INTRODUCTION:

Herbal medicine has been enjoying renaissance among the customers throughout the world. However, one of the impediments in the acceptance of the ayurvedic medicines is the lack of standard quality control profiles. The quality of herbal medicine i.e. the profile of the constituents in the final product has implicationin efficacy and safety. Due to the complex nature andinherent variability of the chemical constituents of plant-based drugs, it is difficult to establish qualitycontrol parameters. To over come these problemsmodern analytical techniques are expected to help in circumventing this problem ^1,2

Rhizomes of Curcuma longa contains curcuminoids (50-60%) which contains curcumin(~6%)⁵ as active constituent responsible for anti rheumatic activity. From the ancient times it is known and used more efffectively for anti inflammatory and anti rheumatic activity. Curcumin shows anti rheumatic activity by inhibiting both 5-lipoxygenase and 12-lipoxygenase activity³, also it inhibits the NF – κB and promotes adhesion of neutrophils to human umblical veins endothelial cells⁴. Nowadays Curcuma longa extract is used in most marketed formulations. Also many phytoconstituents are analysed by HPLC due to its versatility , safety , dependability and sensitivity.Hence HPLC is being used in the laboratories of many pharmaceutical companies for the routine assay of new drugs , as well as substitutes for older, more troublesome assays for marketed drugs.

So, estimation of curcumin in those formulations by using HPLC chromatographic techniques paves major role⁶.

MATERIALS AND METHODS:

Chemical and reagents:

All the formulations were procured from local market of amravati.All the chemicals used in formulatins were of analytical grade.Curcumin ( purity 99 % ) standard was purchased from Natural remedies Pvt Ltd., Bangalore.All the solvents used in experiments were of analytical grade.

Apparatus:

Instrument used : Jasco- Borwin system

HPLC System : Jasco gradient mode HPLC

Pump : Jasco PU 2080 Plus

Column : HiQ Sil C 18 W Size, 4.6 mm × 250 mm i.d.

Wavelength : 420 nm

P^HOf Buffer : 2.7 ± 0.05

Injection volume : 20 µl

Pressure : 16.0-20.0 mpascal

Flow rate : 1.5 ml/min

Temperature : Ambient

Mobile phase : Methanol : 5%Acetonitrile solution in water buffered to p^H 2.7 by 10% Orthophosphoric Acid (90: 10 v/v).

Preparation of standard solutions :

STD solution are prepared by dissolving 5mg of curcumin in 2.5 ml methanol ( HPLC grade ) and sonicate for 15 min and used as a stock solution (2000 μg/ml).Working STD solution are prepared by pippeting 0.1ml of stock solution to 10ml which produce 20.0 μg/ml by making volume with mobile phase from this solution 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 ml were pipetted and volume was made by mobile phase to obtain concentration of 0.4, 0.8, 1.2, 1.6, 2.0, 2.4 μg/ml. is achieved which is then sonicated for about 15 minutes and then used as a working standard.

Table 1 : Validation parameters for estimation of Curcumin

Sr. No.	Parameters	Curcumin
Sr. No.	Parameters	Area
1.	Linear range (mg/ml)	0.4 -2.4
2.	Slope	107581.2
3.	Y-intercept	3.4999
4.	Correlation coefficient (r)	0.9926
5.	LOD (mg/ml)	3.0196
6.	LOQ (mg/ml)	9.1503

Table2: Results of System Suitability Test Parameters

System suitability parameters	Proposed Method
System suitability parameters	Curcumin
Retention Time (t_R)	2.308
Peak Area (µV. sec.)	429432.50
Capacity Factor (k)	4.23
Theoretical plate Number(N)	2833.93
Asymmetry	1.91
Peak Height	25505

Preparation of sample solutions:

Formulation no. 1. Batch no. 1 and 2

For Determination of Curcumin:

Sample preparation is done by evaluating how much active constituent is present in the extracts of rhizome. The amount of Cucumin reported in rhizome extract is ~ 6%w/w. So weight of 1 tablet were taken approximately corresponding to 675 mg and powdered in mortar and pastel to obtain a powdery mass and 38 mg of powder which is then dissolved in mobile phase and final solution was made upto 10 ml by filtration.(2.0 μg/ml)

Formulation no. 2 . Batch no. 1 and 2

For Determination of Curcmin:

Sample preparation is done by evaluating how much active constituent is present in the extracts of rhizome. The amount of β – Boswellic acid reported in resin extract is ~ 6% w/w. So weight of 1 tablet were taken approximately corresponding to 1125 mg ( avg wt is 1183.3 mg and 1170.8 mg respectively for both the batches respectively) and powdered in mortar and pastel to obtain a powdery mass which is then dissolved in mobile phase and final solution was made upto 10 ml by filtration.(2.0 μg/ml).

