Formation and Evaluation of Herbomineral Complex

 

Ms. Shivani Watak, Dr. Mrs. Swati S. Patil

Department of Pharmacognosy and Phytochemistry, Prin. K.M. Kundnani College of Pharmacy, Cuffe Parade, Colaba, Mumbai.

*Corresponding Author E-mail: watak_shivani@rediffmail.com

 

 

ABSTRACT:

Background phenolic compound exhibit strong antioxidant properties but major drawback of phenolic compound is their poor bioavailability. Literature resources reveled that formation of complex of these phenolic compound with mineral will increase bioavailability and free radical scavenging activity. objective it was interesting to investigate their metal-chelating property, which stimulates our interest to evaluate  Zn(II)-chelating ability and the effect of Zn(II) on its radical-scavenging activity The knowledge of those properties can be applied in future studies aimed at elucidating the mechanisms of anticancer activity Material and method Phenolic compounds such as Catechin, Curcumin and polyphenols from Trigonella foenum graecum extract chelate the divalent metal ions Zn2+in weakly acidic aqueous solution. Thus herbomineral complex was formed by the process of chelation. Formed complex was evaluated by Colorimetric assay, UV, IR, NMR and HPLC studies These studies may serve as a guide for the examination of metal chelation by  polyphenols

 

KEYWORDS: poly phenols,  metal chelation,  evaluation.

 

INTRODUCTION:

Phenolic compounds constitute a diversified group of plant secondary metabolites in terms of structure, molecular weight and physicochemical and biological properties. They exhibit strong antioxidant properties but major drawback of phenolic compound is their poor bioavailability. Literature resources reveled that formation of complex of these phenolic compound with mineral will increase bioavailability and free radical scavenging activity. Hence herbomineral complex was formed with a view to increase bioavailability and activity. Phenolic compounds such as Catechin, Curcumin and polyphenols from Trigonella foenum graecum extract chelate the divalent metal ions Zn2+in weakly acidic aqueous solution. Thus herbomineral complex was formed by the process of chelation..

 

 

CHELATION PROCESS

The protonated phenolic group is not a particularly good ligand for metal cations, but once deprotonated, an oxygen center is generated that possesses a high charge density, a so-called "Hard" ligand . Although the pKa value of most phenols is in the region of 9.0-10.0, in the presence of suitable cations for instance iron(III) or copper(H), the proton is displaced at much lower pH values, e.g., 5.0-8.0. Thus metal chelation by phenols can occur at physiological pH values. Aliphatic alcohols do not share this property, as the resulting oxygen anion is not stabilized by the mesomeric effect typical of phenols. In principle, a pyrone oxygen can also bind metal cations due to the partial delocalization of the lone pairs associated with the heteroatom. Such delocalization is more prominent in non fused rings such as maltol, than in compounds with fused rings, e.g., the bicyclic compound

 

Generally, the metal complexes of organic ligands exhibit lightfastness properties better than those of the free ligands. This is due to the effect of coordination with a metal ion reducing the electron density at the chromophore, which in turn leads to improved resistance to photochemical oxidation. In addition, the larger size of the metal ion complex molecules compared with the free ligand generally gives rise to better wash fastness properties through stronger interactions with the fibres(1,2 ).

 

 

Figure 1.1 Chelation process

          

MATERIAL AND METHOD:

Reagents required: HCL, ZnCl2, KCL and Methanol

 

Preparation of test material

HCl buffer: It is prepare by addition of 0.1 M HCl and 0.1M KCl at pH 5.0.

ZnCl2:  ZnCl2 was added in the same buffer at a concentration of 8mM.

Catechin, Curcumin and Trigonella foenum graecum extract solution: Weighed 10mg Catechin, Curcumin and Trigonella foenum graecum extract and dissolved in 10 ml methanol to get 1mg/ml solution, which was further diluted to get a concentration of 10µg/ml.

