1. INTRODUCTION:

Recent years, there is an increasing and popular trend of using a variety of naturally derived materials such as biopolymers, drugs, proteins, vitamins, oils (mineral, fixed and essential) for various biomedical, medicinal as well as pharmaceutical uses [1-5]. Since last few decades’, essential oils are being extracted from the numerous natural sources, especially from plant parts and these extracted essential oils are being utilized in different medicinal and pharmaceutical uses [5-7].

Vetiver oil is an essential oil extracted from the roots of the plant, Vetiveria zizanioides, L. [6]. It is extracted by means of steam distillation process from vetiver roots [7-8]. It is extensively used in perfumery, toiletries and cosmetics [10-11]. Since few decades, it has been recognized as traditional medicine as stimulant, carminative, and diaphoretic [7,9]. It possesses various important medicinal activities like anti-septic, anti-inflammatory, nervine, sedative, tonic, aphrodisiac, cicatrisant, vulnerary, etc [8-9]. Because of its sedative activity, vetiver oil has been traditionally employed in the aromatherapy to relieve stress, anxiety, tensions, depression and insomnia [6]. Vetiver oil contains some sesquiterpenes, like khusinol, khusimol, germacrene D, junipene, γ -muurolene, biclovetivenol, viridiflorene, β-vetispirene, β vetivenene, and β-caryophyllene [9-10]. In view of the reported antidepressant properties of vetiver oil, in the present work, we have planned to predict the possible activity of khusinol, khusimol, germacrene D, junipene, γ-muurolene, biclovetivenol, viridiflorene, β-vetispirene, β-vetivenene, and β-caryophyllene with two cannabinoid receptors, CB1 and CB2 via in silico molecular docking in a way to encapsulate vetiver oil in different gellan gum-based microcapsules. The CB1 cannabinoid receptor is present in the central nervous system and expressed in the brain, generally [12]. On the other hand, the CB2 cannabinoid receptor is generally expressed in the immune system as well as in the hematopoietic cells. Both CB1 and CB2 receptors are responsible for antidepressant activity [12].

Gellan gum, sodium alginate, and karaya gum are the natural biopolysaccharides possessing aqueous solubility, biodegradability and nontoxicity [13-19]. Gellan gum is a linear anionic microbial biopolysaccharide extracted via the fermentation by Pseudomonas elodea [14]. The molecular structure of gellan gum consists of linear repeating units of glucose, glucuronic acid and rhamnose in a molar ratio of 2:2:1 [13-14]. It is commercially available in two different forms: native form of gellan gum and deacylated gellan gum [20]. Because of the ionotropic gelation property, deacetylated gellan gum is capable of producing hydrogels by the influence of divalent and trivalent cations [13]. These ionotropically gelled gellan gum-based beads are used to encapsulate various drugs, enzymes, etc [13-14,20]. Like gellan gum, sodium alginate is also linear anionic biopolysaccharide [21]. Actually, it is sodium salt of alginic acid consists of α-l-guluronic acid residue, β-d-mannuronicacid residue, and regions of interspersedboth the residues [18-19]. Sodium alginate is isolated from the brown marinealgae source [21]. Since past few decades, it has been exploited as the matrix biomaterials for the encapsulations of drugs, enzymes, proteins and peptides, oils, etc [18-19, 21-23]. Sodium alginate possesses the property of to undergo ionotropic gelation to produce gels and hydrogels by the influence of divalent and trivalent cations [21]. On the other hand, karaya gum (also known as sterculia gum) is plant derived biopolysaccharide commercially isolated from the plant, Sterculia urens Roxb. (Family, Sterculiaceae) [15-17]. Karaya gum is an acidic biopolysaccharide, which is structurally branched and partially acetylated [17]. It has been exploited in various pharmaceutical, food and cosmeceutical preparations as emulsifier, stabilizer and thickener [15-17]. In the previous literature, any reports on the development and evaluation of such kinds of vetiver oil-encapsulated polymeric microcapsules are not available, which makes this work novel. In addition, in silico molecular docking studies has not been employed in the preparation of vetiver oil-encapsulated polymeric microcapsules. The main objectives of the current research were: (i) to carry out the in silico molecular docking studies to predict the possible activity of khusinol, khusimol, germacrene D, junipene, γ-muurolene, biclovetivenol, viridiflorene, β-vetispirene, β-vetivenene, and β-caryophyllene with two cannabinoid receptors, CB1 and CB2 responsible for antidepressant activity, (ii) prepare and characterize different vetiver oil-encapsulated gellan gum-based microcapsules made of gellan gum only, gellan gum-sodium alginate blends and gellan gum-karaya gum blends, and (iii) to evaluate the antidepressant activity of vetiver oil-encapsulated gellan gum-based microcapsules was investigated in the male Swiss albino mice.

