INTRODUCTION:

Curcumin a polyphenolic compound, available in the rhizome of Curcuma longa L., has been used traditionally for centuries in Asia for medicinal, dietary and other purposes^[1].It has strong anti-oxidant, anti-inflammatory, anti-carcinogenic, anti-microbial, anti-parasitic and other activities^[2]. Curcumin derived from turmeric (Curcumin longa) is being used form thousands of years as a healing agent for variety of illnesses^[3]. Research over the last few decades has indicated that curcumin is a potent anti-inflammatory agent with strong therapeutic potential against a variety of cancers^[4].

A large number of in vitro and in vivo studies in both animals and man have indicated these activities. Safety evaluation studies indicate that curcumin is well tolerated at a very high dose without any toxic effects^[5]. Most drugs currently available for the treatment of cancer have many constrain due to poor solubility and associated diminished bioavailability that leads to overdose toxicity apart from inefficient therapeutic action. In economic point of view, higher doses cost more expenses in the procurement for the beneficiary. As these drugs are obtained from natural resources, meeting out constant supply against the demand become difficult. Poor yield on purification is another issue that depends on expression of secondary metabolites by plants and physical stability of compounds^[6]. Hence, global community seeks a drug with either high potency or efficient formulation for market driven product than the just known for the pharmacological activity.

It is well established that poor solubility that diminishes the bioavailability of drugs is a major problem for drug development and formulation^[7]. The formulation of poorly water-soluble drugs has always been a challenging problem faced by pharmaceutical scientists. About 40% of the new chemical entities being discovered is poorly water-soluble^[8]. These drugs are poorly soluble in both aqueous and organic media and have a log P value greater than 2. Several approaches to enhance the solubility of curcumin such as chemical derivatisation, complexation or interaction with macromolecules and cyclodextrin have been reported but not successful yet in practical utility.

Nanosuspension is one of the successful methods in improving drug dissolution^[9]. In the recent years, there have been considerable interests in developing nanoparticles as effective drug delivery system that can effectively deliver the drug to a target site and increase the therapeutic effect^[10]. Nanosuspensions can be defined as colloidal dispersions of nano-sized drug particles that are produced by a suitable method and stabilized by a suitable stabilizer^[11]. They are unique because of their simplicity and best over other application strategies^[12].

There are many methods available for the production of nanosuspension formulation like media milling, high-pressure homogenization, dry co-grinding, malt emulsification and so on, but they are expensive^[13].At this backdrop development of a new methodology for the cost effective formulation without compromising the quality and bioavailability become essential. Hence, a study was designed to prepare the nanosuspension formulation by a simple novel procedure and to analyze its quality characters and bioavailability parameters.

MATERIALS AND METHOD:

Preparation of Extract:

The commercially available food graded rhizomes of C. longa (200 g) was purchased from local market. It was broken into small pieces and covered by the filter paper. This material was placed inside the soxhlet extractor. About 500 ml of Acetone was used for this process. The extract obtained from Soxhlet apparatus was transferred to rotary vacuum evaporator. At 40°C solvent was completely removed. The remaining residue was collected aseptically and recrystallized for purity then kept in the airtight container at 4°C.Mass ratio and yield were calculated from the measured weights of raw material and yield. The crystalline nature of curcumin was confirmed by X-ray powder diffractometry (XRPD).

Characterization of physical properties of curcumin:

XRD was performed by X’pert Pro with Spinner PW3071 using Ni filtered, CuKα radiation, a voltage of 40 kV, and a current of 30 mA with a scintillation counter. A portion of recrystallized sample was subjected for Differential scanning calorimeter (DSC) analysis for the presence of curcumin by taking the sample on an open aluminum pan and scanned under a nitrogen purge with a heating rate of 10°C/min from 20°C to 250°C. Each measurement was performed in triplicate and the average and standard deviation were calculated.

