Shri Dev Bhoomi Institute of Education Science and Technology, Dehradun
The study’s objective was to create a unique Smilax China microemulsion medication delivery method for topical use. To prepare the optimal solvent system for the new formulation, methanolic extraction of dried rhizomes from Smilax china will be performed in order to determine the key ingredients present (which will be employed in formulation) and to determine the drug’s solubility with various excipients. To assess the standby status of the formulation using several metrics. To make the medication more soluble and permeable so that it can be applied topically without causing any negative side effects from the drug’s first pass action, also known as hepatic first pass metabolism. The present study focuses on the formulation and evaluation of a topical microemulsion of Smilax china, a medicinal plant renowned for its anti-inflammatory and antioxidant properties. The primary aim is to develop a stable microemulsion system that enhances the percutaneous delivery of active phytoconstituents from Smilax china for effective topical application. The formulation was optimized using various ratios of surfactants, co-surfactants, oil phase, and water phase, determined by constructing pseudoternary phase diagrams. The optimized microemulsion was evaluated for particle size, zeta potential, viscosity, and pH, along with in vitro drug release and ex vivo skin permeation studies using Franz diffusion cells. Stability tests, including centrifugation, and accelerated stability studies, were conducted to ensure the long-term viability of the formulation. The developed microemulsion demonstrated excellent physical stability, improved skin permeation, and significant anti-inflammatory effects, making it a promising candidate for topical therapeutic applications.
Microemulsions are transparent (or translucent), thermodynamically stable, isotropic systems of water, oil and surfactant, usually in combination with a cosurfactant. The droplet size usually in the range of 100-400 nm. They can be classified as oil-in-water (o/w), water-in-oil (w/o) or bicontinuous systems depending on their structure and are characterized by ultralow interfacial tension between oil and water phases. Topical preparations are one of the various dosages forms available for the localized effects to the targeted site i.e., skin.[1] These drugs penetrate the underlying layers of skin or mucous membranes. The key benefit of topical delivery system is that they bypass first pass effect, commonly known as hepatic first pass metabolism. It is patient compliant as it precludes the inconveniences of intravenous therapy and of the unfavorable conditions of absorption, like pH changes, presence of enzymes and gastric emptying time.[2] The topical drug delivery system is widely used for pain management along with contraception, and urinary incontinence or when the other systems of drug administration are ineffective to deliver the therapeutic effect. In past decades, different forms of treatment were used to relieve the patients, which include administration of drugs via various routes such as oral, sublingual, rectal, parental, topical, inhalation etc. Topical drug delivery is the application of a drug directly to skin in order to cure cutaneous disorders (acne) or the general cutaneous manifestations of a disease (psoriasis) with the defined pharmacological or therapeutic effect of the drug on surface of the skin or inside the skin. Topical delivery system may or may not necessitate intra-cutaneous penetration or deposition.[3]
Topical formulations have three main functions: -[4]
Merits of topical medicine delivery: -
Demerits of the topical medicine delivery: -
THE HUMAN SKIN
Anatomy and physiology of skin.
Skin barriers consist of two primary layers: the dermis and the epidermis. The striated, biochemically active cells that make up the epidermal layer lack capillary blood flow. About 100 ?m is its thickness. The epidermis is made up of five layers: spinosum, S. granulosum, S. corneum, S. lucidum, and S. germinativum. The stratum corneum, the outermost layer of the epidermis, is composed of 10 to 15 layers of corneocytes, which are dead, keratinized cells. When the stratum corneum is dehydrated, it is around 10 ?m thick. These closely spaced corneocytes are divided by lipid bilayers made of ceramides and free fatty acids.[9] The simple yet complex structure of the stratum corneum helps protect skin form external environment which also includes preventing loss of water via skin. The cell proliferation cycle takes place at the basal cells of the epidermis which takes around 400 hours for the regeneration of the outer epidermis. Beneath the epidermis layer, lies the dermis which is approximately about 2-3 mm thick and embodies connective tissues.[10]
MECHANISM OF DRUG PENETRATION THROUGH SKIN[11]
Three routes exist for the medication to penetrate a topical formulation:
Direct continuous channel through hair follicles and sweat ducts and sebaceous glands.
