Synthesis of tea tree oil microcapsules via microencapsulation using novel technique

Tea tree oil (TTO) is widely known essential oil extracted from Melaleuca alternifolia leaves naturally having antimicrobial and antibacterial activities. Due to its highly volatile nature it rapidly evaporates causing loss of efficiency and shorten the effects. Microencapsulation technique was incorporated to ensure the core material is being protected from the immediate contact with the environment and offers controlled release. In this study, microencapsulation of Tea Tree Oil was done by employing complex coacervation technique using Chitosan - Gum acacia system as the coating material and utilized tannic acid as the crosslinking agent. All the materials used in this process are from natural sources which are safe for the human and the environment. In designing the operating process condition for TTO encapsulation, we found that wall ratio of 2:5 and 3.6 pH gave the best yield along with better efficiency. The proposed method studied the surface morphology of the microcapsules with an efficiency and yield of 84.50% and 69.9 % respectively.


Introduction
It's an essential oil which is extracted from Melaleuca alternifolia leaves [1].Melaleuca alternifolia is a small tree up to 15m high with papery bark and bushy crown.It is indigenous to Australia and naturally occurs along the northern New South Wales coast, which borders Queensland [2].Native populations in Australia used the extracts of the Melaleuca alternifolia for the treatment of skin infections [3].In 1920s Penfold and Grant published a report on the antimicrobial properties of Australian essential oils, which lead to the commercial production of tea tree oil [3].The studies have suggested that tea tree oil was a more efficacious antibacterial agent than phenol, which was the most common antimicrobial agent during that period.For several decades, tea tree oil was produced by the hand cutting the plant material followed by distillation [4].In 1970s and 1980s the natural products become popular, high density, commercial plantations of tea trees were established in Western Australia, Queensland, and New South Wales that produced the essential oil after extraction from cut and chipped plant material.Extraction was done by steam distillation from terminal branches and leaves of Melaleuca alternifolia.The clear to pale yellow oil is separated from the watery distillate after condensation.The yield from the wet plant material was found to be 1 to 2% [4].After distillation the oil has to be improved in order to raise its commercial worth and conform to criteria.Vacuum distillation is most commonly preferred for the refinement of TTO as it limits the degradation of heat sensitive volatiles [5].According to the regulation for composition by international organization for standardization standard ( ISO4730) Tea Tree Oil (Melaleuca alternifolia) has reported to have around 100 components of various concentration [6].The composition of oil is mainly of terpene hydrocarbons, which majorly consist of monoterpenes, sesquiterpenes and the alcohol that as associated with it [7].Terpin-4-ol is the major component of Tea Tree Oil which has the specific anti-microbial activity [6].The major components of Tea Tree Oil with their maximum allowed concentration terpinen-4-ol(48.0%),-terpinene(28.0%),1,8-cineole(15.0%),-terpinene(13.0%),-terpineol(8.0%),and p-cymene (8.0%).There are different chemotypes of M. alternifolia, which has distinct chemical composition.These includes terpinen-4-ol chemotype, 1,8-cineole chemotype, terpinolene chemotype, among them terpinen-4-ol chemotype is commercially used in Tea Tree Oil production [8].Tea Tree Oil is a transparent, pale yellow to colorless oil which has a characteristic odor.This oil is soluble in no solvents and has a specific gravity ranging from about 0.885 -0.906 g/mL.Although terpinen -4 -ol is soluble in water, α -terpinene & γ -terpinene are poorly water soluble.Tea tree oil undergoes photo -oxidation when exposed to light, moisture, heat, or air during storage [9] Contact allergy has been frequently reported which are caused due to TTO.Compared to all the aromatic oils, it has caused most of the allergic reactions.According to the patch test which was carried by the Australian study it showed that 17 of 41(41%)patients were considered to be relevant.Among the 17 patients only 4 of them (24%) had used cosmetic products which contained tea tree oil, 20% of the patients from 41% specified the application of neat Tea Tree Oil.It was seen that in most of the cases of sensitization, contact allergy the cause was neat application of neat tea tree oil usually for therapeutic purpose.Localized allergic dermatitis, which may blister and ooze, is the main effect of this.It could remain localised or affect the entire body.TTO's capacity to cause sensitization in both people and animals has been thoroughly studied.It was seen that fresh TTO is less to moderately sensitizer, compared to the aged oil because oxidation increases its sensitizing potency.Skin sensitization was seen to enhance due to irritancy.Peroxides, endoperoxides, and epoxides-which are typically produced in small quantities but are made as a result of the oxidation of terpinen-4-ol andterpinene-are produced as a result of the oxidation reaction, which causes alteration in the components.The most frequently reacting sensitizers in TTO appear to be ascaridole, terpinolene, terpinene and its oxidation products, 1,2,4-trihydroxymenthane, -phellandrene, and limonene.Other chemicals that can be responsible for the allergy are mycrene, D-carvone, terpinen-4-ol, aromadendrene, L-carvone, viridiflorene, sabinene, p-cymene and 1,8-cineole.The bulk of sensitizers were found in commercial TTOs at low amounts or not at all, for instance (e.g.ascaridole, which is formed during the oxidation of TTO, and 1,2,4-trihydroxymenthane, which is formed during the ageing process).Microencapsulation is the process of coating or entrapping one or a combination of materials in another material or system.The material used for coating is defined as shell/ wall material/encapsulating agents, while the material which is been coated is defines as active/ core material [10].Under desired circumstances, the core substance progressively diffuses through the capsule walls and provides controlled release properties [11].Type of core material majorly influences the shape of the microcapsules, such as solid or crystal core leads to irregular shape microcapsules whereas liquid core produces spherical capsules.Microcapsules can also be formed of multi-wall or multi-core [12].The physical and chemical properties of microcapsules are influence by the coating material, so certain parameters are required to consider while selecting the coating material, like its physical and chemical properties , its stability , method for encapsulation, etc [13].In general, the method should be straightforward, quick, efficient, and simple to use.There are different microencapsulation techniques which can be used, but the technique used to encapsulate tea tree oil is complex coacervation technique.In this technique two or more biopolymers are used which are oppositely charged.The driving force for this coacervation is attractive force between oppositely charged polymers [14].It majorly depends on pH , ionioc strength and poly ion concentration [15].The concentration of biopolymers, each biopolymer's structure, and the biopolymer network structure formed during coacervation all affect the heat and mechanical properties of coacervates [16].The process is affected by pH, temperature, ionic strength, stirring rate, biopolymer ratio, molecular weight, and biopolymer concentration, is necessary for coacervate synthesis [17].The materials used for walls that are made from various natural and synthetic polymers have been used to encapsulate oils and volatile substances.The choice of wall material is based on a number of factors, including compatibility, control mechanism, economic factors, and the physicochemical properties of the core (such as solubility and porosity) and the wall (such as molecular weight, viscosity, mechanic properties, film-forming and emulsifying properties) [18].Chitin is converted to chitosan (CH), the second most abundant polysaccharide in the world, through alkaline N-deacetylation.Because of its biocompatibility and lack of toxicity, chitosan has a particularly promising future in the food sector.In chitosan, which is a heterogeneous binary polysaccharide, 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose residues make up the majority of the compound.The latter residue gives the compound its cationic charge at acidic pH levels.Chitosan's characteristics in a solution are influenced by its molecular weight, degree of deacetylation, pH, and ionic strength [19] [20].The glucosamine segments' pKa value ranges from 6.3 to 7.35.Chitosan assumes an extended conformation and rapidly rises in intrinsic viscosity at low pH and low ionic strength due to significant electrostatic segment-segment repulsion.The rotational flexibility of its chains is also comparatively high for a polysaccharide polyelectrolyte.Flexibility is however constrained when compared to that of polyelectrolytes with a hydrocarbon backbone because of the bulky sugar rings [20].Due to its high solubility and low viscosity at high concentrations, as well as its favourable emulsifying and microencapsulating capabilities, gum Arabic (GA), a negatively charged polyelectrolyte, is frequently utilised in industry [21].It is an arabinogalactan made up of three separate fractions, each with a different molecular weight and protein concentration [22].GA has a primary galactan chain with extensively branched galactose/arabinose side chains, according to the composition study.D-galactose (40%) L-arabinose (24%), L-rhamnose (13%), and two forms of uronic acids-D-glucuronic acid (21%) and 4-O-methyl-Dglucuronic acid (2%), which give the gum its polyanionic properties-make up the carbohydrate moiety.%).This polysaccharide may have a "wattle blossom"-like structure with a number of polysaccharide units connected to a single polypeptide chain, according to certain theories [23].This characteristic gives it strong surface activity and the capacity to produce viscoelastic films [24].The GA molecule is fairly globular, but depending on how much the uronic acid units or their salts are ionically dissociated, it may have an open shape and even exist in a coiled form.These carboxyl groups will mostly dissolve in the typical salt form at pH values close to neutral, and the consequent Coulombic repulsion of the negatively charged carboxylate groups will result in the molecule taking on an open, highly charged, enlarged shape [25].There were several works reporting microencapsulation of tea tree oil using different technique along with the different wall material, but there not such work reported using chitosan and gum acacia as the wall material for the microencapsulation tea tree oil and even the use of tannic acid as the crosslinking agent is also not have been reported with the above combination of wall material & core material.This study aimed to determine the factors that would produce the best complex coacervation yield between chitosan (CH) and gum acacia (GA), encapsulate tea tree oil (TTO) in complex coacervate matrices, and characterise several physicochemical properties of the complex coacervates and of the tea tree oil microencapsulates, including morphology, solubility, entrapment efficiency, and encapsulation yield.
Tree tea oil microcapsules was prepared according to the method described by with modification.The mass ratio between the tea tree oil and the biopolymer was fixed according to the method.In order to study two system of encapsulation, the tea tree oil was slowly added into the dispersion.Different ratios of two biopolymer i.e Chitsoan and Gum acacia was taken.Chitosan was dissolved in 0.1N acetic acid, 100ML solution was prepared by continuous stirring.Similarly gum acacia solution was prepared by dissolving it in distilled water.The pH was maintain between 4.0-3.6 by adding 0.1N HCL.Chitosan solution and Tea tree oil was slowly drop by drop added to the gum acacia solution with continuous stirring at room temperature.After 1 hr of continuous stirring, the temperature of the solution was gradually decreased by keeping the solution in the ice bath with stirring till the temperature drops below 10ºC.After the temperature goes below the required range crosslinking agent i.e tannic acid(10% w/v) solution was added drop wise for 1hr with continuous stirring.The microcapsules were separated by decanting the supernatant and the coacervates was dried in the oven.

