1. INTRODUCTION:

Turmeric (Curcuma longa L.), family Zingiberaceae, is a medicinal plant widely used worldwide and it possesses a wide range of biological activities against various diseases like antibacterial, anti-inflammatory, antifungal, antiviral, antitumor, hepatoprotective etc. due to naturally occurring abundant compounds known as Curcuminoids.

Curcuminoids consisted of three compounds such as Curcumin (diferuloylmethane or Curcumin I), Demethoxycurcumin (DMC), Bis-demethoxycurcumin (BDMC). The percentage composition of three different curcuminoids may vary as curcumin 62 to 90mg/g, Demethoxycurcumin 9 to 23mg/g and Bisdemethoxycurcumin 0.3 to 14mg/g of turmeric powders respectively¹. The therapeutic effect of turmeric is mostly attributed to curcuminoids which constitute upto 2 to 9%w/w. Esatbeyoglu et al. (2012) further reported that amount of curcuminoids present in turmeric rhizhomes as 80%w/w curcumin, 17%w/w DMC and 3%w/w BDMC. Curcumin (Diferuloylmethane), a yellow colouring compound, is the primary curcuminoids responsible for most of the important pharmacological activities exhibited by Curcuma longa. It was found that a huge variation with respect to the percentage contents of curcuminoids in turmeric rhizomes is reported in may literature which could be largely due to variation in agro-climatic zones². The present work focuses on isolation, characterization of curcuminoids and quantification of curcumin present in crude curcuminoids extract using validated UV Visible spectrophotometer, as there was no detail reports related to characterization of curcuminoids extract obtained from turmeric cultivated in Mizoram.

2. MATERIALS AND METHODS:

2.1. Collection, processing and authentication of plant material:

Curcuma longa rhizomes were collected from Serchhip District, Mizoram. Rhizomes were washed with running water to remove dust particles, sliced and air dried under shed. Dried rhizomes were grinded to obtained coarse powder and ready for extraction process. The standard curcumin was procured from Sigma Aldrich. All the other chemicals used were of analytical grades.

For authentication of the plant, the herbarium specimen was sent to Botanical Survey of India, Eastern Regional Centre, Laitumkhrah, Shillong, Meghalaya.

3. Isolation of Curcuminoids from turmeric:

3.1 Extraction of Oleoresin:

Soxhlet extraction was performed as follows: turmeric was grounded to powder and 700 gm of ground powder was taken, weighed and embedded in a thimble and transferred inside the Soxhlet extractor. Acetone, which is used as extraction solvent was acetone was then poured inside the extraction unit. This procedure was monitored until the yellow colour of the extractions faded. The extraction solvent was then separated after the extraction process is completed by rotary evaporator under vacuum 40˚C and was then concentrated to semisolid brown coloured mass³.

3.2 Extraction of crude Curcuminoids from oleo resin:

The obtained crude extract was soaked with n-Hexane for 24 hrs. After 24 hours, the solution with lump of crude extract was stirred using magnetic stirrer for 4 hrs. The lump was crushed down into fragments and then to powder form with continuous stirring. By centrifugation, the lumps which are suspended at the top was segregated and dried at ambient temperature under the shade. The final crude powder i.e. curcuminoids was characterized and quantified for its curcumin content⁴. The percentage yield of oleoresin and curcuminoids was then determined by using following formula.

% Yield of oleoresin = Weight of oleoresin / Weight of dried rhizomes X 100

% Yield of Curcuminoids = Weight of Curcuminoids / Weight of dried rhizomes X 100

4. Characterization of curcuminoids extract using Thin layer chromatographic technique:

4.1. Sample preparation:

Curcuminoid (1mg) was measured in electronic balance and transferred to a 10ml beaker. Methanol (5mL) was used as a solvent to dissolve the drug, sonicated for three minutes and further adjusted upto the required volume (10mL) with methanol. Samples were applied on Silica gel G (pre-coated) plates using mobile phase Chloroform: Methanol (19:1).

4.2. Application:

Standard borosilicate capillary was employed for applying sample in the plates. These spots are applied at definite distance, allow drying. The prepared plate was placed in development chamber for separation of compounds associated with the curcuminoids extract. After complete separation, the obtained Rf values are compared between the compounds separated on TLC plates and Rf values obtained with pure curcumin determined simultaneously⁵.

