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Annals of Biological Research, 2012, 3 (1):63-72 (http://scholarsresearchlibrary.com/archive.html)

ISSN 0976-1233 CODEN (USA): ABRNBW

Formulation and evaluation of herbal effervescent granules incorporated with Limnophila indica extract for bacillary dysentery
Sandhya S*, Gowthami G, Vinod K.R, Vidya Sravanthi E, Saikumar P, Rao K.N.V and David Banji
Department of Pharmacognosy, Nalanda College of Pharmacy, Cherlapally, Nalgonda, Andhra Pradesh, India

______________________________________________________________________________ ABSTRACT The present research work is based on the formulation of herbal effervescent granules by incorporating the crude extract of Limnophila indica for bacillary dysentery. The folklore of Andhra Pradesh widely use this plant for dysentery. The dried powder of the plant was extracted and then subjected to preliminary chemical tests. Then it was formulated into efferevescent granules and then evaluated for various parameters like angle of repose, dissolution studies, effervescent cessation time, stability studies, FTIR studies and invitro anti bacterial studies against three shigella species and invivo anti diarrheal studies by castor oil induced diarrhea. The preliminary chemical studies sthowe that the extract contains flavonoids, alkaloids and tannins. The formulated effervescent granules exhibited excellent flow properties which showed good angle of repose, carrs index, Hausners ratio, bulk density and Tapped density. The dissolution profile was found to be very good. The FTIR studies showed that no drug polymer interactions were there. The accelerated stability studies revealed that the product will be stable for 57.9 months if stored at 27C. The invitro antibacterial studies proved the formulation to possess more potent anti shigellosis activity than the standard ceftazidime used. The anti diarrheal activity exhibited the herbal effervescent granules to be more potent than the standard loperamide used. Keywords: Shigella, ceftazidime, loperamide, Carrs index, dissolution. ______________________________________________________________________________ INTRODUCTION Nature always stands as a golden mark to exemplify the outstanding phenomena of symbiosis. Natural products from plant, animal and minerals have been the basis of the treatment of human disease[1]. Dysentery is an infectious disease of the large intestine characterized by symptoms of sickness, fever, headaches, abdominal pain and diarrhoea, which occasionally may be bloodstained. Bacillary dysentery is the most common form of dysentery and is caused as a result
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Sandhya S et al Annals of Biological Research, 2012, 3 (1):63-72 _____________________________________________________________________________ of infection by different strains of the bacteria called Shigella. It is an acute infectious enteritis of human and subhuman primates and usually causes frequent passage of small-volume, bloody mucoid stools, accompanied by abdominal cramps and rectal pain[2]. Synthetic treatment includes antibiotics, antidysentric drugs on to which the microorganisms developed the resistance. Effervescent powders used as saline cathartics were available in the eighteenth century and were subsequently listed in the official compendia as compound effervescent powders. Effervescent mixtures have been moderately popular over the years since along with the medicinal value of the particular preparation, they offered the public a unique dosage form that was interesting to prepare. In addition, they provided a pleasant taste due to carbonation which helped to mask the objectionable taste of the drugs [3,4]. Limnophila indica belongs to the family Scrophulariaceae. It is an aquatic or marshy, aromatic short, erect diffusely branched submerged herb [28]. It is highly constituted with flavonoids and essential oils. The plant is widely used as carminative, antiseptic, filariasis, dysentery and dyspepsia[5]. MATERIALS AND METHODS Plant was collected from the forest regions of Thalakona of Chittoor district and was authenticated by Mr. A. Laxma Reddy, Retired Professor, Dept. of Botany, Nagarjuna Government College, Nalgonda. The plant herbarium was prepared and deposited in the Department of Pharmacognosy, Nalanda College of Pharmacy for future reference. The plant was identified as Limnophila indica (Linn.) Druce (Scrophulariaceae)(Voucher No: NCOPNLG/phcog/10-11/038). Procurement of Micro organisms: Shigella flexneri MTCC 1457 was procured as freeze dried form from Microbial type culture collection, Chandighar, India. Shigella dysentery and Shigella boydii were the two pathogenic antibiotic resistant bacterial strains that were procured from Microbiology laboratory of Kamineni Institute of Medical Sciences, Nalgonda, Andhra Pradesh, India. Procurement of animals Wistar Albino Rats of either sex weighing between 150-200g were procured from National Institute of Nutrition, Hyderabad, A.P, India. The experimental protocol was approved from the Institutes animal ethics committee under the reference no. NCOP/IAEC/approval/36/2011 and then experimental studies were undergone according to their rules and regulations. The animals were housed in metabolic cages, bedded with rice husk under standard environmental conditions and had free access to standard pellet diet and water ad libitum. Preparation of extract The 500g of powered plant material was defatted with petroleum ether and then extracted with methanol for 72 h at 45C. The extract thus obtained was then concentrated under vacuum using rotary vacuum evaporator and then subjected to preliminary chemical screening to identify the active chemical constituents[6,7]. Formulation of Herbal Effervescent Granules Herbal Effervescent granules were prepared by wet granulation method. 11.78g citric acid, 23.56g tartaric acid, 40.05g sodium bicarbonate and 24.6g were triturated into fine powder. Then sufficient alcohol was added to make a damp mass. This mass was passed through sieve no 10 to get granules and these granules were dried in hot air oven at 40C and then they were packed in air tight container[3,4].

