Investigating Anti-microbial Properties of Sage

Indigenous people across North America, including the Cree nations across Manitoba have used Sage for millennia as a sacred medicine. It's Uses range from spiritual cleansing through smudging to physical cleansing and healing through teas and poultices made from it. It's also held by many indigenous practitioners that smudging air and surfaces can also kill microbes. In my community and within our culture we use Sage as one of the three sacred medicines, along with Sweetgrass (Hierochloe Ordorata) and Cedar (Juniperus Sp.,) for smudging. We also use Sage oils and extracts for air purification. The knowledge of this plant has accumulated over hundreds of years of observations, trial and error, inference and hypothesis testing; and handed down for countless generations, from knowledge keepers to knowledge keepers (elders).

In this project I set out to use some of the modern science and laboratory tools available to me to investigate the claims about the value of using Sage as a medicine and a cleanser, by testing experimentally it's anti-microbial ability. while there are at least nine varieties of sage reported, this study will focus on white sage (Salvia Apiana), most used in our smudging ceremonies, and as extracts and oils for cleansing.

Four knowledge keepers were consulted during this study, two of whom practice both traditional and western medicine at the Winnipeg Regional Health Authority, in Manitoba.

The input of the traditional knowlegde keepers in this investigation provided me with a solid understanding of the scientific contribution made by our first scientists.

Methodology

Materials:

Glassware, centrifuge+tubes, Spread Sticks, Pipettes+tips, Pseudomonas Sp., Escherichia coli, Streptomyces sp., kanamycin, ethanol, olive oil, Nutrient agar, Sage leaves, bees'wax.

Procedure:

Ethanol Extraction: Add 2.5g of graded sage leaves to 25mL of ethanol.

Olive Oil: Add 57g of leaves to 125mL of olive oil, let sit for three days, heat for two days at 68-79°C

Olive oil+Bees'wax: Identical extraction. Add slowly 16g of bees'wax till dissolved. Incubate in shaker at 37°C, 150 rpm overnight.

Revive cultures and incubate at 30°C, 150 rpm overnight.

Autoclave Nutrient agar and discs at 15 lb pressure and 121°C for 60 min.

Centrifuge 50 ml extract tubes tubes at 12000 rpm for 5 min.

Pipette 1 ml out of each extract, place into 1.5 ml centrifuge tubes.

Transfer 100 microliters of bacterial suspension (Streptomyces sp., Pseudomonas sp., and E.coli) onto plated petri dishes.

Spread liquid bacterial cultures evenly.

Smoke (smudging) Test:

Hold plates with bacteria above burning sage for 1 min, and incubate overnight at 30°C.

Disc and well diffusion methods:

Place sterilized diffusion discs in each extarct of bacterial type (Streptomyces sp., E.coli, Pseudomonas sp.), and plate samples clockwise (2-7). Kanamycin(1) at the center.

Sage extract and ethanol, extracted olive oil, and extracted Olive +bees'wax (2-4).

Controls as only ethanol, Olive oil, and Olive oil+bees'wax(5-7).

For well diffusion, bore wells with with a sterilized borer on the agar.

Add 20 microliters of sample extracts and controls as the disc diffusion method.

Incubate plates overnight at 30°C, and measure the inhibition zone in cm.

Smoking Test

All three trials showed positive results for the presence

of antibacterial properties of burned sage for all three bacteria (Streptomyces sp.,Escherichia coli , and Pseudomonas sp). Burned sage was more effective against streptomyces species, creating larger zones of inhibition compared to the other specimens.

Disk Diffusion Method

Streptomyces sp.

Sufficient results for all three extractions in both trials of investigation 2.

Sage leaves in olive oil worked best against Streptomyces sp.

Pseudomonas sp.

Antibacterial effectiveness shown for all three extractions in the second trial of investigation 2. However, the first trial showed more variability and no inhibition zone for the olive oil and wax extraction. Again, olive oil seems to work best, this time against Pseudomonas sp.

Escherichia coli

All three extracts resulted in inhibitions zones, meaning the antibacterial properties of sage when submersed in these extractions are not effective at stopping the growth of E. coli.

