1. INTRODUCTION:

Curcumin was first isolated in 1815 and formulated into its crystalline form in 1870, and ultimately identified as 1,6-heptadiene-3,5-dione-1,7-bis(4-hydroxy-3-methoxyphenyl)-(1E,6E) or diferuloylmethane.¹ Curcumin (diferuloylmethane) is a polyphenol compound isolated from ground rhizomes of the plant (Curcuma longa) L. belongs to family Zingiberaceae found in South Asia.² It is insoluble in water and ether but soluble in various other organic solvents, such as, methanol, ethanol, glacial acetic acid, dimethylsulfoxide-acetone, and acetone, among others. Curcumin has a melting point of 183°C and acts as strong reducing agent stability, strong colouring (not for protein; once pigmented, it does not fade easily), light-, heat-, and iron ion sensitivity. Curcumin exists in enolic and b-diketonic forms due to keto-enol tautomerism of the molecule.

The absorption spectrum of Curcumin has two strong absorption bands, the first in the visible region of 410nm to 430 nm and the second band in the UV region with a maximum at 265nm.³ There are also reports in the literature indicating therapeutic effects of Curcumin in diseases such as diabetes, HIV infection, multiple sclerosis, rheumatoid arthritis, Alzheimer’s disease, and cystic fibrosis.⁴ Turmeric is widely used in Ayurvedic medicine for its anti-oxidant, antiseptic, analgesic, antimalarial, and anti-inflammatory properties, in addition to its usage as a flavouring and colouring ingredient in formulations. Curcumin has long been used as a dietary supplement and is regarded as pharmacologically safe.⁵ To design and develop an effective pharmaceutical formulation containing Curcumin for its various pharmacological activity and also there is need to develop and validate a newer analytical technique to analyse Curcumin in bulk and its dosage forms. So various analytical methods for estimation of curcumin in bulk and pharmaceutical formulation are estimation by HPLC, estimation by HPTLC, estimation by UPLC, estimation by UV spectrophotometer.

2. DRUG PROFILE:^6–8

Drug	Curcumin
IUPAC Name	1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione
Chemical Formula	C₂₁H₂₀O₆
Molecular Mass	368.39 g/mol
Melting Point	183°C
Physical State	Solid
Appearance	Bright yellow-orange powder
Solubility	Methanol, Ethanol, Glacial acetic acid, Dimethyl sulfoxide-acetone and Acetone, etc.
pKa	8.54

3. STRUCTURE OF CURCUMIN:

4. PHARMACOLOGY^5,9–13

Anti-cancer

Anti-oxidant effect

Anti-inflammatory

Antioxidant

Analgesic

Antiseptic activity

Anticarcinogenic activity

Chemo preventive

Chemotherapeutic activity

Anti-tumour

Antiviral

Antibacterial

Antifungal properties

Antiplatelet activity

Hepatoprotective effect

5. MECHANISM^1,13–18

Down-regulates activity of COX-2, lipoxygenase and inducible iNOS enzymes

Inhibits production of TNF-alpha, IL-1, -2, -6, -8, and -12, MCP, migration inhibitory protein

Down-regulates mitogen-activated and Janus kinases

Inhibits expression of cyclin D1 and CDK₄ via acetylation and upregulation of p53

ATP-competitive inhibitor by down-regulating mRNA and protein expression of cyclin D1

Induction of CDK inhibitors p16(/INK4a), p21(/WAF₁/CIP₁), and p27(/KIP1)

Inhibits cyclin E/cyclin D1 expression

Hyperphosphorylation of retinoblastoma (Rb) protein (CDK2 substrate)

Induction of apoptotic signals

Anti-neoplastic via cell-cycle arrest

Enhances cleavage of procaspases and poly(ADP-ribose) polymerase

Inhibits the TNF-a-induced production of IL-6/IL-8 in HaCaT cells

6. PHARMACOKINETICS:

Curcumin passes through the gastrointestinal tract uncharged in 40-85% of cases, according to animal pharmacokinetic studies, with the most of the absorbed flavonoid being metabolized in the intestinal mucosa and liver. Overall, the data suggest that curcumin has a low absorption and rapid clearance after oral administration. Phospholipid complexation has been proven in several studies to improve the therapeutic impact of drugs with low oral absorption.¹⁹

7. ANALYTICAL METHODS FOR ESTIMATION OF CURCUMIN:

7.1 Estimation of curcumin by HPLC:

High-performance liquid chromatography (High-pressure liquid chromatography, HPLC) is a specific form of column chromatography generally used for analysis to separate, identify, and quantify the active compounds. In pharmaceutical research and development, it is now utilized in the following ways:²⁰

· Purification of synthetic or natural products.

