Green synthesis, Characterization and In vitro Biological Studies of Quercetin complexes with Zn (II) Acetate and N^N Moiety

Tanu Srivastava¹*, S. K. Mishra², O. P. Tiwari³, Kavindra Nath Tiwari⁴, Pradeep Kumar⁴, Jitendra Kumar²,

Amit Kumar Singh², Angaraj Singh⁵, Ashutosh Dwivedi⁵

¹Assistant Professor, SHEAT College of Pharmacy, Varanasi, Uttar Pradesh - 221210, India.

²Assistant Professor, Department of Pharmaceutical, Engineering, and Technology,

Indian Institute of Technology, Banaras Hindu University, Varanasi, Uttar Pradesh - 221005, India.

³Director, Varanasi College of Pharmacy, Varanasi, Uttar Pradesh - 221105, India.

⁴Professor, Department of Botany, MMV, Banaras Hindu University, Varanasi, Uttar Pradesh - 221005, India.

⁵Assistant Professor, Department of Ceramic Engineering, Indian Institute of Technology,

Banaras Hindu University, Varanasi, Uttar Pradesh -221005, India.

*Corresponding Author E-mail: tanusrivns@gmail.com

ABSTRACT:

Green synthesis of two coordination complexes of zinc acetate with N^N moiety on quercetin, which is a flavonoid is carried out. The complexes were obtained in high yields (> 97%) by grinding methods without the involvement of any solvent. Neither catalyst nor any additives were needed to perform the reactions. It was characterized by FT-IR, UV-Vis, NMR, HRMS, and elemental analysis. Antioxidant activity was done through the DPPH method which was compared with ascorbic acid and ligand (Quercetin). The study reveals that Complex 1 (IC₅₀ 163.093µg/ml) has significant free radical scavenging activity as compared to complex 2 (IC₅₀ 258.683µg/ml). Biological activity was performed against microbes (E. coli and S. aureus). MIC value of complex 1 (15.50µg/ml E. coli, 7.18µg/ml S. aureus) was found more significant as compared to complex 2 (22.51µg/ml E. coli, 18.62µg/ml S. aureus) and quercetin.

KEYWORDS: Solid-state, Coordination complexes, DPPH, Minimum inhibitory concentration.

1. INTRODUCTION:

Mostly organic reactions are carried out in the presence of a solvent. The solvents which are used ultimately reduce the yield and also spread hazardous effects on the environment¹. The solvents and catalysts used for the synthesis may be a good source of impurity in the compound. Solid-state synthesis does not require all these additives for the completion of the reaction of so-called green synthesis. Compared to the current conventional organic reaction green synthesis has manifold merits which include high reaction velocity, several steps reduction, cost-effective and green impact on environment².

The green synthetic for novel drug discovery from the natural product has brought different applications³. The flavonoids are obtained from tea, red wine, olive oil, fruits, and seeds of the plant⁴. The flavonoids demonstrate a wide variety of biological activities such as anticancerous, anti-inflammatory, antiplatelet, antithrombotic, antioxidant, antibacterial, cytoprotective activities, etc.^5-14. Quercetin, a flavone class of compounds has different coordination sites in its molecule that may be utilized for complex formation with transition metals. Quercetin has a wide application in health and diseases¹⁵. In this paper, we synthesized a complex with zinc acetate, 2, 2'-bipyridyl, 1, 10-phenanthroline, and quercetin. Metal chlorides (MCl₂) with quercetin and N^N ligand have already been reported in the solvent phase¹⁶ but in this article, we have taken metal acetate (MAcO) coordinated with quercetin and N^N ligand in a solvent-free environment. In coordination, chemistry zinc is a sunshine central metal cation. In human zinc is the most abundant trace metal after iron and this is the only metal cation that is present in all enzyme categories. Zinc performs various physiological functions such as catalytic factor (as a cofactor in six main enzyme classes), as a structural component (zinc fingers), etc.¹⁷. Zinc (II) with quercetin and one of the aromatic observer ligand i.e. 2,2′-bipyridine show different biological activities In vitro¹⁸. The solvents are hazardous; many of them are reported for causing various lethal diseases. We focused on solid-state synthesis with the required ingredients, characterization, and further, the synthesized compounds were tested for antimicrobial and antioxidant activity.

