**1. Introduction**

The genus *Curcuma* is a rhizomatous annual or perennial herb in the Zingiberaceae family comprising about 120 species [1]. Most of the species of *Curcuma* are naturally present in tropical evergreen areas [2,3]. Many members of the genus are well known in traditional medicine. *C. amada* rhizomes are used as an appetizer, carminative, digestive, stomachic, demulcent, vulnerary, aphrodisiac, laxative, diuretic, expectorant, anti-inflammatory and antipyretic [4]. The rhizomes of *C. angustifolia* are used as a demulcent and antipyretic, are effective against gravel Stomatitis and aid in blood coagulation [5]. In combination with astringents and aromatics, the rhizomes of *C. aromatic* are used for bruises, sprains, hiccough, bronchitis, cough, leukoderma and skin eruptions [6]. The rhizome of *C. zedoaria* are used as an appetizer and tonic, and are particularly prescribed to ladies after childbirth. In Ayurveda, it is an ingredient of "Braticityadi kwatha", used in high fever [ ¯ 7].

Ayurvedic systems have widely used *Curcuma longa* (turmeric) for centuries in the treatment of many inflammatory conditions and diseases such as biliary disorders, anorexia, cough, diabetic wounds, hepatic disorders, rheumatism and sinusitis [8]. Turmeric has been used as a remedy for all kinds of poisonous conditions, ulcers and wounds [9]. It gives a good complexion to the skin and is applied to face as a depilatory and facial tonic. The drug is also useful in cold, cough, bronchitis, conjunctivitis and liver affections [10,11]. Several pharmacological studies were conducted to support the use of *C. longa*. Water-

**Citation:** Abdel-Kader, M.S.; Salkini, A.A.; Alam, P.; Alshahrani, K.A.; Foudah, A.I.; Alqarni, M.H. A High-Performance Thin-Layer Chromatographic Method for the Simultaneous Determination of Curcumin I, Curcumin II and Curcumin III in *Curcuma longa* and Herbal Formulation. *Separations* **2022**, *9*, 94. https://doi.org/10.3390/ separations9040094

Academic Editor: Ernesto Reverchon

Received: 16 March 2022 Accepted: 8 April 2022 Published: 10 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and fat-soluble extracts of turmeric and its main active component, curcumin, exhibited strong antioxidant activity, comparable to vitamins C and E. Curcumin was found to be eight times more powerful than vitamin E in preventing lipid peroxidation [12]. Turmeric's hepatoprotective effects have been proven in a number of animal studies. Curcumin has choleretic activity that increases bile output and solubility, which may be helpful in treating gallstones [13]. An open, phase II trial was performed on 25 patients with endoscopically diagnosed gastric ulcer treated with 600 mg of powdered turmeric five times daily. After four weeks, the ulcers had completely healed in 48 percent of patients and the percentage reached 76 after 12 weeks of treatment. No significant adverse reactions or blood abnormalities were noted [14].

The active constituents of *C. longa* are the curcumins [15]. Turmeric contains up to 5% curcumin and up to 5% of essential oils. Other constituents include sugars, proteins and resins [16]. The main oil components were identified as ar-turmerone (31.7%), *α*-turmerone (12.9%), *β*-turmerone (12.0%) and (Z) *β*-ocimene (5.5%), *α*-bisabolene (13.9%), *trans*-ocimene (9.8%), myrcene (7.6%), 1,8-cineole (6.9%), thujene (6.7%) and thymol (6.4%) [17].

Curcumin has anti-inflammatory properties [18] mediated via downregulation of the nuclear factor (NF)-κB [19] and cyclooxygenase 2 (Cox-2) [20]. Animal studies have indicated that oral curcumin possess antinociceptive [21] and indicated the involvement of ATP-sensitive potassium channels [22]. Pilot human studies of curcumin have demonstrated promise for improving the symptoms of rheumatoid arthritis and inflammatory bowel disease [23,24]. Curcumin also demonstrated antiviral, anti-inflammatory, antibacterial, antifungal, antidiabetic, antifertility and cardiovascular protective and immunostimulant activity [11]. Curcumin, one of the most studied chemopreventive agents, allows suppression, retardation or inversion of carcinogenesis. Curcumin is also described as an antitumoral, anti-oxidant and anti-inflammatory agent capable of inducing apoptosis in numerous cellular systems [25]. Both in vitro studies utilizing human cell lines and in vivo studies have demonstrated curcumin's ability to inhibit carcinogenesis at three stages: tumor promotion, angiogenesis and tumor growth [26]. Curcumin exhibits anticoagulant activity by inhibiting collagen and adrenaline-induced platelet aggregation in vitro as well as in vivo in rat thoracic aorta [27].

