Design, Formulation, and Physicochemical Evaluation of Occimum sanctum Containing Honey Based Hydrogel

Background and Aim: The present study aims to formulate a hydrogel containing Tulsi (O. sanctum) and honey, to obtain a pharmaceutical topical preparation having desirable healing effects and antibacterial properties of both the ingredients. Material and Method: Topical honey hydrogel formulation were prepared using 75%honey and 10% Occimum sanctum (Tulsi) with polymer carbopol 934. The prepared formulation was assessed for its pH, spreadability, swelling index and in-vitro release Result: The pH and spreadability were in the range of 4.74 ± 0.02 and 7.75 ± 0.04 cm respectively. Carbopol based Honey –Tulsi Formulation showed a concentration-dependent increase in the amount of honey and O. sanctum diffused through dialysis method. Conclusion: Within the limitations of the study, the results of our present study suggest that the studied Honey –Tulsi based hydrogel could be an economic indigenous substitute which is nontoxic, natural and efficient for clinical application.


INTRODUCTION
Honey is defined as concentrated solution of sugar prepared by bees from the nectar of plants. Humans have consumed honey for thousands of years due to its unending health benefits. Honey is a sweet syrup viscous in nature and contains carbohydrates, proteins, amino acids, vitamins and minerals. In ancient literature it has been well documented for for therapeutic purposes owing to is evidence based antimicrobial [1][2], and analgesic [3] property .It is also used to combat respiratory tract infection [4].
In Ayurveda, Tulsi .i.e. holy basil is considered as "The Queen of Herbs" and also "The mother Medicine of nature". In India, Tulsi has been adopted into daily lifestyle practices and spiritual rituals that provide variety of health benefits to our body [5]. Tulsi .i.e. Oil of O. sanctum possess anti inflammatory properties due to its rich content of vitamin C, beta carotene and calcium. It acts by inhibition of cyclo-oxygenase and lipoxygenase pathways, thereby modulating cyclo-oxygenase pathways and lipo-oxygenase pathway. It helps in modulating humoral and cell mediated immune response [6]. The Leaves of this herb has unique property as they are diaphoretic and antiperiodic so used in patients suffering from bronchitis, gastric and hepatic disorders [7].

I.
anti inflammatory II.
Cardio protective VI.
Anticancer properties [8] due to which it acts as a prospective agent for treating wide array of disease ranging from mild illness to severe conditions.
Honey as we all know is viscous in nature its direct application on an affected area remains complicated. The main disadvantage is leakage and liquefication that leads to difficulty in maintaining the therapeutic concentration for a periodic time over the affected region.
To overcome the above mentioned limitations we incorporated honey in a hydrogel formula that is reported to deliver drug for sufficient time. It can be used for medical purpose, wound management in particular [9][10][11]12] owing to its characteristic property of humid environment and good fluid absorbance property that is essential for successful wound healing process and aids in pain management [9].
Thus the present study focuses at overcoming the disadvantages of direct application of honey by formulation of Tulsi containing honey-based hydrogel, to finally obtain a pharmaceutical honey topical preparation with tulsi for desirable healing effects and antibacterial properties.

Honey Samples
Honey was purchased from local Stores of Nagpur ,Maharashtra, carbopol 934 TM (Goodrich Chemicals,USA) and Triethanolamine (TEA) TM and Methyl paraben TM were purchased from (Fisher Scientific, U.K). All the chemicals used were of analytical Grade.

O. sanctum (Tulsi) Samples
The fresh leaves of tulsi were procured from local market of Nagpur and approved by a botanist from Department of Botany, Dharampeth Science College, Nagpur, Maharashtra.

Occimum sanctum Extract
The Tulsi (O. sanctum) leaves were cleaned under running tap water and shade dried under sunlight for 24 hours. The sun dried leaves were stocked up in an uncontaminated sealed container until further use.
Tulsi extract was prepared by using Soxhlet apparatus by addition of 100gms of dried tulsi in the thimble followed by 500ml of double distilled water as a solvent so as to obtain 10% concentration. The process was continued till the solvent appeared in the thimble, further the extract was placed in a digital water bath till a dark green residue was obtained. The extract was stored in a sterile sealed container until further use [6].

Preparation of 75% Honey Based Hydrogel containing Occimum sanctum
Hydrogel was prepared using cold mechanical method by dissolving a predefined amount of carbopol polymer in purified water with constant stirring using a magnetic stirrer for an hour till its complete dispersion in the water followed by addition of TEA so as to neutralize the pH of the hydrogel.
Methyl paraben was further added as a preservative. The Mixture was then kept in room temperature for 24 h so as to attain equilibration of the polymer and notable puffiness.
Finally 75% honey was added to the aforementioned mixture with constant stirring till it is completely disseminated in the hydrogel [13].
The final weight was completed to 100 g with the aqueous solution. At this point 10% O. sanctum extract was dispersed separately in the aqueous solution and stirred unless a homogenous mixture was obtained.
The final formulation were stored in a glass container and covered with screw plastic lid and sited in refrigerator to complete the formation of Honey -Tulsi hydrogel .

Physicochemical Evaluation of Honey
Hydrogel Formulae

Visual examination
The prepared Honey -Tulsi hydrogel formulation was examined by visual check for their color, consistency, homogeneity and existence of lumps after they were set in the container.

pH determination
One g of the hydrogel formulae was weighed and mixed with 25 ml of purified water. The pH of the formulation was measured using a precalibrated pH meter TM (Orion Research Inc. USA), with buffer solution having pH 4.00/7.00/9.00 (25 °C) .Experiments were carried out for three readings and average values were taken into consideration.

