1. INTRODUCTION:

Thuja occidentalis belongs to the family Cupressaceae known as the tree of life¹. It is mostly known as arbor vitae or white cedar and is widely distributed to North America and is grown as ornamental tree in Europe². The mother tincture diluted or hydroalcoholic have been widely used in homeopathy. Thuja occidentalis used for the treatment of acute and chronic infections of the upper respiratory tract and as an adjuvant to antibiotics for bacterial infections such as bronchitis, angina, pharyngitis etc. In the form of tincture, it is widely used for the treatment of warts, papillomas, condylomas³. The plant contains wide range of active compounds including α-thujone, β-thujone, fenchone, occidentalol, α-pinene, quercetin, rutin, δ-3-carene, α-cedrol, caryophyllene, α-humulene, limonene, α-terpinolene, α-terpinyl acetate and thujone which may be responsible for its widespread activity^1,4,5.

Traditionally, the leaves of this plant have been used for treatment of variety of ailments and well known for its hemostatic and wound healing properties. Medicinal plant has been found to possess pharmacological activities such as antioxidant, antimicrobial activity, neuropharmacological activity of Thuja occidentalis and in-vitro studies, hypolipidaemic activity, anticancer activity^6-10. In recent year advancement of chromatographic and spectral fingerprints plays an important role in the quality control of complex herbal medicines¹¹.

High Performance Thin Layer Chromatography has become a routine analytical technique due to its advantages of reliability in quantification of analytes at micro and even in nanogram levels and cost effectiveness. The major advantage of HPTLC is in reducing analysis time and cost per analysis. Thin Layer Chromatography has been known as the fast tool for the detection of compounds. Another advantage of TLC is the capability to detect more compounds than High Performance Liquid Chromatography, although the resolution is poorer. In this regard, the compounds which cannot be eluted still can be detected. Furthermore, HPTLC image provides extra intuitive parameters of visible colour and/or fluorescence and unlike HPLC and GC, HPTLC can simultaneously determine different samples on the same plate. Such an approach causes the HPTLC method to maintain its innate advantage as well as get over a limitation of developing distance and plate efficiency. The analytical method can be analysed by some techniques such as HS-SPME, GC-MS, NMR^12-14. The determination of quercetin and rutin in selected herbs and pharmaceutical preparations by UV spectrophotometry and HPLC are reported¹⁵.

Literature survey revealed that no method has been reported for quantitation of quercetin from aerial parts extracts of T. occidentalis. Qualitative and quantitative standardization of quercetin was performed using HPTLC but not from this plant. Hence a densitometric HPTLC method has been developed in the present work for quantitation of quercetin from methanolic extract of aerial parts of T. occidentalis. The method was found suitable for rapid screening of plant material for their quantitative assessment and can be performed without any special sample pre-treatment and can be validated as per ICH Guidelines^16,17.

2. EXPERIMENTAL:

2.1. Reagents and standards:

All chemicals and solvents used were of analytical grade and obtained from E-Merck (Darmstadt, Germany). Stock solutions (mg/ml) of standards were prepared daily in methanol immediately before use. From this, solution was applied using Linomat applicator on TLC aluminium plates precoated with silica gel 60 F₂₅₄ (10×10 cm, 0.2 mm thick) obtained from E. Merck Ltd. (Mumbai, India).

2.2. Plant material:

The plant was collected from Medicinal garden, University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur. The plant was authenticated from IIT-BHU, Varanasi. The aerial parts of the plant were manually separated, air dried, powdered, sieved, weighed and stored in air tight container and subsequently referred to as powdered drug.

2.3. Preparation of standard solution:

Quercetin was prepared by transferring 10 mg of quercetin accurately weighed into a 10 ml volumetric flask, dissolving in 10 ml of methanol. It was then sonicated for 10 min and the final volume of the solution was made up to 10 ml with methanol to get stock solution 1mg/ml.

2.4. Preparation of mother tincture:

The dried powdered sample of T. occidentalis (1 g powdered) was refluxed on the water bath with 100 ml of methanol (60-80°C) for 5-6 h. The extract was concentrated under reduced pressure at 50-60°C and finally made up to 10 ml with methanol and ready for HPTLC analysis and labelled as T1 for methanol extract of T. occidentalis.

