|Year : 2016 | Volume
| Issue : 2 | Page : 104-110
Evaluation of antimicrobial efficacy of neem and Aloe vera leaf extracts in comparison with 3% sodium hypochlorite and 2% chlorhexidine against E. faecalis and C. albicans
Sistla Datta Prasad, Prem Chand Goda, Kunam Sashidhar Reddy, Chennuru Sunil Kumar, Melpati Hemadri, Dappili Swamy Ranga Reddy
Department of Conservative Dentistry and Endodontics, C.K.S. Theja Institute of Dental Sciences and Research, Tirupati, Andhra Pradesh, India
|Date of Web Publication||5-Jul-2016|
Prem Chand Goda
Department of Conservative Dentistry and Endodontics, CKS Theja Institute of Dental Sciences and Research, Chadalawada Nagar, Tirupati, Andhra Pradesh - 517 502
Source of Support: None, Conflict of Interest: None
Aim: The aim of study was to determine the antimicrobial efficacy of alcoholic neem and Aloe vera leaf extracts against Enterococcus faecalis and Candida albicans in comparison with 3% sodium hypochlorite (3% NaOCl) and 2% chlorhexidine (2% CHX).
Materials and Methods: In the present study, indigenously prepared neem and Aloe vera leaf extracts were serially diluted to determine the minimum inhibitory concentration (MIC) at which the E. faecalis and C. albicans were sensitive, and compared it with 3% NaOCl and 2% CHX by the agar-well diffusion method.
Results: The MICs of the alcoholic neem extract to E. faecalis and C. albicans were determined as 0.94% and 1.88%, and for Aloe vera extracts they were 1.88% and 3.75%, respectively and the zones of diffusions that were formed around the extracts in the respective agar plates were significantly greater than with 3% NaOCl and 2% CHX within the groups when analyzed statistically by one-way analysis of variance (ANOVA).
Conclusion: The antimicrobial efficacy of extracts was well demonstrated from the present in vitro observation of the agar-well diffusion method, and hence it may be advantageous if we use these extracts as irrigating solutions in endodontics. However, it requires further preclinical and clinical evaluation.
Keywords: Agar-well diffusion method, alcoholic neem extract, Aloe vera extract, C. albicans, 2% chlorhexidine (CHX). E. faecalis, minimum inhibitory concentration, serial broth dilution, 3% sodium hypochlorite (NaOCl)
|How to cite this article:|
Prasad SD, Goda PC, Reddy KS, Kumar CS, Hemadri M, Ranga Reddy DS. Evaluation of antimicrobial efficacy of neem and Aloe vera leaf extracts in comparison with 3% sodium hypochlorite and 2% chlorhexidine against E. faecalis and C. albicans. J NTR Univ Health Sci 2016;5:104-10
|How to cite this URL:|
Prasad SD, Goda PC, Reddy KS, Kumar CS, Hemadri M, Ranga Reddy DS. Evaluation of antimicrobial efficacy of neem and Aloe vera leaf extracts in comparison with 3% sodium hypochlorite and 2% chlorhexidine against E. faecalis and C. albicans. J NTR Univ Health Sci [serial online] 2016 [cited 2019 Oct 14];5:104-10. Available from: http://www.jdrntruhs.org/text.asp?2016/5/2/104/185436
| Introduction|| |
Medicinal plants have been an integral part of human life since the dawn of civilization. Pharmacological studies have acknowledged the value of medicinal plants as potential sources of bioactive compounds.  Medicinal plants are a rich source of novel drugs that are the components of traditional systems of medicine, modern medicines, nutraceuticals, food supplements, and folk medicines. 
Azadirachta indica (neem) is well known in India and its neighboring countries as one of the most versatile medicinal plants with a wide spectrum of biological activity, and most commonly used traditional medicinal plant of India for household remedies against various human ailments, from antiquity. ,, Neem has been extensively used in Ayurveda, Unani, homeopathic, and Siddha medicine and has become a cynosure of modern medicine. 
Aloe vera Linné or Aloe barbadensis Miller is another herbal plant, which consists of thick leaves that supply water for the plant to survive long periods of drought. Aloe gel is used to relieve thermal burn and sunburn, and to promote wound healing;  it has antimicrobial activity and can help stimulate the body's immune system. 