Swelling index
Swelling index of the prepared topical tulsihoney hydrogel, was calculated by soaking one gram of the hydrogel into 5 ml phosphate buffer (pH 5.5) and left in place for a detailed time, followed by removal of excess buffer .The hydrogel was weighted again. This test was done at time intervals after 1 and 3 hours. The following formula was used to calculate the swelling ratio [14].
Swelling ratio = (Ws -W t /W 0 ) × 100 where Ws is the weight of the swollen hydrogel at time t and W 0 is the initial weight.

Spreadability measurement
The spreadability of the hydrogel formulation was determined by the method suggested by M. H. Shukr and G. F. Metwally [15]. According to which, 0.5 g prepared hydrogel was pressed between two horizontal plates (20 × 20 cm), followed by addition of 5 g standardized weight on the upper plate and left for about 5 minutes unless no more spreading was expected. Diameters of spread circles were measured in cm and were taken as comparative values for spreadability. The results obtained were the average of three observations.

In vitro Drug release studies
Honey and O. sanctum release from prepared hydrogels was studied using a dialysis method [16].
Presoaked Dialysis bags (Spectra/PorR Dialysis Membrane, MWCO:3.500, Spectrum Laboratories Inc., USA) were inseretd in distilled water at room temperature for 12 h for removal of any preservative, followed by thorough rinsing with distilled water.
A specific weight of the hydrogel was placed in a measured volume (100 ml) of 5.5 pH phosphate buffer at 37 ± 0.5 °C and the quantity of released honey and O. sanctum was measured at different time intervals by taking the absorbance of test sample using UV-Vis spectrophotometer (Shimadzu 1800, Japan).
The absorbance of the released honey was measured at λ max 340 nm.
All experiments were done in triplicates, the average of results were taken. Blank experiments were done using plain bases.

Visual Examination
The hydrogel appears homogenous and brownish green in colour.

pH Examination
The pH of the hydrogel was found to be 4.74 ± 0.02.

Spreadability
Spreadability of the formulation was found to be 7.75 ± 0.04 cm

In vitro Release
The results (Fig. 1) showed a concentrationdependent increase in the amount of honey and O. sanctum diffused through cellophane bag of the hydrogel formulation. It was also observed that the release of honey and O. sanctum increased with increasing its concentration in the formulation.

DISCUSSION
The present formulation uses Tulsi at 10 % and honey at 75% concentration owing to achieve their utmost antibacterial and enhanced tissue regeneration properties as evident from studied literature [6,14].
Hydrogels have been widely used due to its sustained delivery of a variety of local therapeutic agents [13,14]. It is formulated by cross linking of polymers representing a high water content material. Carbopol, a synthetic polymer has been used in our study, owing to its characteristic controlled delivery of therapeutics and array of biological responses and mechanical strengths [17][18] Our results proved that the process employed to prepare hydrogel formulations in this study was efficient. It was capable of producing formulations with uniform drug content and minimal variability. Both Honey and O. sanctum were dispersed uniformly throughout the hydrogel.
Water-soluble linear polymers like Carbopol are chemically cross-linked to formulate the hydrogel product. The process of making polymer-based physical gel is simple and easy. It involves gentle mixing of the gel components under the appropriate conditions [14].

O. Sanctum
This process has an advantage over other crosslinking methods since,  it can be performed at room temperature,  in physiological pH without using toxic &  hard to remove crosslinking agents [18].
Hence the same method has been employed in our study as it is always safe for clinical applications. Hence we have observed uniform drug content and minimal variability of both Honey and O. sanctum.
The pH of formulated hydrogel was found to be in the range of 4.74, making it slight acidic. This could be due to the acidic property of honey itself; pH of which being between 3.2 and 4.5 [18].
Spreadability is important component in patient compliance as it helps in uniform application of gel to the surface [19]. Spreadability of our formulation was found to be 8.6 ± 0.03 making it an ideal spreadable gel for application.
Swelling index was performed for the formulation initially after 1 h and up to 3 hrs. It was noticed that our formulation showed rapid swelling due to the porous nature of the hydrogels offering large surface area allowing rapid uptake of the solvent but it was found that swelling was reduced. This finding could be attributed to the presence of the honey and carbopol as both the components are viscous in nature. Our findings corelated the relation between swelling and type of the polymer, degree of cross-linking, ionic strength and water content [20].
This is in agreement with our results as the swelling index value was found to be 36% to 44%w/w owing to the presence of carbopol.
In vitro release study showed the release of honey and O. sanctum from the hydrogel resulting in a concentration-dependent increase in the amount of extract diffused through cellophane bag of hydrogel formulation. The results could be attributed to the property viscosity of the gel matrix in the hydrogel.
Our Formulation showed superior and higher sustained drug release, which could be attributed to the property of polymer Carbopol 934. The component is a hydrophilic polyacrylic acid polymer and its carboxyl groups become highly ionized after neutralization with TEA, forming a gel due to electrostatic repulsion among charged polymer [14].

CONCLUSION
Within the limitations of the study, the findings of our in vitro test suggested that the studied Honey -Tulsi based hydrogel could be an economic indigenous substitute which is non-toxic, natural and efficient for clinical application.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.