1. INTRODUCTION:

Ayurveda, taken from the Sanskrit words Ayur meaning life or lifespan and Veda meaning knowledge, originated in India but is now practicized throughout the world ¹. There has been increasing interest in Ayurveda, particularly its botanical remedies, because several Indian traditional herbs have been found to produce potent anti-tumour and immunologic effects ².

Arishtas are an important group of formulations used in Ayurveda. Arjunarishta, also named as Parthadhyarishta, is a poly-herbal hydro-alcoholic formulation. The chief ingredient of Arjunarishta is dried stem bark of Terminalia arjuna Roxb.

It contains hydrolysable tannins, triterpenoid glycosides (arjunglucoside-1, arjunglucoside-2), and sapogenins as arjunic acid, arjunolic acid, cardenolides, phenolics, flavonoids, phyto-sterols, mineral salt and sugar³. It also contains Draksha (fruits of Vitis vinifera) and madhuca flowers (flowers of Bassia longifolia) which are the rich source of phenolic compounds and possess good antioxidant activity. All these ingredients are used as good dietary sources of antioxidants ^4-6 .

The major ingredient of Arjunarishta is stem bark of Terminalia arjuna. The stem bark of Terminalia arjuna has been used for alleviating angina and other cardiovascular conditions⁷. The extract also improved the symptoms of refractory chronic congestive heart failure⁸. Oral administration of Terminalia arjuna bark also prevented ischemic reperfusion injury induced oxidative stress and tissue injury of heart in rabbits indicating its beneficial therapeutic effect in ischemic heart disease⁹. The flavonoids which are found present in Terminalia arjuna bark have shown antioxidant and lipid lowering effect¹⁰. HPLC analysis has been carried out for the quantification of some marker compounds for the standardization of Arjunarishta¹¹. Furthermore, no validated HPTLC method has been reported for the quantification of quercetin and rutin from Arjunarishta.

Standardization is an important aspect for establishing the quality and efficacy of Ayurvedic formulations or any poly herbal formulation. Therefore, a proper scientific validation as chromatographic fingerprinting is required for quantification of marker compounds for quality control purposes.

2. EXPERIMENTAL:

2.1 Preparation of Arjunarishta-T:

The ingredients of Arjunarishta as Arjuna bark (Terminalia arjuna), fruits of Draksha (Vitis vinifera) and madhuca flowers (Bassia longifolia) were procured from local market, Jamnagar. Identification of all the individual plant material was done as per Ayurvedic Pharmacopoeia of India. Authentification of all these ingredients was done in the Department of Botany of Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow. Prepared herbarium has been deposited in the CIMAP for future reference.

It was prepared by traditional method as given in The Ayurvedic Formulary of India, ¹². According to this method, coarsely powdered Arjuna bark (Terminalia arjuna), crushed fruits of Draksha (Vitis vinifera) and madhuca flowers (Bassia longifolia) were placed in polished vessel of brass along with prescribed quantity of water (26L), and allowed to steep overnight. After overnight steeping this material was warmed at medium flame until the water for decoction reduced to one fourth of the prescribed quantity (6.5 L), then the heating was stopped and liquor was filtered in a cleaned vessel and then 2.5 kg of Jaggery (concentrated juice of Saccharum officinarum) was added and mixed properly. This sweet filtered liquor was placed in incubator for 15 days at 33°C ± 1°C after adding Dhataki flowers (Woodfordia floribunda). After 15 days, completion of fermentation was confirmed by standard tests. The fermented preparation was filtered with unstarched muslin cloth and kept in cleaned covered vessel for further next seven days. Then, it was poured in clean amber coloured glass bottles previously rinsed with ethyl alcohol, packed and labelled properly.

2.2 Preparation of Arjunarishta-M:

It was prepared by modern method by carrying slight modifications in the traditional method. Method of preparation was same as followed with Arjunarishta-T, only dhataki flowers were replaced by yeast for inducing fermentation ¹³.

