Bhaskar Rao 2014
Bhaskar Rao 2014
Bhaskar Rao 2014
Full Paper
Synthesis and Biological Evaluation of Glucosyl Curcuminoids
Adari Bhaskar Rao1, Ernala Prasad1, Seelam S. Deepthi1, and Imtiaz A. Ansari1,2
1
Medicinal Chemistry and Pharmacology Division, CSIR – Indian Institute of Chemical Technology, Tarnaka,
Hyderabad, India
2
Department of Chemistry, King Khalid University, Abha, Saudi Arabia
Medicinal plants proved to be a rich source in exploring a variety of lead structures in the development
of new drugs. The natural curcuminoids have gained considerable attention in recent years for
their multiple beneficial pharmacological and biological activities. Clinical application of these
curcuminoids is often impaired due to their poor water solubility, resulting in low in vivo bioavailability
of the active compound in humans. The objective of the present study is to synthesize glucosyl
conjugates of curcumin 1 and tetrahydrocurcumin 4 and to evaluate their biological activities.
The study highlights the synthesis of curcumin-b-di-glucoside 3 (yield 71%) and tetrahydrocurcumin-b-
di-glucoside 6 (yield 64%) in good yields in a biphasic reaction medium using a phase transfer catalyst
under simple and ecofriendly conditions. Both the glucosyl curcuminoids showed enhanced
antioxidant, tyrosinase enzyme inhibitory, antimicrobial and potent cytotoxic activity. The improved
biological activity may be due to the increased solubility of the glucosyl conjugated compounds
compared to the native curcuminoids; this was further confirmed by partition coefficient studies. Thus,
the synthesized glucosyl curcumin may serve as promising future therapeutic molecule in the
management of cancer, whereas glucosyl tetrahydrocurcumin can be a useful ingredient in
achromatic food and in cosmetic applications.
Received: May 15, 2014; Revised: July 8, 2014; Accepted: July 9, 2014
DOI 10.1002/ardp.201400195
methodologies [9, 10]. It is well known that the glycosylation min) 3 and a colorless product tetrahydrocurcumin-di-b-
of hydrophobic phytochemicals has improved the bioavail- glucoside (glucosyl-THC) 6 (Scheme 1).
ability and biological activity of the compounds [11, 12]. In
the present study, it is planned to synthesize glucosyl- Curcumin-4,4-b-di-glucoside (3)
conjugated curcuminoids and to evaluate their biological Yellow colored solid, yield 71%. 1H NMR (DMSO-d6): 3.1–3.4 (m,
activity. 8H), 3.6 (m, 3H), 3.77 (s, 6H, OCH3), 4.31 (br, s, 1H), 4.5 (t, 2H,
J ¼ 5.5Hz), 4.93 (d, 2H, J ¼ 7 Hz), 4.98 (d, 2H, J ¼ 5 Hz), 5.06 (br, s,
Results and discussion 2H), 5.25 (d, 2H, J ¼ 4 Hz), 6.04 (s, 1H), 6.81(d, 2H, J ¼ 16 Hz),
7.05 (d, 2H, J ¼ 8.5 Hz), 7.18 (d, 2H, J ¼ 8 Hz), 7.32 (s, 2H), 7.52 (d,
Chemistry 2H, J ¼ 16 Hz); ESI-MS (m/z): 715 (MþNa)þ.
Synthesis of glucosyl curcuminoids
Synthesis of curcumin-4,4-b-di-D-glucoside(glucosyl-curcumin) Tetrahydrocurcumin-4,4-b-di-glucoside (6)
3 and tetrahydrocurcumin-4,4-b-di-glucoside (glucosyl-THC) 6 Half white colored solid, yield: 64%; m.p. ¼ 184°C, TLC
was achieved in higher yields, in a simple economical process rf ¼ 0.27 (DCM/MeOH, 9.5:0.5), UV (EtOH) lmax 258, 220 nm.
[13]. The glucosyl conjugation reaction was facilitated by a IR(KBr): nmax 3453, 3017, 2927, 2854, 1753, 1605, 1514, 1370,
phase transfer catalyst (tetrabutylammonium bromide), that 1229, 1037, 757 cm1; 1H NMR (DMSO-d6 and D2O): 6.86–6.59
promotes better mass transfer of reactants between two (m, 6H, –CH2); 5.64–5.50 (m, 2H), 5.16 (t, J ¼ 9.8 Hz, 2H), 4.37–
immiscible liquid phases, resulting in high product forma- 4.26 (m, 6H), 4.17–4.10 (m, 4H), 3.87 (s, 6H), 2.86–2.70 (m, 8H);
13
tion with selectivity. The progress of the reaction was C NMR (DMSO): 193.1, 146.3, 143.9, 132.5, 120.7, 114.3,
monitored through HPLC and the single product peak was 110.9, 99.7, 79.1, 76.8, 76.1, 72.6, 70.3, 61.9, 55.8, 40.3, 31.2. LC
obtained at the end of the optimum reaction period. The MS (m/z): 719.13 (MþNa)þ.
