Evaluation of Aromatic Plants and Compounds Used to Fight Multidrug Resistant Infections

Traditional medicine plays a vital role for primary health care in India, where it is widely practiced to treat various ailments. Among those obtained from the healers, 78 medicinal plants were scientifically evaluated for antibacterial activity. Methanol extract of plants (100 μg of residue) was tested against the multidrug resistant (MDR) Gram-negative and Gram-positive bacteria. Forty-seven plants showed strong activity against Burkholderia pseudomallei (strain TES and KHW) and Staphylococcus aureus, of which Tragia involucrata L., Citrus acida Roxb. Hook.f., and Aegle marmelos (L.) Correa ex Roxb. showed powerful inhibition of bacteria. Eighteen plants displayed only a moderate effect, while six plants failed to provide any evidence of inhibition against the tested bacteria. Purified compounds showed higher antimicrobial activity than crude extracts. The compounds showed less toxic effect to the human skin fibroblasts (HEPK) cells than their corresponding aromatic fractions. Phytochemical screening indicates that the presence of various secondary metabolites may be responsible for this activity. Most of the plant extracts contained high levels of phenolic or polyphenolic compounds and exhibited activity against MDR pathogens. In conclusion, plants are promising agents that deserve further exploration. Lead molecules available from such extracts may serve as potential antimicrobial agents for future drug development to combat diseases caused by the MDR bacterial strains as reported in this study.


Introduction
Treatment of infections is compromised worldwide by the emergence of bacteria that are resistant to multiple antibiotics [1]. New and emerging drug resistance bacteria strains, particularly methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), Mycobacterium tuberculosis (MTB), and multidrug resistance (MDR) Gramnegative bacteria, are increasing worldwide and add to the gravity of the situation [2]. S. aureus cause a variety of syndromes such as food poisoning, toxic shock syndrome, skin lesions [3], hyperproliferative skin disease [4], and atopic dermatitis [5,6]. Community-acquired pneumonia caused by Streptococcus pneumoniae, Klebsiella pneumonia, and S. aureus accounts for significant mortality in Southeast Asia [7]. Melioidosis has been recognized as an important human infection caused by Burkholderia pseudomallei in Singapore, Malaysia, Thailand, and Northern Australia [8,9]. Cases have also been reported from some other tropical and subtropical regions like Africa and America, and a number of cases in man has recently been reported to increase in China, Taiwan, and South India [10,11]. Infection with antibiotic resistant bacteria negatively impacts on public health, due to an increased incidence of treatment failure and severity of diseases. Development of resistant bacteria due to the chromosomal mutations is more commonly associated with the horizontal transfer of resistance determinants borne on mobile genetic elements [12]. B. pseudomallei is intrinsically 2 Evidence-Based Complementary and Alternative Medicine resistant to many antibiotics [13,14]. Considering the higher cost for producing synthetic drugs and the various side effects associated with their use, the need to search for alternative agents from medicinal plants and essential oils used in folklore medicine is further justified to overcome these issues. In India, there are about 550 tribal communities covered under 227 ethnic groups residing in about 5000 villages throughout different forest and vegetation regions [15]. India is one of the world's 12 megabiodiversity countries [16,17]. Plant derived medicines have played a major role in human societies throughout the history and prehistory of mankind [18]. The traditional healers (traditional physicians) or medicinemen have a long history of their own diagnostic and treatment system, which they have acquired from their ancestors [19]. Approximately 80% of the world population still relies on traditional medicine for the treatment of common diseases [20][21][22]. Medicinal plants thus offer significant potential for the development of novel antibacterial therapies and adjunct treatments [23]. Plant derived drugs serve as a prototype to develop more effective and less toxic medicines. In previous studies, few attempts were made to confirm the antimicrobial activity of some indigenous medicinal plants [24,25]. Not only extracts of various medicinal plants but also essential oils and their constituents have been investigated for their antimicrobial properties against bacteria and fungi [26][27][28].
The principal compounds from the leaves showed a better antibacterial activity against P. aeruginosa and B. subtilis bacteria and a significant antifungal activity on C. albicans [29]. The essential oil from R. officinalis (alpha pinene/ verbenone/bornyl acetate) was found to be more sensitive to the Gram-positive bacteria (MIC 2.5-4 mg/mL) than to the Gram-negative bacteria [30]. Several essential oils kill bacteria by damaging the cell membrane structure and inhibiting their membrane function [31]. Because of the antimicrobial potency of plant extracts and oils, they become a rich source of raw materials for many biotechnological and pharmaceutical industries for the development of therapeutic drugs. The increasing trend in the use of aromatic plants and essential oils in food, cosmetic, and pharmaceutical industries suggests that a systematic study of traditional medicinal plants is very important in order to find active compounds from such sources [32][33][34]. The purpose of this study is to survey and investigate popular medicinal aromatic plants and their essential oils with a view to fight against multidrug resistant human pathogens. In the present study, 71 plant species were selected on the basis of the available medicinal information and screened for their in vitro antimicrobial efficacy against bacteria.

