INTRODUCTION:

Currently, Curcumin is a crystalline compound with numerous therapeutic effects. It is a polyphenol obtained from herb Curcuma longa mailnly used in food products¹. Its effectiveness is reported as anti-inflammatory, antirheumatic, anti-oxidant, anti-viral, anti-microbial, blood purifier and anti-tumor agent etc.^2-5. THC, the most active analog of curcumin, colorless or off-white colored was first identified in 1978⁶. It is produced by the reduction of curcumin by hepatic reductase in the liver⁷. The pharmacological activities of tetrahydrocurcumin are quite similar to curcumin⁸.

The anti-diabetic effects by increasing the plasma insulin level are shown better by THC compared to curcumin⁹. Curcumin has low bioavailability, in many research piperine has been used in combination with the curcumin to enhance its bioavailability¹⁰. The THC is also less available but its oral bioavailability is higher in tissue and plasma than compared to curcumin¹¹. Many colloidal formulations have been developed in recent years to overcome the problems associated with both the drugs^12-13. Curcumin shows higher solubility in the alkaline medium as compared to the acidic medium, the fact is its solubility increase with an increase in pH¹⁴. Despite the increase in solubility, degradation of curcumin also increases with an increase in pH of solution¹⁵. THC is more stable in 0.1 M phosphate buffers, especially at pH 7.2 (37°C) than curcumin¹⁶. In one of our study we have validated a method for curcumin only using ethanol along with phosphate buffer pH7.4 in ratio 1:1¹⁷. But with the use of ethanol along with buffer solution sink condition can be maitained, bio-comparable surface activity can only be achieved by using surfactant in the low concentration. So, we have tried simulated intestinal fluid pH 7.4 along with a surfactant for the method development of both the drugs on the basis of solubility studies. But later we observed, curcumin was not stable at this pH for long time. It is also reported that, curcumin shows degradation beyond pH 7, so working pH should be maintained below 7¹⁸ and along with solubility, stability should also be taken under consideration for the selection of release medium¹⁹. So, in this study to enhance the stability of medium,a non-ionic surfactant tween 80 was used in varying concentration with the selected release medium. The suitable solvent system having desired concentration of tween 80 was used for method development for the estimation of curcumin and THC using a UV-Visible spectrophotometer. addition of surfactant in the dissolution medium in low concentration achieved bio-comparable surface activity and can be used to estimate the release of drugs precisely and accurately by avoiding sink conditions.

EXPERIMENTAL WORK:

Materials:

Curcumin and THC were kind gifts from Sanat Products Ltd., Delhi and Sunpure Pvt. Ltd. respectively. All other chemicals and reagents used were of analytical grade.

Instrumentation:

All absorbance measurements were done using double beam UV-visible spectrophotometer (Shimadzu,UV-1800) with matched quartz cells.

Selection of media:

Apparent solubility was determined in phosphate buffer pH 6.8, phosphate buffer pH 7.4 and 0.1 N HCl (pH 1.2) using shake flask method²⁰. The excess amount of drug was dispersed into 10ml of solvent and kept in water bath shaker at37ºc, at 20rpm for 48 hrs. After 48 hrs, the solutions were filtered using vacuum filtration through 0.2μm membrane filters and analyzed spectrophotometrically after appropriate dilutions.

Standard stock solution:

1mg/ml drug solutions of curcumin and THC in phosphate buffer pH 6.8 with tween 80 (0.5%, 1%, 2%) were used to prepare stock solutions of concentration 100µg/ml. From stock solutions, standard solutions of concentration from 5 to 60µg/ml were prepared. The concentration of tween 80 which influenced the quality and shape of the peak is selected for further analysis.

Determination of λ_max:

For the determination of λmax, 10µg/ml solutions were prepared for both curcumin and THC from standand stock solution of phosphate buffer pH 6.8 with 2% tween 80 and 1% tween 80 respectively and scanning (200-800nm) was performed in double beam UV-Visible spectrophotometer using their respective solvent systems as a blank.

Analytical method validation:

The ICH guidelines were followed for the evaluation of analytical parameters like linearity, accuracy, robustness, precision, limit of detection (LOD) and limit of quantification (LOQ)²¹.

