Evaluation of Antibacterial Activities and Formulation of Black Mahlab Seeds Aqueous Extract

Murtada Ahmed Oshi

Department of Pharmaceutical Technology, Faculty of Pharmacy, Omdurman Islamic University, Omdurman, Sudan

ABSTRACT:

Black mahlab is an annual herb with a rigid, brown seeds indigenous to Sudan. Its seeds are widely used in Sudanese traditional medicine in the treatment of diarrhea and other abdominal upsets. The objective of the present study is to verify this claim in scientific manner. Therefore, the seeds were tested for in-vitro antibacterial activity and then formulated into a tablet dosage form. The water extract of the seeds was tested against common diarrheal- causing bacteria. Assays were performed using extract concentrations of 25, 50, 100 and 150mg/ml, and agar well diffusion method was utilized. In formulation aspect two formulae were prepared formula-1and formula-2 by using maize starch and polyvinyl pyrrolidine as binder respectively. Starch paste as cost effective binder was evaluated for its binding properties in comparison with polyvinyl pyrrolidine. The results obtained from evaluation of granule properties and quality control tests of the tablets for both formulae were pharmaceutically satisfactory (p ≤0.05). According to pharmacopoeial specifications tablets of formula-1 had less mechanical strength than those of formula-2, but rapidly disintegrated and released the content more successfully than tablets of formula-2. Therefore, tablets of formula-1 were subjected to further stability studies and positive results were obtained. The findings of the work explored tablets of formula-1 were cost- effective; with adequate strength to withstand the shocks during storage, dispensing, and handling; and release its contents for antibacterial activity successfully.

KEYWORDS: Bacteria, Black mahlab, Tablets, Phytochemical constituents

INTRODUCTION:

Plants and plant products have been used extensively throughout history to treat medical problems. A number of traditional herbal medical practices have been adopted for the diagnosis, prevention and treatment of various diseases. Many such practices were experimentally proved depicting the scientific insight behind their traditional uses. Low harm, high activity, patient compliance are the reasons for choosing drug from herbal medicines.

Infections are one of the main causes of death in under- developed countries where it is associated with poverty and malnutrition. A huge number of herbal medicines are used as antimicrobial remedies for many infectious diseases. Numerous studies have been carried out to extract various natural products for screening antimicrobial activity. ^[1]

Black mahlab scientifically named Monechma ciliatum Jacq belongs to Acanthaceae family is an effective antimicrobial herb.

Its seeds (Figure 1) are used widely in Sudanese folksy medicine by traditional healers for gastrointestinal tract upsets e.g. diarrhea. So it was very interesting to select this plant which can help in the treatment of infections.

Figure 1: Black Mahlab Seed

The main objectives of the present study were evaluation of in-vitro antibacterial activity of the dry- aqueous extract (DAE) of seeds of Monechma ciliatum and formulation of the extract to tablet dosage form. Beside, other general objectives were: 1) to entice the pharmacists in particular and other healthcare professionals in general to be active in researching about herbal medicine realm, 2) to fill the fictional gaps between the herbal and synthetic medicines in fields of efficacy and safety, and 3) to establish and implement the most updated scientific knowledge and manufacturing technologies in herbal medicines formulation and manufacturing.

In this study I selected the DAE of seeds of Monechma ciliatum based on a combination of ethnopharmacology and daily healer’s practices. 2g of the seeds powder is usually soaked in 500ml of distilled water overnight and drunk for diarrhea. Although, the seeds were effective but it lacks of standardization and poor-quality presentation. However, to improve its efficacy and patient acceptance, there is need to formulate the seeds into tablet dosage form. The extract used in this research is closely resembled to traditional preparations used by natives in Nubia Mountains regions of Sudan to treat diarrhoea.

MATERIALS AND METHODS:

The seeds

The seeds were purchased from Omdurman Market for Herbal Products, Omdurman, Sudan on august, 2012 and identified at Department of Pharmacognosy, Omdurman Islamic University (voucher number 31). The seeds were separated, dried under shedding and stored in a covered bottle at room temperature until required for use.