Ethnomedicinal Survey and Collection of Plants.
Ethnomedicinal surveys were conducted during March 1998 and July 2001 from various tribal localities (Kolli hills, Kalrayan hills, Pachamalai, Javadi hills, Mundanthurai) of Eastern and Western Ghats, Tamil Nadu, India. For ethnobotanical studies, questioners were used to collect the general information on the tribes, and the key information on medicinal details was collected through interviews. The medicinal plants were identified by a taxonomist using the standard Flora of Tamil Nadu Carnatic [35], and the voucher specimens were deposited in the department's herbarium at the Entomology Research Institute, Loyola College, Chennai, India.

Preparation of Plant Extracts.
Using a Soxhlet apparatus, the shade-dried and powdered plant materials (200 g of each) were extracted with 1000 mL of methanol (CH 3 OH) for 10 h. The collected methanol extracts were filtered (Whatman no. 1 filter paper) and evaporated with a rotary evaporator and freeze dryer (lyophilized) to obtain the crude extracts (Buchi, Labortechnik AG, Switzerland). The dried crude extracts were stored at 4 ∘ C for antimicrobial assays [

Antimicrobial
Activity. The standard bacterial cultures were stored at −70 ∘ C, subcultured on 20 mL MH and TS agar plates (pH 7.4), and incubated overnight at 37 ∘ C prior to use. The antimicrobial property was tested using the discdiffusion method [36]. Five young colonies of each strain of bacteria taken from their respective cultures grown overnight on TS agar plates (Oxoid limited, Wode Road, Basingstoke, Hants, England, UK) were suspended in 5 mL of sterile saline (0.9%), and the density of the suspension was adjusted to approximately 3 × 10 8 colony forming unit (CFU). The swab was used to inoculate the dried surface of TS agar plate by streaking four times over the surface of the agar and rotating the plate approximately 90 ∘ C to ensure an even distribution of the inoculums. The medium was allowed to dry for about 3 min before adding a 6 millimeter in diameter (mm) sterile paper disc (Becton Dickinson, USA) on the surface. Each disc was tapped gently down onto the agar to provide a uniform contact. Lyophilized residue (100 g/mL) of each plant extracts and purified fractions was weighed and dissolved in 1 mL of water, and 20 L of the extracts and oils (containing 100 g of residue) were applied on each disc (3 replicates), while the sterile blank disc served as a normal control. The antimicrobial effect of the extracts on the clinical isolates was determined in comparison with the reference antibiotics (chloramphenicol 30 g/disc and ceftazidime 30 g/disc), which were used as positive controls. The plates were incubated at 37 ∘ C for 24 h, and the inhibition zones were measured and calculated.
Evidence-Based Complementary and Alternative Medicine 3

Minimum Inhibitory Concentrations (MICs) Assay.
MICs were evaluated based on the in vitro screening of 16 purified fractions that were found to have potent antimicrobial activity. Broth dilution method was used for the MIC assay with some slight modifications as recommend by the NCCLS [37]. Two-fold serial dilutions of all the fractionated compounds were made with MH and TS broth in microtiter plate wells to adjust the final concentration from 7.8 to 250 g/mL, while wells containing the broth alone without any sample served as a control. Three replicates ( = 3) were used for each dilution and culture containing approximately 1 × 10 5 CFU/mL. The plates were incubated at 37 ∘ C for 24 h, and the absorbance was measured at 560 nm.
2.6. Cytotoxicity Assay. The cytotoxic effects of various extracts were tested by MTT assay [38] using human skin fibroblast HEPK cells. The toxic effect of plant extracts and essential oils was assayed on human skin fibroblast (HEPK) cell proliferation in 96-well microtitre plates. Confluent cells (5 × 10 6 cells per well) were incubated with extracts and oils for 24 h, and the percentage inhibitory concentration (IC 50 ) was determined.