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Sandhya S et al Annals of Biological Research, 2012, 3 (1):63-72 _____________________________________________________________________________ Evaluation of Formulated Herbal Effervescent Granules[8-14] Angle of repose The fixed funnel method was employed to measure the angle of repose. A funnel was secured with its tip at a given height (h), above a graph paper that is placed on a flat horizontal surface. The blend was carefully pored through the funnel until the apex of the conical pile just touches the tip of the funnel. The radius of the base of the conical pile was measured. The angle of repose ( ) was calculated using the following formula: Tan = h/r , Where, = Angle of repose, h = Height of the cone, r = Radius of the cone base.Values for angle of repose 30 usually indicate a free flowing material and angles 40 suggest a poorly flowing material, 25- 30 show excellent flow properties, 31-35 show good flow properties, 36-40 show fair flow properties and41-45 showing passable flow properties. Bulk Density 15 g powder blend introduced into a dry 100 ml cylinder, without compacting. The powder was carefully leveled without compacting and the unsettled apparent volume, Vo, was read. The bulk density was calculated using the following formula. b = M / Vo Where, b = Apparent bulk density,M = Weight of sample, V = Apparent volume of powder Tapped Density After carrying out the procedure as given in the measurement of bulk density the cylinder containing the sample was tapped 500 times initially followed by an additional taps of 750 times until difference between succeeding measurement is less than 2% and then tapped volume, Vf was measured, to the nearest graduated unit. The tapped density was calculated, in gm per ml, using the following formula. tap = M / Vf Where, tap = Tapped density,M = Weight of sample,Vf = Tapped volume of powder Carrs Index (%) The Compressibility index (Carrs index) is a measure of the propensity of a powder to be compressed. It is determined from the bulk and tapped densities. In theory, the less compressible a material the more flowable it is. As such, it is measures of the relative importance of interparticulate interactions. In a free flowing powder, such interactions are generally less significant, and the bulk and tapped densities will be closer in value. For poorer flowing materials, there are frequently greater inter-particle interactions, and a greater difference between the bulk and tapped densities will be observed. These differences are reflected in the Carrs Index which is calculated using the following formulas: Compressibility index = [(tap - b) / tap] / 100 Where,b = Bulk Density, tap = Tapped Density

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Sandhya S et al Annals of Biological Research, 2012, 3 (1):63-72 _____________________________________________________________________________

Table 1: Compressibility index values Compressibility index 10 11 15 16 20 21 25 26 31 32 37 >38 Properties Excellent Good Fair Passable Poor Very Poor Very Very Poor

