex vivo penetration of an emulgel from sesewanua leaf extracts.

Methods: The sesewanua emulgel was prepared into four formulas, SWE1-SWE4, with different DMSO concentrations: 0%, 3%, 5%, 7%, and compared with QCE, quercetin without DMSO. Further, the penetration test of the sesewanua leaf ethanol extract emulgel was performed by determining the rate and cumulative penetration of quercetin within the skin of the mice by employing the Franz diffusion cell approach.

Results: The SWE4 emulgel containing 7% of DMSO was the best formula that enhanced the penetration of the emulgel, with a cumulative penetration at 331.423 µg/cm2, the penetration rate at 2.762 µg/cm2/minute, and better dispersibility of 4.6 cm and the results of the one-way ANOVA suggested a significant influence (p<0.05). DMSO with a concentration of 7% of the sesewanua emulgel was proven to increase 2,4-fold the rate and cumulative amount penetration. DMSO can be considered as a penetration enhancer of natural compounds for anti-inflammatory treatment.

Conclusion: The use of natural ingredients as topical anti-inflammatory continues to be developed to avoid the first-pass effects. The used of DMSO in topical emulgel preparation become a simple way to enhance the penetrant of active inggredient.

Keywords: Quercetin, Antiinflammation, DMSO, Franz cell diffusion, Flavonoid

© 2022 The Authors.Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2022v14i3.43938. Journal homepage: https://innovareacademics.in/journals/index.php/ijap

INTRODUCTION

Recent advances in science and technology have significantly altered the way we discover, treat, and prevent specific diseases in all aspects of human life [1]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly prescribed medications in the world, but it has a number of side effects, primarily on the gastrointestinal tract, such as ulcers, bleeding, perforation, and obstruction [2]. It is a great need to develop secure and valid drugs for long-term use from natural sources for the establishment of new treatments for clinical use [3].

Sesewanua (Clerodendrum fragrans Wild) been used for the material in bakera spa (a traditional spa of people in North Sulawesi) and as an antipyretic [4–8]. It contains 13.47% of quercetin, a subclass of flavonoid compound, which has anti-inflammatory activities [9] and has moderate anti-cancer potential [10]. The above finding has underpinned the urge to formulate ethanol extracts of sesewanua leaves in the form of topical emulgel.

Emulgel is defined as a multi-drug delivery system comprising emulsion and gel. Several properties of emulgel are thixotropic, greaseless, easily spreadable, moisturizing, long shelf-life, transparent, considerably stuck longer, and easy to wash [11]. Emulgel has been successful in delivering active ingredients to their target, such as clove oil [12], diclofenac sodium [13], methotrexate [14], itraconazole [15], Mimosa pudica polyphenols [16], benzyl benzoate [17], chlorphenesin [18], Mentha essential oil [19], acyclovir [20], tramadol [21], etc.

The outermost layer of skin (stratum corneum) consists of corneocytes enclosed by the lipid bilayers. This is the main barrier to delivering the drug to the skin. When a drug is poorly water-soluble, it can be limited to the superficial stratum corneum (SC) following topical administration [14, 22–25]. For this reason, a specific enhancer is essential in formulating sesewanua ethanol extract emulgel.

Penetration enhancers are substances that aid in the absorption of penetrants through the skin by temporarily thinning the skin's impermeability. Penetrant enhancer has no therapeutic effects, but it helps drug transportation to the skin [26]. Dimethyl Sulfoxide (DMSO) is the most common enhancer that has been proven effective in enhancing drug penetration that capable of changing the conformation of stratum corneum keratin from α–helical conformation to β–sheet conformation and increasing drug fluxes through its interaction with lipid on the stratum corneum and change the protein structure, thereby resulting in changes in the fluxes’ partition coefficient [27, 28].

In this research, Sesewanua has been used historically as an anti-inflammatory was made in a topical dosage form. Emulgel preparations have been selected using DMSO as a penetrant enhancer. The objective is to investigate the effect of various DMSO concentrations on the ex vivo sesewanua extract emulgel and the physical stability of the emulgel formula with the best penetration qualities in terms of cumulative penetration and penetration rate.