PSORIASIS
Psoriasis is a recurring skin condition that involves the formation of minor localized patches to large lesions covering multiple body surface areas. It varies in severity and is suggested to be caused by the sudden immune dysfunction, with increased infiltration of immune cells such as lymphocytes (T cells) and hyper-proliferation of skin cells. The global average prevalence of psoriasis was estimated as 2-4% but American, Canadian, and European populations are mostly affected by it. The occurrence of psoriasis in children ranged from 0% (Taiwan) to 2.1% (Italy). It ranges from 0.92% in the US to 8.45% in Norway for adults. In the United States, the prevalence of the disease ranged from 80.9/100,000 person-years in the United States to 230/100,000 person-years in Italy; data estimates place the prevalence in children at about 42.8/100,000 person-years. According to one study, the likelihood of developing skin psoriasis is 80.21% for those with psoriatic arthritis (PsA). Vulvaria (plaque) psoriasis makes up 90.15% of cases among the seven types of the condition. Psoriasis is not fatal, but studies have indicated that there may be a higher chance of cancer, diabetes, and cardiovascular disease, among other morbidities. Epidemiology Psoriasis affects around 1 to 3% of the world population, with equal gender distribution. Disease encounter rates vary from 50 to 140 new cases per 100,000 people per year.
Patients affected with it encounter high levels of distress and morbidity including general decrease in the quality of life though it is not usually life threatening. But compared to the average population, severe psoriasis Mount increases the chance of death.
HERBAL DRUG USED FOR PSORIASIS
Conferring to World health organization (WHO) notes, 74% of the plant derived medicines are used in modern medicine in a way that their modern application directly correlates with their traditional use as medicines by native cultures. As per another suggestion WHO, 65% to 80% of the world’s population will depend on traditional medicines as their primary form of health care and about 85% of traditional medicines involve the use of herbal preparations.
Table 1: A plant were chosen to create a microemulsion formulation:
Plant profile of Smilax china L. rhizome
Figure 1: Fresh rhizome of Smilax china.
Habit and habitat
A member of the Liliaceae family, which is mostly found in East Asia and is extensively dispersed in both tropical and temperate regions of the world, is Smilax china Linn. With thorny projections and climbing tendrils, it is a perennial plant found in nature. The utilization of Smilax china L. tubers in traditional medicine for the treatment of malignancies, inflammation, gout, and furunculosis is supported by prior study data. Lately, research is also available on the use of Smilax china L. leaves having potential antioxidant, anti-microbial, anti-diabetic, and anti- hyperuricemia effects due to the presence of bio actives like polyphenols such as rutin, kaempferin, and kaempferitrin. Its stem and aculeate skin part are considered as waste and hence not used. However, its thorny vines influence the growth of other plants.
Botanical Description
It is a woody plant with thorn like projection spread all through the stem. Rhizomes are thick, elongated and grey in color. Leaves are veined, simple, alternate, elliptically oblong to sub rounded in shape with a tapering end close to branches. Petioles are adnate speculate; stipules usually stretched up to tendrils. The inflorescence ascends from the upper leaf axils. Flowers are white to yellowish green in color; their pedicels are supported by bracteoles, umbellate. Berries are globular in shape and are in red color when ripe. It is a common mountain climber plant with sturdy stem whose surface is covered with thorns. The leaves are huge, green in color with shining surface. They are globular in shape, resembling to a horse hoof. The plant exhibits yellow flowers trailed by red fruits in the autumn. Its roots are rigid and overlaid with bristle-like trichomes. It is native to China and Japan where it is known as Toojuh. It is not grown in India however it is available in all the local markets. It is believed that the root of Smilax glabra probably constitutes part of the dried tuberous roots. For an extended period, it has been believed to have characteristics like those of Sarsaparilla, the source of multiple native Smilax species found in tropical America. The drug is imported from China to a considerable extent by coating steamers trading with Calcutta and Bombay” (H, 1932). Microemulsions of Smilax china, a plant known for its medicinal properties, have been a subject of interest due to their potential applications in various fields including pharmaceuticals, cosmetics, and food. In this section, we will discuss the results obtained from the formulation and characterization of microemulsions containing Smilax china extract, as well as their implications.
