The R & D thrust in the pharmaceutical sector is focused on development of new indigenous plant- based drugs through investigation of leads from the traditional system of medicine and recognizing the importance of traditional medicines ^[3]. The W.H.O. has created strategies, guidelines and standards for botanical medicines. It is being realized seriously that it is necessary to develop methods for rapid, precise and accurate identification and estimation of active constituents of herbal drugs in order to bring consistency of quality of the formulations. Antihyperglycemic effect of several plant extracts or formulations has been confirmed ^[4].

In connection of formulation development , S. rebaudiana, and A. paniculata have chosen while these herbal drugs have contrast taste as “Natural Sweetener” and “Natural Biter” respectively. A. paniculata invoked as a” Guard of liver” which cures hepatic disorders as well as regulates normal glucose metabolism and homeostasis. Whereas S. rebaudiana, a zero calorie sweetener used as a food additives reported to ameliorate blood sugar level in diabetic individuals ^{[5, 6]}. Recognizing its peculiarity, in December 2008, the United States Food and Drug Administration permitted Rebaudioside-A (A chemical constituent of S. rebaudiana) as a food additive and awarded "no objection" approval for GRAS (Generally recognized as safe) status. Therefore in the light of earlier research and widespread utilization of both plants, current research drew attention at large to unveil the untapped potential of these drugs by paving the way for treatment of Type-2 diabetes. Apart, our proposed study foresees a newer possibility of curing diabetes mellitus, by developing a potential antidiabetic herbal formulation, using bioactive fraction of leaves of S. rebaudiana and whole herb of A. paniculata.

MATERIAL METHODS:

Evaluation of formulated tablets ^[7]

For evaluation of prepared tablets, following parameters were employed.

Weight variation test

With a tablet designed to contain a specific amount of drug in a specific amount of tablet formula, the weight of the tablet being made is routinely measured to ensure that a tablet contains the required amount of drug. In the test, 20 tablets were individually weighed, the average value was calculated and it was compared with individual tablet weights and finally average values were shown (Table 1). According to USP limit for the test, not more than 2 tablets were outside the percentage limit and no tablet differed by more than 2 times the percentage limit (± 5%). The prepared tablets passed the limit.

Hardness Study

Tablets require a certain amount of strength or hardness and friability, to withstand mechanical shocks of handling during transportation. The hardness of tablets for all the formulations was determined by Monsanto hardness tester and the values are shown (Table 2).

Friability Test

The friability of tablets for all formulations was determined by Roche friabilator. The tablets were weighed and put into the plastic chamber and operated for 100 revolutions/min. then the tablets was reweighed. The tablets passed the acceptable limit with less than 1% friability. The percentage friability was calculated for prepared tablets and the values are tabulated as follows (Table 3).

Disintegration Test

The disintegration time of tablet for all formulations was determined using IP Disintegration Apparatus (Indian Pharmacopoeia, 1996). 900 ml of 0.1N hydrochloric acid was used as medium and the time to disintegrate the tablet completely was noted and values are tabulated (Table 4).

Dissolution Time Study

The dissolution time of the prepared tablets was determined by placing the tablet in the basket type dissolution apparatus. The basket was immersed in 0.1N HCl. The mortar was adjusting for 100 rpm and samples were withdrawn at 15, 30, 45, 60 and 120 minutes interval and the amount of drug in solution was determined with the help of marker compound by HPTLC and results are tabulated (Table 5).

Quantification of Stevioside and Rebaudioside-A in SteviTAB and Andrographolide in AndroTAB by HPLC method

Though the tablets were made from the standardized fractions, hence quantification of marker in prepared tablet by HPLC method was desirable to undertake chemical characterization and for evaluating the content uniformity in terms of concentration of marker components.

Standard preparation

Accurately weighed about 15 mg of each standard compound was transferred into a 10 mL of volumetric flask and dissolved in small quantity of ethanol and made up the volume with ethanol.

