1. INTRODUCTION:

There is a long history of traditional system of medicine in Eastern Countries like India. Ayurveda is one of the traditional systems of medicine. The traditional preparations comprise medicinal plants, minerals, organic matter, etc. Despite the use of herbal medicines over many centuries; only a relatively small number of plant species has been scientifically validated. Chromatography and relative techniques are used to evaluate the quality of plant raw materials (1). As the diversity of plant components and their content vary with not only the species but also with the growing conditions, the season when plants are harvested, the process methods and storage duration. In the medicine plant production, it is not always necessary to produce very pure preparations (2). The use of validated methods in the chemical standardization of botanicals and herbal preparations will enhance the quality of the products, assist in pharmacological studies, perform credible clinical trials and propel the move towards evidence based medicine.

Standardized Herbal drugs assure the quality and the quantity of selected component of different preparations of plant origin (3). (Herbal extracts, formulations etc.) In this respect, fingerprint analysis has been accepted by WHO, as a methodology for the assessment of herbal medicines (4). The World Health Organization (WHO) Assembly has emphasized the need to ensure the quality of medicinal plant products by using modern controlled technique and applying suitable standards (5, 6).

Chromatographic profiles (“fingerprints”) are used to help in identification of herbals, and in assessment of their potency and stability. The British Herbal Pharmacopoeia has had an emphasis on using TLC profiles to characterize herbal materials, relying on the use of different spray reagents and TLC profiles to identify characteristic and active principles of herbal materials(7). .Single or multiple markers can be used to ensure that the concentration and ratio of components in an herbal mixture are present in reproducible levels in raw materials, manufacturing intermediates, and in the final dosage forms. Where the active ingredients are not known, a marker substance which should be specific for the botanical could be chosen for analytical purposes (8).

Sitopaladi churna is a most trusted Ayurvedic formulation. It is used for different disorders like Abdominal Lump and colic/pain, tastelessness, digestive impairment, Intercostal neuralgia and pleurodynia. Sitopaladi churna contains roots of Piper longum ( Pipli), fruits of Elettaria cardamomum (Elaichi ) Fruits of Piper longum (Pipali), and the bark of Cinnamomum zeylanicum (Tvak) (9). Cinnamaldehyde is one of the important constituent of this formulation. It has a number of medicinal properties.

The aim of this work was to develop an accurate, specific, repeatable and robust method for the determination of Cinnamaldehyde in this traditional formulation. The proposed method was validated in accordance to ICH guidelines (10).

2. EXPERIMENTAL:

Materials

Standard Cinnamaldehyde (98% pure) was purchased from Sigma–Aldrich Chemicals Pvt., Ltd., India. Cinnamomum zeylanicum was procured as a fresh material from the local suppliers and assessed by the Department of Pharmacognosy. All chemicals and reagents used were of analytical grade and were purchased from Merck Chemicals, India

2.1.1 Preparation of the formulations:

Two laboratory batches of Sitopaladi churna’s (named SCL-I, SCL-II) were prepared in the institutional laboratory according to reported method of Ayurvedic formulary of India. The available commercially brand SCM-A, SCM-B of Sitopaladi churna was procured from local Pharmacy.

2.2 Instrumentation and chromatographic conditions

Spotting device: Linomat V Automatic Sample Spotter; CAMAG (Muttenz, Switzerland)

Syringe: 100 μL Hamilton (Bonaduz, Switzerland)

TLC Chamber: Glass twin trough chamber (20 x 10 x 4 cm); CAMAG

Densitometer: TLC Scanner 3 linked to Win Cats software V.4.06; CAMAG

HPTLC plates: 10 x 10 cm, 0.2 mm thickness precoated with silica gel 60 F₂₅₄; E. Merck KgaA, Cat. no. 1.05548; (Darmstadt, Germany)

Experimental conditions: Temperature 25 ± 2 ºC, relative humidity 40 %

Solvent system: Toluene: Ethyl acetate: Methanol (8:1:1)

Detection Wavelength: 295 nm for Cinnamaldehyde

Slit dimension: 6.00 x 0.20 mm

Scanning Speed: 20 mm/s and source of radiation deuterium lamp.

2.3 Calibration curve of Cinnamaldehyde

A stock solution of Cinnamaldehyde (100 µgmL⁻¹) was prepared in methanol. Different volumes of stock solution 2, 4, 6,8,10,12 µL, were spotted on the TLC plate to obtain concentrations of 200, 400, 600, 800 ,1000,1200 ng spot⁻¹ of Cinnamaldehyde, respectively. The data of peak areas plotted against the corresponding concentrations were treated by least-square regression analysis method validation.

2.4 Precision

Repeatability of the sample application and measurement of peak area were carried out using six replicates of the same spot (600 ng spot⁻¹for Cinnamaldehyde) and was expressed in terms of percent relative standard deviation (%R.S.D.) and standard error (S.E.). The intra- and inter-day variation for the determination of Cinnamaldehyde was carried at three different concentration levels of 200, 400, 600 ng spot−1.

