Formulation Development and Evaluation of Acyclovir Bigel Containing Lemongrass Oil

 

Table 1.1a: Comparison of Conventional vs. Advanced Topical Drug Delivery Systems

 

Table 1.1.b: Selected Experimental and Clinical Findings

 

1.1.2     Advantages of using topical drug delivery

Topical drug delivery has a number of therapeutic and patient benefits over traditional oral and parenteral routes. The main advantage is the possibility of offering a localized effect, which involves high drug concentrations at the point of application, e.g. skin infections or inflammation, and reduced exposure to the system (Bagmar et al., 2024 [2]). Moreover, topical formulae do not have hepatic first-pass metabolism, and therefore, increase the systemic bioavailability of some drugs that would otherwise be heavily degraded in the gastrointestinal tract (Zhao et al., 2024 [60]).

 

Topical preparations have a higher chance of patient compliance, as they are non-invasive, painless, and easy to apply, and thus especially chronic (Alam et al., 2013  [1e]). Sustained drug release can be achieved by a variety of modern formulations such as transdermal patches or nano-based carriers that may help to reduce the frequency of dosing and enhance therapeutic results (Gupta et al., 2022 [12]). Moreover, topical administration is beneficial when it comes to indications like dermatological processes, musculoskeletal issues and mucosal administration, where an immediate targeting of drugs is beneficial (Lopes et al., 2022 [21]). The second significant benefit is the decrease in the gastrointestinal side effects, as topical administration of drugs bypasses the stomach and the intestines, avoiding irritation, nausea, or ulceration that may cause oral therapy (Jain et al., 2021 [14]). Moreover, in cases of drugs that are supposed to produce local effect, e.g., topical anesthetics, the action starts rather quickly, and the syndrome is controlled in the short term (Chen et al., 2022 [5]).

 

1.1.3     Disadvantages of Topical Drug Delivery

Topical and transdermal drug delivery systems have significant limitations in spite of these benefits. The largest obstacle is the skin barrier, especially the stratum corneum that limits the penetration of hydrophilic substances and molecules with a size higher than 500 Da (Zhao et al., 2024 [60]). Consequently, the drugs possessing proper lipophilicity, molecular weight and potency only can be delivered effectively through this route. The second disadvantage is the possibility of local irritation or allergenic reactions to active drugs, excipients, or penetration enhancers, which can result in dermatitis or sensitization when used over a long period of time (Francavilla et al., 2023 [11]). Absorption variance is also an issue, as age, hydration, disease condition, ethnicity, and location of administration can cause a considerable change in permeation rates and treatment efficiency (Lopes et al., 2022 [21]). Furthermore, not all diseases can be treated topically (particularly, systemic diseases or infections), unless special carriers such as nanoparticles or microneedles are used to enhance penetration (Ranade et al., 1991 [31]). The other weakness is that the semi-solid formulations are challenging to precisely dose, and this may result in inconsistency in the dose delivered as well as in the exposure to the drug (Bagmar et al., 2024 [2]). Lastly, topical medications have the risk of unintended movement to clothes, bedding, or even other people, thus losing their efficiency and may be unsafe (Chen et al., 2022 [5]).

 

1.1.4     Essential Elements of Topical Drug Delivery System.

Topical drug formulations are complicated systems that should contain the active drug along with suitable excipients to provide the required stability, penetration, and patient acceptance. All the elements of the formulation contribute to the effect of releasing, absorbing, and therapeutic effects of the drug (Sharma et al., 2021 [42]). Natural penetration enhancers, biocompatible polymers, and multifunctional excipients have also been developed recently, encouraging efficacy and safety (Lopes et al., 2022 [21]; Francavilla et al., 2023 [11]).

