Effects of Antimicrobial activity of Sapindus mukorossi(Reetha): A clinical study

by Organic Kosha March 11, 2021

 The present study was carried out to evaluate the antibacterial activity of Sapindus mukorossi (S. mukorossi).

Treatment Offered    

Plant Materials

Authentic samples of S. mukorossi and R. emodi were obtained from an authorized supplier (M/s Munnalal Dawasas and Co. Hyderabad, Andhra Pradesh, India). The plants were identified and authenticated by experts in the Post Graduate and Research Department of Botany, Anwar-ul-loom College, Hyderabad, Andhra Pradesh, India.

Bacterial strains

Thirty H pylori strains along with two gram +ve (Staphylococcus aureus, Bacillus subtilis) and two gram -ve (Escerichia coliProteus vulgaris) pathogenic bacteria were used in the study. The H pylori strains were isolated from gastric biopsy specimens [15 from duodenal ulcer (DU), 8 from gastric ulcer (GU), 4 from non-ulcer dyspepsia (NUD), 3 from gastric carcinoma (GC)], after informed consents were obtained from patients who underwent upper gastrointestinal endoscopy at Deccan College of Medical Sciences, Hyderabad, India. The other four pathogens were obtained in pure culture form from the Department of Microbiology, Deccan College of Medical Sciences, Hyderabad, A. P, India.

E-test strips

E test strips were obtained from AB Biodisk, Solna, Sweden.

Animals

Male Wister rats (175-200 g) were acclimated to the housing facilities for 5 d before initiation of the study. Free access to standard pellet chow was allowed throughout the experimental protocol, with the exception of overnight fasting before induction of the ulcer. All protocols were approved by the Animal Care and Use Committee of the Deccan College of Medical Sciences and Research Centre, Hyderabad, India where the study was conducted.

Extraction, separation and purification of the compounds

For phytochemical analysis, approximately 100 g of fruit pericarp of S. mukorossi and rhizomes of R. emodi were collected and the materials were chopped, air dried at 35-40°C and pulverized in an electric grinder. The powder obtained was successively extracted in petroleum ether (60-80°C), benzene, chloroform and ethanol.

The extracts were then powdered by using a rotary evaporator under reduced pressure. Fruit pericarp of S. mukorossi yielded 38g, 28g, 34g and 35g of powdered extracts with petroleum ether, benzene, chloroform and ethanol respectively. Rhizomes of R. emodi yielded 19 g, 17 g, 21 g and 22 g of powdered extracts. Extracts obtained by percolation using 70% of ethanol as a solvent at room temperature were processed according to process A of Farmacopeia dos Eastados Unidos do Brasil (1959) (AOAC 1990).

The extracts were evaporated at 40°C under vacuum and the residue was freeze-dried. The dry extracts of the fruit pericarp of S. mukorossi and rhizomes of R. emodi were tested for the presence of saponins and anthraquinones. Each extract of the fruit pericarp of S. mukorossi (SM) and rhizomes of R. emodi (RE) were column chromatographed over a silica gel (200 mesh), eluted with CHCl3-MeOH (70:30, 60:40, 50:50, 25:75) and compound fractions of (250 mL each) were collected and monitored by TLC. These column chromatographed compound fractions were further filtered to yield saponins and anthraquinones, which were separated by paper chromatography and preparative TLC to yield saponins [(SM-A (petroleum ether), SM-B (benzene), SM-C (chloroform) and SM-D (ethanol)] and anthraquinones [(RE-A (petroleum ether), RE-B (benzene), RE-C (chloroform) and RE-D (ethanol)] respectively.

All the filtrates obtained were dried by evaporation (Rotometer, 40°C) and the dried extracts were individually once again dissolved in 10 mL ethanol (95%). Then subjected to a complete drying process and weighed according to the AOAC (1990) method. The products obtained were tested initially for antimicrobial activity against different gram +ve organisms (S.aureus, B.subtilis) and gram -ve organisms (E.coliP.vulgaris). Effective antibacterial activity was noted.

Table 1

Antibiotic sensitivity patterns of H pylori strains as determined by E-test (n = 30)

Antibiotic Sensitive n (%) Resistance n (%) Range (μg/mL) MIC1 (μg/mL)
Metronidazole 7 (23.4) 23 (76.6) 0.0125 - > 256 > 8
Clarithromycin 28 (93.3) 2 (6.7) < 0.016 - > 256 > 2
Amoxicillin 28 (93.3) 2 (6.7) < 0.016 - > 256 > 0.5
Tetracycline 28 (93.3) 2 (6.7) < 0.016 - 2 > 4

Results

In the in vitro study, we found that S. mukorossi and R. emodi extracts exert an inhibitory effect on H pylori (Figure (Figure9).9). Ethanol and chloroform extracts of S. mukorossi and benzene and ethanol extracts of R. emodi with various concentrations ranging from 10-200 μg/mL were tested for their effect on H pylori growth by the disk diffusion method. In the in vivo study, we found that the extracts of S. mukorossi and R. emodi were able to clear H pylori infections in the rat models at concentrations of 2.5 mg/mL and 3.0 mg/mL post-infection.
 

Conclusion 

In conclusion, the results presented here indicate that the extracts of S. mukorossi and R. emodi, which were screened for their anti-bacterial activity against H pylori, are active both in vitro and in vivo. In addition, the in vivo studies also proved to be highly efficient in terms of dosage, tolerability and curing active H pylori infection. Future studies will assess the mechanism by which these extracts effect the survival of H pylori.

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