2. EXPERIMENTAL:

Materials:

The reagents, Quercetin dihydrate (Q), 2, 2’-bipyridine (Bpy), 1, 10- phenanthroline (Phen), and 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) were acquired from the Sigma Aldrich.

Measurements:

Characterization and structure elucidation of the synthesized compounds was done by Elemental analysis (Euro EA Elemental Analyzer), FT-IR spectra (Perkin Elmer Spectrum version 10.4.3 spectrometer as KBr pellet, within the range of 400-4000 cm^-1), UV- visible spectra (UV-1800 Shimadzu ver. 2.52 spectrophotometer using 1 cm path-length quartz cuvettes, in the range of 200-700 nm). NMR study (Bruker Corporation, JNM- ECZ500R/S1), HRMS study (TOF MS, 100-1000). The antioxidant studies (DPPH method), MIC assay against E. coli and S.aureus bacteria were also performed. Here quercetin itself is taken as reference for comparison of the activity of complexes.

2.1 Synthesis of quercetin–zinc (ii) with 2,2'-bipyridal, and 1, 10-phenanthroline complex:

Complex 1 and 2 were synthesized by taking all the ingredients in the molar ratio of 1: 1: 1. Accurately weighed compounds were taken followed by trituration using a clean and dried mortar, pestle at room temperature. Complex 1 (reddish-orange) was synthesized with quercetin (1mmol), 1, 10-phenanthroline (1mmol), and Zn (II) metal ions (1mmol) by the solid-state trituration method using the mortal, pestle. Quercetin (1mmol), 2, 2'-bipyridal (1mmol), and Zn (II) metal ions (1mmol) in the solid-state was taken for complex 2 (reddish- yellow) syntheses by the same process. The change in color of the product shows that the reaction is completed and the product was characterized by different spectroscopic techniques.

Complex1. C₂₉H₂₁ZnN₂O₁₀ (621gmol-1): Yield: 93%, IR (KBr, cm-1): 3363.32 (ƲO-H), 1644.11(ƲC=O), 1416.64 (ƲC-OH), 1265.70 (ƲC-O-C), 1182.56 (ƲCC+ƲCCH), 759.35, 731.72 (ƲNCCH+ƲCCCH). Elemental analysis: Expt. (Calc.) C% 55.87(55.92), H% 3.84(3.40), O% 25.52(25.69), 1H NMR (500 MHz), δ/ppm (DMSO-d6): 6.0 – 6.3 (s, 4H, -OH), 7.918- 9.005(s, 8H, Bpy and s, 5H, -CH), 1.769 (s, 3H, -CH3), HRMS: m/z 624.9856 [(Q-H) + Zn (II) (Phen) (AcO) (H₂O)].

Complex 2. C₂₇H₂₁ZnN₂O₁₀ (597gmol-1): Yield: 95%, IR (KBr, cm-1): 3131.39 (ƲO-H), 1646.43(ƲC=O), 1426.63 (ƲC-OH), 1264.14 (ƲC-O-C), 1168.25 (ƲCC+ƲCCH), 824, 724 (ƲNCCH+ƲCCCH). Elemental analysis: Expt. (Calc.) C% 54.00(54.15), H% 3.45(3.53), O% 26.48(26.72), 1H NMR (500 MHz), δ/ppm (DMSO-d6): 6.107-6.814 (s, 4H, -OH), 7.587- 8.731 (s, 8H, Phen and s, 5H, -CH), 1.809- 2.877 (s, 3H, -CH₃), HRMS: m/z 599.0085 [(Q-H) + Zn (II) (BPY) (AcO) (H₂O)].