Chemicals

Ampicillin (Bristol –Mayers Squibb, USA); Crosscarmellose cellulose (A Johnson Matthey, UK); Gentamicin (Roussel, UK); Lactose (Breckland Scientific Supplier, UK); Magnesium stearate (Breckland Scientific Supplier, UK); Maize starch (A Johnson Matthey, UK); Microcrystalline cellulose (A Johnson Matthey, UK); Mueller-Hinton agar and Mueller-Hinton broth (HiMedia Laboratories Pvt. Ltd, India); Polyvinyl pyrrolidine (Acros Oragnics, Belgium); Talc (A Johnson Matthey, UK).

Bacteria strains used

In this study, the Standard bacteria Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6633), Klebsiella pneumoniae (ATCC 53657), Escherichia coli (ATCC 25922) obtained from Department of Pharmaceutical Microbiology, Omdurman Islamic University, Sudan.

Methods

Extraction procedure

100g of the coarse seed powder was transferred to a flask containing 2000ml of distilled water. The flask was allowed for 24h at room temperature, the content passed through a cotton wool and the solid residue pressed (screw press). The strained and expressed liquid obtained were mixed, put for 12h for clarification and filtrated by Whatman filter paper No 42 (125 mm). Finally, the filtrate was evaporated using rotary evaporator (Stuart Ltd, Germany) under reduced pressure at 40ºC, placed in a Petri dish, and left to dry to constant weight.

Preliminary phytochemical analysis

Qualitative screening of the seeds of Monechma ciliatum was performed for the identification of active phytochemical constituents using standard procedures described by Tiwari et al.^[2]

Preparation of tested bacteria

An overnight culture of tested bacteria were prepared in Mueller Hinton Broth and later adjusted to 0.5 Mc Farland (optical density, OD 0.5) according to method described by Lopez et al.^[3]

Preparation of the extracts for antibacterial testing

The sterile DAE was redissolved in sterile distilled water and serial dilutions of the extract were prepared (25, 50, 100, 150mg/ml).

Preparation of standard drug

Gentamicin and ampicillin were used as standard drugs. Serial dilutions of both were prepared (0.005, 0.01, 0.02 and 0.04 mg/ml) by using sterile distilled water as solvent.

Evaluation of in-vitro antibacterial activity of the extract

The antibacterial testing of the DAE was carried out using the agar diffusion method as described by Lino and Deogracious.^[4]

Minimum inhibitory concentration determination

The Minimum inhibitory concentration of the DAE was determined by using the method described by Fabry et al.^[5]

Preformulation studies

1. pH measurement

The pH of the aqueous solution of DAE was measured by using pH- meter (Wagtech International, UK).

2. Loss on dry

The moisture content was measured at different points during tablets manufacturing steps by loss on dry (LOD) method. Approximately 5g of sample was uniformly placed onto the sample pan of moisture instrument (Kern Ltd, Germany), and the heated. The percentage of moisture was calculated from the weight loss of the sample by heating. The instrument was allowed to cool between tests.

3. Powder flow estimation

According to USP, ^[6]the flow were measured at different points within the manufacturing process by using angle of repose method. 30g of tested material was poured manually into funnel hanged at 3cm height, and then the angle was measured.

4. Powder Density

According to USP, ^[6]the bulk density, tapped density and Carr's index of final- blended granules were measured. The bulk and tapped densities of final- blended granule were determined according to the following method: a 50ml glass cylinder was weighed and filled with 30g of powder and reweighed. The opening of the cylinder was secured and gently reversed once, and the powder carefully levelled without compacting. Bulk volume was determined after one mechanical tap. Tapped volume was measured after 500taps.

Formulation of the tablets

Technique of tablets preparation

Two formulae were prepared by using wet granulation tabletting technique. Formula- 1 by using starch as binder and formula- 2 by using polyvinyl pyrrolidine as binder.

Calculation of the extract dose per tablet

150mg of the extract was incorporated into the tablet, depending on the traditional use of the seeds for diarrheal and abdominal upsets.

· 100g of the seed powder give 9.5g of the extract (Table 2)

· 2g (the weight of seed powder used in traditional preparations) gives 150mg of the extract.