 

Table 1.2: Basic Components of Topical Drug Delivery Systems (with Explanations & Recent Examples)

 

The API is the focal ingredient, but its effectiveness highly depends on the base/vehicle selected that either makes the formulation act as an ointment, cream, gel, or patch. Bigel systems have been of interest in recent years because of their ability to combine the solubility benefits of hydrophilic (through the use of hydrogels) and lipophilic (through the use of organogels) drugs (Rocha-Guzmán et al., 2025 [35]). Penetration enhancers play an important role in the penetration of the stratum corneum barrier. But traditional enhancers, e.g. oleic acid and propylene glycol are still popular, yet more recent studies emphasize the use of essential oils and nanocarrier-based systems to enhance skin penetration in a safer way than previously used drug (Das et al., 2023 [7]). The use of supportive excipients like humectants, emulsifiers, thickeners, and stabilizers provides stability of formulations, cosmetic acceptability and controlled release profiles. In the meantime, preservatives and antioxidants ensure microbial safety and degradation prevention, but the tendency is moving toward the use of natural or biocompatible excipients in the topical formulations of the day (Sharma et al., 2021 [42]; Francavilla et al., 2023 [11]).

 

1.1.5     Bigels: An Overview

Bigels consist of a hydrophilic hydrogel, and a lipophilic organogel biphasic semisolid combination to create a stable and homogeneous matrix. This amphiphilic property enables the concomitant entrapment of hydrophilic and lipophilic drugs, increasing the controlled release, biocompatibility, and spreadability (Samui et al., 2021 [37]). Bigels are drawing interest in anti-inflammatory, antifungal, antimicrobial agent delivery and are better tolerated by patients than more conventional creams or ointments (Shakouri et al., 2023 [41]).

 

1.1.6     Preparation of Bigels

The overall process consists of three major steps:

1.      Hydrogel Preparation Disperse a natural or synthetic polymer (e.g., carbopol, HPMC, chitosan) in water, swell, neutralize or cross-link.

2.      Preparation of the Organogel: Dissolve in an oil phase the organogelator (fatty acid, wax, lecithin, sorbitan ester) heating it above the melting point and allowing it to solidify to a gel network.

3.      Preparation of the Bigel: Add both phases in a certain proportion (typically 70:30 or 50:50 hydrogel:organogel) and stir it carefully to keep it unseparated in phase (Sreekumar et al., 2020 [53]).

 

Table 1.6— Preparation Methods and Key Conditions for Bigel Components

 

Table 1.7 - Recent Studies on Bigel-Based Topical Formulations (2014 – 2024)

List of Chemicals: The drugs and excipients used for the purpose of research includes following:

 

1.1         List of chemicals used

Table 4.1 Chemical used

 

1.2         List of Equipments and instruments:

 

The instruments and Equipments used are:

Table 4.2 List of Equipment’s and instruments used

 

1.3         PROFILE OF EXCIPIENTS

1.3.1     Olive oil

Table 4.3 Details of Olive oil

 

1.3.2     Beeswax

Table 4.4 Details of Beeswax

 

1.3.3     Carbopol 934

Table 4.5 Details of Carbopol 934

 

1.3.4     Lemongrass oil

Table 4.6 Details of Lemongrass oil

 

1.4         Preformulation studies

Prior to starting pre-formulation studies, it is important to have a solid understanding of the drug's properties, dose, potency in comparison to dosage form. We should also conduct a literature search for information on stability and degradation, the suggested route of administration, formulation strategies, and the bioavailability and pharmacokinetics of drugs that are chemically related. By ascertaining the kinetic rate profile, compatibility with other ingredients, and physicochemical properties, a pre-formulation study seeks to develop a stable, elegant, safe, and effective dosage form.

 

The following pre-formulation studies were carried out on-

1.            Organoleptic properties

2.            Melting point 

3.            Partition coefficient

4.            TLC

5.            Solubility

6.            Preparation of standard curve

7.            Preparation of calibration curve

8.            pH

9.            FTIR

 

1.4.1     Organoleptic properties

The drug was observed for its appearance, color and odour.

 

1.4.2     Melting point

By using the open capillary method and the melting point apparatus, the melting point of acyclovir drug was determined.

