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RESEARCH POSTER PRESENTATION DESIGN © 2012 www.PosterPresentations.com OBJECTIVE: In order to compare the treatment used by the curanderos (native healers) with Western medicine practice, the antibacterial activity of plant extracts (Acanthoxanthium spinosum, Borago officinalis, Desmodium mollicum, Phyllantus niruri, Picrosia longifolia, Uncaria tomentosa, and Mentha spicata) was measured.. HYPOTHESIS: The plant extracts will have antibacterial activity. METHODS: Plants were purchased, dried, and ground. Alcohol extracts were prepared, concentrated, dried, re-suspended in boiling water, and sterilized. Bacterial growth inhibition against S.aureus and E.coli was measured spectrophotometrically at various extract concentrations. RESULTS: Data was normalized to percent growth and IC50 values were calculated. Plant extracts were more efficacious against S. aureus than E.coli. The IC50 of plants used for infections against S.aureus was 0.15-6.5 mg/mL. Two of these had IC50 values against S. aureus <1 mg/mL. CONCLUSION: Antibacterial activity was observed in all plants. Further study of plants with high anti-bacterial activity could identify new antibiotic compounds. ABSTRACT BACKGROUND REFERENCES SUNY at Buffalo SMBS, Buffalo, NY 1 ; SUNY at Buffalo Honors College, Buffalo, NY 2 , Chapman University, CA 2 ,University of California, Berkeley, Berkeley, CA 3 , and Universidad Nacional de Trujillo, Trujillo, Peru 4 ,Columbia University, New York, NY 5 Lauren Carnevale 1,2 , Emily Frisch 3 , Abelardo Arellano 4 , Jessica Oritz 4 , Thomas Cleland 1 , Kristen Brooks 1,2 , Giselle Rodriguez 4 , Emily Bakaj 5 Gail R. Willsky 1 Antibacterial Activity of Medicinal Plants Used to Treat Infectious Disease in Northern Peru Northern Peru is thought to be the center of the “Central Andean Health Axis,” an area with a rich supply of potential pharmaceutical plants stretching from Ecuador to Bolivia. 1 With its diverse climates (Fig. 1) there has been a high degree of biodiversity (Figure 2) among the flora native to Northern Peru. Native healers of Northern Peru have a long history of using plants to combat infectious disease. 3 Traditional medicine is still practiced in Northern Peru and is a critical component of daily life. Since the late 1900s, hundreds of plants used by the curanderos have been taxonomically classified. 5 In addition, many of these plants have been identified for use in medicine to treat a variety of conditions including bacterial infections. Today, modern technology has allowed scientists, to explore the chemical composition of medicinal plants. This project was designed to study plants for the treatment of diseases believed by allopathic medicine to be caused by bacterial infections. Through the evaluation of antimicrobial activity of selected plant extracts and plant mixture extracts, we were able to observe antimicrobial activity of plants used by curanderos to treat infectious disease. Figure 1: Research area and ecosystems of Peru. 1 Figure 2: Plant biodiversity of northern South America. 1 Figure 3: Number of patients treated with plants at a Peruvian medical clinic. 