6Departments of Food Technology, School of Applied and Life Sciences (SALS),
Uttaranchal University, Dehradun.
*Corresponding Author E-mail: bhavya.trivedi25@gmail.com
ABSTRACT:
The disc diffusion method, MIC and MBC were used to test the antibacterial activity of methanol, aqueous, ethyl acetate extract and essential oils of Cymbopogon citratus leaves, and Syzygium jambolanum seeds against Escherichia coli and Bacillus cereus, which were isolated from herbal medicines. Methanolic extract of Syzygium jambolanum was highly active against Bacillus cereus and E. coli. Chemical analysis was done by GC- MS. All the plant extracts showed different kinds of phytochemicals like terpenoids, flavonoids and steroids etc.
Graphical Abstract:
KEYWORDS: Cymbopogon, Essential oil, In vitro, Disc diffusion, Phytochemicals etc.
INTRODUCTION:
Herbal medication safety concerns have grown significantly in recent decades as their use has increased. Due to handling by workers who have been exposed to harmful microorganisms during the processes of harvesting, post-harvest processing, and medication production, herbal pharmaceuticals can become contaminated by a variety of pathogenic and non-pathogenic microorganisms both before and after harvest. The microbiological quality of herbal medicines was evaluated by Limyati and Juniar in 1998, Bahri et al. in 2001, and Govender in 2006. Their research revealed that the analysed samples contained both bacteria and fungus1,2,3. In commercial herbal oral medicinal solutions, microbial contamination was investigated by Kulkarni et al. (2010). The microbiological limitations set by the WHO were not met by any of the ten samples of herbal medicinal drink. The limit tests for Pseudomonas could not be passed by Amla juice, Ginger, lemon juice, or Punarwin liquid, While Ginger Lemon drink, Amla juice, and Compound Adulsa syrup all failed the Staphylococcus test, respectively4. Actinomycetes were recovered from ayurvedic medicines by Singh et al. in 20125. Despite the fact that chemical preservatives can lessen these contaminations, repeated exposure to these chemicals may cause bacteria to develop resistance to them, and their accumulation may be harmful to humans6. In order to extend the shelf life of medicines, natural preservatives, which may be derived through plant extracts and are regarded to be safe, swiftly biodegradable, and have antibacterial and antioxidant properties, are currently the main emphasis. Many researchers have shown that plant extract has antibacterial action against different infection7,8,9. Lemon grass's antifungal and antibacterial properties have been examined by Tzortzakis and Economakis (2007) and Oloyede (2009). The plant extracts shown inhibitory actions against a number of pathogens that have been linked to a number of disorders. Researchers evaluated the antibacterial capabilities of medicinal plants10,11,12. Their research focused on the medicinal plants' inhibitory effects on both Gram-positive and Gram-negative bacteria. The MBC ranged between 0.02 and 2.5mg/ml, while the MIC of the crude extracts varied between.01 and 2.5mg/ml. The antibacterial and antifungal activity of medicinal plants was studied by various researchers13,14,15. As a result, the goal of the current study was to determine the chemical make-up of active extract and the antibacterial capacity of medicinal plants.
2. MATERIALS AND METHODS:
2.1 Plant material Collection:
Using herbarium, taxonomic literature and standard flora, aat the library of Gurukul Kangri University, Haridwar, the leaves of Cymbopogon citratus and Syzgium jambolanum seeds were taken from the nursery and identified.
2.1.1. Preparation of plant extract:
An altered extraction procedure was used. The plant materials underwent cleaning, disinfection, rinsing, and room-temperature shade drying lastly13. The samples were milled into a fine powder. In a soxhlet device, 50g of powdered plant material was mixed with distilled water for 72hours, and the mixture was then filtered using a buchner funnel and whatman no. 1 filter paper. Using a rotary evaporator, the filtrate was concentrated to 50ml12. For analysis, the extracts were subjected to refrigeration and kept at a temperature of 4°C. Plant extracts have been developed using water, methanol, and ethyl acetate as the solvents.
2.1.2. Extraction of essential oil:
In a modified Clevenger apparatus, 250grams of the powdered leaves were treated to hydro distillation for 8 hours after being combined with 1000millilitres of distilled water16. Before use, the recovered oil was stored in the refrigerator at 40C and dried over anhydrous sodium sulphate. Following equation was used for calculation yield (%)17.
Weight of extract residue after solvent removal (g)
% Yield of extract = -------------------------------------------------- × 100.2
Weight of dried sample powder (g)
Bacterial strains were isolated from herbal syrups and crude herbs and selected on the basis of their occurrence and pathogenicity. After that isolates were identified up to the species level through 16SrDNA sequencing. Escherichia coli strain KR4.11 and Bacillus cereus strain Fd (GenBank Accession Numbers: KC510288.1 and JQ912539.1, respectively) were the two bacteria that were employed.
