INTRODUCTION:

Antibiotic treatment is associated with common adverse effect known as diarrhea. Antibiotic associated diarrhea occurs in about 5-30% of patients.¹Almost all antibiotics, particularly those that inhibit anaerobes, can cause diarrhoea, but the risk is higher with broad spectrum antibiotics.^2,3Host factors for antibiotic associated diarrhoea include age (elderly patient over 65), immunosuppression, being in an intensive care unit, and prolonged hospitalisation.⁴

Clinical manifestation of antibiotic associated diarrhoea range from mild diarrhoea to fulminant pseudomembranous colitis. The latter is characterised by watery diarrhea, fever, leucocytosis, and the presence of pseudomembranes observed on endoscopic examination.

As antibiotic associated diarrhea mostly results from disequilibrium of the normal intestinal flora, therefore research has oriented on the benefits of administering living organisms (probiotics or biotherapeutic agents) to restore the normal flora. Many studies reported numerous probiotics such as Lactobacillus acidophilus, Lactobacillus casei GG, Lactobacillus bulgaricus, Bifidobacterium bifidum, B longum, Enterococcus faecium, Streptococcus thermophilus, or Saccharomyces boulardii have been tested for the treatment and prevention of antibiotic associated diarrhea.⁵

The Food and Agriculture Organization of the United Nations-World Health Organization (FAO-WHO), defined probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”.⁶Probiotics are known to offer various benefits, such as it keeps the gut microflora healthy, relieving constipation, enhance the gastro-intestinal immune system, inhibit the growth of pathogenic bacteria, helps in vitamin synthesis, control of lactose intolerance symptoms, reduction of blood cholesterol levels and improve absorption of calcium.⁷

Probiotics bacteria are reported and claimed to mitigate and prevent bowel disorders such as diarrheal diseases (antibiotics-associated diarrhea (AAD), infectious diarrhea), lactose intolerance, allergy, as well as stimulation of the immune system and emerging evidence support their role in lowering of serum cholesterol, reduction of the risk associated with mutagenicity and carcinogenicity and inhibition of pathogens.^8,9Probiotics must withstand during long term storage and upon oral administration withstand to different physicochemical, enzymatic, and microbial stresses throughout the gastrointestinal (GI) transit before they reach to their site of action that is colon.¹⁰

Therefore, maintaining viability of probiotics during storage and importantly during passage through gastric harsh conditions following oral administration is observed as major challenge with marketed formulations of probiotics.^10,11

To overcome the problem of probiotics viability, encapsulation methods were developed and reported by many authors. The potential of microencapsulation in improving probiotic viability during their storage, gastrointestinal transit and also release of probiotics at desired site (colon) of GI tract have been mentioned in various published reports.^12,13In present study extrusion or ionotropic gelation method of encapsulation was selected as it is claimed to be a simple and cheap method that does not involve deleterious solvents and can be done under aerobic and anaerobic conditions and uses a gentle operation which causes no damage to probiotics cells and gives a high probiotics viability.^14,15Moreover, there is scarcity of efficacy trial of encapsulated probiotics in wistar albino rats model.

Therefore, present study was designed to assess the efficacy of encapsulated Lactobacillus rhamnosus GG probiotics for the treatment of antibiotic associated diarrhea in wistar albino rat model.

MATERIALS:

Freeze dried Lactobacillus Rhamnosus GG was procured from National Dairy Institute, Karnal, India. Cellacefate (Cellulose acetate phthalate, CAP) was purchased from Ranbaxy Fine Chemicals, New Delhi, India. Low methoxy pectin and sodium carboxymethyl cellulose (NaCMC), calcium chloride (CaCl₂), aluminium chloride (AlCl₃), Polyethylene glycol (PEG) were obtained from Central Drug House Pvt. Ltd., New Delhi, India. Phosphate buffer saline (pH 7.4), Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Loba Chemical Pvt. Ltd., Mumbai, India. Pepsin and bile salt were purchased from SD Fine Chemicals, Mumbai, India. Amoxicillin was purchased of Sun Pharmaceuticals, Delhi. All other chemicals used are of analytical grade.

