Research Article - International Research Journal of Plant Science ( 2023) Volume 14, Issue 4
Received: 01-Aug-2023, Manuscript No. 109481; Editor assigned: 03-Aug-2023, Pre QC No. 109481; Reviewed: 17-Aug-2023, QC No. 109481; Revised: 21-Aug-2023, Manuscript No. 109481; Published: 28-Aug-2023, DOI: http:/dx.doi.org/10.14303/irjps.2023.27
The present study aimed to identify the effect of Carissa roots on its antibacterial potential. Six medically important bacterial strains were selected to identify the antibacterial potential by measuring their zone of inhibitions using agar well diffusion method. Antimicrobial activity of roots has been evaluated against various diseases causing organisms (Escherichia coli, Salmonella typhi, Staphylococcus aureus, Agrobacterium tumefaciens, Erwinia carotovora, Entarococcus faecalis) has shown positive activity against some of the tested extracts. Ethanol and methanol extracts of roots of all the four species have shown very promising activity againt Escherichia coli and Entarococcus faecalis. For ethanol extract the Carissa carandas L. has shown the highest activity against Escherichia coli and Carissa congesta Wight and Carissa spinarum L. has shown the highest activity against Entarococcus faecalis. In methanol extract Carissa carandas L. and Carissa opaca Stapf ex Haines have shown very promising activity againt Staphylococcus aureus. Thus, the study proves significant difference between the antibacterial activities of roots of Carissa species.
Antibacterial activity, Carissa L., Roots, Underutilized.
‘Kasamarda’ had been included in ten drugs which are cordials (Charak Samhita, Su. 4/10). The same had been mentioned in Amalakagana of Sushrut Samhita (Su. 10/26-26). Usage of roots leaves and fruits of Carissa congesta Wight, Carissa carandas L., Carissa opaca Stapf ex Haines and Carissa spinarum L. for number of ailments had been reported in ethnobotany literature (Jain, 1991). Roots are anthelminitic, stomachic and antiscorbutic, and are useful in stomach disorders, intestinal worms, scabies and pruritus (Anonymous, 1993). Roots of Carissa spinarum L. pounded and mixed with country liquor made from Madhuca longifolia flowers are applied on sore for removing the worm, Body pains, cuts, injuries. Paste with cold water is administered orally in fever (Kirtikar and Basu, 1935). Roots of Carissa opaca Stapf ex Haines are purgative, also used in worm infested sores of animals; juice of roots in wounds of cattle (Anonymous, 1976). This review reveals that roots of four species viz. Carissa carandas L., Carissa congesta Wight, Carissa opaca Stapf ex Haines and Carissa spinarum L. are used as medicine. These ethno-medico-botany claims however, needs validations.
Plant Extract
The root material collected was cleaned, dried in shade and grounded in powder form. The powder was stored in air tight bottle. This powder was used for further extraction, cold extraction was done for 12 hours at room temperature and then the extract was filtered through Whatman filter paper. Different solvents, viz. petroleum ether, ethanol 38 and Methanol were used for extraction. The extracts were dissolved in Dimethyl Sulfoxide (DMSO) except for the petroleum ether extract as it is not soluble in DMSO, and therefore dissolved in Hexane. These extracts were used for the antimicrobial assay.
Preparation of Inoculum
Microorganisms were grown in Muller Hinton broth at 37°C to a cell density of approximately 105–106 CFU/ml and used for the antimicrobial activity assay.
Test Organism
For the antimicrobial activity screening, bacterial strains used in this study was Escherichia coli (MCMB-813), Salmonella typhi (MCMB-814), Staphylococcus aureus (MCMB-818), Agrobacterium tumefaciens (MTCC-413), Erwinia carotovora (MTCC-1428) and Entarococcus faecalis (MCMB-812). All these stains were acquired from MACS collection of Microorganisms (MCM) at ARI:
Agar well Diffusion Method This method was adopted to determine the antibacterial activity of root extract against the test organisms.The Muller Hinton Agar (3.8 g/100 ml) weighed and dissolved in 100 ml of distilled water in a sterile conical flask. The medium was sterilized by autoclaving and allowed to cool at 50oC temperature. The medium was poured into the sterile petri plate. The antimicrobial activities of methonal, ethanol and Petroleum ether extracts were determined by agar well diffusion method (Ojala et al., 2000) with slight modification to evaluate the antibacterial activity. Minimum Inhibitory Concentrations (MICs) of the extract and of Carissa was determined using the agar-well diffusion method. All the extracts were diluted in Dimethyl Sulfoxide (DMSO) and hexane to obtain series of concentrations of 100, 40, 20 μg /500μl. The MIC was taken as the lowest concentration of extract that caused a clear to semi-clear inhibition zone around the well after 24 h incubation at 37°C. Sterile petri dishes were prepared with a base layer of Muller-Hinton agar. Bacterial cultures at density of 106-108 cfu (100 μl) were inoculated on solid agar. Wells (6mm) were made in the agar with a sterile cork borer and filled with 50 μl of different dilutions of the extract and fractions. Petri dishes were incubated at 37°C for 24 h. The diameters of the circular inhibition zones obtained were measured. Commercial antibiotics namely Gentamicin and tetracycline were used as positive controls. DMSO 25% and hexane were used as a negative control. The whole experiment was carried out under aseptic conditions in the laminar flow.