Botanical parameters
Macroscopically the rhizomes tubers were about the average size 6 to 12 cm (length) and 2 to 4 cm (width), the fresh rhizome are brownish yellow in color externally, and brown in color internally (Figure 2).
Figure 2: Macroscopic characters of S. china Rhizomes
MATERIALS AND METHODS
CHEMICAL LIST
The list of chemicals which is needed in proposed research work is mentioned below in the table 3.
EXTRACTION OF PLANT MATERIAL
By the process of grinding or triturating the Dried rhizomes of Smilax China 50gram of fine powder was packed in filter bag (called thimble) and then assemble it in Soxhlet Apparatus (in Figure 3). 200mL of Methanol (70%) used as solvent to be added for the process of extraction within the temperature between 50? to 60?. Extraction process was completed after approximately 6 hours (22 Cycles) which was confirmed by the transparency of siphoned liquid.
Figure 3: Extraction of Smilax China using Soxhlet Apparatus.
RESULT AND DISCUSSION
Construction of Ternary Phase Diagram
The ternary phase diagram was constructed by titration of homogeneous mixture of oil, surfactants and co-surfactant with water at room temperature. Oil, surfactants and co-surfactant at different ratios was mixed into the different beakers (100 ml). Using a burette, water was added dropwise to each beaker holding the liquid combinations to titrate each one. After addition of water, the mixture was homogenized by magnetic stirrer at a speed of 200- 400 rpm and the sample was monitored visually for any change of optical transparency. After the addition of 100 ml of dropwise water in the mixtures, these solutions were analyzed by UV-vis spectrophotometer in order to check the transmittance at 650 nm. The titrations used to generate the data to construct the ternary phase diagram. Phase diagrams can be used to show microemulsions, which are created using the spontaneous emulsification method (also known as the phase titration method). Phase diagram construction is a helpful method for researching the intricate web of interactions that might happen when several substances are combined. Depending on the chemical makeup and concentration of each component, different association structures such as emulsion, micelles, lamellar, hexagonal, cubic, and other gels as well as oily dispersion are created along with microemulsions. Determining the phase boundaries and comprehending their phase equilibria are crucial components of the research. Pseudotentary phase diagrams are frequently created to identify the various zones, including the microemulsion zone, in which each corner of the diagram represents 100% of the specific component, while quaternary phase diagrams (four component systems) are laborious and challenging to comprehend. The area can be classified as w/o or o/w microemulsion based solely on its composition, i.e., whether it is rich in water or oil. Care should be taken while making observations to ensure that no metastable systems are mentioned. The ternary phase diagram was constructed using Chemix School software. The transparent zone generated by the mixing of oil and Smix (surfactant: co-surfactant) is identified using a ternary phase diagram. A superior microemulsion is defined by a clear region, whereas a bad microemulsion is defined by a turbid region. Orange oil and Smix (Tween 80: PEG 400) were selected for Table 5.9 based on their solubility. For the oil phase, there were fourteen distinct weight ratios and the following 42 Smix ratios: 1:1, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 9:1, 7:3, and 6:4.The results indicated that although increasing the concentration of surfactant or co-surfactant creates clear microemulsion, increasing the concentration of oil forms murky nano emulsion. Three duplicates of each experiment were conducted.
Figure 4: Ternary phase diagram of orange oil and Smix (1:1)
Figure 5: Ternary phase diagram of orange oil and Smix (2:1) Figure 5: Ternary phase diagram of orange oil and Smix (2:1)
Figure 6: Ternary phase diagram of orange oil and Smix (3:1)
IDENTIFICATION OF DRUG
Determination of melting point
The medication was stored in a digital melting point apparatus and filled into an open capillary tube with one end. It has been noted what temperature the medication begins to melt. The method was explained. 123?C was determined to be the melting point of Smilax china.