2.5 Robustness of the method

By introducing small changes in the mobile phase composition, mobile phase volume, duration of mobile phase saturation and activation of pre washed TLC plates with methanol; the effects on the results were examined. Robustness of the method was done in triplicate at a concentration level of 600 ng spot−1 for Cinnamaldehyde and the % R.S.D and S.E. of peak areas was calculated.

2.6 Limit of detection and limit of quantification

In order to estimate the limit of detection (LOD) and limit of quantitation (LOQ), blank methanol was spotted six times following the same method as explained in Section and the signal-to-noise ratio was determined. LOD was considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally verified by diluting the known concentrations of Cinnamaldehyde until the average responses were approximately 3 or 10 times the standard Deviation of the responses for six replicate determinations.

2.7 Recovery

The pre-analyzed samples were spiked with extra 50, 100 and 150 % of the standard Cinnamaldehyde and the mixtures were reanalyzed by the proposed method. The experiment was conducted six times. This was done to check for the recovery of the cinnamaldehyde at different levels in the formulations.

2.8 Ruggedness

A solution of concentration 1000 ng spot⁻¹ was prepared and analyzed on day 0 and after 6, 12, 24, 48 and 72 h. Data were treated for % R.S.D. to assess ruggedness of the method.

2.9 Specificity

The specificity of the method was confirmed by analyzing the standard drugs and extract. The spot for Cinnamaldehyde in the sample was confirmed by comparing the Rf values and spectra of the spot with that of the standard. The peak purity of the Cinnamaldehyde was assessed by comparing the spectra at three different levels, viz. peak start (S), peak apex (M) and peak end (E) positions of the spot.

2.10 Analysis of Cinnamaldehyde in crude drug extracts

To determine the content of Cinnamaldehyde in crude drug extract, 1 gram was transferred into a 100mL volumetric flask containing 25mL methanol, sonicated for 30 min and diluted to 50mL with methanol. The resulting solution was centrifuged at 3000-rpm for15 min and the supernatant was analyzed for the drug content. Six microliters of the filtered solution was applied on the TLC plate followed by development and scanning. The analysis was repeated in triplicate. The possibility of interference from other components of the extract in the analysis was studied.

2.11 Analysis of cinnamaldehyde in Lab and marketed formulations

An accurately weighed quantity of formulation equivalent to about 100 ng of Cinnamaldehyde was extracted with 25 mL methanol by sonication for 20 min. This extract was centrifuged at 10,000 rpm for 10 min at 8 ⁰C. The supernatant was filtered and the filtrate was dried to constant weight at room temperature. The residue was again dissolved in 5mL of methanol. Six micro liters of the filtered solution was applied on the TLC plate followed by development and scanning .The analysis was repeated in triplicate. Combination of active components was also analyzed separately to study the interference of each active component.

3 RESULTS AND DISCUSSION:

3.1. Development of the optimum mobile phase

The TLC procedure was optimized with a view to quantify the formulation. Initially and toluene: ethyl acetate: (8:1) gave good resolution with Rf 0.0.55 for Cinnamaldehyde. However, typical peak shape was missing. Then , the Mobile phase : Toluene: Ethyl acetate: Methanol consisting of (8:1:1 v/v/) gave a sharp and well-defined peak at Rf 0.55 well defined spots were obtained when the chamber was saturated with mobile phase for 25 min at room temperature (figure 1).

3.2 Calibration curves

The developed HPTLC method for estimation of Cinnamaldehyde showed a good correlation coefficient (r² = 0.996± 0.002) respectively in concentration range discussed under section 2.3 with respect to the peak area. The mean value (±S.D.) of slope and intercept were 4.263 ± 0.0011, 3145.42 ± 0.8165 for cinnamaldehyde (Table-1). No significant difference was observed in the slopes of standard curves.

Table 1: Linear regression data for the calibration curve (n=6)

Linearity

range (ng/spot)

r ± S.D.

Slope ± S.D.

Intercept ± S.D.

200-1200

0.9964 ±

0.000358

4.263 ± 0.0011

3145.42 ± 0.8165

3.3 Method validation

The % R.S.D. for repeatability of sample application and measurement of peak areas were found to 0.0140 %. The measurement of the peak area at three different concentration levels showed low values of S.E. and % R.S.D. (<1%) for inter- and intra-day variation, which suggested an excellent precision of the method (Table 2). The low values of S.D., % R.S.D. and S.E. obtained after introducing small changes in the developed HPTLC method indicated the robustness of the method (Table 3). Detection limit and quantification limit with signal-to-noise ratio of 3:1 and 10:1 were found to be 1.56 and 4.23 ng for Cinnamaldehyde, which indicates the adequate sensitivity of the method .The proposed method when used for extraction and subsequent estimation of cinnamaldehyde from the formulation afforded recovery of 99.088 – 100.11% as listed in Table (6). Low % R.S.D value of 0.18303 between the peak area values proved the ruggedness of the method indicating the stability during the extraction procedure as well as during analysis. The peak purity of cinnamaldehyde was assessed by comparing the spectra at peak start, peak apex and peak end positions of the spot. Good correlations were obtained between the standard and the sample-overlain spectra of cinnamaldehyde.

