INTRODUCTION:

Curcumin 1,7–bis (4-hydroxy-3-methoxyphenyl) hepta-1,6-diene-3,5-dione is a dietary phytochemical obtain from the dried rhizomes of the turmeric plant (curcuma longa). Turmeric rhizomes, one of the nature most powerful healers, is consumed either fresh or in dried form for its colouring, aroma and medicinal properties.[1]The plant is indigenous to the south and southeast Asian region, needs temperature between 20°c to 30°c.[2] It is a dried ground rhizome obtained from perennial herb curcuma longa belonging to family Zingiberaceae.

[3]Curcumin is a diferuloymethane present in extracts of the plant. The Turmeric contains wide variety of Phytochemical including but not limited to curcumin, demethoxycurcumin, bisdemethoxycurcumin, zingiberene, curcumenol, curcumol, eugenol, tetrahydrocurcumin, triethylcurcumin, turmerin, turmerones, and turmeronols. Extensive research within the last half century has proven that most of these activities, once associated with turmeric are due to curcumin.[4] It make ups 2 to 5 % of spice. Turmeric contains protein (6.3%), fat (5.1%), minerals (3.5%), carbohydrates (69.4%), moisture (13.1%) and essential oil (5.8%).[5]Chemically, curcumin is a bis-α,β-unsaturated β diketone. As such, curcumin exists in equilibrium with its enol tautomer. Inflammation is part of a complex biological response that is induced by harmful stimulation, such as burns, pathogens, and chemical irritants. Over-expressed inflammatory cytokines are involved in the pathological processes of a number of diseases.A daily dose of 2 g of Curcuma domestic extract was found to provide pain relief that was equivalent to indomethacin. Curcumin extract acts by inhibiting the secretion of mediators like IFN-γ(cytokine), IL-8 (chemokine), prostaglandins like PGE₂, leukotrienes, nitric oxide, TNF-α, IL-1, histamine. [4,5]

Experimental work

MATERIAL AND METHODS:

Melting points were determined using a Veegomake microprocessor based melting point apparatus having silicone oil bath and are uncorrected. IR spectra (wave numbers in cm⁻¹) were recorded on a Bruker Alpha FT-IR spectrophotometer using Potassium bromide discs. NMR spectra were recorded on Bruker AvanceII 400 MHz instrument in CDCl₃ with TMS as internal standard for ¹H NMR. Chemical shift values are mentioned inδ, ppm. Mass spectra were recorded on Advion Expression, CMS, USA at SynZeal Research Solutions, Gandhinagar. Chromatographic separations were performed on columns using silica gel 100–200 mesh. The progress of all reactions was monitored by TLC on 2 cm X 5 cm pre-coated silica gel 60 F₂₅₄ (Merck) plates of thickness of 0.25 mm. The chromatograms were visualized under UV 254 nm and/or exposure to iodine vapours. All reagents used were of analytical reagent grade, obtained from LOBA chemicals, SDFCL and Spectrochem. Chemicals and solvents were purified by general laboratory techniques before use. All moisture free operations were performed in oven dried glasswares and under nitrogen atmosphere.

Extraction of Curcumin:

Literature studies how shown that the extraction of curcumin from turmeric could be done in several different ways. In the present study extraction of curcumin from turmeric powder was done by Soxhlation. The curcuminoids are not water-soluble and therefore extractions have to be made in non–polar solvents. In previous studies hexane, acetone, ethylene dichloride and different alcohols have been reported as solvents for extraction. It has been shown that the best yield has been obtained from the attempts where the extraction is made by acetone, hence the solvent used in present study for extraction is acetone. Before extraction of curcumin, turmeric powder was defatted. Defating was done by soaking turmeric powder in hexane overnight. Later solvent was recovered and turmeric powder was air dried. A large filter paper was used in instead of paper and was then placed in the soxhlet apparatus. 200ml acetone was heated and refluxed for extraction of the “filter paper thimble”. The procedure was monitored until the yellow colour of the extraction faded after 5h. An advantage with soxhlet extraction was that no further filteration was needed before it was concentrated. The obtained extract gave a crude yield of 2.6g. [7]