Partition coefficient
The MESC sample’s log partition coefficient at 37 °C was determined to be 2.694. Drugs that have a partition coefficient larger than 1 are lipophilic. The medications are lipophilic, according to the results. Therefore, topical drug delivery systems are appropriate for the medication MESC.
Determination of ?max by UV Spectroscopy
In methanol, the Smilax china ?max (320 nm) was identified. The UV spectra of Smilax china are shown in the figure 7.
Figure 7: UV spectra of MESC drug sample.
FTIR spectroscopy
FTIR analysis of methanolic extract of pure drug sample was carried out to confirm the purity of drug sample as shown in figure 8. The main functional groups present in the MESC chemical structure are –OH, Aromatic C-H stretching, C=O stretching, C-H bend (methylene -CH2) stretching, Aromatic C=C stretching, C-O stretching (sp2 hybridized), C-O stretching (sp3 hybridized) and C-Cl stretching. The IR spectrum obtained for the MESC sample is denoted in Fig.6.4. All these major functional groups clearly showed their infrared absorption peaks at their designated regions. The observed IR absorption peaks are 3423.44 cm-1 (O-H groups), 2938.73 cm-1 (Aromatic C-H Str), 1639.01 cm-1 (C=O stretching), 1405.62 cm-1
Figure 8: FTIR spectrum of MESC
ANALYTICAL METHODS
UV calibration curve of MESC in methanol
The UV calibration curve of MESC in methanol at concentration range of 20- 100µg/ml was found to be linear (Figure 9). The observed correlation coefficient was 0.9943. The regression line equation was y = 0.009x + 0.111.
Figure 9: Calibration curve of MESC in methanol.
UV calibration curve of MESC in octanol
The UV calibration curve of MESC in octanol at concentration range of 20-100 µg/ml was found to be linear (Figure 10). The observed correlation coefficient was 0.9886. The regression line equation was y = 0.0109x + 0.1015.
Figure 10: Calibration curve of MESC in octanol.
UV calibration curve of MESC in PBS (pH 7.4) with methanol
The UV calibration curve of MESC in PBS (pH 7.4) with methanol at concentration range of 20-120 µg/ml was found to be linear (Figure 11). The observed correlation coefficient was 0.9969. Equation y = 0.0076x + 0.0392 represented the regression line.
Determination of solubility of Smilax China in different oils, surfactant and co-surfactant
An essential component of keeping the medication stable in its lipophilic environment is the oil system. As a result, various oils were selected for our study, and displays their solubility. Orange oil was shown to be the most soluble in smilax china (89.648±2.22) when compared to other oils (in figure 12), making it the ideal carrier for preparing microemulsions. An essential component of keeping the medication stable in its lipophilic environment is the oil system. As a result, various oils were selected for our study, and displays their solubility. Orange oil was shown to be the most soluble in smilax china (89.648±2.22) when compared to other oils, making it the ideal carrier for preparing microemulsions. While choosing the oil, surfactant, and co-surfactant, the necessary hydrophilic-lipophilic balance (HLB) for the creation of an o/w (oil in water) microemulsion was also taken into account. Orange oil, Tween 80, and cosurfactant PEG 400 all have higher solubility levels for Smilax china (in figure 12, figure 13 and figure 14. It displays the solubility of smilax china in various oils as well as surfactant and cosurfactant solutions, respectively. For blending water and oil into the microemulsion, Tween 80, a nonionic surfactant with high hydrophilic-lipophilic balance (values), is a useful guide. The demonstrates that tween 80 was chosen as a surfactant because of its greater capacity for drug solubilization, biocompatibility, and reduced irritability potential in comparison to other surfactants. It was also chosen because it could generate a stable and transparent microemulsion formulation.
Figure 12: Graph showing solubility of drug in oils.
Figure 13: Graph showing solubility of drug in surfactant
Figure 14: Graph showing solubility of drug in co-surfactant.
Drug-excipient compatibility studies
Room temperature was used for the studies. Physical contact was noted during a month of room temperature storage. The excipients used to prepare the product must guarantee that it is compatible with the medicine being used before any formulation can be done. Consequently, during preformulating, drug excipient interaction investigations are crucial.