 

1.4.3     Partition Coefficient Determination in Octanol-Water

To determine partition coefficient, 50 mg of drug was dissolved in 20 ml solvent (10 ml n-octanol and 10 ml water). Then the flasks were shaken vigorously, and the two phases were allowed to separate. The two phases were collected separately and solutions were filtered, diluted and were analyzed using UV spectrophotometer.     

Po/w = Co / Cw

 

Where Co = concentration of drug in n-octanol, Cw = concentration of drug in water

 

Thin layer chromatography

According to British Pharmacopeia, TLC is a separation method that may be used to separate and identify various substances or components in a combination.

 

‘Mobile phase – n butanol : glacial acetic acid : water - 15 ml : 9ml : 6 ml 

Stationary phase – silica gel

RF value -0.50-0.62

 

Procedure –

We prepare a solution of mobile phase

Then take TLC plates and prepare silica gel in motar pestle

After preparation, we spread silica gel on TLC plate and keep it 24 hrs

Next day take tween chamber and marked TLC plate upto 2 cm and spoting drug on TLC and keep on chamber for 75 min

 

After 75 min we perform on uv chamber to observe      

Figure 4.1 TLC plate of Acyclovir

 

Solubility

Take excess amount of drug and cleaned culture tube contain 10 ml of various solvent ethanol, phosphate buffer (6.8), water, and DMSO add 100 mg drug .tube securely sealed and shaken for 24 hours at room temperature on a water bath shaker set 25˚c then after shaking each sample was centrifuged at 15000 rpm for 24 hours to separate supernatant was carefully removed and filtered appropriately dilute and concentration using spectrophotometry to determine solubility of drug in each solvent Identification of drug by UV spectrophotometry Preparation of standard curve Preparation of stock solution 100mg of drug was weighed and dissolved in 100ml distilled water. From the above solution, 1000μg/ml solution was prepared by taking 1ml from above solution and diluting up to 10ml with distilled water. The resultant solution was scanned in the UV region (400nm) using UV spectrophotometer (Shimadzu, UV-1800240.

 

Standard curve of acyclovir in water

To make standard dilutions of 2, 4, 6, 8, 10, 12, 14, and 20 μg/ml from 1000 μg/ml solution, aliquots of 0.2, 0.4, 0.6, 0.8, 0.10, 0.12, 0.14, and 1 ml were transferred into 10 ml volumetric flasks, and the volume was adjusted to the mark.  After measuring the absorbance, a graph was created that showed the absorbance against concentration.

 

Figure 4.2 UV spectrum of acyclovir in water with λmax 253nm

 

Calibration curve of acyclovir in phosphate buffer 7.4

For 250 ml – 6.8045 gm KH2PO4 are dissolved in distilled water and dilute 250ml distilled water For 250 ml – 2gm NaOH are dissolved in distilled water and diluted 250 ml From 1000μg/ml solution, standard dilutions of 2, 4, 6, 8,10,12,14 and 20 μg/ml were prepared by transferring aliquots of 0.2, 0.4, 0.6, 0.8,0.10,0.12,0.14 and 1ml in 10ml volumetric flasks and volume was made up to mark. The absorbance was measured and graph was plotted between and concentration and absorbance.

 

Figure 4.3 UV spectrum of acyclovir in phosphate buffer 7.4 with λmax 253nm

 

1.4.7     pH

The pH of drug solution in water was measured using a digital pH meter.

 

1.4.8     Fourier Transform Infrared Spectroscopy of acyclovir

In a glass mortar and pestle, the medication and potassium bromide were combined separately and then triturated.  Using an infrared spectrophotometer (Shimadzu, IR affinity-1), the triturated mixture was compressed and then processed for FTIR spectra by scanning in the 4000-400 cm-1 range.  The spectra offer comprehensive details on the structural configurations of molecular compounds since FTIR is associated with covalent or hydrogen bonds.  FTIR is used to verify the drug's functional identification and identify any interactions it may have with excipients.