2.3 Materials:

All solvents used were of analytical grade and were purchased from Merck. Quercetin (purity98%) and rutin (purity 98%) were purchased from Yucca Enterprises, Mumbai, India.

2.4 HPTLC:

Chromatography was performed on 20 x 10 cm HPTLC plates coated with 0.25 mm layers of silica gel 60 F₂₅₄ (Merck, Darmstadt, Germany). Before use the plates were washed with methanol and activated at 110ºC for 5 min. Samples were applied as bands 4 mm wide and 6 mm apart by use of Desaga (Ziegel Wiesen, Germany) AS 30 Win sample applicator equipped with a 100 µL syringe. A constant application rate of 10 µL s^-1 was used. The mobile phase for quercetin was toluene-ethyl acetate-methanol-formic acid, 6 + 3 + 0.2 + 0.4 (v/v), while for rutin was ethyl acetate-n- butanol-formic acid-water, 10 + 6 + 2 + 2 (v/v), were used for chromatography. Linear ascending development was performed in a Camag 20 cm x 10 cm glass twin-trough chamber. Before insertion of the plate the chamber was saturated with mobile phase vapor for 20 min at room temperature (25 ± 2ºC) and relative humidity 60 ± 5% by lining the TLC chamber on three sides with filter paper, also placed in the mobile phase. The development distance was 8 cm. After development the TLC plates were dried in a current of air by means of an air dryer. Densitometric scanning was performed with a Desaga TLC scanner CD 60 in reflectance absorbance mode at λ = 300 nm for quercetin and λ = 280 nm for rutin controlled by ProQuant software (v1.06; Desaga) resident in the system. The slit dimensions were 4 x 0.02 mm and the scanning speed 100 nm s^-1. The radiation source was a deuterium lamp emitting continuous UV radiation between 190-360 nm. The amounts of the compounds chromatographed were determined from the intensity of diffusely reflected light.

2.5 Standard Solutions and Calibration Plots:

2.5.1 Quercetin:

Stock solution of 1000 µg mL^-1 of quercetin was prepared by dissolving 50 mg of accurately weighed quercetin in methanol and making the volume of solution up to 50 mL with methanol in volumetric flask. The aliquots (0.5 to 2.5 mL) of stock solutions were transferred to 10 mL volumetric flasks and the volume of each was adjusted to 10 mL with methanol, to obtain standard solutions containing 50, 100, 150, 200 and 250 µg mL^-1 of quercetin, respectively. 10µL each of the standard solutions of quercetin (500-2500 ng spot^-1) were applied as bands 4 mm wide and 6 mm apart in triplicate on a TLC plate using an automatic sample spotter (AS 30 Win). Linear regression data for the calibration plot are listed in Table 1. A good linear relationship between response (peak area) and amount was obtained over the range 500-2500 ng/band.

2.5.2 Rutin:

Stock solution of 800 µg mL^-1 of rutin was prepared by dissolving 40 mg of accurately weighed rutin in methanol and making the volume of solution up to 50 mL with methanol in volumetric flask. The aliquots (0.5 to 2.5 mL) of stock solutions were transferred to 10 mL volumetric flasks and the volume of each was adjusted to 10 mL with methanol, to obtain standard solutions containing 40, 80,120,160 and 200 µg mL^-1 of rutin, respectively. 10µL each of the standard solutions of rutin (400-2000 ng spot^-1) were applied as bands 4 mm wide and 6 mm apart in triplicate on a TLC plate using an automatic sample spotter (AS 30 Win). Linear regression data for the calibration plot are listed in Table 1. A good linear relationship between response (peak area) and amount was obtained over the range 400-2000 ng/band.

2.6 Sample Preparation:

1 g (equivalent to 0.94 mL) of each of the test formulation of Arjunarishta as Arjunarishta-T, Arjunarishta-M and its marketed formulation was dried on water bath for half an hour to remove the alcohol. Then, each of the test sample of Arjunarishta was diluted with methanol up to 10 mL and sonicated for 15 min and centrifuged at 3200 rpm to settle down the precipitated sugars. 1 mL of supernatant was passed through 0.45 µm filter (Millipore) and 10µL of each of the test formulation was applied as band on plate for quantification.