Purification of Curcumin by Crystallization:

Crystallization of curcumin was performed by dissolving a 2.6g sample on 200 ml of methanol at 60⁰C. After dissolution, 10-15ml of distilled water was added, and the mixture was kept at 5⁰C for 2 h. The curcumin crystals were separated from the mother liquor by filtration. The first crystallization of curcuminoid pigments resulted in crystals containing 56.9% of curcumin and other curcumin pigments. Successive crystallizations improved the purity of curcumin, however,there was a loss in yield. In the third successive crystallization, a 40% yield of 92% pure curcumin was obtained with no BDMC detected. Detection of curcumin pigments was carried out by TLC method followed by its IR. [8]

Synthesis and Spectral Data

Figure 1: Scheme for the synthesis of Novel Curcumin derivatives.

Preparation of substituted Acyl Chloride:

Substituted benzoic acids were heated with excess of thionyl chloride for two hours in the presence dry dimethylformamide on a water bath and then excess of thionyl chloride was distilled off under reduced pressure. These prepared acyl chlorides were used as such without further purification for preparation of ester derivatives of curcumin. Formation of acyl chlorides was monitored by converting into esters with methanol using TLC technique. [9]

1,7 –Bis (4-benzoyloxy-3-methoxyphenyl) hepta-1,6-diene-3,5-dione (2a)

Curcumin (0.5g)was dissolved in dry pyridine (20ml) followed by the addition of DMAP and benzoyl chloride. The reaction mixture was sonicated for 8h. Maintaining the temperature 40⁰C-50⁰C. After completion of the reaction, chilled water (5ml) was added and stirred for 10 min. Further,the mixture was extracted with (75 ml) diethyl ether. The organic layer was collected and the excessive solvent was recovered. The residue was collected and allowed to get dried at room temperature. The dark red solid was then recrystallized from ethanol and compound was confirmed by spectroscopic methods like UV, IR and NMR.

R_f: 0.54, %yield: 65.38, M.P.(⁰C): 198-200 nm, λmax(MeOH) : 418 IR (cm^-1) : 3050, 2931, 1692, 1600, 1450 and 1261 NMR (δ,ppm) : 3.731 (s,6H;-CH3), 4.597 (s,2H;-CH2), 6.671 (d,2H;-CH), 6.880-6.992(m,6H;Ar-H),7.416 (m,4H;Ar-H),7.417-8.142 (m,8H;Ar-H)

1,7 –Bis (3-chloro)benzoyloxy-3-methoxyphenyl) hepta-1,6-diene-3,5-dione (2b)

3-chlorobenzoyl chloride was prepared as per procedure mentioned in previous reaction. To it a solution of curcumin (0.5g) in dry pyridine (20ml) was added followed by the addition of DMAP. The reaction mixture was sonicated for 8h. Maintaining the temperature 40⁰C-50⁰C. After completion of the reaction, chilled water (5ml) was added and stirred for 10 min. Further,the mixture was extracted with (75 ml) diethyl ether. The organic layer was collected and the excessive solvent was recovered. The residue was collected and allowed to get dried at room temperature. The dark red solid was then recrystallized from ethanol and compound was confirmed by spectroscopic methods like UV, IR and NMR. [10]

R_f: 0.30, %Yield: 63.21, M.P.(⁰C): 222-224 nm, λmax (MeOH) :275,IR(cm ^-1): 3046, 1679, 1600, 1465 and 1100

1,7–Bis (4-chloro)benzoyloxy-3-methoxyphenyl) hepta-1,6-diene-3,5-dione (2c)

Curcumin (0.5g) was dissolved in dry pyridine (20ml) followed by the addition of DMAP and benzoyl chloride. The reaction mixture was sonicated for 8h. Maintaining the temperature 40⁰C-50⁰C. After completion of the reaction, chilled water (5ml) was added and stirred for 10 min. Further,the mixture was extracted with (75 ml) diethyl ether. The organic layer was collected and the excessive solvent was recovered. The residue was collected and allowed to get dried at room temperature. The dark red solid was then recrystallized from ethanol and compound was confirmed by spectroscopic methods like UV, IR and NMR. [10]