5. FT-IR study:

The FT-IR study of the curcuminoids extract was performed. The infra- red spectrum of both standard curcumin and curcuminoids extract was recorded and compared. For this study, standard curcumin and curcuminoids extract (0.1mg) were mixed with 100mg of KBr so as to get a pellet which was further used for analysis at 400–4000 cm-1 wavelengths^6.

6.UV-visible spectrophotometric method development for curcumin and Validation:

6.1. Method development:

6.1.1 Primary stock solution preparation:

Primary stock solution of Curcumin (100µg/mL) was prepared with methanol as a dissolving solvent. It was done by weighing 10mg of Curcumin and transferred into 100ml volumetric flask. The solvent was added to the flask so as to get the required volume of solvent^7.

6.2 Measurement of maximum absorption wavelength (λmax):

The primary stock solution of curcumin was diluted so as to get a dilute concentration 10µg/mL. The resulting solution was in the cuvette for measurement of λmax by scanning in UV-visible spectrophotometer at wavelength ranges from 200 to 800nm. The wavelength at which maximum absorption takes place in UV detector was selected for further analysis^8,9.

6.3 Preparation of standard calibration curve:

For preparation of standard calibration curve, a series of dilute concentrations of curcumin (1-5µg/mL) was prepared form the primary stock solution. Absorbance was measured for these solutions at λmax of 424nm using UV spectrophotometer at 424nm. All the readings were taken in triplicate. The observed absorbance was then plotted on y-axis against the concentration on x-axis⁹.

6.4 Validation:

ICH Q2(R1) guidelines was followed for the UV visible spectrophotometric method development of curcumin and its validation for the following parameters.

6.4.1 Linearity and Range:

This is performed by measuring the absorbance of Curcumin at different standard concentrations at which was prepared previously at 424nm against Methanol as blank. All the experiments were done in triplicate.

6.4.2 Precision:

Precision was evaluated by using repeatability and intermediate 2 precision. Repeatability was assessed by measuring the absorbance of three replicates of previously prepared 2µg/ml at three different times on the same day (intra-day). Intermediate precision was evaluated using a similar procedure over three separate days (inter-day)⁹.

6.4.3 Accuracy:

Determination of accuracy was performed by using recovery studies. It was done by adding known concentrations of pure Curcumin (8, 10, 12μg/mL) to the pre-analyzed sample solutions (10μg/mL). The method was repeated for three times for each concentration¹⁰.

6.4.4 Robustness:

To assess the robustness, 2µg/ml Curcumin solutions were analyzed six times at two different temperatures (room temperature and 9ºC).

6.4.5 Limit of Detection (LOD):

It refers to the smallest quantity of analyte in a sample that can be detected, while the Limit of Quantitation (LOQ) is the minimum amount of analyte that can be quantitatively measured with acceptable precision and accuracy. Calculation for LOQ and LOD was done by using the following equation¹¹.

LOQ = 10 s/m and LOD = 3.3 s/m

Here, s represents the standard deviation of the response, while m denotes the slope of the calibration curve obtained from repeated measurements.

7.Quantitative determination of curcumin:

A preparation of sample solution of curcuminoids crude extract was carried out by dissolving approximately 100 mg of the sample (crude powder) in 50mL methanol in a 100mL volumetric flask (100mL). The resulting solution was then sonicated for 10 mins. The final volume was adjusted to 100mL with methanol and then filtered. The filtrate was used for quantification of the curcumin content in the crude curcuminoids extract using UV-Visible spectrophotometer at 424nm. The experiments were performed in triplicates⁴.

8. RESULTS AND DISCUSSION:

8.1 Plant authentication:

The plant authentication was done at Botanical Survey of India, Eastern Regional Centre, Laitumkhrah, Shillong, Meghalaya. and confirmed the plant to be Curcuma longa L. (Zingiberaceae) with the identification number BSI/ERC/Tech/2022-23/350

8.2 Extraction of crude curcuminoids from Curcuma longa:

Curcuminoids, the principal phytochemical compounds in turmeric, can be isolated by various methods, ranging from traditional techniques like Soxhlet extraction and maceration to more advanced methods such as microwave-assisted extraction, Super critical fluid, pressurized liquid extraction, ultrasound-assisted extraction etc. Among these, Soxhlet extraction method has been reported to provide the most promising extraction methods giving highest yields compared to modern techniques¹².

Curcuminoids exhibit variations in their chemical structures, physicochemical properties, and functional characteristics. Regarding the solubility of the principal phytochemical components, curcuminoids have low solubility in hydrocarbon solvents. Organic solvents including acetone, hexane, acetone, alcohols (ethanol, methanol), isopropanol, ethyl acetate etc. are commonly used for extracting oleoresins from spices. Among these, acetone has been suggested to be highly effective as compared to alcohol and other solvents, as a result of providing higher content of curcuminoids, indicating selective extraction.