 

1.5         Formulation Development

1.5.1     Screening of hydrophilic polymer for hydrogel

Separately, Carbopol 934, HPMC K100M, and Carbopol 940 were soaked in distilled water for 24 hours at varying concentrations (1%, 1.5%, and 2%).  Viscosity, spreadability, and visual examination were used to screen the hydrogel.

 

Figure 4.4 Different compositions of hydrogels

 

1.5.2     Screening of oils for organogel

Based on the drug's solubility in oils, a variety of oils were evaluated.  The drug's solubility in lemon, peppermint, olive, eucalyptus, and sesame oils was examined.  Acyclovir's solubility in oil served as the basis for the selection. For seventy-two hours, excess acyclovir was poured to each capped glass vial that held five milliliters of oils on a motorized mixer.  For ten minutes, the resultant dispersion was centrifuged at 15,000 rpm.  Filtrates were tested after being suitably diluted.  Using the same medium as the blank, the absorbance of solutions was measured at 253 nm using a double beam UV spectrophotometer.

 

1.5.3     Screening of thickening agent for organogel

The thickening ingredient was chosen based on the organogel's appearance, viscosity, and spreadability.  Different types of beeswax, span 60 (sorbitan monostearate), stearic acid, and olive oil in varying concentrations (5%, 10%, and 15%) were used to create organogels.

 

1.5.4     Screening of ratio of hydrogel and organogel for bigel formulation

Hydrogel and Organogel swere screened according to their appearance, viscosity, and spreadability.  Different ratios of hydrogel and organogel (50:50, 60:40, 70:30, 80:20, and 90:10) were used to create the bigel.  In hydrogel, the medication (%) was distributed.

 

Figure 4.5 Different compositions of Organogels

 

1.6         Preparation of acyclovir loaded bigel containing lemongrass grass oil

Step 1 – Preparation of Hydrogel

Firstly, we prepared a hydrogel, take a weighed quantity of ingredients, drug and essential oil. Then Acyclovir drug was dissolved in water and stir for few minutes after that hydrophilic polymer Carbopol 934, propylene glycol was added and keep it aside for few minutes then added lemongrass oil and triethanolamine dropwise in hydrogel at room temperature.

 

Step 2 – Preparation of Organogel

Take olive oil for organogel and add thickening agen beeswax and heat the solution at 50 to 70˚c with continuous stirring.

 

Step 3 – Preparation of Bigel

After making hydrogel and organogel separately, we slowly added organogel in hydrogel dropwise with continuous stirring.

 

Table 4.7 Composition of Bigel

 

1.7         Evaluation of bigel

●             Viscosity

Using spindle number 63, the Brookfield viscometer was used to evaluate the viscosity of the formulated bigel.  For every bigel, the process was carried out three times.  The angular velocity was maintained at 50 rpm for the test, which was carried out at room temperature.

 

●             Spreadability

To assess spreadability, excess bigel was sandwiched between two glass slides and crushed to a uniform thickness by holding a weight (in grams) for five minutes.  The pan was filled with weight (in grams).  Spreadability may be measured by measuring the amount of time needed to separate the two slides, which is the time for the upper glass slide to move across the bottom plate.  For every bigel, the process was carried out three times.

S= (m×l)/t

“S= Spreadability

l=Length moved on upper glass slide

m=Weight tied to upper slide.

t=Time taken”

 

●             pH of bigel

The precisely weighed bigel was dissolved in 10 milliliters of pure water, and the pH of the resulting mixture was then measured at 25 °C using a digital pH meter.  For every formulated bigel, the process was carried out three times.

 

●             Drug content

A ‘UV visible double beam spectrophotometer’ was used to measure the filtered material at 253 nm after 1g of the bigel was dissolved in 25 ml of phosphate buffer pH 5.5 and the proper dilutions were prepared using the same buffer solution.