In endodontics, the most common pathway for microorganisms from the normal oral flora to the dental pulp is through an open cavity caused by dental caries, and then the root canal becomes a privileged sanctuary for microorganisms and their byproducts, and the degradation products of both the microorganisms and the pulp tissue. 
Unlike primary endodontic infections, which are polymicrobial in nature and dominated by Gram-negative anaerobic rods, the microorganisms involved in secondary infections are composed of one or a few bacterial species. ,, Enterococcus faecalis has been frequently found in obturated root canals exhibiting signs of chronic apical periodontitis, has been isolated in 23-70% of the positive cultures, and often occurs in monoculture. ,,
Fungi constitute a small part of the oral microbiota. The largest proportion of the fungal microbiota is made up of Candida species. The incidence of Candida albicans in the oral cavity has been reported to be 30-45% in healthy adults  and 95% in patients infected with human immunodeficiency virus.  The presence of C. albicans is evident in both primary infections (5-20%) and persistent infections (25%) of root canal systems. 
Root canal irrigation plays an important role in the debridement and disinfection of the root canal system and is an integral part of root canal preparation procedures. The most frequently used irrigants are sodium hypochlorite (NaOCl) and hydrogen peroxide, or the combined use of both (Grossman 1981).
The concentration of the irrigants is still a matter of debate and remains controversial: Many authors recommend a 5.25% concentration of NaOCl (Harrison 1984), while others prefer a lower concentration of 3% or even 0.5% (Spangberg et al. 1973, Baumgartner and Cuenin 1992). NaOCl has been demonstrated to be an effective agent against a broad spectrum of bacteria and to dissolve vital as well as necrotic tissue (Senia et al. 1975).
However, it has also been shown that NaOCl has toxic effects on vital tissues, resulting in hemolysis, skin ulceration, and necrosis (Pashley et al. 1985). It has a pH value of approximately 11-12 and causes injury primarily by the oxidation of proteins. 
CHX gluconate has been recommended as a root canal irrigant because of its broad-spectrum antimicrobial action, substantivity, and low toxicity. However, CHX being incapable of tissue dissolution has been pointed out as its major disadvantage. Some attempts have been made to evaluate the activity of CHX in dissolving organic matter, demonstrating that both preparations of this substance, aqueous solution and gel, are not able to dissolve pulp tissues. ,,
It is advantageous to use herbal extracts as root canal irrigants after proper cytotoxic and clinical trial exposure is done because nontoxic medicinal plant products have better biocompatibility than chemicals. Hence, the current study was designed to test the null hypothesis that the neem and Aloe vera leaf extracts do not have any antimicrobial activity against E. faecalis and C. albicans when compared by the agar-well diffusion method, and there was no significant difference between the extracts 3% NaOCl and 2% CHX in their activity.
| Materials and methods|| |
0Indigenous preparation of neem leaf extract
Mature, fresh A. indica leaves were collected, washed in sterilized distilled water, and air-dried for 2 weeks at room temperature. Then the leaves were ground into fine powder and packed in a muslin cloth bag for the process of extraction through a hot continuous pressurized technique. We have used ethanol (absolute alcohol 99.99% v/w) as a solvent for the extraction procedure. After obtaining the crude form of neem extract, we dissolved 150 mg of crude form in 10 mL of dimethyl sulfoxide (DMSO) and made it 15% to use as a stock solution.
Indigenous preparation of Aloe vera extract
Aloe vera leaves along with gel were collected, kept in a hot air oven at 80°C for 48 h, and then the dried leaves were grounded into fine powder. The powder was packed in a muslin cloth bag and the extraction process was carried out through a hot continuous pressurized extraction technique. After obtaining a crude form of the Aloe vera extract, we dissolved 150 mg of this crude form in 10 mL of DMSO and made it 15% to use as a stock solution.