 

Methodology

HCl buffer (0.1M)  is prepared with addition of 0.1M KCl at PH 5.0. ZnCl2 was dissolved in the same buffer at a concentration of 8mM. Catechin, Curcumin and fenugreek extract solution was prepared in methanol i.e (10µg/ml). 1ml of Catechin ,Curcumin and Trigonella foenum graecum extract was mixed with 3ml salt solution  and kept aside for few hours. After few hours precipitates were formed which were settle down at the bottom of the test tube. Herbomineral complex of Catechin, Curcumin and Trigonella foenum graecum extract with zinc was formed in the form of precipitates, solvent was evaporated in evaporating dish and dried precipitates were collected.(3,4)

 

METAL CHELATING ACTIVITY ASSAY

Reagents required :Murexide, methanol, Deionized water, Zinc chloride

 

Preparation of test material

Catechin, Curcumin and Trigonella foenum graecum extract solution: 1mg/ml solution of Catechin, Curcumin and Trigonella foenum graecum extract was prepared, were reconstituted in methanol to get different concentration like 10, 40, 80 and 100µg/ml, used for assay.

 

Methodology

The chelating activity of the extracts for zinc ions was measured. To 0.5 mL of extract, 1.6 mL of deionized water and 0.1 mL of ZnCl2 (8 mM) was added. After 30 second, 0.1 mL murexide (5mM) was added. Murexide reacted with the divalent zinc to form stable magenta complex species that were very soluble in water. After 10 min at room temperature, the absorbance of the zinc complex was measured at 562 nm. The chelating activity of the extract for Zn2+ was calculated as (5)

 

 

Chelating rate (%) = (A0 - A1) / A0 × 100

Where, A0 was the absorbance of the control (blank, without extract) and A1 was the absorbance in the presence of the extract

 

Table 1.1 Metal chelating Activity of   Catechin

Concentration(µg/ml) of Catechin

Chelating rate(%)

IC50

10

49.19

 

40

51.61

20.92

80

58.46

 

100

61.53

 

 

 

Figure 1.2 plot of % chelation rate Vs concentration of catechin

 

Table 1..2 Metal chelating   Activity of  Curcumin

Concentration(µg/ml) of Curcumin

Chelating rate(%)

IC50

10

20.56

 

40

45.56

76.49

80

49.19

 

100

57.25

 

             

 

 

Figure 1.3  Plot of % chelation rate Vs concentration of Curcumin

 

 

Table 1.3 Metal chelating Activity of Trigonella foenum graecum extract

Concentration (µg/ml) of Trigonella foenum graecum extract

Chelating rate( %)

IC50

10

37.5

 

40

49.59

70.87

80

50.4

 

100

54.03

 

  

Figure 1.4 Plot of % chelation rate Vs concentration of Trigonella foenum graecum extract

 

 

RESULT AND DISCUSSION:

Catechin and Curcumin possess good anticancer activity but both have poor bioavailability. From the literature search it was revealed that formation of herbomineral complex will increase the bioavailability and antioxidant activity of drug. A few studies found that zinc levels in serum and/or inside white blood cells were often lower in patients with head and neck cancer or childhood leukemia. Zinc helps to decrease oxidative stress and improves immune function and has important role in DNA, RNA synthesis and healing of damage cells thus improves overall cell function, is possible mechanisms for cancer preventive activity. Hence herbomineral complex was formed to increase activity of Catechin, Curcumin and Trigonella foenum graecum extract. Zinc was selected for formation of complex because of its above benefits

 

Chelating rate (%) evaluation by metal chelating activity assay revealed that, Catechin has highest chelation activity. This greater affinity of Catechin for metal ion may be due to large number of hydroxyl group in the molecule.

Literature survey revealed that curcumin has less affinity for Zinc ion, also it has less number of hydroxyl group in their molecule, due to which it showed less chelation activity than catechin

 

Trigonella foenum graecum seed extract (methanolic) contains flavonoids which may undergo chelation, and are responsible for its metal chelating activity.