The aim of the current study is to develop and validate the high-performance thinlayer chromatography (HPTLC) method for the quantification of curcumins I–III in different extracts of *C. longa* and formulation. There are several advantages of using HPTLC for the analysis, such as the ability to analyze crude samples containing multi-components. Several samples can be separated parallel to each other on the same plate, resulting in a high output and a rapid low-cost analysis. The choice of solvents for the HPTLC development is wide, as the mobile phases evaporated before the spot detection. Spray reagents can be used to detect separated spots [28]. The HPTLC method uses much less amounts of the mobile phase, minimizing exposure to organic solvents and reducing environment pollution [29,30].

#### **2. Materials and Methods**

#### *2.1. Chemicals and Plant Materials*

A standard curcumin mixture was procured from Sigma-Aldrich, St. Louis, MO, USA. All other reagents utilized for the extraction and method development were of analytical grade. The rhizomes of *Curcuma longa* and its herbal formulation containing 500 mg of curcuma extract per capsule were obtained randomly from the hypermarket in Al-Kharj, Saudi Arabia. Chromatographic and analytical grade reagent (AR) were used for the extraction and method development. Centrifugal preparative TLC (CPTLC) was preformed using a chromatotron (Harrison Research Inc. model 7924, Harrison Research, Palo Alto, CA, USA): a 4 mm silica gel P254 disc. Pre-coated, glass-baked TLC plates obtained from E. Merck (silica gel-60F254; thickness: 0.2 mm; area: 20 × 10 cm) were used for quantification.

#### *2.2. Purification of Individual Curcumins*

The standardly composed mixture of the three major curcumins was purified to obtain the individual compounds for the quantification. From the standard mixture, 100 mg were separated using CPTLC on 4 mm silica gel GF254 disks (solvents: 0.5% MeOH in CHCl3). Three zones were collected from the chromatotron and corresponded to curcumins I–III (Figure 1).

**Figure 1.** Structures of Curcumins I–III.

#### *2.3. Charaterization of Curcumins*

NMR data (Figures S1 and S2) were measured using a Bruker UltraShield Plus 500 MHz spectrometer (Bruker, Fällanden, Switzerland) at the NMR Unite, College of Pharmacy, Prince Sattam Bin Abdulaziz, and was operated at 500 MHz for protons and 125 MHz for carbon atoms, respectively. Chemical shift values were reported in δ (ppm) relative to the residual solvent peaks. HRESIMS (Figures S3–S5) were determined by direct injection using a Thermo Scientific UPLC RS Ultimate 3000-Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, United States) combined with the high-performance quadrupole precursor selection with high resolution and accurate-mass (HR/AM) Orbitrap™ detection.

#### *2.4. Extraction Procedure*

Fresh and dried curcuma purchased from the local market and a formulation containing 500 mg of curcuma extract per capsule were extracted with MeOH by maceration (×3) at room temperature, using the soxhlet apparatus for 6 h to obtain the corresponding six extracts, two for each sample. All extracts were filtered and dried using the rotary vacuum evaporator to obtain the corresponding dry extracts.

#### *2.5. HPTLC Conditions*

HPTLC (Camag, Muttenz, Switzerland) was performed on glass-backed TLC silica-gel plates (20 cm × 10 cm). Purified curcumin I, curcumin II and curcumin III and sample solutions were separately spotted with nitrogen flow on a plate with a 6-mm-wide band at 8-mm from the bottom by using the automatic Camag-TLC (Linomat V, Camag, Muttenz, Switzerland) applicator. The (linear ascending) development of TLC plates was performed in a twin glass Camag chamber (automatic ADC2, Camag). The chamber was filled with 20 mL of mobile phase for a fixed time of 15 min.

#### *2.6. Method Validation*

Different validation parameters for the simultaneous determination of curcumin I, curcumin II and curcumin III were determined as per the ICH-Q2 (R1) guidelines [31].

#### 2.6.1. Accuracy

Accuracy was determined by the standard addition method. The preanalyzed samples of curcumin I, curcumin II and curcumin III were spiked with the extra 0, 50, 100 and 150% of the standard curcumin I, curcumin II and curcumin III, and the solutions were reanalyzed in six replicates by the proposed method. The accuracy of the HPTLC technique for the

simultaneous determination of curcumin I, curcumin II and curcumin III was estimated as % recovery.