EVAULATION OF MICROEMULSION
Figure 17: Prepared microemulsion
Viscosity
The viscosity of the formulated microemulsion was determined by using spindle no. 62 of Brookfield viscometer.
Viscosity of the formulation was determined 758.63 cps.
pH
By utilizing a pH meter at room temperature, the pH of the prepared microemulsion was confirmed. pH of the formulation was determined 5.78.
Drug entrapment
The drug contained in the formulation can be identified by looking at the proportion of drug entrapment. By dissolving 100 mg of microemulsion in methanol and adding water to generate up to 10 ml, drug entrapment was ascertained. After that, the stock solution was appropriately diluted, and absorbance was measured using UV-visible spectrophotometry at ?max 258 nm. Next, absorbance was entered into the methanol straight-line equation, and the result was converted to milligrams by adding a dilution factor. It was discovered that 99.45% of the formulation’s drugs were entrapped.
In vitro Drug Release
A magnetic stirrer was used to conduct in-vitro drug release experiments of the prepared microemulsion in phosphate buffer pH 5.5: methanol (1:1). One gram of the formulation was put into a dialysis bag and submerged in 150 milliliters of pH 5.5 phosphate buffer with methanol (1:1) at 37±0.5 °C. After extracting an aliquot, the medium was replaced with new one and scanned at ?max 254.9 nm. Comparative outcomes, or 20% in 7 hours, were obtained using microemulsion.
In vitro Drug Permeation
The Franz Diffusion equipment was used to carry out the formulations’ in vitro diffusion. The membrane that separated the donor compartment from the receptor was made of dialysis membrane. pH 5.5 phosphate buffer: The solvent in the receptor compartment was methanol. The cumulative amount of medication penetrated per unit area dialysis membrane and flux at six hours after formulation. Cumulative drug permeability after six hours of formulation. Comparative data, or 38% in 6 hours, was achieved using Smilax China glycerosomes.
Skin irritation study
The erythema scores achieved for control group, positive control group and optimized MESC were0.0±0.0,3.0±0.42and0.5±0.06, while that of edema were 0.0 ± 0.0, 3.5 ± 0.58 and 0.75 ± 0.14. The score attained for erythema and edema were added to compute primary irritation index (PII) and the final score [PII: 6.5 and 1.25] was achieved for standard irritant formalin solution and MESC, respectively. Materials scoring two or lower are considered non-irritating to the skin. The skin irritation study’s data, which showed a score of less than two, support the formulation’s non-irritating characteristics. Therefore, it can be said that the developed dermal MESC formulations do not cause skin irritation.
CONCLUSIONS
It was found that the orange oil-created microemulsion was an effective carrier for the Smilax china microemulsion. A useful dosing form for the weakly water-soluble drug Smilax China is a microemulsion made of orange oil, tween 80, PEG 400, and water. The phase diagram construction showed that the optimal microemulsion results were obtained at a ratio of 3:1. In the refined formulation employed in this study, B-11 was a transparent liquid with simple flowability. The best formulation, B-11, was found to be a w/o microemulsion with a pH of 5.78, a viscosity of 758.63 cps, and 99.45% of the medicines in the formulation were entrapped. It was found that a w/o microemulsion with a high viscosity that is easily absorbed by the skin is produced by a high oil content.
The ternary phase diagram, created using Chemix School software, identifies the transparent zone created by mixing oil and Smix (surfactant: co-surfactant). A superior microemulsion is defined by a clear region, while a bad microemulsion is a turbid region. Orange oil and Smix were chosen based on their solubility. The oil phase had 14 weight ratios and 42 Smix ratios. Results showed that increasing the concentration of surfactant or co-surfactant created clear microemulsions, while increasing oil concentration formed murky nano emulsions. Three duplicates of each experiment were conducted.
REFERENCES
Ashutosh Lakhera, Meenakshi Kandwal, Shivanand Patil, Formulation And Evaluation Of Topical Microemulsion Of Smilax China, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 10, 1423-1438. https://doi.org/10.5281/zenodo.13995988
10.5281/zenodo.13995988