Phytochemical Analysis of Plant Extracts.
The most active extracts were used for purification of antimicrobial compounds [39]. 100 g each of the plant powder was percolated with 500 mL of 4% aqueous HCl (adjusted to pH 2) and heated at 50 ∘ C for 3 h. The extract was washed with 2×250 mL of diethyl ether, and the organic phase evaporated to dryness using vacuum rotary evaporator. The dark brown gummy residues obtained by acid hydrolysis were chromatographed on silica gel column 60 × 3.2 cm (60-120 mesh, pH 7, Merck) by gradually eluting with n-hexane/ethyl acetate (8 : 2; 6 : 4; 3 : 7 and 1 : 1) and chloroform/methanol (3 : 2). The aliquots of each fraction were subjected to thin layer chromatography (TLC) on silica gel coated TLC plate (1 mm Merck) using the solvent system consisting of 20% (v/v) n-hexane/ethyl acetate. The chromatograms were detected using 50% H 2 SO 4 solution as a spray reagent [34]. The individual fractions were collected and concentrated by vacuum rotary evaporator at 40 ∘ C. All the purified compounds recovered from the silica gel column were monitored by reading the absorbance at 190-350 nm (UV spectrophotometer, Hitachi, Japan). The active fractions were further purified, and the final yields of the compounds were recorded. The lyophilized pooled concentrated compounds were then assayed (100 g/mL) against bacteria. The phytochemical screening was done on the pure compounds using the chemical method previously reported for the detection of secondary metabolites [40]. The different chemical constituents tested include alkaloids, flavonoids, glycosides, polyphenols, saponin, sterols, triterpenes, tannins, reducing sugars, gallic acid, catechol, and aglycones.

Statistical
Analysis. The bacterial growth inhibitory activity (inhibition zones millimeter in diameter) was compared for significant differences within the bacterial strains. One way analysis of variance was performed (mean ± SD, = 3 replicates) using GraphPad Prism 4, USA. * < 0.01 was considered statistically significant (inhibition zones of extracts/fractions versus antibiotic drugs).

Study Area for the Collection of Aromatic Medicinal Plants.
The Western and Eastern Ghats were selected for the present study with the cite map showing the landmarks (Figures 1(a)-1(b)). Kalrayan hills are situated north of Attur taluk (Salem district), one of the major range of hills in the Eastern Ghats of Tamil Nadu (Figure 1(c)). Pachamalai hills are situated to the north of Thuraiyur taluk of Tiruchirappalli district. The rich biodiversity part of Eastern Ghats lies between latitudes 11 ∘ 09 00 to 11 ∘ 27 00 N and longitudes 78 ∘ 28 00 to 78 ∘ 49 00 E, and occupies an area of about 527.61 square km. It is located near 11 ∘ 11 N 78 ∘ 21E/11.18 ∘ N 78.35 ∘ E/11.18; 78.35 (Figure 1(d)). Mundanthurai is located nearly 45 km west of Tirunelveli district, TN, between latitude 8 ∘ 25 and 8 ∘ 53 N and longitude 77 ∘ 10 and 77 ∘ 35 E. This is the only area of Western Ghats that has the longest raining period of about 8 months and forms the catchment area for 14 rivers and streams (Figure 1(e)). Kolli Malai is a small mountain range located in Namakkal district. The mountains are about 1000-1300 m in height and cover an area of approximately 280 km. The Kolli hills are part of the Eastern Ghats, which is a mountain range that runs mostly parallel to the east coast of Tamil Nadu in South India (Figure 1(f)). Javadi hills are one of the largest in the Eastern Ghats in Vellore district in the northern part of the state of Tamil Nadu. They consist of bluish gray hills, with peaks averaging 3600-3800 feet or 1100-1150 meter (Figure 1(g)). Based on the vegetation type (Figures 2(a)-2(d)), the study area consists of (i) dry, deciduous, (ii) moist deciduous, and (iii) rain forests and diverse proportion of plant parts in abundance (Figures 2(e)-2(f)). Three different types of tribes (i.e., Kani, Malayali and Paliyan tribes) inhabit in the hill ranges. The Kani tribes, located at Mundanthurai, raise different types of vegetables in their own fields, while the Malayali tribes cultivate rice. They all engage not only in the agricultural work but also are involved in silvicultural work assigned by the forest department, Government of TN, India.

Medicinal Plants Glory. Western Ghats (Mundanthurai)
and Eastern Ghats (Kolli hills, Javadi hills, Kalrayan hills, Pachamalai hills) possess a rich diversity of medicinal plants that are used as food and drug by different groups of tribal communities. Urbanization, habitat degradation, and fragmentation of these forests have resulted in the depletion of natural resources on which these tribes used to depend for their livelihoods. It has become increasingly difficult for them to live in their traditional way. In addition, the impact of modernization and urbanization has encroached in and around tribal settlements, thus changing their lifestyles.