2.5. Chromatography conditions:

Chromatography was performed on a 10×10 cm preactivated HPTLC Silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany). Aliquot of the extracts was applied (Samples and standard) to the plate as 6 mm wide band with Linomat V with N₂ flow (CAMAG, Switzerland). Densitometric scanning was performed on CAMAG scanner III at 420nm. The plates were prewashed by methanol and activated at 60°C for 5 min prior to chromatography. The slit dimension was kept at 5×0.45 and 40 mm s^-1 scanning speed was employed. The mobile phase consisted of Chloroform: Ethyl acetate: Methanol: Glacial acetic acid (5:2:1.5:0.02% v/ v) and 10 ml of mobile phase was used per chromatography. Linear ascending development was carried out in 10×10 cm twin glass chamber saturated with the mobile phase.

2.6. Detection and quantitation:

After development, plates were dried with hair dryer and observed after 30 min under CAMAG UV cabinet (254 and 366 nm). Quantitative analysis of the compounds was done by scanning the plates at 420nm using CAMAG TLC scanner III equipped with visionCATS software. The identification of quercetin was confirmed by superimposing the UV spectra of the samples and standards within same Rf 0.5 window (Fig. 1).

Figure 1: HPTLC of quercetin and methanol extracts of Thuja occidentalis (S1, S2, S3, S4, T1, M1) under 254 nm

2.7. Calibration curve of quercetin:

The content of quercetin compound was determined by using a calibration curve established with a standard concentration range from 100-400ng/spot. A stock solution of standard quercetin (1mg/ml) was prepared in methanol. The different volumes of stock solution 10,20,30,40 µl were spotted on HPTLC plate to obtained concentration 100, 200, 300 and 400 ng/spot, respectively (band width 6 mm, distance between tracks 12 mm) using automatic sample spotter. Each concentration peak area was plotted against the concentration of quercetin spotted.

The linear regression of standard curve was determined with r² ± SD = 0.9981 ± 5.5. The linear regression line is y=4.139x + 623.5 (Fig. 2). The regression data have shown a good linear relationship over the concentration range of 100-400 ng/spot. The SD for intercept value is noticed to be less than 2% (RSD 0.28%).

Figure 2 Calibration curve for standard quercetin

2.8. Validation:

2.8.1. Precision:

Intra-day assay precision was evaluated by analysis of replicate (n=6) applications of freshly prepared standard solution of same concentration (100-400 ng/spot), on the same day. Inter-day precision was evaluated by analysis of replicate (n = 6) applications of standard solution of the same concentration (100-400 ng/spot) on six different days. The repeatability of sample was expressed in terms of % CV.

2.8.2. LOD and LOQ:

For the evaluation of LOD and LOQ different concentrations of the standard solutions of quercetin were applied along with methanol as blank and determined on the basis of signal to noise ratio. LOD was determined at an S/N of 3:1 and LOQ at an S/N of 10:1.

2.8.3. Specificity:

The specificity of the method was ascertained by analyzing standard quercetin and extracts. The spot for quercetin in the sample was confirmed by comparing the Rf and spectra of the spot with that of sample.

2.8.4. Robustness:

The estimation was performed by varying the selected parameters (mobile phase composition, mobile phase volume and duration of mobile phase saturation) within certain limits (±10%) and there has been no notable alteration found in method performance and in results obtained. The results were indicated by the %RSD between the data at each variable condition.

2.8.5. Accuracy:

The accuracy of the method was measured by performing recovery experiments at three different levels (50%, 100% and 150% addition of quercetin) using the standard addition method. The known amounts of quercetin standard (100 ng/spot) were added by spiking. The values of % recovery and average value of % recovery for quercetin were calculated.

2.9. Method applicability:

2.9.1. System suitability:

System suitability tests were performed to verify whether resolution and repeatability were adequate for the analysis. System suitability was determined by applying freshly prepared standard solution of quercetin, concentration 100 ng/spot, 6 times to the same chromatographic conditions then scanned and densitograms were recorded. The measured peak areas for quercetin and their retention factor were noted for each concentration of quercetin and values of the mean peak area, the standard deviation (SD) and the %CV were calculated.

2.9.2. Estimation of quercetin in mother tincture:

To determine the content of quercetin in the mother tincture, 1 g of powder of plant parts is extracted with 50 ml of methanol then concentrated and finally diluted with methanol. The resulting solution is centrifuged at 3000 rpm for 15 min and the supernatant is analyzed for quercetin content. The filtered solution is applied on the TLC plate followed by development and scanning. The analysis is repeated for 6 times and the possibility of interference from other components of extract in the analysis is studied. The spot at Rf = 0.5 corresponding to quercetin was observed in the chromatogram of the extracts along with other components. There was no interference from other components present in the chromatogram (Figs. 2-4).