 

●             In vitro drug release

A modified in vitro permeation equipment and an egg membrane was used to perform in vitro drug release. The study's dissolving media was phosphate buffer, which has a pH of 7.4.  The study's egg membrane was immersed in phosphate buffer throughout the whole night.  After precisely weighing two grams, the bigel was put over the middle section of the egg membrane, which was then secured to one of the opening ends of the hollow glass cylinder.  The glass cylinder was then attached to the iron shaft and dipped until the membrane just touched the 50 milliliters of phosphate buffer. Throughout the investigation, a magnetic stirrer set at 50 rpm was used to keep the dissolving medium at 37 +/- 0.5 ºC. This temperature was maintained until the experiment's conclusion.  Five milliliter aliquots of the receptor medium sample were taken out and filtered within a predetermined time frame.  After diluting each filtered sample, the absorbance at 253 nm was determined using a UV spectrometer (Elias, 2019 [9]).

 

1.2 Rationale of the work

Present work deals with acyclovir drug bigel  system for the treatment of herpes simplex virus so as to gain various advantages such as improved bioavailability, patient-compliance, sustained and controlled delivery of drugs.

 

The vast family of herpesviruses includes the Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2).  Together with varicella-zoster, they are further classified in the Alpha herpes virinae subfamily.  These viruses can establish latency within sensory ganglia, have a brief reproductive cycle, and destroy the host cell quickly. HSV-1 is widely known for producing encephalitis and orofacial lesions in both adults and children, albeit these conditions are not exclusive.  Aseptic meningitis, genital herpes, and severe infections in newborns are all brought on by HSV-2.  The diagnosis and treatment of many illnesses have advanced significantly (Singh et al., 2024 [49]).

 

Acyclovir is a widely used antiviral drug, primarily indicated for the treatment of herpes simplex virus (HSV) infections, including genital herpes, cold sores, and varicella zoster virus (VZV) infections. While oral administration of acyclovir is common, it is associated with systemic side effects, and for topical treatments, the drug’s limited skin permeation significantly reduces its therapeutic efficacy. Achieving an effective topical concentration of acyclovir at the site of infection is a key challenge in the development of dermatological formulations (Klysik et al., 2020 [18]).

 

To enhance the transdermal delivery of acyclovir, a topical drug delivery system that can overcome the stratum corneum barrier (the outermost layer of the skin) is essential. One promising approach is the formulation of bigels, which combine the benefits of hydrogels and oleogels. Bigels are known for their superior skin penetration, controlled release, and high bioavailability of active ingredients due to their dual-phase structure. The oleophilic (oil) and hydrophilic (water) components in the bigel matrix facilitate the release and absorption of drugs, especially those with both hydrophilic and lipophilic properties.

 

However, even with bigels, penetration enhancement remains a key challenge in transdermal drug delivery. Lemongrass oil (Cymbopogon citratus) has garnered attention for its ability to enhance the permeability of the skin. Rich in citral, lemongrass oil is known to disrupt the skin’s lipid barrier and increase drug diffusion across the epidermis, making it a suitable candidate for use as a penetration enhancer in topical formulations. Unlike synthetic enhancers, lemongrass oil is biocompatible, non-toxic, and naturally derived, aligning with the increasing demand for green pharmaceutical formulations.

 

Incorporating lemongrass oil into a bigel formulation for acyclovir could significantly improve the drug’s permeability and bioavailability at the site of action, thus enhancing its therapeutic efficacy for topical antiviral treatment. Despite the potential of combining bigels and lemongrass oil, limited research has been conducted on their synergistic effects, particularly in the case of acyclovir.

 

DISCUSSION

The current research was done to prepare and evaluate a topical delivery system of acyclovir using a bigel as the base and integrate lemongrass oil, a natural penetration enhancer, so as to enhance drug release and penetration and retain appropriate physicochemical properties to be used topically. Biphasic systems composed of hydrogel and organogel networks (Bigels) combine the advantages of lipophilic systems and hydrophilic systems and are particularly successful with topical delivery of drugs with low skin penetration such as acyclovir.

