In vitro evaluation of the antimicrobial activity of methanolic and aqueous extract of Acorus calamus L. and Tinospora cardifolia L.
Shobha KL1, Prathibha Maria D Almeida2*, Sreedhara R Pai3
1Professor and coordinator, Division of Microbiology, Department of Basic Medical Sciences,
Manipal Academy of Higher Education, Manipal, Karnataka, India.
2Associate professor, Division of Physiology, Department of Basic Medical Sciences,
Manipal Academy of Higher Education, Manipal, Karnataka, India.
3Professor, Department of Pharmacology, Manipal College of Pharmaceutical Sciences,
Manipal Academy of Higher Education, Manipal, Karnataka, India.
*Corresponding Author E-mail: prathibha.almeida@manipal.edu
ABSTRACT:
Medicinal plants are used as therapeutic agents as well as nutritive supplements by traditional practitioners. There is need for the study to substantiate the use of herbs in combating diseases caused by microbes. Hence study has undertaken to identify the antibacterial antifungal effect of aqueous and methanolic extract of Acorus calamus [AC] rhizome and Tinospora cardifolia [TC] stem. Results: At 100 μg/ml concentration, methanolic extract of TC and methanolic extract AC and aqueous extract of AC have shown the zone of inhibition observed against Staphylococcus aureus (ATCC 25923) was 18mm,19mm 15mm; Escherichia coli (ATCC 25922) was 24mm,20mm 14mm; Pseudomonas aeruginosa (ATCC 27853) 19mm, 16mm, 13 mm; Candida albicans (ATCC 90028) 7mm,10mm, respectively. But, aqueous extract of TC did not show zone of inhibition against any of above mentioned microbes. Both the AC rhizome and TC stem extracts did not show any zone of inhibition to MDR strains. Conclusion: Study shows that both aqueous and methanolic extracts of AC are effective antimicrobial effect whereas only the methanolic extract of TC has shown the antimicrobial effect. But both the extracts of AC and TC were not effective against MDR strain. Study substantiates the use of Acorus calamus [AC] rhizome, Tinospora cardifolia [AC] against the infectious diseases by traditional practitioners.
KEYWORDS: Acorus calamus L., Tinospora cardifolia L., Antimicrobial activity, Zone of inhibition, Methanolic extract, Aqueous extract.
INTRODUCTION:
Ayurveda claims that the rhizomes of AC can be used in improving memory power, intellect and due to its strong aroma, it is used in the treatment of epilepsy. It is also used to prepare aromatic oils. It is a strong nasal stimulant, has emetic property, used as an expectorant, aphrodisiac. It produces laxative and diuretic effect, relives spasmodic pains, colic, and anthelmintic effect 3. Traditional practitioners’ advice daily application a small drop of paste of AC rhizome on new-borns tongue which helps to improve the speech and gives antiepileptic effect, reduces the fever 4,5.
Recent studies have shown that the essential oil prepared from rhizomes have antifungal6, antibacterial effect against E. coli7, anti-cellular and immunosuppressive effects8. Studies also established the presence of active compounds in rhizome are safrol 9 ,10, alpha and beta -asarone 11. Studies have shown that, beta and alpha-asarone have genotoxic and mutagenic effect if they are used in high dosages12.
Tinospora cordifolia [TC] of family Menispermaceae13. Its common names are Amritha, Guduchi, Giloe. TC is a climber, stem is grey to brown-black in colour and bitter in taste 14, stem shows cartwheel appearance when observed in transvers section. This character help in identification of the stem. All the parts of TC like the leaves, stem, and roots, have shown healing and remedial properties. In Ayurveda, TC is considered as one among the rasaynas [ provides the general health]. Various formulations prepared out of TC used to treat chronic fever, respiratory disturbances, improves appetite, fall in blood glucose concentration and in treatment of liver disorders14. TC is well studied for its medicinal benefits, that establishes its usage against diabetes, inflammations, arthritis, allergies, stress and reactive oxygen species 15.
Studies have shown that, many chemicals have been isolated from TC16, Stem of TC contain alkaloid, Berberine, Palmatine, glycoside, 18-norclerodane glucoside, Furanoid diterpene glucoside, steroid- Hydroxy ecdysone, sesquiterpenoid, Tinocordifolin16 also contain immunomodulatory protein17. TC was found to have the compounds due to which it shows the immunomodulatory effect18. At higher concentrations the active components have shown to increase phagocytosis activity in neutrophils, decreased the production of superoxide and increased the antibody production in B cells18 additionally, α-d-glucan, a compound isolated from the stem found to kindle the immune system by stimulation of TLR6 signalling mechanism, translocation of NF-κB and cytokine production in macrophages. 19,20.
