Journal of Biomedicine and BiotechnologyVolume 2012 (2012), Article ID 106746, 4 pagesdoi:10.1155/2012/106746

Research Article

Cytotoxicity of Selected Medicinal and Nonmedicinal Plant Extracts to Microbial and Cervical Cancer Cells

Gary M. Booth,¹ Robert D. Malmstrom,¹ Erica Kipp,² and Alexandra Paul¹

¹Department of Plant and Wildlife Sciences, Brigham Young University, Provo, UT 84602, USA² The New York Botanical Garden, 200th Street and Kazimiroff Boulevard, Bronx, NY 10458-5126, USA

Received 2 August 2011; Accepted 16 December 2011

Academic Editor: Ikhlas A. Khan

Copyright © 2012 Gary M. Booth et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

This study investigated the cytotoxicity of 55 species of plants. Each plant was rated as medicinal, or nonmedicinal based on the existing literature. About 79% of the medicinal plants showed some cytotoxicity, while 75% of the nonmedicinal plants showed bioactivity. It appears that Asteraceae, Labiatae, Pinaceae, and Chenopodiaceae were particularly active against human cervical cancer cells. Based on the literature, only three of the 55 plants have been significantly investigated for cytotoxicity. It is clear that there is much toxicological work yet to be done with both medicinal and nonmedicinal plants.

1. Introduction

There is a one-in-four chance that a drug used from any pharmacy has an active ingredient derived from a plant [1]. Indeed, the international consumer market for medicinal herbs and botanicals is estimated to be at about US $18 billion [2]. Hence, in our technological age, plants continue to play a significant role both medically and economically.

Even the most ancient written records of human civilization tell of humans using plants in everyday life. For centuries plants have been used to feed, clothe, and heal families. Examples of medicine that contains plant derivatives include aspirin, used for pain relief and inflammation reduction; physostigmine and pilocarpine, used for glaucoma control; quinidine, which has saved the lives of many heart attack victims.

The principal goal of this study was to determine if extracts from selected medicinal and nonmedicinal plants were cytotoxic; often, the difference between a therapeutic and a toxic extract or compound is simply the dose level. Our hope is that these survey data can be used as early indicators of some plants that may have therapeutic activity. Moerman has done extensive screening studies on a variety of medicinal plants [3]. From his investigation, we selected 55 plants representing 37 different species from 8 families. The four principal families, Asteraceae, Labiatae, Ranunculaceae, and Pinaceae, represent the first, third, fourth, and fifth families with the most medicinal species. It was hoped that our data might show some trends of toxicity within medicinally rich families.

The toxicity of each extract was determined in both prokaryotic and eukaryotic cells. Prokaryote cells included Staphylococcus aureus, a gram-positive cocci responsible for infections of the skin and respiratory tract, food poisoning, and toxic shock; Salmonella choleraesuis, a gram-negative facultative aerobe responsible for food poisoning; Pseudomonas aeruginosa, a gram-negative rod that causes infections in wounds. For the eukaryotic system, HeLa cells, an epithelial carcinoma of the cervix, were used.

2. Materials and Methods

2.1. Plant Extraction

(i)50 g of plant tissue were collected and dried at 45°C.(ii)The plant was ground in a Wiley Model no. 4 plant mill.(iii)The ground material was then extracted in methanol for twenty-four hours.(iv)The samples were filtered in glass-fiber filters fitted with coarse pore discs, and rotary evaporated down to 20 mL of extract on a Buchi RE111 Rotary Evaporator.

2.2. Microbial Bioassay

(i)Twenty-four hours before the assay, each of the three bacteria were grown in a culture tube with 5 mL of tryptic soy broth without dextrose and incubated at 35°C.(ii)(14.5 cm) Petri dishes were previously prepared with a coat of Muller Hinton Medium (agar). The cultures were checked on a spectrophotometer to ensure the proper growth (20% transmittance at 600 nm). A lawn was then spread in the petri dish. Six 1.4 cm circles of filter paper were then coated in plant extract, three with 20 μL and three with 30 μL, and placed on the plate. A disk with 20 μL of water was added to the plate for a negative control and to S. aureus, S. choleraesuis, 10 μL of Ampicillin (BBL Sensi-Disc (Becton Dickinson)) was added as a positive control. The plates were incubated overnight at 35°C.(iii)The plates were then collected the next day and the zones of inhibition were measured.