2. MATERIALS AND METHODS:

2.1 Materials:

Gellan gum (Yarrow Chem Products, India) and karaya gum (Yarrow Chem Products, India), sodium alginate (Karnataka Fine Chemicals, India), vetiver oil (Falcon, India), sodium citrate tribasic dihydrogen (Sisco Research Laboratories Pvt. Ltd, India) and n-hexane (Merck Specialities Private Limited, India) were purchased. All other reagents used in this work obtained commercially and were of analytical grade. Throughout the course of investigation, double distilled water was used.

2.2 Molecular docking studies:

The chemical structures of a series of sesquiterpenes from literature such as khusinol, khusimol, germacrene D, junipene, γ -muurolene, biclovetivenol, viridiflorene, β-vetispirene, β vetivenene, and β-caryophyllene were selected for the in silico molecular docking study using Auto Dock Vina program and Auto Dock Tools (The Scripps Research Institute, San Diego, US ). The in silico molecular docked by these selected molecules present in the vetiver oil with CB1 and CB2 receptors were searched.

2.3 Preparation of gellan gum solution:

To prepare gellan gum solution, required percentage of gellan gum (0.9%) was solubilised in double distilled water by using a magnetic stirrer (Remi Motors, India) at 600 rpm.

2.4 Preparation of gellan gum-karaya gum solutions:

To prepare gellan gum-karaya gum solutions, required percentages of gellan gum (0.7-0.8%) and karaya gum (0.1-0.2%) were solubilised in double distilled water by using a magnetic stirrer (Remi Motors, India) at 600 rpm.

2.5 Preparation of gellan gum-sodium alginate solutions:

To prepare gellan gum-sodium alginate solutions, required percentages of gellan gum (0.7-0.8 %) and sodium alginate (0.1-0.2 %) were solubilised in double distilled water by using a magnetic stirrer (Remi Motors, India) at 600 rpm.

2.6 Preparation of oil in water (o/w) emulsions:

Vetiver oil (20 ml) and aqueous solutions of gellan gum, gellan gum-sodium alginate and gellan gum-karaya gum aqueous solutions (10 ml) were mixed separately in beakers and subjected for homogenization using a digital high shear homogenizer (Ultra-Turax, Model T-25, IKA, India) at 6000 rpm speed for 20 min to prepare oil in water (o/w)typeemulsions.

2.7 Determination of emulsion stability:

Prepared emulsions (50 ml) were left standing for 1 h to determine the rate of phase separation, if any. Emulsion stability of these prepared o/w type emulsions was determined depending on the position of phase separation interface. This measurement was done based on the volume of initial emulsion and on the volume of remaining emulsion.

2.8 Encapsulation of vetiver oil:

The gellan gum-based o/w type emulsions made of vetiver oil were extruded via a needle having 0.55 mm into the aqueous solution of calcium chloride (4 %) to form vetiver oil-encapsulated gellan gum-based microcapsules. The tip of the extruder needle was fixed at 15 cm height above the surface of the calcium chloride aqueous solution. The calcium chloride solutions were stirred gently by using a magnetic stirrer (Remi Motors, India) for protecting the prepared microcapsules from sticking. The formed gellan gum-based microcapsules were retained in the aqueous solution of calcium chloride (4 %) for 30 min and dried over night at the room temperature. The formulation chart for vetiver oil-encapsulated gellan gum-based microcapsules is described in Table 1.

2.9 In vitro release study:

In vitro release study of these vetiver oil-encapsulated gellan gum-based microcapsules was performed by a dissolution apparatus of paddle type equipped with six baskets (Electro Lab TDT-08L, India) in phosphate buffer (pH, 7.4). In vitro release was measured at the temperature of 37 ± 0.5°C under 75 rpm paddle speed. Aliquots were withdrawn at customary time intervals, and estimated by using a UV-VIS spectrophotometer (UV-1800, Shimadzu, Japan) at 235 nm (λ_max). Vetiver oil released (%) from the vetiver oil-loaded gellan gum-based microcapsules was plotted as a function of time.