In this study, oleoresins were first isolated from the rhizomes powder of turmeric using. acetone Separation of crude curcuminoids from the acetone extract was done using n-hexane through a simple continuous stirring process. At the time of stirring process, volatile oils along with other resinous impurities were eliminated from the acetone extract, resulting in the formation of yellow curcuminoid crystals. The resultant crude curcuminoid powder was then subsequently purified by recrystallization in a mixture of hexane and isopropyl alcohol (1:1.5).

From 700g of turmeric, 52.344g of curcuminoids crystals was obtained which gives 7.47% yield. %yields (Oleoresin): 13.07% w/w, %yields (Curcuminoids): 7.47% w/w

8.3 Analysis of extracted Curcuminoids using Thin layer Chromatographic technique:

A number of studies are undertaken to separate Curcuminoid pigments such as Thin layer Chromatography, High Performance Thin Layer Chromatography and column chromatography. Silica gel is commonly used as a stationary along with different solvent systems including benzene, ethyl acetate, ethanol, chloroform, acetic acid, hexane, and methanol for chromatographic separations ⁵.

TLC technique is a simple and most reliable techniques for identifying and separating compounds, owing to low cost of use, ease of use, rapid output of analysis, high sensitivity with excellent reproducibility. This method allows simultaneous spatial separation of multiple substances. The RF (retardation factor, ratio of fronts or retention index) value is the standard measure of retention. Curcuminoids complex viz. Curcumin, Demethoxycurcumin (DMC) and Bisdemethoxycurcumin (BDMC) were effectively separated by TLC. Three spots were detected both for standard curcumin and crude curcuminoids. The calculated Rf value for crude curcuminoids matched with standard curcumin which helps to confirm the identity and purity of the curcuminoids. The obtained Rf value for sample and standard curcumin were 0.75,0.52,0.32 and 0.76,0.56,0.34 for curcumin, DMC and BDMC respectively. Figure 1 depicted the TLC photographs.

Figure 1. TLC plate of standard curcumin and Curcuminoids extract

8.4. FT-IR study:

The IR spectra show stretching vibrations at 3300 – 3500 cm^-1 in both curcumin and curcuminoids extract attributed to – OH groups. Also, the band at 1504 cm^-1 for curcumin and 1512 cm^-1 for curcuminoids extract indicates C=O and C=C stretching vibrations. The stretching vibrations at 1273 cm^-1 for curcumin, and 1265 cm^-1 attributed to the aromatic C-O vibrations for curcumin and curcuminoids respectively. Therefore, it is further suggested that structural similarities existed between curcumin and curcuminoids extract.

Figure 2. FT-IR spectra of: - a) Curcumin b) Curcuminoids extract

8.5 Development and Validation of a UV-Visible Spectrophotometric Method for quantification of Curcumin:

An easy, accurate, and precise UV-Visible spectrophotometric method for curcumin estimation was developed and validated in accordance with ICH guidelines. The established method showed a reliable, accurate and precise, linearity and suggested to be was robust and rugged in nature.

8.6 Determination of Maximum Absorption wavelength: After the scanning for maximum absorption wavelength(λmax) using UV visible spectrophometer, the λmax was identified as 424nm.

Figure 3. UV spectra of Curcumin

8.7 Preparation of Standard Calibration Curve:

The standard calibration curve was successfully established, showing an R² value of 0.999 and a slope of y = 0.1922x - 0.0312, as illustrated in Figure 4.

Table 1. Linearity and range

Concentration(µg/ml)	Absorbance
1	0.159 ± 0.051
2	0.743 ± 0.036
3	0.541 ± 0.015
4	0.743 ± 0.036
5	0.927± 0.040

9. Method Validation

9.1 Linearity and Range:

The calibration curve for curcumin in a methanol solution demonstrated linearity, with a correlation coefficient of 0.999, as presented in Table 1: Linearity and Range (results expressed as mean ± SD).

Figure 4. Standard calibration curve (curcumin)

9.2 Precision:

Precision results in terms of mean absorbance values, SD and % RSD for intra- and inter-day precision study are depicted in table 3, 4 and 5 respectively. Overall, % RSD values less than 2 showed the reliability of precision study.

