|Year : 2019 | Volume
| Issue : 1 | Page : 30-34
In vitro antimicrobial activity of aqueous extracts of Ocimum suave Willd., Plectranthus barbatus andrews and Zanthoxylum chalybeum Engl. against selected pathogenic bacteria
Joseph Mwanzia Nguta1, Micheni Ndei Kiraithe2
1 Department of Public Health, Pharmacology and Toxicology, College of Agriculture and Veterinary Sciences, Nairobi, Kenya
2 School of Biological Sciences, University of Nairobi, Nairobi, Kenya
|Date of Submission||12-Nov-2018|
|Date of Decision||04-Jan-2019|
|Date of Acceptance||07-Jan-2019|
|Date of Web Publication||13-Mar-2019|
Dr. Joseph Mwanzia Nguta
Department of Public Health, Pharmacology and Toxicology, College of Agriculture and Veterinary Sciences, University of Nairobi, P. O. Box: 29053 – 00625, Nairobi
Source of Support: None, Conflict of Interest: None
Background: There is an urgent need for new antimicrobials, due to the increase in drug resistance. Current studies suggest that, by 2050, in the absence of major improvements in drug discovery, more individuals will die from drug-resistant bacterial infections than from cancer. This will result in a cumulative effect on the global gross domestic product of as much as 100 trillion dollars. To discover new drugs, new targets, and lead compounds are badly needed. Natural products of plant biodiversity are a key source of starting points for novel antimicrobial agents with activity against sensitive and resistant bacterial strains. The current study is timely and of a high impact, since it sought to validate anecdotal efficacy of aqueous extracts from selected medicinal plants conventionally used against microbial infections, namely, leaves from Ocimum suave Willd. (Lamiaceae), roots from Plectranthus barbatus Andrews (Lamiaceae), and roots from Zanthoxylum chalybeum Engl. (Rutaceae). Methods: The study plant parts were collected from Msambweni Sub-county, Kwale County, Kenya. Agar well diffusion method was used to evaluate the antimicrobial activity of the extracts against methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 1385), Escherichia coli (ATCC 25922), S. aureus (ATCC 25923), and Bacillus cereus (ATCC 11778). Results: Root extract of P. barbatus was found to be the most active of the studied extracts exhibiting mean inhibition zone values of 18.67 mm, 20.00 mm, and 25.33 mm in S. aureus, MRSA and B. cereus strains, respectively, after 24 h of incubation. All the studied extracts did not exhibit activity against E. coli. In addition, the aqueous leaf extract from the leaves of O. suave did not display antimicrobial activity against the tested organisms. Conclusion: These findings justify the continued ethno pharmacological utilization of P. barbatus and Z. chalybeum extracts against bacterial infections in traditional herbal medicine by various local communities. Furthermore, the current findings lay a strong foundation for further investigation of extracts from P. barbatus and Z. chalybeum for isolation, identification, and characterization of bioactive molecules responsible for the observed antimicrobial activity. These molecules could serve as templates for the discovery of a new class of antimicrobial agents for the management of economically important bacterial infections.
Keywords: Agar well diffusion, antimicrobials, aqueous extracts, bacterial infections, ethnopharmacology, medicinal plants, msambweni, natural products
|How to cite this article:|
Nguta JM, Kiraithe MN. In vitro antimicrobial activity of aqueous extracts of Ocimum suave Willd., Plectranthus barbatus andrews and Zanthoxylum chalybeum Engl. against selected pathogenic bacteria. Biomed Biotechnol Res J 2019;3:30-4
|How to cite this URL:|
Nguta JM, Kiraithe MN. In vitro antimicrobial activity of aqueous extracts of Ocimum suave Willd., Plectranthus barbatus andrews and Zanthoxylum chalybeum Engl. against selected pathogenic bacteria. Biomed Biotechnol Res J [serial online] 2019 [cited 2019 Jul 22];3:30-4. Available from: http://www.bmbtrj.org/text.asp?2019/3/1/30/254093
| Introduction|| |
Throughout history, microbial infections have been a major threat to human and animal health and prominent cause of morbidity and mortality leading to reduced work productivity and long-term poverty. The problem of microbial infection in humans is made worse by the fact that almost all groups of important antibiotics such as tetracyclines, cephalosporins, aminoglycosides, and macrolides are in danger of losing their efficacy due to increase in microbial resistance. Today, cases of multi-drug resistant microbial strains have become a global public health concern. Consequently, there is a critical need for more research, especially geared toward the development of new antimicrobial agents that are effective, safe, and with novel mechanisms of action. Plants represent an important source of novel antimicrobial compounds. Plants are always exposed to diverse groups of microorganisms in their environment enabling them to produce a wide spectrum of compounds with antimicrobial activity.