Microbial collection and preparation
E. faecalis (ATCC® 29212™) and C. albicans (ATCC® 14053™) were the strains used in the present study.
The respective bacterial and candidal strains from the stock were revived by plating on blood agar medium. After overnight incubation at 37°C, isolated colonies were selected and the identities of the organisms were confirmed. Isolated colonies were transferred to sterile brain-heart infusion (BHI) broth and Sabouraud's dextrose agar (SDA) for the bacterial and candidal strains respectively, and once again incubated overnight. The growth concentration was adjusted to 10 5 organisms/mL by using the 0.5 McFarland turbidity standard.
Determination of minimum inhibitory concentration (MIC) of neem and Aloe vera extract
The extract of 15% concentration was used as the stock solution. Two hundred (200) μL of the BHI broth was added in each of 10 MIC tubes per bacterial strain. For the Candida strain, 200 μL of the SDA was added in each of 10 MIC tubes. For both strains, 200 μL of stock was added in the first MIC tube containing 200 μL of the respective broth. After mixing well, 200 μL from the first tube was transferred to the second MIC tube. This was continued until the last (10th) tube. From the last tube, 200 μL final solution was discarded. By following this serial dilution, the concentrations of the neem/Aloe vera extracts achieved were the following: 15%, 7.5%, 3.75%, 1.88%, 0.94%, 0.47%, 0.23%, 0.12%, 0.06%, and 0.03%.
The tubes were then incubated for 24 h at 35°C. After intubation, the MIC values were examined by visual inspection of the tubes. With each batch of tests, positive and negative controls were put up. Positive control containing broth plus bacterial/candidal strain showed turbidity, and negative control containing only broth appeared clear. In each series of tubes, the last tube with clear supernatant was considered to be without any growth and considered the MIC value. Turbidity in the MIC tube indicated growth of the bacterial/candidal strain, implying that the organisms were resistant to the neem/Aloe vera extract.
After 24 h of incubation based on turbidity, on visual examination we found that for the neem extract, sensitivity started from 0.94% for E. faecalis and from 1.88% for C. albicans. As for the Aloe vera extract, sensitivity started from 1.88% to E. faecalis and from 3.75% to C. albicans. We used these base-level sensitivity concentration values of both extracts and compared them with 3% NaOCl and % CHX solution (EndoCHX; Essential Dental Systems; United States).
E. faecalis was grown overnight at 37°C in BHI broth on a rotary shaker at 150 rpm, with bacterial growth checked by changes in turbidity at 24 h, and C. albicans was grown overnight at 28°C in SDA broth on a rotary shaker at 150 rpm, growth checked by changes in turbidity at 24 h.
Agar-well diffusion method
After confirming the growth of respective microorganisms in the broth, it was mixed with agar, plated, and complete solidification allowed. After complete solidification, 6 mm (both in depth and width) wells were prepared on the surface of the agar, and 200 uL of the respective extracts at the predetermined concentrations along with 200 uL of 3% NaOCl and 2% CHX was placed. The control here was DMSO, used for dissolving the crude form of extracts.
In the first group of agar plates, the activity of the extracts was examined with 3% NaOCl against E. faecalis and C. albicans. In the second group of agar plates, the activities of the extracts were examined with 2% CHX against E. faecalis and C. albicans. The plates were incubated for 24 h and examined for zones of inhibition, and the procedure was triplicated.
Zones of inhibition
After 24 h of incubation, we calculated the zones of inhibitions by using the Hi zone antibiotic scale; (HiMedia Laboratories Pvt. Ltd., Mumbai, India); they did not change significantly even after 48 h. The mean values of the zones of inhibition around the extracts with respect to 3% NaOCl and 2% CHX were tabulated [Table 1].
One-way analysis of variance (ANOVA)/Kruskal-Wallis test (P = 0.000) [Table 2] and intercomparison between the groups [Table 2] were done.
|Table 2: Multiple Comparison Done By Post Hoc Test (Mean Difference Significant At 0.05)|
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| Results|| |
The zones of inhibition that were formed around the extracts were significantly greater than those of 3% NaOCl and 2% CHX in the respective groups of agar plates. The study was triplicated and the mean values were tabulated accordingly.