Further studies required to substantiate the antimicrobial activity of medicinal plants, to identify a potent agent that can deal effectively with increasing antimicrobial resistance. These studies also provide a potential source for the development of newer therapeutic strategies for controlling infections and hence present study has been undertaken.
Antimicrobial activity of methanolic extract and aqueous extract of Acorus calamus rhizome and Tinospora cardifolia stem were used against various antimicrobial strains such as Staphylococcus aureus (ATCC 25923) Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Candida albicans (ATCC 90028) and clinical strain of multidrug resistant Escherichia coli.
MATERIALS AND METHODS:
Collection of Acorus calamus rhizome and Tinospora cardifolia stem:
Both the medicinal plants were collected from herbal garden of department of Pharmacology. After identification and confirmation of the plant species by the botanist, the samples were placed in the herbarium of department of Pharmacology. Stem of Tinospora cardifolia and rhizome of Acorus calamus were cleaned well and allowed to shade dried for 15 days or till they become brittle. Then both the samples were pounded into powders separately and preserved them separately using airtight containers till they are used for extract preparation21.
Methanolic extract preparation:
Dry powder of Tinospora cardifolia stem and Acorus calamus rhizome was continuously refluxed with methanol using soxhlet apparatus at 40ºC-50ºC for 72 hours. The solvent extract was then stored in airtight container at 4ºC till further use21.
Aqueous extract preparation:
Fresh stems of Tinospora cardifolia [TC]and Acorus calamus [AC] rhizome were collected, cleaned and sunshade dried for a period of 15 days. After drying, samples were made into small pieces and ground into powder form. To prepare an aqueous solution one part of dry powder was mixed 10 parts of distilled water [1:10 ratio]. Mixture cooked using a low flame till it reduced to one forth. Mixture was allowed to cool. The supernatant was allowed to centrifuge about 3000 [5 minutes]. Thus, obtained supernatant was evaporated using water bath to get a thick extract. Extract was stored in a desiccator till the usage 21.
Antimicrobial activity:
Antibacterial as well as antifungal susceptibility study was performed using Agar well diffusion technique 15. Antimicrobial strains used in the present study to be determined antibacterial susceptibility are; The American Type Culture Collection (ATCC) strains of bacteria used were Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Candida albicans (ATCC 90028). To study multidrug resistance clinical strain of multidrug resistant Escherichia coli was used. Procedure of the experiment was followed as per previous study21.
Antibacterial activity:
About 20ml of Mueller-Hinton Agar (MHA) was transferred into Petri dishes. over the surface of the MHA plate spread the bacterial culture used for this study. Test procedure was followed as per the previous study 21. Tests repeated for three times and the average value obtained from the three tests was considered for the study21.
Antifungal activity:
Antifungal activity studied by spreading Candida albicans organism over the surface of the SDA plate which contain 20ml of SDA in each petri dishes. Test procedure was followed as per the previous study 21. Tests repeated for three times and the average value obtained from the three tests was considered for the study21.
RESULTS:
Antimicrobial effect of the extracts was studied by determining the distance of the zone of inhibition. Methanolic extract Tinospora cardifolia [TC] stem alone has shown antimicrobial effect whereas methanolic as well as aqueous extract of Acorus calamus [AC] rhizome has shown antimicrobial effect. It was observed that maximum diameter of zone of inhibition was in methanolic extract of TC stem and methanolic and aqueous extracts of AC rhizome when used at concentration of 100 μg/ml. Aqueous extract of TC did not show any zone of inhibition
The methanolic extract of TC, aqueous extract and the methanolic extract of AC had antibacterial activity against all the ATCC bacterial strains as given in Tables 1, 2 and 3.
Table 1: Diameter of zone of inhibition observed in methanolic extract of Tinospora cordifolia when used against various organisms
|
Name of the organism |
100μg/ml |
75μg/ml |
50μg/ml |
25μg/ml |
10μg/ml |
|
Staphylococcus aureus (ATCC 25923) |
18 mm |
17mm |
10mm |
7mm |
- |
|
Escherichia coli (ATCC 25922) |
24mm |
22mm |
18mm |
10mm |
7mm |
|
Pseudomonas aeruginosa (ATCC 27853) |
19mm |
19mm |
16mm |
8mm |
- |
|
Escherichia coli MDR strain |
- |
- |
- |
- |
- |
|
Candida albicans ATCC 10231 |
7mm |
- |
- |
- |
- |
MDR: Multidrug resistant strain. (-) : Indicates no zone of inhibition.