MATERIALS AND METHODS

Materials

The materials encompassed sesewanua leaf simplicia collected from Tilango District, Gorontalo Regency in January 2020, SWR/J mice aged 6 to 8 w weighing at 20 to 30 g, DMSO (CV. Chem-Mix P. A Grade), Veet® (fur remover), ethanol 96%, quercetin (CV. Chem-Mix P. A Grade), Carboxy Methyl Cellulosa (CMC) Sodium (CV. Chem-Mix P. A Grade), Nipagin (CV. Chem-Mix P. A Grade), Nipasol (CV. Chem-Mix P. A Grade), Poly Ethylene Glicol (PEG) 400 (CV. Chem-Mix P. A Grade), metabisulfite sodium (CV. Chem-Mix P. A Grade), tween 80 (CV. Chem-Mix P. A Grade), span 80 (CV. Chem-Mix P. A Grade), and aquadest (Dept. Pharmacy Chemistry Laboratory Polkesgo, Pharmaceutical Grade).

Ethical approval

All research procedures have received ethical approval from the Health Polytechnic Ethics Commission of the Ministry of Health of Gorontalo with No. LB.01.01/KEPK/52/2020

Preparation of sesewanua extract emulgel

Table 1: Emulgel formulation

Materials SWE1 SWE2 SWE3 SWE4 QCE
Oil phase emulsion
Sesewanua extract 5% 5% 5% 5% -
Quercetin - - - - 5%
Olive Oil 10% 10% 10% 10% 10%
Poly Ethylene Glycol 400 25% 25% 25% 25% 25%
Cetyl Alkohol 2% 2% 2% 2% 2%
Span 80 2.028% 2.028% 2.028% 2.028% 2.028%
Nipasol 0.02% 0.02% 0.02% 0.02% 0.02%
Water phase emulsion
DMSO 0% 3% 5% 7% 0%
Tween 80 0.972% 0.972% 0.972% 0.972% 0.972%
Aquadest 14.34% 14.34% 14.34% 14.34% 14.34%
Metabisulfit Sodium 0.1% 0.1% 0.1% 0.1% 0.1%
Gels
CMC Sodium 1% 1% 1% 1% 1%
Nipagin 0.18% 0.18% 0.18% 0.18% 0.18%
Poly Ethylene Glycol 400 25% 25% 25% 25% 25%
Aquadest 14.34% 14.34% 14.34% 14.34% 14.34%

SWE1: Sesewanua Emulgel with 0% DMSO, SWE2: Sesewanua Emulgel with 3% DMSO, SWE3: Sesewanua Emulgel with 5% DMSO, SWE4: Sesewanua Emulgel with 7% DMSO, QCE: Quercetin Emulgel

The concentration of DMSO made refers to previous research [29–31]. The materials for the emulgel formula were grouped into three parts, namely oil-phase emulsion, water-phase emulsion, and gel base. All of them were firstly mixed according to the respective part. Cetyl alcohol melted and mixing with olive oil, PEG 400, span 80, nipasol, and extract at 60 °C. Meanwhile, the water phase (tween 80, Metabisulfite sodium, water, and DMSO) mixed was done by mixing the materials in a beaker using a stirrer. In this phase, DMSO was added according to the concentrations that had been determined. Following the process was the preparation of gel components by heating the water, adding and mixing CMC Sodium until well-hydrated. Moreover, the nipagin and PEG 400 were added to form a gel base. After the formation of the gel base, all three components from the oil phase, water phase, and gel were mixed using a stirrer set at the speed of 2000 RPM, resulting in four emulgels with the DMSO concentration SWE1-SWE4 and emulgels with quercetin QCE.

Analyzing the penetration of sesewanua extract emulgel

Quercetin standard curve

The absorption of quercetin solutions at the concentration of 20 ppm, 40 ppm, 60 ppm, 80 ppm, 120 ppm, 140 ppm, 160 ppm, and 180 ppm was measured at the wavelength (λ) of 415 nm using a UV-Vis spectrophotometer. After that, we the drawing of the calibration curve and its regression equation.

Recovery test

The absorption of the level of quercetin, each at 120 PPM, was identified at the wavelength of 415 nm using a UV-Vis spectrophotometry. The content of the quercetin was measured using the equation of the quercetin standard curve.

Preparing the mice skin

Mice were purchased from Rizky Agency in Gorontalo Province, Indonesia. Mice were housed in cages, in controlled temperature (20-22 °C) and room under a 12 h day/night cycle. The animals were used in the study after acclimatizing for a week to allow free access to water and food. The mice were euthanatized before carefully removing their fur. The abdomen skin was cut to the size of the diameter 0.03 cm and membrane area 0.002826 cm2 and then soaked in phosphate buffer pH 7.4 for 30 min.

