Activities of Guatemalan medicinal plants against cancer cell lines and selected microbes : Evidence for their conservation

College of Life Sciences, Brigham Young University (BYU), Provo, UT USA. 2 Secretaría General del Consejo Superior Universitario Centroamericano (CSUCA), Ave. Las Americas 1-03, Zona No. 14, Interior Club Los Arcos, Guatemala City, Guatemala. Benson Agriculture and Food Institute, Brigham Young University (BYU), Provo, UT USA. Facultad de Agronomia, Universidad de San Carlos de Guatemala (USAC), Guatemala City, Guatemala. Benson Institute Guatemala, Chiquimula, Guatemala. Facultad de Odontología, Area Socio-Preventiva, Universidad de San Carlos de Guatemala (USAC), Guatemala City, Guatemala.


INTRODUCTION
Well documented is the use and value of the earth's medicinal resources with regard to primary health care for the human population.For example, Kingston (2011) and Newman and Cragg (2007) suggest that up to 50% of the drugs now available to treat human diseases are related to natural products.For anticancer, anti-migraine, and other drugs the estimate is well over 50% (Newman and Cragg, 2012;Butler, 2008;McChesney et al., 2007).However, Newman et al. (2008), Adams and Hawkins (2007), and Chaudhuri (2007) noted that global access to these types of drugs is highly variable.The result is that traditional remedies support the health care of over 65% of the world population (Fabricant and Farnsworth, 2001), and in rural communities the estimate is 75 to 90% (Chivian and Bernstein, 2008;Fowler, 2006), depending on the geographical area.
An additional consideration is that traditional knowledge and the biodiversity that supports that knowledge and the development of new drugs are being lost (Cordell and Colvard, 2012;Strobel et al., 2004).These in combination with the evolution of drug resistance (Lambert et al., 2011) contribute to the increased awareness to conserve these valuable plant resources (Siwach et al., 2013;Kingston, 2011).Another concern regarding the primary health care of people in rural communities worldwide is the lack of information on the role of medicinal plants to improve oral hygiene (Colvard et al., 2006), For example, Kufer et al. (2005) in their study on the use of medicinal plants in the Ch'orto' area in southeastern Guatemala listed about 41 plants that were used to treat gastrointestinal illnesses, 34 species used for fever and pain, 38 for women's remedies, 25 for respiratory illnesses, but only seven for oral health problems.Of these seven, three were used in prevention and all seven were used for toothaches.Rural family members in southeastern Guatemala near Esquipulas who were suffering from toothache or orofacial pain resorted to using nine herbals but no traditional remedies were noted to prevent cavities or other oral cavity diseases (Hunter and Arbona, 1995).Consequently, a need exists to find medicinal plants that have potential to prevent and treat periodontal diseases and other oral health issues.
These concerns are relevant to the health care of villagers in Guatemala and therefore formed the basis for this study.The first objective was to evaluate the in vitro growth inhibition of acetone and methanol extracts from 63 plant species against breast, cervical, skin, and tongue cancer cell lines and a non-cancerous line.For those extracts that were inhibitory at 60% or greater IC 50 and CC 50 values were determined.Secondly, in vitro growth inhibition of these extracts against Staphylococcus aureus, Streptococcus mutans, Escherichia coli, Lactobacillus acidophilus, and Candida albicans were determined.For those active at 60% or greater minimum inhibitory concentrations (MIC) were obtained.All 63 species are noted in Guatemalan health care pharmacopoeias and about half of these species are used for oral health care.Consequently, activity against Streptococcus mutans, Lactobacillus acidophilus, Candida albicans and the tongue cancer cell line was of particular interest due to their association with dental plaque, caries, and other oral cavity health issues (Kleinberg, 2002).

Plant collection, tissue preparation, cell lines and microbial cultures
Eighteen species were collected from the Museo Odontológico de Guatemala y Jardín Botánico Maya, Guatemala City, Guatemala, 20 species from Colección y Huerto Productivo de Plantas Medicinales, Facultad de Agronomía, Guatemala City, and 25 from the communities of Olopa and San Juan Ermita in southeastern Guatemala.Aids in identifying species other than vouchers and digital pictures were the Vademecum National de Plantas Medicinales (Cáceres, 2009), the guide to medicinal plants by Arevalo and Dieseldorff (2005), and a species list for the Museo Odontologico de Guatemala y Jardin Botánico Maya.Voucher specimens are located in the herbaria at the Centro Universitario de Oriente, Universidad de San Carlos de Guatemala, Chiquimula, Guatemala (CUNORI) and at Brigham Young University (BRY), Provo, UT.Each sample from the 63 species analyzed consisted of tissue (Table 1) collected from three or more individuals that was mixed, then bagged, labelled, and stored at -80 o C (Isotemp Basic, Thermo Electron Corporation, Asheville, NC USA) at BYU. Acetone and methanol extracts derived from five grams of plant tissue were eventually dissolved in double-distilled water at a final concentration of 8 mg/ml.The human cancer cell lines used were breast (ATCC HTB-22, breast mammary gland adenocarcinoma; ATCC, Manassas, VA), HeLa (ATCC CCL-2, cervix epithelial adenocarcinoma; ATCC), skin (ATCC CRL-2095,epithelial malignant melanoma; ATCC), and tongue (ATCC CRL-2095, human epithelial squamous carcinoma; ATCC).Cytotoxicity was determined using a non-cancerous Vero cell line (ATCC CRL-1586, epithelial kidney monkey; ATCC).Staphylococcus aureus (ATCC 6538P; Becton Dickinson Laboratories, Cockeysville, MD), Escherichia coli (ATCC 11229; ATCC) oral isolates of Streptococcus mutans (ATCC 33402, ATCC), Lactobacillus acidophilus (ATCC 11975, ATCC) and Candida albicans (ATCC 90028, ATCC) were used to determine the antimicrobial activity of acetone and methanol extracts.Methods for culturing cancer cell lines, the non-cancerous cell line, and microbes are described by Cates et al. (2013).