2.2 In vitro antioxidant activity (DPPH method):

Antioxidant activity of the compounds (Complex 1, 2) was checked by an earlier reported method of Brand Williams et.al with slight modification¹⁹. Different concentrations of complex 1, 2 (30µL) were taken with 3ml of DPPH (0.004% of methanolic solution) followed by stirring. The mixture was incubated for a quarter-hour at room temperature. The absorbance of the incubated mixture was taken at 517nm using spectrophotometer and results are expressed as inhibition percentage of free radical by the compounds.

[DPPH free radical scavenging activity (%) =

Ao-At/Ao× 100]

Where Ao = absorbance of the control and A_t= absorbance of the test /standard sample

2.3 In vitro antibacterial activity:

Antibacterial activity of Complex 1 and complex 2 was tested in-vitro and MIC was determined by using the micro-dilution method²⁰ against Gram-positive i.e. Staphylococcus aureus (S. aureus, ATCC 29213) and gram-negative i.e. E.coli strains (ATCC 25922). Cultures were adjusted to 0.5 Mc Farland standards, i.e. microbial suspension of roughly1.5 × 10⁸ CFU/mL. MHA (Mueller Hinton agar) plates were swabbed with bacterial inoculums of S. aureus and E. coli separately. Antibacterial activity and MIC of compounds were evaluated after 24 hrs at 37ºC by taking absorbance.

3. RESULTS AND DISCUSSION:

Reddish yellow-colored compounds were obtained and characterized by photo physics, FT-IR spectroscopy, NMR, HRMS, and elemental analysis. The proposed chemical structure of the complexes as in Scheme 1 was drawn based on spectroscopy and analytical methods. ¹H NMR spectra were recorded on a Bruker Advance III 500 spectrometer with standard pulse programs at 500.10 MHz (¹H) in DMSO-D6 at 302°K with TMS as an internal standard. HRMS and FT-IR data supported well with the proposed stoichiometry. The compounds are soluble, only in DMSO <10-3mol/L. The in-vitro biological activities as antioxidant assay, antibacterial activities (E. coli and S. aureus) were performed. The activity of complexes synthesized has been compared with quercetin as reported in earlier studies²¹.

3.1 Infrared analysis:

The assignments of the wavenumbers of selected hand-picked bands from the FT-IR spectra of quercetin and its metallic element i.e. zinc bipyridal and phenanthroline were tabulated in Table 1. FTIR analysis of the residue obtained after heating of complex 1 and 2 above 800⁰C showing bands at 478 cm-1 and 475 cm^-1 Fig.1 (a) and (b)²². These bands are characteristics of (ZnO) left after heating indicate the presence of Zn in reported complexes (1 and 2).

Fig.1: FT-IR spectrum of residue (ZnO) for complex 1 Fig. (a) and Complex 2 (b).

3.4 High-Resolution Mass Spectroscopy Study:

Mass spectral study of the complexes was performed to determine the molecular weight of the complexes. [Fig.4 (a)] HRMS spectra of complex 1 were taken in positive mode the peak at m/z (Calc.) 624.878 and (Exp.) 624.9856 [(Q-H) + Zn (II) (Phen) (AcO) (H₂O)] corresponds to the formation of QZnPhenAcOH₂O with a stoichiometry of 1:1:1 and [Fig.4 (b)] HRMS spectra of Complex 2 were also taken in positive mode and the peak at m/z (Calc.) 599.858 and (Exp.) 599.0085 [(Q-H) + Zn (II) (BPY) (AcO) (H2O)] corresponds to the formation of QZnBPYAcOH₂Owith a stoichiometry of 1:1:1.

3.5 Antioxidant activity (DPPH method):

The antioxidant activity of quercetin, complex 1, and 2 were determined by the radical scavenging DPPH method. The percentage of free radical inhibition activity is summarized in Table 2. It is observed that quercetin (IC₅₀ 38.1427µg/ml), complex 1(IC₅₀ 163.093µg/ml) have significant free radical scavenging activity as compared to complex 2 (IC₅₀ 258.683µg/ml). Complex 1 has more potent antioxidant activity as compared to complex 2.