· To identify the metabolites

· To assay active ingredients, impurities, degradation products and in dissolution assays. Studies on pharmacodynamics and pharmacokinetics

Sr. No	Paper title	Description	Ref.
1	Simultaneous estimation of paclitaxel and curcumin in nano-formulation: Stability analysis of drugs, optimization and validation of HPLC method	1. Mobile phase: Methanol and water with 0.05% of O-phosphoric acid 2. Flow rate: 0.8 ml/minutes 3. Detection: 370 nm 4. Retention Time: 8.9 minutes 5. Limit of detection: 1.02 µg/ml 6. Limit of quantification: 3.41 µg/ml 7. Column: Phenomenex Luna C-18, 5 µm, 100Å, 4.6 × 150 mm	21
2	Analytical method for the determination of curcumin entrapped in polymeric micellar powder using HPLC	1. Mobile phase: water (1%, v/v acetic acid) and acetonitrile in a ratio of 50:50 v/v 2. Flow rate: 1.0 mL/min 3. Detection: 421 nm 4. Column C18 column (250 × 4 mm, 5 µm)	22
3	Development and Validation of a RP-HPLC Method for Simultaneous Quantitation of Resveratrol and Curcumin: Application to Nanolipid Gel Formulation	1. Mobile phase: Acetonitrile: 0.01 M phosphate buffer pH adjusted to 4.0 ± 0.05 using 1 % v/v o-phosphoric acid (60:40, v/v) 2. Flow rate: 1 ml/min 3. Detection: 345 nm 4. Retention Time: 6.58 min 5. Limit of detection: 0.29 µg/ml 6. Limit of quantification: 0.90 µg/ml 7. Calibration curve: 10-60µg/ml 8. Percentage recovery: 99.01- 101.05 % 9. Column: Hiber C-18 column, (4.6 mm × 250 mm, 5 μm)	23
4	Isocratic high-performance liquid chromatography (HPLC) for simultaneous quantification of curcumin and piperine in a microparticle formulation containing Curcuma longa and Piper nigrum	1. Mobile phase: acetonitrile-methanol-water of 65:5:35 % 2. Flow rate: 1 mL/min 3. Detection: 353 nm 4. Retention Time: 12.54 min 5. Limit of detection: 0.42μg/mL 6. Limit of quantification: 1.41 μg/mL 7. Calibration curve: 2.5–30μg/mL 8. Percentage recovery: 99.14% 9. Column: C18 column (250×4.6 mm, Eurospher 100 with 5μm	24
5	Quality assessment and RP-HPLC method development for estimation of curcuminoids in Curcuma longa: A Quality by Design approach	1. Mobile phase: Acetonitrile and Water (0.02%OPA) in the ratio of 55:44 2. Flow rate: 1 mL/min 3. Detection: 425 nm 4. Retention Time: 9.86 min 5. Limit of detection: 0.45μg/ml 6. Limit of quantification: 1.38μg/ml 7. Calibration curve: 2-10 µg/ml 8. Percentage recovery: 105.46% 9. Column: Reversed-phase C-18 column (5μm, 4.6mm ×250 mm, ZORBAX)	25
6	Development and Validation of RP–HPLC Method for Estimation of Curcumin from Nanocochleates and Its Application in in–vivo Pharmacokinetic Study	1. Mobile phase: Methanol and water (80:20 v/v) was adjusted to 4.5 with acetic acid 2. Flow rate: 1.0 mL/min 3. Detection: 421 nm 4. Limit of detection: 0.09 µg/mL 5. Limit of quantification: 0.34 µg/mL 6. Column: HIQ SIL C18 (250 mm × 4.6 mm,5 μm))	26
7	Development and validation of HPLC method for the determination of curcuminentrapped in polymeric micellarpowder	1. Mobile phase: water (1%, v/v acetic acid) and acetonitrile (50:50, v/v) 2. Flow rate: 1.0 mL/min 3. Detection: 421 nm 4. Retention Time: 5,363/5.355 min 5. Percentage recovery: 104.56 ± 4.51/82.86 ± 3.15% 6. Column: C18 column, 250 x 4 mm, 5 µm	27
8	A simple reversed phase high-performance liquid chromatography (HPLC) method for determination of in situ gelling curcumin-loaded liquid crystals in in vitro performance tests	1. Mobile phase: acetonitrile and water (50:50 v/v) acidified with 2% acetic acid 2. Flow rate: 1.2 mL/min 3. Detection: 425 nm 4. Retention Time: 11.5 min 5. Limit of detection: 11.61 µg/mL 6. Limit of quantification: 0.5 µg/mL 7. Column: Reverse-phase C18 250 mm x 4.6 mm, 5 µm	28
9	Determination Of The Curcumin Pigment In Extract Curcuma Domestica Val From South Sulawesi, Indonesia, By High Performance Liquid Chromatography	1. Mobile phase: acetonitrile: acetic acid: Aquabides 50:1:49% 2. Flow rate: 1 mL/min 3. Detection: 425nm 4. Retention Time: 7.04 min 5. Column: Column C18 (250x4.6mm)	29
10	Development of a liquid chromatographic method for the simultaneous quantification of curcumin, b-arteether, tetrahydrocurcumin and dihydroartemisinin. Application to lipid-based formulations	1. Mobile phase: methanol-ammonium acetate (pH 4; 10 mM) (80:20, v/v) 2. Flow rate: 0.45mL/min 3. Detection:230 nm 4. Retention Time: ~3 min 5. Column: C18 column (125 mm × 4 mm, 5µm)	30