3 METHODS RESULTS Antimicrobial Activity of Plant Extracts: ACKNOWLEDGEMENTS The study was originated by Drs. Rainer Bussman (Missouri Botanical Garden) and Douglas Sharon (anthropologist) as a training site for an NIH MHIRT grant. Drs. Gail Willsky (Biochemistry) of UB School of Medicine and Biomedical Sciences (SMBS) and Douglas Sharon were co-directors of the MHIRT lab. Dr. John Crane (Infectious Disease and Microbiology) of UB SMBS was the infectious disease consultant. Dr. Bussmann was the ethnobotany consultant. Manuel Isaías Vera Herrera and Prof Alberto Eduardo Quezada Alvarez arranged for us to work in the Chemical Engineering Faculty of the Universidad Nacional de Trujillo (UNT) and Mario Alva and José Alfredo were our links to the Chemistry Department there. Major funding for this project is through a NIH Minority Health Initiative Training Grant (MHIRT) awarded to Dr. Dena Plemmons at San Diego State University. Jessica Oritz, Giselle Rodriguez, Abel Arellano, and Maria Perez were MHIRT students. Inés Yolanda Castro was the prior MHIRT lab manager. Alejandro Piña Iturbe was the 2015 lab manager. A special thank you to the University of Buffalo’s Honors College for funding my trip to Peru through the Research and Creativity Fund. Concentration-response curves were used to show the effects of various plant extract concentrations on the bacterial growth of E.coli and S.aureus. In all 12 experiments of Figure 7, bacterial growth was seen at lower extract concentrations, and in all experiments, bacterial inhibition was seen at higher extract concentrations. Bacterial growth at lower extract concentrations is thought to be due to the natural chemical nutrients in plants which stimulate the growth of bacteria rather than inhibit their growth. Achicoria (7B and 7G), Mentha (7D and 7I), and Alanso (7E and 7J) are said to be noninhibitory (NI) since their concentration-response curve did not show any inhibition of bacterial growth. In all 10 experiments, plant extracts screened against S.aureus showed a greater inhibition of bacterial growth at lower extract concentrations then their corresponding experiments in E.coli. RESULTS and DISCUSSION Plant Collection: Voucher specimens used in this study were purchased at a local market (Fig. 4), identified by botanists, and stored at the Universidad Nacional de Trujillo, Peru and the Missouri Botanical Garden, MO, USA. Figure 4: A plant vender’s stall in Chiclayo, Peru. Extract Preparation: Plants purchased from the market were dried, ground, and incubated in 1L ethanol for one week to extract chemical components. Extracts made from single plants contained 50g of plant material in 1L of ethanol. Extracts were then filtered to remove plant debris, dried by rotary evaporation, transferred into a glass vial, and stored at -30°C. As needed, frozen extracts were thawed, resuspended in boiling water, and filter sterilized. . Extract dry weights were used to determine concentration. Figure 5: A hand-crank maize grinder was used to homogenize dried plants. Bacterial Growth Assay (BGA): Bacterial strains Escherichia coli ATCC 25922 and a clinical isolate of Staphylococcus aureus were obtained in Peru. A single colony was inoculated into 2 mL of Müeller-Hinton Media and incubated for 24 hours at 37°C. Fresh overnight bacteria were prepared for each experiment from this master stock which was made fresh each week. Data were normalized to percent survival or percent growth, in comparison to negative controls (without extract). Data were then plotted as concentration-response curves and the section of the curves showing maximum change was fit to a line (y=mx+b). The concentration which caused 50% inhibition of bacteria (IC 50 ) were calculated where y=50. [1] Bussmann, R.W.; Glenn, A.; Meyer, K.; Kuhlman, A.; Townesmith, A. Herbal mixtures in traditional medicine in Northern Peru. Ethnobiology and Ethnomedicine. 2010, 6-10. [2]. Bussmann, R.W.; Sharon, D. Shadows of the colonial past-diverging plant use in Nothern Peru and Southern Ecuador. Ethnobotany and Ethnomedicine. 