In vitro Assessment of Antimicrobial potential of Siddha Polyherbal Formulation Tulasi oil against RTI pathogens
S. Yavanarani1*, R. Selvakumar2*
1Yavanarani Siddha Clinic, Pillaiyarpalayam, Kanchipuram, Tamilnadu, India - 631501.
2Siddha Government Hospital, Tiruttani, Tiruvallur, Tamilnadu, India – 631209.
*Corresponding Author E-mail: eshaayy@yahoo.com
ABSTRACT:
In Siddha medication, along with herbal formulations, some mineral formulations are used clinically to prevent or cure infectious diseases. The therapeutic values of some siddha formulations have been well documented earlier, but a huge number of them remain unexplored in terms of safety and efficacy. The aim of this study was to screen the antimicrobial potential of Siddha polyherbal formulation Tulasi oil (SPHTO) against respiratory tract infection (RTI) causing pathogens. The antimicrobial activity of SPHTO was examined by using the methods of agar well diffusion, Minimum inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), Minimum Fungicidal Concentration (MFC) and Microbicidal action. Ciprofloxacin (bacteria) and Amphotericin B (fungi) were used as the positive control for this study. The potassium leakage was determined using a flame emission and atomic absorption spectroscopy. All tested concentrations gave notable antimicrobial activity against all tested pathogens. The 100mg/ml concentration showed the highest activity against E.coli followed by 200mg/ml against K. pnuemonia and C.neoformans when compared to 50 and 25mg/ml. For MIC, the highest activity was registered at 150mg/ml concentration against E. coli, while MBC/MFC was 200mg/ml concentration has shown the better activity against K. pneumonia and C. neoformans. Tulasi oil induced leakage of potassium ions from cells of E.coli and K.pneumonia suspended in glycil-glycine buffer solution and the leakage started within 10 minutes of contact of drugs with target cells. From this study, it may support the clinical claims of Tulasi oil to have potent antibacterial and antifungal activities and offer profound therapeutic benefits against respiratory tract infection.
KEYWORDS: Tulasi oil, Siddha polyherbal formulation, Antimicrobial, MIC, K+ leakage.
INTRODUCTION:
Infectious diseases are a leading cause of illness and death in humans at global1. In recent days, the emergence of number of multidrug resistance microbial strains and the strains with low sensitivity to antibiotics continue to increases. This increase is due to the improper functioning of broad-spectrum antibiotics, immunosuppressive agents, intravenous catheters, organ transplantation and the epidermis of human immunodeficiency virus infections2,3.
Synthetic drugs are not only expensive but also inadequate to treat diseases, but they are often accompanied by impurities and side effects. Hence, there is a need to search for new infection-fighting strategies to control microbial infections4. Finding new sources of antibiotics is a global challenge for research institutes, pharmaceutical companies and educational institutions as many infectious agents are resistant to synthetic drugs5. Traditional Systems of medicines are constantly performing a crucial role in meeting the global health care needs. India has various recognized systems of medicine like, Siddha, Ayurveda, Unani, Yoga, Naturopathy, Homoeopathy and Sowa Rigpa. Among them Siddha is a unique medicine originated from the state of Tamil Nadu, which claims to cure the root cause of diseases by maintaining the ratio of Vatham, Pitham and Kapham. The origin of the Siddha system of medicine is attributed from ancient saint called Siddhars6.
Thulasi oil (TO) is a polyherbal formulation prescribed in the management of childhood asthma. Literature review of the ingredients of Tulsai oil revealed that the drugs are having good expectorant, analgesic, anti-allergic, antidiabetic agent, anti inflammatory, antioxidant, antidote, anti-pyritic, carminative and antimicrobial agent7. It contains various phytoconstituents i.e., Eugenol, Naphthalene, Oleic acid and Ricinoleic acid derivatives which possess the actions of broncho dialotor, anti-inflammatory, anti-tussive, anti-asthmatics, anti-oxidant, anti-viral, antibiotics etc8. Since not much research had been done to evaluate the biological activity of the Tulasi Oil, the aim of the present study was to assess the antimicrobial activity against clinically isolated RTI pathogens.
MATERIAL AND METHODS:
Preparation of drug:
The ingredients; Ricimus communis (352ml), Ocimum sanctum (160ml), O. Prostratum (160ml), O. canum (160ml), Taxus buccata (160ml), Aegle marmelos (160 ml), Allium cepa (160ml), Zingiber officinale (8g), Piper longum (8g), Piper nigrum (8g) were powdered separately and mixed well together then taken in a tightly closed container. This powder was mixed with honey (160ml)7. The medicine will be prepared in Gunapadam lab of National Institute of Siddha after proper purification. All the ingredients mentioned in the formulation are purified as per the direction described in the Siddha literature.
