A Novel Proniosomal Platform for Delivery of Lantana Camara Extract as A Natural Antimicrobial Agent

Figure 1: Structure of Proniosome

PREPARATION OF PRONIOSOMES

Figure 2: Preparation of Proniosomes

MATERIALS USED FOR THE PREPARATION OF PRONIOSOMES

SURFACTANT: 

Surfactants, especially non-ionic surfactants are the key structural components in the preparation of proniosomes. These surfactants do not have any charge as they possess a polar head and non-polar tail. So, their stability, toxicity and compatibility is higher than other surfactants. The non-ionic surfactants have wet and emulsifying effects by which they improve the solubility and permeability of drugs. The hydrophilic-lipophilic balance (HLB) value is critical for selecting surfactants and HLB value between 4 and 8 is compatible with vesicle formation by proniosomes. It is difficult for hydrophilic surfactants to achieve a high concentration because of the high liquid solubility of hydrophilic surfactants.

CHOLESTEROL: 

Cholesterol can interact with non-ionic surfactants and regulates the physical and structural properties of proniosomes. It improves the stability and rigidity of the proniosomal membrane and controls drug permeation through the membrane. Depending on the HLB value of the surfactants, the amount of cholesterol required for the preparation of proniosomes is determined. When the HLB value is above 10, the amount of cholesterol to be increased to cover the larger groups. But entrapment efficiency (EE) of the prepared formulation is decreased above a certain level of cholesterol, possibly due to a decrease in volume diameter.

 HYDRATION MEDIUM: 

Generally, the hydration medium used in proniosomes is phosphate buffer. Depending on the solubility of the encapsulated drug, the pH of the buffer is selected. Ascertained that drug leakage increased with the increase in the volume of hydration medium but simultaneously, EE increases, when the hydration time was increased from 20 to 45 min.

ORGANIC SOLVENT: 

The solvent can act as a penetration enhancer. It also greatly affects the size of the vesicles formed. The size of the vesicle and permeation rate of the drug in a proniosomal formulation are influenced by the type of alcohol. Different sized vesicles are formed using different alcohols as they have the order: >> isopropanol < butanol < propanol < ethanol. [3]

METHOD OF PREPARATION OF PRONIOSOMES

COACERVATION PHASE SEPARATION METHOD:

In this method, accurately weighed amount of surfactant, carrier (lecithin), cholesterol and drug are taken in a clean and dry wide mouthed glass vial (5 ml) and solvent is be added to it followed by simple mixing. To prevent the loss of solvent, the open end of the glass vial can be covered with a lid and heated over water bath at 60-70ºC for 5 minutes until the surfactant dissolved completely. The mixture should be allowed to cool down at room temperature till the dispersion gets converted to a proniosomes. [4]

LANTANA CAMARA

Plants generally produce many secondary metabolites which are biosynthetically derived from primary metabolites and constitute an important source of chemicals which are used as pharmaceuticals, agrochemicals, flavours, fragrances, colours, biopesticides, and food additives. [5]

 Lantana camara Linn, belongs to family Verbenaceae, commonly known as wild or red sage, is the most widespread species of this genus and regarded both as a notorious weed and a popular or ornamental garden plant. [6]

L. camara oil and extracts are used in herbal medicine for the treatment of various human diseases such as skin itches, leprosy, cancers, chicken pox, measles, asthma, ulcers, tumours, high blood pressure, tetanus, rheumatism etc.[7]

Lantana camara L. (Verbenaceae) is a woody straggling plant, commonly known as red or wild sage, with various flower colours, leaves with rounded tooth edges which comprises 650 species and indigenous to tropical and subtropical America.[8]

L. camara grows in tropical, subtropical, and temperate regions at a high altitude up to 2000 m. The plant has a woody stem with several different colours of flowers i.e., red, white, pink as well as the plant contain spines or prickles.

Lantana camara possesses therapeutic potential because of various bioactive components, including steroids, triterpenoids, oligosaccharides, iridoid glycosides, naphthoquinones, and phenylpropanoid glycosides. [9]

Lantana camara is an erect vigorous shrub that usually grows up to a height of 4 meters. The leaf is ovate or ovate-oblong shaped with a size 2-10 cm (length) and 2-6 cm (width). Leaves are green and tough with fine hairs and have a pungent odour, and it have the ability to climb up to 15 meters with the help of support. It can easily grow in favourable conditions, and flowers usually appear in the month of March and August. The colour of the fruit is green and drupaceous with two nutlets. Mature plants produce up to 2000 seeds annually. The roots of  lantana  camara are very strong, having a main taproot with many small side roots.