Procedure of tablets preparation

For tablets of formula-1

According to formula in table 1, DAE was weighed precisely, transferred to a beaker and labelled (A). A specified amount of mixture of lactose, MCC and half amount of maize starch was transferred to another beaker and labelled (B). Both (A) and (B) were mixed together adequately by mortar and kneaded to a wet mass by using starch paste 10% (w/v). The wet mass was forced manually through a No. 10 mesh screen to form granules, which were placed in a hot air oven at 40ºC for 12h. The dried granules were resized using No. 32 mesh screen to get uniform- sized granules. The dried granules were remixed with reminder of maize starch, magnesium stearate and talc for 5min. The final lubricated- granule was transferred to a hopper of a single- punch tabletting machine (Erweka, Germany) and tabletted using die No. 10. The weight and the pressure of the machine were adjusted to obtain a tablet of 500 mg.

For tablets of formula-2

For tablets of formula-2, polyvinyl pyrrolidine solution 1% (w/v) was used instead of starch paste 10% (w/v) and the same procedure steps were carried out.

Table 1: Constituent of tablet formula

Composition	Formula-1		Formula-2
Composition	Weight in tablet (g)	Conc. in tablet (Wt %)	Weight in tablet (g)	Conc. in tablet (Wt %)
DAE (Active ingredient)	150	30	150	30
Lactose (Filler)	142	28.4	125	25
MMC (Filler)	125	25	142	28.4
Starch paste 10% w\v (Adhesive)	q.s.	-	-	-
PVP 2% w\v (Adhesive)	-	-	-	q.s.
Maize starch (Disintegrant)	50	10	50	10
Mg stearate (Lubricant)	5	1	5	1
Talc (Glidants)	25	5	25	5
Total weight	500	100	500	100

Evaluation of prepared tablets

1. Weight variation test

According to USP, ^[6]20 tablets were selected randomly from each batch and weighed individually. The average weight and deviation of each tablet from the mean were calculated. The standard deviation and percentage deviation were calculated and compared with standard.

2. Hardness test

The test was done according to method described by Kiran et al.,^[7]10 tablets were placed between jaws of hardness test apparatus (Erweka, Germany) and the force required breaking the tablet was noted.

3. Friability

The friability test was carried out according method described in USP. ^[6]20 tablets were weighed, placed in friability tester (Erweka, Germany) and rotated up to 100 revolutions. The tablets were reweighed after removal of fines and the percentage of weight loss was calculated.

4. Disintegration test

The disintegration test was carried out according method described in USP, ^[6]under botanical dosage forms. Six tablets were placed individually in each tube of disintegration test apparatus (G.B. Caleva, England). The water was maintained at a temperature of 37°±2°C and time taken for the entire tablet to disintegrate completely was noted.

5. Dissolution test

5.1. Screening of the extract

The whole extract was selected as a marker. 0.2g of the extract was placed in two a 100ml flasks and 100ml of 0.1M HCl and distilled water was added separately. The flasks were shaken, and then filtered through a Whatman filter paper. The absorption spectrum of the both solutions of the extract was recorded using a UV spectrophotometer (Jenway Ltd, UK) and the wavelength for maximum absorption was determined.

5.2. Drawing of the calibration curve of the extract

Different concentrations of the extract were prepared to contain between 0.01 to 0.2mg/ml of extract in 0.1M HCl and distilled water separately. The absorbance of each concentration in each case was taken at wavelength 275nm (maximum absorption wavelength) and plotted against the various concentrations to obtain the calibration curve for the extract for both solvents.

5.3. Dissolution test procedure

The dissolution test was carried out according method described in USP, ^[6]under botanical dosage forms. The dissolution tester (G.B. Caleva, England) was filled with 900ml of 0.1M HCl and water separately as medium, and allowed to warm up to 37±0.5ºC. 6 tablets were selected randomly and individually introduced into dissolution tester (apparatus 2, 75rpm). Then the machine was operated for 60min. 10ml of sample was withdrawn in different time intervals (10, 20, 30, 40, 60 min), filtered and assayed using UV method with reference to the standard.

