Abstract. The purpose of this experiment was to determine the effect of herbal decoctions on indigenous oral microbiota. Decoctions of Salvia officinalis, Thymus vulgaris, Tanacetum vulgare, Mentha piperita, and Oreganum vulgare were prepared according to traditional herbal recipes. These aqueous solutions were then tested for their activity against a mixed culture of microbes which was established by sampling ten members of the local human population. A simple disk-diffusion method was employed. Overall, the decoctions exhibited a positive effect on the bacteria by increasing colony growth. Increased growth was determined by inflated colony size and number. Elevation of growth of normal bacterial flora by phytochemicals could have an effect on disease causing organisms. Through competition, a healthy indigenous microbe population could aid its host organism in combating infection.

The effects of herbal compounds and phytochemicals on pathogenic and economically important bacteria have been well studied. For example, Sato et al. have illustrated the antibacterial properties of methanolic compounds on cariogenic bacteria (1996). Many other researchers have conducted similar studies on other plants and microorganisms (Khalid et al. 1997, Binutu 1997, and Sampietro et al. 1997). Additionally, the effects that antibiotics have on the indigenous microbiota of the human body are well known. Generally, antibiotics have the same effect on normal flora as on the pathogens they are meant to inhibit (Haahr-Vera et al. 1997).

Indigenous oral microbiota commonly include such genera as Staphylococcus, Stomatococcus, Streptococcus, Eubacterium, Lactobacillus, Camphylobacter, Porphyromonas, and many others (Leung et al. 1998). At this time, the effects of herbal compounds on indigenous microbiota have not been extensively studied. Also, when evaluating the efficacy of phytochemicals concerning the growth of pathogenic and economic bacteria, extracts of the herbs typically have been used. These are normally obtained by means of steam-distillation, dichlorIomethane extraction (Laenger et al. 1996), maceration, sonication, Soxhlet, and supercritical fluid extraction with hexane (Vilegs-Janete et al. 1997). These types of preparations are normally unavailable to persons in a domestic setting for purposes of self-medication. Instead, aqueous solutions (e.g., infusions and decoctions) that are prepared in the home according to recipes found in herbals (e.g., a work which details common uses and properties of herbaceous plants) are most frequently used. Certainly, the compositions and concentrations of compounds within these distinct types of preparations would differ (Lienert et al. 1998, Oszagyan et al. 1996, Ross-Samir et al. 1996, Laenger et al. 1996, Vigiles-Janete et al. 1997, and Nass-Reinhold et al. 1998). The efficacy of an herbal remedy may in turn be altered depending on the preparation method employed. Given these considerations, the effects of five traditionally prepared aqueous herbal solutions on the growth of microorganisms commonly found in the human oral cavity were studied. Salvia officinalis (sage), Thymus vulgaris (thyme), Tanacetum vulgare (tansy), Mentha piperita (peppermint), and Oreganum vulgare (oregano) were the herbs under consideration in this study.

Plant Collection and Storage:

Herbal material was collected between the first of June and the last day of August, 1998. Only aerial parts (leaves, stems, flowers, shoots) were included. The plant material from each species was then placed in separate one-gallon storage bags and frozen until the time of the experiment.

Establishment of a Mixed Indigenous Microbe Culture:

Microbes were collected, using dacron swabs, from the buccal cavities of ten Middle Tennessee State University students of varying age, sex, tobacco use, and ethnicity. These were mixed in a tryptic soy broth tube and the entire culture was incubated at 37^oC for 48hrs.

Preparation of Herbal Decoctions:

Decoctions of sage, thyme, tansy, oregano, and peppermint were prepared by boiling 1/4 cup (approximately 59.1mL by volume) of thawed plant material in 1 cup (approximately 236.6mL) of deionized, distilled water for 5 minutes. The vessel containing the decoction and herb was then covered and removed from the heat and allowed to cool for 5 minutes. The plant material and liquid was then strained through cheese cloth and the resulting decoction placed into 100mL reagent bottles which had been autoclaved.

Inoculation and Experimentation:

Each tryptic soy plate was inoculated with the established mixed culture so as to obtain confluent growth. Next, three blank filter paper circles which had been soaked in different decoctions or the control (deionized, distilled water) were placed equidistant from one another and from the edges of each plate. Care was taken to randomize the combinations of treatments per each plate to reduce the possibility of interactions between treatments. Additionally, a plate was cultured directly from each of the decoctions to check for contamination. All plates were then incubated for 48hrs at 37^oC.

