Qualitative Determination of the Secondary Metabolites And

Journal of Pharmaceutical Research International

23(2): 1-12, 2018; Article no.JPRI.41205 ISSN: 2456-9119 (Past name: British Journal of Pharmaceutical Research, Past ISSN: 2231-2919, NLM ID: 101631759)

Qualitative Determination of the Secondary Metabolites and Evaluation of the Antimicrobial Activity of Leaf Extracts from Different Plant Families (Boraginaceae, Fabaceae, Lamiaceae and Lauraceae) against Microorganisms of Clinical Importance

Ruby A. Ynalvez1*, Kassandra L. Compean1 and Alfred Addo-Mensah1

1Department of Biology and Chemistry, Texas A&M International University, USA.

Authors’ contributions

This work was carried out in collaboration among all authors. Author RAY designed the study, did literature searches, researched and modified protocols accordingly, wrote the first draft of the manuscript and edited the manuscript to its final version. Author KLC did literature searches, performed all experiments with statistical analysis and wrote the first draft of the manuscript. Author AAM helped out in the phytochemical screening and analysis. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/JPRI/2018/41205 Editor(s): (1) Dr. Emmanuel Christy Jeyaseelan, Department of Botany, University of Jaffna, Sri Lanka. Reviewers: (1) Maciej Tadeusz Strzemski, Medical University of Lublin, Poland. (2) P. Rameshthangam, Alagappa University, India. (3) Daohong Chen, China. Complete Peer review History: http://www.sciencedomain.org/review-history/25634

Received 31st March 2018 Accepted 15th July 2018 Original Research Article st Published 21 July 2018

ABSTRACT

Aims: This study was conducted to investigate the antimicrobial activities of Sassafras albidum (Nutt.), Ehretia anacua (Terán & Berl.), Melissa officinalis (Linn.), Eysenhardtia texana (Scheele), and Melissa odorata. Specifically, this study aims to evaluate the antimicrobial potential and to qualitatively determine presence of secondary metabolites in the different leaf extracts. Place and Duration of Study: Plant leaves were collected from the San Antonio Botanical Garden in Texas. The microbial assays and chemical analysis were done at the Department of Biology and

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*Corresponding author: E-mail: [email protected];

Ynalvez et al.; JPRI, 23(2): 1-12, 2018; Article no.JPRI.41205

Chemistry, Texas A&M International University, Laredo Texas. This study was done from October 2013 to May 2016. Methodology: Leaves were collected and aqueous, acetone, diethyl ether, and ethanol leaf extracts were prepared. Antimicrobial activity against bacteria and fungi were investigated via disc diffusion assay. Phytochemical screening was done to qualitatively determine secondary metabolites. Results and Conclusion: The ethanol and diethyl E. anacua (Boraginaceae) extracts showed a statistically significant antimicrobial activity against S. aureus. Although the values, 7.4 mm and 7.5 mm for the ethanol and diethyl ether extracts could be low values for zone of inhibitions, the potential for E. anacua for anti-S. aureus activity cannot be undermined. Phytochemical analysis showed detectable presence of alkaloids, diterpenes, and phenols in the ethanol and diethyl E. anacua extracts. Results of this study, although preliminary, demonstrated the potential of E. anacua as a new source of bioactive metabolites. Further investigations are needed in order to specifically identify, quantify, and isolate the bioactive compounds that might act against S. aureus associated skin infections.

Keywords: Antimicrobial; Ehretia anacua; leaf extracts; S. aureus; secondary metabolites.