Sr. No.	Stationary Phase	Mobile phase	Detection wavelength	Saturation Time	Ref.
1	Precoated silica gel 60F₂₅₄ TLC aluminium sheets	Chloroform: Ethanol: Glacial acetic acid (90: 5 :1)	425 nm	-	31
2	silica gel 60 F254 Thin Layer Chromatographic (TLC) plate (Merck)	Toluene: acetic acid (8:2 v/v)	540nm	20 min	32
3	20 × 10 cm, 0.2 mm thickness precoated with the silica gel 60 F254 (E-Merck	Chloroform: Methanol (97:3v/v)	420 nm.	-	33
4	Silica gel 60 F254 TLC plates (20×10 cm, layer thickness 0.2 mm, E. Merck, Germany)	Chloroform: Ethyl acetate: Formic acid (7.5 mL+ 6 mL + 0.5 mL)	254 nm	20 min.	34
5	TLC plates (60 F254, 20 cm × 10 cm, 250m thickness, Merck, Darmstadt, Germany)	n-Hexane: Ethyl acetate: acetic acid: methanol (7:2:0.5:0.5 v/v/v)	404 nm	30 min	35
6	TLC aluminium plates silica gel 60F254	Chloroform: methanol (9.25:0.75 v/v)	430 nm	10 min	36
7	TLC plates pre-coated with 0.2-mm layer of silica gel 60 GF254 (20 cm×10 cm)	Chloroform: methanol (48:2 v/v)	425 nm	30 min	37
8	LiChrosphere Si HPTLC plates precoated with 0.2-mm layer of silica gel 60 F254 (20×20 cm)	Chloroform: methanol (98:02 v/v)	366 nm	30 min	38
9	Kiesel gel HPTLC plates pre-coated with 0,2-mm layer of silica gel 60 F254 (10 cm ×10 cm)	Toluene: glacial acetic acid (8:2 v/v)	425 nm	20 min	39
10	TLC plates pre-coated with 0.2-mm layer of silica gel 60 F254 (20 cm ×10 cm)	Toluene: chloroform: methanol (5:4:1 v/v/v)	430 nm	15 min	40
11	TLC plates pre-coated with 0.2-mm layer of silica gel 60 F254 (20 cm ×10 cm)	Dichloromethane: methanol (99:1 v/v)	427 nm	10 min	41