Ocimum suave Willd. is a herb that belong to Lamiaceae family and Ocimum genus. This plant is a native of mainly Africa and India. It is commonly known by common names such as African basil, clove basil, and wild basil. The plant has an erect, round-quadrangular and much-branched stem, which can be smooth or hairy and woody at the base and can grow to a height of around 1–3 m in height. The leaves are green in color, narrow and oval in shape growing 5–13 cm in length, 3–9 cm in width and arranged opposite. The flower is pale yellow in color with stalk that is acuminate, sessile egg-shaped spreading or ascending and slightly curved. The flowers give out a sweet scent of camphor. Plectranthus barbatus is a perennial branched aromatic herb, that also belongs to Lamiaceae family and genus Plectranthus that contains >300 species distributed mostly in tropical Africa, Asia, Australia, and Brazil. It is an herbaceous plant with stems that grow up to 1–2 feet with inflorescence and flowers that are typical of the family. Its roots are fasciculate, thick, succulent, and contain the unique chemical known as forskolin. The wide range of pharmacological properties and ethnomedicinal uses of P. barbatus are attributed to the presence of forskolin whose adenylyl cyclase activation properties are documented. Zanthoxylum chalybeum is a deciduous spiny shrub or tree that may stand at 1.5–10 m tall that is widely distributed in the East and Central Africa.
The three plants have a wide usage, especially in traditional medicinal practices of many local communities in East Africa. O. suave locally known as Murihani in Msambweni is a plant of great ethnomedicinal importance. It is conventionally used by the Msambweni community to treat malaria. An infusion of leaves of the plant is used as a disinfectant and insecticide. In Tanzania, a mixture of O. suave and Zingiber officinale Roscoe (Zingiberaceae) is used in the treatment of oral candidiasis. The dried leaves of the plant are snuffed to alleviate headaches and fever among other uses. The essential oils of O. suave have been found to be active against some bacterial and fungal strains, especially those belonging to genuses Listeria, Shigella, Salmonella More Details, Proteus, Trichophyton, Cryptococcus, Penicillium, and Candida. P. barbatus which is locally known as Mraga dare in Msambweni is widely used by various local communities of Africa in the treatment of various ailments such as malaria. HIV/AIDS and related conditions Stomachache, wounds, and ringworms. Z. chalybeum locally known as Mjafari by the local communities of South Coast Kenya is a plant of great medicinal importance where its root extracts are used for the treatment of malaria. The plant is also used in the wider East Africa for treatment of snake bite, kwashiorkor, chest pain, stomach pains, fever, and sore throat.
In the present study, aqueous plant extracts from leaves of O. suave Willd., roots of P. barbatus Andrews, and roots of Z. chalybeum Engl. were screened for antibacterial activity against four bacterial strains namely; methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 1385), Escherichia More Details coli (ATCC 25922), S. aureus (ATCC 25923), and Bacillus cereus (ATCC 11778).
| Methods|| |
Plant collection and identification
The plant materials were collected from Shimoni area within Msambweni Sub-county, Kwale County, Kenya in June 2018 with the guidance of an identified traditional health practitioner. Before collection of the plant materials, consent was sought and obtained from the local community representative. The plant parts collected were leaves from O. suave and root bark from both P. barbatus and Z. chalybeum. In addition, voucher specimens were collected and pressed for taxonomic identification and authentication of the study plants and deposition at the University of Nairobi herbarium. Identification and authentication of the collected voucher specimens were performed by a plant taxonomist at the School of Biological Sciences, University of Nairobi and voucher specimens MNK 01/2014, MNK 02/2014, and MNK 03/2014 for Z. chalybeum, O. suave, and P. barbatus, respectively, deposited at the University's herbarium. This was done in accordance with the international code of botanical nomenclature. The study was approved by the Faculty of Veterinary Medicine (FVM), University of Nairobi ethics committee
Preparation of crude extracts
The plant parts were dried at room temperature under shade for 1 month chopped into small pieces (≈1 centimeter) and pulverized using a laboratory mill. An aqueous hot infusion of each plant part was prepared as follows: 50 g of the ground plant material was dissolved and properly stirred in 500 ml of distilled water and heated at 60°C in a water bath for 1 h. The extracts were then filtered using a muslin cloth (gauze), and the filtrate kept in a deep freezer until frozen in preparation for lyophilization. The filtrate was lyophilized into dry powder using a freeze drier. The dry extracts were stored in stoppered sample vials at 4°C until the time when they were required for antimicrobial bioassay.