METHODOLOGY:

Enapsulation of LR: The L Rencapsulated microbeads were developed by ionotropic gelation technique using LMP and NaCMC as encapsulating polymers. The microbeads were prepared by dispersing LR in LMP solution using LR: LMP ratio as 1:1, 1:2 and 1:3. The above dispersion was added slowly with the help of syringe (10ml, 24 G needle) into the beaker filled with solution (100ml) containing calcium chloride (5% w/v). This gelling solution was stirred gently at 100rpm using a magnetic bar and beads formed were allowed to harden for 30 minutes. Similarly, the microbeads of LR were also prepared by using aqueous solution of NaCMC through ionotropic gelation method. LR was dispersed in NaCMC solution using LR: NaCMC ratio as 1:1, 1:2 and 1:3.The above dispersion was added slowly with the help of syringe (10ml, 24 G needle) into the beaker filled with solution (100ml) containing aluminum chloride (20% w/v).^14,15This gelling solution was stirred gently at 100 rpm using a magnetic bar and beads formed were allowed to harden for 30 minutes. The beads were rinsed with distilled water. The entire process was carried out aseptically on the horizontal laminar flow bench.

Coating of encapsulated LR cells: Aqueous solution of CAP (5% w/v) was prepared using phosphate buffer at pH 6.8. To improve the efficacy of CAP coating, polyethylene glycol 200 (20% dry weight of CAP) was incorporated into the cellulose acetate phthalate solution as a plasticizer. The solution was filtered aseptically using a 0.3μm membrane filter.¹⁶Similarly, aqueous solution of Eudragit S 100 was prepared using phosphate buffer at pH 7.0. Five gm of Eudragit S 100 was dissolved in 100ml of phosphate buffer solution with continuous stirring. Previously encapsulated microbeads were dispersed in the enteric coating solution at 400rpm for 30 minutes at room temperature.^17,18 The coated microbeads were filtered with filter paper, spread on a petri dish and dried for 2 days at room temperature. Coated microbeads were kept in a desiccators for 24 hours, then transferred aseptically into a sterile glass vial, sealed and were stored in the refrigerator for further studies. The entire process was carried out aseptically.¹⁹

In vivo aassessment of anti-diarrheal effect of LR cells:

Albino wistar rats with weight range of 150-200gm were used in present study. The animals were allowed to adapt to the experimental conditions for 7 days. The standard laboratory rodent diet and water were provided ad libitum and were housed in cages under natural light/dark cycle. The experiments were conducted in accordance with CPCSEA guidelines for proper care, handling and animal experimentation. This study protocols were approved by the Institutional animal Ethics Committee (IAEC), Banasthali University, Rajasthan. (Ref.No.BU/BT/396/13-14).

Ninety-six animals were divided into 8 experimental groups as follow and comprise of 6 animals in each group:

Treatment:

Induction of diarrhea and pharmacological treatment:

The animals were treated with amoxicillin 150mg/kg po OD for 2 weeks to induce diarrhea in all groups except naïve control group. After induction of diarrhea, animals were treated with pharmacological agents as mentioned below²⁰

1. Naive Control:

Animals without any interventions were kept under experimental condition and were provided food and water ad libitum.

2. Diarrhea Control:

Animals were treated with amoxicillin 150mg/kg po OD for 2 weeks to induce diarrhea.

3. Active Control:

Animals were treated with marketed product of LR twice daily for six days or till reversal of diarrhea.

4. Negative Control 1:

Animals were treated with vehicle named euragit S 100 coated pectin microbeads twice daily for six days.

5. Negative Control 2:

Animals were treated with vehicle named cellulose acetate phthalate coated pectin microbeads twice daily for six days.

6. Negative Control 3:

Animals were treated with vehicle named Eudragit S 100 coated NaCMC microbeads twice daily for six days.