Sr. No.	Instruments	Description	Ref.
1	A Shimadzu UV-visible spectrophotometer 1601	Solvent: Simulated intestinal fluid Detection wavelength: 421nm LOD: 0.16137µg/ml LOQ: 0.489001µg/ml	44
2	UV-Spectrophotometer of Shimadzu UV-1900 Shimadzu UV-1800	Solvent: Methanol Detection wavelength: 422nm LOD: 0.34µg/ml LOQ: 1.03µg/mL	45
3	UV-visible spectrophotometer (Shimadzu UV-1800)	Solvent: Methanol Detection wavelength: 423 nm LOD: 0.092µg/ml LOQ: 0.280µg/ml	46
4	Sican 2301 UV-Spectrophotometer	Solvent: Methanol Detection wavelength: 421nm LOD: 0.225µg/ml LOQ: 0.68µg/ml	47
5	Jasco double beam UV-Vis spectrophotometer (Model V-630)	Solvent: ethyl acetate Detection wavelength: 418 nm	43
6	V-Spectrophotometer of Shimadzu make and 1800 model	Solvent: Methanol: Water (50:50% v/v) Detection wavelength: 430 nm LOD: 0.24 µg/mL LOQ: 0.73 µg/mL	48
7	Double beam UV Spectrophotometric (UV 2375	Solvent: phosphate buffer and ethanol (1:1) mixture Detection wavelength: 429 LOD: 0.861 µg/ml LOQ: 2.872 µg/ml	49
8	double beam UV-VIS spectrophotometer (UV-1700, Shimadzu, Japan)	Solvent: Methanol Detection wavelength: 423 nm LOD: 0.18 ± 0.004 µg/ml LOQ: 0.65 ± 0.010 µg/ml	50
9	Double beam UV Visible Spectrophotometer (UV SHIMADZU 1800	Solvent: Methanol Detection wavelength: 422 LOD: 0.28 µg/ml LOQ: 0.87 µg/ml	51

7.5 Estimation of curcumin by RP-UFLC:

Phytoconstituents are mostly methanol soluble and this solvent creates a back pressure exceeding 4000 psi in a conventional HPLC pump hence UFLC have been selected for the study which can withstand pressure up to 6800 psi. Hence a simple, rapid and cost-effective simultaneous RP-UFLC method was developed for the estimation of curcumin⁵².

Paper Title

Description

Ref.

Development and validation of simultaneous estimation method for curcumin and piperine by RP-UFLC

Column: C8 column (250 x 4.6 mm, 5µ)

Mobile Phase: 25 mM potassium dihydrogen ortho phosphate buffer (pH 3.5) and acetonitrile (30: 70 v/v)

Detection wavelength :280 nm

Flow Rate: 1 ml/min

LOD :6 ng/ml

8. VALIDATION PARAMETER:

The developed method should be validated as per ICH Guidelines by using the various validation parameters such as,

1. Specificity

2. Linearity

3. Limits of Detection (LOD)

4. Limits of Quantitation (LOQ)

5. Range

6. Accuracy

7. precision

· Repeatability

· Intermediate precision

· Reproducibility

8. Robusteness

9. Ruggedness

10. Sensitivity

9. CONCLUSION:

The relevance of studies aimed at developing and improving methodologies for estimating curcumin in various matrices, whether for quality control in pharmaceutical or to ensure the efficacy of this chemical in pharmaceutical, is astounding. The literature has a vast range of techniques, and it is crucial to note that the best option should consider the analyst's needs as well as the available resources to prevent wasting solvents and reagents. Various chromatographic methods and procedures are discussed in detail in this review.

10. CONFLICT OF INTEREST:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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Received on 26.03.2022 Modified on 09.04.2022

Asian J. Pharm. Ana. 2022; 12(2):142-148.

DOI: 10.52711/2231-5675.2022.00025