3. RESULTS AND DISCUSSION:

3.1 Selection of the Optimum Mobile Phase:

In an attempt to optimize mobile phase, toluene-ethyl acetate-methanol-formic acid mixtures in different proportions were investigated. Use of toluene-ethyl acetate-methanol-formic acid 6 + 3 + 0.2 + 0.4 (v/v) resulted in sharp, well defined quercetin peaks of R_F0.36 ± 0.02 while solvent system ethyl acetate-n-butanol-formic acid-water 10 + 6 + 2 + 2 (v/v) resulted in sharp rutin peaks of R_F0.59 ± 0.02. Well defined bands were obtained only when the chamber was saturated with the mobile phase for 30 min at room temperature before plate development.

3.2 Validation of the Method:

ICH guidelines were followed for the validation of the analytical methods developed. The developed HPTLC method was validated in terms of precision, accuracy, LOD, LOQ, specificity, robustness and ruggedness¹⁴.

3.2.1 Instrumental precision:

Instrumental precision was checked by repeated scanning (n = 6) of the same spot of quercetin (500 ng spot^-1) and rutin (400 ng spot ^-1) expressed as relative standard deviation (% RSD) as shown in Table 1.

3.2.2 Repeatability:

The repeatability of method was affirmed by analysing 500 ng spot^-1 of quercetin and 400 ng spot^-1 of rutin individually on TLC plate (n = 6) and expressed as % RSD as shown in Table 1.

3.2.3 LOD and LOQ:

The limit of detection and quantification were determined by visual evaluation. The detection and quantification limits obtained by this method for quercetin were 180 and 500 ng, respectively while for rutin detection and quantification limit were 120 and 400 ng respectively as shown in Table 1 which indicates that the sensitivity of the method is adequate.

3.2.4 Intra-day and Inter-day Precision:

The intra-day and inter-day precision of the method were estimated by analysing aliquots of standard solution containing 500, 1500, 2500 ng spot^-1 of quercetin and 400, 1200, 2000 ng spot^-1 of rutin on the same day (intra-day precision) and on different days (inter-day precision) and the results were expressed as % RSD in Table 2 ¹⁵.

3.2.5 Specificity:

The specificity of the method was ascertained by analyzing reference standard and samples. The bands for quercetin and rutin from Arjunarishta-T, Arjunarishta-M and its marketed formulation were confirmed by comparing the R_F of the separated bands with those from the standard.

Table 1: Method validation parameters for the quantification of quercetin and rutin in Arjunarishta-T, Arjunarishta-M and its marketed formulation.

Parameter	Quercetin	Rutin
Instrumental Precision (% RSD, n = 6)	0.61	0.73
Repeatability (% RSD, n = 6)	0.64	0.76
LOD (ng)	180	120
LOQ (ng)	500	400
Linear range (n = 3)	500-2500 ng/band	400-2000 ng/band
Correlation coefficient (r)	0.9996	0.9995
Linearity (Regression equation)	y = 2381x + 2205.2	y = 1065.2x + 2262.1

Table 2 : Intra-day and Inter-day precision of the HPTLC method.^a)

Marker	Amount [ng/band]	Intra-day precision		Inter-day precision
Marker	Amount [ng/band]	Mean area [AU]	RSD [%]	Mean area [AU]	RSD [%]
Quercetin	500	3383.37	0.67	3382.29	0.77
	1500	5781.48	0.40	5780.74	0.48
	2500	8113.15	0.33	8112.40	0.41
Rutin	400	2687.21	0.78	2686.43	0.86
	1200	3527.19	0.66	3526.37	0.71
	2000	4379.15	0.37	4378.53	0.44

^a) n = 6

Table 3 : Results of recovery study of Quercetin from Arjunarishta-T, Arjunarishta-M and its marketed formulation (n = 3).