 

EVALUATION OF HERBOMINERAL COMPLEX

Herbomineral complex of Catechin, Curcumin and Trigonella foenum graecum extract was formed with zinc. To determine how much quantity of zinc chloride was needed for converting drug into its complex form. Where the zinc was actually attach, how it was attach what was the structure of parent drug after formation of complex, these types of various questions come in mind. Hence it intends to offer critical evaluation of existing herbomineral complex. Complex formed was evaluated by following methods

·        Colorimetric assay

·        Ultraviolet spectroscopy

·        Infrared spectroscopy

·        Nuclear magnetic resonance

·        High performance  liquid chromatography

 

COLORIMETRIC ASSAY

Introduction

Heterocyclic azo dyes have been used as chromogens in spectrophotometric determination of metal ions. This method showed a good sensitivity and high selectivity for zinc(II) ions as most of the common metal ions. A sensitive and selective spectrophotometric method is proposed for the rapid determination of zinc(II) using an 8-hydroxyquinoline derivative, 7-(4-nitrophenylazo)-8-hydroxyquinoline-5-sulfonic acid (p-NIAZOXS), as a new spectrophotometric reagent. The reaction between the p-NIAZOXS and zinc (II) is instantaneous at pH 9.2 .and the absorbance remains stable for over 24 h. The method allows the determination of zinc over the range of 0.05-1.0 mg mL-1 with a molar absorptivity of 3.75x104 L mol-1 cm-1 and features a detection limit of 15 ng mL-1. The proposed method has been successfully applied to the determination of zinc in several pharmaceutical preparations

 

The 8-hydroxyquinoline (oxine) behaves as a bidentate (N,O-) univalent ligand toform chelates with several metal ions. Cations with n charge and 2n coordination number form the so-called "coordination saturated uncharged chelates" which are insoluble in water, but easily soluble in organic solvents. The reagent was a dark red powder, with a low solubility in water and organic solvents but very soluble in alkaline solutions. The p-NIAZOXS reacts immediately with zinc forming an orange-yellow complex in aqueous medium and the absorbance reached its maximum within 5 min and remained stable, for at least, 24 h. The structure of p-NIAZOXS is as follows. (6)

                               

Structure of 7-(4-nitrophenylazo)- 8-hydroxyquinoline-5-sulphonic acid.

 

 

Reagents required

7-(4-nitrophenylazo)-8-hydroxyquinoline-5-sulfonic acid (p-NIAZOXS)

Preparation of reagents and test material

·        PNIAZOXs solution – 100mg of PNIAZOXs was dissolved with 250ml of 0.01M NaOH (1month stable)

·        HCl buffer- 0.1 M HCl buffer is prepared with addition of 0.1M KCl at PH 5.0.

·        Stock solution of Zncl2 – 50mg/500ml

 

Methodology

Into six different test tubes transfer 0.5ml, 1, 2, 3, 4, 5ml of Zncl2 from stock solution. Add 0.4ml of PNIAZOXs solution and 1.25ml of HCl buffer solution. Make up the volume to 10 ml. And measured the Absorbance at 430nm.Standared curve for ZnCl2 was  ploted between concentration and absorbance.

 

Transfer a portion of ZnCl2 in increasing concentration from 0.5--------5ml in six different test tubes. Add 1ml of Catechin (10µg/ml) to each test tube. Add and 1.25ml of Hcl buffer solution. Kept  aside for 1 hr. Which allowed formation Catechin zinc complex in the form of precipitates? Then add 0.4ml of PNIAZOXs solution in each test tube. Make up the volume upto 10ml. PNIAZOXs reagent form yellowish orange colored complex with zinc. Test tubes are centrifuged at 4000 RPM for 10 minutes. Absorbance of supernatant was recorded spectrophotometrically at 430nm.Same procedure is repeated for Curcumin and Trigonella foenum graecum extract

 

Figure 1.5 Colour reaction of ZnCl2

       

Figure 1.6 Colour reaction of Catechin

  

Figure 1.7 Colour reaction of Curcumin

 

Figure 1.8 Colour reaction of Fenugreek extract

 