R_f: 0.65, %Yield: 56.32, M.P.(⁰C): 218-220 nm, λmax(MeOH) :416 ,232, IR(cm ^-1): 3050, 1679, 1600, 1475 and 1123, NMR(δ,ppm): 3.731 (s,2H;-CH2), 6.671 (d,2H;-CH), 6.880-6.992 (m,6H;Ar-H),7.416 (m,4H;Ar-H);7.417-8.142(m,8H;Ar-H)

1,7–Bis(4-(2-chloro-5-nitro)benzoyloxy -3-methoxyphenyl) hepta-1,6-diene-3,5-dione (2d)

Curcumin (0.5g) was dissolved in dry pyridine (20ml) followed by the addition of DMAP and benzoyl chloride. The reaction mixture was sonicated for 8h. Maintaining the temperature 40⁰C-50⁰C. After completion of the reaction, chilled water (5ml) was added and stirred for 10 min. Further,the mixture was extracted with (75 ml) diethyl ether. The organic layer was collected and the excessive solvent was recovered. The residue was collected and allowed to get dried at room temperature. The dark red solid was then recrystallized from ethanol and compound was confirmed by spectroscopic methods like UV and IR. [10]

R_f: 0.65 , %Yield : 93.93 , M.P.(⁰C): 256-258 nm, λmax (MeOH) :424 ,278, IR(cm ^-1) : 3050, 1712, 1604, 1531,1473,1326,1214 and 1110

1,7 –Bis (4-(3-amino)benzoyloxy -3-methoxyphenyl) hepta-1,6-diene-3,5-dione (2e)

Curcumin (0.5g) was dissolved in dry pyridine (20ml) followed by the addition of DMAP and benzoyl chloride. The reaction mixture was sonicated for 8h. Maintaining the temperature 40⁰C-50⁰C. After completion of the reaction, chilled water (5ml) was added and stirred for 10 min. Further,the mixture was extracted with (75 ml) diethyl ether. The organic layer was collected and the excessive solvent was recovered. The residue was collected and allowed to get dried at room temperature. The dark red solid was then recrystallized from ethanol and compound was confirmed by spectroscopic methods like UV and IR. [10]

R_f: 0.57, %Yield: 51.21, M.P.(⁰C): 208-210 nm, λmax (MeOH):424 ,358, IR(cm ^-1): 3450, 3050, 2992, 1691,1600,1557,1475,1265 and 1161

1,7 –Bis (4-(4-amino)benzoyloxy -3-methoxyphenyl) hepta-1,6-diene-3,5-dione (2f)

Curcumin (0.5g) dissolved in dry pyridine (20ml) followed by the addition of DMAP and benzoyl chloride. The reaction mixture was sonicated for 8h. Maintaining the temperature 40⁰C-50⁰C. After completion of the reaction, chilled water (5ml) was added and stirred for 10 min. Further,the mixture was extracted with (75 ml) diethyl ether. The organic layer was collected and the excessive solvent was recovered. The residue was collected and allowed to get dried at room temperature. The dark red solid was then recrystallized from ethanol and compound was confirmed by spectroscopic methods like UV, IR and NMR. [10]

R_f: 0.41, %Yield: 53.65, M.P.(⁰C): 216-218 nm, λmax (MeOH):358 ,272, IR(cm ^-1): 3459, 3050, 1697, 1608,1562,1473,1353, and 1222 ,NMR (δ,ppm): 3.731 (s,6H;-CH₃), 4.597 (s,2H;-CH₂), 6.671 (d,2H;CH), 6.880-6.992 (m,6H;Ar-H), 7.417-8.142 (m,8H;Ar-H)

1,7–Bis (4-(3-hydroxy)benzoyloxy -3-methoxyphenyl) hepta-1,6-diene-3,5-dione (2g)

Curcumin (0.5g) dissolved in dry pyridine (20ml) followed by the addition of DMAP and benzoyl chloride. The reaction mixture was sonicated for 8h. Maintaining the temperature 40^oC-50⁰C. After completion of the reaction, chilled water (5ml) was added and stirred for 10 min. Further,the mixture was extracted with (75 mL) diethyl ether. The organic layer was collected and the excessive solvent was recovered. The residue was collected and allowed to get dried at room temperature. The dark red solid was then recrystallized from ethanol and compound was confirmed by spectroscopic methods like UV, IR. [10]