Hausners Ratio Hausners ratio is an indirect index of ease of powder flow. It is calculated by the following formula. Hausners Ratio=Tapped density(t) / Bulk density(b ) Where t tapped density and b is bulk density. Lower Hausners ratio (<1.25) indicates better flow properties than higher ones, between 1.25 to 1.5 showing moderate flow properties and more than 1.5 poor flow. Dissolution Studies The effervescent granules were placed inside the dissolution vessel. Samples of 1ml were withdrawn at time intervals of 5, 10, 15, 20, 25,30,35,45,60,75,90,105 and 120minutes. The volume of dissolution fluid adjusted to 900 ml by replacing 1ml of fresh dissolution medium after each sampling. The release studies were conducted with 3 doses of effervescent granules, & the mean values were plotted versus time. Each sample was diluted to 10 ml and analyzed at 217nm using double beam UV and Visible Spectrophotometer against reagent blank. All dissolution runs were performed in triplicate. Fourier Transform Infrared Spectroscopy (FT-IR) FTIR spectra was obtained on a Perkin Elmer FTIR spectrometer (OU,Hyd) in the transmission mode with the wave number region 2,000500 cm-1. KBr pellets were prepared by gently mixing 1 mg sample powder with100 mg KBr. Effervescent cessation time 100ml of distill water was taken in 250ml beaker, one dose of effervescent granules were poured in to the beaker, effervescent cessation time and effervescent production was observed. Stability studies The formulation was subjected to accelerated stability study by Arrhenius method. Log % retained was calculated and a graph was plotted between log % retained and time in hours. As it is a straight line, it is following first order reaction. From the graph the k values are calculated for 50C and 70C. Invitro anti bacillary dysentery activity Kirby-Bauer method was followed using the standardized sterile paper disc-agar diffusion method. All Petri dishes and graduated measuring pipettes were heat sterilized in an autoclave at 120C for 1hr. Media were steam sterilized at 121C (15psi) for twenty minutes in an autoclave. All plates were prepared with equal thickness of nutrient agar. The plates were inoculated with S.
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Sandhya S et al Annals of Biological Research, 2012, 3 (1):63-72 _____________________________________________________________________________ flexineri, S. dysentery and S.boydii. Sterile discs were impregnated with the herbal effervescent granules (test), control as placebo (effervescent granules devoid of herbal extract) and Ceftazidime(Standard).All the samples concentration was 1mg/ml. It was then incubated at 37C for 48h.The experiment was performed in triplicates[15,16]. In vivo Anti Diarrheal Activity The invivo anti diarrheal screening by castor oil induced diarrhea in wistar albino rats was performed. Animals were housed individually with free access to food and water under standard condition and the basal food intake, body weights to the nearest gram were noted. The animals were starved 18 hr prior to starting biological activity. The rats were grouped into four groups of six animals each according to their weights and housed in separate locally fabricated metabolic cages. Group I designated as control received placebo(effervescent granules without the plant extract), group II standard received Loperamide 3mg/kg and group III received herbal effervescent granules (200mg/kg) (p.o). All the samples were given 1 hour before the oral administration of cathartic agent castor oil (2 ml per rat). Oral administration of castor oil was facilitated by the use of a stomach tube. The animals were monitored for 12 hours for consistency of stool and frequency of defecation. At the end of this period, the total number of faecal matter for each group and the number of diarrhoeic (wet) faeces were recorded and the mean value for each group calculated[17,18]. Statistical analysis: Experimental data were expressed as meanS.D. The difference between experimental groups was compared by One-way Analysis of Variance (ANOVA) followed by Dunnet Multiple comparison test (control vs. test) using the soft ware Graph Pad Instat. The differences were considered to be statistically significant when** P<0.01. RESULTS AND DISCUSSION The methanolic extract of L.indica after extraction gave %yield of 28.5%w/w. When subjected to preliminary chemical screening showed the presence of flavonoids, essential oil, tannins and alkaloids. A suitable oral dosage form was formulated by taking into consideration of pediatric and geriatric groups. A herbal effervescent granules was prepared as this particular formulation can be easily administered by all age groups. The colour of the granules was olive green in colour with citrus odour. The frictional force in a loose powder can be measured by the angle of repose (). It is defined as, the maximum angle possible between the surface of the pile of the powder and the horizontal plane. If more powder is added to the pile, it slides down the sides of the pile until the mutual friction of the particles producing a surface angle , is in equilibrium with the gravitational force. Density is defined as weight per unit volume. Bulk density, b, is defined as the mass of the powder divided by the bulk volume and is expressed as gm/cm. The bulk density of a powder primarily depends on particle size distribution, particle shape and the tendency of particles to adhere together. Bulk density is very important in the size of containers needed for handling, shipping, and storage of raw material and blend. It is also important in size blending equipment. The Compressibility index (Carrs index) is a measure of the propensity of a powder to be compressed. It is determined from the bulk and tapped densities. In theory, the less compressible a material the more flow able it is. As such, it is measures of the relative importance of inter particulate interactions. In a free flowing powder, such interactions are generally less significant, and the bulk and tapped densities will be closer in value. For poorer flowing materials, there are frequently greater inter-particle interactions, and a greater difference between the bulk and tapped densities will be observed. Hausners ratio is an indirect index of ease of powder flow. The effervescent granules were evaluated for angle of repose, Hausners
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Sandhya S et al Annals of Biological Research, 2012, 3 (1):63-72 _____________________________________________________________________________ ratio, and compressibility index[3,4]. The angle of repose was found to be 31.This value suggest that the formulated herbal effervescent granules have very good flow property. The bulk density and tapped density was found to be 0.43 and 0.5, respectively.The Hausners ratio obtained was 1.16, which indicates a very good flow property. The compressibility index was 14 which denote a good flow property for the prepared herbal granules. The dissolution testing is performed to provide invitro drug release profile for quality control purpose. The datas obtained for the drug dissolution results suggest the safety and efficacy of the product. The principle function of the dissolution test is optimization of therapeutic effectiveness during product development and stability assessment, routine assessment of production quality to ensure uniformity between production lots, assessment of bioequivalence, that is to say, production of the same biological availability from discrete batches of products from one or different manufacturers, prediction of in-vivo availability. The in vitro dissolution testing was performed and the results of the effervescent granules are expressed in Table.The release of plant extract from effervescent granules was studied using USP dissolution apparatus II (ELECTROLAB TDT-08L). The %drug release from the formulation and extract was found to be 83.08% and 31.64% in 2 hrs respectively(Figure 1). Hence comparatively the formulation had shown better drug release.
92 88 84 80 76 72 68 64 60 56 52 48 44 40 36 32 28 24 20 16 12 8 4 0 0 5 10 15 20 25 30 35 45 60 75 90 105 120