1. INTRODUCTION inflammatory, and antimicrobial properties; these properties are attributed to the presence of Traditional medicinal plants serve as potential naphthoquinones, alkaloids, flavonoids, sources of new antimicrobial agents, since they polyphenols, phytosterols, terpenoids, and/or possess natural products, including secondary allantoin [10,12,13,14,15,16]. Likewise, several metabolites and their derivatives [1,2,3]. plants from the family Fabaceae have been Secondary metabolites could include alkaloids, reported to possess potential of antimicrobial flavonoids, tannins, terpenes, quinones, and activity [2,17]. For example, methanol and ethyl resins. These plant secondary metabolites serve acetate bark extracts of Adenanthera pavoina L as defense mechanisms against many showed antimicrobial activity against gram- microorganisms, insects, and herbivores [4]. positive, i.e. S. aureus, and gram-negative Secondary metabolites are the subject for many strains, i.e. E. coli. The antimicrobial activity research studies since these compounds exhibit could be attributed to the reported presence of many biological activities. These include saponins, alkaloids, tannins, flavonoids, and antibacterial, antifungal, anticancer, and anti- steroids in both methanol and ethyl acetate inflammatory. extracts [18]. Sideritis species, which belongs to the family Lamiaceae, has been used for Antimicrobial properties are important for future centuries for their anti-inflammatory and pharmacological uses. There has been a antimicrobial properties [2,8]. Some of the continuous interest in the therapeutic use of chemical constituents in Sideritis identified to be natural products because of availability, less side responsible for pharmacological activity are: effects, and less abusive potential [5,6,7]. Over terpenes, flavonoids, essential oil, iridoids, the years, many scientists have performed coumarins, lignanes, and sterols [8]. Under the research on different plant families to be able to Lauraceae family, the leaves of Cinnamomum identify antimicrobial activities and secondary species are used in traditional medicine for metabolites [2,8,9,10,11]. Phytochemical pains, while the leaves of Laurus nobilis L. have screening of different plants has revealed been known to contain medicinal compounds numerous bioactive compounds including (tannins, acidic materials, and volatile oil) [11]. alkaloids, tannins, flavonoids, glycosides, and saponins. Drug-resistant pathogens’ infections are reported at 700,000 annual deaths and are estimated to Previous studies reported plants belonging to increase to 10 million by 2050. Included in these Boraginaceae, Fabaceae, Lamiaceae, and numbers is the contribution of antibiotic Lauraceae to have medicinal properties. In this resistance in addition to failure of anti-malarial regard, representative plants from these families drugs and antiviral therapy [19,20]. It is widely were used in this study. The Boraginaceae family known that there is an increasing and continuous has been found to have biological activities that prevalence of antibiotic-resistant bacteria include antioxidant, wound healing, anti- emerging from the extensive use of antibiotics.

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This situation may render the current that can cause bovine mastitis and septicemia antimicrobial agents insufficient to control at least in immunosuppressed mammals [23]. some bacterial infections [21]. According to the Vancomycin-resistant E. faecium (VREF) are 2012 World Health Organization (WHO) report, linked with infections of the urinary tract, wound, antibiotic resistance should be regarded as a and bloodstream; enterococci are the second global threat comparable to climate change and most common nosocomial pathogen and terrorism [22]. The use of traditional medicinal the pathogenic cause of bloodstream plants to search for new antimicrobial agents is infections in patients in intensive care units. an important line of research because of the Bloodstream infections due to vancomycin- resistance acquired by several pathogenic resistant Enterococcus faecium (VREF) are microorganisms. associated with substantial morbidity [25]. There has been a significant increase in resistance to The microorganisms used in this study namely, antibiotics, specifically vancomycin, thus the Staphylococcus aureus, Methicillin-resistant S. management of enterococcal infections has been aureus (MRSA), Pseudomonas aeruginosa, challenging [26]. Escherichia coli, Yersinia enterocolitica, Serratia marcescens and Vancomycin-resistant The fungal microorganisms used for this study Enterococci faecium (VREF) are of clinical were Candida albicans, Aspergillus niger, and importance. S. aureus is a major component of Trichophyton mentagrophytes. C. albicans the normal microflora of animals and humans, (candidiasis), inhabits the mucous membranes of colonizing the nasal cavity, naso-pharynx, skin, most mammal and bird species. The disease is and mucous membranes. However, related to immune and hormonal inadequacies, staphylococci can occasionally cause and reduced colonization resistance; these opportunistic infections in many animal species, conditions account for susceptibility of infants, including mastitis and suppurative conditions diabetics, subjects on antibiotics and steroids, [23]. S. aureus is also a common pyogenic agent patients with catheters, and mammary glands of in humans and several animal species, such as lactating cows [24]. A. niger is a ubiquitous mold abscess formation [24]. with opportunistic pathogenic patterns. It is present in the soil, vegetation, feed, and P. aeruginosa is an opportunistic microorganism secondarily in air, water, and exposed items. commonly found in wound infections in animal species e.g. canine otitis externa and cystitis, the Aspergillus spp. can cause disease in several uteri of mares, and the eyes of horses with ways, including mycotoxicoses and can be corneal ulcers. In humans, P. aeruginosa can involved in allergic reactions to humans; it can cause bacteremia in people with burns, also cause aspergillosis, which is acquired from leukemia, or cystic fibrosis [23,24]. Most strains environmental sources, generally by inhalation or are resistant to common antimicrobial agents and ingestion [23,24]. T. mentagrophytes, a are therefore difficult to eliminate. Another dermatophyte, is a mold capable of parasitizing organism used in this study is E. coli. It is an only keratinized epidermal structures (i.e., skin, opportunistic pathogen in many animal species hair, nails). Dermatophyte infection, also known and humans, causing urinary tract disease, as ringworm, is a zoonotic disease; the zoophilic abscess, and pneumonia. E. coli can also cause dermatophytes are obligate pathogens, primarily septicemic disease in foals, calves, piglets, parasitizing animals but also capable of infecting puppies, and lambs; enterotoxigenic diarrhea in humans [24]. newborn farm animals; and of edema disease in pigs [24]. This study aims to evaluate the antimicrobial potential of different leaf extracts and to identify Y. enterocolitica is commonly found in animal their secondary metabolites. In this study, products (i.e., milk and pork) and may be a gram-positive and gram-negative bacteria were source of human infection. It can cause acute used to examine the broad-spectrum antibiotic gastroenteritis, terminal ileitis (inflammations of potential of the leaf extracts understudy. the small intestine, specifically the ileum), and Secondary metabolites are known to be more septicemia in other primates [24]. This active against gram-positive bacteria and less microorganism is responsible for a zoonotic active against gram-negative bacteria [27]. In infection known as yersiniosis, which causes addition, the leaf extracts were used to determine food poisoning; pigs in particular are carriers of their antifungal activities. The rising frequency of Y. enterocolitica strains [23]. On the other hand, fatal mycoses associated with the increasing use S. marcescens is an environmental organism of immunosuppressed medical therapies have