Diffusion test

The penetration test was performed utilizing the Franz diffusion cell [32–35]. The receptor compartment was filled with phosphate buffer 7.4 pH, with a temperature kept at 37±1 °C, and stirred at a constant speed. Further, the mice's skin, with the stratum corneum part facing upward, was placed between the donor and receptor compartment. The 1g sample was then applied to the skin. After every 0, 30, 60, 90, and 120 min, 3 ml of sample was removed from the receptor compartment using a syringe and then replaced with the 7.4 pH phosphate buffer in the same volume. The absorbance of each sample was measured on the wavelength of 415 nm using a UV-Vis spectrophotometer.

Preparation evaluation

Homogeneity test

SWE1-SWE4 and QCE were weighed as much as 0.1 grams and smeared evenly on the slide, no coarse grains should be seen.

Spreadability test

SWE1-SWE4 and QCE were weighed as much as 0.5 grams, placed on a transparent glass lined with graph paper covered with transparent glass, and given a load of 150 g. The diameter of the distribution was measured 4 times.

Stability test

SWE1-SWE4 and QCE were placed in a glass container, placed alternately at 4 °C and 40 °C for 24 h for 7 cycles. Emulgel stability was observed.

Data analysis

The correlation between each sesewanua extract emulgel and the cumulative penetration was analyzed using the one-way ANOVA with SPSS 16.

RESULTS

Recovery test

The accuracy test using a percentage recovery method was aimed at determining the accuracy of the method, i.e. whether or not the method, in determining the level, was capable of depicting the actual level of the substance of the quercetin within the emulgel. Provided in table 2 are the values of the percentage of recovery method.

Table 2: Quarcetine standard absorbance data

Concentration (µg/ml) Absorbance (A) Deviation standard Equation of line

60

80

100

120

140

160

180

0.240

0.346

0.415

0,470

0.565

0.657

0.786

0.0006

0.0012

0.0010

0.0010

0.0006

0.0010

0.0015

r = 0,9878

a = 0,0196

b = 0,0043

y = 0,0043x-0,0196

Fig. 1: Graph of quercetin's standard curve, data represents mean±SD (n=3)

Table 3: Percentage value of recovery

Teoritic value (µg/ml) Replication A µg/ml % Recovery Mean of % recovery
120 1 1 0.490 118.51 98.7583 99.5990±0.995
2 0.493 119.21 99.3411
3 0.500 120.84 100.6976
2 1 0.494 119.44 99.5348 99.2118±0.296
2 0.492 118.97 99.1472
3 0.491 118.74 98.9534
3 1 0.490 118.51 98.7583 98.9529±0.512
2 0.489 118.27 98.5658
3 0.494 119.44 99.5348
4 1 0.487 117.81 98.1782 98.7204±0.524
2 0.496 119.91 99.2248
3 0.490 118.51 98.7583
5 1 0.490 118.51 98.7583 99.0175±0.623
2 0.495 119.67 99.7286
3 0.489 118.27 98.5658

n=3

Diffusion test

Fig. 2: Cumulative penetration amount of emulgel preparation Sesewanua extract optimization DMSO; Data represents mean, (n=3)

Fig. 3: Total cumulative amount of penetrated sesewanua extract emulgel; Data represents mean±SD, (n=3)

Fig. 4: Flux penetration rate of emulgel, Data represents mean, (n=3)

Fig. 5: Penetration rate of sesewanua extract emulgel, Data represents mean±SD, (n=3)

Table 4: One way ANOVA statistical test cumulative penetration amount and penetration rate emulgel preparation 120 min DMSO optimization

Criteria Formula
SWE1 SWE2 SWE3 SWE4 QCE
Cumulative Amount Penetrated (µg/cm2) 136,99±21,1-* 173,76±57,3-** 59,81±23,6+ 331,42±29-*** 65,71±26,3-
Penetration Rate (µg/cm2/min) 1,142±0,005-* 1,448±0,004-** 0,498±0,003+ 2,76±0,005-*** 0,547±0,006-

Description: -***Very Significantly Different Increase Against QCE,-**Different Moderate Significant Increase Against QCE,-*Significantly Different Increase Against QCE,+No Significant Difference in Its Decrease Against QCE, Data represents mean±SD (n=3)

Evaluation of emulgel

Homogeneity and stability

Table 5: Homogeneity and stability/freezthaw test of sesewanua emulgel

Test SWE1 SWE2 SWE3 SWE4 QCE
Homogeneity Homogenous Homogenous Homogenous Homogenous Homogenous
Stability/Freezthaw No phase separation occurs No phase separation occurs No phase separation occurs No phase separation occurs No phase separation occurs

n=3

Dispersibility test

Table 6 below shows the dispersibility of sesewanua extract emulgel.