Figure 1.9  Plot of Absorbance Vs Concentration of Zinc chloride

 

Table 1.4: Results of Colorimetric assay by using P-NIAZOXs indicator, Absorbance of blank= 0.1913                                                                                                                   

 

 

 

Concentration of ZnCl2(50mg/500ml)

stock solution

Absorbance of supernantant

Absorbance of zinc which form complex with drug

 

 

 Zinc chloride (A1)

0.5ml

0.1968

 

1ml

0.2286

 

2ml

0.2581

 

3ml

0.3561

 

4ml

0.4087

 

5ml

0.4864

 

 

 

 

       (A1-C1)

 

 

1ml of

Catechin(10µg/ml) C1

0.5ml

0.0204

0.1764

1ml

0.0408

0.1878

2ml

0.0603

0.1978

3ml

0.1003

0.2558

4ml

0.1542

0.2545

5ml

0.2285

0.2579

 

 

 

(A1-C2)

 

1ml of

Curcumin(10µg/ml) C2

0.5ml

0.0312

0.1656

1ml

0.0674

0.1612

2ml

0.0825

0.1756

3ml

0.1145

0.2416

4ml

0.1621

0.2466

5ml

0.2408

0.2456

 

 

 

(A!-C3)

 

0.5ml

0.0432

                0.1536                          

1ml of Trigonellafoenumgraecumextract

C3

1ml

0.0689

             0.1579

2ml

0.0898

   0.1683

3ml

0.1076

0.2485

4ml

0.1465

0.2631

5ml

0.2198

0.2670

 

 

Figure 1.10 Plot of Absorbance Vs Concentration of catechin

 

Figure 1.11  plot of Absorbance Vs Concentration of  curcumin

 

Figure 1.12  plot of Absorbance Vs Concentration of Trigonella foenum graecum extract

 

ULTRAVOILET SPECTROSCOPY

Preparation of test solution:

10mg of Catechin, Curcumin, Trigonella foenum graecum extract was weighed and dissolved in 10ml methanol to get a stock solution of 1mg/ml. It was further diluted to get a concentration of 10µg/ml

 

Methodology

0.1 M HCL buffer is prepared with addition of 0.1M KCL at PH 5.0.ZnCl2 was dissolved in the same buffer at a concentration of 8mM. Catechin, Curcumin, Trigonella foenum  graecum extract solution was prepared 10µg/ml. 1ml of Catechin solution was  take in test tube  and added salt solution  1ml, 2ml,3ml……in increasing concentration. UV spectra was taken in the range of 200-400nm after adding each ml of salt solution. Same procedure was repeated for Curcumin and Trigonella foenum graecum extract

 

Instrument used -Jasco UV-550 UV/VIS spectrophotometer.

 

 Figure 1.13 UV scan of Catechin (10µg/ml)

 

 

 Figure 1.14UV scan of Catechin1ml+ 1ml salt solution

 

 Figure  1.15 UV scan of Catechin1ml+ 2ml salt solution

 

 

 Figure 1.16  UV scan of Catechin1ml+ 3ml salt solution

 

 Figure 1.17  UV scan of Curcumin (10µg/ml)

 

 

 Figure 1.18 UV scan of Curcumin1ml+ 1ml salt solution

 

 Figure 1.19 UV scan of Curcumin1ml+ 1.5 ml salt solution

 

 Figure 1.20 UV scan of Curcumin1ml+ 2ml salt solution

 

 Figure  1.21  UV scan of Trigonella foenum graecum extract (10µg/ml)

 

Figure  1.22  UV scan of Trigonella foenum graecum extract1ml+ 1ml salt solution

 

 

Figure 1.23 UV scan of Trigonella foenum graecum extract 1ml+ 1.5 ml salt solution

 

 

Figure 1.24  UV scan of  Trigonella foenum graecum extract1ml+ 2 ml salt solution

 

 Figure 1.25 UV scan of  zinc chloride

 

 Figure 1.26 UV scan of  buffer 

 

Figure 1.27  UV scan of methanol

       Figure 1.28 UV scan of buffer + methanol

             Figure 1.29  UV scan of ZnCl2+buffer + methanol

 

INFRARED SPECTROSCOPY

Methodology

IR spectrum of Catechin, Curcumin and Trigonella foenum graecum extract and their herbomineral complex was recorded using SHIMADZU IRAffinity-1. These samples were examined by mixing the sample with KBr in the Ratio of 1:100 and applying the principals of diffuse reflectance spectral measurement.