2.3. HeLa Assay

(i)HeLa cells were maintained and assayed in MEM with α modification (Sigma M-0894) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1x MEM-nonessential amino acids (Sigma M-7145), 2 mM L-glutamine, and gentamicin at 50 μg/mL.(ii)Each extract was dried down and 2 mg/mL solutions were made using 10 mM Tris buffer at pH 7.4.(iii)150 μL of a solution of suspended HeLa cells diluted with 15 mL of α-MEM is added to each well of a 96 well plate and incubated overnight at 37°C and 5% CO₂.(iv)The next day 75 μL, 50 μL, 25 μL with 25 μL of α-MEM, 12.5 μL with 37.5 μL of α-MEM, or 6 μL with 44 μL of α-MEM of the 2 mg/mL extracts was added to 9 wells as a control. The prepared plate was incubated overnight.(v)The cells were arrested the next morning with 0.4 N perchloric acid. The perchloric acid is removed, and the cells were stained in 4% sulforhodamine B in 1% acetic acid and then washed in 1% acetic acid. The dye was allowed to dry and 150 μL of 10 mM Tris base unbuffered was then added to each well, and the absorbance of each well was read using a spectrophotometer at 570 nm.(vi)The percent viability was calculated as the ratio of absorbance of the treated sample over the average of the controls. These values were then plotted and analyzed for a dose response.

3. Results

3.1. Microbial Assay

Of the 55 plants tested, only four, Pinus monticola, Abies procera, Salvia vaseyi, and Salvia apiana, inhibited the growth of S. aureus. The remaining microorganisms were unaffected by the extracts. However, the zones of inhibition were quite small, only about 1 cm each. The assay is rather a crude test when compared with the HeLa cell assay. This is understandable because the zone of inhibition is directly proportional to the concentration of the biologically active agent and its diffusibility, so the possibility of active compounds not showing a positive response could be expected if the active ingredients did not diffuse. Due to the screening nature of this procedure and small sample size, the quantitative analysis of the size of the rings of inhibition was quite subjective.

3.2. HeLa Cell Assay

The LC-50 s were calculated for each of the samples. Some of the extracts were so toxic to the HeLa cells that very low doses of 0.0l and 0.001 mg/mL were studied in order to establish an LC-50. The LC-50 s were calculated from least squares regression using the LINEST function on Microsoft Excel 2000 over the dose response range or the whole data set in the case of nontoxic extracts to get a rough quantitative value in order to assess cytotoxicity. Tris buffer, the control, gave an average 92% viability with no dose response. All values were adjusted up by 8% accordingly.

We experienced four general trends in the data. The first two types we labeled “A” for active. The first type was a clear dose-response over the full range of concentrations. Type two followed a steep dose-response over the initial range of concentrations while the lower concentrations did not. Type two was the most cytotoxic. Type three was labeled with an “M” for mildly active. These showed a weaker dose-response only at the higher concentrations. Type four was labeled “N” for not active. These samples showed no dose response and only marginal mortality. These trends were then evaluated over medicinal and family lines (Table 1).

Table 1: Cytotoxicity of selected plant extracts to bacterial cells and HeLa cancer cells.

Family
Genus
Species
Plant part
MD
BA
HA
LC 50 (mg/mL)
(slope of the line)
(-intercept)

Asteraceae
Acanthospermum
australe
Whole
Y
N
M
0.191
−38.817
57.414
0.112

Asteraceae
Ambrosia
ambrosioides
Areal
Y
N
M
−2.199
−13.656
19.970
0.469

Asteraceae
Ambrosia
ambrosioides
Leaf
Y
N
A
−2.446
−11.722
21.327
0.412

Asteraceae
Ambrosia
ambrosioides
Stem
Y
N
A
0.466
−152.941
121.230
0.857

Asteraceae
Ambrosia
ambrosioides
Root
Y
N
A
0.439
−124.376
104.617
0.817

Asteraceae
Ambrosia
deltoidea
Stem
Y
**
A
0.500
−83.064
91.544
0.293

Asteraceae
Hieracium
caespitosum
Whole
Y
N
M
0.669
−95.238
113.737
0.546

Asteraceae
Anaphalis
margaritacea
Whole
N
N
N
1.195
−51.360
111.373
0.530

Asteraceae
Gutierrezia
microcephala
Areal
Y
N
A
0.470
−143.776
117.641
0.969

Asteraceae
Pyrrhopappus
carolinianus
Whole
Y
N
N
0.117
−1052.510
173.100
0.623

Asteraceae
Silphium
compositum
S/L/Fl/R
Y
N
A
1.388
−49.271
118.400
0.134

Asteraceae
Tetragonotheca
helianthoides
Root
Y
N
A
0.436
−137.978
110.164
0.760

Asteraceae
Tetragonotheca
helianthoides
S/L/Fl/R
Y
N
M
0.357
−157.865
106.395
0.844

