1. INTRODUCTION:

Plants having medicinal importance have been used from long time for the treatment of various diseases. Phyto-constituents although present in various plant parts but they have limited therapeutic utility due their large size and poor absorption therefore resulting in poor bioavailability¹. Problem of bioavailability can be resolved by formulating the plant extract in lipid carrier like liposomes and Phytosomes. Phytosomes are vesicular system in nano range that binds plant extract with phospholipid to formulate a complex that is fat soluble to increase its bioavailability and therapeutic availability. Phytosomal composition offers better availability of plant extract in terms of its absorption through intestinal lipoidal membrane than the herbal extract itself. Andrographis paniculata is one of most widely used plant for treatment of various illnesses like common cold, fever and liver infection².

All parts of the plants are used for extraction of therapeutically potential phytoconstituents, the major bioactive constituent present in almost all parts of the plants particularly in leaves is andrographolide having broad therapeutic potential including anti-inflammatory, antidiabetic activity, anti-allergic, antiplatelet aggregation and hepatoprotective activity. In biological system andrographolide proposed to interact with inter and intracellular constituent to produce multiple biological responses. One of the prime reasons of serious health issues and transience in individuals is liver disease due to number of reasons including alcohol consumption and drug induced liver toxicity, due to lack of safer hepatoprotective drugs in modern medicine one has to rely on herbs obtained from plants for the management of liver disorders³. Andrographis paniculata is claim to have hepato stimulant and protective activity and is constituent of numbers of polyherbal formulation used as liver tonic. In the proposed research work an attempt have been done to formulate Phytosomes of methanolic extract of Andrographis paniculata for its improved availability for hepatoprotective activity. Phytosomes are vesicular drug delivery systems used for forming complex between phospholipid and standardized plant extract for the improvement of bioavailability. Phospholipids used in the formulation of Phytosomes are cellular component which are biodegradable and is reported to have hepatoprotective activity⁴.

2. MATERIALS AND METHODS:

Materials:

The leaves of Andrographis paniculata were collected from local area of University Institute of Pharmaceutical Sciences, Chandigarh and were identified by Dr. Alok Gupta of NIPER, Mohali with accession number NIP- H-302.

Chemical and reagents:

The chemicals and reagents used in the given research work were of analytical grade and procured from SD Fine Chemicals, Mumbai. Andrographolide standard 95% (Batch No - T12G001) was procured from Natural Remedies, Bangalore.

Methods:

Extraction of method:

250gm of dried leaves of Andrographis paniculata were placed in a thimble and 1000ml of methanol solvent was used for extraction and was placed in a round bottom flask. The Soxhlet extraction was done for 18 hours till liquid in a siphon tube was become a clear solution. The extracted solvent was evaporated under reduced pressure and obtained as dried powder of extract⁵.

Standard calibration curve:

For the preparation of standard calibration curve, 100 mg of andrographolide dried powder was dissolved in 100ml of 50:50 mixture of methanol and water with concentration (100µg/ml), from the prepared standard stock solution working solutions were prepared in concentration ranging from 50-250µg/ml and the absorbance of resulting solutions were assessed at 321 nm using double beam UV Spectrophotometer⁶.

Formulation of Phytosomes:

The Phytosomes were prepared by thin film hydration method. Accurately weighed amount of soya phospholipid and methanolic drug extract were mixed in different molar ratios as given in the composition (Table 1). This was dissolved in mixture of 20ml dichloromethane and 30ml of methanol as solvent. This was refluxed for 2hrs at 60ºC until a clear solution was obtained; the solution was then transferred to round bottom flask to evaporate the solvent using Rota-evaporator to form a thin film. The film so formed was then hydrated using 25ml of phosphate buffer pH 7.4 to form suspension of phyto-phospholipid complex. The complex so formed was then freeze dried and stored in amber colored bottle for further evaluation⁷.

Table: 1 Composition of Phyto-phospholipid complex of Andrographis paniculate leaves extract

Formulation Code	Methanol (ml)	Dichloromethane (ml)	Drug extract: Soya-lecithin molar ratio	Phosphate buffer pH 7.4 (ml)
PF1	30	20	1: 0.5	25
PF2	30	20	1: 1	25
PF3	30	20	1: 1.5	25
PF4	30	20	1: 2	25
PF5	30	20	1: 2.5	25

Characterization of Phytosomes:

FTIR analysis of Complex:

FTIR spectral analysis was taken to study the structural and chemical stability of extract, Phospholipid and phytosomal complex formed. Spectral scanning was done in the range between 4000 - 500 cm^-1. using Bruker Alpha T instrumentation following KBR pellet method.

Percentage Yield of the phyto-phospholipids complex:

The percentage yield of the Phyto-phospholipid complex was calculated by considering the difference in weight of initial Phyto-constituent and free Phyto-constituent after complex formation. It gives the amount of the Phyto-constituents involved in complex formation.

Initial Weight - Free Weight

% Yield = ------------------------------------X100

Initial Weight

Particle Size and Drug entrapment efficiency (EE):

The particle size and size distribution of Phytosomes was assessed. The average particle size of Phytosomes ranges from 50nm to 100μm. The particle size and size distribution were assessed by zeta potential value which measured with the help of Malvern particle size analyzer. Drug entrapment efficiency was found by weighing 100mg of Phytosomes after drying which was dispersed in a 50ml of distilled water and stirred for 2 hours; it was then filtered off using Whatman filter paper of pore size 0.45μm. It was then suitably diluted and analyzed using double beam UV spectrophotometer at λ _max321nm.

Differential Scanning Calorimetry (DSC):

The DSC defines as amount of heat energy absorbed and released by a sample on heating, cooling or held at a constant temperature. The sample was sealed in aluminum crimp cell and heated at the rate of 10ºC/min at the temperature ranging from 0-400ºC and peak transition temperature of Phyto-phospholipid complex formed was read⁸.