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stimulated research directed towards the enterocolitica 330, Candida albicans 925, discovery of novel antifungal agents [28]. Due to Aspergillus niger 922 and Trichophyton the complexity of the fungal cell the pace of mentagrophytes 1047. antifungal activity discovery and development is exceedingly slow [29], thus the determination of 2.4 Antibacterial Susceptibility Testing antifungal activity of a leaf extract would be noteworthy. The turbidity of an overnight broth culture was adjusted to a level of A= 0.132 ± 0.005 at 625 2. MATERIALS AND METHODS nm. This level is optically comparable to the 0.5 McFarland standards. A spectrophotometer 2.1 Plant Collection (Bausch and Lomb, Model Spectronic 20) was used to adjust the absorbance of the suspension. Plant leaves were collected from labeled This yields a bacterial suspension of 8 plants/trees growing in the San Antonio Botanical approximately 0.5-1.0 x 10 CFU/mL. Agar Garden in Texas. Table 1 presents the plant plates were inoculated with 100 μL of the species collected for this study. The plant respective adjusted bacterial suspension. The material was ground to a fine powder. disk diffusion method [30] with modifications, was used to determine zone of inhibition values for 2.2 Plant Extracts the plant extracts. Mueller Hinton agar was prepared according to the manufacturer’s In the preliminary study, aqueous and ethanol instructions. Sterile Whatman antibiotic assay extracts of Sassafras albidum (Nutt.), Ehretia discs were impregnated with 20 μL of the plant anacua (Terán & Berl.), Melissa officinalis (Linn.), extracts or DMSO. The following antibiotic discs Eysenhardtia texana (Scheele), and Melissa were used as antibiotic positive controls: (1-2) S. odorata were prepared by mixing the powdered aureus and P. aeruginosa- 10 μg of samples in sterile distilled water or 70% ethanol, streptomycin; (3) MRSA- 30 μg of novobiocin respectively. They were then allowed to incubate and 10 units of penicillin; (4) VREF- 30 μg of in a heating water bath shaker for 18 h at 30°C. chloramphenicol and 30 μg of vancomycin; (5-6) Following filtration and centrifugation, the E. coli and S. marcescens- 5 μg of ciprofloxacin; supernatant was collected, and lyophilized using and (7) Y. enterocolitica- 30 μg of a freeze dry system (LabconcoTM). Thereafter, chloramphenicol. The plates were incubated at each stock was diluted using dimethyl sulfoxide 37°C for 18-20 h. After incubation, zone of (DMSO) to 66.7, 50, and 25 mg/mL. inhibition (largest and smallest) measurements were taken using a vernier caliper. The test was Preliminary results of the antimicrobial assay done in three replications and each replication only showed E. anacua extract to have was carried out in triplicate. antimicrobial activity. In this regard, only E. anacua leaves were used in soxhlet extraction 2.5 Antifungal Susceptibility Testing (ChemglassTM) procedure. Ehretia anacua’s diethyl ether, acetone, and ethanol extracts were Two methods of antifungal susceptibility testing prepared separately. Each extract was further were employed, depending on the morphology of concentrated by a rotary evaporator (Heidolph the fungi being tested. C. albicans’ morphology North America) and a freeze-drier system includes convex colonies which were similar to (Labconco). The extracts were prepared to 25 bacterial colonies, thus the same antimicrobial mg/mL. susceptibility testing was used, with a few modifications. The modifications were: PDA 2.3 Microorganisms media, 100 units of Nystatin, and incubation for 72 h at 28°C. The test was done in three The microorganisms used in this study were replications and each replication was carried out isolates and strains provided by American Type in triplicate. Culture Collection (ATCC) and Presque Isle Culture: Staphylococcus aureus ATCC 700699 For filamentous fungi producing septate hyphae, (MRSA), vancomycin-resistant Enterococci PDA and SDA media was used for A. niger and faecium ATCC 700221 (VREF), Staphylococcus T. mentagrophytes, respectively. The bottom of aureus 4651, Pseudomonas aeruginosa 99, each agar plate was marked 1.5 cm from the methicillin-resistant Escherichia coli strain B 337, perimeter to create a new circular circumference. Serratia marcescens 4651, Yersinia A loop-full of fungal sample was transferred from