The Gram negative bacterial strain S. typhi was found to be one of the high resistant bacterial strains, since only ethanolic extracts of Carissa carandas L. and Carissa spinarum L. at their highest concentration (50 μl) showed an inhibitory zone of 4.5 and 3.3 mm respectively, while S. typhi was highly resistant to Carissa congesta Wight and Carissa opaca Stapf ex Haines which showed no inhibitory zone at other concentrations. In contrast, both Carissa congesta Wight and Carissa opaca Stapf ex Haines exhibited higher zone of inhibiton (4.8 and 4.3 mm respectively) with methanolic extracts. In addition Carissa spinarum L. also showed inhibitory zone of 4.5 mm with a higher concentration of 50 μl, while S. typhi proved to be highly resistant and showed no inhibitory zone with lower concentrations of 10 and 20 μl methanolic extracts of all Carissa sp (Table 1). S. aureus, Gram positive bacterial strain proved to be highly susceptible against alcoholic extracts of all Carissa sp. except for Carissa carandas L. and Carissa spinarum L., each at 10 μl. Besides, the highest zone of inhibition (6.8 μl) was observed with Carissa carandas L. extract at 50 μl. Similarly, all methanolic extracts showed higher zone of inhibition against S. aureus except for Carissa opaca Stapf ex Haines at 10 μl concentration. Also highest inhibitory zone was observed (9 mm) with Carissa carandas L. and Carissa opaca Stapf ex Haines extract at 20 and 50 μl respectively.
| Extract | Strains used | C. car | C. con | C. opa | C. spi | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Concentrations | Concentrations | Concentrations | Concentrations | ||||||||||
| 10 µl | 20 µl | 50 µl | 10 µl | 20 µl | 50 µl | 10 µl | 20 µl | 50 µl | 10 µl | 20 µl | 50 µl | ||
| Zone of Inhibition (mm) | Zone of Inhibition (mm) | Zone of Inhibition (mm) | Zone of Inhibition (mm) | ||||||||||
| CRE | E. coli | 3.75 | 4.75 | 9.5 | - | 6 | 7 | 2 | 5.25 | 8.5 | 3.75 | 4.25 | 8.25 |
| S. typhi | - | - | 4.5 | - | - | - | - | - | - | - | - | 3.75 | |
| S. aureus | - | 4.5 | 6.75 | 5.25 | 5.25 | 4.75 | 6.5 | 6.25 | 4.25 | - | 4.75 | 3.75 | |
| A. tumefaciens | 3.5 | 5.25 | 6.75 | - | 5.75 | 5.5 | 5.75 | 5.25 | 6.25 | 5 | 4.75 | 4 | |
| E. carotovora | - | 4.5 | 5 | 3.25 | 4.5 | 4.5 | 3.75 | - | - | 3.5 | 5.25 | - | |
| E. faecalis | - | 3.5 | 5.25 | 5 | 5.25 | 7.75 | 4.75 | 4.5 | 6.25 | 6.5 | - | 7.25 | |
| CRM | E. coli | - | 5.5 | 5.25 | - | - | 4 | - | - | 4 | - | - | 5.75 |
| S. typhi | - | - | - | - | - | 4.75 | - | - | 4.25 | - | - | 4.5 | |
| S. aureus | 4 | 9 | 7 | 4.75 | 7 | 6.25 | - | 8.5 | 9 | 3.75 | 6.75 | 5.5 | |
| A. tumefaciens | 3.5 | 4.25 | 5.75 | - | 3.25 | 4.75 | 4.5 | 5 | 6 | 4 | 4.25 | 5.25 | |
| E. carotovora | 7 | 5.75 | 5.25 | - | 4.25 | 4.75 | 5 | 3.5 | 4.75 | - | 3.5 | 4.75 | |
| E. faecalis | - | - | 5.25 | - | - | 5.75 | - | - | 4.5 | - | - | 4.75 | |
| Standard Tetra cyclin 30 µg |
E. coli | 8.5 | |||||||||||
| S. typhi | 15 | ||||||||||||
| S. aureus | 15 | ||||||||||||
| A. tumefaciens | 12 | ||||||||||||
| E. carotovora | 10 | ||||||||||||
| E. faecalis | 10 | ||||||||||||
Among all the alcoholic extracts tested against Agrobacterium tumefaciens, the results showed greater susceptibility with higher zone of inhibition using 50 μl extract of Carissa carandas L., while no inhibitory zone was observed using Carissa congesta Wight at 10 μl. Similarly, methanolic extract of Carissa congesta Wight at 10 μl showed no inhibitory zone, while Carissa opaca Stapf ex Haines at 50 μl showed a inhibition zone of 6 mm.