352ml of Ricimus communis oil was mixed with the juice of 160ml of Ocimum sanctum, O. prostratum, O. canum, Taxus buccata, Aegle marmelos, Allium cepa in a mud vessel. 8gms of dried ginger, long pepper, black pepper were fried them and make them in fine powder and this powder was mixed with 160ml of honey and heat it till muster form appears and filter it. The filtrate was mixed with TO and stored in clean and dry glass container7.
Microorganisms used:
The RTI bacteria, Streptococcus pneumonia, Staphylococcus aureus, Escherchia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, and fungi, Aspergillus niger, Candida albicans, Cryptococcus neoformans, Mucor sp were used for the experiment. The bacterial strains were maintained in Nutrient agar slants (Hi-media Laboratories Pvt. Ltd., Mumbai) and the strains were allowed to grow for 24hours and stored at 4°C for further studies. The fungal strains were maintained on Potato dextrose agar slants (Hi-media Laboratories Pvt. Ltd., Mumbai) and the strains were allowed to grow for five days and stored at 4°C for further studies.
Antibacterial activity:
Agar well diffusion method:
The antimicrobial activity of the Tulasi oil was determined using the agar-well diffusion method with slight modification as described by Priyanka et al9. Sterile Mueller Hinton agar plates were flooded with appropriately diluted organism (inoculum was adjusted according to McFarland standards, 0.5%), and allowed to drying at room temperature. After drying, the wells were made in the culture seeded plates with the help of a sterile well borer (6mm diameter). After 25, 50, 100 and 200mg/ml concentrations of Tulasi oil were filled into separate wells. Ciprofloxacin (15mg/ml) and Amphotericin B (15mg/ml) were used as a positive control. The plates were allowed a pre-diffusion time of 1h at room temperature and then incubated at 37°C for 24h after which zones of inhibition were read to the nearest millimeter. Each experiment was carried out in triplicates.
Minimum inhibitory concentration (MIC):
Minimum inhibitory concentration of Tulasi oil extracts were in Mueller Hinton broth for bacteria by Broth Dilution method10. The drug concentrations ranged from the Tulasi oil were 400, 350, 300, 250, 200, 175, 150, 125, 100, 75, 50 and 25mg/ml of standardized suspension (0.5% Macfarland turbidity) of the test organism was transferred into each tube. The control tube contained only organism and devoid of Tulasi oil. The culture tubes were incubated at 37°C for 24h. The lowest concentrations which did not show any growth of tested organism after microscopic evaluation were determined as MIC. The assay was repeated three times.
Minimum Bactericidal and Fungicidal Concentration (MBC/MFC):
The MBC/MFC of the Tulasi oil were determined by Anuradha et al11 plating a loopful of bacterial sample from each MIC assay tube with growth inhibition into freshly prepared MH broth and the plates were incubated at 37°C for 24hr. The MBC values were recorded as the lowest concentration of the extracts that did not permit any visible bacterial colony growth on the agar plate during the period of incubation. The assay was repeated three times.
Potassium leakage:
The highly sensitive pathogens i.e., E. coli and K. pneumonia and from antimicrobial studies were selected for microbicidal mechanism studies. Potassium leakage was determined as previously described by Rodriguez et al12 with a minor variation. In brief, the pathogens were cultured on Trypticase soy agar and incubated at 370C for 12-24hours. The pathogens washed with Ringer’s solution, three times and they were centrifuged at 5000 rpm for 30min at 150C. The pellets were resuspended in 25ml of 1mM glycil-glycine buffer solution, pH 6.8 to obtain a cell density of 3.0X 108cfu/ml. The pathogens were treated with the tulasi oil at the MIC and at 1.5 X MIC for 30min. Samples (5ml) of cell suspension were removed at 10, 25, 50 and 100min, diluted and filtered through a 0.2µm pore-size membrane to remove bacteria. Test drug free controls were prepared in the same conditions to determine normal pottasium flux over the time course of the experiment. Thermal treatment was conducted by incubating the cell suspension in a water bath at 70°C. The potassium concentration in the supernatant was measured using an atomic absorption spectrophotometer.
Statistical analysis:
Results were represented as mean ± standard error. One-way analysis of variance was used for statistical significance of intergroup differences and the Duncan Multiple Range Test (DMRT) was used for comparison of means. The Statistical Package for Social Sciences (SPSS) version 16.0 was used to perform all analysis.