In the first group: Around the E. faecalis, the activity of 0.94% neem extract >1.88% Aloe vera extract >3% NaOCl; around the C. albicans, the activity of 3.75% Aloe vera extract >1.88% neem extract >3% NaOCl [Figure 1] and [Figure 2].
|Figure 1: Zone of inhibition of extracts in comparison with 3% NaOCl against E. faecalis|
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|Figure 2: Zone of inhibition of extracts in comparison with 3% NaOCl against C. albicans|
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In the second group: Around the E. faecalis, the activity of 0.94% neem extract >1.88% Aloe vera extract >2% CHX; around the C. albicans, the activity of 1.88% neem extract >3.75% Aloe vera extract >2% CHX [Figure 3] and [Figure 4].
|Figure 3: Zone of inhibition of extracts in comparison with 2% CHX against E. faecalis|
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|Figure 4: Zone of inhibition of extracts in comparison with 2% CHX against C. albicans|
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There was a significant statistical difference between the groups and within the groups.
| Discussion|| |
According to the results of the present in vitro observations, the null hypothesis was rejected. The agar-well diffusion method was employed in the study to evaluate the activity of extracts, which is one of the most commonly used methods for the evaluation of antibacterial activity. , The disadvantage associated with the employed technique is that it does not differentiate between bactericidal or bacteriostatic activity of the extracts. However, we used 3% NaOCl in the present study, which showed bactericidal activity from all the strength of >2.5%,  and 2% CHX to show bactericidal activity,  employing them in the study for comparison with the extracts at the base-level sensitivity value. A significant difference between their activities was seen.
NaOCl has various disadvantages, such as toxic effects on vital tissues resulting in hemolysis, skin ulceration, and necrosis. NaOCl in contact with the patient's or operator's eyes results in immediate pain, profuse watering, intense burning, and erythema. Additionally, when extruded through the apical foramina, it causes periapical swelling and edema of the tissues. ,
The use of 2% CHX has disadvantages, such as the lack of tissue-dissolving capacity. Although sensitivity to CHX is rare, contact dermatitis is another common adverse reaction (Krautheim et al. 2004). Apart from this, CHX may have a number of rare side effects, such as desquamative gingivitis, discoloration of the teeth and tongue, and dysgeusia (distorted taste). Contact with the conjunctiva can cause permanent damage, and accidental contact with the tympanum can cause ototoxicity. 
However, these irrigating solutions require the use of 17% ethylenediaminetetraacetic acid (EDTA) for removal of the smear layer. In addition, drug-resistant strains may develop because of continuous and long-term use of these irrigating solutions. The use of neem as an irrigating solution has advantages because of its antibacterial activity,  anti-adherent property, , and chelative property.  These properties will be helpful in microbial eradication and removal smear layer from the root canal with advent use of neem extract as irrigating solution where it is not possible with conventional irrigants as a single component. Thus, the use of herbs with a wide range of properties led to the development of new irrigating solutions and intracanal medicaments.
Rajshekharan et al. concluded that the leaf extracts of A. indica exhibited significant antibacterial activity against all the test microorganisms. However, the inhibitory activities of the leaf extracts were both organism- and solvent-dependent. The leaf extracts limited the growth of both Gram-positive and Gram-negative bacterial species. 
Siddiqui in 1942 isolated nimbin, the first bitter compound to be isolated from neem oil. More than 135 compounds have been isolated from different parts of neem, and several reviews have been published on the chemistry and structural diversity of these compounds. The compounds have been divided into two major classes: Isoprenoids and nonisoprenoids. 
In addition, Botelho et al. (2002) and Behl et al. (2008) concluded from their experiments and trials that A. indica is highly efficacious in the treatment of periodontal disease, thus exhibiting its biocompatibility with human periodontal ligament (PDL) fibroblast; it has anti-adherence activity by altering bacterial adhesion and has been helpful in the prevention of colonization by microorganisms inside the root canals. ,
The use of neem as an endodontic irrigant may be advantageous because it is not likely to cause severe injuries to patients such as might occur via NaOCl accidents. In our study, neem extract at a concentration of 0.94% against E. faecalis and at 1.88% against C. albicans showed significant antibacterial activity when compared with 3% NaOCl and 2% CHX.