Table 3: Intraday precision

Concentration (µg/ml)	Absorbance 1(10:00am)	Absorbance 2(1:00pm)	Absorbance 3(4:00pm)	Average% RSD
2	0.356	0.360	0.358	0.918
	0.348	0.354	0.356
	0.362	0.357	0.360
%RSD	1.614	0.686	0.456

Table 4: Interday precision

Concentration (µg/ml)	Absorbance 1(Day1)	Absorbance 2(Day2)	Absorbance 3(Day3)	Average % RSD
2	0.354	0.358	0.350	1.277
	0.347	0.352	0.349
	0.362	0.360	0.358
%RSD	1.729	0.96	1.144

Table 5: Accuracy

Labeled claim (µg/ml)	Addedamount of Curcumin (µg/ml)	Predicted conc. (µg/ml)	Observed concentration (μg/mL)	Average % recovery	Average % RSD
10	8 (80%)	18	17.607 ± 0.220	97.82 ± 1.22	1.254
	10 (100%)	20	20.60 ± 0.198	103.00 ±0.99	0.962
	12 (120%)	22	22.057 ± 0.294	100.26 ±1.33	1.335

9.3 Accuracy:

From the accuracy studies, it can be concluded that the UV method highly accurate with the percentage recovery ranging from 97.82% to 103%, and %RSD > 2%.

9.4 Robustness:

Results of robustness are shown in Table 7% RSD value below 2 showed that the developed method is robust in nature.

Table 7: Robustness

Concentration (µg/ml)	Absorbance	Statisticalanalysis
Temperature at 25ºC		Mean=0.355 SD=0.005 %RSD=1.613
2	0.356
2	0.348
2	0.362
Temperature (2-8ºC)		Mean =0.358 SD = 0.003 %RSD=1.026
2	0.359
2	0.354
2	0.363

9.5 LOD and LOQ:

The LOD and LOQ were determined to be 0.0899± 0.872µg/mL and 0.272±1.05µg/mL respectively (Table 11). The lower value suggested that the suitability of developed method for analysis of samples with even small amounts of curcumin.

Table 8: Summary of validation parameters of the proposed UV method

Parameter	Result
Absorptionmaxima	424 nm
Beerslawrange	1-5µg/ml
Correlationcoefficient(R²)	0.999
Regressionequation	0.1922x-0.0312
Slope	0.192
Intercept	0.031
Accuracy	97.82%-103.00%
Precision	Intraday – 0.918% RSD, Interday - 1.277% RSD
LOD	0.089 µg/ml
LOQ	0.272µg/ml

10. Quantification of total curcumin content:

To confirm the presence as well as quantification of the curcumin from extracted sample, UV-vis spectroscopy analysis was conducted. The absorption spectra of standard and extracted Curcuminoids were scanned within the range of 200 to 800 nm. The standard curcumin represented an absorption peak at around 424 nm. This characteristic peak is also noted in the spectrum of crude curcuminoids and recrystallized curcuminoids, as given in Figure 5.

The amount of Curcumin content from crude curcuminoids powder was found to be 71.72±1.58% w/w, while the purity increased to 89.76±1.82% w/w in the recrystallized curcuminoids powder.

Fig 5. UV-vis spectra of Standard Curcumin, Crude Curcuminoids and Recrystallized curcuminoids.

11. CONCLUSION:

Curcuminoids, phenolic compounds found in the rhizomes of Curcuma longa, were successfully extracted using the Soxhlet extraction method. The three curcuminoids were efficiently separated by TLC, and the calculated Rf value for crude curcuminoids matched with standard curcumin which helps to confirm the identity and purity of the curcuminoids. The FT-IR study was also conducted to confirm the structural similarity between standard curcumin and the extract, it is further suggested that structural similarities existed between curcumin and curcuminoids extract. A UV-Visible spectrophotometric method was developed and validated as per ICH Q8 guidelines for estimating the curcumin content in the curcuminoid extract. The amount of Curcumin content from crude curcuminoids powder was 71.72% w/w, while the purity increased to 89.76% w/w in the recrystallized curcuminoids powder assuming a curcuminoid content of 7.47% in the turmeric rhizomes. This indicates that the amount of curcumin present in turmeric cultivated in this region is relatively high and thus represents the quality and purity as well.

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Received on 17.10.2024 Revised on 27.01.2025

Accepted on 04.04.2025 Published on 01.10.2025

Available online from October 04, 2025

Research J. Pharmacy and Technology. 2025;18(10):4871-4876.

DOI: 10.52711/0974-360X.2025.00702

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