structures of the glucosyl-conjugated curcuminoids were Natural curcuminoids are yellow colored compounds
confirmed by spectral analysis. From 1H NMR studies, it was resulting in staining of the skin and dress materials during
confirmed that the configurations of the anomeric carbons topical application. Tetrahydrocurcumin is a colorless metab-
were defined as b for all the glucose molecules therefore the olite of curcumin that exhibits pharmacological and biologi-
conjugated glucosylations of the two curcuminoids show b- cal activities similar to that other curcuminoids. It is planned
configuration. The SN2 nucleophilic substitution at the to synthesize the conjugated glucosyl-curcumin and glucosyl-
anomeric C-1 position of the activated sugar moiety resulted tetrahydrocurcumin compounds to increase hydrophilicity of
in the formation of curcumin-di-b-glucoside (glucosyl-curcu- these molecules and improve their biological activity.
O Br
AcO
KOH
O O AcO OAc O O
MeO OMe OAc TBAB CH 3 ONa MeO OMe
2
HO OH 45°C/2 h O O
1
OH OH
O O
OH
3 OH
OH OH OH OH
O Br
AcO
KOH
O O AcO OAc O O
MeO OMe OAc TBAB CH3 ONa MeO OMe
5
HO 4 OH 45°C/2 h O O
OH OH
O O
OH OH
OH OH
6
OH OH
Figure 2. IC50 values of curcuminoids on DPPH radical scavenging and tyrosinase inhibition activity.
confirmed by low MIC values, this may be due to the increased cells for curcumin 1 was 66.94 mM, THC 4 it was 77.17 mM
aqueous solubility of these hydrophilic curcuminoids. whereas for curcumin-b-di-glucoside 3 and for THC-b-di-
glucoside 6 IC50 values were 41.56 mM and 32.13 mM. Thus, it
Cytotoxicity was confirmed from the results that on glucosidation of the
The cytotoxicity of the four curcuminoids curcumin 1, THC 4, curcuminoids the inhibition of colon cancer cells (HT-29)
curcumin-b-di-glucoside 3, and THC-b-di-glucoside 6 were activity was increased by twofolds when compared with free
studied in selected human cancer cell lines (HT-29 colon curcuminoids (Fig. 4). The conjugated curcumin has also
cancer, A549 lung cancer, MCF-7 breast cancer) by MTT assay. showed a noticeable inhibition activity on breast cancer cells
From the results, it was observed that all the tested (MCF-7), whereas no significant activity was found in liver
curcuminoids have shown cytotoxic activity on the four cell cancer cells (A549). Curcuminoids possess a wide spectrum of
lines and compounds 4 and 6 exhibited marked activity on antitumor properties but, due to its poor bioavailability the
colon cancer cell line (HT-29). The IC50 value on colon cancer curcuminoids are not yet been clinically used routinely in
Figure 4. Evaluation of cytotoxicity (IC50 mM) of curcuminoids on different cancer cell lines by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) assay method.
anticancer chemotherapy [16–18]. Glycosylation of hydropho- L-DOPA, tyrosinase T-3824, 25 KU were procured from Sigma
bic curcuminoids increases solubility of the compound and Chemical Co. (St. Louis, MO, USA). The cancer cell lines A549 (lung
thus improving the anticancer activity as confirmed by their cancer), MCF-7 (breast cancer) and HT-29 (colon cancer) used were
obtained from National Center for Cell Science (NCCS), Pune,
lower IC50 values. India. All the microbial strains and the Mueller Hinton broth,
Natural products are known to possess potent anticancer Dulbeccos modified Eagles agar medium (DMEM) and other
activity and are also used topically against widespread skin media components used in these studies were procured from
diseases and cosmetics. Curcuminoids attracted considerable Hi Media Ltd (Mumbai, India). All the reagents and solvents
attention in the recent years due to their broad pharmaco- (n-octanol) used in the experiments were of analytical grade and
were purchased from Merck (India).
logical activities. The poor circulating bioavailability of these
curcuminoids attributed due to low water solubility thus
Equipment
impairing the compounds biological activity. The synthesized Spectrophotometric analyses were carried out using UV visible
glucosyl-conjugated compounds curcumin-b-di-glucoside 3 spectrophotometer (Perkin Elmer). 1H and 13C NMR spectra for
and tetrahydrocurcumin-b-di-glucoside 6 have shown potent the products were recorded on a 300, 500 MHz NMR spectrometer
antioxidant and cytotoxic activities when compared to native (Bruker, Avance, Germany). Mass spectral analyses of compounds
curcuminoids. The results obtained clearly substantiate the were performed using MS (Waters Q-Tof Ultima, Manchester, UK)
in the ESI positive mode.