2009, 6. [3]. Bussmann, R.W.; Sharon, D.; and Lopez, A. Blending Traditional and Western Medicine:Medicinal plant use among patients at Clinica Anticona in El Porvenir, Peru. Ethnobotany Research & Application. 2007, 5, 185. [4] Bass MS, Finer M, Jenkins CN, Kreft H, Cisneros-Heredia DF, et al. Global Conservation Significance of Ecuador’s Yasuní National Park. PLoS ONE 5(1): e8767. doi: 10.1371/journal.pone.0008767 (2010). [4]. Caner, H.; Groner, E.; Levy, L. Trends in the Development of Chiral Drugs. Drug Discovery Today. 2004, 9, 105. [5.] McManis, C. R. Biodiversity and the law: Intellectual Property, Biotechnology andTraditional Knowledge; London: Earthscan, 2008. [6.] The University at Mississippi School of Pharmacy: What Is Pharmacognosy? http://www.pharmacy.olemiss.edu/pharmacognosy/ (accessed February 28, 2014). CONCLUSIONS The plant extracts that were determined to be NI have IC 50 values that are too high to be therapeutically relevant (IC 50 ≥ 10 mg/mL). Ideally, IC 50 values are minimized (i.e., a lower concentration is needed to kill bacteria). • Antibacterial activity was seen in all of the plant extracts, used by the curanderos to treat infectious disease • Antibacterial activity for plants used to treat infectious disease by the curanderos show similarity to allopathic medicine therapies Health Condition Number of Respondents Cough/Cold related 44 Digestive 34 Headache/pain 28 Kidneys 18 Infections 14 Bones 11 Respiratory (bronchios) 10 Data Analysis: Plant extracts were diluted 1:2 into media, and the diluted extracts were serially diluted (1:4). Each dilution is subsequently aliquoted into four tubes to give an n=4. The bacteria were added to each tube. Controls are prepared for media alone, for extract alone, for total growth with no extract, and with an antibiotic for growth inhibition. Next 4 steps are visualized below. Samples are centrifuged & decanted Samples are resuspended in 0.9 NaCl Spectrophotometer used to take OD_600 readings of the serial diluted plant extracts against bacteria Samples after 24-36 hrs incubation at 50°C Latin Botanical Name Common Name IC50 (mg/mL) S.Aureus IC50 (mg/mL) E.coli Acanthoxanthium spinosum Alanso 18.3 ± 0.3 (4) NI: 0.0376-9.8438 (8) Borago officinalis Borraja 7.0 ± 2.0 (8) 20.7 ± 0.2 (4) Desmodium mollicum Manayupa 0.38 ± 0.05 (7) 29 ± 4 (8) Phyllantus niruri Chancapiedra 0.15 ±0.02 (8) 18 ± 3 (8) Picrosia longifolia Achicoria 13 ± 1 (12) NI: 0.0635 - 16.250 (4) Uncaria tomentosa Uña de Gato 0.56 ± 0.01 (8) 9.6 ± 0.1 (4) Mentha spicata Menta 5.0 ± 0.1 (8) NI: 0.0261-8.1927 (8) -20 0 20 40 60 80 100 120 0 5000 10000 15000 % Growth Resuspended Extract Conc. (ug/mL) Figure 7A: Individual Curves (Screening) – (276) Uña de Gato S. aureus -20 0 20 40 60 80 100 120 0 2000 4000 6000 8000 10000 12000 14000 % Growth Resuspended Extract Conc. (ug/mL) Figure 7F: Individual Curves (Screening) – (279) Uña de Gato E.coli 0 50 100 150 200 250 0 1000 2000 3000 4000 5000 % Growth Resuspended Extract Conc. (ug/mL) Figure 7G: Individual Curves (Screening) – (275) Achicoria E. coli 0 20 40 60 80 100 120 140 160 180 0 10000 20000 30000 40000 % Growth Resuspended Extract Conc. (ug/mL) Figure 7H: Individual Curves (Screening) –(222) Manayupa E.coli 0 10 20 30 40 50 60 70 80 90 100 0 10000 20000 30000 40000 % Growth Resuspended Extract Conc. (ug/mL) Figure 7C: Individual Curves (Screening) – (223) Manayupa S.aureus 0 20 40 60 80 100 120 140 0 2000 4000 6000 8000 % Growth Resuspended Extract Conc. (ug/mL) Figure 7D: Individual Curves (Screening) – (256) Mentha S.aureus 0 20 40 60 80 100 120 