Stability study of tablets of formula-1

Both disintegration and dissolution tests were done after 3, 6, 9 and 12 months of storage of the tablets in glass bottle at room temperature 30± 2°C and relative humidity 60 ± 5.

Statistical Evaluation

The data were statically analyzed by Students t-test and values were considered significant. And value were expressed as p<0.05.

RESULTS:

The results of antibacterial activity of the extract were summarized in table 2.

Table 2: Bioactivity of DAE and standard antibiotics against standard bacteria

Extract	Yield (±0.5)	Conc. (mg/ml)	Mean diameter of growth inhibition zone (MDIZ)
Extract	Yield (±0.5)	Conc. (mg/ml)	S. aureus	B. subtilis	K. pneumoniae	E. coli	P. aeruginosa
Water	9.50	25	-	16	12	-	–
		50	13	16	15	15	–
		100	18	25	18	17	–
		150	20	33	21	18	–
Gentamicin		0.005	12	16	12	11	–
		0.01	14	16	15	15	–
		0.02	16	22	19	18	–
		0.04	20	30	33	22	–
Ampicillin		0.005	–	12	–	–	–
		0.01	–	13	–	–	–
		0.02	–	14	–	–	–
		0.04	–	16	–	–	–
D. Water		–	–	–	–	–	–

Data are presented as mean MDIZ (mm) of three replicates; Inhibition zone 15mm: sensitive; 14-15mm: intermediate; <15mm: resistant; - : no inhibition zone

The minimum inhibitory concentrations of the extract were 12.5mg/ml for Bacillus subtilis and 25mg/ml for Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli. The results were detailed in table 3.

Table 3: Minimum inhibitory concentration of the DAE

Conc. mg/ml	Medium (ml)	Inoculum (ml)	Water extract
Conc. mg/ml	Medium (ml)	Inoculum (ml)	Sa	Bs	Kp	Ec
3.125	20	0.01	+	+	+	+
6.25	20	0.01	+	+	+	+
12.5	20	0.01	+	*	+	+
25	20	0.01	*	-	*	*
50	20	0.01	-	-	-	-

(+): Growth; (-): No growth; (*): MIC; All determinations were done in triplicates. Sa: Staphylococcus aureus; Bs: Bacillus subtilis; Kp: Klebsiella pneumoniae; Ec: Escherichia coli

Phytochemical analysis of the Monechma ciliatum seeds showed the presence of flavonoids, tannins, anthraquinones, unsaturated sterols and triterpens (Table 1). These phytochemical compounds have been reported in other studies to elicit antimicrobial and astringent effects.

Table 4: Phytochemical test results of seeds of Monechma Ciliatum

Test Name	Observation	Result
Test for alkaloids	No color change	-
Test for anthraquinones	Pink precipitate	++
Test for coumarins	No color change	-
Test for cyanogenic glycosides	No color change	-
Test for flavonoids	Red colouration	++
Test for saponins	No color change	-
Test for tannins	Green precipitate	+
Test for triterpens	Pink to purple precipitate	+++
Test for unsaturated sterols	Green to purple precipitate	+++

(+ + +): appreciable amount; (+ +): moderate amount; (+): minute amounts; (-): not detected

The granules obtained for the both formulae were satisfactory. No rat holing was observed during the flow of granules from the hopper and no capping and sticking was not observed. The results were provided in table 5.

Table 5: Precompression (Preformulation) study results

Measured properties	Formula-1	Formula-2
pH of the DAE	7.5	7.5
Moisture content (%) of wet- mass	18.8	13.7
Moisture content (%) of dry- granule	1.2	1.4
Moisture content (%) of final- granule	1.5	1.6
Angle of repose (°) of dry-granule	20.47	16.38
Angle of repose (°) of final- granule	19.62	16.64
Bulk density (g/ml)	0.52	0.53
Tapped density (g/ml)	0.61	0.62

Figure 2: Weight variation statistical analysis tablets of both formulae by SPSS- computer- program

The tablet post- compression parameters (weight variation, friability, hardness and disintegration tests) were measured and the results were given in table 6 and figures 2 and 3.