2.10 Size analysis:

To analyze microcapsule size, phase contrast microscope (Ernst Leitz GMBH, Wetzlar, Germany) was used. Microcapsule samples of vetiver oil-encapsulated gellan gum-based microcapsules were placed on a glass slide and observed by the phase contrast microscope.

2.11 Scanning electron microscopy (SEM):

The microcapsule shape as well as surface morphological features of vetiver oil-encapsulated gellan gum-based microcapsules were examined using SEM (JEOL-Datum, JSM-6390, Tokyo, Japan), where an accelerated voltage of 5 KV was used.

2.12 In vivo evaluation of antidepressant activity:

Swiss albino mice (weighing around 24-35 g) were used for the in vivo evaluation of antidepressant activity of vetiver oil-encapsulated gellan gum-based microcapsules of F5 formulation. From an authorized animal breeders and suppliers, animals were procured for the use in this current study. The mice were randomly divided into 5 groups and housed in polyacrylic cages (38 cm×23 cm×10 cm) maintained under standard laboratory environment and standard pellet diet and water ad libitum. Prior to the commencement of in vivo evaluation, the mice were allowed to acclimatize in the laboratory environment for a period of 12 days. This in vivo evaluation using Swiss albino mice was approved by the Institutional Animal Ethics Committee (Protocol No. Sl. no.KCP/IAEC/2/17-18/13/15-04-17; dated: 20.04.2017) and was conducted according to the regulations of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

2.12.1 Forced swimming test:

Swiss albino mice were randomly divided into five groups. After 30 min, the treatment with the vetiver oil-encapsulated gellan gum-based microcapsules of F5 formulation (63 and 82 mg, p.o.) or vetiver oil (68.53 mg) or diazepam (1 mg/kg, i.p.), a standard drug, or distilled water (0.1 mL/mouse, p.o.), mice were individually placed in an open cylindrical container (45 cm height × 20 cm diameter) containing 17 cm of water at 25 °C ± 1 for 5 min. Mice were recorded as immobile when floating motionless or making only those movements necessary to keep the head above water [24].

2.12.2 Tail suspension test:

Swiss albino mice were randomly divided into five groups. the treatment with the vetiver oil-encapsulated gellan gum-based microcapsules of F5 formulation (63 and 82 mg, p.o.) or vetiver oil (68.53 mg) or diazepam (1 mg/kg, i.p.), a standard drug, or distilled water (0.1 mL/mouse, p.o.), mice were suspended 50 cm above the floor using adhesive tape placed approximately 1 cm from the tip of their tails. The duration of immobility time was recorded for 6 min. The mice were considered immobile when they passively hung or stayed motionless [24].

2.13 Statistical analysis:

The data was analyzed by one way ANOVA followed by Turkey’s multiple comparison tests. All the values were expressed as mean± S.D. (standard deviation).

3. RESULTS AND DISCUSSION:

3.1 Molecular docking:

The in silico molecular docking procedure is a computational analysis, which is performed to predict and explore the affinity of the selected molecules to the receptors responsible for specific pharmacological actions [25]. The selected molecules present in the vetiver oil (such as khusinol, khusimol, germacrene D, junipene, γ-muurolene, biclovetivenol, viridiflorene, β-vetispirene, β vetivenene, and β-caryophyllene) were searched for molecular docked pose with cannabinoid receptors (CB1 and CB2) using Auto Dock Vina program and Auto Dock Tools. The results of the docking analyses demonstrated that khusinol, germacrene D, and γ-muurolene were docked in the CB1 and CB2 receptors, molecularly (Fig. 1). This indicated that the vetiver oil-encapsulated formulation could be effective for antidepressant action.

Fig. 1: Overlay of the docked poses of (a) khusinol, (b) germacrene D, and (c) γ-muurolene with that of the CB1 and CB2 receptors

3.2 Characterization of emulsion:

In the current research, gellan gum-based o/w emulsions were prepared using vetiver oil (20%) and polymer (s) (0.9%). Polymer(s) used for the preparation of various gellan gum-based o/w emulsions were gellan gum (only), gellan gum-alginate blends and gellan gum-karaya gum blends. By the o/w emulsification process, the formation of emulsions has been confirmed using light microscopy. The light microscopic images of o/w emulsions for F3 microcapsules (prepared using 0.7% gellan gum and 0.2% karaya gum) and F5 microcapsules (prepared using 0.7% gellan gum and 0.2% sodium alginate) were presented in Fig. 2 (a and b, respectively).