RESULTS:
Agar Well diffusion method:
The Tulasi oil revealed the highest inhibitory activity against E.coli (25.39±0.10 mm) at the concentration of 100 mg/ml followed by S. pneumoniae (16.8 ±0.33mm) and C. neoformans (18.3 ±0.11 mm) at the concentration of 200 mg/ml and the minimum amount of inhibitory activity was observed against P. aeruginosa and Mucor sp (3.8±0.05 and5.7 ±0.07 mm, respectively) at 200 mg/ml concentration tested. The test concentration of 25 mg/ml did not exhibit any antimicrobial activity against P. aeruginosa (Fig 1).
Minimum inhibitory concentration:
The MIC of Tulasi oil was screened against pathogenic bacteria and the results are presented in Table 1. The E.coli showed MIC at 150 mg/ml whereas, for C.neoformans it was 200 mg/ml and for S. aureus and S. pneumoniae, the MIC was at 250 mg/ml. The least value of MIC was registered in P. aerogenosa and Mucor sp at 350 mg/ml.
Minimum Bactericidal concentration:
The MBC/MFC of Tulasi oil was screened against RTI pathogens and the results are presented in Fig 2. The E.coli showed MBC at 200 mg/ml whereas; for K. pnemoniae was registered the MBC level of 250 mg/ml. Tulasi oil has registered the least value of MBC/MFC against P. aeruginosa and Mucor sp (400 mg/ml).
Fig 1: Antimicrobial activity - plate assay Fig 2: MBC/MFC potential of Tulasi oil
Values represented mean ± SE of three independent experiments with three replicates each
Table 1: MIC of Tulasi oil against tested RTI pathogens
|
Drug conce. (mg/ml) |
S. aureus |
S. pneumoniae |
E coli |
K.pnemoniae |
P. aerogenosa |
C.albicans |
C.neoformans |
Mucor sp |
|
25 |
GS |
GS |
GS |
GS |
GS |
GS |
GS |
GS |
|
50 |
GS |
GS |
GS |
GS |
GS |
GS |
GS |
GS |
|
75 |
GS |
GS |
GS |
GS |
GS |
GS |
GS |
GS |
|
100 |
GS |
GS |
GS |
GS |
GS |
GS |
GS |
GS |
|
125 |
GS |
GS |
GS |
GS |
GS |
GS |
GS |
GS |
|
150 |
GS |
GS |
NG |
GS |
GS |
GS |
GS |
GS |
|
175 |
GS |
GS |
-- |
GS |
GS |
GS |
GS |
GS |
|
200 |
GS |
GS |
-- |
NG |
GS |
GS |
NG |
GS |
|
250 |
NG |
NG |
-- |
-- |
GS |
NG |
|
GS |
|
300 |
-- |
-- |
-- |
-- |
GS |
-- |
-- |
GS |
|
350 |
-- |
-- |
-- |
-- |
NG |
-- |
-- |
NG |
|
400 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
GS- growth seen NG- No growth
Table 2: Effect of tulasi oil on potassium leakage in RTI pathogens (mm/l)
|
Time interval (Min) |
Pottasium leakage (mm/l) |
|
|
E.coli |
K.pneumonia |
|
|
10 |
1.5±0.04 |
1.4±0.05 |
|
25 |
4.2±0.05 |
3.8±0.02 |
|
50 |
5.1±0.06 |
4.3±0.07 |
|
100 |
6.9±0.05 |
6.1±0.04 |
Values represented mean±SE of three independent experiments with three replicates each
Microbicidal action:
Different concentrations of tulasi oil have registered potassium ions leakage from E. coli and K. pneumonia at dose dependent manner. The lowest leakage of pottasium i.e., 2.6mm/l and 3.3mm/l of potassium leaked from E. coli and K. pneumonia at 10 minutes interaction. The maximum leakage of pottasium was recorded at the time 100minutes i.e., 6.9±0.05 and 6.1±0.04 against two bacteria tested (Table 2).
DISCUSSION:
The development of resistance against currently available antibiotics creates the need to rediscover new antimicrobial agents in traditional medicine. Siddha medicine is considered to be the oldest form of medicine known to mankind. There are plentiful medicinal plants and traditional formulas are available in Siddha system to treat various human ailments because they contain elements of therapeutic value13-18. The results obtained show that as expected all the extracts, exhibited a level of antimicrobial activity which generally increased with increasing concentration. The degree of antibacterial activity varied according to the concentrations and the type of bacteria.