Preparation of Nanosuspension:

Bottom up method:

In bottom up technology the poor water soluble drug is dissolved in a nonpolar solvent then added to polar solvent to form precipitate. This experiment prepared this nanosuspension by bottom up method using saturation solubility with acetone as nonpolar solvent and water as polar solvent. About 200 mg of curcumin crystal was dissolved in 10 ml of acetone. Then the solution was added drop wise to 100 ml of distilled water which was kept on a magnetic stirrer for continuous stirring.

Standardization of formulation with different sonication methods:

Ultrasonic homogenizing is a mechanical means of breaking micro particles to nano and sub-nano size levels. The specific size and particles uniformity depends on the conditions followed during this process. The prime factors that maintain this characteristic feature of nanosuspension formulation are intensity and pulse rate of ultrasonic wave at a given period of time. In this experiment, different level of intensities, pulse rate of ultrasonic wave and preset time were set using (Ultrasonic homogenizer model – 3000). The resultant nanosuspension obtained was subjected for characteristic analysis such as particle size by and stability. The process was repeated many times till the desired quality of nanosuspension is achieved. The parameters and conditions were noted for the reproducibility of nanosuspension preparation for further applications.

Characterization of Nanoparticles:

The particle size and distribution of particles of prepared curcumin nanosuspension was measured by Dynamic Light Scattering principle using a particle size analyzer Malvern MAL1054413 - Zetasizer Ver. 6.20 at CARISM - SASTRA University, Thanjavur. The stability of the nanosuspension was observed by Zeta potential analyzer.

Pharmacokinetics and bioavailability of prepared nanosuspension:

Materials:

The experiment was conducted in CPCSEA (Committee for the purpose of control and supervision of experiments on animals) approved animal house after obtaining the prior approval from the Institutional Animal Ethics Committee (IAEC). Approval Number: PRIST/IAEC/Ph.D./CRD/01-2013-2014. Two weeks old, healthy, laboratory bred, Swiss albino Wister rats (Species: Rattusnorvegicus) of either sex were maintained under standard laboratory conditions with acclimation period about 7 days. Body weight about 180g ± 10% and the living accommodation with temperature 22 ± 3° C, Humidity 30 – 60 %, Light / dark cycle 12 / 12 h with Ad libitum access to food (commercially available Normal feed) and (RO) water were maintained.

As the curcumin has well established data for toxicity and effective dose it was considered for the present experiment in order to avoid animal usage, save money and labour^[14]. In general, any formulation needs toxicity and effective dose data for pharmacokinetic study. In this study, it is not necessary because, the study is designed to ensure not to reach lethal dose even if the formulation achieve 100% efficiency and did not allow to take any chemical modification to key ingredient curcumin.

In vivo studies:

The aim of this experiment is to study the rate of bioavailability and level of curcumin in serum after a single oral dose. There are two different test materials given orally to two different groups having 12 animals each shown in Table 1.

A common vehicle used for the curcumin administration was aqueous media. It is distilled water which contains 300mg/dl SDS. 300 mg/dl of SDS is about 200 times lesser than the LD50 (Rat: 1288 mg/Kg body weight) dose of SDS were added to 100 ml distilled water to prepare the stabilizer with concentration having 300mg/dl of SDS.

Group – I (Non formulated Curcumin), in this group 1.0 ml aqueous media loaded with 250 mg / kg body weight of raw (purified crystalline) curcumin was given presented in Table 2.

Table 2: Dosage Calculation of Non-Formulated Curcumin

Weight of the animal	Calculated Dose 250mg/kg	Stock (100mg/ml) solution (ml)	Normal saline (ml)	Total Volume (ml)
172	43	0.43	0.57	1
174	43.5	0.43	0.57	1
178	44.5	0.44	0.51	1
182	45.5	0.45	0.55	1
175	43.75	0.43	0.57	1
180	45	0.45	0.55	1
185	46.25	0.46	0.54	1
187	46.75	0.46	0.54	1
177	44.25	0.44	0.56	1
179	44.75	0.44	0.56	1
183	45.75	0.45	0.55	1
186	46.5	0.46	0.54	1