#### 2.6.2. Precision

Precision of the proposed method was determined at two levels i.e., repeatability and intermediate precision. Repeatability was determined as the intraday precision, whereas the intermediate precision was determined by carrying out an inter-day variation for the determination of curcumin I, curcumin II and curcumin III. The precision of the HPTLC technique for the simultaneous estimation of curcumin I, curcumin II and curcumin III was expressed as the percent relative standard deviation (% RSD).

#### 2.6.3. Robustness

The robustness of the proposed HPTLC method was determined to evaluate the influence of small deliberate changes in the chromatographic conditions during the determination of curcumin I, curcumin II and curcumin III. The robustness of the HPTLC technique for the simultaneous determination of curcumin I, curcumin II and curcumin III was evaluated by introducing small deliberate changes in the composition of mobile phase components, total run length, saturation time and detection wavelength.

#### 2.6.4. Limit of Detection and Quantification (Sensitivity)

The sensitivity of the HPTLC technique for the simultaneous determination of curcumin I, curcumin II and curcumin III was determined in terms of detection (LOD) and quantification (LOQ) limits. The limit of detection (LOD) and limit of quantification (LOQ) were determined by the standard deviation (SD) method. They were determined from the slope of the calibration (S) curve and SD of the blank sample using the following equations:

$$\text{LOD} = 3.3 \times \text{SD/S}$$

$$\text{LOQ} = 10 \times \text{SD/S}$$

#### 2.6.5. Specificity

The specificity of the proposed TLC densitometric method was confirmed by the Rf and spectra of the spot with that of the standards of curcumin I, curcumin II and curcumin III.

#### *2.7. Quantification of Curcumins I–III in C. longa and Herbal Formulation*

Curcumin I, curcumin II and curcumin III peaks in *C. longa* and herbal formulation were identified by comparing their spots at that of the standard curcumin I, curcumin II and curcumin III. The amount of curcumin I, curcumin II and curcumin III present in the *C. longa* and herbal formulation was quantified from the regression equation obtained from the calibration plot of HPTLC.

#### **3. Results**

*3.1. Method Validation*

3.1.1. Linearity

Calibration curves for curcumins I–III are presented in Figures S6–S8. The results for the least square regression analysis of the calibration curves (CCs) of curcumins I–III are included in Table 1.


**Table 1.** Linear regression data for the calibration curve of curcumins I–III (*n* = 6).

#### 3.1.2. Accuracy

The accuracy of the HPTLC technique for the simultaneous determination of curcumins I–III was estimated as % recovery, and the results are included in Table 2.

**Table 2.** Accuracy of the proposed method of curcumins I–III (*n* = 6).


#### 3.1.3. Precision

The precision of the HPTLC technique for the simultaneous estimation of curcumin I, curcumin II and curcumin III was estimated in terms of instrumental and intra/inter-assay precision and expressed as the percent relative standard deviation (% RSD). The results of intra/inter-assay precisions for the simultaneous determination of curcumins I–III using the HPTLC technique are included in Table 3.

**Table 3.** Precision of the proposed method of curcumins I–III.



**Table 3.** *Cont.*

#### 3.1.4. Robustness

The robustness of the HPTLC technique for the simultaneous determination of curcumins I–III was evaluated by introducing small deliberate changes in the composition of mobile phase components, total run length, saturation time, and detection wavelength. The results of the robustness analysis after changing the mobile phase components are included in Table 4.

**Table 4.** Robustness of the proposed HPTLC method of curcumins I–III.


#### 3.1.5. Sensitivity

The sensitivity of the HPTLC technique for the simultaneous determination of curcumin I, curcumin II and curcumin III was determined in terms of detection (LOD) and quantification (LOQ) limits.

#### 3.1.6. Specificity

The specificity of the proposed TLC densitometric method was confirmed by the Rf at 0.45 ± 0.02, 0.52 ± 0.02 and 0.61 ± 0.04 and the spectra of the spot with that of the standards curcumins I–III (Figure 2). The proposed method was found to be specific by comparing the Rf of *C. longa* rhizomes, herbal formulation and standard, as well as the overlaid spectra at peak start, peak apex and peak end position of the spot, showing λmax 423 nm for curcumin I, curcumin II and curcumin III (Figure S9).

**Figure 2.** HPTLC densitogram of standard curcumins I–III.
