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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, NY1; SUNY at Buffalo Honors College, Buffalo, NY2, Chapman University, CA2 ,University of California, Berkeley, Berkeley, CA3,
and Universidad Nacional de Trujillo, Trujillo, Peru4, Columbia University, New York, NY5
Lauren Carnevale1,2, Emily Frisch3, Abelardo Arellano4, Jessica Oritz4, Thomas Cleland1, Kristen Brooks1,2, Giselle Rodriguez4, Emily Bakaj5
Gail R. Willsky1
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:
ACKNOWLEDGEMENTSThe 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 foreach 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 (IC50) 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 IC50 values that are too high to be
therapeutically relevant (IC50 ≥ 10 mg/mL). Ideally, IC50 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 Manayupa0.38 ± 0.05 (7) 29 ± 4 (8)
Phyllantus niruri Chancapiedra
0.15 ±0.02 (8) 18 ± 3 (8)
Picrosia longifolia Achicoria13 ± 1 (12) NI: 0.0635 - 16.250 (4)
Uncaria tomentosa Uña de Gato0.56 ± 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%
Gro
wth
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
% G
row
th
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
% G
row
th
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
% G
row
th
Resuspended Extract Conc. (ug/mL)
Figure 7H: Individual Curves (Screening) –(222)Manayupa E.coli
0102030405060708090
100
0 10000 20000 30000 40000
% G
row
th
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
% G
row
th
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
% G
row
th
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
% G
row
th
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 IC50 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 IC50 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 IC50). This observation shows bias between bacterial strains
that are incubated with plant extracts of varying concentrations.
Analysis of IC50:
0
20
40
60
80
100
120
140
0 5000 10000 15000 20000 25000
% G
row
th
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
% G
row
th
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 IC50 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 + 110.8585859R² = 0.9060355
y = -0.007x + 101.26R² = 0.7909
y = -0.0086281x + 132.0707071R² = 0.9976287y = -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
% G
row
th
Resuspended Extract Conc. (ug/mL)
Figure 8B: Individual Curves (Screening) – (281) Borraja S.aureus modified curve
ATubes:
BTubes:
CTubes:
DTubes:
y = -0.0072381x + 105.9941520R² = 0.9203607
y = -0.007x + 100.66R² = 0.8419 y = -0.0070452x + 112.4933546
R² = 0.8956089y = -0.0071496x + 114.4271664R² = 0.8921032
-20
0
20
40
60
80
100
120
140
0 5000 10000 15000 20000
% G
row
th
Resuspended Extract Conc. (ug/mL)
Figure 8A: Individual Curves (Screening) – (281)Borraja S.aureus unmodified curve
ATubes:
BTubes:
CTubes:
DTubes:
Figure 8C