 Parts Used: Apart from the whole plant, seeds, stem, root, leaves and flowers are also used.

LANTANA CAMARA LEAVES

Figure 3: Lantana camara leaves

Although all the samples seemed to present homogeneous profile, a quantitative chemical variability was observed, with a significant variation of the contents of the major components. The chemical compositions of the samples were characterized by a high proportion of sesquiterpene hydrocarbons such as (E)-b- caryophyllene (40.8%) and a-humulene (21.2%), monoterpene hydrocarbons such as sabinene (up to 9.0%).It is noteworthy that the oil of Blokauss was poor in sabinene (0.4%) in opposition to others samples. Four other components were present at appreciable contents: bicyclogermacrene (7.9%), germacrene D (6.9%) and a- pinene (4.4%) as well as b-elemene (up to 3.5%). By contrast, oxygenated compounds are poorly represented: linalool (0.4–1.9%), sesquithuriferol (0.3–1.7%), panthenol (0.2–1.5%), (E)-nerolidol and t-cardanol (0.0–1.0%, respectively). The contents of caryophyllene oxide reached 4.9% in one sample.[10]

METHODOLOGY

COLLECTION OF PLANT MATERIAL

SOXHLET EXTRACTION PROCESS:

The leaves were dried under shade for 8 Days, done powder by using Mortar and Pestle and we passed leaves powder in Sieves (Mesh No 22).

• Preparation of Extracts

The lantana camara leaves collected weighed (100gm), grinded leaves powder are added into the thimble apparatus and add (1000ml) methanol in a round bottom flask. Set up for Soxhlet apparatus for  extraction  and started to boil up to 40^oc.

        The set up Soxhlet apparatus for 6 to 7 cycles repeatedly until the complete extraction was done. Content was filtered out; filtrate was allowed to evaporate in evaporating pan until the desired concentration of extract was obtained.[11]

UV Spectroscopic Analysis: Selection of analytical wavelength of Lantana camara extract: With appropriate dilution of the standard stock solution with methanol, the solution was scanned using the double beam UV visible spectrophotometer (Labman LUV-2000T) in the spectrum mode between the wavelength ranges of 600 nm to 200 nm and the spectrum was recorded. From the spectrum, the wavelength of maximum absorbance (λmax) of the drug was identified against the blank. The λmax of pure drug was found to be 240 nm

Figure 4: UV graph of lantana camera methanol extract

FORMULATION:

Table 1: Development of Formulation

COACERVATION PHASE SEPARATION METHOD

• In a beaker, the surfactant and cholesterol were mixed with 0.5 ml of absolute ethanol and then 25 mg of drug was added.
• Then, the beaker is covered with a lid to prevent the loss of solvent and the beaker was warmed in a water bath [55˚C - 60˚C ]for 5 min while shaking until the complete dissolution of cholesterol.
• Then, about 0.16 ml of hot distilled water [55˚C - 60˚C] was added while warming in water bath for 3-5 min till a clear or transulent solution was produced.
• The mixture was allowed to cool at room temperature until the dispersion was converted to gel.[12]

EVALUATION OF PRONIOSOME

Organoleptic evaluation

Physical parameters such as colour, appearance and texture were carried out. Colour and texture were evaluated by vision and touch sensation respectively. For odour evaluation a team of five odour sensitive persons were selected. [13]

Physicochemical evaluation

Physicochemical parameters were determined, including the determination of pH, surface morphology and particle size by optical microscope, zeta potential, viscosity and spreadablity. [14]

• Surface morphology & particle size by optical microscope

The proniosomes were mounted on glass slides and viewed under a microscope. The microscope has a magnification of 10x/40x used for morphological observation. The photomicrograph of the preparation was obtained from the microscope by using a digital Single lens reflex (SLR) camera. The particle size analysis of the formulations was performed using an optical microscope with eye micrometer. [15]

• Viscosity

About 10 g of proniosomal was taken in a tube and 10 ml of distilled water was added on it at temperature 28.2℃ and spindle type 2 and 12 RPM. Proniosomal gel viscosity was determined by LMDV60 Viscometer. [16]

• Spreadability

For the determination of spreadability, excess of sample was applied between the two glass slides and was compressed to uniform thickness by placing 500 gm weight for 5 min. Weight (50 gm) was added to the pan. The time required separating the two slides, i.e. the time in which the upper glass slide moves over the lower plate was taken as measure of spreadability (S).