Table 3: Dosage Calculation of Formulated Curcumin

Weight of the animal	Calculated Dose 250mg/kg	Stock (100mg/ml) solution (ml)	Normal saline (ml)	Total Volume (ml)
181	45.25	0.45	0.55	1
184	46	0.46	0.54	1
173	43.25	0.43	0.57	1
177	44.25	0.44	0.56	1
180	45	0.45	0.55	1
186	46.5	0.46	0.54	1
183	45.75	0.45	0.55	1
173	43.25	0.43	0.57	1
175	43.75	0.43	0.57	1
178	44.5	0.44	0.56	1
188	47	0.47	0.53	1
187	46.75	0.46	0.54	1

Group – II [Formulated Curcumin], in this group 1.0 ml aqueous media loaded with 250 mg / kg body weight of formulated (Nanosuspension)curcumin was given presented in Table 3.

Analytical Procedure:

A validated sensitive and selective method of High-performance liquid chromatography (Shimadzu LC 8A with variable wavelength UV detector) was used for the determination of curcumin. A reversed phase C 18 analytical column (4.6 × 250 mm, particle size 5 μm), with mobile phase consists of 10 mM ammonium acetate pH 4.5 and pure Acetonitrile. The ratio of ammonium acetate ranging from 95% to 55% with the flow rate 1 mL/min at 45 ± 2°C was maintained for 20 min. The elute was monitored at 420 nm. Retention time for standard curcumin at the condition was standardized, it was 7.5 min. The recorded absorbance at 420 nm was considered as reference concentration. Thus, calibrated HPLC was used for the determination of serum curcumin obtained from pharmacokinetic experiment.

Statistical evaluation:

Curcumin in the blood samples were quantified by HPLC. The Area Under the Concentration time curve (AUC) was drawn and evaluated statistically using unpaired ‘t’ test for significant improvement in bioavailability of formulation.

RESULTS:

Curcumin yield:

About 10.122 g of curcumin was obtained from 200g of dried rhizome of C. longa.

Physical parameters:

X-ray powder diffraction (XRPD):

The obtained curcumin was crystalline in nature according to 2(θ) value (18°) of XRD pattern. The crystalline size (D = 200 nm) was calculated using Scherer’s formula D = 0.9λ/β cos θ (Fig.1).

Figure 1: X-ray powder diffraction of curcumin

Differential scanning calorimetry:

Melting point was found to be 183°C by differential scanning calorimetry. That confirmed the presence of curcumin (Fig. 2).

Figure 2: Differential scanning calorimetry of curcumin

Standardization of formulation with different sonication methods:

The formulation was standardized with fixed frequency and intensity of sonication wave which was able to produce by the instrument (Ultrasonic homogenizer model – 3000) at the mid-level performance. This condition was constantly maintained throughout the experiment. The pulse rate of ultra-sonication treatment was set at a striking/silence about 10/10 sec for a one complete cycle. Among the number of trials like 10, 20, 30, 40, and 50 cycles the 30 cycle (about10 min) was found to be optimum for the desired particle size (193 nm) given in Table 4.

Table 4: Particle Size Optimization

Pulse rate (Striking / silence for one complete cycle)	Cycles (Number of trails)	Particle size (nm)
10/10 sec	10	100
	20	160
	30	193
	40	200
	50	250

Characterization of Nanoparticles - Particle size Analysis and Zeta potential analysis:

The particle size of the prepared suspension had the mean diameter of 193 ± 122.8 (1SD) nm. The entire suspension had the particle size range 60 - 200 nm (Fig. 3).

Figure 3: Particle size analysis of formulated curcumin

The zeta potential of curcumin nanosuspension was -16.1 ± 2.8 mV respectively (Fig. 4).

Figure 4: Zeta potential analysis of formulated curcumin

HPLC analysis:

The retention time of standard curcumin was 7.5min at 44551mV (Fig. 5).