Sample preparation

Ten tablets of SteviTab and AndroTab each were taken and powdered separately in pestle and mortar and per tablet weight was taken and dissolved in small quantity of ethanol in 100 mL volumetric flask and made up the volume. The standard and sample were filtered through 0.22 µm filter paper and the filtrate was transferred to HPLC vial and placed inside the HPLC auto sampler chamber. The system was standardized for the HPLC instrument and the analysis was accomplished and results were recorded (Table 6; 7 and 8; Figure 1 and 2).

Accelerated stability studies of prepared tablets

The prepared tablets were examined for the characteristic features i.e. color, mottling, weight variation, hardness and to estimate the amount of the marker compound present in tablet. Accelerated stability studies have been performed by incubating samples at 35⁰C±2⁰C/ 65% RH±5% for a period of six months^[8] (Table 9; 10; 11 and 12).

RESULTS AND DISCUSSION:

Evaluation of formulated tablets

Therefore prior to development of formulation as a tablet dosage form, characterizing the spray-dried powder of both plant materials was revealed their hygroscopic nature. Therefore in respect of moisture pick up tendency of plant material microcrystalline cellulose was incorporated and direct compression method was chosen for tablets preparation. After preparing the tablets, they were evaluated for drug content, weight variation, hardness, friability, disintegration time, and dissolution time and marker quantification.

In our study mono herbal tablet formulations were prepared using the butanol fraction of ethanol extract of S. rebaudiana and chloroform fraction of ethanol extract of A. paniculata for which 200 mg of the each fraction incorporated with 250 mg of excipients amounting to total 450 mg weight of one tablet, named as SteviTab and AndroTab accordingly.

The weight variation test was performed by weighing 20 tablets for each formulation. The variation in the weight of the individual tablet recorded for tablets SteviTab and AndroTab was 0.42% and 0.58% respectively, which was found within the limit of Indian Pharmacopoeia (The variation was in range of 0.10 to 0.60%) (Table 1).

Table 1: Average weight (mg) variation test of 20 tablets

Parameter	SteviTab	AndroTab
Mean ± SD	449.8± 1.03	451.3± 2.24
% Weight variation	0.42	0.58

The tablets require a certain amount of strength or hardness and resistance or friability to withstand mechanical shocks of handling in all processes. The hardness of tablets was determined by Monsento hardness tester which was observed 5.3±0.01% in SteviTab and 4.9±0.03% in AndroTab respectively. The hardness of tablets was within the Indian Pharmacopoeia limit for all the tablet formulations (Table 2).

Table 2: Hardness test study of prepared tablets

Formulation code	Hardness (kg/cm²)
Formulation code	T1	T2	T3	T4	T5	Mean±SD
SteviTab	5.2	5.7	5.8	4.6	5.4	5.3±0.01
AndroTab	5.2	4.3	5.0	5.2	5.1	4.9±0.03

The friability of tablets was determined by Roche friabilator. It was found to be 0.29±0.003% in SteviTab and 2.27±0.004% in AndroTab. The range of percentage friability was within the pharmacopoeial limit for all the tablet formulations (Table 3).

Table 3: Friability test study of prepared tablets

Formulation code	Friability (%)
Formulation code	T1	T2	T3	T4	T5	Mean± SD
SteviTab	0.29	0.31	0.28	0.29	0.32	0.29 ± 0.003
AndroTab	0.28	0.29	0.31	0.27	0.23	0.27± 0.004

The disintegration time of tablets of all the formulations was determined using IP disintegration test apparatus. The original rational for using tablet disintegration tests was the facts that as the tablet break down into small particles, it offers a greater surface area to the dissolving media and ensured related bioavailability of the drug to the body compartment system. The disintegration time for SteviTab and AndroTab was found to be 36.4±0.08 min and 32.5±0.09 min subsequently (Table 4).