Sulforhodamine B assay and neutral red (NR) assay
The sulforhodamine B assay used to determine the level of inhibition of extracts against cancer cell lines followed Skehan et al. (1990) and Donaldson et al. (2004) as described by Cates et al. (2013).Inhibition activity against cell lines was determined in triplicate at 200, 100, and 50 µg/ml of extract.Results in Table 2 are reported only for the 200 µg/ml concentration.The NR assay followed Putnam et al. (2002) and was used on all extracts that showed 60% or greater inhibition in the sulforhodamine assay.Serial dilutions of 200, 100, 50, 25, 12.5 and 6.25 µg/ml of each plant extract were run in triplicate against each cell line ( Cates et al., 2013).Additional concentrations of extract were included in the NR assay so that more data would be available for accurate calculation of half-maximum inhibitory concentrations  To determine which extracts exhibited inhibition against the pathogens a microwell dilution bioassay was performed using 1000, 500, and 250 µg/ml of extract following Shrestha and St. Clair (2013).Each extract was tested in triplicate and only percent inhibition at the 1000 µg/ml concentration was reported (Table 4).For plant extracts that were inhibitory at 60% or greater (Table 4) MICs were determined using a microwell dilution bioassay.Concentrations of 1000, 500, 250, 125, 62.5, and 31.25 µg/ml were tested in triplicate against the microbes.The MIC was defined as the lowest concentration of extract at which no reduction of piodonitro-tetrazolium violet dye (Sigma-Aldrich) was observed.MICs were not calculated for S. mutans and L. acidophilus due to irregular growth and clumping.Details of these two assays are found in Cates et al. (2013).

Data analysis
Data were coded by species and fraction and statistical significance (P ≤ 0.001) between control vs. inhibition values were determined by ANOVA (R Core Team, 2013).Results from the 200 µg/ml concentration used against cancer cell lines and the 1000 µg/ml concentration used against the microbes are the only results reported (Tables 2 and 4).This is because these concentrations yielded the maximum number of active plant species.Consequently, any extract showing greater than 60% inhibition for the acetone or methanol extracts at the 200 µg/ml level for any cancer cell line, and at the 1000 µg/ml for any microbial species, was considered active and worthy of neutral red or MIC analysis.An additional criterion was that if the inhibition level of a cancer cell line was two to three times that of the Vero line then those extracts were considered active.

Neutral red (NR) assay for inhibition and cytotoxicity
The methanol extract from B. simaruba and the acetone extract from T. peruviana were highly inhibitory at low concentrations (IC 50 = 75 µg/ml and 30 µg/ml, respectively) against the breast and HeLa cancer cell lines, respectively (Table 3).They also yielded low inhibition at high concentrations against Vero cells (CC 50 > 800 µg/ml and 663 µg/ml, respectively).The acetone extract from L. guatemalensis, and to some extent the acetone extract from P. americana, showed moderate activity against the breast and HeLa lines (IC 50 = 226 µg/ml and 387 µg/ml, respectively), and low inhibition at high concentrations against the Vero line (CC 50 > 800 µg/ml).The other species showed high IC 50 and/or low *All comparisons between values at 60% or greater inhibition and their controls were significantly different at P ≤ 0.001.Genus/Species % Inhibition (1000 µg/ml)*   4).

Minimum inhibitory concentrations (MICs)
The acetone extracts of M. jalapa, P. dioica, and R. villosus yielded MIC values of 250 μg/ml against S. aureus (Table 5).The methanol extract of B. simaruba produced an MIC of >1000 μg/ml against S. aureus, and a MIC of 500 μg/ml against E. coli (Table 5) even though it was not inhibitory to E. coli in the inhibition assay (Table 4).
Extracts from E. japonica and P. dulce yielded extracts with a MIC of 1000 µg/ml; all other extracts yielded MIC values >1000 μg/ml and were not considered inhibitory.