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Table 1. Plant species used in this study

Scientific Name Common Name Family Ehretia anacua (Terán & Berl.) Anaqua, Knockway Boraginaceae (borage) Eysenhardtia texana (Scheele) Texas kidneywood Fabaceae (pea) Melissa odorata Lemon Balm Lamiaceae (mint) Melissa officinalis (Linn) Common balm Lamiaceae (mint) Sassafras albidum (Nutt.) Sassafras, Cinnamon Wood, Ague Tree Lauraceae (laurel) *This table was created with the information provided by the United States Department of Agriculture: Natural Resources Conservation Service and Lady Bird Johnson Wildflower center at the University of Texas at Austin. the culture to the respective media plate, within 3. RESULTS AND DISCUSSION the boundaries of the newly drawn boundary. Sterile water was pipetted onto the 3.1 Antimicrobial Activity of Aqueous and center of the media plate and was mixed with the Ethanol Extracts from Different Plant fungal hyphae to spread it evenly within the new Families (Boraginaceae, Fabaceae, drawn boundary. Plates were incubated at 28 C  Lamiaceae, and Lauraceae) for 24 h (A. niger) or 7-8 days at room temperature (T. mentagrophytes). Assay discs Fig. 1 demonstrates the mean zone of inhibitions were impregnated with 20 μL of the plant (ZOI) against S. aureus of all aqueous and extracts or DMSO; they were then placed on the ethanol leaf extracts from the five plant species perimeter of the newly drawn boundary. The understudy. It demonstrates that the ethanol E. negative control was DMSO, while positive was anacua extracts were found to be significantly 100 units of Nystatin [31]. The plates were then different (p<0.05), in terms of mean ZOI (9.3 mm, incubated, for 48 h at 28°C and 9 days at room 13.0 mm, and 12.2 mm at 25 mg/mL, 50 mg/mL, temperature for A. niger and T. mentagrophytes, and 67 mg/mL respectively), from the negative respectively. After the second incubation, each controls (6 mm, water, and DMSO), for the anti- disc was observed for a zone of inhibition (length S. aureus activity. Results showed evidence that and height) using a vernier caliper. The test was the ethanol E. anacua extracts possess anti-S. done in three replications and each replication aureus activity. This is the first report on the was carried out in duplicate. antimicrobial activity of E. anacua leaf extract.