Fig. 2: The light microscopic images of o/w emulsions for (a) F3 microcapsules (prepared using 0.7 % gellan gum and 0.2 % karaya gum) and (b) F5 microcapsules (prepared using 0.7 % gellan gum and 0.2 % sodium alginate)

All the prepared gellan gum-based o/w emulsions of vetiver oil (20%) were found stable. These also displayed excellent stability even after storing overnight at the room temperature. The attainment of the excellent stability of these may be because of the higher viscosities produced by the use of highly viscous hydrophilic gum solutions covering the oil phase by means of controlling the movements and fusing the oil droplets with each other.

3.3 Preparation of vetiver oil-encapsulated gellan gum-based microcapsules:

Vetiver oil-encapsulated gellan gum-based microcapsules prepared using gellan gum-based o/w emulsions were prepared using vetiver oil (20%) and polymer (s) (0.9%), where the polymer(s) used for the preparation of various gellan gum-based o/w emulsions were gellan gum (only), gellan gum-alginate blends and gellan gum-karaya gum blends. The aqueous solutions of calcium chloride (4%) were used to prepare different vetiver oil-encapsulated gellan gum-based microcapsules via the o/w emulsification-ionotropic crosslinking gelation methodology. For the preparation of vetiver oil-encapsulated gellan gum-based microcapsules, the prepared gellan gum-based o/w emulsions were extruded into the aqueous solutions of calcium chloride (4%) via a needle (having a 0.55mm opening channel). The extruded droplets were dripped into the calcium chloride (4%) aqueous solutions, where divalent calcium cations acted as ionotropic crosslinker to form vetiver oil-encapsulated gelled gellan gum-based microcapsules made of gellan gum (only), gellan gum-alginate blends and gellan gum-karaya gum blends.

3.4 Oil encapsulations:

The vetiver oil encapsulation in the gelled gellan gum-based microcapsules was accomplished via the o/w emulsification-ionotropic crosslinking gelation methodology. The vetiver oil encapsulation was calculated and found to be ranged in-between 29.61± 3.18% and 52.69±3.31% (Table 1). From the vetiver oil encapsulation results, it was noticed that the vetiver oil encapsulated microcapsules (F5) prepared using 0.7% gellan gum and 0.2% sodium alginate exhibited highest oil encapsulation (52.69±3.31%) than other formulations. The gellan gum-based microcapsules (F1) prepared using 0.9% gellan gum exhibited lowest oil encapsulation (29.61±3.18%) than other formulations. It was also noticed that with the increment of concentration other blend polymers (karaya gum and sodium alginate), the oil encapsulation was found augmented. This occurrence could be due the fact of viscosity increment that highly viscous layer over the surface of the gelled mucocapsules have been produced a barrier to leach out the encapsulated oil during crosslinking. Moreover, addition of the sodium alginate to prepare vetiver oil-encapsulated gellan gum-alginate microcapsules could have been supported by the availability of more crosslinking sites for ionotropic crosslinking gelation and this fact might be one of the prime causes for the oil encapsulation enhancement when gellan gum-sodium alginate blends were used.

3.5 In vitro release study:

In vitro release study was performed in phosphate buffer (pH, 7.4) at the temperature of 37±0.5°C. The in vitro releases of encapsulated vetiver oil from gellan gum-based microcapsules were found to be sustained over 8 h. The percentage cumulative vetiver oil release vs. time graph is presented in Fig. 3. The vetiver oil encapsulated microcapsules (F1) prepared using 0.9% gellan gum exhibited in vitro releasing of almost all encapsulated vetiver oil (99.64±4.07%) than other formulations. The vetiver oil encapsulated microcapsules (F5) prepared using 0.7% gellan gum and 0.2 % sodium alginate exhibited more sustained in vitro releasing of vetiver oil (72.15±3.27%) than other formulations. The addition of the sodium alginate to prepare vetiver oil-encapsulated gellan gum-alginate microcapsules could have been supported by the availability of more crosslinking sites for ionotropic crosslinking gelation. This could have been produced the more sustained releasing effect than the other formulations. With the increment of concentration of sodium alginate), the oil encapsulation in the vetiver oil-encapsulated gellan gum-alginate microcapsules was almost similar. The vetiver oil encapsulated microcapsules (F5) prepared using 0.7% gellan gum and 0.2% sodium alginate.