 

 

Figure 1.30  I.R. spectra of Catechin

 

Table 1.5  Analysis of  I.R. spectra of   of Catechin

C-H stretching

3035.12

O- H stretching(free)

3650.44,3580.04

- C-O stretching

1362.77

C=C stretching

1623.17, 1520.94

Benzene ring

1287.54, 1147.89, 1031.00,

 

 

 

 

 

                            Figure 1.31   I.R. spectra of hrebomineral complex of Catechin

Table  1.6  Analysis of I.R. spectra of hrebomineral complex of Catechin

C-H stretching

3250.05

O –H stretching (free)

disappear

Benzene ring

1105.21

C=Cstrething

1612.49,

-C-O stretching

1303.88, 1402.25

 Chelated compound

3271.27, 3250.05,2912.51,2845.00

                 

                

                Figure.1.32  I.R .spectra of Curcumin

Table 1.7  Analysis of I.R. spectra of Curcumin

O-H(free) stretching

3565.54,3678.11

C=C stretching

1507.44,

C-H stretching

3185.58

C= O stretching

1616.42,

 C-O-C (H3CO) methoxy

1283.68

                        

.

Figure 1.33  I.R. spectra of herbomineral complex of Curcumin

 

Table 1.8  Analysis of I.R. spectra of herbomineral complex of Curcumin

O- H (free) stretching

disappear

C=C stretching

1507.44,

C – H stretching

3022.58

C= O(α- β unsaturated acyclic)

1624.13,, 1680.07

Chelated compound

2902.99, 2848.98, 

 C- O-C  (H3CO ) methoxy

1283.68

                     

 

Figure 1.34  I.R. spectra of Trigonella foenum graecum extract

Table 1.9  Analysis of I.R. spectra of Trigonella foenum graecum extract

C-H stretching

3008.96, 2922.18

O-H (free)stretching

3624.25,3583.74

Benzene ring

1232.62,1213.23,1043.49

C=C stretching

1504.48,1651.70

-C-O stretching

1444.89

C =O stretching

1745.58

           

            

       Figure 1.35  I. R. spectra of herbomineral complex of  Trigonella foenum graecum extract

 

Table 1.10 Analysis of I. R. spectra of herbomineral complex of  Trigonella foenum  graecum  extract

C-H stretching

 3219.20, 2941.45

O- H (free) stretching

disappear

C=C stretching

1612.49

-C-O stretching

1408.04

C =O stretching

 disappear

Chelated compound

3219.20,2941.45,2910.59,

 

      

NUCLEAR MAGNETIC RESONANCE 

Methodology

Nuclear Magnetic Resonance (NMR) spectroscopy was done by recording the spectra using CDCl3 by 1HNMR on Joel FT/NMR 300MHz analyzer.

    

      Figure 1.36  NMR spectra of Catechin      

 

 Figure 1.37  NMR spectra of herbomineral complex of Catechin

 

Table 1.8 Analysis of NMR spectra of Catechin complex

Catechin

Catechin complex

Multiplicity

Chemical shift(ppm)

groups

 Height/frequency

 

Double dublet

8.9

-OH

39.8 (2686.135)

disappear

Double dublet

6.7

-OH

19.8 (2012.365)

disappear

Dublet

6.65

-OH

24.3(1999.168)

disappear

Dublet

2.72

-OH

4.7 (805.226)

disappear

Dublet

2.27

-OH

4.6 (689.387)

disappear

 

 