Table 6: Post- Compression study results

Test name	Formula-1	Formula-2
% Weight Variation	1.2	2.1
% Friability	0.1	0.4
Hardness (kg/cm²)	6.5	8.4
Disintegration time (min: sec)	8.3	11.7

Figure 3: Hardness statistical analysis for tablets of both formulae by SPSS- computer- program

As a drug efficiency depend upon its bioavailability, which is affected by dissolution of the formulated drug tablet.^[8]Formula-1 tablets were selected for further dissolution and stability studies. As the formulated material was from natural plant origin that needs unusual methods to assay as stated in USP, ^[6] under the article of botanical origin require more vigorous procedure, marker selection method was used for the dissolution test using the UV spectrophotometer at wavelength 275nm for the calibration curve. The absorbencies obtained from the tablets were referred to the curves (Table 7 and Figures 4, 5 and 6), and the percent dissolved was calculated in different time intervals (Table 8).

Table 7: The absorbance data by using HCl and water as dissolution medium

No.	Concentration (mg/ml)	Absorbance in 0.1HCl	Absorbance in distilled water
1	0.03	0.179	0.164
2	0.06	0.319	0.301
3	0.09	0.474	0.422
4	0.12	0.621	0.601
5	0.15	0.763	0.671

Table 8: The mean absorbance of six tablets in different time intervals by using 0.01N HCl and distilled water as dissolution medium

Time of dis. test	Hydrochloric acid			Distilled water
Time of dis. test	Average absorbance	Extract amount	Percent dissolved	Average absorbance	Extract amount	Percent dissolved
After 10 min	0.572	0.747	74.77	0.490	0.724	66.97
After 20 min	0.580	0.758	78.86	0.518	0.737	71.41
After 30 min	0.598	0.781	81.21	0.588	0.745	72.78
After 40 min	0.601	0.792	82.87	0.590	0.764	84.57
After 60 min	0.700	0.936	90.21	0.600	0.779	85.41

DISCUSSIONS:

The bacteria selected in the study were common diarrheal- causing pathogens. The extract inhibited Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae and Escherichia coli with inhibition zone diameter 13 to 20, 16 to 33, 12 to 21 and 15 to 18mm respectively. The antibacterial activity of the extract showed the highest activity against Bacillus subtilis compared to the other bacteria used. Collectively, the growth of all bacteria used was inhibited effectively and to different extent by the extract at different concentrations; and were comparable to that exhibited by reference antibacterial drug (as positive control) and distilled water (as negative control).Wendakoon et al.,^[9]report the greater the inhibition zone (measured in mm) indicates the higher antibacterial effect. Pseudomonas aeruginosa neither was sensitive to the extract nor to standard antibiotics used gentamicin and ampicillin. Omolola,^[10]state resistance of Pseudomonas aeruginosa to extracts may be due to its the permeability barrier afforded by its outer membrane lippolysaccharide and its tendency to colonize surfaces in the biofilm form makes the cells impervious to therapeutic concentrations of antibiotics.

The relatively low MIC values against tested bacteria means that the extract has the potential to treat any ailments associated with these bacterial pathogens effectively and with high potency. From the MIC results, it can be observed that Gram positive bacteria are more susceptible than Gram negative bacteria to the extract. In Gram negative bacteria, the outer phospholipidic membrane carrying the structural lipopolysaccharide components makes the cell wall impermeable to lipophilic solutes.^[11] Gram positive bacteria having only an outer peptidoglycan layer, thus is not an effective permeability barrier.^[12]

The antibacterial activity test results were coincided with phytochemical screening results (Table 4) and with the native people traditional practices that use primarily water as the solvent for extraction of the plant seeds for diarrhea. This study found that water provided more consistency in antibacterial activity. The antibacterial activity of the extract was attributed to flavonoids and tannins, that acted by different mechanisms comparable to the standard antibiotics, and with large margin of safety. Doss et al.,^[13]report tannins have antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. Rhama and Madhavan,^[14]report flavonoids have antibacterial activity against Escherchia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.