Sample	Amount of drug added [%]	Theoretical content [ng]	Recovery [%]	RSD [%]
Arjunarishta-T	50	39	99.83	0.83
	100	52	99.87	0.68
	150	65	100.16	0.56
Arjunarishta-M	50	35	99.81	0.71
	100	46	100.02	0.77
	150	58	99.83	0.61
Marketed Arjunarishta	50	38	99.91	0.80
	100	50	100.26	0.75
	150	63	100.16	0.58

Table 4 : Results of recovery study of Rutin from Arjunarishta-T, Arjunarishta-M and its marketed formulation (n = 3).

Sample	Amount of drug added [%]	Theoretical content [ng]	Recovery [%]	RSD [%]
Arjunarishta-T	50	88	99.92	0.37
	100	118	99.72	1.78
	150	147	99.91	0.26
Arjunarishta-M	50	83	100.02	0.44
	100	110	99.70	1.88
	150	138	100.03	0.22
Marketed Arjunarishta	50	85	99.92	0.37
	100	114	99.71	1.82
	150	142	100.12	0.23

Table 5: Robustness of the method for Quercetin.^a)

Condition	Arjunarishta-T		Arjunarishta-M		Marketed Arjunarishta
Condition	Recovery [%]^b)	RSD [%]^b)	Recovery [%]^c)	RSD [%]^c)	Recovery [%]^d)	RSD [%]^d)
Mobile phase composition^e)
T-EA-M-FA (6 + 3 + 0.2 + 0.4)	99.91	0.79	99.84	0.84	100.18	0.68
T-EA-M-FA (5.9 + 3.1 + 0.2 + 0.4)	99.87	1.04	99.79	0.95	100.10	1.09
Development distance
8 cm	99.93	0.73	99.83	0.81	100.16	0.69
9 cm	99.89	0.99	99.76	0.97	100.09	1.12

^a)n = 6: ^b)Average for two amounts, 39 and 65 ng/band: ^c) Average for two amounts, 35 and 58 ng/band

^d) Average for two amounts, 38 and 63 ng/band: ^e)T, toluene; EA, ethyl acetate; M, methanol; FA, formic acid

Table 6: Robustness of the method for Rutin.^a)

Condition	Arjunarishta-T		Arjunarishta-M		Marketed Arjunarishta
Condition	Recovery [%]^b)	RSD [%]^b)	Recovery [%]^c)	RSD [%]^c)	Recovery [%]^d)	RSD [%]^d)
Mobile phase composition^e)
EA-n-Bu-FA-W (10 + 6 + 2 + 2)	99.76	0.84	99.89	0.92	99.94	0.68
EA-n-Bu-FA-W (9.9 + 6.1 + 2 + 2)	99.72	0.96	99.85	0.90	99.88	0.89
Development distance
8 cm	99.78	0.82	99.86	0.94	99.91	0.72
9 cm	99.74	0.94	99.82	0.88	99.85	0.93

^a)n = 6

^b)Average for two amounts, 88 and 147 ng/band: ^c) Average for two amounts, 83 and 138 ng/band

^d) Average for two amounts, 85 and 142 ng/band: ^e)EA, ethyl acetate; n-Bu, n butanol; FA, formic acid; W, water

Table 7: Ruggedness of the method for Quercetin.^a)

Variable	Arjunarishta-T		Arjunarishta-M		Marketed Arjunarishta
Variable	Recovery [%]^b)	RSD [%]^b)	Recovery [%]^c)	RSD [%]^c)	Recovery [%]^d)	RSD [%]^d)
Analyst I	99.93	0.74	99.82	0.81	100.14	0.71
Analyst II	99.84	0.98	99.73	0.92	100.05	1.08

^a)n = 6

^b)Average for two amounts, 39 and 65 ng/band: ^c) Average for two amounts, 35 and 58 ng/band

^d) Average for two amounts, 38 and 63 ng/band: ^e)T, toluene; EA, ethyl acetate; M, methanol; FA, formic acid

Table 8: Ruggedness of the method for Rutin .^a)