Gram negative bacterial strain Erwinia carotovora was recorded to be highly susceptible against most of the alcoholic extracts tested, except for extracts of Carissa carandas L. (10 μl), Carissa opaca Stapf ex Haines (20 and 50 μl) and Carissa spinarum L. (50 μl), while extract of Carissa spinarum L. at 20 μl showed highest inhibitory zone of 5.3 mm. However methanolic extract of Carissa carandas L. at 10 μl demonstrated highest inhibitory zone of 7 mm, while extracts at other concentrations of Carissa sp. exhibited some inhibition zone except for Carissa congesta Wight and Carissa spinarum L. at 10 μl each.
Enterococcus faecalis, the Gram positive bacterial strain showed remarkable difference between the alcoholic and methanolic extracts used. Alcoholic extract of Carissa sp. showed highest zone of inhibition of 7.8 mm with 50 μl extract of Carissa congesta Wight, while no inhibitory zone was observed with 10 and 20 μl extract Carissa carandas L. and Carissa spinarum L. respectively. In contrast, the methanolic extract at 50 μl concentration inhibited the growth of Enterococcus faecalis at the highest concentration tested (50 μl) using all Carissa sp. tested, while other extracts of all species at lower concentrations showed no activity.
When compared to the different polar system extracts used, alcoholic extracts proved to be the more effective than methanolic extracts. Among all the Carissa sp. used, Carissa carandas L. showed higher inhibition against five bacterial strains, while Carissa congesta Wight, Carissa opaca Stapf ex Haines and Carissa spinarum L. showed higher inhibition against only two bacterial strains used. Bacterial strains, E. coli and S. aureus both were highly susceptible, while bacterial strain S. typhi was the most resistant bacterial strain. The lowest concentration at which the only bacterial strain that showed highest zone of inhibition (7 mm) was Erwinia carotovora with 10 μl methanolic extract of Carissa carandas L.
For ethanol extract the Carissa carandas L. shows the highest activity against Escherichia coli and Carissa congesta Wight and Carissa spinarum L. shows the highest activity against Entarococcus faecalis. In methanol extract Carissa carandas L. and Carissa opaca Stapf ex Haines showed very promising activity againt Staphylococcus aureus.
Dhanushka (2006) demonstrated activity of compounds volatile 2’-hydroxy acetopheneone, lignan carinol and eudesmane sesquitepene carissone from Carissa lanceolata root against Gram positive and Gram negative organisms viz. E. coli, S. aurues, B. substilis and P. aeruiginosa. The results obtained in present investigation are more or less similar to above mentioned reference. Presence of carissone and carinol have been reported in roots of Carissa congesta Wight (Joshi and Boyce, 1957; Morton and Miami, 1987); in Carissa carandas L. (Rastogi et al., 1966, 1967; Satyavati (1987); Bhaduri et al., 1968; Hettiarachchi (2006); Jagadeeshwar Rao (2005); Naim et al., 1988; Wangteeraprasert & Likhitwitayawuid, 2009); Warrier (1993), in Carissa spinarum L. (Rao et al., 2005). The antimicrobial activity therefore, may be due to presence of compounds carissone, carinol in respective species.
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