Table 4: Disintegration time study of prepared tablets

Formulation code	Disintegration time (min.)
Formulation code	T1	T2	T3	T4	T5	Mean± SD
SteviTab	34.2	37.8	39.1	33.5	37.5	36.4± 0.08
AndroTab	32.3	35.6	31.5	30.5	32.8	32.5± 0.09

The dissolution time of the prepared tablets was determined by placing the tablets in the basket type dissolution apparatus. The dissolution time was determined at 100 RPM. The amount of drug released was determined on the basis of marker compound by HPTLC at different time intervals (15, 30, 45, 60 and 120 minutes). According to Indian Pharmacopoeia to pass the test, 70% of the drug should go into the solution after 45 minutes at 100 rpm. In our studies for SteviTab and AndroTab tablets more than 70% of the drug goes into the solution after 45 minutes at 100 rpm and after 120 minutes at same rpm contents of both tablets was found to dissolve precisely (Table 5).

Table 5 : Dissolution time study of prepared tablets

Time (minutes)	% Dissolution of active constituents/markers
	SteviTab		AndroTab
	Stevioside	Rebaudioside-A	Andrographolide
15	47.46	36.21	30.13
30	64.24	64.30	68.14
45	91.36	78.52	80.12
60	91.87	96.44	92.38
120	99.87	98.99	99.99

Quantification of marker(s) in prepared tablets by HPLC

The presence of markers in the developed mono herbal formulations was quantified by HPLC analysis. Quantification of markers i.e. stevioside and rebaudioside-A, in SteviTab and andrographolide in AndroTab were evaluated.

Moreover 20 ml amount of SteviTab comprises, stevioside (0.037 µg) and rebaudioside-A (0.024µg). Thus 450 mg weight of one SteviTab containing 200 mg of butanol fraction, ascertained 469.1 mg of stevioside and 320.9 µg of rebaudioside-A collectively. On the basis of HPLC study of butanol fraction of ethanol extract of S.rebaudiana leaves, in SteviTAB active constituents i.e stevioside (RT; 10.396) and rebaudioside (RT; 15.188) was confirmed in HPLC chromatogram (Table 6 and 7; Figure 1).

Table 6 : Estimation of Stevioside in SteviTab tablet by HPLC

Replicate	Sample area	Stevioside (µg)
1	1178542	0.037
2	1147152	0.037
3	1179698	0.037
Average	1168464±10661	0.037± 0.0

Mean ± SD, n=3

Peak	RT	Area	Height
1	10.396	1178542	617213
2	15.188	524312	318015
Total	-	1702854	935228

RT; retention time (Stevioside; 10.396) and (Rebaudioside-A; 15.188)

Figure 1: HPLC chromatogram of SteviTab

Similarly 20 ml amount of AndroTab was found 0.019 µg of andrographolide, hence 450 mg weight of AndroTab possessed 200 mg of chloroform fraction was contained 141.98 µg of andrographolide. Further with reference of HPLC study of chloroform fraction of ethanol extract of A.paniculata (whole herb), chromatogram study of AndroTab confirmed the presence of andrographolide (RT; 6.968) and results was shown (Table 8; Figure 2).

Table 8: Estimation of Andrographolide in AndroTab tablet by HPLC

Replicate	Sample area	Andrographolide (µg)
1	639146	0.020
2	632564	0.019
3	638792	0.020
Average	636834±2137	0.019± 0.0

Peak	RT	Area	Height
1	6.986	639146	312018

RT; retention time (andrographolide; 6.968)

Figure 2: HPLC chromatogram of AndroTab

Stability studies of prepared tablets

Stability of an herbal formulation is an important criterion, which determines the efficacy and safety of the product. In our study we examined the stability of developed mono herbal formulations. The developed mono herbal antidiabetic formulations i.e. SteviTab and AndroTab were subjected to stability studies, to monitor efficacy of the tablets on a long-term basis. Accelerated stability at (35⁰C±2⁰C/ 65% RH±5%) for six months were monitored to check the stability of the tablet for parameter such as color, weight variation, hardness and the amount of the marker compound (Table 9).