In-vitro dissolution studies:

In-vitro dissolution studies were performed on Phytosomes at 37°C (±0.5°C) at rpm of 100 using USP Dissolution Assembly II paddle type (Electro-lab). An accurately weighed amount of phytosomal suspension was taken in the cellophane bag which was suspended in dissolution media which consists of 900ml of phosphate buffer of pH 7.4. The aliquot (5ml) of dissolution solutions were withdrawn at a specified interval of time to examine the amount of drug released using spectrophotometrically at a wavelength of 312nm⁹.

Scanning Electron Microscopic Analysis (SEM):

The Phytosomes were coated with gold palladium for 1100 seconds to get a thin film of 20nm under an inert atmosphere (Coater polaron, 18mA at 1.4 kV). The coated samples were examined by using scanning electron microscope (Variable Pressure Scanning Electron Microscope, Hitachi S3400N) for studying surface morphology^10,11.

Stability Studies:

The stability studies of optimized phytosomal formulation were done. The prepared Phytosomes were subjected to stability studies at a temperature and relative humidity of 40±2°C/75± 5% RH and 32±2°C/60± 5% as per ICH guidelines for the time period of 3 months¹².

RESULTS AND DISCUSSION:

Standard calibration curve:

Standard calibration curve of methanolic extract of Andrographolide paniculate leaves was plotted as shown in Fig 1. With R² value 0.9817.

Figure 1: Standard calibration curve andrographolide paniculate leaves extract

Evaluation of Phytosomes:

FTIR analysis:

The formation of Phyto-phospholipid complex is demonstrated by comparison of FTIR spectra of phospholipid, Phyto-constituent alone and physical mixtures to that of complex formed as given in Fig. 2. FTIR spectra of Phyto phospholipid complex PF4 formulation indicated no physical interaction between the phytoconstituent and phospholipid.

Percentage Yield, Particle size and Entrapment efficiency (EE):

The percentage yield of different formulations of Phyto-phospholipid complexes was estimated. The average percentage yield of all the formulations was found to be in the range of 73.18 - 88.43%, with PF4 formulation having maximum percentage yield of 88.43%.The results particle size, Percentage yeild and polydispersity index of different phytosomal formulations are mentioned Table 2. The formulation PF4 has good particle size of 145.6nm and polydispersity index (PDI) of 0.247. Value of PI less than 0.5 suggest good stability of phytosomal complex formed.

Table 2: Percentage yield, Particle size and Polydispersity Index of different Phytosomal Batches (PF1- PF5)

Formulation Code	Percentage yield	Particle size	Polydispersity index (PDI)	Zeta Potential (mV)
PF1	87.23 %	129.8 nm	0.411	-28.3±0.8
PF2	73.18%	122.8 nm	0.320	-29.0±0.51
PF3	85.34%	134.7 nm	0.340	-30.1±0.23
PF4	88.43%	145.6 nm	0.247	-31.5±0.12
PF5	84.23%	125.7nm	0.342	-27.4±0.26

The optimum zeta potential value for stable phytosomal complex is greater than ±30 Mv. The zeta potential value of PF4 is -31.57mV as given in Fig. 4 confirms the stability of Phytosomes and poly dispersity index (PDI) values 0.247. The value lies in the given range indicated that Phytosomes are highly stable. The entrapment efficiency of Phytosomes formulations PF1, PF2, PF3, PF4 and PF5 was found to be 72.01±0.2%, 75±0.8%, 81.75±0.23%, + 83.3±0.23% 70.34±0.2% respectively.

Differential scanning calorimetry (DSC):

DSC thermogram of andrographolide extract and that Phyto-phospholipid complex formed is shown in Fig.3 respectively. DSC thermogram of methanolic extract of andrographolide showed sharp endothermic peak at 135ºC which is a characteristic of amorphous material. Whereas there is no such peak was observed in DSC thermogram of Phyto-phospholipid complex formulation indicating the complete dissolution of andrographolide in phospholipid complex.

Figure 3: DSC thermogram of Phyto- phospholipid complex

SEM studies:

Surface morphology of andrographolide’s phytosomal formulation assessed by SEM as given in Fig. 4 indicated the spherical shape of the vesicles and size in accordance with particle size determination data.

Figure 4: Surface morphology of Andrographolide’s phytosomal formulation PF4

In vitro Drug Release:

In vitro drug release profile of PF1- PF5 Phytosomal formulations as shown in Fig. 5 indicated that PF4 formulation with 1:2 drug to phospholipid ratio gives sustained release of 50.34 % in 180 minutes.

Figure 5: in vitro drug release of phytosomal formulations (PF1-PF5)

Stability Studies:

Stability testing performed on formulation PF4 showed no significant difference in in-vitro drug release characteristics for the period of 3 months on withdrawal of samples after, 15,45,60 and 90 days and was found to be stable.

CONCLUSION:

The results of various experimental finding indicated that Phytosomes formulation (PF4) having ratio 1:2 was stable with uniform particle size distribution. Various analytical studies including FTIR, SEM confirm the formation of stable phytosomal complex. In vitro dissolution study of the selected formulation showed extended-release pattern for 180 minutes giving 50.34 % release. Therefore, it is concluded the Phyto-phospholipid complex of Andrographis leaves extract have got potential for the improvement of bioavailability of andrographolide for which detailed pharmacokinetic studies need to be carried out so that novel formulation with improved therapeutic potential can be explored and developed.

CONFLICT OF INTEREST:

Declared None.

ACKNOWLEDGMENT:

The profound gratitude to Chitkara College of Pharmacy, Chitkara University, Punjab, India, for providing essential services to carry out study.

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