Curcumin, a polyphenolic compound obtained from turmeric and has been used for centuries as a spice and coloring agent¹. It has a lot of medicinal activities like anti-microbial, anti-inflammatory, anti-oxidant, anti-viral, wound healing, blood purifier and anti-tumor activity^2-5. Because of its radiosensitization property, it was used by Minafra et al., in the treatment of breast cancer⁶. In turmeric, out of its three curcuminoids namely curcumin, demethoxycurcumin and bisdemethoxycurcumin⁷.

Unfortunately, the curcumin is poorly absorbed into the bloodstream but research supports that combining the piperine in black pepper with the curcumin in turmeric enhances its absorption⁸. It shows higher solubility in the alkaline medium as compared to the acidic medium⁹. THC, a colorless or white color was first detected in 1978 by holder et al.,¹⁰. The pharmacological activities of tetrahydrocurcumin are quite similar to curcumin¹¹. It shows better anti-diabetic effects in rats by increasing their plasma insulin level¹². It is produced by the reduction of curcumin by hepatic reductase in the liver¹³. The oral bioavailability of THC is higher in tissue and plasma as compared to curcumin¹⁴. It was reported that in 0.1 M phosphate buffers, especially at pH 7.2 (37°C) THC is more stable than curcumin¹⁵. To overcome the problems related to oral bioavailability of curcumin and THC, many colloidal formulations have been developed^16-17. Different solubility of curcumin and tetrahydrocurcumin is obtained by varying in the composition of simulated intestinal fluid (SIF)¹⁸. So, in our study solubility of both the drug was determined in the simulated gastric fluid (SGF) pH 1.2, SIF pH 6.4 and SIF pH 7.4, the solvent system in which the higher solubility obtained was used for method development using a UV-Visible spectrophotometer.

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:

A Shimadzu UV-visible spectrophotometer 1601 (Kyoto, Japan) with 10mm path length, matched quartz cells was used for all absorbance measurements.

Selection of solvent:

Apparent solubility was determined in SIF pH 6.8, SIF pH 7.4 and SGF pH 1.2 using shake flask method. The excess amount of drug was dispersed into 10ml of solvent and kept in a water bath shaker (37ºc), at 20rpm for 48 hrs. After 48hrs, the solutions were filtered through 0.2μm membrane filters by using vacuum filtration, then diluted accordingly and analyzed spectrophotometrically.

Standard stock solution:

The maximum solubility of drugs was observed in SIF pH 7.4. Stock solutions containing 1mg/ml drug was prepared in 50ml SIF pH 7.4 with tween 80 (0.5%, 1%, 2%) is used to make the concentration of 100µg/ml. From stock solutions, different 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 wavelength of maximum absorption

A 10 µg/ml solution was prepared for both curcumin and THC from stock solution of SIF pH 7.4 with 1% tween 80 and 0.5% tween 80 respectively and scanning (200-800 nm) was performed by using UV-Visible spectrophotometer in their respective solvent systems as a blank to determine the λmax of curcumin andTHC.

Analytical validation:

Analytical parameters like linearity, accuracy, robustness, precision, limit of detection and limit of quantification were evaluated as per the ICH guidelines¹⁹.

Linearity:

The linearity of the analytical procedure was performed for different concentrations of curcumin (5µg/ml to 40µg/ml) and THC (5µg/ml to 25µg/ml) in SIF pH 7.4 with 1% tween 80 and SIF 7.4 phosphate buffer with 0.5 % tween 80 respectively. The obtained data was used to determine the linearity curve, regression equation and correlation coefficient equation²⁰.

Precision:

The precision of the developed method was evaluated in terms of intra-day and inter-day variations. Perfection levels were determined for the three different known concentrations of curcumin (15, 25 and 35µg/ml) and THC (5, 15 and 25µg/ml) which were prepared from the stock solutions. For determining intra-day veracity, the absorbance of the three concentrations of both curcumin and THC was measured three times a day in triplicate and for inter-day veracity, the absorbance of both curcumin and THC was determined daily for 3 days in triplicate. The percent relative standard deviation (% RSD) was calculated for three different concentrations by using the linearity curve²¹.

Robustness:

The robustness of the developed method was determined by analyzing the 20µg/ml concentration of the curcumin and THC by changing the wavelength from 421 nm and 280nm respectively. The absorbance of both curcumin and THC was determined six times²².

Accuracy:

Accuracy is defined as the closeness of agreement between the true value and the analytical value. The known amount of standard stock solution was considered at different levels 50%, 100%, and 150% as compared to the pre-analyzed solution of curcumin and THC. The solutions were analyzed by the proposed method. Using the linearity curve, sample concentration was recalculated in triplicate²³.

LOD and LOQ:

The Limit of detection (LOD) and Limit of quantification (LOQ) of the developed methods were determined. LOD and LOQ were calculated by using the standard deviation of the response 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 curve²⁴.

RESULTS AND DISCUSSION:

Selection of solvent

Different media were used to perform solubility studies of curcumin and THC (table 1). Out of which, SIF pH 7.4 was selected. Further solubility studies were conducted by using SIF pH 7.4 with tween 80 (0.5 %, 1 % and 2 %).