Table 6: Dispersibility of sesewanua extract emulgel

Formula Dispersibility (cm)
SWE1 4.025±0.05
SWE2 3.700±0.18
SWE3 3.625±0.05
SWE4 4.600±0.00
QCE 4.15±0.01

Data represents mean±SD, (n=3)

DISCUSSION

DMSO is one of the sulfoxide chemical penetrant enhancers. DMSO was chosen because it is cheap, easy to obtain, and has proven successful in increasing the penetration of the active ingredient [27, 28]. Chemical penetration enhancers are done in one of three ways: by chemical interaction with intracellular proteins, by improving the distribution of drugs in the stratum corneum, or by destroying highly ordered lipids. stratum corneum [36]. There were variations of the DMSO, 3%, 5%, and 7%, because DMSO showed a better action at that concentration [29–31], listed in table 1.

Based on quercetin standard absorbance data (table 2), the recovery test (table 3) and graph of quercetin’s standard curve (fig. 1) result of five replications of the test reported that the percentage recovery met the prerequisite of 120 ppm formulated and recovered quercetin using the raw curve drawn for at least 99%. Such a result is consistent with the study by [37] suggesting that the compound level examined using a percentage recovery at>1% has the range of percentage recovery from 97 to 103%. The notion signifies that the method is accurate in determining quercetin's content in the penetration of sesewanua extract emulgel.

The diffusion test parameters in the present work consisted of the cumulative compound penetration (Q) and fluxes (J). The amount of cumulative penetration for SWE1, SWE2, SWE3, and SWE4 are displayed in fig. 2 and 3. According to the curve, we can see the concentration of penetrant enhancer DMSO in the sesewanua extract emulgel formula, compared to SWE1, contributes to an increase in the quercetin of the emulgel passed through the membrane. Jatav et al., [38] show that such an increase in DMSO can lead to a rise in the cumulative penetration amount of active, hydrophilic substances. DMSO is capable of speeding up drug penetration, either hydrophilic or lipophilic, by its mechanisms of extracting the fat layer through the transcellular pathway. The process results in the replacement of the bonding of water through the intracellular pathway. Consequently, the quercetin possessing polar properties can pass through the skin layer, precisely the thick stratum corneum, through two different pathways [39, 40].

A diffusion test result showing a contrasting result is seen in SWE3, where SWE3 experiencing a decline in its cumulative penetration amount is due to the consistency of the sesewanua extract. In formula SWE3, the extract had a different thickness than those used in other formulas, i.e., SWE1, SWE2, and SWE4. Anita Sukmawati and Suprapto [41] opine that increased viscosities may result in a drop in transdermal drugs' total cumulative chemical penetration. This condition is the cause of the drop in the rate and cumulative penetration in SWE3 after 120 min. In the present study, the most significant increase in the cumulative of sesewanua extract emulgel penetration is seen in SWE4 with DMSO 7%. Simply put, the higher the DMSO concentration is, the greater the cumulative amount of emulgel penetrated. DMSO has successfully acted as a penetrant enhancer with a concentration of 7%. It was proven to increase 2,4-fold the rate and cumulative amount penetration of the ethanolic extract of Sesewanua leaves in the emulgel preparation. This is similar to the research of Damayanti in that DMSO increased the theophylline transport from 17,9 µg to 139,1 µg [31]. But this finding is not similar to Jantharaprapap, that found DMSO did not improve the permeability of meloxicam emulgel [30] and Bhutkar found that found Aloe vera gives a better permeability than DMSO in lidocaine emulgel [29].

Other results indicated that the cumulative amount of sesewanua emulgel penetration in SWE1 without DMSO was greater than QCE, with the pure quercetin material functioning as the active substance material (and later serving as the target in determining the cumulative penetration). This situation occurs because the quercetin contained in the extract had been mixed with other compounds with oil properties, thus causing a longer quercetin contact in the extract; this is in line with the physical and chemical properties of skin that consists of several fat layers [27]. Such a notion corresponds to the results seen in Ramadon’s study [42] that compare the cumulative of polar and non-polar carriers penetration. As a result, the non-polar carriers have greater cumulative penetration than the positive polar.