Inoculums preparation:
Muller Hinton broth was inoculated with selected microorganisms. The turbidity of the broth was tested against the 0.5 McFarland standard by incubating it at 35–37°C for 2–8hours.
Antibacterial activity through disc diffusion method:
One hundred microliter of viable culture was transferred onto Muller Hinton plate. Utilising the spread plate approach, the culture was infected, and it was left to dry for 5–15 minutes. Plant extracts have been employed to impregnate sterilized paper discs of 6mm fabricated from Whatman Number 1 filter paper. On seeded agar plate, a disc was now positioned. At this point, plates were incubated for 24hours at 37°C after incubation zone of inhibition was measured. A positive control was given by taking streptomycin (10mg/ml). As a negative control, distilled water was used.
To each well, 50 microlitre of inoculum were added. A 96-well microtiter plate was used for the broth dilution test. Each well received the same quantity (150μl) of broth. Now, selected plant extracts were diluted twice in a row in broth with concentrations ranging from 0.15 to 80mg/ml. The final well volume remained 200μl. At 37 °C, the Plate was incubated. MIC of plant extract that prevents microbial growth was identified after incubation. The amount of plant extract (MIC) that prevents microbial development was identified.
Determination of Minimum Bactericidal Concentrations (MBCs) of effective plant extract:
The freshly prepared Mueller Hinton Agar plates were then sub-cultured onto the surface of all the wells utilised in the Minimum Inhibition Concentration study that exhibited no growth of bacteria, and they were incubated in incubators at 37°C for 24hours. The MBC was identified as the lowest concentration of extract that, after the incubation period, prevented any discernible bacterial growth on the agar plate.
Phytochemical screening of Syzygium jambolanum seed extract:
Additionally, GC-MS was used to analyse the chemical composition of plant extracts. The GC-MS Shimadzu MS 2010 instrument with an AB innowax column (60 0.25mm id, film thickness 0.25m) was used to analyse the extracts by GC-MS. After three minutes of keeping the oven temperature at 50°C, the temperature gradually raised to 310°C after 30 minutes, and 0.2 l of the sample was injected for analysis. The carrier gas used was helium. Helium gas was flowing at a rate of 1.2ml/min. Throughout the duration of the experiment, the split ratio was kept at 10, the mass transfer line and sample injector temperatures were both set at 250°C. The energy used for the ionisation mass spectroscopic study was 70 eV. Over the course of 60 minutes, mass spectra were captured in the 40 to 650m/z region.
Identification of compound:
The different constituents were qualitatively identified using computer-aided comparison of their mass spectra with those of computer library searches, such as NIST08, WILEY8, and Flavour and Fragrance Natural and Synthetic Compounds, and validated by comparing the retention indices of the test samples to those of real substances or data from the library.
An analysis of variance (one-way ANOVA) was used to statistically analyse the data. Richard Lowry's web-based ANOVA calculator" was used to do a One-way ANOVA test.
3.1. Out of 36 samples, 20 samples were contaminated with bacteria (64.5%). Identification of bacteria was carried out by Gram staining and biochemical characteristics. The frequency of occurrence of Bacterial isolated is shown in Fig.1, where E. coli and Bacillus cereus was found to be highest occurrence i.e. 32% and 38.7% respectively while by contrast the least occurrence were of Shigella sp. 0.5%, and Salmonella sp. 0.1%. Dominating bacteria were further identified up to the species level by the 16SrDNA technique. The high bacterial counts found in the herbal drugs in this investigation may be the result of poor hygiene, the use of polluted water for washing and preparation, the use of contaminated equipment, and the use of contaminated packaging materials, the personnel who handle the raw material during processing are another potential source of contamination. It has been observed that these bacteria are present in herbal remedies18,19,20,21
3.2 Antibacterial activity through disc diffusion method:
Antibacterial activity of various extracts of Cymbopogon citratus and Syzygium jambolanum against both pathogens has been shown in Fig 2. It was found that the Essential oil oil of C. citratus showed the highest activity (18mm) against Bacillus cereus followed by ethyl acetate extract (1.3mm). A Negligible zone was observed in the case of aqueous extract. While methanolic extract of C. citratus showed the highest activity against E. coli (7.6mm) followed by ethyl acetate (4.6mm) and then essential oil (4.3mm). In case of aqueous extract, no zone was observed. Antibacterial activity of C. citratus have been reported in various studies22,23 .