HPLC analysis for the stability study

The tablet samples were taken at periodical three-month time interval for six months period of time and HPLC analysis was performed to estimate the marker compound. Following to periodical three month of evaluation stability study of SteviTab showed no significant difference or content loss of stevioside and rebaudioside-A, whereas three months periodical stability study of AndroTab, with respect to 0 month content value (0.019 µg) was found to exhibit a gradual content loss of andrographolide by 0.014 µg at 3 months and 0.012 µg at 6 months respectively (Table 10; 11 and 12).

Table 10: Quantitation of Stevioside in SteviTab tablet by HPLC

Replicate	Stevioside (µg/tablet) (0 month)	Stevioside (µg/tablet) (3 months)	Stevioside (µg/tablet) (6 months)
1.	0.037	0.036	0.036
2.	0.037	0.037	0.037
3.	0.037	0.037	0.036
Average	0.037±0.0	0.036± 0.00	0.036± 0.00

Table 11: Quantitation of Rebaudioside in SteviTab tablet by HPLC

Replicate	Rebaudioside-A (µg/tablet) (0 month)	Rebaudioside-A (µg/tablet) (3 months)	Rebaudioside-A (µg/tablet) (6 months)
1.	0.025	0.024	0.024
2.	0.024	0.024	0.023
3.	0.025	0.024	0.024
Average	0.024±0.0	0.024± 0.00	0.023± 0.00

Table 12: Quantitation of Andrographolide in AndroTab tablet by HPLC

Replicate	Andrographolide (µg /tablet) (0 month)	Andrographolide (µg /tablet) (3 months)	Andrographolide (µg /tablet) 6 months)
1.	0.020	0.014	0.012
2.	0.019	0.015	0.012
3.	0.020	0.015	0.013
Average	0.019±0.0	0.014± 0.00	0.012± 0.00

CONCLUSION:

Finally study concludes that prepared herbal formulations have passed through various studied parameters significantly. Thus prepared herbal tablets could prove as potential candidates for treatment of diabetes and its complications. Further biopharmaceutical parameters including pharmacokinetic study are needed for wide therapeutic value at clinical perspective so that economic exploitation of SteviTab and AndroTab can be established.

REFERENCES:

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2. Handa, S.S. Quality Control and Standardization of Herbal Raw Materials and Traditional Remedies.1995;Pharmatimes., 13-21.

3. Patwardhan, B., Vaidya, A.B. and Chorghade, M. Ayurveda and natural products drug discovery. 2004; Current Science., 86 (6): 789 -799.

4. Sharma, S., Batra, A. and Mehta, B.K. Indian Journal of Chemistry. 1991b; Indian J. Chem., 30; 715-716.

5. Visen, P.K., Shukla, B., Patnaik, G.K. and Dhawan, B.N. Andrographolide protects rat hepatocytes against paracetamol- induced damage.1993; J. Ethnopharmacol., 40 (2) :131-136.

6. Mantovaneli, I.C.C., Ferretti, E. C., Simoes, M.R. and Da Silva, F.C. The effect of temperature and flow rate on the clarification of the aqueous stevia-extract in a fixed-bed column with zeolites. 2004; Brazilian J. Chem. Engineering., 21: 449-458.

7. L. Lachman, H. Liberman, J. Kanig; The Theory and Practice of Industrial Pharmacy, Varghese Publishing House, New Delhi,1987;3^rd ed: pp 293-373.

8. Pingle, S.S., Pokharakar, B.D. and Pingale M.S. (2008). Pharmacology Online., 1; 20-23.

Received on 26.12.2012

Modified on 05.01.2013

Accepted on 15.01.2013

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(1): January- February, 2013, 17-21

Table 9: Stability studies of prepared tablets

Colour