In the present investigation, Tulasi oil has shown good antimicrobial activity against all tested pathogens. All tested concentrations showed considerable antimicrobial activity but 200mg/ml has shown the better activity followed by 100mg/ml when compared to 50 and 25 mg/ml. The highest antimicrobial activity of Tulasi oil was registered against E. coli followed by K. pneumonia and C. neoformans. These results evidently indicate that among the four concentrations were used for the study, the activity of 200 and 100mg/ml were registered excellent activity. This variation in the effectiveness of the different extracts against different microorganisms depends upon the chemical composition of the extracts and membrane permeability of the microbes for the chemicals and their metabolism. It has been suggested that the antimicrobial activity is mainly due to the presence of active principles of drugs19. Similarly, Sivakumar et al20 reported that the promising antibacterial activity of Sirattai thylam has registered against bacterial strains i.e., Staphylococcus aureus (26mm), B-Haemolytic Streptococci (21mm), E. coli (28mm), Klebseilla pneumoniae (23mm) and Pseudomonas aegruinosa (24mm).
The determination of minimum inhibitory concentration (MIC), also known as the MBC or MFC, is the most common estimation of bactericidal or fungicidal activity. The MIC value of tulasi oil against bacteria, the better MIC and MBC/MFC values were recorded against E. coli, K. pneumoniae, C. neoformans and S. pneumoniae. Similarly, Kajaria et al21 investigated that the antimicrobial and bronchodialator effect of hydroalcholic extract of polyherbal drug Shirishadi against RTI pathogens and shown the MIC of 6.25 mg/mL against Staphylococcus aureus and 12.5mg/mL for Escherichia coli with strong antifungal activity.
Potassium is a primarily essential in intracellular cation that acts a crucial task in the production of membrane potential, activating cytoplasmic enzymes and maintaining turgor pressure in cells. The potassium leakage into the extracellular space is considered an indicator for an increase in membrane permeability and ultimate loss of viability for the cell22. As shown in the present studies, tulasi oil induced leakage of potassium ions from cells of E.coli and K.pneumonia suspended in glycil-glycine buffer solution and the leakage started within 10 minutes of contact of drugs with target cells. In accordance to this result with previous studies that, Walsh et al23 reported that, the phytocomponenets of Oregano essential oil were found to dissipate the potassium gradient in B. cereus, S. aureus and E. coli. The phytoconstituents of tulasi oil could act on the membrane to cause the bacterial cell to lose the ability to regulate potassium transfer across the membrane, leading to an outpouring of potassium from the cell and a subsequent loss of viability.
CONCLUSION:
Our findings suggest that Tulsi oil shows broad spectral antibacterial activity against the tested organisms of which E.coli, followed by K. pneumonia, C.neoformans and S. pneumoniae had significant sensitivity against the test drug. Hence it can be acknowledged as a potential drug with antimicrobial activity against RTI pathogens and would be a worthwhile agent in the management of Respiratory tract infection. More researches may be warranted at the molecular level and also the pharmacodynamic targets of these ancient Siddha medicines should be identified to optimize clinical success in the management and prevention of infectious diseases.
ACKNOWLEDGEMENT:
The Authors gratefully acknowledge the Director and faculties, Govt. Siddha Medical College, Chennai for their extensive support and advice throughout this study.
CONFLICT OF INTEREST:
The authors have declared that there is no Conflict of Interest.
REFERENCES:
1. Guerrant RL. Why America must care about tropical medicine: threats to global health and security from tropical infectious diseases. Am J Trop Med Hyg. 1998; 59(1):3-16.
2. Gonzalez CE, Venzon D, Lee S, Mueller BU, Pizzo PA, Walsh TJ. Risk factors for fungemia in children infected with human immunodeficiency virus: a case-control study. Clin Infect Dis. 1996; 23:515-21.
3. Rajesh D, Amita G, Mandal TK, Desh DS, Vivek B, Gurav AM, et al. Antimicrobial activity of some Indian medicinal plants. Afr J Trad CAM. 2007; 4:313-8.
4. Sieradzki K, Wu SW, Tomasz A. Inactivation of the methicillin resistance gene mecA in vancomycin-resistant Staphylococcus aureus. Micro Drug Resist. 1999; 5(4):253–257.
5. Latha PS, Kannabiran, K. Antimicrobial activity and phytochemicals of Solanum trilobatum Linn. African Journal of Biotechnology. 2006; 5(23), 2402-2404.
6. Puranam Pichaiah. Pulippani vaidya sastramu. 1st ed. 1915; 1-3
7. Yavanarani S, Sathiyabama M, Mirunaleni P, Suresh K, Meenakshi Sundaram M, Banumathi V. Therapeutic effectiveness of Siddha formulation Thulasi Ennai: A Review. Int J Curr Res Chem Pharm Sci. 2021; 8(4):9-15.