Emulsion-based systems containing sesewanua extract do not adhere well to the skin due to their liquid state and have poor, short-term effects. Therefore, to solve the problem and improve retention, an emulsion was formulated into an emulsion gel formulation, same as curcumin [43]. The results of the one-way ANOVA revealed that the cumulative amount in minute 120 of the emulgel formula SWE1, SWE2, and SWE4 increased significantly compared to QCE. In other words, the emulgel and the other emulgel with enhancer DMSO can further improve the total cumulative penetration of the sesewanua emulgel extract. Different results were shown in the result of the SWE3 test, where it had positive values, contrasting with QCE with its negative values (table 4). Such a result implies that the decline in the cumulative amount in SWE3 is insignificant statistically.

A rise in the cumulative amount penetrated is proportional to the flux value (penetration rate), thereby increasing the flux value of the emulgel with DMSO and allowing the emulgel to pass through the skin layer. The increase in the flux is displayed in fig. 4 and 5.

In the evaluation of emulgel, the dispersibility indicates that all emulgels have met the requirement of good dispersibility, as displayed in table 6. Such a result resonates with the one found by [44], claiming that emulgel with good dispersibility is the one with dispersibility from 5 to 7 cm. Referring to the data, we can see that SWE4 emulgel with DMSO at 7% has the widest dispersion, thus increasing the cumulative total of the penetration of sesewanua extract to the skin.

CONCLUSION

The use of natural ingredients as topical anti-inflammatory continues to be developed to avoid the effects of the fist pass. The use of DMSO in topical emulgel preparation has become a simple way to enhance the penetrant of natural active ingredients.

ACKNOWLEDGMENT

The authors would like to thank the support from the Health Polytechnic of Gorontalo, the Health Ministry of Indonesia for funding support in this study.

AUTHORS CONTRIBUTIONS

All authors have contributed equally.

CONFLICT OF INTERESTS

The authors declared no conflict of interest in this study.

REFERENCES

  1. Ahuja NK, Rajawat JS. Novel nano therapeutic materials for the effective treatment of rheumatoid arthritis-recent insights. Int J App Pharm. 2021;13:31-40. doi: 10.22159/ijap.2021v13i6.42912.

  2. Rohmah A, Prabowo HA, Setiasih S, Handayani S, Jufri M, Hudiyono S. The evaluation of activity and stability of isolated bromelain from pineapple cores (Ananas comosus [L.] Merr) and in vitro penetration test of nanoemulsion topical base. Int J App Pharm. 2021;13:336-42. doi: 10.22159/ijap.2021v13i5.41769.

  3. Vargas RA, Guerrero RV, Petricevich VL. Evaluation of anti-arthritic potential of partitioned extracts of Bougainvillea X Buttiana (Var. Rose) Holttum and Standl. Int J Pharm Pharm Sci. 2018;10(3):117. doi: 10.22159/ijpps.2018v10i3.24015.

  4. Simbala H. Floristic diversity and its use as medicinal plants in conservation area II Bogani Nani Wartabone national park (Bolaang Mongondow regency, North Sulawesi). Bogor Agricultural University; 2007.

  5. Sangi M, Runtuwene MRJ, Simbala HEI. Phytochemical analysis of medicinal plants in North Minahasa regency. Chem Prog. 2008;1:47-53.

  6. Moot CL, Bodhi W, Mongi J. Test of antipyretic effects of infusion of Sesewanuwa leaves (Clerodendron squamatum Vahl.) against male rabbits induced by DTP HB vaccine. Pharmacon. 2013;2:58-61.

  7. Irawati D, Kinho J. Diversity of medicinal plants in the coastal forest of Tangkoko Nature Reserve. J Wasian. 2015;2:1-8.

  8. Nonke E, Kaunang S, Semuel MY. Botanical and phytochemical constituents of several medicinal plants from mount Klabat north Minahasa. J Med Plants Stud. 2017;5:29-35.

  9. Sapiun Z, Pangalo P, K Imran A, Wicita PS, Astuti Daud RP. Determination of total flavonoid levels of ethanol extract Sesewanua leaf (Clerodendrum fragrans wild) with maceration method using UV–vis spectrophotometry. Pharmacogn J. 2020;12(2):356-60. doi: 10.5530/pj.2020.12.56.