R_f: 0.40, % Yield: 35.36, M.P.(⁰C): 190-192 nm, λmax(MeOH): 299, IR (cm^-1) : 3620, 3050, 2850, 1695, 1600, 1475 and 1250

1,7–Bis (4-(3-hydroxy)benzoyloxy -3-methoxyphenyl) hepta-1,6-diene-3,5-dione (2h)

Curcumin (0.5g) dissolved in dry pyridine (20ml) followed by the addition of DMAP and benzoyl chloride. The reaction mixture was sonicated for 8h. Maintaining the temperature 40^oC-50⁰C. After completion of the reaction, chilled water (5ml) was added and stirred for 10 min. Further,the mixture was extracted with (75 mL) diethyl ether. The organic layer was collected and the excessive solvent was recovered. The residue was collected and allowed to get dried at room temperature. The dark red solid was then recrystallized from ethanol and compound was confirmed by spectroscopic methods like UV, IR. [10]

R_f: 0.53, %Yield:21.95, M.P.(⁰C): 184-186 nm λmax (MeOH); 348, IR (cm^-1): 3630, 2916, 1697, 1600, 1475, and 1263

1,7–Bis (4-(3-hydroxy)benzoyloxy -3-methoxyphenyl) hepta-1,6-diene-3,5-dione (2i)

Curcumin (0.5g) dissolved in dry pyridine (20ml) followed by the addition of DMAP and benzoyl chloride. The reaction mixture was sonicated for 8h. Maintaining the temperature 40^oC-50⁰C. After completion of the reaction, chilled water (5ml) was added and stirred for 10 min. Further,the mixture was extracted with (75 mL) diethyl ether. The organic layer was collected and the excessive solvent was recovered. The residue was collected and allowed to get dried at room temperature. The dark red solid was then recrystallized from ethanol and compound was confirmed by spectroscopic methods like UV, IR. [10]

R_f: 0.33, % Yield: 30.09, M.P.(⁰C): 232-234 nm, λmax(MeOH) : 362, IR (cm^-1) : 2884,1697, 1604, 1450, 1234, 1114 and 1045

1,7–Bis (4-(3-hydroxy)benzoyloxy -3-methoxyphenyl) hepta-1,6-diene-3,5-dione (2j)

Curcumin (0.5g) dissolved in dry pyridine (20ml) followed by the addition of DMAP and benzoyl chloride. The reaction mixture was sonicated for 8h. Maintaining the temperature 40^°C-50^°C. After completion of the reaction, chilled water (5ml) was added and stirred for 10 min. Further,the mixture was extracted with (75 mL) diethyl ether. The organic layer was collected and the excessive solvent was recovered. The residue was collected and allowed to get dried at room temperature. The dark red solid was then recrystallized from ethanol and compound was confirmed by spectroscopic methods like UV and IR. [10]

R_f: 0.61, %Yield: 21.77, M.P.(⁰C): 178-180 nm,λmax(MeOH): 420, 299, IR (cm^-1): 2880,1695, 1626, 1506, 1450, 1320 and 1240

Docking study

Docking of compounds was carried out by using VLife MDS software. Based upon the dock scores the derivatives were sorted out. Among15 derivatives, 10 derivatives with good dock score were selected for synthesis. Standard drug used for docking is selective COX-2 inhibitor i.e Indomethacin. The enzyme used for docking was 3NL1 enzyme. From dock score we can say that these compounds have a probability of having good anti-inflammatory activity. The dock score and binding interaction of derivatives (Compound 2a-2j) is shown in Table 1.

Table 1: Docking Score and Binding Interactions on COX-2 of conformers

S.N	Compound	Dock score(KJ/mol)	Binding Interaction on COX-2
			H-Bond	Charge	Hydrophobic	Vdw
1	2a	-3.196716	----	----	31	182
2	2b	-3.094188	----	----	31	202
3	2c	-2.568591	----	----	29	177
4	2d	-2.398632	1	----	29	195
5	2e	-4.000001	1	----	30	192
6	2f	-3.475532	----	----	31	208
7	2g	-4.008340	1	----	31	209
8	2h	-3.130361	----	----	29	188
9	2i	-2.2957622	----	----	41	215
10	2j	-3.957622	2	----	32	242
11	Celecoxib	-4.323335	----	----	17	115

Table 2: % Inhibition of Inflammation.