The granules produced were of acceptable quality. The goals of preformulation studies are to choose the correct form of the drug substance, evaluate its physical and chemical properties, and generate a thorough understanding of the material’s stability under the conditions that will lead to the development of a practical drug delivery system.^[15]

In the process of formulation of an effective tablet dosage form from the DAE, the wet granulation technique was used. Wet granulation technique was used due to the extract high dose and poor compatibility of its powder. These two factors are not favourable for the manufacture of tablet by other techniques (direct and dry granulation). Majekodunm et al., ^[16]report in preparation of tablet from natural products wet granulation appears to produce tablets of acceptable mechanical and drug release properties than those prepared by direct compression. The whole plant extract was chosen for the formulation due to the probability of synergistic action. Plant metabolites are present in complex mixtures, each containing various functional groups; a phytochemical matrix will exhibit multiple functionalities and bioactivities.^[17]

Two formulae were designed, one containing maize starch as a binder (formula- 1), and other containing PVP (formula-2). Both formulae contain MCC/maize starch, magnesium stearate, and talc as the disintegrant, lubricant, and glidant respectively at recommended levels. The batch size was maintained at 250g for each trial. Each granulation batch used the same quantity of water for granulation, theoretically, 16% or 40ml. The use of colouring and flavouring agents were not needed as the extract was mutually coloured and flavoured (brown colour with aromatic flavour), and the presence of high content of lactose in the both formulae masked the bitter taste of extract. The tablets were compressed at the specified weight (500mg).

The higher weight variation of the tablets was 2% for tablets of formula-2 but still within the acceptable weight variation range of ± 5%. Tablets of both formulae passed the weight variation test with p<0.05 (Table 6 and Figure 2). The variation of the weight of individual tablet is a valid indication of the corresponding variation in the drug content. ^[18]Hardness of tablets of both formulae was complied with the requirements stated in official pharmacopeias with p<0.05 (Table 6 and Figure 3). Hardness indicates the capability of a tablet to withstand mechanical shocks during handling in manufacturing, packaging and shipping.^[19]The tablets of both formulae disintegrated before 20min. The high hardness value and long disintegration time of formula-2 tablets was attributed to polymer used. Hirasawa et al., ^[20]conclude their research use of PVP as tablets binder increase the hardness of tablets.

0.01M HCl was better than distilled water as dissolution medium (Table 8 and Figure 6), this may be attributed to the relatively weak alkalinity of the extract (pH 7.5). Therefore, 0.01M HCl was proposed as dissolution medium for further stability tests. Both disintegration and dissolution tests were carried out after storage at room temperature, for three months, six months, and nine months. Tablets of formula- 1 were stable for nine months as shown in tables 9 and 10. The increasing of disintegration time (Table 9) and decreasing of percent dissolved in dissolution test (Table 10) may be attributed to the increase of hardness of tablets by loss of moisture via evaporation.

Tablet of both formulae complied with the standard requirements regarding their weight variation, friability, hardness, disintegration, and dissolution. A cost effective formulation was adopted using starch as binder. The dissolution test of tablets of formula-1 was carried out to assay the amount of the dissolved ingredients, by measuring the amount of the dissolved ingredients in different time intervals, which gave positive scientific prediction about the pharmacokinetics and bioavailability. Tablets of formula- 1 were stable for twelve months as shown in the ongoing stability results.

ACKNOWLEDGEMENT:

I thankful to Omdurman Islamic University, Department of Pharmaceutical Technology for providing the laboratory facilities to perform the research work.

REFERENCES:

1. Abu- Shanab B, Adwan G, Abu- Safia D and Adwan NJ. Antibacterial Activities of Some Plant Extracts Utilized in Popular Medicine in Palestine. Turkey Journal of Biology. 28; 2004: 99-102.

2. Tatiya A, Tapadiya G, Kotecha S and Surana S. Effect of Solvents on Total Phenolics, Antioxidant and Antimicrobial Properties of Bridelia retusa Spreng Stem Bark. Indian Journal of Natural Product Research. 2(4); 2011: 442-447.