  10. Yudistira A. Anti-breast cancer activity test of sesewanua leaf extract (Clerodendron squmatum Vahl.) against T47D breast cancer cells. Pharmacon. 2017;6:45-51.

  11. Mehta M, Shah N, Shah S. Gellified emulsion: a review of state of art and recent. Advances 2016;6:420-6.

  12. Ibrahim I, Hashim A. Clove oil. Definitions. 2020:2171-95. doi: 10.32388/appqcm.

  13. Pich J. Treatment for superficial thrombophlebitis of the leg. Br J Community Nurs. 2019;24(6):263-4. doi: 10.12968/bjcn.2019.24.6.263, PMID 31166771.

  14. Das S, MS, Venkatesh MP, Sahoo S, Tripathy J, Gowda DV. Formulation and evaluation of topical nanoemulgel of methotrexate for rheumatoid arthritis. Int J App Pharm. 2021;13:351-7. doi: 10.22159/ijap.2021v13i5.41026.

  15. Mubeen F. Formulation and evaluation of Topival itraconazole Emulgel. Int J Pharm Ther. 2013;3:211-6.

  16. Ijaz S, Shoaib Khan HM, Anwar Z, Talbot B, Walsh JJ. HPLC profiling of Mimosa pudica polyphenols and their non-invasive biophysical investigations for anti-dermatoheliotic and skin reinstating potential. Biomed Pharmacother. 2019;109:865-75. doi: 10.1016/j.biopha.2018.10.089, PMID 30551540. biopha.2018.10.089.

  17. Hamid KA, Ibrahim SI, Hashim MA, Salama M. Formulation and evaluation of Benzyl Benzoate Emulgel. IOSR JPBS Ver. 2015(10):2319–7676. doi: 10.9790/3008-10310609.

  18. Mohamed MI. Optimization of chlorphenesin emulgel formulation. AAPS J. 2004;6(3):e26. doi: 10.1208/aapsj060326, PMID 15760111.

  19. Srivastava N, Patel DK, Rai VK, Pal A, Yadav NP. Development of emulgel formulation for vaginal candidiasis: pharmaceutical characterization, in vitro and in vivo evaluation. J Drug Deliv Sci Technol. 2018;48:490-8. doi: 10.1016/j.jddst.2018.10.013.jddst.2018.10.013.

  20. Dixit AS, Charyulu N, Nayari H. Design and evaluation of novel emulgel containing acyclovir for herpes simplex keratitis. Lat Am J Pharm. 2011;30:844-52.

  21. Saraswathi B, Shabana S, Sri DR, Lakshmi DA, Shivaji M, Satyanarayana T. Formulation and characterization of tramadol Emulgel. Res J Top Cosmet Sci. 2017;8(2):53. doi: 10.5958/2321-5844.2017.00006.1.

  22. Ahmad N, Ahmad R, Al-Qudaihi A, Alaseel SE, Fita IZ, Khalid MS, Pottoo FH, Bolla SR. A novel self-nanoemulsifying drug delivery system for curcumin used in the treatment of wound healing and inflammation. 3 Biotech. 2019;9(10):360. doi: 10.1007/s13205-019-1885-3, PMID 31544014.

  23. Van Staden D, du Plessis J, Viljoen J. Development of topical/transdermal self-emulsifying drug delivery systems, not as simple as expected. Sci Pharm. 2020;88(2). doi: 10.3390/scipharm88020017.

  24. Andleeb M, Shoaib Khan HM, Daniyal M. Development, characterization and stability evaluation of topical gel loaded with ethosomes containing Achillea millefolium L. extract. Front Pharmacol. 2021;12:603227. doi: 10.3389/fphar.2021.603227, PMID 33912036.

  25. Cheng YC, Li TS, Su HL, Lee PC, Wang HD. Transdermal delivery systems of natural products applied to skin therapy and care. Molecules. 2020;25(21):1-21. doi: 10.3390/ molecules25215051, PMID 33143260.

  26. Kumar D, Singh J, Antil M, Kumar V. EMULGEL-novel topical drug delivery SYSTEM–A comprehensive. Int J Pharm Sci Res. 2016;7:4733-42. doi: 10.13040/IJPSR.0975-8232.7(12).4733-42.