Plants Valued as Edibles.
Various types of plant parts are collected during different seasons, cooked, and eaten along with boiled rice (Table 1). For example, Solanum nigrum leaf is most commonly used in all the four regions. There are    -     /disc)  33  16  22  19  16  25  21  20  12 15 * Bacteria (+/−). Results obtained in the disc diffusion assay; antibacterial activity is expressed as the mean ± SD ( = 3), of the inhibition by the extract and its diameter around the discs. One way analysis of variance was performed (mean ± SD, = 3 replicates). Size of inhibition zones were including the sterile blank discs 6 millimeter (mm) in diameters. Absence of bacterial inhibition indicates (-), antibiotic disc (30 g/disc).

Phytochemical Screening of Plants.
The results obtained from the phytochemical screening as shown in Table 1 indicate the presence of various types of secondary metabolites such as polyphenols, tannins, saponins, alkaloids, and glycosides/polysaccharides. Most of the plant extracts relatively rich in alkaloids, phenols, flavonoids, polyphenols, tannins, sterols, and terpenoids were found to inhibit the growth of organisms.   On the other hand, compounds from C citrates, O. sanctum, E. caryophyllus, and Z. zizanioide, showed some promising effect only against S. aureus ( Figure 5). The activity of the compounds were pronounced more than that of the oil yielding plant fractions.   and C. citratus fractions also showed antimicrobial activity (MICs of 31.25-125 g/mL) only at higher concentrations against the tested bacteria. In addition to that, higher concentrations (>250 g/mL) (of Vetiveria fractions) were required to inhibit Vibro species, and others (including E. globulus fractions) failed to show any effect at tested concentrations (7.8-125 g/mL). However, the purified fractions (from most active medicinal plants) showed strong bacteriostatic inhibition against the tested organisms (Table 3).  Figures S3 and S4) compounds. The toxicity was found to be concentrationdependent when the skin fibroblasts (HEPK) cells were The bacterial growth inhibitory activity was compared for significant differences within the bacterial strains by broth-dilution method at 250, 125, 62.5, 31.25, 15.6, and 7.8 g/mL. F: fractions.

Cytotoxic Effects of
exposed to various compounds. The cell proliferation was increased by the influence of the plant components at the lower concentrations. Whereas the oil yielding plant compounds showed inhibition of cell proliferation and toxicity at higher doses 250-1000 g/mL.