% D r u g r e l e a s e

Extract Formulation

Time(min)

.
Figure1: Cumulative % drug release of effervescent granules and extract.

The FTIR studies were performed for polymer, formulated herbal effervescent granules, plant extract and physical mixture, to analyse if there were any kind of polymer-drug interactions. The report obtained revealed no radical interactions (Figure 2).This showed that the drug and polymer are compatible to develop a stable product. The invitro anti bacterial screening on three shigella species was performed for the excipients, formulation and ceftazidime. It was observed that the formulation showed a much better activity than the standard. The placebo also produced a mild antibacterial activity (Table 2). The three shigella species used in the evaluation were found to be highly susceptible to the formulation than the standard ceftazidime. Among the three two species viz; S.boydii and S. dysentery were
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Sandhya S et al Annals of Biological Research, 2012, 3 (1):63-72 _____________________________________________________________________________ pathogenic bacterial strains which were antibiotic resistant strains. These two strains were found to be highly sensitive to the formulation.

Figure 2: FTIR studies.A-placebo; B-herbal effervescent granules; C- crude extract; D-physical mixture. Table 2: Anti shigellosis activity of herbal effervescent granules Zone of inhibition (mm) Placebo(Control) Herbal effervescent granules(Test) Ceftazidime(Standard) 1mg/ml 1mg/ml 1mg/ml S. dysentery 12 45 25 S. boydii 8 35 18 S. flexineri 4 12 12 Zone of inhibition reported is exclusive of disc diameter which is 6mm. Shigella species

The effervescent cessation time was found to be 3minutes which denotes an optimum time. Accelerated stability studies were performed where by the stability of the formulated herbal effervescent granules was found to be 57.8 months when stored at 27C (Figure 3,4).

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Sandhya S et al Annals of Biological Research, 2012, 3 (1):63-72 _____________________________________________________________________________

log %ug concentration Dr VsTime

2 1.995 1.99 1.985 1.98 1.975 1.97 1.965 1.96 1.955 0 1 2 months 3 4 5 log% Drug concentration

y = -0.001x + 1.998 R = 0.962 y = -0.006x + 2.001 R = 0.966 y = -0.007x + 1.993 R = 0.934 log 27 log 50 log 70 Linear (log 27) Linear (log 50) Linear (log 70)

.
Figure 3: Log %drug concentration Vs Time

log K Vs 1/T
-1.78 0.00280.00290.0030.00310.00320.00330.0034 -1.8 -1.82 -1.84 log K -1.86 -1.88 -1.9 -1.92 -1.94 -1.96 y = -370.8x - 0.711 R = 1 1/T . Figure 4: LogK Vs 1/T log K values Linear (log K values)

The invivo anti diarrheal activity screened revealed that the formulated herbal effervescent granules produced an excellent activity which was determined to be more potent than the standard loperaminde used (Figure 5). To evaluate whether the excipients played any role in the activity a control group treated with placebo was performed. The results displayed that the excipients used in the formulation of herbal effervescent granules did not possess any antidiarrheal activity. The total number of defecation observed till 12h of observation was found to be 70.4472, 2.20.5831** and10.3162** for placebo(standard), loperamide 3mg/kg(standard) and herbal effervescent granules 200mg/kg(test). The total number of wet motions observed were found to be 5.20.2, 0.60.2449** and 00 for control, standard and test. p<0.01 was considered to be significant.