Variable	Arjunarishta-T		Arjunarishta-M		Marketed Arjunarishta
Variable	Recovery [%]^b)	RSD [%]^b)	Recovery [%]^c)	RSD [%]^c)	Recovery [%]^d)	RSD [%]^d)
Analyst I	99.72	0.81	99.87	0.91	99.92	0.71
Analyst II	99.68	0.98	99.81	0.82	99.83	0.92

^a)n = 6

^b)Average for two amounts, 88 and 147 ng/band: ^c) Average for two amounts, 83 and 138 ng/band

^d) Average for two amounts, 85 and 142 ng/band: ^e)EA, ethyl acetate; n-Bu, n butanol; FA, formic acid; W, water

3.2.8 Ruggedness:

Ruggedness is a measure of the reproducibility of a test result under normal, expected operating conditions from instrument to instrument and from analyst to analyst. Ruggedness was tested by analysis of quercetin 39 and 65 ng per band from Arjunarishta-T, 35 and 58 ng per band from Arjunarishta-M and 38 and 63 ng per band from its marketed formulation; the results are listed in Table 7 ¹⁶. Similarly, ruggedness was measured by performing the analysis of rutin 88 and 147 ng per band from Arjunarishta-T, 83 and 138 ng per band from Arjunarishta-M and 85 and 142 ng per band from its marketed formulation as shown in Table 8.

3.3 Estimation of Quercetin and Rutin in Arjunarishta-T, Arjunarishta-M and its Marketed Formulation:

The suitability of the method was examined by estimation of quercetin in Arjunarishta-T, M and its marketed formulation. Bands of R_F0.36 ± 0.02 were observed in the densitogram for quercetin standard (Figure 1) while the bands of same R_F were observed in the densitogram obtained from the quercetin isolated from Arjunarishta-T, M and its marketed formulation (Figure 2). Similarly, rutin was also estimated in Arjunarishta-T, M and its marketed formulation. Bands of R_F0.59 ± 0.02 were observed in the densitogram for rutin standard (Figure 3) while the bands of same R_Fwere observed in the densitogram obtained from rutin isolated from Arjunarishta-T, M and its marketed formulation (Figure 4). Quercetin was found to be 0.00260, 0.00234 and 0.00252% in Arjunarishta-T, M and its marketed formulation respectively while rutin was found 0.00585, 0.00553 and 0.00568% in Arjunarishta-T, Arjunarishta-M and in marketed Arjunarishta respectively as showed in Table 9.

Figure 1: Overlay HPTLC densitogram of Quercetin standard.

Table 9: Estimation of Quercetin and Rutin from Arjunarishta-T, Arjunarishta-M and its marketed formulation by proposed HPTLC method.

Sample	Gallic acid (% w/w)^a)	Ellagic acid (% w/w)^a)
Arjunarishta-T	0.00260±0.0002	0.00585±0.0002
Arjunarishta-M	0.00234±0.0005	0.00553±0.0001
Marketed Arjunarishta	0.00252±0.0003	0.00568±0.0003

^a) Mean ± SD, n = 3

Figure 2: Overlay HPTLC densitogram of Quercetin from samples of Arjunarishta.

a, Arjunarishta-T; b, Arjunarishta-M; c, marketed Arjunarishta

(the mobile phase was toluene-ethyl acetate-methanol-formic acid, 6+3+0.2+0.4)

Figure 3: Overlay HPTLC densitogram of Rutin standard.

Figure 4: Overlay HPTLC densitogram of Rutin from samples of Arjunarishta.

a, Arjunarishta-T; b, Arjunarishta-M; c, marketed Arjunarishta

(the mobile phase was ethyl acetate-n-butanol-formic acid-water, 10+6+2+2)

4. CONCLUSION:

This HPTLC technique is precise, specific, robust and accurate and could find application in routine quality-control analysis of Ayurvedic formulations.

5. REFERENCES:

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Received on 07.05.2011 Modified on 12.05.2011

Asian J. Research Chem. 4(6): June, 2011; Page 1019-1024