3.6 Selection of the best formula:

For further investigation, best formula of vetiver oil-encapsulated gellan gum-based microcapsules was selected. The selection of the best formula was done on the basis of oil encapsulation efficiency and sustained releasing encapsulated oil from the microcapsules. The selected vetiver oil encapsulated microcapsules formulation was F5 microcapsules prepared using 0.7% gellan gum and 0.2% sodium alginate. These vetiver oil-encapsulated gellan gum-alginate microcapsules (F5) exhibited highest oil encapsulation (52.69±3.31%) and more sustained in vitroreleasing of vetiver oil (72.15± 3.27%) than other vetiver oil-encapsulated microcapsules formulations.

Fig. 3: In vitro release of encapsulated vetiver oil from different gellan gum-based microcapsules in phosphate buffer (pH, 7.4) at the temperature of 37 ± 0.5°C. Values are expressed as mean ± SEM (n = 6).

3.7 Size analysis:

The microcapsule size of the vetiver oil-encapsulated gellan gum-alginate microcapsules (F5) was measured by using a phase contrast microscope (Ernst Leitz GMBH, Wetzlar, Germany). The mean diameter of the F5 microcapsules prepared using 0.7% gellan gum and 0.2% sodium alginate was 650µm.

3.8 SEM analysis:

The surface morphology of vetiver oil-encapsulated gellan gum-alginate microcapsules (F5) prepared using 0.7% gellan gum and 0.2% sodium alginate was characterized by SEM analysis. The SEM images of F5 microcapsules at 100 x magnification indicated their spherical shape. The surface morphology of these microcapsules as evidenced in the micrographs at the magnifications of 250 x, 500 x and 1000 x revealed some pores or channels (Fig. 4).

Fig. 4: SEM images of F5 microcapsules indicating spherical shape (a) and surface morphology with pores or channels (b, c and d).

3.9 In vivo evaluation of antidepressant activity:

3.9.1 Forced swimming test:

The forced swim test is a well-known rodent behavioral study generally applied for the pharmacological screening of antidepressant agents, antidepressant effectiveness of newer agents, as well as experimental manipulations, which are intended at rendering or preventing the depressive-like states in the treated animals [26]. In the current study, in forced swimming test, vetiver oil-encapsulated gellan gum-alginate microcapsules (F5)at the dose of 63mg/kg body weight (low dose) and 82mg/kg body weight (high dose) significantly increased (p<0.05) the immobility time (101.15±3.82 sec and 152.45±4.14 sec, respectively) as compared to that of the control (73.59±3.12 sec) (Table 2).

Group	Dose/kg body wt	Immobility time (sec)^#
Control	Vehicle	73.59 ± 3.12
Diazepam	1 mg	196.82 ± 5.24***
Encapsulated polymeric microcapsules	63 mg	101.15 ± 3.82**
Encapsulated polymeric microcapsules	82 mg	152.45 ± 4.14***
Vetiver oil	2 ml (68.53 mg)	165.26±4.91***

^#Values are expressed as mean ± SEM (n = 6). EPM: Encapsulated polymeric microcapsules *p < 0.05; p<0.01; p<0.001 compared to control (One way ANOVA followed by Tukey’s multiple comparison test)

However, the diazepam (1mg/kg) as standard drug showed high immobility time (196.82±5.24 sec), while vetiver oil (2ml/kg) significantly increased the immobility time (165.26±4.91 sec) as compared to that of by vetiver oil-encapsulated gellan gum-alginate microcapsules (F5)at the dose of 63mg/kg body weight (low dose) and 82mg/kg body weight (high dose). The comparative results of forced swimming test are presented in Fig. 5.

Fig. 5: Immobility time (sec) attained in the forced swimming test of vetiver oil-encapsulated gellan gum-alginate microcapsules (F5)

3.9.2 Tail suspension test:

The tail-suspension test is a well-known mouse behavioral study widely employed in the pharmacological screening of antidepressant agents evaluating other manipulations that are anticipated to influence the depression associated behaviors in mice [27]. In the tail suspension test, vetiver oil-encapsulated gellan gum-alginate microcapsules (F5)at the dose of 63 mg/kg body weight (low dose) and 82 mg/kg body weight (high dose) significantly increased (p< 0.05) the immobility time (122.39±4.21 sec and 162.28±5.15 sec, respectively) as compared to that of the control (91.25± 3.53 sec) (Table 3).