Source and maintenance of test bacteria
The test bacteria used were: MRSA ATCC 1385 which was obtained from the Centre for Microbiology Research, Kenya Medical Research Institute, E. coli ATCC 25922, S. aureus ATCC 25923, and B. cereus ATCC11778 which were obtained from the Department of Public Health, Pharmacology and Toxicology, University of Nairobi. The test microorganisms were maintained at −20°C on nutrient agar (Oxoid®), and their identity confirmed based on cultural, morphological, and biochemical characteristics.
Retrieval of test pathogens and inocula preparation
Stock culture of each of the test bacterial strains stored at −20°C was retrieved by sub-culturing on Mueller-Hinton agar plates (MHA) (Himedia®). For each bacterial strain, three pure colonies of the same morphological type were selected from plates and aseptically transferred into test tubes containing 10 ml of Mueller-Hinton broth (MHB) (Himedia®) using a sterile loop. The culture tubes were then incubated at 37°C for 24 h to obtain fresh cultures. Bacterial suspensions were standardized with sterile saline to turbidity equivalent to 0.5 McFarland (prepared by mixing 0.05 ml of 1% barium chloride dehydrate with 9.95 ml of 1% sulfuric acid). Exactly, 0.5 McFarland standard is approximately 1 × 108 colony forming units/ml for bacteria.
Agar well diffusion assay
Agar well diffusion method as previously described, was used to assess the antimicrobial activity of the plant extracts against the test pathogens. Twenty milliliters of molten MHA was poured into sterile Petri dish More Detailses (9 cm in diameter) and allowed to set. Fifty microliters of standardized inoculum were swabbed uniformly to solidified MHA plates using sterile cotton swabs and allowed to dry for 5 min. Holes with a diameter of 6 mm were punched aseptically with a cork borer on freshly seeded plates. Aliquots of 25 μl of plant extracts dissolved in distilled water at a concentration of 500 mg/ml were dispensed into respective wells using a micropipette. Streptomycin at a concentration of 30 mg/ml was used as a positive control. Streptomycin is a broad-spectrum aminoglycoside antibiotic which is active against a wide range of both Gram-positive and Gram-negative bacteria. Distilled water was used as a negative control. The plates were labeled and incubated upside down at 37°C for 24 h. Antimicrobial activity was detected by measuring zones of inhibition using a transparent ruler at the end of the incubation period. An extract was considered to be active against a microorganism if it produced a mean inhibition zone of ≥8 mm. All the tests were run in triplicate in a biological safety cabinet and in accordance with the protocols of the Clinical and Laboratory Standards Institute formerly National Committee for Clinical Laboratory Standards.
Data were analyzed using Microsoft Excel® 2010. The inhibitory effects of the plant extracts against the test pathogens were expressed as mean ± standard error of the mean inhibition zones diameter (mm).
| Results|| |
The current study investigated the in vitro antimicrobial activity of three selected medicinal plants conventionally used against bacterial infections by the Msambweni community of Kenyan South Coast [Figure 1].
|Figure 1: Morphological features of Ocimum suave (a), Plectranthus barbatus (b) and Zanthoxylum chalybeum (c)|
Click here to view
Antimicrobial investigations conducted revealed that among the three plant extracts studied, only two showed significant activity against at least one of four test bacteria (MRSA), E. coli, S. aureus and B. cereus). P. barbatus root extract at a concentration of 500 mg/ml exhibited antimicrobial activity against S.aureus, MRSA and B. cereus, whereas the root extracts from Z. chalybeum were active against B. cereus and MRSA [Figure 2].