Evaluation of Plates:

After incubation, the zones of increased growth around each filter paper disk were measured and the results reported in millimeters. A zone was considered to begin at the edge of the filter paper and end at the margin of increased colony growth. Observations on colony appearance and number were recorded. Finally, statistical analysis was carried out using the Kruskal-Wallis test.

Variable colony morphologies indicated the presence of several different microbes in the mixed culture tube. This was taken to be representative of the normal flora that can be found in the general population.

All plates used to check for contamination of the decoctions and the control were negative for growth. No contamination of the treatments is said to have occured.

The most striking result of this experiment was the zones of increased growth around some of the filter paper circles which had been treated with sage, tansy, oregano, and thyme decoctions. Inhibited growth or no effect at all is typically expected. Only the control and peppermint treatments failed to show increased growth around at least some of the filter paper circles (Table 1). The greatest of these was oregano, which exhibited an average zone of increased growth of 6.4mm (Figure 1). Oregano was followed by tansy (3.95mm), sage (1.65mm), and thyme (1.05mm) in descending magnitude of effect. The effect of the decoctions on the microbes was found to be statistically significant (corrected H = 59.099, c² = 11.07, P < 0.05). Judging from the data, the effect was in a positive direction with respect to colony size, number, and overall health.

In a study such as this, a few short comments concerning the methods employed are needed. First, cup measurements were used as opposed to the more scientific metric ones because this is the manner in which the recipes are described in traditional herbals. Second, recipes for herbal decoctions vary slightly from reference to reference. In an effort to create a preparation that would be representative of many different ones in common use today, an approximate "average" of several different recipes was used. Third, the specific identities of the microbes in the mixed culture were not determined because this information was not considered especially relevant to the study, especially considering the great variety of microbes that were apparent. The mixed culture was thought to be fairly representative of the normal flora present in the general population. Fourth, the plant material was collected over the course of the summer in an effort to ensure the representativeness of the constituents contained therein. Many studies have illustrated the differences in composition and concentrations of phytochemicals among plants of the same species that are collected at different times of the year or in different geographic and climatic conditions (Laenger et al. 1996, Marotti et al. 1994, McGimpsey et al. 1994, Piccaglia and Marotti 1993, Saez 1995, Svoboda and Deans 1992).

The growing concern over the advent of increasingly antibiotic resistant organisms has prompted many individuals to look for alternative treatments. One such alternative is herbal remedies, which are being marketed with mounting zeal. However, many are skeptical concerning the efficacy of these remedies. Hence, researchers have been testing herbs for activity against pathogenic organisms. The antibacterial effects of thyme (Alippi et al. 1996 and Tabak et al. 1996), oregano (Plaster et al. 1995 and Sivropoulou et al. 1996), sage (Vokou et al. 1993 and Pavlenko et al. 1989), peppermint (Tassou et al. 1995) have all been documented. Invariably, these studies have been carried out on pathogenic bacteria. It would be expected that the activity of these herbs against indigenous microbiota would be similar- that is, detrimental. However, the results of this study point to the opposite effect. If this effect is shown to be consistent after repeated experimentation, many questions about the nature of herbal remedies could be raised. Why would herbal compounds increase the growth of indigenous microbiota but inhibit that of pathogenic bacteria? Possibly, the phytochemicals in herbs are similar to those which would be found in our regular diet and as such would not inhibit the growth of indigenous microbes due simply to their continued exposure to such compounds. If herbal remedies are in fact found to be effacious in combating disease, perhaps this is due more to aiding normal flora as opposed to hindering pathogens. In that instance, disease alleviation through the use of herbs would be a classic matter of competition between bacteria as opposed to a drug-pathogen interaction. Indeed, a few studies have recently described the effects that some normal flora organisms have on pathogenic bacteria (Fujimori et al. 1996 and Vieira et al. 1998). These studies show that a healthy bank of indigenous microbiota helps to fight infection.

Certainly, further study is indicated here. Different forms of herbal remedies (infusions, tinctures, pills, syrups, salves, etc.) should be tested as well as different recipes for decoctions. Also, these preparations should be tested on each indigenous organism separately to determine their individual effects.

I would like to thank the following for their invaluable help and advice: Dr. Stephen Howard, Dr. Philip Mathis, Dr. Stephen Wright, Becca DuBose, Lori Ray, Curtis Scott, Candice Matheny, Mindy Sands, John Addison, and Magnus Evertson. Each contributed in multifarious and essential ways.

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