Group	Dose/kg body wt	Immobility time (sec)^#
Control	Vehicle	91.25 ± 3.53
Diazepam	1 mg	231.69 ± 6.55***
Encapsulated polymeric microcapsules	63 mg	122.39 ± 4.21**
Encapsulated polymeric microcapsules	82 mg	162.28 ± 5.15***
Vetiver oil	2 ml (68.53mg)	181.35±5.99***

^#Values are expressed as mean ± SEM (n = 6). EPM: Encapsulated polymeric microcapsules *p < 0.05; p<0.01; p<0.001 compared to control (One way ANOVA followed by Tukey’s multiple comparison test)

However, the diazepam (1 mg/kg) as standard drug showed high immobility time (231.69±6.55 sec), while vetiver oil (2ml/kg) significantly increased the immobility time (181.35±5.99 sec) as compared to that of by vetiver oil-encapsulated gellan gum-alginate microcapsules (F5)at the dose of 63mg/kg body weight (low dose) and 82mg/kg body weight (high dose). The comparative results of forced swimming test are presented in Fig. 6. In terms of the immobility time, vetiver oil-encapsulated gellan gum-alginate microcapsules (F5) prepared using 0.7 % gellan gum and 0.2% sodium alginate has shown significant antidepressant activity.

Fig. 6: Immobility time (sec) attained in the tail suspension test of vetiver oil-encapsulated gellan gum-alginate microcapsules (F5)

Values are expressed as mean ± SEM (n = 6). EPM: Encapsulated polymeric microcapsules *p < 0.05; p<0.01; p<0.001 compared to control (One way ANOVA followed by Tukey’s multiple comparison test)

4. CONCLUSION:

The current investigation demonstrated the prediction of the possible activity of khusinol, khusimol, germacrene D, junipene, γ-muurolene, biclovetivenol, viridiflorene, β-vetispirene, β-vetivenene, and β-caryophyllene with two cannabinoid receptors, CB1 and CB2 via in silico molecular docking in a way to encapsulate vetiver oil in different gellan gum-based microcapsules for antidepressant activity. The results of the docking analyses demonstrated that khusinol, germacrene D, and γ-muurolene were docked in the CB1 and CB2 receptors, indicating that the vetiver oil-encapsulated formulation could be effective for antidepressant action. Different vetiver oil-encapsulated gellan gum-based microcapsules were prepared via the o/w emulsification-ionotropic crosslinking gelation methodology. In vitro release study of vetiver oil-encapsulated gellan gum-based microcapsules exhibited sustained oil releasing pattern over 8 h. On the basis of oil encapsulation efficiency and sustained releasing encapsulated oil from the microcapsules, the vetiver oil-encapsulated gellan gum-alginate microcapsules (F5) prepared using 0.7% gellan gum and 0.2% sodium alginate was selected as best formulation for further study. These vetiver oil-encapsulated gellan gum-alginate microcapsules (F5) exhibited highest oil encapsulation (52.69±3.31%) and more sustained in vitro releasing of vetiver oil (72.15± 3.27%) than other vetiver oil-encapsulated microcapsules formulations. The in vivo antidepressant activity of vetiver oil-encapsulated gellan gum-alginate microcapsules (F5) prepared using 0.7% gellan gum and 0.2% sodium alginate was evaluated by forced swimming test and tail suspension test using Swiss albino mice. The results have shown significant antidepressant activity of vetiver oil-encapsulated gellan gum-alginate microcapsules (F5). These vetiver oil-encapsulated gellan gum-alginate microcapsules (F5) were found capable to release the encapsulated oil in a sustained manner over a prolonged period, in vivo, indicating the significant antidepressant potential for the sustained effects.

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Received on 19.10.2019 Modified on 23.12.2019

Research J. Pharm. and Tech. 2020; 13(7): 3135-3142.

DOI: 10.5958/0974-360X.2020.00554.5

Formulation	Gellan gum (%)	Karaya gum (%)	Sodium alginate (%)	Calcium chloride solution (%)	Vetiver oil (%)	Oil encapsulation (%)*
F1	0.9	-	-	4	20	29.61 ± 3.18 %
F2	0.8	0.1	-	4	20	30.69 ± 3.54 %
F3	0.7	0.2	-	4	20	37.69 ± 3.03 %
F4	0.8	-	0.1	4	20	50.38 ± 3.37 %
F5	0.7	-	0.2	4	20	52.69 ± 3.31 %