2.6 Statistical Analysis E. anacua belongs to Boraginaceae family, results of this study is similar to a study on C. A single factor Analysis of Variance (ANOVA) gilletii and L. erythrorhizone, also belonging to and Post Hoc Bonferroni test were performed, in the Boraginacae family. C. gilletii and L. order to determine which treatment group’s mean erythrorhizone extracts also exhibited anti-S. zone of inhibition was significantly different, in aureus activities [38, 39, 40, 41]. On the other terms of zone of inhibition, from the respective hand, similar to C. odontophyllum, both extracts negative and positive controls. All analyses were were specific only for anti-S. aureus activity [42]. carried out using IBM SPSS Statistics Version The antibacterial activities of hexane, acetone, 20™ software, provided by Texas A&M methanol, and aqueous leaf extracts from C. International University. odontophyllum were tested against two-gram positive bacteria (S. aureus and Bacillus cerus) 2.7 Phytochemical Screening and two gram-negative bacteria (P. aeruginosa and E.coli). The acetone and methanol C. Phytochemical analysis was performed on the odontophyllum extracts exhibited anti-S. aureus plant extracts found to have a potential for activity; all plant extracts were found to not have antimicrobial activities. The plant extracts were antimicrobial potential for the other subjected to standard chemical tests to microorganisms tested. From phytochemical determine which secondary metabolites are screening, they detected the presence of present in the plant species [32,33,34,35, te rpenoids, tannins, flavonoids, and phenols in C. 36,37]. Chemical tests were done in three odontophyllum extracts. The authors attributed replications and each replication was carried out the anti-S. aureus activity to the secondary in duplicate. metabolites present in both plant extracts [42].

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In our study all 5 aqueous plant extracts and 4 officinalis, it is possible that the antimicrobial out of the 5 plant species for the ethanol plant secondary metabolites, or if at all present, are in extracts were found to be not significantly concentrations that cannot exhibit antimicrobial different (P = 0.05), in terms of mean zone of activity detectable by the assays performed. In inhibition, to the negative control, for anti-S. addition, MRSA and VREF bacteria have aureus activity (Fig. 1). In addition, all aqueous developed mechanisms to become resistant to and ethanol plant extracts for the plant species antibiotics; the secondary metabolites extracted tested, were found to not have a significant from the ethanol E. anacua extract could have antimicrobial potential for P. aeruginosa, MRSA, not been present in high enough concentrations or VREF activity (data not shown). All aqueous to have antimicrobial activity against MRSA nor and ethanol plant extracts for the plant species VREF. It could also be likely that the secondary tested, were found to be significantly different metabolites in E. anacua are not inhibitory to any from the positive control, Streptomycin. This is of the biological processes that allow MRSA or not surprising since most secondary compounds VREF to survive. Some plant extracts have been with anti-microbial activities have been reported found to have anti-P. aeruginosa and MRSA to be extracted by ethanol as compared to activity, but it could be attributed to the flavonoids aqueous extraction. Ethanol extraction has been present as secondary metabolites [44, 45]. Since shown to be the most effective extraction method there was no detectable flavonoids in the ethanol for isolating the bioactive phytocompound [43]. extract of E. anacua, this could be one of the On the other hand, for the ethanol extracts of E. possibilities why E. anacua extracts did not texana, S. albidum, M. odorata, and M. exhibit anti-P. aeruginosa and MRSA activity.

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Fig. 1. Determination of anti-Staphylococcus aureus activity against all plant extracts tested *denotes significance at 5% level

Ynalvez et al.; JPRI, 23(2): 1-12, 2018; Article no.JPRI.41205

3.2 Antimicrobial Activity of E. anacua activity will provide the basis for future research Extracts activities. This could include antimicrobial activity against other microorganisms; antimicrobial Since the ethanol E. anacua extract was found to activity with extracts from other parts of E. have an anti-S. aureus activity, E. anacua leaves anacua plant i.e. stem, bark, flower; and were subjected to soxhlet extractions using antimicrobial assays using different extracts, i.e. different solvents. As demonstrated in Fig. 2, chloroform, hexane, and ethyl acetate. both ethanol (7.4 mm) and diethyl ether (7.5 mm) E. anacua extracts, and streptomycin (17.3 mm) The acetone, diethyl ether, and ethanol E. were found to be significantly different (P= 0.05), anacua extracts were found not to have a in terms of mean zone of inhibition, to the significant antimicrobial potential against P. negative control (6.0 mm); therefore, compared aeruginosa, Y. enterocolitica, E. coli, S. to the acetone extract, the ethanol and diethyl E. marcescens, C. albicans, A. niger, and T. anacua extracts showed a statistically significant mentagrophytes (data not shown). It is not antimicrobial activity against S. aureus. Although surprising that the ethanol and diethyl ether E. the values, 7.4 mm and 7.5 mm for the ethanol anacua extract possessed anti-S. aureus activity and diethyl ether extracts could be low values for but did not exhibit antimicrobial activity against ZOI, the potential for E. anacua for anti-S. aureus the gram-negative bacterium and fungi. S. activity cannot be undermined. In this regard, our aureus is a gram-positive bacterium, while P. results will give rationale for the isolation, aeruginosa, S. marcescens, Y. enterocolitica and purification, and identification of the bioactive E. coli are gram-negative bacteria. It has been compound in the E. anacua extracts that is shown that secondary metabolites are more responsible for the observed anti-S. aureus active against gram-positive than gram-negative activity. The isolation and purification of the bacteria. This difference has been attributed to bioactive compound in the E. anacua extracts will the different cell wall and membrane structure be an exciting and impacting research endeavor between the gram-positive and gram-negative especially for scientists in search for and/or in bacteria. The secondary metabolites can need of a natural product against S. aureus. In penetrate the peptidoglycan envelope and reach addition, with the anti-S. aureus activity found in the cell membrane of gram-positive bacteria E. anacua, although not as promising compared easier than in gram-negative bacteria, which are to other plant extracts, the presence of such an protected by a lipopolysaccharide envelope [46].

