INTRODUCTION:

Curcumin an active ingredient of a much known spice, turmeric (Curcuma longa), used in cooking in whole India and also other regions of Asiawhich belongs to ginger family (Zingiberaceae) having number of pharmacological effects including anti-inflammatory, antioxidant, anti proliferative and antiangiogenic activities. Commercially curcumin contains approximately 77% diferuloylmethane, 17% demethoxy curcumin and 6% bisdemethoxycurcumin ^1-7.

In spite of that the efficacy and safety of curcumin has not yet been approved as a therapeutic agent, and the relative bioavailability of curcumin has been highlighted as a major problem for this.

The reasons for low bioavailability of any agent inside the body are low intrinsic solubility, poor absorption, and high rate of metabolism, inactivation of metabolic products and/or rapid elimination and clearance from the body^8-10. Regarding the distribution of curcumin, it showed its accumulation in the intestine, colon and liver, so this is one of the major reasons why it is showing most promising in-vivo effects in gastrointestinal diseases when compared with other organ systems. The liver, and to a lesser extent the intestinal mucosa are the major organs responsible for metabolism of curcumin. On absorption, curcumin gets converted into metabolite form i.e., glucuronides and sulfates or it is reduced to hexahydrocurcumin^11,12. How to improve the bioavailability of curcumin is a burning issue. The roles of adjuvant which can block metabolic pathways of curcumin is one of the major means that are being used to improve its bioavailability^13,14. Nanoparticles, liposome, micelles, and phospholipids complexes are other promising new formulations, which appear to provide better bioavailability of curcumin. The main goal of any drug delivery system is to achieve desired concentration of the drug in blood or tissue, which is therapeutically effective and non toxic for a prolonged period ¹⁵.

MATERIALS AND METHOD:

Soy lecithin was purchased from HiMedia laboratories pvt. Ltd., cholesterol from lob a chemie pvt. Ltd. curcumin from Natural remedies Bangalore, piperine from Alfa Aesar, Uk and all other chemicals are of AR grade and HPLC grade and used as received.

Preparation of liposome:

Liposomal containing curcumin were prepared according to the thin film hydration method of Bangham et al ^4,16,17. In this method the curcumin, piperine, soya lecithin and cholesterol were dissolved in 10 ml methanol and chloroform mixture (9:1 ratio) and the solution was taken in a round bottom flask. The flask was kept in a rotary shaker in low speed so that upon evaporation of solvent a thin film was formed. To ensure complete evaporation of organic solvents the flask was kept overnight. Then to form the vesicles the dried film was hydrated with 30 ml of normal saline solution and vortexed for one hour (above the gel – liquid transition temperature of the soya lecithin). Upon hydration a milky suspension was formed. To reduce the vesicle size the liposomal suspension was exposed to ultrasonic irradiation with an output of 50 watt for 3 cycles of 1 minute each.

Table:1 Composition of liposome

Compositions of liposomal formulation (mg)	Formulation code
Compositions of liposomal formulation (mg)	F1	F2
Curcumin	250
Curcumin + Piperine (10:1)		250
Soya Lecithin	900	900
Cholesterol	200	200
Methanol : Chloroform (v/v)	9:1	9:1
Normal Saline Solution (ml)	30	30
Method Used	Thin film hydration	Thin film hydration

Mean particle size and Zeta potential measurement:

Mean vesicle size and zeta potential of prepared liposomes was measured by using Zeta Sizer 300HSA (Malvern Instrument, Malvern, UK).

Drug Entrapment Studies:

The percentage drug entrapped (PDE) was determined by Ultra-centrifugation. The liposomal formulations were subjected for ultracentrifugation (Ultra Centrifuge. Remi laboratories, Mumbai, India) at 11000 rpm for 30 min in an ultracentrifuge in order to separate the entrapped drug from the free drug. Then the clear supernatant was separated and analyzed for drug content after appropriate dilution by UV-Visible Spectro photometer. This indicates amount of free drug. The liposomal pellet was redispersed in methanol and analyzed for drug content after appropriate dilution by UV-Visible Spectrophotometer ^18-20. This indicates amount of drug entrapped. The entrapment capacity of liposomes was calculated as follows.

PDE = [(T-C)/T] ×100

Where T is the total amount of drug that is detected both in the supernatant and sediment, and C is the amount of drug detected only in the supernatant.

In-vitro drug release studies:

In-vitro release studies were performed using dialysis membrane method. It was soaked in warm water at 45° C for 30 minutes before using it for release study. This membrane was then carefully clamped to one end of the hollow glass tube and considered as the donor compartment. The dissolution medium i.e., phosphate buffer solution pH 7.4 (200ml) was taken into the receiver compartment. The donor compartment was immersed into the receiver compartment so that the edge just touched the receiver compartment. Before the release test, 0.5 ml of curcumin formulation was diluted to 3 ml in release medium and placed into the hollow glass tube as the donor compartment at 37⁰c at 100 rpm by using magnetic stirrer and bead. Samples (5 ml) were removed from the receptor compartment at predetermined intervals and replaced with fresh medium immediately. The samples were analyzed using UV-visible spectrophotometer at 420 nm. Drug release was monitored for 12 hrs ^20-22.

Pharmacokinetic study for determination of curcumin:

In-vivo study was investigated in Sprague dawley rat. All the animal’s experiments were conducted according to the rules and guidelines of committee for the purpose of control and supervisions of experiments on animals (CPCSEA).The study was approved by Institutional animal ethical committee.