Figure 5: HPLC of standard curcumin

Statistical evaluation:

Statistical evaluation showed that there is a significant (P < 0.05) improvement in bioavailability and specific pharmacokinetic property for the prepared formulation. It reveals that curcumin nanosuspension has more bioavailability than the non-formulated curcumin. A polynomial trendline was drawn in such a way to have an average for the results obtained against the time of sample taken for analysis using MS-excel program (Fig. 6).

Figure 6: Pharmacokinetics of curcumin

DISCUSSION:

Modern world is often looking for chemically synthesized antibiotics to eradicate infections but many viruses challenge the health sector of the world such as Influenza, Ebola, Dengue, Chikungunya and so on. At the same time, if we are able to increase the efficacy of some of the natural products like curcumin as equivalent to the efficacy of such chemically synthesized antibiotics that will minimize the toxicity and cost of the drug.

Nanosuspension was prepared by means of bottom up technology^[15,16,17]. The bottom-up technology is an assembling method from molecules to nanosized particles. It is widely accepted in the area of nanoscience and nanotechnology^[18,19]. The basic challenge of this technique is that during the precipitation procedure the growth of the crystals needs to be controlled by the addition of stabilizers to avoid Agglomeration or Aggregation that develop the formation of microcrystals^[20]. The determination of the zeta potential of a nanosuspension is essential as it gives an idea about the physical stability of the nanosuspension^[21].The zeta potential of a nanosuspension is governed by both the stabilizer and the drug. It is an index of the magnitude of the electrostatic repulsive interaction between particles^[22,23]. Pharmacokinetics has a strong influence in the absorption, distribution, metabolism and excretion properties of the drug. Bioavailability refers to the extent and rate at which a drug or substance becomes available to the target tissue after administration^[24,25]. The oral route is preferred for drug delivery because of its numerous well-known advantages^[26,27]. Because the oral route is the most convenient, safest, least expensive and requires no special training^[28,29]. The efficacy or performance of the orally administered drug generally depends on its solubility and absorption through the gastrointestinal tract^[30,31]. This enhancement in bioavailability will lead to a subsequent reduction in drug dose and rendering the therapy cost effective^[32,33,34].

To determine the role of particle size influences in the bioavailability enhancement, formulations containing different size of nanoparticles may be prepared by standardization protocol developed in this study. Pharmacokinetic study may be performed for bioavailability, which will explain the relationship between the particle size and bioavailability on comparison of formulations efficiency. In order to avoid numerous such trials, the efficiency achieved by this experiment (for mean particle size 193 nm) was compared with other experimental data (for mean particle size 200, 250, 306, 375 and 633 nm) already available in literature for a similar oral test dose of 250 mg/Kg body weight. Among all of these, the prepared formulation in this study showed better bioavailability.

We conclude that the prepared formulation has achieved the nanosize and the characterization studies were proved to be good and it has good stability. The present work clearly demonstrated that curcumin nanosuspension has better bioavailability than the non-formulated curcumin. The data obtained from this pharmacokinetic study make it worth for further exploration in many previous efficacy study outcomes and application strategy in new dimension

ACKNOWLEDGEMENT:

We sincerely acknowledge the Center for Research and Development, PRIST University and Centre for Fundamental Cognizance and Logical Science, Thanjavur (CFCL) for the utilization of research facilities.

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Received on 09.09.2018 Modified on 10.11.2018

Research J. Pharm. and Tech. 2019; 12(6): 2615-2620.

DOI: 10.5958/0974-360X.2019.00438.4

Time (min) After oral dose	Group I -Non formulated Curcumin	Group II -Formulated Curcumin	Blood collection
0	Animals 1-12	Animals 1-12	1-2 ml blood collected by retro-orbital puncture
30	Animals 1and 2	Animals 1 and 2
60	Animals 3 and 4	Animals 3 and 4
90	Animals 5 and 6	Animals 5 and 6
120	Animals 7 and 8	Animals 7 and 8
150	Animals 9 and 10	Animals 9 and 10
180	Animals 11 and 12	Animals 11 and 12