In case of Syzygium jambolanum, The largest zone diameter was seen in methanolic extract (18.6mm), followed by ethyl acetate (7.6mm) and aqueous extract (5mm), in that order. The activity of the essential oil was the lowest (1.3mm), indicating that B. cereus was resistant to it. In the case of E. coli, methanolic extract (10mm) and ethyl acetate (6mm) had the highest levels of activity. Essential oil and aqueous extract exhibited no action. These findings coincide with prior studies on this plant's antibacterial properties. In practise, Syzygium jambos' bark, leaves, and seeds' aqueous and acetone extracts previously demonstrated antibacterial activity against delicate strains of Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Enterococcus gallinarum Salmonella typhi, Pseudomonas aeruginosa, Vibrio cholera etc24. Additionally, Staphylococcus aureus, Alcaligenes faecalis, Aeromonas hydrophilia and Bacillus subtilis, and were also amenable to the antibacterial consequences of leaf extract in methanol25. The results of the current study can be compared to those of other investigations. The antibacterial effectiveness of S. jambolanum methanolic and ethanolic extracts against Gram-negative and Gram-positive bacteria was investigated by Kothari26. The results showed that both extracts had a wide range of bacteriostatic effects on both Gram-positive and Gram-negative bacteria. The ZOI against E. coli and V. cholerae that were obtained using the methanol extract of S. cumini seeds at 110mg/mL and its ethanol extract at 250mg/mL were of the same diameter. This indicates that methanol extract is 2.27 times more effective against these organisms than ethanol extract. To eliminate an infectious organism a microbicidal agent is required which cannot be identified by diffusion test. Hence dilution susceptibility tests were performed to determine MIC and MBC values. Hence further studies were concluded to determine their MIC and MBC against both microorganisms.
3.3. Determination of Minimum Inhibitory Concentrations (MICs) of effective plant extract:
It was observed that MIC and static growth were found to be the same, while MBC was recorded as the concentration just higher than the concentration which exhibited MIC. For Bacillus cereus, the methanolic extract of S. jambolanum exhibited the lowest MIC (2.5 mg/ml) and MBC (5mg/ml) while MIC and MBC of ethyl acetate extract of C. citratus against Bacillus cereus was 40mg/ml and 80mg/ml respectively.
Figure 2: Comparative analysis of the antibacterial activity of C. citratus and S. jambolanum against test bacterial isolates.
For E. coli, the methanolic extract of S. jambolanum exhibited lowest MIC (5mg/ml) and MBC (10mg/ml) while MIC and MBC of methanolic extract of C. citratus against E. coli was 40mg/ml and 80mg/ml respectively. Results can be comparable to the former studies conducted by various researchers. A very modest inhibitory impact was observed against Escherichia coli at the highest extract concentration utilised (400mg/ml concentration) since Jafari et al. (2012) demonstrated that the growth inhibitory effect of methanol extract of lemon grass leaves on tested Gram-negative bacteria was very low. Lethal concentration of this extract against Bacillus cereus was obtained at 12.5mg/ml27. The active ingredients in it, which also include terpenoids, alkaloids, and steroids, tannins, eugenol, citral, saponin, flavonoids and phenolic compounds may be responsible for this inhibitory action. Hence the chemical compositions of plant extracts were determined by photochemical and GC-MS analysis.
Considering the above antimicrobial activity, Methanolic extract of S. jambolanum was worthy for further investigation. Hence it was analysed by phytochemical test and GC-MS technique to determine its main components. In MSE, total 71 compounds were identified. Major compounds found in this extract Furfural (4.21%), 2 furancarboxaldehyde,5-(hydroxymethyl) (47.20%), Caryophyllene (3.27%), 1,4,8-)-cycloundecatriene, 2,6,6,9-tetramethyl-(4.53%), 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy- 6-methyl-(11.38%), Juniper camphor (2.18%). Other minor phytoconstituents are intermediol (1.44%), hexadecanoic acid (1.32%), beta selinene (1.33), alpha selinene (1.02%) etc. The components which were identified in the present study have various characteristic features. Compounds present in S. jambolanum have the following properties such as 2 furancarboxaldehyde,5-(hydroxymethyl) used in pharmaceuticals., caryophyllene is used as an antitumor, fungicidal, antibacterial., Germacene as flavoring agent, hexadecanoic acid as antibacterial and α pinene as an antifungal agent etc.
4. CONCLUSION:
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Received on 05.08.2023 Modified on 07.12.2023
Accepted on 12.03.2024 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(6):2476-2480.
DOI: 10.52711/0974-360X.2024.00387