beneficial effects of glucosyl-curcumin as a potent anticancer
agent and colorless glucosyl-THC shows superior antioxidant
HPLC
and inhibit tyrosinase activity thereby helping in lightening The concentration of curcumin, THC, glucosyl-curcumin, and
the skin tone. Therefore, the conjugated THC acts as useful glucosyl-THC were quantified through HPLC (Gilson LC), using
ingredients in anti-ageing and other cosmetic topical reverse phase analytical C18 column (150 mm 3.9 mm, 5 mm
formulations designed to maintain general skin health [19]. particle size). The system was eluted isocratically using mobile
phase acetonitrile/water (85:15 v/v; pH adjusted to 3.0 with 0.01%
acetic acid) at a flow rate of 1 mL/min and the sample detection
Conclusion was at 280 nm. The mobile phase was filtered through 0.5 mm
nylon membrane filter and the solvent was degassed ultrasoni-
The study confirms a novel synthesis of curcumin-b-di-glucoside cally before use. Salbutamol (0.2 mg/mL) was added to each test
3 and tetrahydrocurcumin-b-di-glucoside 6 in good yields and sample as an internal standard. Peak area was used as a measure
selectivity (b-form) in simple and eco-friendly reaction to calculate the respective concentrations.
conditions. The results confirm the increased hydrophilicity
and biological activity of glucosyl conjugated curcuminoids Chemistry
when compared to their native un-conjugated curcuminoids; General procedure for the synthesis of glucosyl-
this was further confirmed by partition coefficient studies. The curcuminoids
The general method used for synthesis of glucosyl conjugated
data also reveal the compounds glucosyl-curcumin and
compounds 3 and 6 are summarized in Scheme 1. A modified
glucosyl-THC showing enhanced antioxidant, antimicrobial, procedure of Koenings–Knorr was followed in synthesis of
and tyrosinase inhibition activities, when compared to non- glucosyl-conjugated curcuminoids [20, 21]. The reaction was
conjugated curcuminoids studied. In summary, the glucosyl- initiated by adding aqueous solution of KOH (10.8 mmol) to 20 mL
curcumin shows potent antioxidant and cytotoxic activity, and of DCM solvent containing disallowed compounds 1 or 4
(2.71 mmol), followed by the addition of 2,3,4,6-tetra-O-acetyl-b-
thus the molecule may be of therapeutic importance against
D-glucopyranosyl bromide (5.41 mmol) and molecular sieves (4 A).
several types of cancer diseases. Whereas, the non-staining The reaction mixture was stirred for 3 h at 45°C under the
glucosyl-conjugated THC molecule acts as a potent ingredient nitrogen atmosphere. On completion of the reaction, the
in achromatic food and in cosmetic applications. compounds curcumin-b-di-glucoside tetraacetate 2 or tetrahy-
Thus, the study concludes both the synthesized conjugated drocurcumin-b-di-glucoside tetraacetate 5 were isolated and
these compounds were deacetylated by adding 2% sodium
curcuminoids, help in improving the medical value of the
methoxide in dichloromethane. The two products formed were
natural curcuminoids and hope that future clinical trials will isolated and purified through column chromatography silica gel
lead to the development of potent preventive and therapeutic (100–200 mesh). The purity of the final products was determined
agent for skin photo aging and UV induced cancer diseases. by HPLC and the structures of the compounds obtained
curcumin-b-di-glucoside 3 or tetrahydrocurcumin-b-di-glucoside
6 were confirmed by NMR (1H, 13C), mass and LC–MS studies.
Experimental
Partition coefficient (log P value)
Materials and methods The concentration of curcuminoids present in two-phase n-
Chemicals and materials octanol/water system was determined by HPLC [22]. The
Curcumin and THC were gift from Ashian Herbex Ltd., curcuminoids 50 mM (curcumin, THC, glucosyl-curcumin, and
Hyderabad, India. DPPH, and bovine serum albumin (BSA), glucosyl-THC) were dissolved in known volumes of n-octanol (2, 4,
6, 8, and 10 mL)/water (O/W ratio) in a shake-flask and incubated New Delhi for award of fellowship; the authors also thank MD, Ashian
at 37°C for 2 h. Lipophilicity of the compound quantified by the Herbix Ltd, Hyderabad for gift sample of curcuminoids.
logarithm of the 1-octanol/water partition: log P ¼ log[Co/Cw],
where Co was the concentration of compound in the octanol
phase and Cw its concentration in the aqueous water phase, when The authors have declared no conflict of interest.
the system was at equilibrium. log P values presented were an
average of three measurements. References
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