Aloe vera has been shown to have anti-inflammatory activity (Afzal et al., 1991; Malterud et al., 1993), immunostimulatory activity (Ramamoorthy and Tizard, 1998), and cell growth-stimulatory activity (Tizard et al., 1994), Furthermore, activity against a variety of infectious agents has been attributed to Aloe vera, for instance antiviral (Khalon et al., 1991) and antifungal (Kawai et al., 1998). Specific plant compounds such as anthraquinones (Gracia-Sosa et al., 2006; Dabai et al., 2007), dihydroxyanthraquinones (Wu et al., 2006), and saponins (Reynolds and Dweck, 1999) have been proposed to have direct antimicrobial activity.
The presence of coumaric acid was reported, in the studies of Baranowski et al., to increase the lag phase of the microorganisms, and it was also able to inhibit the enzymatic activity of the microorganisms.  Cinnamic acid in A. vera gel, it is suggested, can inhibit glucose uptake and adenosine triphosphate (ATP) production in the resting cells of bacteria. 
In our study, the efficacy of the Aloe vera extract both at 1.88% concentration against E. faecalis and at 3.88% concentration against C. albicans was significantly greater than 3% NaOCl and 2% CHX.
- Phytochemical analysis of neem and Aloe vera extracts has to be done through high-performance liquid chromatography (HPLC) to quantify the individual components.
- The ability of extracts should be evaluated on removal of the smear layer and elimination of biofilm from the root canal dentin.
- Proper cytological evaluation and clinical trial exposure have to be done to determine the biocompatibility and effective use of the solution as an irrigant in endodontics.
| Conclusion|| |
According to the proposed methodology and on the basis of the present study's results, it may be concluded that the antimicrobial activity of both neem and Aloe vera leaf extracts against E. faecalis and C. albicans is quite effective. The extracts appear to be promising for use as irrigating solutions in endodontics.
We would like to thank Dr. M. Anjana, Associate Professor, Krishna Theja Institute of Pharmacy, Tirupati, A.P and Dr. Kishore, clinical microbiologist, for providing facilities their their help in completing our study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Prusti A, Mishra SR, Sahoo S, Mishra SK. Antibacterial activity of some Indian medicinal plants. Ethnobot Leaflets 2008;12:227-30.
Ncube NS, Afolayan AJ, Okoh AI. Assessment techniques of antimicrobial properties of natural compounds of plant origin: Current methods and future trends. Afr J Biotechnol 2008;7:1797-806.
Parrotta JA, Chaturvedi AN. Azadirachta Indica A. Juss. Neem, Margosa. Meliaceae. Mahogany Family: USDA Forest Service, International Institute of Tropical Forestry; 1994. p. 1-8.
Biswas K, Chattopadhyay I, Banerjee RK, Bandyopadhyay U. Biological activities and medicinal properties of neem (Azadirachta indica). Curr Sci 2002;82:1336-45.
Puri HS. Neem the Devine Tree: Azadirachta Indica. The Netherlands: Harwood Academi Publishers; 1999. p. 182.
Varma GS. Miracles of Neem Tree. New Delhi, India: Rasayan Pharmacy; 1976.
Foster S. Aloe Vera: The Succulent with Skin Soothing Cell Protecting Properties. Health World Online. Herbs for Health; 1999.
Davis HR. Aloe Vera: A Scientific Approach. New York: Vantage Press; 1997. p. 3-5.
Naidorf IJ. Inflammation and infection of pulp and periapical tissues. Oral Surg Oral Med Oral Pathol 1972;34:486-97.
Molander A, Reit C, Dahlén G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1-7.
Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth With failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93.
Hancock HH 3 rd
, Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after unsuccessful endodontic treatment in a North American population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:579-86.
Flahaut S, Hartke A, Giard JC, Auffray Y. Alkaline stress response in Enterococcus faecalis: Adaptation, cross protection, and changes in protein synthesis. Appl Environ Microbiol 1997;63:812-14.
Giard JC, Hartke A, Flahaut S, Benachour A, Boutibonnes P, Auffray Y. Starvation-induced multiresistance in Enterococcus faecalis JH2-2. Curr Microbiol 1996;32:264-71.