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
SGF pH 1.2	0.0064	Practically insoluble	0.0053	Practically insoluble
SIF pH 6.8	0.109	Very Slightly soluble	0.120	Very slightly soluble
SIF pH 7.4	0.156	Very Slightly soluble	0.169	Very Slightly soluble
SIF pH 7.4+ tween 80 (0.5%)	28.429	Sparingly Soluble	35.099	Soluble
SIF pH 7.4 + tween 80 (1%)	35.708	Soluble	38.058	Soluble
SIF pH 7.4 + tween 80 (2%)	43.535	Soluble	47.859	Soluble

Optimization of tween 80 concentration:

SIF pH 7.4 with tween 80 at 1% concentration satisfied all the conditions relative to peak quality at the stated wavelength for curcumin and SIF pH 7.4 with tween 80 at 0.5% concentration satisfied all the conditions relative to Peak quality at the stated wavelength for tetrahydrocurcumin. The in-vitro testing, while using low concentration of surfactant achieved biocomparable surface activity²⁵. The low concentration of tween 80 used for THC is may be due to its high aqueous solubility and stability in intestinal fluids compared to curcumin²⁶.

Method validation:

Linearity studies:

Certain readings of wavelength at which maximum absorption takes place is 421nm for curcumin and 280 nm for THC (tetrahydrocurcumin). The linearity regression data for calibration curve of curcumin (figure 1) and THC (figure 2) in selected media showed good relationship in the range of 5 to 40µg/ml and 5 to 25µg/ml respectively. The linear regression equation for curcumin and THC was found to be Y=0.025X-0.011 and Y=0.043X-0.058 respectively.

Precision:

The % RSD value were found to be less than 2 in curcumin and THC, which indicates this method is precise (table2 and table 3).

Figure 1: calibration curve for curcumin in SIF pH 7.4 with 1% tween 80

Figure 2: Calibration curve for THC in SIF pH 7.4 with 0.5 % tween 80

Table 2: Intraday studies of curcumin and THC

Drug solution	Concentration (µg/ml)	Avg. absorbance	Std. dev.	%RSD
Curcumin	15	0.36	0.001	0.2777
	25	0.630	0.00152	0.24220
	35	0.892	0.002	0.2242
THC	5	0.161	0.001	0.6211
	15	0.590	0.002082	0.3524
	25	1.032	0.00916	0.888096

Table 3: Interday studies of curcumin and THC

Drug solution	Concentration (µg/ml)	Avg. absorbance	Std. ev.	%RSD
Curcumin	15	0.358	0.002082	0.58039
	25	0.629	0.002646	0.4206
	35	0.888	0.005568	0.627
THC	5	0.158	0.002358	1.0488
	15	0.585	0.002263	0.3865
	25	0.985	0.002828	0.28715

Robustness:

The variation in wavelength with in limit of ± 2 nm for both curcumin and THC gave %recovery lying from 99.48 to 100.25 and 99.45 to 101.424 respectively with the maximum confidence level of±2%. The results are shown in table 4.

Table 4: Results of Robustness (Curcumin)

	Curcumin			THC
S. NO.	λ max.(nm)	Conc. (µg /ml)	% Assay	λ max.(nm)	Conc. (µg /ml)	% Assay
1	423	20	99.48	279	20	100.240
2	421	20	99.67	280	20	101.424
3	419	20	100.25	283	20	99.45

Table 5: Accuracy results

Recovery level	Conc. of Std. drug	% Recovery	Conc. of Std. drug	% Recovery
50	10	98.9556	5	101.8807
100	20	100.7722	15	99.556
150	30	99.8713	25	99.9174
Average		99.8663		100.4513

Table 6: LOD and LOQ for curcumin and THC

	Curcumin			THC
Concentration	LOD	LOQ		LOD	LOQ
5	0.03567	0.108108		0.042262	0.128067
10	0.07426	0.225045		0.093122	0.282188
15	0.08978	0.272066		0.074265	0.225045
20	0.0742	0.225045		0.086091	0.260882
25	0.12863	0.38978		0.342808	1.038813
30	0.2028	0.614729	Average	0.12771	0.38699
35	0.0713	0.216216
40	0.6141	1.86100
Average	0.16137	0.489001

Accuracy:

The mean % recovery for curcumin and THC was found 99.8663 and 100.4513 respectively (table 5).

LOD and LOQ:

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

CONCLUSION:

The UV-Visible spectrophotometric method has been developed for the quantification of curcumin and tetrahydrocurcumin. The results from solubility studies show that curcumin has a high solubility in simulated intestinal fluid pH 7.4. For estimation of both the drugs, tween 80 in 0.5%, 1% and 2% concentration was used with SIF pH 7.4. All the conditions related to peak quality at the specified wavelength were satisfied by a simulated intestinal fluid pH 7.4with 1% tween 80 for curcumin and 0.5% tween 80 for tetrahydrocurcumin. The less anount of tween 80 was required for THC due to its high aqueous solubility and stability inintestinal fluids compared to curcumin. The selected solvent systems were validated and can be used in routine for analysis of curcumin and tetrahydrocurcumin in SIF pH 7.4 especially in oral formulations.

CONFLICT OF INTEREST:

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

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Received on 03.02.2021 Modified on 20.03.2021

Research J. Pharm. and Tech. 2021; 14(6):2971-2975.

DOI: 10.52711/0974-360X.2021.00520