DISCUSSION
Our study along with Kufer et al. (2005) and Comerford (1996) note a wide variety of uses for the medicinal plants selected for this study (Table 1).This suggests that these resources are valuable to rural Guatemalans and need to be conserved.Overall, 16 (25.4%) of 63 species were inhibitory to one or more cancer cell lines and/or one or more microbes at the 60% or greater level.Eight species were inhibitory to one or more cancer cell lines and eight were inhibitory to one or more microbes (Tables 2 and 4).Of those active against cancer cells, extracts from B. simaruba and L. guatemalensis demonstrated significant inhibition at low concentrations (IC 50 75 and 226 μg/ml, respectively) against the breast cell line and showed low inhibition at high concentrations (CC 50 >800 μg/ml) against the noncancerous Vero cells (Table 3).The acetone extract from T. peruviana also demonstrated significant activity against the HeLa cell line (IC 50 30 μg/ml vs CC 50 663 μg/ml).P. americana showed some activity against the HeLa line and with further fractionation this species might prove effective against this line.For the eight species that were active against one or more microbes three (M.jalapa, P. dioica and R. villosus) registered a MIC of 250 μg/ml against S. aureus.B. simaruba was inhibitory to S. auerus (Table 4) but the MIC for the methanol extract was >1000 μg/ml (Table 5).Interestingly the methanol extract from B. simaruba was almost significant at 54% inhibition to E. coli (Table 4) and that level of inhibition was reflected in a moderately inhibitory MIC of 500 μg/ml against E. coli (Table 5).Extracts from C. odorata, C. longirostrata, B. simaruba, P. americana, and P. dulce were inhibitory to both cancer cell lines and microbes (Table 2 and 4).However, extracts from these five species did not demonstrate significant IC 50 , CC 50 , or MIC values (Tables 3 and 5).The stated uses of these species by villagers did not include cancer and microbial diseases (Table 1) so likely the ethnomedical use will not change.
Even so, because these species were active against cancer cells and microbes further study of these species may yield promising results.
One focus was to identify medicinal plant species that might be used to improve oral hygiene.Specific emphasis was on plant species demonstrating activity against S. mutans and L. acidophilus both of which may contribute to cavity formation, and those active against the tongue cancer cell line.S. purpurea and P. dulce demonstrated significant inhibitory activity against S. mutans (Table 4).C. longirostrata was inhibitory to the tongue cancer cell line (Table 2), and this species along with A. millefolium (and P. lappulacea was almost inhibitory at 59% inhibition) were active against L. acidophilus.These species merit further investigation as to their efficacy to prevent or treat diseases of the oral cavity.
Several species reported in this study have been reported elsewhere to have activity against human diseases.For example, Johnson (1999) refers to extracts from B. simarubra and P. americana as being used to treat stomach cancer and tumors, respectively, and in our study these species were active against breast and cervical cancer cells, respectively.Additionally, S. umbellatum is an important medicinal plant in some cultures but was not reported to have activity against cancer cell lines (Johnson, 1999).
However, in our study this species was active against cervical cancer cells.In summary, data from this study yielded 11 significantly active species and Cates et al. (2013) noted seven additional active species.Miller (2014) found 11 other Guatemalan species that produced essential oils which were highly active against the same set of microbes used in this study which brings the total to 29 active medicinal plant species.Future work is needed to determine the pharmacological activity and cytotoxicity of active components.For example, T. peruviana was active against the HeLa cell line but is well known for its cytotoxicity (Bandara et al., 2010).Additional studies of the active species might include characterizing the active compounds, and in vitro and in vivo investigations of their cytotoxicity, mechanism(s) of action, and ultimately their efficacy in preventing and treating diseases.

Conclusion
Sixteen species of medicinal plants were found to be inhibitory to one or more cancer cell lines and/or microbes.Based on cytotoxicity to the Vero cell line, high IC 50 values and low CC 50 values, and high MIC values several of these species may not merit further study.However, seven species (B.simaruba, E. japonica, L. guatemalensis, M. jalapa, P. dioica, R. villosus, T. peruviana) merit additional investigation based on their inhibition, IC 50 /CC 50 values, and MIC values.With regard to oral hygiene four species (A.millefolium, C. longirostrata, P. dulce, S. purpurea) merit further fractionation and testing against various oral diseases.

*
All comparisons between values at 60% or greater inhibition and their controls were significantly different at P ≤ 0.001 except for S. purpurea** which was significantly different at P ≤ 0.03.

Table 1
cont'd Solanum umbellatum were not analyzed for activity against microbes due to lack of tissue.†Medicinal use not clearly defined at time of collection.‡Local villagers referred to V. zizanioides as Valeriana and F. pycnocephala as violet.(IC50 ) and half-maximum cytotoxicity concentrations (CC 50 ).The IC 50 and CC 50 values were obtained using dosage response curves.

Table 2 .
The effect of acetone and methanol extracts on cancer cell lines.

Table 3 .
Half-maximum inhibitory concentration (IC 50 ) for cancer lines and half-maximum cytotoxicity concentration (CC 50 ) for the Vero cell line.

Table 4 .
The effect of acetone and methanol extracts on microbes.

Table 5 .
Minimum inhibitory concentrations (MIC) for Guatemalan medicinal plants that showed greater than 60% inhibition against microbes.
*A=acetone extract; M=methanol extract; blank space indicates no inhibition per Table4.