Well Diffusion Method

Streptomyces sp.

Using wells, the ethanol extraction containing sage leaves showed the greatest results of inhibiting the growth of Streptomyces sp., in both trials of investigation 2. Compared to the disc diffusion method, diffusing using wells created smaller zones of inhibition.

Pseudomonas sp.

No zones of inhibition for the first trial of investigation 2 for ethanol and olive oil extractions. Small area of clearing for the olive oil + wax extraction in the first trial. In the second trial of investigation 2, all three extractions showed similar sizes of inhibition zones, meaning all three had effectiveness against growth of pseudomonas sp. The difference between the first and second trial could be due to differences in the technique of creating the wells.

Escherichia coli

When diffusing using wells, only the ethanol extract showed a zone of clearing when it was tested against E. coli. There was no inhibition using olive oil and olive oil + wax extractions, like what resulted using the disc diffusion method.

(The graph for this piece will be available at the display board)

Discussion and Conclusion:

According to the results obtained in the smudging test, there is an expression of anti-microbial activity by the three bacterial samples. Streptomyces sp. holds the highest evidence of it, with the largest diameter zones of inhibition. This confirms my hypothesis that white sage leaves smudge has antimicrobial properties.

As for the three different types of extraction solvents, olive oil demonstrated a strong action against streptomyces sp., and Psuedomonas sp. On the E.Coli culture , olive oil did not produce results in either methods (disc diffusion and well diffusion).

The results from the extractions doesn't agree with my hypothesis that alcohol was the best solvent for the sage leaves extraction.

What is Next:

Throughout the period of my investigation, many of my questions were answered, but much more were raised and needed to be answered.

For my next steps in this research, I would like to extend my investigation by preparing extractions using bear fat, and geese fat as solvents.

In addition, I wish to run tests with more strengths of disease-causing bacteria against the different types of extractions and smoke exposure.

Furthermore, I wish to investigate the efficacy of oils and ointments with sage extracts.

I would like to thank the following people:

Dr. Levin provides me with the opportunity to work in his lab to pursue my investigation.

Mrs. Sarita Shresha, who worked very hard teaching and guiding me throughout my investigation at Dr. Levin's laboratory.

Ms. Shayla Fossen for support during my visits to the laboratory at the University Of Manitoba.

Elders: Cathy Bird, Cari Bird, Dan Thomas, Vanessa Lillie, Ronnie Beardy, William Osborne, and Elaine Beardy, for the foundational information provided on the legacy of indigenous cultures.

References:

Cordova-Guerrero, I., Aragon-Martinez, O.H., Diaz-Rubio, L., Franco-Cabrera, S., Serafin-Higuera, N.A.; Pozos-Guillen, A.; Soto-Castro, T.A.; Martinez-Morales, F.; and Isiordia-Espinoza, M. Actividad Antibacteriana y Antifungica de UN Extracto de Salvia apiana frente a Microorganismos de Importancia Clinica. Revista Argentina de Microbiologia. 2016;48(3);217-221.

Geertsma, I.P., Zandstra, B. E., Stefanaki, A., and van Andel, T.R. Commercialized "smudge Sticks" Used as Incense in the Netherlands: An Inventory of Plants and Trends Behind a New Age Fashion. Plants 2024, 13,3003. (https://mdpi.com/journal/plants)

Conde-Hernandez, L.A.; Luna-Guevara, M.L; Luna-Guevara, J.; Perez-Vazquez, J.; and Aranda-Garcia, R.J. Mexican Sage (Salvia Officinalis) Extraction Using Factorial Design and Its Effect on chemical ad Antibacterial Properties. Journal of chemistry, 2021

Elders: Carl Bird and Cathy Bird from Peguis First Nations in Manitoba. Both practice traditional and western medicine, and work at the Winnipeg Regional Healthy Authority.

Dan Thomas, a Midewiwin Knowledge keeper.

Vanessa Lille, a First Nation Knowledge keeper at the University of Manitoba.

William Osborne, Knowledge Keeper at Cross Lake, Manitoba.

Ronnie and Elaine Beardy, knowledge keepers at Cross Lake, Manitoba.