Discussion
The Western Ghats is considered as one of the richest biodiversity hotspots in the world [41]. In this survey, we collected nearly 78 medicinal plants from Western and Eastern Ghats that are edible and popularly used for curing various ailments including snakebite. Traditional remedies have a long-standing history in many tribal settlements in TN, India, and they continue to provide useful and applicable tools for treating ailments [42]. The ingredients that make up the "Vishakallu" stone, which is used as an antidote for snakebite, are different herbs and pebbles available from the river banks. Likewise, aqueous paste and decoction obtained from the leaves of A. paniculata are widely used for snakebite treatment by indigenous people [43]. Previous studies have reported that ethnomedicine plays major roles in conserving the disappearing knowledge of tribal communities [44][45][46][47].
The traditional beliefs of reliance on a rich diversity of ethnomedicinal plants located at different settlements have also been confirmed in another study [48,49]. Herein, we explored the various types of traditional practices reported by the primitive tribal communities with a view to gain further knowledge from such studies.
In the present investigation, potentially rich sources of tribal medicine (71 plants) were scientifically evaluated for their antibacterial activity against the MDR bacteria, and the accumulated data was disseminated for the first time to the scientific community. Out of the 71 medicinal plants screened for the antibacterial activity, 10 of them (T. involucrata, C. acida, A. marmelos, A. vasica, C. procera, A. paniculata and M. piperita, A. indica, S. indicus, and E. cardamomum) displayed the highest antibacterial activity against the multidrug resistant B. pseudomallei (KHW and TES) and S. aureus strains. The antibacterial activity of those crude plant extracts was as equally effective as that of the standard drugs. Our findings corroborated with the previous reports made on the antistaphylococcal activity of tribal medicinal plants [34,50,51]. On the other hand, isolated components from the most active extracts of T. involucrata, shellsol, and C. acida exhibited the most potent action against the antibiotic resistant B. pseudomallei (KHW), K. pneumoniae, and S. aureus strains. These results further confirmed our previous findings on the leaves of T. involucrata and its compounds hydrocarbon ester-like shellsol, which displayed a high antibacterial effect against the different bacterial strains, especially that of S. aureus [34]. Eugenol and caryophyllene are the active agents contained in the M. piperita [52] and O. sanctum plants, which are believed to be mainly responsible for the antimicrobial properties of these plants [53]. Interestingly, the compounds obtained from the aromatic plants such as C. zeylanicum and R. officinalis were also found to be very effective against the multidrug resistant human pathogen K. pneumoniae, S. aureus, S. typhi, and B. pseudomallei (KHW) that causes melioidosis.
The inhibitory potential determined for the shellsol (T. involucrata), vaseline (A. vasica), C. acida, and C. zeylanicum indicates that the MIC of 7.8-31.25 g/mL found against the B. pseudomallei of KHW, K. pneumoniae, K. pneumoniae, S. pyogenes, and V. damsel, and S. pneumoniae was quite low. Similarly, lower MIC values were found for M. piperita (MIC of 1.13-2.25 mg/mL) against the above bacterial strains [52]. MIC was found for the most active alcohol extracts of A. salvifolium (MIC 0.034-0.263 mg/mL) on S. aureus [54], and the MIC for S. trilobatum aqueous extracts determined against the tested organisms ranged from 0.06 to 0.5 mg/mL [55]. C. zeylanicum was found to have an effective antibacterial activity (MIC 64 g/mL) against P. aeruginosa, E. coli, B. subtilis, and S. aureus [56]. Previously, several investigators have demonstrated that active agents exert interesting activity against bacteria even at lower concentrations tested [57,58]. The bacteriostatic mechanism involves damage to the cell walls of bacteria, followed by inhibition of protein synthesis that ultimately leads to bacterial death [59]. The most active plants are widely used by various tribes as traditional treatment (i.e., cut wounds, skin infection, and scabies), thus indicating the potential for further development into promising drugs. In order to ascertain the safety and efficacy of the most active compounds, their effect on human skin fibroblast cells was evaluated. Chemical constituents of E. cardamomum, T. involucrata, S. indicus, C. acida, A. vasica, A. marmelos, A. indica, and A. paniculata plants failed to produce any noticeable toxicity up to 1000 g/mL. Although some of the tested compounds exhibited a slight reduction of cell proliferation and some minor morphological changes, such changes were insignificant at lower doses and became evident only at higher doses. However, certain aromatic compounds of O. sanctum, E. globulus, V. zizanioides, C. citratus, and E. globulus plants showed reduction of cell proliferation against HEPK cells.
Our phytochemical screening also provides evidence of the presence of several types of compounds that are mainly responsible for the remarkable antibacterial effect of these plants. The differences noted for the bactericidal activity of various plant extracts in this study appears to be directly related to the diversity of compounds (shown in parentheses) that are accumulated in the following plants (e.g., A. marmelos and O. umbellate) [60][61][62]. Compounds like tannins, phenol, and polyphenols can bind the Gramnegative bacteria to form a heavy soluble complex on the cell surface, which subsequently disturbs the availability of receptor on cells and kills the bacteria [63]. Several species having wide spectra of antimicrobial activity mainly due to the active constituents such as essential oil, phenolic compounds like thymol, carvacrol in oregano and thyme, eugenol in clove, and cinnamon were also identified previously [64,65]. Essential oils degrade the cell wall, interact with the cell components, and then disrupt the cytoplasmic membrane [66]. The antimicrobial effect of phenolic compounds may involve multiple modes of action, including damage to the membrane protein, interference with membrane integrated enzymes [67], causing leakage of cellular components, coagulation of cytoplasm, depletion of the proton motive force, alteration of fatty acid and phospholipid constituents, impairment of enzymatic mechanisms for production and metabolism, alteration of nutrient uptake and electron transport [68], influencing the synthesis of DNA and RNA, and destroying protein translation and the function of the mitochondrion in eukaryotes [69]. The mode of action of antimicrobial agents depends on the type of microorganism and is mainly related to their cell wall structure and the outer membrane arrangement. Most of the plant spices and herbs contain complex phenolics (i.e., phenolic acids, flavonoids, tannins, lignans, coumarins, quinines). In addition, the mechanisms of action of each phenolic compound against various bacteria are also very complicated [26,70]. Further investigation is therefore required to understand the relationship between the antimicrobial action and the chemical structure of every phenolic compound in the tested extracts. The information available from previous pharmacological sources combined with the findings herein reported on the medicinal plant extracts may serve as essential data for future drug development to combat diseases caused by the MDR bacterial strains.