  27. Trommer H, Neubert RHH. Overcoming the stratum corneum: the modulation of skin penetration. A review. Skin Pharmacol Physiol. 2006;19(2):106-21. doi: 10.1159/000091978, PMID 16685150.

  28. Shembale AI, Borole DK, Lohiya R. Useful permeation enhancers for transdermal drug delivery: A revew. Int J Pharm Res Dev. 2010.

  29. Bhutkar MA, Bhinge SD, Randive DS, Nayakal O, Patil P. Evaluation of dimethylsulfoxide and Aloe vera as penetration enhancers for cutaneous application of lidocaine. Ars Pharm. 2019;60:85-92.

  30. Jantharaprapap R, Stagni G. Effects of penetration enhancers on in vitro permeability of meloxicam gels. Int J Pharm. 2007;343(1-2):26-33. doi: 10.1016/j.ijpharm.2007.04.011, PMID 17531412.

  31. Damayanti RA, Yuwono T. Dimethylsulfoxide as an enhancer of transdermal transport of theophylline in gel dosage forms. J Ilm Kefarmasian. 2013;3:61-9.

  32. Jufri M, Rachmadiva RR, Gozan M, Suyono EA. Formulation, stability test and in vitro penetration test of emulgel from tobacco leaves extract. J Young Pharm. 2018;10(2s):S69-72. doi: 10.5530/jyp.2018.2s.13.

  33. Salamanca CH, Barrera Ocampo A, Lasso JC, Camacho N, Yarce CJ. Franz diffusion cell approach for pre-formulation characterisation of ketoprofen semi-solid dosage forms. Pharmaceutics. 2018;10(3):1-10. doi: 10.3390/pharmaceutics10030148, PMID 30189634.

  34. Kandalkar A, Singh N. Effect of permeation enhancer on bioavailability of formulated patches of glibenclamide. Int J Pharm Investig. 2021;11(2):214-9. doi: 10.5530/ijpi.2021.2.38.

  35. Thakker KD, Chern WH. Development and validation of in vitro release tests for semisolid dosage forms-case study. Dissolution Technol. 2003;10(2):10-5. doi: 10.14227/DT100203P10.

  36. Kumar M, Prashar D. Pharmaceutical world of permeation enhancers. Int J Curr Pharm Sci. 2021;13:14-7. doi: 10.22159/ijcpr.2021v13i5.1894.

  37. Harmita H. Petunjuk pelaksanaan validasi metode dan cara perhitungannya. MIK. 2004;1(3):117-35. doi: 10.7454/psr.v1i3.3375.

  38. Jatav VS, Saggu JS, Sharma AK, Sharma A, Jat RK. Design, development and permeation studies of nebivolol hydrochloride from novel matrix type transdermal patches 2013. Adv Biomed Res. 2013;2:62. doi: 10.4103/2277-9175.115813, PMID 24223377.

  39. Damayanti RA, Yuwono T. Dimetilsulfoksid sebagai enhancer transpor transdermal Teofilin Sediaan gel. Pharmaciana. 2014;3(1):263-7. doi: 10.12928/pharmaciana.v3i1.419. v3i1.419.

  40. Trommer H, Neubert RHH. Overcoming the stratum corneum: the modulation of skin penetration. A review. Skin Pharmacol Physiol. 2006;19(2):106-21. doi: 10.1159/000091978, PMID 16685150.

  41. Sukmawati A, Suprapto MRMAP. Effects of various penetration enhancers on percutaneous penetration of diclofenac sodium gel in vitro. Pharmacon. 2009;10:7-12.

  42. Anwar E, Ramadon D, Harmita. Formulation and evaluation of gel and emulgel of chili extract (Capsicum frutescens L.) as topical dosage forms. Int J Pharm Pharm Sci. 2014;6:13-6.

  43. Bairagi GR, Patel VP. Formulation and development of curcumin based Emulgel in treatment and recurrence of vaginal candidiasis. Int J Curr Pharm Sci. 2021;13:89-99. doi: 10.22159/ijcpr.2021v13i5.1900.

  44. Yati K, Jufri M, Gozan M, Dwita LP. Effect of variations in concentration of hydroxypropyl methylcellulose (HPMC) on physical stability of tobacco extract gel (Nicotiana tabaccum L.) and its activity against streptococcus mutans. Pharm Sci Res. 2018;5:133-41. doi: 10.7454/psr.v5i3.4146.