Determination of Encapsulation Yield
The complicated coacervation process depends in part on the biopolymer mixing ratio.By measuring coacervate yield, the impacts of GA/CH ratios on the development of complex coacervation were assessed.By preparing the biopolymer dispersions separately and setting their pH to the ideal interaction pH, which would encourage quick electrostatic contact between the biopolymers, the mixing procedure was used to create the coacervates.The ideal pH values that were employed to produce the greatest amounts of GA/CH complex coacervates were fixed at 3.75 and 3.6, respectively.Vacuum filtration was used to separate the coacervate phase, and the separated coacervate was then collected and dried at 70 °C until the weight remained constant.The coacervate yield was calculated according to the following equation: Encapsulation yield (%) =   × 100 Where, Mo is the total biopolymers weight used to make the biopolymers solutions and Mi is the weight of the dried coacervate phase.

Determination of Encapsulation Efficiency
The encapsulation efficiency is one of the important parameter which was calculated using the techniques mentioned in literature.Initially, the dried microcapsule weight was determined by immediately drying 1 gm of sample microcapsules at 70 °C until the weight remained constant.To wash the liquid that was outside the microcapsule, 1ml of n-hexane is applied to 1g of sample weight for the washed microcapsules.After shaking on a tube stirrer for five minutes, the liquid was centrifuged at 5000 rpm.The microcapsules were collected and dried at 70°C until the weight remained constant after being washed three times with deionized water.Encapsulation Efficiency = Where, Md is weight of the dried microcapsules and Mw is weight of the washed microcapsules.

Moisture Content
By oven-drying the microparticles at 105 °C until they reached a consistent weight, the moisture content of the particles was quantified gravimetrically.

Morphological Studies
Morphological studies are usually done to study the shape, particle size, etc of the microcapsules.In morphology of the microcapsules its Particle Size and size distribution was analysed using optical microscope.

Results and Discussion
The results for the morphological study are shown below along with the optical images of the microcapsules.The coacervate yield and the encapsulation efficiency is calculated according to the mentioned formula.

Determination of Encapsulation Yield
The table 1 below shows the weight of the dried microcapsules along with the weight of the total biopolymer used i.e weight of chitosan and weight of gum acacia.So with the help of the formula mentioned above coacervate yield was calculated, there is increase in the yield, which showed that there is electrostatic interaction between the essential oil and the biopolymer materials (Fig 3

Determination of Encapsulation Efficiency
Table 2. listed the weight of the dried microcapsules along with the weight of the washed microcapsules.With the help of the formula mentioned above encapsulation efficiency was calculated and was plotted which showed that, there is increase in the encapsulation efficiency as shown in(fig.4).

Conclusion
For centuries, due to their diversity in composition and biological activity, essential oils have provided some of the most important health benefits to people all over the world.Until now, essential oils have been used in a variety of industries as a natural and healthy alternative to synthetic chemicals.One of such essential oil is tea tree oil.It has been used for the treatment of skin infections before the arrival of Europeans.After the publication, reported by Penfold and Grant, people started to know more about its broad spectrum antimicrobial activity.Because of which, it was used directly into natural products.But after doing more research it was found that tea tree oil causes contact dermatitis which was caused majorly because of the direct application as well as from cosmetic application, due to this tea tree oil can be encapsulated using various techniques.By encapsulating, the properties of tea tree oil are improved as well as its activity is improved.In this study coacervation techniques was used along with the different ratios of the wall materials(CH:GA): TTO, the studies showed that (2:5):2 (w/w) gave the maximum encapsulation yield as well as better encapsulation efficiency.The morphological studies showed that the microcapsules that are formed are spherical in shape and the size is also in the required range.The microcapsules found to be stable at room temperature.This encapsulated tea tree oi microcapsules can be further used in the application of cosmetic, food, and pharmaceutical fields.

4. 1 .
MorphologyOptical microscopy was used to evaluate and compare the morphological structure with TTO.All the images showed that the microcapsule are spherical in shape as shown in Fig1 and 2. The images showed no evidence of cracking or fissures in the microparticles superficies.The below images show the microcapsules at 10X and 45X of optical microscope.

No Weight of dried microcapsules Weight of total Encapsulation Yield
). Table1.Encapsulation Yield of the microcapsules along with the weights of the dried microcapsules and the weight of the biopolymers i.e Chitosan and Gum Acacia.Sr.

Table 2 .
Encapsulation efficiency of the microcapsules are calculated with the help of the weight of the dried microcapsules along with the weight if the washed microcapsules.