|Figure 2: Growth inhibition activity of extracts of Plectranthus barbatus against Staphylococcus aureus (a), methicillin-resistant Staphylococcus aureus (b), and Bacillus cereus (c); and extracts of Zanthoxylum chalybeum against Bacillus cereus (d) and methicillin-resistant Staphylococcus aureus (e) and the controls (Streptomycin)|
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The results revealed that there were variations in the antibacterial activity of the three plant extracts among the bacterial strains. Root extracts of P. barbatus were active against S. aureus, B. cereus and MRSA with mean inhibition zones of 18.67, 25.33, and 20.00 mm, respectively. Z. chalybeum root extracts, on the other hand, produced mean inhibition zones of 21.67 and 24.33 in B. cereus and MRSA, respectively [Table 1].
|Table 1: Inhibition zones (mm) of Plectranthus barbatus, Zanthoxylum chalybeum and Ocimum suave extracts and streptomycin (control) on four test bacteria after 24 h incubation period|
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Leaf extracts of O. suave were not active against any of the four test bacteria. One of the most significant findings in this study was that none of the three tested plant extracts was able to inhibit the growth of E. coli at the studied concentration of 500 mg/ml. Streptomycin which was used a positive control at a concentration of 30 mg/ml revealed strong antibacterial activity against the test bacteria with the highest activity observed in S. aureus and E. coli with mean inhibition zones of 39.67 mm and 36.67 mm, respectively. Streptomycin had the highest activity against all the test bacterial strains in comparison to all the studied plant extracts.
| Discussion|| |
The current study was designed to evaluate the in vitro antimicrobial activity of three ethnopharmacologically important medicinal plants in South Coast Kenya. The test plant parts have been used since time immemorial against microbial infections. We believed that the local communities had good knowledge on the medicinal utilization of natural products of plant biodiversity which inhabit their locality. The study is timely and of key significance since microorganisms resistant to the conventional antimicrobials has developed, and hence, search and development of new drugs with novel mechanisms of action are urgent. Medicinal plants have continued to serve as a major source of molecules with activity against many illnesses. With the ever increasing incidences of drug-resistance to the commonly available antimicrobials, plant and plant products are being investigated as potential sources for the discovery of new antimicrobial agents for the management of common bacterial infections. Lamiaceae (the mint family) and Rutaceae are some of the most important medicinal plants families whose members are widely used in various traditional medicinal practices to treat a wide range of ailments. The current study evaluated the antibacterial activity of aqueous crude extracts from the leaves of Ocimum suave (Lamiaceae), the root barks of P. barbatus Andrews (Lamiaceae) and Z. chalybeum Engl. (Rutaceae) against three clinically important Gram-positive and one Gram-negative bacterial strains.
Out of the three plant extracts, P. barbatus was active against three of the four test bacteria while Z. chalybeum exhibited antibacterial activity in two test bacterial strains. Crude aqueous and organic extracts from different parts of P. barbatus have been reported to have antibacterial activity against a wide range of bacteria such as B. cereus, E. coli, S. aureus, S. epidermidis, and S. pneumonia. These observations are in agreement with our findings. Earlier reports have indicated that organic and aqueous extracts from Z. chalybeum have antimicrobial activity against some bacterial and fungal pathogens such as Candida albicans, Pseudomonas aeruginosa, B. cereus, and S. aureus. In the current study, none of the extracts showed antimicrobial activity against E. coli. These findings are in agreement with earlier studies that reported in-activity of both aqueous and organic extracts of Z. chalybeum against E. coli. The observed low susceptibility of E. coli to the plant extract could be because E. coli is a Gram-negative bacterium. It has been reported in the literature that Gram-negative bacteria are more resistant to the plant-based extracts including essential oils in comparison to Gram-positive bacteria. The difference in susceptibility of the two groups of bacteria to plant extracts and indeed to other antibacterial agents is as a result of the fundamental differences in the structural composition of the cell wall of the two groups of bacteria. Gram-negative bacteria have an outer lipopolysaccharide layer that restricts the penetration of antimicrobial agents hence offering greater protection of Gram-negative bacteria against many antimicrobials. Organic and aqueous leaf extract of O. suave have been found to have numerous biological activities such as anti-ulcer and antioxidant action, antipyretic properties, and antibacterial activity. The failure of the aqueous leave extract of O. suave to inhibit any of the test bacteria in the current study despite the use of the plant in ethnomedicine among the people of Msambweni could be attributed to a number of factors. First, traditional practitioners rarely practice monotherapies, and it is possible that O. suave when used in combination with other plants can elicit antimicrobial effect through synergism. Furthermore, it could be possible that large doses of ethno-formulations are used during preparations of concoctions/decoctions. There is also a possibility that aqueous extraction was unable to extract any active principle from the studied plant.