8. Yavanarani S, Mirunaleni P, Sathiyabama M, Suresh K, Meenakshi Sundaram M, Banumathi V. GCMS analysis of Thulasi ennai – A Siddha polyherbal formulation. Int J Adv Multidiscip Res. 2018; 5(7):65-69.
9. Priyanka A, Yogananth N, Syed Ali M, Anuradha V, Antibacterial and antifungal activity of Solanum torvum Sw: An Ethnomedicinal Plant. International Journal of Comprehensive Research in Biological Sciences. 2014; 1(3):6-12.
10. Sathyanarayanan M, Seeni Mubarak M, Mohamed Rabeek S, Syed Ali M, Anuradha V, Yogananth N. Characterization and antibacterial activity of newly synthesized heterocyclic compound. European Journal of Biomedical and Pharmaceutical Science. 2018; 5(3):575-580.
11. Anuradha V, Shankar P, Bhuvana P, Syed Ali M, Yogananth N. Terminalia arjuna bark assisted biosynthesis, Characterization and bioactivity of metal oxide nanoparticles. Journal of Chemical and Pharmaceutical Research. 2017; 9(3):34-46
12. Rodriguez E, Seguer J, Rocabayera X, Manresa A. Cellular effects of monohydrochloride of L-arginine, Na-lauroylethylester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureus. J Appl Microbiol. 2004; 96:903–912.
13. Mankar SD, Shaikh SB, Tamboli, AA. Formulation of Herbal Tablet with the help of Tulsi and Turmeric Extract which Showing Antimicrobial Activity. Research J. Science and Tech. 2020; 12(1): 69-73.
14. Badmanaban R, Patel CN, Devi P, Sen DJ, Modh KM. Studies on antimicrobial and anti-inflammatory activity of the siddha formulation (Thailam – Medicated oil). Research J. Pharmacology and Pharmacodynamics. 2010; 2(1):83-88.
15. Nidhi Rao, Sandhya Mittal. An in vitro Evaluation of the Antimicrobial Activity of Curcuma longa against selected pathogenic microorganisms. Research J. Science and Tech. 2014; 6(2): 71-74.
16. Chaudhari MA, Sarode SM, Chaudhari YA, Vidyasagar G, Kale MK. Antimicrobial studies on selected medicinal plants in Khandesh Region, Maharashtra, India. Research J. Pharmacognosy and Phytochemistry. 2010; 2(5): 386-390.
17. Rukhsana A Rub, Aaisha Sagri, Vandana Joshi, Jasmin Momin, Nusrat Shaikh. Evaluation of antimicrobial potency and synergistic activity of nine traditionally used Indian medicinal plants. Research J. Pharmacognosy and Phytochemistry. 2010; 2(2): 131-135.
18. Anil Kumar A, Subodh P, Sadahev Y, Umesh P, Snehendu B. Evaluation of antimicrobial activity of Casuarina equisetifolia Frost (Casuarinaceae). Research J. Pharmacognosy and Phytochemistry. 2009; 1(1): 64-68.
19. Rojas R, Bustamante B, Bauer J, Fernandez I, Alban J, Lock O. Antimicrobial activity of selected Peruvian medicinal plants. J Ethnopharmacol. 2003; 88:199–204.
20. Sivakkumar SU, Juliet. Anti-bacterial activity of topical Siddha medicine Sirattai thylam against selected human pathogens. World Journal of Pharmaceutical Research. 2015; 4(10): 2859.
21. Kajaria DK, Gangwar M, Kumar D, Kumar Sharma A, Tilak R, Nath G, Tripathi YB, Tripathi JS, Tiwari SK. Evaluation of antimicrobial activity and bronchodialator effect of a polyherbal drug-Shrishadi. Asian Pacific Journal of Tropical Biomedicine. 2012; 2(11):905–909.
22. Lambert PA, Hammond SM. Potassium fluxes, first indications of membrane damage in micro-organisms. Biochem Biophys Res Commun. 1973; 54:796–799.
23. Walsh SE, Maillard JY, Russell AD, Catrenich CE, Charbonneau DL, Bartolo RG. Activity and mechanisms of action of selected biocidal agents on Gram positive and negative bacteria. Journal of Applied Microbiology. 2003; 94:240– 247.
Received on 14.12.2022 Modified on 03.03.2023
Accepted on 12.06.2023 ©Asian Pharma Press All Right Reserved
Asian J. Res. Pharm. Sci. 2024; 14(1):1-5.
DOI: 10.52711/2231-5659.2024.00001