7. Negative Control 4:

Animals were treated with vehicle named cellulose acetate phthalate coated NaCMC microbeads twice daily for six days.

8. LR3 Treated Group:

Animals were treated with LR3 microbeads (dose: 3.5 × 10⁸ CFU in 2ml normal saline, po) per day for six days or till reversal of diarrhea.

9. LR6 Treated Group:

Animals were treated with LR6 microbeads (dose: 3.5 × 10⁸ CFU in 2ml normal saline, po) per day for six days or till reversal of diarrhea.

10. LR9 Treated Group:

Animals were treated with LR9 microbeads (dose: 3.5 × 10⁸ CFU in 2ml normal saline, po) per day for six days or till reversal of diarrhea.

11. LR12 Treated Group:

Animals were treated with LR12 microbeads (dose: 3.5 × 10⁸ CFU in 2ml normal saline, po) per day for six days or till reversal of diarrhea.

Assessment of anti-diarrheal activity:

a) Effect on frequency of defecation:

The frequency of defecation was assessed by counting the fall of fecal material at base of metabolic cage.

b) Fluid content of fecal material:

The fluid content of fecal material was calculated by following formula:

Fluid content of fecal material (mg) = Weight of wet fecal material (mg) - Weight of dry fecal material (mg)

The fresh fecal material was collected and weighed and to assess weight of dried fecal material it was oven dried for 15 minutes and weighed. The difference in their weight was assessed as fluid content of fecal material.

c) Fecal consistency:

Fecal consistency was observed and scored as

1: Solid stool (formed, stool maintains its shape);

2: Semisolid stool (semi-formed or soft, does not pour) and

3: Watery stool (liquid, pours more easily).

RESULT:

1. Effect on frequency of defecation:

The frequency of defecation with treatment of vehicle in all negative control groups (negative control group 1-4) was found to be similar to diarrhea control group up to five days of treatment. This, inference that vehicle of test drug did not possess antidiarrheal activity. However, the frequency of defecation was significantly reduced at 36 hours with treatment of active control group (available marketed preparation of standard treatments), as compared to diarrhea control group and negative control group. The onset of significant reduction of defecation frequency with LR3, LR6, LR9 and LR12 microbeads was observed at 12 hours of treatment as compared to active control group, diarrhea control group and negative control group. Therefore, our selected LR microbeads (LR3, LR6, LR9 and LR12) have shown antidiarrheal activity similar to available standard marketed preparation. However, LR3, LR6, LR9 and LR12 test preparations were found to be more favourable as these has shown their antidiarrheal effect by 12 hours of treatment as compared to antidiarrheal effect by 36 hours of treatment by available standard marketed formulations (Figure1 and 2).

Figure 1: Effect of various pharmacological treatments (LR microbeads, Standard treatments and vehicle) on number of defecation (frequency) of at different time intervals (from zero to 30 hours)

Each group (n=6) represents mean ± standard deviation. Two-way ANOVA followed by Tukey's multiple comparisons test, F (10, 660) = 427.P < 0.0001. ^ap < 0.05 frequency of defecation (antidiarrheal effect) at 0 hour Vs frequency of defecation (antidiarrheal effect) at 12 hours, ^bp < 0.05 frequency of defecation (antidiarrheal effect) at 0 hour Vs frequency of defecation (antidiarrheal effect) at 30 hours.