Figure 1.38  NMR spectra of Curcumin

 

Figure 1.39  NMR spectra of herbomineral complex of Curcumin

 

 

Table 1.11  Analysis of NMR spectra of Curcumin complex

Curcumin

Curcumin complex

Multiplicity

Chemical shift(ppm)

groups

 Height/frequency

 

singlet

9.8

-OH

51.2 (2901.316)

disappear

 singlet

10

-OH

5.5 (3018.987)

disappear

            

 

 

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Reference compound and reagents

Catechin hydrate was procured from sigma Aldrich lab (China) and Curcumin was procured from LOBA chem Laboratory, Mumbai (India)

 

 

 

Preparation of standard solutions

10 mg of standard Catechin, Curcumin and Trigonella foenum graecum extract was accurately weighed and was added to 10 ml HPLC grade methanol (Stock solution 1mg/ml). This stock solution was further diluted to get different concentrations ranging from 100μg/ml HPLC grade methanol.

 

Preparation of sample solution

10 mg of herbomineral complex of Catechin, Curcumin and Trigonella foenum graecum extract was accurately weighed and dissolved in 10 ml HPLC grade methanol to get solution of 1mg/ml. This solution was further diluted to get concentration of 100μg/ml.

 

Chromatographic Conditions:

Table 1.12 Optimized chromatographic conditions for the analysis using HPLC.

 

Chromatographic Mode

Chromatographic Condition

Standard solution

100µg/ml in methanol

HPLC System

Jasco HPLC system

Pump

JascoPU 2080 PLUS Intelligent HPLC Pump

Detector

Jasco MD-2010 PLUS multiwavelength Detector

Data processor

chrompass Software

Stationary phase

 Tsk gel silica-60 no. 6SLMO112

Mobile phase

Catechin

  Acetonitrile: water: ortho phosphoric acid

(15: 84.9: 0.1)

 

 

Curcumin

Methanol: water ( 70:30)

Fenugreek extract

Water: methanol ( 80:20)

Detection

wavelength

Catechin

280nm

Curcumin

425nm

Fenugrrk extract

270nm

Flow rate

0.5 ml/min

Volume of injection

20µl

 

 

 Figure  1.40  HPLC chromatogram of Catechin

 

 

 

Figure 1.41   HPLC chromatogram of  chelated Catechin

 

  Figure 1.42   HPLC chromatogram of Curcumin

 

 

Figure 1.43 HPLC chromatogram of Chelated Curcumin

 

 Figure 1.44 HPLC chromatogram of Trigonella foenum graecum extract

 

 

 Figure 1.45  HPLC chromatogram of chelated fenugreek extrac

Table 1.14 HPLC analysis of drug and their herbomineral complex

component

Retention time

λmax

Catechin

3.067

278

Catechincomplex

7.34

208

Curcumin

6.173

425

Curcumin complex

6.4

208

Fenugreek extract

5.26

272

Fenugreek ext. complex

5.413

212

 

RESULT AND DISCUSSION:

Hydroxyl azo dye i.e. P-NIAZOXs react with Zn+2 ion and forms orange yellow complex.  The standard curve for zinc chloride showed that, as the concentration of zinc increases corresponding absorbance increases, where as drug zinc complex, was settle down at the bottom. Hence, absorbance of super supernatant was taken. These reading for supernatant were minus form the readings of zinc chloride of same concentration which gives absorbance of zinc chloride present in the complex. The graphs for zinc chloride and supernatant liquid of complex are straight line graphs where as the graph of complex showed increase in concentration initially but remain stable form 3ml concentration of zinc chloride.

 

From this colourimetric assay it was found that for 1ml of (10µg/ml) of drug solution approximately 3ml of 8mM ZnCl2 is sufficient for chelation.

