INTRODUCTION:

Senna leaves and its preparation used through the world for its Purgative action in habitual constipation. Senna contains mainly two anthraquinone glycosides called as Sennoside A and Sennoside B, which account for its purgative property.

Sennoside A and Sennoside B are stereoisomer of each other. They are dimeric glycosides with rhein anthrone as aglycone. Chemically it is 10, 10’bis (9, 10-dihydro-1-8-dihydroxy-9-oxoannthracene-3 carboxylic acid). It also reported that, sugar part of this glycoside has transporting function for the aglycone up to large intestine and also protective action so that oxidation of aglycone to other less active anthraquinone is prevented.^{1, 2}

Many analytical methods HPLC, HPTLC are developed for the identification and estimation of Sennoside B from the extract and little work had done on estimation of Sennoside B from the pharmaceutical dosage forms.^{3, 4, 5}

The objective of the present work was to develop an accurate, specific and reproducible method for the quantitative estimation of Sennoside B from pharmaceutical dosage forms.

MATERIALS AND METHODS:

A Camag HPTLC system comprising of Linomate V automatic sample applicator, Hamilton Syringe, Camag TLC Scanner-3, Camag Win CAT software, Camag Twin trough chamber and stationary phase precoated silica gel 60F ₂₅₄ were used. Referance STD of Sennoside B was purchased from Subhod Trading Company, Pune. The capsule containing Senna leaves are purchase from the local market. All chemicals were used are of AR grade.

TABLE 1: ANALYSIS OF SENNOSIDE B

Sr. No.	Label Claim (mg Extract / Capsule)	%of Drug Found	Average %	% R.S.D
1	500	98.33	97.84	1.75
2	500	97.80
3	500	98.12
4	500	99.2
5	500	97.86
6	500	95.73

Chromatographic Conditions:

Stationary phase : HPTLC precoated, silica gel 60, F₂₅₄(Merck)

Thickness : 0.2 mm

Mode of application : Band

Band width : 8 mm

Separation technique : Ascending

Temperature : 25 ± 3°

Saturation time : 25min.(10min.chamber alone+15min.with plate)

Migration distance : 70 mm

Measurement mode : Absorbance / Reflectance

TABLE 2: RESULTS AND STATISTICAL DATA FOR RECOVERY STUDY

Sr. No.	Sample applied (ug)	Sennoside B in sample (ng)	STD Added (ng)	Total Amount Added (ng)	*Amount recovered (ng)** ±SD	% Recovery	Mean %	% RSD
1	50	1428.39	50	1478.39	1463.32±0.43	98.97±0.39	98.22	0.73
2	100	2941.68	100	3041.68	2986.28±0.73	98.17±0.71
3	150	4415.37	150	4565.37	4452.67±0.60	97.53±0.27

Slit dimension : 6.0 x 0.30 mm

Scanning mode : Single level

Scanning wavelength : 308 nm

Detection TLC : UV-Densitometric scanning (CAMAG 3 Scanner)

Preparation of Sample and STD stock solution:

Twenty capsules were weighed and the total weight was determined. The capsule granules are subjected to fine powder with the help of mortal and pestle. The 1250 mg of powder sample was refluxed with methanol and final volume was made to 25 ml to get concentration of 50 mg/ml and same used in through out the study.

STD solution was prepared by dissolving 10mg of STD Sennoside B in 10ml methanol and sonicate for 15 min to get the concentration of 1mg/ml and used as a stock solution.

Preparation of Mobile Phase:

Mobile Phase was prepared by mixing 2-propanol, ethyl acetate, water and ammonia in the proportion of 50:35:25:2 v/v.

Calibration Curve:

Aliquots of STD solution of Sennoside B 0.5ul, 1ul, 1.5ul, 2ul, 2.5ul were applied in duplicates on the silica gel 60F ₂₅₄plate. The plate was developed and analyzed as described earlier.

RESULTS AND DISCUSSION:

Development of Optimum Mobile phase:

A number of different individual solvents as well as combination of solvents were tried to obtain a well separated, resolved and stable peak. During the selection and optimization of mobile phase it was observed that due to similar Rf value of other constituent (which might be dye or other constituent), the peaks were overlapped.

TABLE 3: RESULTS AND STATISTICAL DATA FOR PRECISION STUDY

S. NO.	Amount of sample applied(ug)	Amount of Sennoside Estimated(ug)	Mean	% R.S.D.
1	500	14.17	14.15	1.41
2	500	14.11
3	500	13.98
4	500	14.46
5	500	14.28
6	500	13.91

The mobile phase 2-propanol, ethyl acetate and water gave good resolution with improved characteristics with Rf value 0.33. In order to reduce band broadening and to make difference in Rf, ammonia solution was added and tailing problem are overcome by increasing the proportion of water. Finally the mobile phase 2-propanol, ethyl acetate, water and ammonia in the proportion of 50:35:25:2 v/v gave sharp and symmetrical peaks. The well defined spot was obtained when the chamber was saturated with mobile phase 10 min. at room temp.