Figure 2: Effect of various pharmacological treatments (LR microbeads, Standard treatments and vehicle) on number of defecation (frequency) of at different time intervals (from 36 to 120 hours)

Each group (n=6) represents mean ± standard deviation. Two-way ANOVA followed by Tukey's multiple comparisons test, F (10, 660) = 427. P < 0.0001.^ap < 0.05 frequency of defecation (antidiarrheal effect) at 0 hour Vs frequency of defecation (antidiarrheal effect) at 12 hours, ^bp < 0.05 frequency of defecation (antidiarrheal effect) at 0 hour Vs frequency of defecation (antidiarrheal effect) at 30 hours

Effect on fluid content of fecal material: The fluid content of fecal material with treatment of vehicle in all negative control groups (negative control group 1-4) was found to be similar to diarrhea control group up to five days of treatment. This inference that vehicle of test drug did not possess antidiarrheal activity. However, the onset of significant reduction of fluid content of fecal material was observed at 48 hours with treatment of available marketed preparation of LR (active control group) as compared to diarrheal control group and negative control group. The onset of significant reduction of fluid content of fecal material with treatment of LR3, LR6, LR9 and LR12 microbeads was observed at 12 hours of treatment as compared to active control groups, diarrhea control group and negative control groups. Therefore, our selected LR microbeads (LR3, LR6, LR9 and LR12) have shown antidiarrheal activity similar to available standard marketed formulations. The LR3, LR6, LR9 and LR12 microbeads were found to be more favourable as these has shown their antidiarrheal effect by 12 hours of treatment as compared to antidiarrheal effect by 36 hours of treatment by available standard marketed preparations (Figure 3 and 4).

Figure 3: Effect of pharmacological treatments (LR microbeads, Standard treatments and vehicle) on fluid content of fecal material at different time intervals in wistar albino rats (from zero to 30 hours)

Each group (n=6) represents mean ± standard deviation. Two-way ANOVA followed by Tukey's multiple comparisons test; F (10, 660) = 228.9, p< 0.0001. ^cp < 0.05 fluid content of fecal material at 0 time Vs fluid content of fecal material at 12 hours; ^dp < 0.05 fluid content of fecal material at 0 time Vs fluid content of fecal material at 30 hours

Figure 4: Effect of pharmacological treatments (LR microbeads, Standard treatments and vehicle) on fluid content of fecal material at different time intervals in wistar albino rats (from 36 to 120 hours)

Each group (n=6) represents mean ± standard deviation. Two-way ANOVA followed by Tukey's multiple comparisons test; F (10, 660) = 228.9, p< 0.0001. ^bp < 0.05 Fluid content of fecal material of active control compared with zero hour

Effect on fecal consistency:

The fecal consistency score with treatment of vehicle in all negative control groups (negative control group 1-4) was found to be similar to diarrhea control group up to five days of treatment. Like other antidiarrheal parameters, this is also inference that vehicle of test drug did not possess antidiarrheal activity. However, the onset of improvement in fecal consistency score was observed by 12 hours of treatment with marketed formulation (active control group) as compared to diarrhea control group and negative control group. The fecal consistency as solid stool for all animals was achieved in 72 hours in active control group. The LR3, LR6, LR9 and LR12 microbeads also has shown the onset of improvement of fecal consistency score by 6 hours of treatment as compared to diarrhea control group and negative control groups. The fecal consistency as solid stool for all animals of LR3, LR6, LR9 and LR12 test preparations was achieved in 24 hours. Therefore, our LR3, LR6, LR9 and LR12 microbeads has shown antidiarrheal activity similar to available standard marketed formulations. However, LR3, LR6, LR9 and LR12 microbeads were found to be more favourable as these has shown their antidiarrheal effect (solid stool of all animals) by 24 hours of treatment as compared to antidiarrheal effect by 72 hours with available standard marketed formulations (Figure 5).

Figure 5: Effect of various pharmacological treatments (LR microbeads, Standard treatments and vehicle) on consistency of fecal material at different time intervals in wistar albino rats

Each group (n=6) represents mean ± standard deviation.