Linearity:

The different concentrations of curcumin (5µg/ml to 55µg/ml) and THC (5µg/ml to 40µg/ml) in phosphate buffer pH 6.8 with 2% tween 80 and phosphate buffer pH 6.8 with 1% tween 80 respectively were used to perform the linearity. The absorbance was measure in triplicate. The linearity curve, regression equation and correlation coefficient were determined using the obtained data²².

Precision:

The precision of the developed method was evaluated in terms of intra-day and inter-day variations. The results were expressed in terms of percent relative standard deviation (% RSD) for three different concentrations²³. For determining the perfection levels three different known concentrations of curcumin (10, 20 and 30µg /ml) and THC (10, 20 and 30µg/ml) were prepared from the stock solutions. Intra-day precision was determined by measuring the absorbance of the three concentrations of both curcumin and THC three times a day in triplicate. The absorbance of both curcumin and THC was determined daily for 3 days in triplicate for inter-day precision.

Robustness:

The robustness of the developed method of the curcumin and THC was determined by changing the wavelength from 421nm and 280nm respectively for analyzing a fixed (25µg/ml) concentration. The absorbance was determined in triplicate at each wavelength²⁴.

Accuracy:

The closeness of agreement between the true value and the mean value was determined in terms of accuracy²⁵. The three different levels (75%, 100%, and 125%) of known amount of standard stock solution were compared to the pre-analyzed solution of curcumin and THC. The solutions were analyzed by the proposed method, sample concentration was recalculated in triplicate using the linearity curve.

LOD and LOQ:

LOD and LOQ were calculated by using the standard deviation of the responses and the slope of the corresponding curve by using the following equations: LOD: 3.3 (σ/S); LOQ: 10 (σ/S). Where σ represents the standard deviation of absorbance of sample and S represents the slope of the calibration curve²⁶.

RESULTS AND DISCUSSION:

Selection of media:

Slubility studies of curcumin and THC were performed in various media of physiological pH levels (table 1). Out of which, phosphate buffer pH 6.8 was selected. Although solubility of both the drugs were higher in phosphate buffer pH 7.4, but due to degradation of curcumin from pH 7-10, the working pH was maintained below 7¹⁸. To ensure the stability at 6.8 pH, solubility studies were conducted with phosphate buffer pH 6.8 using tween 80 (0.5%, 1% and 2%). In our study, concentration of tween 80 was used only upto 2% because whenever we conduct the in-vitro dissolution testing, the low concentration of surfactant achieved biocomparable surface activity²⁷.

Table 1: Solubility studies of Curcumin in different solvents

	Curcumin		THC
Media	Solubility (mg/ml)	Term to be used	Solubility (mg/ml)	Term to be used
0.1 N HCl pH 1.2	0.042	Practically insoluble	0.073	Practically insoluble
Phosphate Buffer pH 6.8	0.174	Very Slightly soluble	0.286	Very slightly soluble
Phosphate Buffer pH 7.4	0.203	Very Slightly soluble	0.493	Very Slightly soluble
Phosphate Buffer pH 6.8 + tween 80 (0.5%)	23.26	Sparingly Soluble	30.62	Sparingly Soluble
Phosphate Buffer pH 6.8 + tween 80 (1%)	30.08	Sparingly Soluble	34.58	Soluble
Phosphate Buffer pH 6.8 + tween 80 (2%)	41.39	Soluble	46.19	Soluble

Optimization of tween 80 concentration:

all the conditions relative to peak quality at the stated wavelength were satisfied by the phosphate buffer pH 6.8+tween 80 (2 %) for curcumin and phosphate buffer pH 6.8+tween 80 (1%) for tetrahydrocurcumin. As compared to curcumin low concentration of tween 80 was found sufficient in solvent system of THC, this may be due to its high aqueous solubility and stability in intestinal fluids than curcumin²⁸.

Analytical Method validation:

Linearity studies: λ_max: For curcumin and tetrahydrocurcumin was observed at 421nm and 280 nm respectively. The best linear regression data for calibration curve of curcumin (figure 1) and THC (figure 2) in selected solvent system was found in range of 5 to 55 µg/ml with equation y=0.0201x-0.0061 and 5 to 40µg/ml with equation y = 0.0301x – 0.0994 respectively.