3. RESULTS AND DISCUSSION:

3.1. TLC Fingerprint and co-chromatography:

Quercetin standard was quantitated accurately using silica gel F₂₅₄ HPTLC pre-coated plates with mobile phase Chloroform: Ethyl acetate: Methanol: Glacial acetic acid (5:2:1.5:0.02% v/ v), the Rf value was about 0.5. The chromatographs of quercetin (S1-S4) and methanolic extract of mother tincture (T1) and marketed mother tincture (M1) of T. occidentalis are shown in Fig. 2-4.

Figure 3: Densitometric chromatogram of quercetin in T. occidentalis (S1)

Figure 4: Densitometric chromatogram of marketed mother tincture of T. occidentalis (M1) after derivatization 420 nm HPTLC fingerprint patterns have been evolved for extracts of T. occidentalis.

3.2. TLC densitometric quantification of quercetin using HPTLC:

The well-defined spots were obtained when the chamber was saturated with the mobile phase Chloroform: Ethyl acetate: Methanol: Glacial acetic acid (5:2:1.5:0.02% v/ v) for 20 min at room temperature. The TLC plate was visualized at 420 nm. A photograph of a TLC plate after chromatography of quercetin standard and marketed T. occidentalis mother tincture and the identity of quercetin bands in the sample chromatogram were confirmed by the chromatogram obtained from the sample with that obtained from the reference standard solution (Fig. 1) and by comparing retention factor of quercetin from sample and standard solution. The peak corresponding to quercetin from the sample solution had same retention factor as that from quercetin standard at Rf 0.5.

The TLC densitometric method was validated in terms of precision, repeatability, and accuracy (Tables 1). The linearity range for quercetin was 100-400 ng/spot with correlation coefficient, intercept and the slope 0.9981, (sdv=1.99), 4.13 and 623.5, respectively (Y = 4.139x + 623.5). The percentage recovery was in the range of 96.77-99.74%. The measurement of the peak area at 5 different concentrations levels showed low values of %CV (<2%) for inter-day (0.46-1.75) and intra-day (0.44-1.8) variation for different concentrations of quercetin which suggested an excellent precision and reproducibility of the method.

Table 1: Method parameters for quantification of quercetin by HPTLC method

Parameters	Method (quercetin)	Acceptance criteria (maximum acceptable)
Selectivity	Selective
Specificity	Specific	No interference observed
Linear range (ng/spot)	100-400	Linearity, accuracy and precision over the range
Correlation coefficient (r²)	0.9981	Within 0.9–1.1
Linear regression equation Y = mx + c	y = 4.139x + 623.5	–
LOD (ng/spot)	23.05	–
LOQ (ng/spot)	69.87	–
% Recovery	98.13-99.42	Within 90–110%
Repeatability (%RSD, n = 6)	0.27-0.42	%RSD≤ 2%
Precision (%CV)		%CV≤ 2%
Intraday (n = 6)	0.15-0.38
Interday (n = 6)	0.20-0.25

Table 2 Amount of quercetin found in T. occidentalis in mg/ 100 mg

Thuja occidentalis

Quercetin (mg/100 mg)

(mean ± SD) (n = 3)

Sample (T1)

1.5586 ± 0.003

Marketed (M1)

1.1383 ± 0.003

Robustness tests examine the effect of operational parameters on the analysis by introducing small changes in mobile phase composition. The contents of quercetin quantified using TLC densitometric methods were found to be 0.1545 ± 0.003 and 0.1383 ± 0.003% w/w, respectively in sample and marketed samples of T. occidentalis (Table 2).

4. CONCLUSION:

A rapid, simple, precise, accurate and specific HPTLC method for quantitative estimation of quercetin present in T. occidentalis has been developed and validated. A comparative study of this biomarker compound has shown that it is present in higher amount in the sample as compared to marketed mother tincture. The activity of a plant extract is always influenced by the quantity of active principle present in the extract.

5. ACKNOWLEDGEMENTS:

Authors thankfully acknowledge IIT-BHU, Varanasi for authentication of plant material, also thankful to University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur for providing the facilities for work.

6. CONFLICTS OF INTEREST:

No conflicts of interest.

7. REFERENCES:

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Received on 06.06.2018 Modified on 28.07.2018

Research J. Pharm. and Tech 2019; 12(9):4102-4106.

DOI: 10.5958/0974-360X.2019.00707.8