Table 2: Diameter of zone of inhibition of aqueous extract of Acorus calamus when used against various organisms
|
Name of the organism |
100μg/ml |
75μg/ml |
50μg/ml |
25μg/ml |
10μg/ml |
|
Staphylococcus aureus (ATCC 25923) |
15mm |
13mm |
10mm |
8 mm |
- |
|
Escherichia coli (ATCC 25922) |
14 mm |
13mm |
9mm |
- |
- |
|
Pseudomonas aeruginosa (ATCC 27853) |
13mm |
10mm |
8mm |
- |
- |
|
Escherichia coli MDR strain |
- |
- |
- |
- |
- |
|
Candida albicans ATCC 10231 |
- |
- |
- |
- |
- |
MDR: Multidrug resistant strain. (-): Indicates no zone of inhibition.
Table 3: Diameter of zone of inhibition of methanolic extract of Acorus calamus when used against various organisms
|
Name of the organism |
100μg/ml |
75μg/ml |
50μg/ml |
25μg/ml |
10μg/ml |
|
Staphylococcus aureus (ATCC 25923) |
19mm |
17mm |
15mm |
14mm |
8mm |
|
Escherichia coli (ATCC 25922) |
20mm |
19mm |
16mm |
13mm |
7mm |
|
Pseudomonas aeruginosa (ATCC 27853) |
16mm |
15mm |
13mm |
9mm |
- |
|
Escherichia coli MDR strain |
- |
- |
- |
- |
- |
|
Candida albicans ATCC 10231 |
10mm |
9mm |
- |
- |
- |
MDR: Multidrug resistant strain. (-): Indicates no zone of inhibition
DISCUSSION:
Present years research is aimed to produce effective medication that have less side effects and cost effective. These days plants are the source of inspiration for preparation of novel drug compounds in the developing countries. The drugs derived from plants contribute significantly towards human health as they are used as therapeutic as well as nutritive supplements. Their antibacterial, antifungal, and antiviral activity is significant22. The antibacterial effect against both gram positive and gram-negative bacteria, suggests that the plant is the good a source of the bioactive substance which has broad spectrum of activity23. Drugs derived from plants were reported to be safe and without effects 24,25 hence, the natural products are given more attention as new healing agents26. Therefore, extract and the biologically active compounds isolated from the plants have gained importance in the recent days.
Our study showed methanolic extract of Tinospora cordifolia [TC] and aqueous extract and methanolic extract of Acorus calamus [AC] were effective in inhibiting the test pathogens - Staphylococcus aureus (ATCC 25923) Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Candida albicans (ATCC 90028), whereas, aqueous extract of TC did not have any antimicrobial activity. Study done by Ezhilarasu et al 27 was in concordance with our study showed no zone of inhibition against aqueous extract. There was no zone of inhibition was observed in TC stem and AC rhizome extract against MDR strains. Study done by Nath H et al 28 using extracts of TC against Pseudomonas aeruginosa showed antibacterial effect which vouches our study. In the present study, it was observed that methanolic extract had a better antimicrobial activity than aqueous extract of AC rhizome, and only methanolic extract of TC having antimicrobial activity. This substantiates the use of Amritha arista, a fermented preparation of TC is effectively used in traditional medicine for treating chronic fever, respiratory disturbances and as an appetiser. Oils prepared out of AC rhizome used in the treating rhinitis, chronic sinusitis, headache and stuffy nose. Study done by Rose MF et al 29 revealed the presence active phytocompound in hot and cold methanol extracts of TC roots have shown the antioxidant property30,31,32. AC rhizome contains phenyl propanoids, sesquiterpenes and monoterpenes as well as xanthone, glycosides, flavones, lignans, steroids alkaloids and inorganic constituents33. Methanolic extract and alkaloid isolated from rhizomes of AC showed anti-cancer activity when tested on onion cell and yeast cell models34. Also, alcoholic extract and alpha- asarone, an active principle of AC rhizome has showed neuro protect effect in restrained stressed rats 35.
Further work can be carried out on the isolation procedure for finding out the exact phytocompound responsible for the antimicrobial activity of TC and AC.
CONCLUSION:
Study shows that both aqueous and methanolic extracts of AC are effective antimicrobial effect whereas only the methanolic extract of TC has showed the antimicrobial effect. But both the extracts of AC and TC are not effectives against MDR strain. Study substantiates the use of Acorus calamus [AC] rhizome, Tinospora cardifolia [AC] against the infectious diseases by traditional practitioners.
CONFLICT OF INTEREST:
Authors have no conflict of interest.
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Received on 17.04.2024 Revised on 11.07.2024 Accepted on 18.09.2024 Published on 24.12.2024 Available online from December 27, 2024 Research J. Pharmacy and Technology. 2024;17(12):5991-5995. DOI: 10.52711/0974-360X.2024.00909 © RJPT All right reserved
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