Hartke A, Giard JC, Laplace JM, Auffray Y. Survival of Enterococcus faecalis in an oligotrophic microcosm: Changes in morphology, development of general stress resistance, and analysis of protein synthesis. Appl Environ Microbiol 1998;64:4238-45.
Arendorf TM, Walker DM. The prevalence and intra-oral distribution of Candida albicans in man. Arch Oral Biol 1980;25:1-10.
Dupont B, Graybill JR, Armstrong D, Laroche R, Touzé JE, Wheat LJ. Fungal infections in AIDS patients. J Med Vet Mycol 1992;30(Suppl 1):19-28.
Ashraf H, Samiee M, Eslami G, Ghodse Hosseini MR. Presence of Candida Albicans in root canal system of teeth requiring endodontic retreatment with and without periapical lesions. Iran Endod J 2007;2:24-8.
Hülsmann M, Hahn W. Complications during root canal irrigation--literature review and case reports. Int Endod J 2000;33:186-93.
Gomes BP, Ferraz CC, Vianna ME, Berber VB, Teixeira FB, Souza-Filho FJ. In vitro
antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis
. Int Endod J 2001;34:424-8.
Jeansonne MJ, White RR. A comparison of 2.0% chlorhexidine gluconate and 5.25% sodium hypochlorite as antimicrobial endodontic irrigants. J Endod 1994;20:276-8.
Marley JT, Ferguson DB, Hartwell GR. Effects of chlorhexidine gluconate as an endodontic irrigant on the apical seal: Short-term results. J Endod 2001;27:775-8.
Georgopoulou M, Kontakiotis E, Nakou M. In vitro
evaluation of the effectiveness of calcium hydroxide and paramonochlorophenol on anaerobic bacteria from the root canal. Endod Dent Traumatol 1993;9:249-53.
Lui JN, Sae-Lim V, Song KP, Chen NN. In vitro
antimicrobial effect of chlorhexidine-impregnated gutta percha points on Enterococcus faecalis. Int Endod J 2004;37:105-13.
Berber VB, Gomes BP, Sena NT, Vianna ME, Ferraz CC, Zaia AA, et al
. Efficacy of various concentrations of NaOCl and instrumentation techniques in reducing Enterococcus faecalis within root canals and dentinal tubules. Int Endod J 2006;39:10-7.
Wang Z, Shen Y, Haapasalo M. Effectiveness of endodontic disinfecting solutions against young and old Enterococcus faecalis biofilms in dentin canals. J Endod 2012;38:1376-9.
Mohammadi Z. Yazd. Sodium hypochlorite in endodontics: An update review. Int Dent J 2008;58:329-41.
Behl H, Sidhu O, Kumar V, Singh D, Saimbi C. Efficacy of Neem Active Metabolite for Prevention of Dental Plaque and Gingivitis. Neem Foundation; 2002.
Botelho M, Santos AD, Martins J, Carvalho C, Paz M, Azenha C, et al
. Efficacy of a mouthrinse based on leaves of neem in the treatment of patients with chronic gingivitis. J Med Plants Res 2008;2:341-6.
Kizhedath A, Suneetha V. Estimation of chlorophyll content in common household medicinal leaves and their utilization to avail health benefits of chlorophyll. J Pharm Res 2011;4:1412-3.
Rajasekaran C, Meignanam E, Vijayakumar V, Kalaivani T, Ramya S, Premkumar N, et al
. Investigations on antibacterial activity of leaf extracts of Azadirachta indica A.Juss (Meliaceae): A traditional medicinal plant of India. Ethnobot Leaflets 2008;12:1213-7.
Kraus W. The Neem tree: Schmutterer H, editor. Source of Unique Natural Products for Integrated Pest Management, Medicine, Industry and Other Purposes. Germany: VCH Weinheim 1995. p. 35-88.
Weir TL, Park SW, Vivanco JM. Biochemical and physiological mechanisms mediated by allelochemicals. Curr Opin Plant Biol 2004;7:472-9.
Kouassi Y, Shelef LA. Inhibition of lysteria monocytogenes
by cinnamic acid: Possible interaction of the acid with cysteinyl residues. J Food Safety 1998;18:231-42.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]