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Fig. 2. Determination of anti-Staphylococcus aureus activity against Ehretia anacua leaf extracts *denotes significance at 5% level

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Flavonoids and saponins have been studied to secondary metabolites. For the ethanol E. be able to disrupt cell membrane integrity [47]. anacua extract - - alkaloids, cardiac glycosides, Our phytochemical analysis results (Table 2) of diterpenes, and phenols were present, while for the ethanol and diethyl ether E. anacua extracts the diethyl ether E. anacua extract - - alkaloids, did not possess detectable flavonoids and diterpenes, and phenols were present. These saponins. This could also be the reason that the findings are novel as no other studies has yet E. anacua extracts did not exhibit antifungal reported the secondary metabolites present in E. activity against C. albicans, A. niger, and T. anacua extracts. mentagrophytes. Cardiac glycosides are naturally occurring Acetone, diethyl ether, and ethanol E. anacua compounds with a steroidal framework and are extracts, were found to be significantly different used for the treatment of congestive heart failure (P= 0.05) from the positive control, Streptomycin. and as anti-arrhythmic agents. One example of a In addition, all crude plant extracts were found to cardiac glycoside was extracted from the Digitalis be significantly different than the respective lanata leaf. The cardiac glycoside is a digoxin, a antibiotic used for each tested microorganism. moderately polar compound used for the The commercial antibiotics are expected to be treatment of different heart conditions and not more potent than the crude plant extract since reported for any anti-bacterial activity; therefore, the antibiotic is a pure compound. Plant crude this secondary metabolite present in the ethanol extracts contain many compounds, and could E. anacua extract is probably not the compound include compounds that will act as antagonists to exhibiting the anti-S. aureus activity [48, 49]. the compounds that are exhibiting the antimicrobial activity; this could by why the E. Diterpenes are a class found in one of the major anacua extracts where found to be not as potent, group of secondary metabolites, known as compared to Streptomycin, for anti-S. aureus terpenoids; they contain 4 isoprene units creating activity. a 20-carbon compound [50]. The terpenoids are diverse substances, ranging in polarity where 3.3 Phytochemical Analysis most of the classes are insoluble in water. Nonpolar components would be extracted with Since the ethanol and diethyl ether E. anacua the nonpolar solvent, diethyl ether. Several extracts were found to have anti-S. aureus compounds in the E. anacua diethyl ether extract activity, these extracts were subjected to could be compounds of the class diterpenes. On chemical tests in order to determine the classes the other hand, terpenes could also be present in of secondary metabolites present. As shown in the ethanol E. anacua extract. Terpenes are Table 2, both ethanol and diethyl ether E. anacua known to defend many plants against herbivores, extracts were positive for several classes of by being toxins and feeding deterrents to many

Table 2. Determination of the presence of secondary metabolites for the ethanol and diethyl ether Ehretia anacua extracts through qualitative phytochemical tests

Chemical tests for Ethanol Diethyl Observations secondary metabolites extract ether extract Alkaloids Positive Positive et: +++ (heavy flocculation with both reagents) de: ++ (turbidity with Wagner’s reagent) Diterpenes Positive Positive emerald green solution Sterols/ Triterpenes Negative Negative et: beige in chloroform phase de: 4 phases present (beige to dark green) Saponins Negative Negative no foam present Phenols Positive Positive black solution Flavonoids Negative Negative dark olive green/ brown Anthranol glycosides Negative Negative Solution olive solution with white precipitate Tannins Negative Negative no precipitate present Resins Negative Negative no precipitate Cardiac glycosides Positive Negative et: dark emerald green solution de: light olive green solution Cyanogenic glycosides Negative Negative yellow picrate paper *Observations: et=ethanol E. anacua extract; de=diethyl ether E. anacua extract