Figure 1: Calibration curve for curcumin In Phosphate Buffer Ph 6.8 + Tween 80 (2%)

Figure 2: Calibration curve for THC In Phosphate Buffer Ph 6.8 + Tween 80 (1%)

Precision:

The %RSD value were found to be less than 2 in intra-day and inter-day precision of curcumin and THC, which indicates the preciseness of this method (table2 and table 3).

Table 2: Intra-day studies of curcumin andTHC

Drug solution	Concentration (µg/ml)	Avg. absorbance	Std.dev.	% RSD
Curcumin	10	0.194	0.001	0.515
	20	0.401	0.0005	0.143
	30	0.588	0.001	0.170
THC	10	0.188	0.001	0.531
	20	0.492	0.004	0.885
	30	0.812	0.008	1.060

Table 3: Inter-day studies of curcumin andTHC

Drug solution	Concentration (µg/ml)	Avg. absorbance	Std.dev.	%RSD
Curcumin	10	0.196	0.002	1.281
	20	0.409	0.007	1.86
	30	0.587	0.004	0.780
THC	10	0.186	0.003	1.639
	20	0.477	0.006	1.332
	30	0.864	0.009	1.145

Robustness:

The variation in % Assay was lying from 99.12 to 101.51 and 98.834 to 100.385 for curcumin and THC respectively with variations in wavelength with in limit of ± 2nm. Which proves the robustness of developed method. The results are shown in table 4.

Accuracy:

The mean % recovery for curcumin and THC was found 99.775 and 99.279 respectively (table 5).

LOD and LOQ:

The LOD and LOQ for curcumin and THC in slected solvent systems were found within range (table 6).

Table 4: Results of Robustness (Curcumin and THC)

	Curcumin			THC
S.NO.	λ max. (nm)	Conc. (µg /ml)	% Assay	λ max. (nm)	Conc. (µg /ml)	% Assay
1	423	25	101.51	278	25	98.834
2	421	25	100.18	280	25	100.385
3	419	25	99.12	282	25	99.012

Table 6: LOD and LOQ for curcumin and THC

	Curcumin			THC
Concentration	LOD	LOQ		LOD	LOQ
5	0.377	1.144		0.179	0.544
10	0.560	1.699		0.183	0.554
15	0.727	2.205		0.188	0.572
20	0.397	1.203		0.183	0.554
25	0.188	0.572		0.196	0.595
30	0.229	0.695		0.272	0.825
35	0.762	2.311		0.188	0.572
40	0.897	2.720		0.356	1.081
45	0.517	1.568	Average	0.218	0.662
50	0.535	1.627
55	0.532	1.612
Average	0.520	1.577

CONCLUSION:

The UV-Visible spectrophotometric method development and validation has been done for the estimation of curcumin and tetrahydrocurcumin in routine in-vitro dissolution testing. The results from solubility studies show that curcumin has a high solubility in simulated phosphate buffer pH 7.4. But to avoid the high degradation, phosphate buffer pH6.8 was used for the method development. Further stability at pH 6.8 was improved by using tween 80 in 2 % and 1 % concentrationfor the estimation of curcumin and THC respectively. Less amount of tween 80 was found sufficient for THC, this may be due to its high aqueous solubility and stability in intestinal fluids compared to curcumin. The developed methods were validated according to ICH guidelines and can be used in routine in-vitro testing of curcumin and tetrahydrocurcumin in phosphate buffer pH 6.8.

CONFLICT OF INTEREST:

The author(s) confirm that this article content has no conflict of interest.

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Received on 02.03.2021 Modified on 05.06.2021

Research J. Pharm.and Tech 2022; 15(2):650-654.

DOI: 10.52711/0974-360X.2022.00107

	Curcumin		Tetrahydrocurcumin
Recovery level	Conc. of Std. drug	% Recovery	Conc. of Std. drug	% Recovery
75	30	99.353	15	99.313
100	40	100.758	20	99.069
125	50	99.213	25	99.455
Average		99.775		99.279