BIOLOGICAL ACTIVITY OF THE INDIGENOUS FRUITS AND VEGETABLES OF THE INDIAN HIMALAYAN REGIONHTML Full Text
BIOLOGICAL ACTIVITY OF THE INDIGENOUS FRUITS AND VEGETABLES OF THE INDIAN HIMALAYAN REGION
Murtaza Gani * and Tanveer Alam
Department of Chemistry, KLDAV PG College Roorkee, Uttarakhand, India.
ABSTRACT: This work aims to evaluate the antimicrobial potential of ethanol, methanol, and water extracts of cherry (Prunus avium) and acetone extracts of quince (Cydonia oblonga), hexane & ethyl acetate extracts of Handh (Taraxacum officinale), acetone & methanol extracts of sustchal (Malva neglecta) of the selected minor fruits and vegetables of Indian Himalayan Region. Agar well diffusion method has been used to determine the antimicrobial activities and minimum inhibitory concentrations (MIC) of different plant extracts against Gram-positive (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two strains of fungus (A. niger, A. flavus). The extracts exhibited both antibacterial and antifungal activities against tested microorganisms. The concentration increase of the extracts resulted in the increase of inhibition zone values resulting in the increase in the antimicrobial activities of the extracts. Both pulp and peel of Quince (Cydonia oblonga) exhibited significant antimicrobial activity. Acetone Quince (Cydonia oblonga) and Handh (Taraxacum officinale) extract showed significant antibacterial activity (P < 0.05) against all tested bacterial strains.
Keywords: Antibacterial, Antifungal, Minor, extracts, Indigenous
INTRODUCTION: Food spoilage is one of the big problems faced by the food industry. The growth of microorganisms predominantly causes food spoilage or deterioration 1. Most of the bacterial strains got resistant to certain antibiotics due to mutations in genes, changes in their structures, and indiscriminate use of antibiotics to treat infectious diseases, which led to most antibiotics' ineffectiveness.
This generated a renewed interest in herbal medicines 2. For the genus Taraxacum, only a few studies concerning its antimicrobial properties have been reported, which is due to the presence of terpenoids, triterpenoids, steroids, coumarins, phenols, saponins, flavonoids, flavones, flavonols, chalcones, phlorotannins and cardiac glycosides in antimicrobial extracts. Still, neither compound isolation nor further identification was performed 3.
In the case of Malva neglecta, the entire plant exhibits therapeutic properties, but in general, the pharmacological effects of Malva are assigned to the leaves and flowers, especially due to the presence of some flavonoids and mucilages in these parts 4. For Cydonia oblonga, studies on antimicrobial activity of different aerial parts of the quince plant demonstrated that its leaf and fruit are effective against gram-positive and gram-negative bacteria 5. The crude extract of quince fruit polyphenols showed antibacterial activity against the Gram-negative bacterium E. coli. For Prunus avium, it has been shown that its extract can exhibit antimicrobial activity against pathogenic oral bacteria 6. In addition, it has been shown that a blend of cherry has effects of natural antimicrobials on the inhibition of Listeria monocytogenes 7. The aim of the present work was to evaluate the microbial activity of the selected minor fruits and vegetables of the Indian Himalayan Region.
MATERIAL AND METHODS:
Antimicrobial Activity: To assess the antimicrobial activity of the extracts, the following microorganisms were used, Staphylococcus aureusfor Gram-(+) bacteria, Escherichia coli, Pseudomonas aeruginosa, Salmonella sp. strain for Gram-(-) bacteria, the yeast Candida albicans and the mould Aspergillus niger, while fungal strains Aspergillus flavus and Aspergillus niger were used for antifungal activity. Susceptibility of the test organism to the extract was determined by employing the standard disk or well diffusion technique 8, 9. Briefly, the bacterial suspension in potato count broth (PCB), adjusted to 0.5 McFarland turbidity and evaluated using a serial 10-fold dilution method, was spread plated on count agar medium (PCA) in order to give a population of 108 colony-forming units (cfu)/ plate.
For the disk diffusion test, sterile paper discs (6 mm) were added of the test sample (20 µL) and placed onto the inoculated agar surface. After cultivation at 37 (bacteria)/ 27 °C (C. albicans) for 24 h or 22 °C for 4-12 days (A. niger), the resulting inhibition zone diameters were measured.
A 100 µL amount of the diluted working extract or pure phenolic solution and 100 µL of the bacterial suspensions (5 × 105cfu/mL) were added in the microwells. The plates were incubated aerobically at 37 °C for 24 h. Bacterial growth was revealed by the presence of turbidity and a pellet on the well bottom. MICs were determined as the first well in ascending order that did not produce a pellet. To confirm MIC and establish MBC, 25 µL of broth was removed from each well and inoculated on PCA plates.
After overnight incubation at 37°C, the number of surviving organisms was determined (MBC was determined when 99.9% of bacteria were dead).