Table 2: Intra- and inter-day precision of HPTLC method (n=6)

Principle constituent	Amount	Intra -day precision		Inter-day precision
Principle constituent	(ng/spot)	S.D. of area	R.S.D. %	S.D. of area	R.S.D. %
Cinnamaldehyde	200	0.8134	0.02099	0.78929	0.01836
	400	0.9993	0.01427	0.74478	0.01062
	600	0.5009	0.00447	0.55813	0.00498

Table 3: Robustness testing (n=6)

Parameter	Cinnamaldehyde
Parameter	S. D.^a of Peak area	R.S.D. %
Mobile phase composition	0.8584	0.11852
Amount of mobile phase	0.5478	0.14572
Temperature	1.0527	0.29635
Time from spotting to chromatography	0.45495	0.12852
Time from chromatography to scanning	0.29745	0.03578

Estimation of Cinnamaldehyde

HPTLC plates (10 x 10 cm), 0.2 mm thickness precoated with silica gel 60 F₂₅₄; E. Merck KgaA, were used for characterization by narrow particle size, narrow pore volume distribution, standardized surface area, better resolution and capacity A single spot at Rf = 0.55 was observed in the chromatogram of the Cinnamaldehyde, isolated from extract along with other components. There was no interference in analysis from the other components present in the extracts. These components appear in the chromatogram at significantly different Rf values. The total Cinnamaldehyde content for (in cinnamomum zeylanicum), SCL-I, SCL-II, SCM-A, SCM-B, was reported in (Table-4).

3.6 Recovery studies

A single spot at Rf = 0.55 was observed in the chromatogram of Cinnamaldehyde extracted from formulation. There was no interference from active components present in the herbal formulation. The % recovery of the Cinnamaldehyde from the formulation was found to be 99% (Table-5) and it was well within the limits.

Figure 1. HPTLC chromatogram of standard cinnamalehyde

4. CONCLUSION:

In the present study, an original, simple and accurate HPTLC method was developed for the quantitative determination of Cinnamaldehyde in the formulation containing dried extracts of the plants traditionally used for Intercostal neuralgia. Several parameters were studied in order to perform the best separation and the chromatographic system was optimized in order to improve the selectivity of the method. The method is sensitive and precise, and may be of value in standardization of preparations containing the cinnamomum zeylanicum and for routine determination of cinnamaldehyde.

5. REFERENCES:

1- Lianga YZ, Xieb P, Chanc K. Quality control of herbal medicines Journal of Chromatography B 812; 2004: 53–70.

2- Shukla SS, Saraf Swarnlata, Saraf S, J. Res. Educ. Indian Med. 15( 1);2009: 25-32.

3- Shukla K, Saraf Swarnlata, Saraf S. Development of Quality Control Parameters of Bhaskar Lavan Churna: A Traditional Ayurvedic Formulation59 (2) ;2007: 47-56

4- Jain V, Saraf Swarnlata, Saraf S . Asian Journal of Chemistry 19(7); 2007: 5331-5335.

5- World Health Organization, Quality Control Methods for Medicinal Plants Materials: Geneva 1998.

6- Jain V. Vyas A., Singh D., Singh M., Shukla S.S., Pandey R., Saraf Swarnlata and Saraf S., TLC Densitometric Method for the Estimation of piperine in Ayurvedic formulation Trikatu Churna, Oriental Journal of Chemistry 27 (1) ;2011: 40-44.

7- British Herbal Pharmacopoeia. Guildford and King’s Lynn: British Herbal Medicine Ltd; (1996 )11

8- J. Natalie Lazarowych , Pekosuse P. Fingerprinting and marker compounds for identification and standardization of botanical drugs: strategies for applying pharmaceutical hplc analysis to herbal products Drug Information Journal. 32; 1998: 497–512.

9- Ayurvedic formulary of India Part-2., ( 2^nd edition). Government of India, Ministry of Health and family Planning, Department of Indian System of Medicine and Homeopathy, Delhi 2000.

10- Q2A, ICH, Q2A (R1).validation of analytical procedures: text and methodology, international conference on harmonization, Geneva.(November2005), 1URL (http://www.ich.org/LOB/ media/ MEDIA417.pdf)

Received on 09.10.2011 Modified on 11.11.2011

Asian J. Research Chem. 4(12): Dec., 2011; Page 1953-1956

Principle constituents	Excess drug added to the analyte (%)	Recovery (%)	S.D.	R.S.D. (%)
Cinnamaldehyde	0	199.08888	0.19254	0.31258
	50	249.26268	0.82737	0.67482
	100	298.1124	0.7814	0.39867
	150	349.89521	0.56257	0.14536

Sample	Cinnamaldehyde content [Mean ± SD; n=3] (mg/g)
SCL-I	14.10 ± 0.35874
SCL-II	14.10 ± 0.11052
SCM -A	13.58 ± 0.01856
SCM -B	12.73 ± 0.02758
Cinnamomum zeylanicum	16.58 ± 0.17854