DISCUSSION:

Probiotics are live microorganisms are primarily used for prevention and treatment of antibiotic associated diarrhea (AAD), Clostridium difficile infections (CDI) and chemotherapy induced diarrhea etc.^5,21

In present study, colon targeted microbeads of LR were prepared and assessed for their prevention from upper GI harsh conditions. In this study, ion gelation microencapsulation method was used to prepare colon targeted microbeads of probiotics. This method offers advantages over other methods of microenapsulations such as simple technique and requires no use of organic solvent. The technique involves interaction of a cation (or an anion) with an ionic polymer to generate a highly cross linked structure. However, microbeads of LRwith LMP and NaCMC were not previously prepared by ion gelation method. Therefore, in this study, LMP and NaCMC were used as microencapsulating polymers for preparing microbeads of LR with ion gelation method.

In previous studies it is also reported that to an extent, the LMP microbeads encapsulation properties in aqueous media may not be efficiently able to prevent them from avoiding drug release during transit through the upper gastrointestinal tract. Thus making the combined use of other strategies, such as coating with pH-sensitive polymers become an unmet need. Therefore, in our study, we have used Eudragit S 100 and CAP as coating material for colon targeted LMP and NaCMC microbeads. These coating polymers are reported to dissolve at colonic pH and allow releasing drug at their target site i.e. colon.¹³It would be logical to expect a size increase with Eudragit S 100 and CAP coating. However, the increase in microbeads size with coating will not be considered significant as the coating will not be thick enough.

The results of antidiarrheal assessment have shown that the onset of antidiarrheal activity of our test formulations (LR3, LR6, LR9 and LR12) was significantly earlier than free probiotics (active controls). This early onset of antidiarrheal activity of test formulations seems to be due to lesser/negligible degradation of probiotics and release of maximum amount of viable probiotics at colon. However, the delayed onset of antidiarrheal activity of free probiotics (marketed formulation) might be due to degradation of viable probiotics in upper GI harsh condition and reaching lesser amount of probiotics at colon. These findings indicate that our microbeads formulation offers protection to probiotics against gastric harsh conditions and thereby reaching maximum amount of viable probiotics at their site of action (colon).

CONCLUSION:

In present study, microbeads of LR encapsulated LMP and NaCMC microbeads were developed using the ionotropic gelation method. Microencapsulation of probiotics was done for their protection from the harsh gastric environment and release of the entrapped probiotics at the colonic site. The prepared microbeads were coated with Eudragit S 100 and CAP separately to ensure the prevention of probiotics release during transit of upper gastrointestinal region. The study showed that the Eudragit S 100 and CAP coating provides protection to the loaded probiotics from the gastric acidic environment. The microencapsulated probiotics has shown greater anti-diarrheal activity assessed in terms of quick onset of action than marketed preparation. The quick onset of anti-diarrheal action might be due to the fact of lesser degradation of probiotics microbeads in upper GI harsh conditions. Also among formulated microbeads LR3, LR6, LR9 and LR12 has shown promising result as compare to other formulated microbeads. Therefore, it was concluded that the encapsulation of probiotics in coated microbeads (LR3, LR6, LR9 and LR12) could be a promising strategy to deliver them safely and effectively into the targeted colonic region.

REFERENCES:

1. Wistrom J, Norrby SR, Myhre EB, Eriksson S, Granstrom G, Lagergren L, Englund G, Nord CE, Svenungsson B. Frequency of antibiotic-associated diarrhoea in 2462 antibiotic-treated hospitalized patients: a prospective study. J Antimicrob Chemother. 2001; 47(1): 43-50.

2. Barbut F, Meynard JL, Guiguet M, Avesani V, Bochet MV, Meyohas MC, Delmée M, Tilleul P, Frottier J, Petit JC. Clostridium difficile-associated diarrhea in HIV-infected patients: epidemiology and risk factors. J Acquir Immune Defic Syndr Hum Retrovirol. 1997; 16(3): 176-181.

3. McFarland LV, Surawicz CM, Stamm WE. Risk factors for Clostridium difficile carriage and C. difficile-associated diarrhea in a cohort of hospitalized patients. J Infect Dis. 1990; 162(3): 678-684.