RESULT AND DISCUSSION:
Taraxacum officinale (Handh): The antibacterial effects of the extracts of T. officinale were evaluated against four uropathogenic bacteria (Table 1). Evaluation of the Taraxacum extracts revealed that the Hexane extract inhibited the growth of S. aureus by 85% at 200 µg/mL and was a more effective inhibitor for the Gram-positive than for the Gram-negative strains (Table) 1.
K. pneumoniae was the lowest inhibited by the Hex extracts. EtOAc extract showed low activity against E. coli with 1600 µg/mL and growth inhibition with a 92% value. However, this extract did not exert an antibiotic effect on Gram-positive bacteria S. aureus in the concentrations tested (Table 1). Similar results have been reported where the ethanol extract of leaves of T. officinale had low antimicrobial activity against S. aureus, E. coli, and Salmonella abony 10. In addition, it was observed that as the extract concentration increased, so does the bacteria growth inhibition. This inhibitory effect may be due to phenolic compounds, terpenes, tannins, flavonoids, alkaloids, and/or proteins in the plant extracts. Such compounds had been reported to have an active effect on the bacterial cell membrane, which may destroy these microorganisms 11, 12.
In studying the antipathogenic properties of genus Taraxacum to combat infectious diseases, T. officinale is the most studied species, but it has shown various results depending on the extraction characteristics or on the bioassay performed. Omit lines 32-39.
Micrococcus luteus and Vibrio cholera with MIC values of 1.0 mg/mL and 12.5 mg/mL, respectively, but did not show any activity against S. aureus, Enterobacter faecalis, Enterococcus bacteria, V. cholera, Bacillus subtilis, Pseudomonas aeruginosa, K. pneumonia, or E. coli 13. This work provides preliminary information for the development and use of natural medicine with extracts of T. officinale in the control of disease against uropathogenic bacteria as antimicrobial agents.
TABLE 1: ANTIBACTERIAL ACTIVITY OF N-HEXANE AND ETHYL ACETATE EXTRACT OF TARAXACUM OFFICINALE
|Percentage of growth inhibition (%)/MIC (µg/mL) at 24 h∗|
|Extracts||E. coli||S. aureus||K. pneumoniae||P. mirabilis|
|Hex||69 ± 2.1||85± 3.3||49 ± 0.0||68 ± 0.8|
|EtOAc||92 ± 0.9||0 ± 0.0||-||-|
|Chloramphenicol||89 ± 0.0||-||91 ± 0.0||86± 0.0|
|Streptomycin||-||82 ± 1.1||-||-|
∗Mean of triplicates ± standard deviation of three replicates; (-) not tested.
Malva neglecta (Sustchal): The antimicrobial activities of M. neglecta extracts against different microorganisms were assessed according to inhibition zone diameter. Results are presented in to change Table 2. The acetone and methanol extracts were active on all microorganisms tested with different zone diameters indicating weak (inhibition zone < 12) and moderate antimicrobial activity (inhibition zone < 20-12).
The acetone extracts of M. neglecta showed moderate activity against all microorganisms tested. These results are in lieu of Mansour et al. 13. The most effective reason for the selected plants is the presence of certain phytochemicals like glucosinolates, alkaloids, flavonoids, rhamnose, galactose, galacturonic acid, glucuronic acid, phenolic acids, tannins and volatile oils.
TABLE 2: ZONES OF GROWTH INHIBITION (MM) SHOWING THE ANTIMICROBIAL ACTIVITY OF THE MALVA NEGLECTA
|Inhibition Zone Diameter (mm)|
|Extract||Concentration||E. coli||S. pyogenes||S. aureus||P. aeruginosa||C. albicans|
|30 mg/ml||15±0.5||13 ±0.5||12±0.4||13±0.4||18±0.4|
Values are means ± SD of three separate experiments done in triplicate.
Cydonia oblonga (Quince): The consistent and reproducible results were obtained using the standard disk and well diffusion techniques.
The antimicrobial activity was highest against the Gram-(+) S. aureus and the Gram-(-) P. aeruginosa bacteria, somewhat weaker against the E. coli and the yeast C. albicans, while with the Salmonella sp. and the mould A. niger no inhibition was obtained (Table 3).
S. aureus was the most sensitive microorganism to the extracts examined in this study. Quince pulp extracts showed both bacteriostatic and bactericide activities. MICs and MBCs for peel extracts were found to be equal in (Table 3).
The agar diffusion assay showed that both quince pulp and peel extracts exhibited antimicrobial potentials with clear cut inhibition zones Fig. 1. The diameters of these inhibition zones increased with the concentration of polyphenolic compounds indicating that these molecules were responsible of the antimicrobial effects. The Omit sensitivity to the whole extracts or pure phenolic compounds varied widely. Rodriguez Vaquero et al reported that P. aeruginosa (ATCC 27853) and E. coli (ATCC 25922) were the most sensitive and the most resistant to flavonoid compounds than the other tested bacteria 14.