Asteraceae
Erigeron
pumilus
Whole
Y
N
A
0.366
−142.337
102.035
0.895

Asteraceae
Liatris
secunda
Whole
Y
N
M
0.531
−156.829
133.257
0.638

Asteraceae
Cirsium
undulatum
Areal
Y
N
M
0.884
−61.281
104.180
0.642

Asteraceae
Thelesperma
filifolium
Areal
Y
N
A
0.635
−99.503
113.172
0.950

Asteraceae
Helianthus
nuttallii
Stem
Y
N
A
0.539
−135.164
122.791
0.755

Asteraceae
Helianthus
nuttallii
Twig
Y
N
A
0.399
−181.986
122.686
0.944

Asteraceae
Haplopappus
annuus
Whole
Y
N
N
0.918
−77.726
121.359
0.803

Asteraceae
Antennaria
parvifolia
Whole
N
N
A
0.273
−81.056
72.160
0.497

Asteraceae
Hymenopappus
filifolius
Areal
Y
N
A
0.112
0.167
38.910
0.611

Asteraceae
Centaurea
maculosa
Twig/Fl
N
N
M
0.247
−113.161
77.895
0.863

Asteraceae
Scorzonara
laciniata
Root
N
N
N
1.838
−28.633
102.630
0.188

Boraginaceae
Echium
candicans
Stem
Y
N
M
0.394
−124.333
99.049
0.853

Chenopodiaceae
Atriplex
confertifolia
Areal
N
N
A
0.127
−1116.726
192.195
0.694

Chenopodiaceae
Atriplex
confertifolia
Rhizome
N
N
A
0.317
−142.791
95.259
0.838

Euphorbiaceae
Bernardi
myicifolia
Stem
N
N
A
0.488
−144.404
120.448
0.863

Labiatae
Salvia
vaseyi
Root
Y
N
A
0.105
−1109.581
166.468
0.981

Labiatae
Salvia
vaseyi
Stem
Y
N
A
0.399
−177.100
120.693
0.783

Labiatae
Salvia
vaseyi
Twig/L
Y
Y
A
0.218
−120.339
76.261
0.537

Labiatae
Salvia
vaseyi
Flowers
Y
N
A
0.231
−103.088
73.816
0.589

Labiatae
Salvia
apiana
Root
Y
Y
N
0.112
−1229.526
187.967
0.765

Labiatae
Salvia
dorrii
L/T/FloBu
Y
N
A
0.512
−125.827
114.434
0.852

Labiatae
Lavandula
stoechas
Root/Fl
Y
N
A
0.110
−1069.264
167.838
0.927

Labiatae
Lavandula
stoechas
Stem/L
Y
N
A
0.440
−196.726
136.532
0.804

Labiatae
Lycopus
asper
Stem
Y
N
A
1.043
−39.514
91.202
0.094

Labiatae
Marrubium
vulgare
Areal
Y
N
A
0.241
−112.266
77.093
0.768

Labiatae
Satureja
douglasii
Whole
Y
N
A
0.293
−102.001
79.891
0.665

Malvaceae
Sphaeralcea
angustifolia
Whole
N
N
A
0.304
−129.152
89.243
0.658

Pinaceae
Pinus
monticola
Bark/St
Y
Y
A
0.346
−175.851
110.896
0.764

Pinaceae
Pinus
monticola
Twig/L
Y
N
M
0.438
−78.452
84.370
0.396

Pinaceae
Pinus
monticola
Root
Y
N
A
0.573
−95.050
104.422
0.606

Pinaceae
Picea
sitchensis
Root
Y
N
N
0.496
−125.394
112.182
0.847

Pinaceae
Picea
sitchensis
Stem
Y
N
M
0.583
−118.928
119.326
0.562

Pinaceae
Picea
sitchensis
Bark
Y
N
M
0.366
−134.091
99.034
0.684

Pinaceae
Picea
sitchensis
Twig/L
Y
N
N
0.526
−121.778
114.038
0.835

Pinaceae
Picea
sitchensis
Cone
Y
N
A
0.438
−59.520
76.046
0.326

Pinaceae
Abies
procera
Root
N
Y
N
0.381
−155.741
109.290
0.978

Ranunculaceae
Delphinium
geyeri
Areal
Y
N
M
1.306
−29.140
88.057
0.173

Ranunculaceae
Aquilegia
fromosa
Root
Y
N
M
1.532
−25.856
89.605
0.222

Ranunculaceae
Aquilegia
fromosa
Flowers
Y
N
N
0.612
−82.675
100.574
0.534

Ranunculaceae
Aquilegia
fromosa
Leaf
Y
N
N
0.521
−143.711
124.894
0.784

Ranunculaceae
Delphinium
glareosum
Whole
Y
N
N
0.824
−91.704
125.548
0.428

Ranunculaceae
Delphinium
nuttallianum
Areal
Y
N
N
1.319
−35.840
97.287
0.350

MD: medicinal plant; Y: medicinal plant; N: non-medicinal plant

BA: bacterial assay; Y: inhibition; N: no inhibition; **sample lost

HA: HeLa cell assay; A: active; M: mildly active; N: not active

Plant part; S/St: stem Fl: flower; FloBu: flowering bush; A: areal R: root; L: leaf; T: twig.