Calibration curve for Curcumin :

20 μl of standard solution of Curcumin was injected in injection port of HPLC system by using mobile phase Methanol : 5%Acetonitrile solution in water buffered to P^H2.7 by 10% Orthophosphoric Acid (90: 10 v/v) on HiQ Sil C 18 W Size, 4.6 mm × 250 mm i.d. column with a flow rate of 1.5 ml/min at a pressure of 16-20 m pascal at ambient temperature.The peak was detected at 420nm. The peak areas were recorded and calibration curve was prepared by plotting peak areas Vs concentration applied.

Figure 1 Structures of analogues of curcuminoids .

Figure 2 a) overlay chromatogram of std.curcumin

b) curcumin chromatogram of formulation 1 Batch no.1

c) curcumin chromatogram of formulation 1 Batch no.2

d) curcumin chromatogram of formulation 2 Batch no.1

Table 3: Results of Method and Intermediate Precision for Curcumin

Formulations	Parameters	Method Repeatability Mean ± SD	Intermediate Precision
Formulations	Parameters	Method Repeatability Mean ± SD	Interday	Intraday	Different Analyst
Formulation 1Batch .no.1	% Estimation	98.13±0.174	98.47±0.4417	98.56±0.1533	98.83±0.2149
Formulation 1Batch .no.1	% RSD	0.177	0.4485	0.1556	0.2175
Formulation 1Batch .no.2	% Estimation	98.69±0.1067	98.89±0.2073	99.21±0.1140	99.31±0.1479
Formulation 1Batch .no.2	%RSD	0.1081	0.2096	0.1149	0.1489
Formulation 2Batch .no.2	% Estimation	99.89±0.15	99.64±0.1774	99.59±0.1398	99.72±0.1271
Formulation 2Batch .no.2	% RSD	0.1501	0.1762	0.1404	0.1275
Formulation 2Batch .no.2	% Estimation	101.41±0.4878	100.68±0.1387	100.27±0.08786	100.62±0.1447
Formulation 2Batch .no.2	% RSD	0.48	0.1392	0.08762	0.1438

^*Mean ± S.D(n=3)

Table 4: Results of Recovery Study For Curcumin

Formulations	% Level	Added Concentration ( μg)	Recovered (μg)	% Recovery	Mean ± SD	% RSD
Formulation 1Batch .no.1	80	1.6	1.58	98.75	97.22±1.9679	2.0241
	100	2.0	1.9	95
	120	2.4	2.35	97.91
Formulation 1Batch .no.2	80	1.6	1.54	96.25	97.36±1.0470	1.0754
	100	2.0	1.95	97.5
	120	2.4	2.36	98.33
Formulation 2Batch .no.2	80	1.6	1.55	96.87	98.34±1.2784	1.2999
	100	2.0	1.98	99
	120	2.4	2.38	99.16
Formulation 2Batch .no.2	80	1.6	1.56	97.5	97.39±0.6717	0.6897
	100	2.0	1.96	98
	120	2.4	2.32	96.67
Average % Recovery					97.57 ±0.5137	0.5264

^*Mean ± S.D(n=3)

e) curcumin chromatogram of formulation 2 Batch no.2

Quantification of Curcumin:

20 μl of sample solution of Curcumin was injected in injection port of HPLC system by using mobile phase Methanol: 5%Acetonitrile solution in water buffered to P^H2.7 by 10% Orthophosphoric Acid (90: 10 v/v) on HiQ Sil C 18 W Size, 4.6 mm × 250 mm i.d. column with a flow rate of 1.5 ml/min at a pressure of 16-20 m pascal at ambient temperature.The peak was detected at 420 nm.Peak areas and absorption chromatogram were recorded.To check the identity , peak chromatogram of Standard was overlayed with the corresponding chromatogram of sample solution.The amount of

Curcumin in the sample was calculated by using the formula :

The amount of each drugs estimated in laboratory mixture was calculated by using following formula.