Quantitative Estimation:

In the chromatogram of the drugs extracted from the capsule, many well resolved spots were observed, out of these spots one spot matches with the Rf value of shown by Standard Sennoside B and having the same λmax (308nm). The drug content as per label claim was found to be 97.84 % with % R.S.D. of 1.75. The low %R.S.D values as shown in (Table 1) indicated the suitability of this method for routine analysis of Senna in Pharmaceutical dosage form.

Fig.1: Densitogram of Sennoside B in STD

VALIDATION OF METHOD:

Calibration Curves:

Calibration graph was found to be linear over the concentration range 50-600ng/spot. Linearity was evaluated by determining seven standard working solutions in duplicate. The peak area and concentration was subjected to least square linear regression analysis to calculate the calibration equation (Y=6.60+10.15X) and regression coefficient (r²=0.999).⁶

Accuracy (Recovery Studies):

To study accuracy of the developed method, recovery studies were carried out using standard addition method at four different level and the % recoveries were calculated. The average % recoveries were 98.22 % and % R.S.D is 0.73 (Table 2). The result revealed that was no interference of excipients.^{7, 8}

Fig.2: Densitogram of Sennoside B in Capsule

TABLE 4: RESULTS AND STATISTICAL DATA OF LOD and LOQ BY SIGNAL TO NOISE RATIO

Sample	Average Area of Blank (15 noise Peaks)	SD	LOD	LOQ
Sennoside B	31.37	23.51	10.08	25.6

Precision:

The repeatability of sample application and measurement of peak area were expressed in terms of % R.S.D. Precision studies were carried out by using the capsule solution. Six spots of 10µl of capsule solution were applied on the plate and the plate was scanned at 308nm after development. The amount of Sennoside B present in per track was calculated by using regression equations(Y=6.60+10.15X). The results were revealed that the % R.S.D. was found to be <2% (Table 3).^{7, 8}

Limit of Detection (LOD) and Limit of Quantification (LOQ):

LOD is the amount of applied sample producing the peak area which is equal to the sum of the mean blank area (15 noise peak) and three times of its standard deviation.^{7, 8}

LOQ is the amount of applied sample producing the peak area which is equal to the sum of the mean blank area (15 noise peak) and ten times of its standard deviation.

The LOD and LOQ were found to be 10.08 and 25.6 respectively (Table 4).

Fig.3: Calibration Graph for Sennoside B

Ruggedness and Robustness:

The study of ruggedness and robustness was carried out by keeping all the parameters constant except for the time, day and analysts.^{7, 8} the results were shown in (Table 5 and 6)

The developed HPTLC technique is simple, precise, specific and accurate. Statistical analysis proved that the method is repeatable and selective for the estimation of Sennoside B from pharmaceutical dosage form without any interference from the excipients.

Fig.4: Overlain Spectrum of Sennoside B in Standard and Capsule

Parameter	Initial condition	Change in condition	Effect
Mobil phase composition	2-propanol:ethyl acetate:water:ammonia (50:35:25:2)	2-propanol :ethyl acetate:water: ammonia (55:40:25:2)	No proper resolution
DevelopmentDistance	7 cm	6 cm and 8 cm	No effect on RF
Saturation Time	25 min	Without saturation	Band get curved and tailing
Temperature	25±3 ºC	40±3 ºC	Band get curved
Extraction Time	20 min	30 min	No change in conc.

REFERENCES:

1. Kokate CK and Purohit AP. Pharmacognosy.20^th Edition, Nirali Prakashan, Pune 174.

2. Evans WC. Pharmacognosy. Harcourt Brace and Company Asia PTE LTD.,14^th Edition, 231

3. The Merck Index. Merck and Co., INC., Whitehouse Station. NJ, 13^th Edn.,1516

4. Indian Pharmacopoeia, 1996. Vol.II Govt. of India, Ministry of Health and family welfare, Publish by Controller of Publication, New Delhi, 675

5. Stahl E. Thin Layer Chromatography A laboratory Hand Book, II^ndedition, 694, 706-709.

6. Wragner H and Bladt S. Plant Drug Analysis, A Thin Layer Chromatography ATLAS., Springer, Publication, 2^ndedition, 68.

7. ICH guidelines, Validation of Analytical Procedure.www.nihs.go.jp.

8. Sethi PD. HPTLC Quantitative Analysis of Pharmaceutical Formulation, CBS Publicatin, 53-55

Received on 08.09.2008 Modified on 25.10.2008

Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 97-100

Analytes	Sample	Amount of sample applied (ug)	Amount found(ug)	Mean	% Amount estimated	% RSD
I	1	250	7.26	7.32	97.66	1.24
I	2	250	7.39	7.32	97.66	1.24
II	3	250	7.46	7.37	98.33	1.64
II	4	250	7.29	7.37	98.33	1.64