4. Discussion and Conclusions

Of the 46 medicinal plant extracts, 54% were active, 26% were mildly active, and 20% were not active against HeLa cells. Thus, 80% of the medicinal plant extracts showed some type of cytotoxicity. This strongly suggests that there may be some connection between plants known from indigenous cultures to have medicinal properties compared to empirically determined cytotoxicity. Our eight non-medicinal plants also tended to be bioactive, with 50% active, 13% mildl, and 37% not active. Only four samples showed antibacterial activity, which was only in S. aureus, and all these extracts were from medicinal plants. Thus, only 14% of the medicinal plants showed limited antibiotic activity.

Asteraceae, the sunflower family and one with the highest medicinal activity rating in Moerman’s paper [3], was the only family from which we had a fairly large sample, 15 medicinal plants. Extracts from Asteraceae tended to be quite active and followed the general trends of medicinal plant bioactivity as stated above with 54% active, 29% mildly active, and 17% not active. The mint family, Labiateae, also tended to be cytotoxic with 86% of the plants showing bioactivity. Because only seven plants were tested, more data should be collected from this family before a general conclusion can be made about its cytotoxicity. Of the nine Pinaceae plant extracts, 67% showed some bioactivity. Additional work is needed to determine which plant parts tend to have the highest bioactivity. The least active of our five medicinal families was Ranunculaceae with two out of six plant extracts (33%) showing mild activity. Overall these data clearly suggest that non-medicinal as well as so-called medicinal plants should be used in general cytotoxicity screening evaluations. In fact, de Oliveira Maria et al. [4] also found significant bioactivity in 12 species of Amazonian plants which were non-medicinal.

Though this work proved to be insightful, future studies should be undertaken in order to get a clearer picture of the evolutionary relationship of bioactivity and medicinal ranking of plants. From the literature, it appears that only three plants from our group, Ambrosia ambrosioides [5, 6], Gutierrezia microcephala [7], and Atriplex confertifolia [8] have had extensive research on their cytotoxicity. Hence, there is a great deal of toxicology work yet to be done on the remainder of the plants shown to be bioactive in our investigation.

Acknowledgments

The author would like to thank Jeff Nackos, Nathan Ruben, Eric Jacobsen, and Malia Price for their work on the project; the staff of Dr. Leo Vernon’s laboratory for their help with the HeLa cell assay; the New York Botanical Garden for providing the extracts; Brigham Young University for supplying personnel and resources.

References

M. J. Balick and P. A. Cox, Plants, People and Culture: The Science of Ethnobotany, Scientific American Library, New York, NY, USA, 1996.

B.-E. van Wyk and M. Wink, Medicinal Plants of the World Portland, Timber Press, London, UK, 2004.

D. E. Moerman, “The medicinal flora of Native North America: an analysis,” Journal of Ethnopharmacology, vol. 31, no. 1, pp. 1–42, 1991.

V. de Oliveira Maria, B. A. Carneiro Lucia, B. de Cauper, G. Socoiro, and A. Martin, “In vitro screening of Amazonian plants for hemolytic activity and inhibition of platelet aggregation in human blood,” Acta Amazonica, vol. 39, no. 4, pp. 973–980, 2009.

R. W. Doskotch and C. D. Hufford, “Damsin, the cytotoxic principle of Ambrosia ambrosioides (Cav.) Payne,” Journal of Pharmaceutical Sciences, vol. 58, no. 2, pp. 186–188, 1969.

R. W. Doskotch and C. D. Hufford, “The structure of damsinic acid, a new sesquiterpene from Ambrosia ambrosioides () Payne,” Journal of Organic Chemistry, vol. 35, no. 2, pp. 486–490, 1970.

X.-P. Dong, C.-T. Che, and N. R. Farnsworth, “Cytotoxic flavonols from Gutierrezia microcephala,” Journal of Natural Products, vol. 50, no. 2, pp. 337–338, 1987.

C. J. Capua, N. P. Hopson, C. M.M. Stewart et al., “Cytotoxicity of atriplex confertifolia,” Journal of Toxicology, vol. 2010, Article ID 976548, 2010.