Table2.Antioxidant data of the ligand and their complexes.

	Complex 1	Q	Complex 2
conc.	% Inhibition	% Inhibition	% Inhibition
20	19.2	30.8	17.233
50	25.5	63.1	23.786
100	39.7	71.4	25.728
200	55.3	77.2	43.042
300	79.5	77.7	54.531
400	80.8	78.3	60.841
500	81.3	78.7	61.327
IC₅₀	163.093 µg/ml	38.142 µg/ml	258.683 µg/ml

3.6 In vitro bactericide activity (MIC):

The minimum inhibitory concentration of ligand and its complexes (1,2) were studied on the growth of tested microorganisms (Table 3). The MIC data indicated that synthesized complexes have high potential to restrict the growth of tested microbes as compared to the free ligand. Complex 1(MIC15.50µg/ml) was found more active as compared to complex 2 (MIC 22.51µg/ml) and ligand (MIC 33.50µg/ml) against E. coli (ATCC 25922). S. aureus (ATCC 29213) was found more susceptible for complex 1 (MIC 7.18µg/ml) as compared to complex 2 (MIC18.62µg/ml) and ligand (MIC 31.25µg/ml). Complex 1 was found more active in both the microbes as compared to complex 2 and quercetin.

Table3. Minimum Inhibitory Concentration of the ligand and their complexes

Complexes	*S. aureus* MIC(µg/ml)	*E. coli* MIC(µg/ml)
Q	31.25	33.50
complex 1	7.18	15.50
complex 2	18.62	22.51

4. CONCLUSION:

Solid-state synthesis of the complex based on quercetin ligand with Zn (II) acetate incorporated with N ^ N moiety and spectral data (FTIR, UV, NMR, HRMS, and elemental analysis) analysis we may conclude the participation of ring c i.e. carbonyl group of quercetin coordinate through the 5-hydroxyl group with metal [Zn(II)] acetate. Both the complexes are only soluble in DMSO and DMF solvents. Quercetin itself has low bioavailability but when it will coordinate with metal forming complexes its bioavailability increases which may show wide applications in human diseases. From the above conclusion, we may say that complexes (1, 2) safe and showing good antibacterial activity. The study confirms that the newly synthesized compounds are very much active as compared to the quercetin. The complexes may further be studied for detailed biological activity and mechanism of action.

5. ACKNOWLEDGMENTS:

The authors are thankful to Dr. S. K. Mishra Department of Pharmaceutics IIT-BHU for his moral support, encouragement.

6. CONFLICT OF INTEREST:

All the authors declare that there’s no conflict of interest regarding the publication of this paper.

7. ABBREVIATIONS:

DPPH (1, 1-Diphenyl-2-picrylhydrazyl), FT-IR (Fourier Transformation – Infra-Red Spectroscopy), HRMS (High-Resolution Mass Spectroscopy), Zn²⁺ (Zinc ions), ZnO (Zinc oxide), AcO (Acetate ions), DMSO (Dimethyl sulfoxide)

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Received on 24.06.2020 Modified on 17.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3585-3590.

DOI: 10.52711/0974-360X.2021.00620

Complexes Assignment (cm-1)
	Ʋ-OH	Ʋ-C=O	Ʋ-C-OH	Ʋ-CC+ Ʋ –NC Ʋ-CCH	Ʋ –C-O	Ʋ-CC+ Ʋ-CCH	Ʋ-NCCH+ Ʋ-CCCH	Ʋ-ZnO
Q	3410.46	1666.65	1463.00	1521.1	1263.13	1169.49	-	-
Complex 1	3131.39	1646.43	1426.63	1599.07	1264.14	1168.25	824, 724	478
Complex 2	3363.32	1644.11	1416.64	1599.42	1265.70	1182.56	759, 731	475