Members of Lamiaceae and Rutaceae families are known to have a wide spectrum of biological activities in addition to a diverse ethnobotanical usage in many countries, especially in traditional medicinal practices. Although phytochemical screening of secondary metabolites from the plant extracts was not carried out in this study, previous studies have shown organic and aqueous extracts of P. barbatus and Z. chalybeum to be rich in terpenoids, triterpenes, phenolics, tannins, flavonoids, saponins, sterols, alkaloids, and coumarins. Many of these compounds have been found to have a wide range of biological activities such as antibacterial, antifungal, antiprotozoal, larvicidal, and antioxidant activities. The observed antibacterial activity of the two plant extracts in this study could, therefore, be attributed to the presence of one or more of the aforementioned compounds either working alone or in synergism.
| Conclusion|| |
Increasing cases of antimicrobial resistance to currently available drugs have elicited interest among researchers in natural products of plant biodiversity and other natural products as a source of new drugs. The search for new plant-based drugs has been greatly enhanced by exploitation of the richness of ethnobotanical and ethnopharmacological survey data from various local communities around the world. The current study revealed that root extracts of P. barbatus inhibited the growth of S. aureus, B. cereus, and MRSA while extracts of Z. chalybeum were active against the latter two microbes. This study thus provides the scientific basis for the continued use of these plants in traditional medicinal practices by the local communities. Further studies, especially on the chemical structure, determination of active compounds, mechanism of action, in vivo activity and toxicity tests are recommended to position these plants as future sources of affordable, safe, and effective antibacterial agents.
The authors are grateful to the School of Biological Sciences, the University of Nairobi for offering space and facilities to carry out this study and the people of Msambweni for accepting to share with us their ethno knowledge which is often jealously guarded.
Financial support and sponsorship
The authors are grateful to Bill and Melinda Gates Foundation, through Grant No. (#OPP52155) for funding the current study.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ventola CL. The antibiotic resistance crisis: Part 1: Causes and threats. P T 2015;40:277-83.
Okach D, Nyunja A, Opande G. Phytochemical screening of some wild plants from Lamiaceae
and their role in traditional medicine in Uriri district-Kenya. Int J Herb Med 2013;1:135-43.
Ortíz-Castro R, Contreras-Cornejo HA, Macías-Rodríguez L, López-Bucio J. The role of microbial signals in plant growth and development. Plant Signal Behav 2009;4:701-12.
Chogo JB, Crank G. Chemical composition and biological activity of the Tanzanian plant Ocimum suave
. J Nat Prod 1981;44:308-11.
Langer R, Langer R, Hill D. Agricultural Plants. Cambridge: Cambridge University Press; 1991.
Lukhoba CW, Simmonds MS, Paton AJ. Plectranthus
: A review of ethnobotanical uses. J Ethnopharmacol 2006;103:1-24.
Alasbahi RH, Melzig MF. Plectranthus barbatus
: A review of phytochemistry, ethnobotanical uses and pharmacology – Part 1. Planta Med 2010;76:653-61.
Ruffo C, Birnie A, Tengnäs B. Edible wild Plants of Tanzania. Nairobi: Regional Land Management Unit; 2002.
Kiraithe MN, Nguta JM, Mbaria JM, Kiama SG. Evaluation of the use of Ocimum suave
willd. (Lamiaceae), Plectranthus barbatus
andrews (Lamiaceae) and Zanthoxylum chalybeum
engl. (Rutaceae) as antimalarial remedies in Kenyan folk medicine. J Ethnopharmacol 2016;178:266-71.
Kokwaro J. Medicinal Plants of East Africa. 3rd
ed. Nairobi: University of Nairobi Press; 2009.