Spreadability (g.cm/s) (S) = M×L/T

Where M = weight tied to upper slide, L = length moved on the glass slide, T= time taken.

• Zeta Potential

 Sample preparation: dilute approximately 3 ml of proniosome dispersion in 10 ml of distilled water. A zeta sizer to determine the surface charge [zeta potential] of the vesicles . Zeta potential value of + or – 30 mV or higher typically indicates good colloidal stability.

• Drug Entrapment Efficiency

The formulation was taken in a test tube and reconstituted with 10ml of PBS [ph=7.4]. This aqueous solution was sonicated in a sonicator bath for 10 minutes; the drug containing proniosomes were separated from the unentrapped drug by centrifugation at 25,000 rpm for 30 min at 20 degree. The supernatant was removed and diluted with PBS. The drug concentration in the resulting solution was removed was assayed by the UV SPECTROSCOPY at 240 nm.

The percentage of drug encapsulation was calculated by using the following equation.

EP% = [Ct –Cr]  \ [Ct] *100   

      Ct =is the concentration of total drug .

Cr = is the concentration of free drug.

Preparation of phosphate buffer of [ph 7.4]

Dissolve 6.8 g of potassium dihydrogen orthophosphate,1.56 g of sodium hydroxide was adjusted to 1000 ml with distilled water.

In Vitro Diffusion Study

 The vitro release study using Franze diffusion cell assembly

It consists of two compartments , one of the receptor chamber contains a phosphate buffer of saline ph 7.4 and another donar compartment containing proniosomal gel of 25 mg of drug .

A dialysis membrane [molecular size 12000 -14000] was previously soaked for 24 hours.

The dialysis membrane was placed in contact with PBS filled in the receptor compartment to avoid disruption in the on going process. It was ensured that no air bubbles were seen between the dialysis membrane and the liquid surface of PBS.

The temperature was maintained at 37ºC + 0.5 at 50 rpm using a magnetic stirrer. 0.5 ml of sample was withdrawn from the receptor chamber slide tube at the time interval of 1 hr, 2 hr ,4 hr,6 hr ,8 hr, 12 hr and 24 hours and equilibrated with a new fresh dissolution medium to main to a sink state. Suitable dilution was carried out and spectroscopically analyzed at lamda max of 240 nm using UV visible spectroscopy.[16]

RESULT AND DISCUSSION

ORGANOLEPTIC EVALUATION RESULTS

Table 2: Organoleptic Evaluation Results

Proniosome was evaluated for organoleptic parameters showed in the Table 2. The colour of formulation was Light greenish. The odour of prepared formulations was pleasant. Appearance was smooth, Texture and smoothness was acceptable as per requirement of cosmetic formulations.

PHYSICOCHEMICAL EVALUATION RESULTS

Table 3: Physicochemical Evaluation Results

Proniosome was evaluated for physicochemical parameters showed in the Table 3.

The pH of formulations was found close to neutral.

Surface morphology was observed through optical microscope, vesicles were observed in all formulations, understood that drug was entrapped clearly into vesicles. The particle size was measured by stage micrometer and size was found in the range of   2 - 16μm.

Spreadability is good in all the formulations.

     Viscosity was found in the range of 2400 to 2712 mPas.

Zeta potential was found in the range of -5.4 mV to -17.2 mV suggests that the proniosome gel particles have a negative surface charge.

      Surface morphology images:

LC1

LC2

LC3

LC4

Figure 8 : Proniosome vesicles under optical microscope

ZETA POTENTIAL GRAPHS:

Figure 9: Zeta potential graph of LC1 Formulation

Figure 10: Zeta potential graph of LC2 Formulation

Figure 11:  Zeta potential graph of LC3 Formulation

Figure 12:  Zeta potential graph of LC4 Formulation

DRUG ENTRAPMENT

Table 4: Results of Drug entrapment efficiency Study

Entrapment efficiency of proniosomes formulations ranged from 42.12% to 56.21 %. Proniosomes formed from span 60 and tween 80 combination (1:1) proniosomal gel exhibits higher EE.

Figure 13:  Drug entrapment efficiency graph

IN VITRO DIFFUSION STUDY

Table 5: Results of In-vitro Diffusion study

The results of In Vitro diffusion were shown in Table 5. The percentage drug release of all prepared formulation is in the ranging from of 94.771 to 99.649% of 24 hr.

Figure 14: In vitro diffusion release graph