S.N	Compounds	Dose (mg/kg)	% Inhibition of paw edema
			0hr	1hr	2hr	3hr
1	2a	10	-	27.20	29.23	44.61
2	2b	10	-	19.15	25.76	33.07
3	2c	10	-	11.49	18.84	26.53
4	2d	10	-	14.55	18.07	33.40
5	2e	10	-	15.70	21.53	41.92
6	2f	10	-	19.15	25.38	26.53
7	2g	10	-	10.34	19.23	54.61
8	2h	10	-	22.98	26.92	36.95
9	2i	10	-	11.49	18.84	19.23
10	2j	10	-	15.33	17.69	50.46
11	Celecoxib	10	-	19.54	26.15	53.46

Anti-inflammatory Activity:

Anti-inflammatory Screening of synthesized compounds was carried out by carrageenan induced rat paw edema method. Thickness of paw was measured using plesthymometer and expressedin mL Indomethacin was used as standard drug. The lowest dose of Celecoxib i.e. 10 mg/kg was selected. The same dose 10mg/kg was given to the test animals of that of test compound. [11] The % inhibition was calculated. Indomethacin showed 53.46% inhibition of paw edema. Among the synthesized compounds 2g showed 54.61% inhibition and is the most active compound amongst ten derivatives synthesized. Also compounds 2j (50.46%), 2a (44.61%) and 2e (41.92%) showed moderate anti-inflammatory activity.

Figure 2: Graphical Representation of % Inhibition of Compounds at Various Time Intervals

RESULT AND DISCUSSION:

All the Curcumin derivatives were synthesized as per the designed scheme which has been started by substituted Benzoic acid after getting a docking score, hydrogen bonding interaction, hydrophobic interaction and van der waals interaction. Molecular docking was performed by MDS VLife software. All the designed compounds and standard drug Indomethacin were docked against Cyclooxygenase enzyme on PDB Id 3NL1. All the compounds were synthesized with satisfied yield, and characterized by IR, MASS and H¹NMR.

Derivatives were screened for anti-inflammatory activity by using carrageenan induced paw edema method. The lowest dose of Celecoxib i.e. 10mg/kg was selected. The same dose 10mg/kg was given to the test animals of that of test compounds. Celecoxib Showed 53.46% inhibition of paw edema. Among the synthesized compounds 2g Showed 54.61% and 2j showed 50.46% inhibition of inflammation.

Figure 3: Hydrophobic interaction and H-Bond interaction in cavity 1of compound 2g

Figure 4: Van der Waals interaction in cavity 1 of compound 2g

Application:

The Curcumin derivatives which are prepared in this study are useful in the treatment of inflammation.

CONCLUSION:

A series of curcumin derivatives (1,7-bis(4-(3-substituted) benzoyloxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione) have been synthesized as potential anti-inflammatory.All the novel compounds were evaluated for anti-inflammatory activity by Carrageenan induced rat paw edema model. Compounds (2g and 2j)exhibited good anti-inflammatory activity whereas compounds (2a and 2e)moderate bioactivity comparable to the reference drug Celecoxib. The anti-inflammatory activity and docking results highlighted the fact, that the synthesized compounds 2g and 2j could be considered as possible hit for the development of new anti-inflammatory agents.

ACKNOWLEDGMENT:

The authors are thankful to the Head, Department of Pharmaceutical Sciences, RashtrasantTukadoji Maharaj Nagpur University, Nagpur for providing necessary facilities and other technical supports during the preparation of this research article.

CONFLICT OF INTEREST STATEMENT:

The authors report no conflict of interest.

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Received on 20.12.2018 Modified on 21.02.2019

Asian J. Research Chem. 2019; 12(2):49-54.

DOI:10.5958/0974-4150.2019.00011.7