4. Bignardi GE. Risk factors for Clostridium difficile infection.J Hosp Infect. 1998; 40(1): 1-15.

5. Marteau PR, de Vrese M, Cellier CJ, Schrezenmeir. Protection from gastrointestinal diseases with the use of probiotics. J Am J Clin Nutr. 2001 Feb; 73(2 Suppl): 430S-436S.

6. Lokhande S, More S, Raje V. A Systematic Study of Probiotics- An Update Review. Asian J. Pharm. Tech. 2018; 8 (3):149-157.

7. Swathi KV. Probiotics - A Human Friendly Bacteria. Research J. Pharm. and Tech 2016; 9(8): 1260-1262.

8. Devi NKD, Sujitha K, Madhavi BR, Mrudula BS, Janaki MN, Ramya VS. Symbiosis - A Friendly Relationship between Man and Microbes. Research J. Science and Tech. 2010; 2 (1): 1-7.

9. NKD Devi, K Sujitha, BR Madhavi, BS Mrudula, MN Janaki, V Sri Ramya. Symbiosis – A Friendly Relationship between Man and Microbes. Research J. Science and Tech. 2010; 2(1): 1-7.

10. Thantsha M, Mamvura C, and Booyens J. Probiotics-what they are, their benefits and challenges. In: Brzozowski, T, editors. New advances in the basic and clinical gastroenterology, Croatia: InTech Publisher 2012, p. 22-50.

11. Rodklongtan A, La‐ongkham O, Nitisinprasert S, Chitprasert P. Enhancement of Lactobacillus reuteri KUB‐AC5 survival in broiler gastrointestinal tract by microencapsulation with alginate-chitosan semi‐interpenetrating polymer networks. J Appl Microbiol 2014; 117(1): 227-238.

12. Gambhire MS, Lilke S, Gambhire VM. Microencapsulation- An Overeview. Research J. Pharma. Dosage Forms and Tech. 2010; 2(4): 270-276.

13. Akel H, Madani F, Ibrahim W. Preparation of Paracetamol Microcapsules by Complex Coacervation and Studying the effect of some Factors Influencing Microencapsulation Yield and Efficacy. Research J. Pharm. and Tech 2017; 10(10): 3271-3275.

14. Salve PS. Development of Controlled Release Floating Beads of Ibuprofen using Ionotropic Gelation Technique. Research J. Pharma. Dosage Forms and Tech. 2011; 3(6): 260-268.

15. Mali SD, Khochage SR, Nitalikar MM, Magdum CS. Microencapsulation: A Review. Research J. Pharm. and Tech. 6(9): 2013; 954-961.

16. Willliams RO, and Liu J. Influence of processing and curing conditions on beads coated with an aqueous dispersion of cellulose acetate phthalate. Eur J Pharm Biopharm 2000; 49(3): 243-252.

17. Arica B, Arica MY, Kas HS, Hincal AA, Hasirci V. In vitro studies of enteric coated Diclofenac sodium-carboxymethylcellulose microspheres. J Microencapsul 1996; 13(6): 689-699.

18. U. D. Shivhare, G. S. Chhabra, V. B. Mathur, S. B. Patel. Microencapsulation of Acyclovir into Eudragit S100 Using Emulsion-Solvent Evaporation Method. Research J. Pharm. and Tech. 4(4): April 2011; Page 652-658.

19. Jawed Akhtar, Saurabh Kanoongo, Deepak K. Mittal, Rajesh Sharma, Vaibhav Solanki. The New Developed Concepts and Trends in Microencapsulation. Research J. Pharma. Dosage Forms and Tech. 2011; 3(3): 75-83 .

20. Fiol, F.D.S.D., Tardelli, A.C.M., Marciano, J.J., Marques, M.C., Sant'Ana, L.L. Obesity and the Use of Antibiotics and Probiotics in Rats. Chemotherapy. 2014; 60: 162-167.

Received on 31.05.2019 Modified on 21.10.2019

Research J. Pharm. and Tech. 2020; 13(12):5736-5742.

DOI: 10.5958/0974-360X.2020.00999.3