No inhibition of the mould A. niger growth was obtained at any concentration of the quince extracts in comparison to control culture. This finding may be expected since microbiological analyses of quince jams, which contain mainly the same phenolics as the fruit, revealed that some samples presented many yeasts and moulds 15.
The polyphenols, especially the chlorogenic acid, act in synergism with other components of the extracts to exhibit their total antimicrobial activities.
FIG. 1: S. AUREUS (ATCC6538) GROWTH INHIBITION: 20 AND 100 µL OF THE ACETONE QUINCE PULP EXTRACT WERE USED IN DISK (I) AND WELL (II) DIFFUSION ASSAYS, RESPECTIVELY
TABLE 3: ANTIBACTERIAL ACTIVITY OF QUINCE PULP AND PEEL EXTRACTS
|Microorganism||S. aureus (ATCC6538)||P. aeruginosa
|Technique||Disk Diffusion||Well Diffusion||Disk Diffusion||Well Diffusion||Disk Diffusion||Well Diffusion||Disk Diffusion||Well Diffusion|
|Diameters of the Inhibition Zone (mm)|
|Pulp Extract||16.5 ± 1.3||20.2 ± 1.4||11.4 ± 0.1||16.2 ± 0.4||W||08.2 ± 0.9||N||W|
|Peel Extract||22.3 ± 2.7||27.1 ± 2.6||15.5 ± 0.8||18.3 ± 0.9||08.6 ± 1.0||10.5 ± 1.1||W||09.6 ± 0.|
|Pulp Extract||10 2||5 × 10 2||10 3||5 ×10 3|
|Peel Extract||10 2||10 2||5 × 10 2||5 × 10 3|
|Pulp Extract||5 ×10 2||10 3||5 ×10 3||10 4|
|Peel Extract||10 2||10 2||5 × 10 2||5 × 10 3|
Values are means ± SD of three separate experiments done in triplicate. N= no antimicrobial activity, Ø) 6 mm; W= weak antimicrobial activity, 6 mm < Ø < 8 mm.
Prunus avium (Cherry): The results are shown in Fig. 2. The lowest value of extract that exhibited the MIC80 effect was 0.5 mg/ml (A. haemolyticum) and the highest value for the breakpoint effect was 21.13 mg/ml (S. typhymurium).
Coccia et al., assumed that bactericidal effect was expressed only at concentration higher than double MICs 16 tested bacteria is understandable, considering it is a conventional antibiotic.
Antibiotics, as well as synthetic substances, exhibit a much higher antibacterial effect than natural products, but they also exhibit side effects 17.
We also found that some concentrations of extract exhibit a beneficial effect on bacterial growth. Our study, as well as theirs, showed no antifungal activity 16. The results of the present study show that cherry extract caused MBC, MIC99, MIC90, and MIC80 effects in lower concentrations on E. coli. This coincides with the findings of Kirsch et al., but they used sour cherry methanol extract and water extract 17.
Kołodziejczyk et al., investigated the activity of cherry pomace ethanol extracts against Salmonella choleraesuis and E. coli and showed a reduction of bacteria numbers at doses higher than 2,500 µg/ml.
They noticed that extracts showed bactericidal activity at concentrations higher than 10,000 µg/ml 18.
Omit lines 12-15 MIC99 concentrations for S. enteritidis and E. coli were 5.60 mg/ml and 5.08, while MBC values were 6.07 mg/ml and 8.19 mg/ml, respectively.
FIG. 2: ANTIMICROBIAL ACTIVITY OF CHERRY EXTRACT
CONCLUSION: The scope of the experiments included analysis of the antimicrobial activity of ethanolic, methanolic, ethyl acetate, hexane, acetone, and aqueous extracts against bacterial and fungal cultures and determination of the minimum inhibitory concentration of plant extracts tested microbial growth.
These results suggest that extracts have the potential as antibacterial uropathogenic disease agents. The findings suggest that these native plants have good antibacterial and antifungal properties and can be used for infection control and treatment of various diseases. It can also be a new source for antibiotics discovery and infection treatment.
Further studies are necessary to isolate and characterize the active components of the extractsfractions and also to elucidate their mechanisms of action for this activity.
ACKNOWLEDGMENT: The authors are highly thankful to the Department of Chemistry, KL DAV PG College Roorkee Uttrakhand India for providing facilities of the current work.
Contribution of Authors: We declare that this work was done by the authors named in this article and the authors will bear all liabilities about claims relating to the content of this article.
Funding Statement: The research work did not receive any funding from any source. This work was a part of research work for awarding a Ph.D. degree.
CONFLICTS OF INTEREST: The authors declare that there are no conflicts of interest regarding the publication of this paper.
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How to cite this article:
Gani M and Alam T: Biological activity of the indigenous fruits and vegetables of Indian Himalayan region. Int J Pharm Sci & Res 2022; 13(7): 2697-02. doi: 10.13040/IJPSR.0975-8232.13(7).2697-02.
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