140 0 2000 4000 6000 8000 % Growth Resuspended Extract Conc. (ug/mL) Figure 7I: Individual Curves (Screening) – (257) Mentha E.coli Figure 7. Plant extracts of Uña de Gato (A,F), Achicoria (B,G), Manayupa (C,H), Mentha (D,I), and Alanso (E,J) were tested against S. aureus (A-E) and E.col (F-J). Numbers in parentheses are used to indicate specific experiments performed by the lab. 0 20 40 60 80 100 120 140 0 1000 2000 3000 4000 5000 % Growth Resuspended Extract Conc. (ug/mL) Figure 7B: Individual Curves (Screening) – (272) Achicoria S.aureus Figure 8: (A) Unmodified screening curves for the inhibition of S. aureus growth by Borraja. (B) IC50 values were calculated by modifying the initial curve to contain a negative slope. The line of best fit for the modified graph is shown. (C) IC50 values were calculated by interpolating the concentration that results in 50% growth, using the line of best fit The IC50 values for each trial were then averaged and the standard deviation was calculated. Table 1 shows the IC 50 values of the two plant sets. The same volume of resuspended plant extract was used for each set to eliminate potential confounding variables. For Manayupa, it is important to note that only 3 replicates were used due to the absence of a S.aureus pellet post- centrifugation. The concentration-response curve for Chancapiedra against S.aureus had to be modified twice to determine the corresponding IC 50 values. According to Table 1, all plant extracts tested had at least one bacterial strain that it could inhibit. For all of the plant extracts there was a much lower concentration required to inhibit the growth of S.aureus than required to inhibit the growth of E.coli (lower IC 50 ). This observation shows bias between bacterial strains that are incubated with plant extracts of varying concentrations. Analysis of IC 50 : 0 20 40 60 80 100 120 140 0 5000 10000 15000 20000 25000 % Growth Resuspended Extract Conc. (ug/mL) Figure 7E: Individual Curves (Screening) – (231) Alanso S.aureus 82 84 86 88 90 92 94 96 98 100 102 0 2000 4000 6000 8000 10000 12000 % Growth Resuspended Extract Conc. (ug/mL) Figure 7J: Individual Curves (Screening) – (230) Alanso E.coli To solve the linear equation y = mx + b, where y = 50: Variables Constants A B C D m -0.0076310 -0.007 -0.0086281 -0.0087814 b 110.859 101.260 132.071 134.596 x (IC50) (ug/mL) = Resupended extract conc. at 50% growth Mean SD 7975 7323 9512 9634 8610.9 1143.24 Figure 6: Steps used in monitoring bacterial growth TABLE 1: Summary of Extract IC 50 values against S.aureus and E.coli Numbers in parentheses are meant to show either n=4, 7, 8 or 12. Plant extracts that did not inhibit growth (non-inhibitory) at the between the indicated concentrations are marked as “NI”. y = -0.0076310x + 110.8585859 R² = 0.9060355 y = -0.007x + 101.26 R² = 0.7909 y = -0.0086281x + 132.0707071 R² = 0.9976287 y = -0.0087814x + 134.5959596 R² = 0.9960430 -20 0 20 40 60 80 100 120 140 0 2000 4000 6000 8000 10000 12000 14000 16000 % Growth Resuspended Extract Conc. (ug/mL) Figure 8B: Individual Curves (Screening) – (281) Borraja S.aureus modified curve A Tubes: B Tubes: C Tubes: D Tubes: y = -0.0072381x + 105.9941520 R² = 0.9203607 y = -0.007x + 100.66 R² = 0.8419 y = -0.0070452x + 112.4933546 R² = 0.8956089 y = -0.0071496x + 114.4271664 R² = 0.8921032 -20 0 20 40 60 80 100 120 140 0 5000 10000 15000 20000 % Growth Resuspended Extract Conc. (ug/mL) Figure 8A: Individual Curves (Screening) – (281) Borraja S.aureus unmodified curve A Tubes: B Tubes: C Tubes: D Tubes: Figure 8C
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