3. Lopez CM, Nitisinprasert S, Wanchaitanawong P and Poovarodom N. Antimicrobial Activity of Medicinal Plant Extract against Food Borne Spoilage and Pathogenic Microorganisms. Kasetsart Journal of Natural Science. 37; 2003:460-467.

4. Lino A and Deogracious O. The In-vitro Antibacterial Activity of Annona senegalensis, Securidacca longipendiculata and Steganotaenia araliacea Ugandan Medicinal Plants. African Health Science. 6(1); 2006: 31-35.

5. Fabry W, Okemo P, Ansorg R. Fungistatic and Fungicidal Activity of East African Medicinal Plants. Mycoses. 39(1-2); 1996: 67–70.

6. United State Pharmacopeia (USP). The United States Pharmacopoeial Convention. 2007.

7. Kiran NR, Palanichamy S, Rajesh M, Rajadhas TG, Anusha V and Parasakthi N. Formulation and Evaluation of Orodispersible Piroxicam Tablets. Journal of Pharmaceutical Science and Research. 2(10); 2010: 615-621.

8. Murphy R and John R. Current St John s wart research from mode of action to clinical efficacy. Pharmacology Research. 47(2); 2003: 101-109.

9. Wendakoon C, Calderon P and Gagnon D. Evaluation of Selected Medicinal Plants Extracted in Different Ethanol Concentrations. Journal of Medicinally Active Plants. 1(2); 2012:60-68.

10. Omolola O. The Isolation of Pseudomonas aeruginosa From Septic Sore Using Some Biological Tests. Internet Journal of Food Safety. 13; 2011:188-90.

11. Adwan K and Abu-Hasan N. Gentamicin Resistance in Clinical Strains of Enterobacteriaceae Associated with Reduced Gentamicin Uptake. Folia Microbiology.43;1998: 438-440.

12. Scherrer R and Gerhardt P. Molecular sieving by the Bacillus megaterium cell wall and protoplast. Journal of Bacteriology. 107(3); 1971:718-735.

13. Doss A, Mohammed M and Dhanabalan R. Antibacterial Activity of Tannins from the Leaves of Solanum trilobatum Linn. Indian Journal of Science and Technology. 2(2); 2009: 41-43.

14. Rhama S and Madhavan S. Antibacterial Activity of the Flavonoid, Patulitrin Isolated from the Flowers of Tagetes erecta L. International Journal of Pharmaceutical Technology Research. 3(3) 2011:1407-1409.

15. Niazi SK. Hand book of preformulation chemical, biological and botanical drugs. Informa health care, New York. 2006.

16. Majekodunmi SO, Adegoke OA and Odeku OA. Formulation of the extract of the stem bark of Alstonia boonei as tablet dosage form. Tropical Journal of Pharmaceutical Research. 7 (2); 2008: 987-994.

17. Cseke LJ, Kirakosyan A, Kaufman P, Warber S, Duke J and Brielmann HL. Natural Products from Plants. CRC Press, New York. 2006.

18. Rawlins EA. Bentley’s text book of pharmaceutics. Bailliere Tindal, London. 1977.

19. Banker GS, and Anderson NR. Tablets. In: The theory and practice of industrial pharmacy, Edited by Lachman L and Lieberman HA. CBS Publishers, Delhi. 2006: pp. 229.

20. Hirasawa N, Ishise S, Miyata H and Danjo K. Application of nilvadipine solid dispersion to tablet formulation and manufacturing using crospovidone and methylcellulose as dispersion carriers. Chemical and Pharmaceutical Bulletin. 52(2); 2004: 244-247.

Received on 26.03.2013

Modified on 01.04.2013

Accepted on 05.04.2013

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(2): March- April, 2013, 99-106

Test no.	Time of disintegration the test	Time (min: sec)
1	At zero time	7.4
2	After three month	10.41
3	After six month	12.61
4	After nine month	14.58

No.	Time of the test	Absorbance after 1h	Amount of the drug	Percent dissolved
1	At zero time	0.610	0.8357	83.57
2	After 3 months	0.605	0.82885	82.85
3	After 6 months	0.598	0.81926	81.92
4	After 9 months	0.580	0.7946	79.46
5	After 12 months	0.576	0.7679	77.57