 

Ultra violet spectroscopy, showed shifting of λ- max value,   λ-max of Catechin was 280 nm which was shifted to 216 nm after adding 3ml of salt solution in case of Catechin complex. Curcumin  showed three peaks with λ-max value at 268nm, 261nm and 226nm whereas Curcumin complex showed  shifting to 208 nm after adding 2ml salt solution   curcumin required less amount of salt solution as compared to catechin and TFG extract because as compared to both these curcumin has less number of hydroxyl group. Trigonella foenum graecum extract showed three peaks with λ-max value at  330nm, 270nm and 224nm, whereas Trigonella foenum graecum extract complex showed shifting to 210nm.

 

UV scan of Zinc chloride showed λ-max value at 216nm, where as buffer has no λ-max value.  From these values it indicate that λ-max of Catechin, Curcumin and Trigonella foenum graecum extract was shifted to 216nm, 208nm and 210nm respectively, which was near to the λ-max of zinc chloride i.e. 216nm, hence it indicate that there is formation of drug zinc complex .This shifting required 2-3ml of salt solution, hence 2ml for curcumin and TFG extract  and 3ml of salt solution for catechin  is sufficient for (1ml of 10µg/ml of drug) complete formation of herbomineral complex with zinc.

 

Presence of a broad band at (3500-3650 cm-1) in Catechin and Curcumin is attributable to the –OH group. Absence of this broad spectrum in herbomineral complex indicates that the complexation involve the phenolic and enolic –OH groups. Also indicates that Zn+2 attach to Catechin and Curcumin by replacing -OH group. Similarly, incase of Trigonella foenum graecum extract presence of broad band at (3500 -3650 cm-1) is attributable to the –OH group. Absence of this broad spectrum in herbomineral complex indicates that the complexation involve the phenolic and enolic –OH group. And Zn+2 attach to Trigonella foenum graecum extract by replacing -OH group. Also absence sharp peak at 1745.58 cm-1(attributable to the C=O) in herbomineral complex indicates that it is also involved in complexation

 

From NMR spectra of catechin and its herbomineral complex with zinc showed that , five prominent peaks catechin i.e. 8.9dd, 6.7 dd, 6.65 d, 2.72 d, 2.27 d ( attributable to –OH group) were disappear or the integration of these prominent peaks goes very down in catechin complex. This indicates that Zn +2ion attach to catechin by replacing –OH group.

 

In case curcumin, four prominent peaks i.e. 6.9 d, 7 d, 7.2 s, 7.5 d were remain as it is in curcumin complex. Where as 6 s, was shifted to 5.8 in curcumin complex. Two prominent e. 9.8 s, 10s ,(attributable to –OH)  which may come under acidic region are completely disappear in curcumin complex. Hence Zn +2ion attach to curcumin by replacing –OH group.

 

The probable structure of catechin and curcumin from NMR after complexation may be as given below

 

 (Catechin complex)

 

 (curcumin complex )

 

 

TSk gel column are used to analyze compounds depending upon the molecular weight on same reverse phase silica gel column. Lower molecular weight compound elute first in these column higher molecular weight later. The HPLC chromatogram of catechin depicted retention time at 3.06 mins at 278nm and catechin complex depicted retention time at 7.34 at 208 nm. The HPLC chromatogram of curcumin depicted retention time at 6.1 mins at 425 nm and curcumin complex depicted retention time at 6.4 at 208nm. The HPLC chromatogram of Trigonella foenum graecum extract depicted retention time at 5.26 mins at 272nm and its complex depicted retention time at 5.4 at 212 nm. Hence from the above values of retention time showed that there may be increase in molecular weight of drug after formation of complex with zinc. It also showed shifting of Λ-max values which indicate that herbomineral complex was formed.

 

CONCLUSION:

Herbomineral complex formed was evaluated by colorimetric assay, UV, IR, NMR and HPLC. Colorimetric assay confirmed presence of zinc in the complex and also helps to find out amount Zinc chloride required for formation of complex. From IR and NMR analysis it was confirmed that complex was formed by replacing OH group.  HPLC study indicates increase in molecular weight. Thus these above observation confirmed formation of complex.

 

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Received on 13.04.2012       Accepted on 24.05.2012     

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