Where,

Ew = Drug estimated in sample weight, mg

Cs = Concentration of standard, µg/ml

Au = Area of unknown

As = Area of standard

D = Dilution factor

The results indicate that the laboratory mixture containing( α + β) Boswellic acid and Curcumin can be estimated accurately by proposed method; so it was thought to apply the same procedure for marketed formulation.

Amount of drug present in average weight of tablet as per of labeled claim was calculated using following formula:

Where,

Ew = Amount estimated, mg

W_AV = Average weight of tablet, mg

Ws = Sample weight, mg

Lc = Labeled claim, mg/tablet

Validation of method :

The method was validated for precision, repeatability and accuracy. The method repeatability was carried out by injecting five replicates of sample solution (2 mcg/ml).

Variability of the results was studied by injecting sample solution on same day( intra day), on different days ( inter day) and by different analyst.

Accuracy of the method was tested by performing recovery studies at three levels (80%,100%,120% addition). The percent recovery as well as average percent recovery was calculated. Limit of detection and Limit of quantitation was evaluated by slope and standard deviation of the response.

Table 5: Estimation of Curcumin from the formulations 1and2

Formulations	% Curcumin (w/w)
Formulation 1 batch no.1	99.70 ±0.1529
Formulation 1 batch no.2	100.85±0.4882
Formulation 2 batch no.1	98.50±0.1024
Formulation 2 batch no.2	98.70± 0.1067

^*Mean ± S.D(n=3)

RESULTS AND DISCUSSION:

Formulations1and2 contains Curcuma longa extracts which contains curcumin as major actives. For quantification of this marker from these formulations HPLC method was developed. Mobile phases were optimized and extraction was carried out by using petroleum ether solvent for better resolution of marker compound from the other components of the sample extracts.Of the various mobile phases tried ,the one containing mobile phase Methanol: 5%Acetonitrile solution in water buffered to P^H2.7 by 10% Orthophosphoric Acid (90: 10 v/v) gave best resolution at retention time 2.333 for curcumin respectively in the presence of other compounds in the sample extract and enabled the quantification of the marker compounds. To check the identity and purity, peak chromatogram of Standard was overlayed with the corresponding chromatogram of sample solution.The method was validated in terms of precision , repeatability, accuracy studies.( Table 3and4).

The relationship between the concentrations of standard solutions and peak response was linear within the concentration range of 0.4 -2.4 mcg/ml with a correlation coefficient of

0.9926 for curcumin respectively.The average percent recovery at three different levels was found and results are presented in (Table 4).The content of curcumin in the formulations was estimated by proposed method ( Table 5). The content of curcumin in. in all these batches of formulations is found as per the labeled claim.The method was found to be suitable for estimation of marker compounds from herbal tablets.

We established HPLC method for the quantification of Curcumin from the formulations. The method were found to be simple, precise, specific, sensitive and accurate and can be used for their quantification in the plant materials and also in routine quality control of the raw materials as well as formulations containing any or all of these compounds.

REFERENCES

1. L.V. Asokar, K.K. Kakkar and O.J. Chakra, Glossary of Indian medicinal plants with active principles, (Publication and Information Directorate, New Delhi, 1992) pp. 122.

2. M.N. Ravishankara, N. Shrivastava, H. Padh and M. Rajani. HPTLC method for the estimation of alkaloids of Cinchona officinalis stems bark and its marketed formulations. Planta Med. 67: 294-296 (2001).

3. Ammon. H.P , Safayhi H, Mack T, Sabieraj J.Mechanism of anti-inflammatory activities of curcumin and Boswellic acids, Journal of Ethnopharmacology, 1993 Mar; 38(2-3); 113-119.

4. Madan B, Gade W N, Ghosh B. Curcuma longa inhibits the NF – κB and promotes adhesion of neutrophils to human umblical veins endothelial cells, Journal of Ethnopharmacology,2001 April,75(1); 25-32.

5. Indian herbal Pharmacopoeia, revised new edition, 2002, pp.169-76

6. S.B.Gokhale , C.K.Kokate , A P Purohit, Textbook of Pharmacognosy , First edition , 2002, pp.118-119.

Received on 04.07.2009 Modified on 09.08.2009

Asian J. Research Chem. 2(3): July-Sept., 2009, page 340-343