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Sandhya S et al Annals of Biological Research, 2012, 3 (1):63-72 _____________________________________________________________________________

Figure 5: Anti diarrheal activity of herbal effervescent granules

The formulated herbal effervescent granules has shown a potent antibacterial and antidiarrheal activity which was observed to be much better than the standard drug used. The potency of the prepared formulation can be attributed to the excellent flow properties, dissolution profile and the powerful phyto constituents present in the extract. The effervescent dosage forms usually provide substantial immediate release of pharmaceutical ingredient present in it. Another advantage of the effervescent granules is that it can provide effective taste masking of poor tasting compounds[19]. Since dysentery is an infectious disease which requires immediate treatment this can be a very good choice due to its potential advantages. As these problems generally occur more in paediatric age groups the taste masking advantage of the effervescent granules will benefit. Since these granules will have to dispersed in water and taken it can be considered as a most promising oral dosage form for all age groups for bacillary dysentery. CONCLUSION It is well documented that most of the micro organisms causing dysentery have developed resistance to synthetic medications including antibiotics. Hence it can be suggested that the formulated herbal effervescent granules can be a very good alternative as it has proved to be very effective for antibiotic resistant pathogenic shigella species. The synthetic anti diarrheal drugs like loperamide generally possess side effects and cannot be taken by children under the age to two. Since L.indica possess no side effects it can be considered to be the best alternative as it has proved be more effective. Acknowledgement The authors express their gratitude to MTCC and Microbiology department of Kamineni Medical sciences, for providing the required microorganisms. They are highly grateful to the Management of Nalanda College of Pharmacy for providing necessary help and laboratory facilities in performing these studies. REFERENCES [1] S. Verma, S.P Singh, Veterinary World, 2008, 1,11, 347-350. [2] C. Cobra David, D. A. Sack, The Control of Epidemic Dysentery in Africa, SD Publication Series,Africa, 1996,1-5.
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Sandhya S et al Annals of Biological Research, 2012, 3 (1):63-72 _____________________________________________________________________________ [3] H.A Lieberman, L. Lachman, J.B Schwartz, Pharmaceutical Dosage Forms: Tablets, Vol.1 and 2, Marcel Dekker Inc. New York, 1993, 285. [4] H.C Ansel, N.G Popovich, L.V Allen, Pharmaceutical Dosage Forms and Drug Delivery Systems, B. I. Waverly, New Delhi ,1999,6, 469-471. [5] K.C Madhava, K. Sivaji, K.R Tulasi, Flowering Plants of Chitoor Dist A.P. India, Students Offset Printers,Tirupati, 2008, 236. [6] K.R Khandelwal, Practical Pharmacognosy,19th ed: Nirali Prakashan, Pune 2009,146-165. [7] C.K Kokate, Practical Pharmacognosy, 4th ed, Nirali Prakashan,Pune, India. 2008, 10-27. [8] W. James, Pharmaceutical preformulation: the physicochemical properties of drug substances: Aulton ME. Pharmaceutics the science of dosage form design, Churchill livingstone, Spain, 2006, 2, 113-138. [9] G.S Banker, N.R Anderson, Tablets: Lachman L, Lieberman H, The theory and practice of Industrial Pharmacy, CBS publishers, New Delhi, 2009, 293-345. [10] D. Peter, Oral solid dosage forms: Gibson M. Pharmaceutical preformulation and formulation a practical guide from candidate drug selection to commercial dosage form, Interpharm/CRC, New York, 2008, 379-432. [11] M. Raymond, Effervescent tablets: Lieberman HA, Lachman L, Schwartz TB. Pharmaceutical dosage forms, Marcel Dekker, Inc., New York, 2008,2,1, 285-328. [12] J.K Guillory, I.P Rolland, Chemical kinetics and drug stability: Modern Pharmaceutics, Marcell Dekker Inc., New York, 2005, 4, 121, 139-163. [13] B. Suresh, E. Chandramohan, T. Ashok, Y. Madhusudhan Rao, Acta Pharm, 2010, 60, 8997. [14] B. Prakash, K. Ashok, V.S. Snehith, C. Ramesh, ARS Pharmaceutica,2009, 50,1, 8-24. [15] M. Panghal, V. Kaushal, J.P. Yadav, Ann Clin Microbiol Antimicrob., 2011, 20, 10-21. [16] F. Sahin, I. Karaman, M. Gulluce, H. Ogutcu, M. Sengul, A. Adiguzel, S. Ozturk, R. Kotan, J Ethnopharmacol., 2003, 87,5, 6165. [17] B.S. AdzuS, S. Amos, M.B. Amizan, K.S. Gamaniel, Acta Tropica.,.2003, 6,1,1-5. [18] Bhanu Pratap, Der Pharmacia Lettre, 2010, 2,3, 309-314. [19] Effervescent granules and methods for their preparation. http://www.patentstorm.us/patents/6071539/description.html.retrieved on 20/11/11.

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