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herbivorous insects and mammals [50]. It is then pharmaceuticals, including morphine, codeine, possible, that diterpenes present in the ethanol caffeine, and nicotine [50]. The role of chemical and diethyl ether E. anacua extracts, could be defense for plant alkaloids is supported by their the main secondary metabolites exhibiting the wide range of physiological effects on animals anti-S. aureus activity. As most terpenes are and by the antibiotic activities that many alkaloids lipophilic, they readily interact with membrane possess [53]. Several plant extracts, such as proteins. Thus, terpenes can increase the fluidity aqueous Tamarindus indica and ethanol Morinda and permeability of the membranes, which can citifolia, have exhibited antimicrobial activity, lead to uncontrolled efflux of ions and including S. aureus, where phytochemical metabolites and even to cell leakage, resulting in screening finds that their major constituents are necrotic or apoptotic cell death [51]. The alkaloids [54,55]. Phytochemical analysis from mechanism of action of terpenes for antimicrobial the chemical tests showed that the ethanol and activity is not fully understood, but is thought to diethyl ether E. anacua extracts contain involve membrane disruption due to their alkaloids. It is also possible that the alkaloids lipophilic nature, consequently they can modulate contributed to the observed anti-S. aureus the activity of membrane proteins and receptors activity. In particular, the pyrrolizidine alkaloids, or ion channels [51,52]. frequently found in members in the Boraginaceae family, could be exhibiting this antimicrobial Plants produce a large variety of secondary activity. However, these alkaloids render most compounds that contain a phenol, which are plants toxic to mammals [51]. The mechanism of classified as phenolics. Plant phenolics are a action of highly aromatic alkaloids is attributed to chemically heterogeneous group of nearly their ability to intercalate with DNA [56]. 10,000 compounds. Some are soluble only in organic solvents, while others are water-soluble Therefore for both the ethanol and diethyl ether carboxylic acids. This explains the potential of E. anacua extracts, we propose that alkaloids, phenol compounds to be extracted in both diterpenes, and phenols contribute to the S. nonpolar diethyl ether and polar ethanol E. aureus activity. In this regard, our preliminary anacua extracts. Phenolics have been known to results will provide a basis for further play a variety of roles in plants such as: serving investigations that will include quantitative as defenses against herbivores and pathogens; phytochemical analysis of the secondary attracting pollinators; absorbing harmful metabolites, HPLC (high performance liquid ultraviolet radiation; or reducing the growth of chromatography) for the isolation and purification nearby competing plants. Many simple phenolic of the bioactive compound in E. anacua and compounds are important in plants as defenses mass spectrometry for the identification of the against insect herbivores and fungi [50]. specific phytochemical responsible for the anti-S. Consequently, phenols present in both ethanol aureus activity in E. anacua. The isolation, and diethyl ether E. anacua extract could be purification, and identification of the bioactive contributing to the anti-S. aureus activity. The compounds in the E. anacua extracts will be an mechanism proposed for phenolics includes: exciting and impacting research endeavor phenolic toxicity to microorganisms via enzyme especially for scientist in search for and/or in inhibition by the oxidized compounds; their need of natural product against S. aureus. reaction with sulfhydryl groups; or by building hydrogen, hydrophobic and ionic bonds, thus 4. CONCLUSIONS modulating their 3D structures and in consequence their bioactivities [51,52]. Results showed evidence that the ethanol and diethyl E. anacua extracts possess anti S. aureus Alkaloids are organic heterocyclic nitrogen activity. Phytochemical analysis demonstrated compounds, which make up 15,000 secondary that ethanol E. anacua extract was positive for metabolites [50]. They contain nitrogen, which is alkaloids, cardiac glycosides, diterpenes, and usually derived from an amino acid. Alkaloids are phenols, while diethyl ether E. anacua extract of considerable interest because of their was positive for alkaloids, diterpenes, and medicinal properties. Since most alkaloids are phenols. This is the first report on E. anacua’s alkaline, the nitrogen atom is protonated; they antimicrobial activity and their secondary are positively charged and are generally water metabolites. Alkaloids, diterpenes, and phenols soluble [50]. As expected, both ethanol and either individually or synergistically are likely diethyl ether E. anacua extracts extracted the responsible for the observed antimicrobial alkaloid compounds. Many alkaloids are used as activity.