Nguta JM, Mbaria JM, Gakuya DW, Gathumbi PK, Kiama SG. Traditional antimalarial phytotherapy remedies used by the South Coast community, Kenya. J Ethnopharmacol 2010;131:256-67.
Iwu M. Handbook of African Medicinal Plants. Boca Raton, Floridamm: CRC Press Inc.; 1993.
Begum J, Yusuf M, Chowdhury U, Wahab M. Studies on essential oils for their antibacterial and antifungal properties. Part 1. Preliminary screening of 35 essential oils. J Sci Ind Res 1993;28:25-34.
Kapewangolo P, Hussein AA, Meyer D. Inhibition of HIV-1 enzymes, antioxidant and anti-inflammatory activities of Plectranthus barbatus
. J Ethnopharmacol 2013;149:184-90.
Abdel-Mogib M, Albar H, Batterjee S. Chemistry of the genus Plectranthus
. Molecules 2002;7:271-301.
Twaij H, Kery A, Al Khazraji N. Some pharmacological, toxicological and phytochemical investigations on Centaurea phyllocephala
. J Ethnopharmacol 1983;9:299-3214.
Kokwaro J. Medicinal plants of East Africa. 2nd
ed. Nairobi: Kenya Literature Bureau; 1993.
Balouiri M, Sadiki M, Ibnsouda S. Methods for in vitro
evaluating antimicrobial activity: A review. J Pharm Biomed Anal 2016;6:71-9.
Venkateshappa S, Sreenath K. Potential medicinal plants of Lamiaceae
. Am Int J Res Form Appl Nat Sci 2013;3:82-7.
Sasikala T, Prabakaran R, Sathiyapriya S. Evaluation of antimicrobial activities of Plectranthus barbatus
L. tuber. Int J Ayurveda Herb Med 2014;4:1506-601.
Kaigongi M, Dossaji S, Nguta J, Lukhoba C, Musila F. Antimicrobial activity, toxicity and phytochemical screening of four medicinal plants traditionally used in Msambweni district, Kenya. J Biol Agric Health 2014;4:6-12.
Cock I. Antimicrobial activity of Leptospermum
bracteata and Leptospermum juniperium
methanolic extracts. Pharmacogn Commnu 2013;3:45-52.
Parekh J, Chanda S.In vitro
antibacterial activity of the crude methanol extract of Woodfordia fruticosa
Kurz. flower (Lythraceae). Braz J Microbiol 2007;38:204-7.
Tan P, Mezui C, Enow-Orock G, Agbo G. Antioxidant capacity, cytoprotection, and healing actions of the leaf aqueous extract of Ocimum suave
in rats subjected to chronic and cold-restraint stress ulcers. Ulcers 2013;2013:1-23.
Makonnen E, Debella A, Zerihun L, Abebe D, Teka F. Antipyretic properties of the aqueous and ethanol extracts of the leaves of Ocimum suave
and Ocimum lamiifolium
in mice. J Ethnopharmacol 2003;88:85-91.
Musau J, Mbaria J, Gakuya D. The antibacterial activity of some medicinal plants used in Meru central district, Kenya. Kenya Vet 2011;35:18-24.
Sharma U, Agnihotri R, Ahmad S, Mahajan S, Sharma R. Antibacterial activity of some medicinal plants of family Lamiaceae from Braj region. Glob J Res Med Plants 2013;1:72-6.
Balakumar S, Rajan S, Thirunalasundari T, Jeeva S. Antifungal activity of Ocimum sanctum
linn. (Lamiaceae) on clinically isolated dermatophytic fungi. Asian Pac J Trop Med 2011;4:654-7.
Severino VG, Cazal Cde M, Forim MR, da Silva MF, Rodrigues-Filho E, Fernandes JB, et al.
Isolation of secondary metabolites from Hortia oreadica
(Rutaceae) leaves through high-speed counter-current chromatography. J Chromatogr A 2009;1216:4275-81.
Emam A, Swelam E, Megally N. Furocoumarin and quinolone alkaloid with larvicidal and antifeedant activities isolated from Ruta chalepensis
leaves. J Nat Prod 2009;2:10-22.
Erdemoglu N, Turan NN, Cakici I, Sener B, Aydin A. Antioxidant activities of some Lamiaceae
plant extracts. Phytother Res 2006;20:9-13.
[Figure 1], [Figure 2]