 

Pre formulation studies validated the identity, purity and physicochemical suitability of acyclovir to be used as a topical formulation. The melting point of Acyclovir (254-256˚C) was observed to be very consistent with the values provided in the literature implying stability of the drug and purity. The studies of the partition coefficient revealed moderate lipophilicity, which means that intrinsic skin penetration cannot be performed easily and that a penetration-enhancing delivery system is needed. The necessity of a bigel system together with penetration enhancers to enhance the topical bioavailability can also be justified by the solubility studies that demonstrated that acyclovir is highly soluble in DMSO and only slightly soluble in water and buffer.

 

Acyclovir's FTIR analysis verified the existence of distinctive functional groups, and drug-excipient compatibility tests revealed no appreciable changes or disappearance of peaks, indicating absence of chemical interaction between acyclovir and formulation excipients such as Carbopol 934 and preservatives. This confirms the chemical stability of the drug within the bigel matrix.

 

Assessment of Bigel Formulations

There were no indications of phase separation or grittiness, and all of the bigels that were created were found to be smooth, homogeneous, and physically stable. All of the formulations' pH values fell between 5.5 and 6.3, which is within the acceptable range for topical application and is not likely to irritate the skin.

 

Measurements of viscosity showed that an increase in Carbopol concentration led to an increase in viscosity, which is consistent with densification of the polymer network. Viscosity and spreadability values were inversely correlated; formulations with moderate viscosity were more spreadable. For topical formulations to guarantee patient compliance and ease of application, this balance is essential.

 

Although TBG1 had the highest drug content, indicating better drug distribution within the matrix at lower polymer, drug content analysis showed acceptable uniformity across all formulations.

 

In Vitro Drug Release Studies

The in vitro drug release experiments showed that all bigel preparations had a controlled and sustained release profile in 180 minutes. TBG1 showed the best cumulative drug release as compared to TBG2, TBG3, TBG4, and TBG5. This decreasing release rate with increasing polymer concentration can be explained by the fact that the higher the concentration of polymer, the higher the viscosity and the polymeric networks the slow diffusion of drugs.

 

This increase in the drug release could be attributed to a good balance between the hydrogel and organogel phases, reduced polymer density, and efficient penetration enhancement with lemongrass oil in TBG1. The occurrence of the organogel phase may have enabled the better partitioning of acyclovir into the lipid domain whereas the existence of the hydrogel phase ensured the hydration of stratum corneum, which along with the above-mentioned feature enhanced the diffusion of the drug.

 

The kinetic analysis of release indicated that the release of the drug was based on the diffusion-controlled action, which is characteristic of semi-solid polymeric system. The following system of bigel was, therefore, effective in giving sustained release and keeping the drug level sufficient at the point of application.

 

The results obtained in this research paper show clearly that bigels can be used as a promising topical delivery system of acyclovir. The use of lemongrass oil as a natural penetration enhancer played a significant role towards increased performance of the drug release. Compared to all formulations, TBG1 was the optimized formulation with good physicochemical properties, maximum drug content, and good in vitro drug release.

 

The study helps to justify the effectiveness of partnership between natural bioactive product and modern drug delivery methods to promote effectual therapy with preserving formulation safety and patient tolerance.

 

The kinetic analysis of release indicated that the release of the drug was based on the diffusion-controlled action, which is characteristic of semi-solid polymeric system. The following system of bigel was, therefore, effective in giving sustained release and keeping the drug level sufficient at the point of application. 

Among the various formulations developed, TBG1 emerged as the most effective based on its superior drug release profile and good physicochemical stability. Characterization studies revealed that TBG1 possessed desirable rheological properties, homogeneity, and compatibility between the phases, making it a promising carrier for acyclovir. The inclusion of lemongrass oil not only contributed to the improved penetration of the drug through the skin but also offered additional antimicrobial and soothing properties, supporting its use in dermatological applications.

 

This research supports the potential of bigel systems, especially those incorporating natural bioenhancers, as efficient and sustainable alternatives to conventional topical therapies. The findings contribute valuable insights to the growing field of novel drug delivery systems and pave the way for future studies focusing on clinical validation and broader therapeutic applications.

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