Construction of curcumin standard graph:

For the evaluation of pharmacokinetics of the curcumin formulations in rat a standard graph of curcumin was developed by following method. 100 mg of pure curcumin was dissolved in 100 ml of methanol and sonicated for 10 mins to form stock solution. From that 10 ml was taken and diluted with same solvent methanol up to 100 ml and again it was sonicated to form standard solution. From standard solution further dilution were performed i.e., 0.5 ml was transferred from the standard solution into test tubes and diluted to 10 ml with methanol to form 5micorgram/ml. The solutions so prepared (5microgram/ml, 10microgram/ml, 15 microgram/ml, 20microgram/ml and 25 microgram/ml). The samples were analyzed in HPLC at 420 nm wavelength taking acetic acid: acetonitrile (90:10) ratio as a mobile phase at a flow rate of 0.5 ml /min using C18 column. The retention time and peak area are noted and data obtained through HPLC analysis was further used to interpolate the experimental peak area values to get the corresponding concentration of curcumin in plasma.

Animals were divided into 4 groups

Group 1 received: - liposomal curcumin

Group 2 received: - liposomal curcumin+ pure piperine

Group3 received:- liposomal curcumin: piperine formulation

Group 4 (standard) received: - pure curcumin

Blood samples (0.5ml) from the experimental rats were collected by retro orbital plexus technique into a series of micro centrifuge tubes containing 0.3 ml of sodium citrate solution. Blood samples were collected at different time intervals like 30 mins, 1 hr, 2hrs, 3hrs, 6 hrs, 12hrs, 18 hrs, 24 hrs. The collected blood sample were centrifuged at a speed of 5000 rpm for 10 mins and plasma was separated into another micro centrifuge tube by using micropipette and stored in deep freeze until analysis. The drug was extracted out from plasma by adding in a methanol solvent and it was centrifuged for 10 mins from which the organic layer of drug comes out other than sediment. The organic layer of drug was then injected into the HPLC system for the further processing of determination of plasma drug concentration and other parameters i.e., AUC, Cmax etc to report the oral bioavailability enhancement of the drug ^16-18,19.

RESULT AND DISCUSSION:

Surface charge:

1 particles, as the zeta potential increases, the repulsive interactions will be larger leading to the formation of more stable particles with a more uniform size distribution^14,17.

Table: 2 evaluation parameters of prepared liposome.

Formulation code	Zeta potential (mV)	Mean Particle size (nm)	% Encapsulation efficiency
F1	-26.15 ±0.231	198.26±3.56	66.51±1.28
F2	-26.30 ±0.243	235.56±4.69	68.35±1.95

The in-vitro drug release of drug curcumin from liposome formulations was carried out by using dialysis membrane in 7.4 pH phosphate buffer for 12 hrs. The in-vitro release profile of obtained for formulation, are shown in Table-3 and in Fig-1, respectively.

Table:3 In-vitro release profile of liposome formulation F1 and F2

S. No	Time (hr)	Cumulative percentage Drug Release
S. No	Time (hr)	F1	F2
1	0.5	2.87	2.52
2	1	4.35	4.54
3	1.5	7.93	8.06
4	2	10.34	10.58
5	3	16.74	18.08
6	4	25.51	26.74
7	6	37.38	35.16
8	8	46.13	48.67
9	10	59.391	62.32
10	12	74.752	76.86

Fig:1 Drug Release profile

In-vivo study:

Standard graph of curcumin by HPLC:

Table no:4 Standard graph of curcumin

S.No.	Concentration (μg/ml)
1.	5	1837.65
2.	10	3786.3
3.	15	5412.85
4.	20	7138.4
5.	25	9087.15

Fig:2 Standard graph of curcumin for pharmacokinetic evaluation

Fig: 3 Plasma drug concentration vs time profile.

From the in-vivo study its very much clear that there are certain scope to improve the bioavailability of curcumin. As compare to pure curcumin liposomal curcumin shows better bioavailability. Which shows that novel drug delivery system have the potential to improve the bioavailability. When adjuvant was added with it then also AUC value increased. In next step piperine was also entrapped in liposome along with curcumin and found that it shows better bioavailability and improved C_max value. Which indicates that liposomal drugs release it slowly thus metabolic pathway is also blocked for longer period of time thus circulation time for curcumin was increase.

CONCLUSION:

The present work deals with development of liposomal drug delivery system for curcumin, an isolate from curcuma longa. Liposomes were prepared by using phospholipid as carrier. From all the formulations, F2 was selected as best formulation due to its ideal particle size high entrapment efficiency and desirable drug release. Further there is a need to improve the dissolution rate of the curcumin liposomes. When it used with adjuvant it shows better in-vivo parameters. One such ideal approach is to use an effective bioenhancers in the formulation. Hence in future, further work has to be carried out by applying multiple concepts like application of novel drug delivery system and aid of adjuvant in curcumin formulation.

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Received on 30.03.2016 Modified on 28.04.2016

Research J. Pharm. and Tech 2016; 9(8):1059-1063.

DOI: 10.5958/0974-360X.2016.00200.6

Parameters	Curcumin liposome formulation	Curcumin liposome + pure piperine	Curcumin and piperine liposome	Pure Curcumin
Cmax(μg/ml)	3.362	14.724	20.17	2.501
Tmax(h)	6	6	6.7	3
AUC0∞ (μg/ml/h)	51.3	223.373	419.409	47.724
MRT	13.813	15.22	20.52	13.261
Kel’	0.0723	0.0657	0.04873	0.0754