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CONSENT (Wall.). J App Pharm Sci, 2016;6(05):163- 170. As per international standard or university 7. Yalavarthi C, Thiruvengadarajan VS. A standard, patient’s written consent has been review on identification of strategy of collected and preserved by the author(s). phytoconstituents present in herbal plants. Int J Res Pharm Sci. 2013;4(2):123-140. ETHICAL APPROVAL 8. González-Burgos E, Carretero ME, Gómez-Serranillos MP. Sideritis Spp.: As per international standard or university Uses, chemical composition and standard, written approval of Ethics committee pharmacological activities—a review. J has been collected and preserved by the Ethnopharmacol. 2011;135:209-25. author(s). 9. Amor I, Boubaker J, Sgaier MB, Skandrani I, Bhouri W, Neffati A, Kilani S, Bouhlel I, ACKNOWLEDGMENTS Ghedira K, Chekir-Ghedira L. Antimicrobial activity of essential oils isolated from The authors would like to thank Ms. Sasha Kodet Phlomis crinite Cav. ssp/ mauritanica and the staff of the San Antonio Botanical Munby. J Am Oil Chemists’ Society. 2008; Garden. The authors also would like to thank 85:1-5. TAMIU GREAT Program (funded by Title V 10. Papageorgiou VP, Assimopoulou AN, PPOHA Program United States Department of Ballis AC. Alkannins and shikonins: A new Education Award #P031M105048) for funding class of wound healing agents. Curr Med this research. Chem. 2008;15:3248-3267. 11. Balpinar N, Okmen G. The biological COMPETING INTERESTS activities of Moltkia aurea Boiss., an endemic species to Turkey. Afr J Tradit Authors have declared that no competing Complement Altern Med. 2017;14:60–64. interests exist. 12. Digrak M, Alma MH, Ilçim A. Antibacterial and antifungal activities of Turkish REFERENCES medicinal plants. Pharm Biol, 2001;39: 346-350. 1. Gupta PD, Birdi TJ. Development of 13. Dresler S, Szymczak G, Wójcik M. botanicals to combat antibiotic resistance. Comparison of some secondary metabolite Journal of Ayurveda and Integrative content in the seventeen species of the Medicine, 2017;8:266-275. Boraginaceae family. Pharm Biol. 2017; 2. Hossain K, Hassin M, Parvin N, Hasan M, 55:691–695. Islam S, Haque A. Antimicrobial, cytotoxic 14. Gharib A, Godarzee M. Determination of and thrombolytic activity of Cassia senna secondary metabolites and antioxidant leaves. J Appl Pharm Sci, 2012;2:186-190. activity of some Boraginaceae species 3. Ramawat KG. Secondary plant products in growing in Iran. Tropical J Pharm Res. nature, in: Ramawat, KG, Merillon, JM 2016;15:2459-2465. (Eds.). Biotechnology secondary 15. Oza MJ, Kulkarni YA.Traditional uses, metabolites plants and microbes. Enfield: phytochemistry and pharmacology of the Science Publishers. 2007;21-58. medicinal species of the genus Cordia 4. Vaghasiya Y, Dave R, Chanda S. (Boraginaceae). J Pharm and Pharmacol. Phytochemical analysis of some medicinal 2017;69:755-789. plants from Western region of India. Res. 16. Sousa C, Moita E, Valentão P, Fernandes J. Med. Plant. 2011;5:567-576. F, Monteiro P, Andrade PB. Effects of 5. Chandra H, Bishnoi P, Yadav A, Patni B. colored and non-colored phenolics of Antimicrobial resistance and the alternative Echium plantagineum L. bee pollen in resources with special emphasis on plant- Caco-2 cells under oxidative stress based antimicrobials—a review. Plants. induced by tert-butyl hydroperoxide. J 2017;16:1-11. Agric Food Chem. 2015;63:2083-2091 6. Tania UH, Hassan MR, Eshita NJ, Akhter 17. Duraipandiyan V, Ayyanar M, Ignacimuthu R and Shahriar M. Evaluation of In vitro S. Antimicrobial activity of some Antioxidant and In vivo Pharmacological ethnomedicinal plants used by Paliyar Activity of Leaf Extracts of Hoya parasitica tribe from Tamil Nadu, India. BMC

Ynalvez et al.; JPRI, 23(2): 1-12, 2018; Article no.JPRI.41205

Complementary and Alternative Medicine. new naphthoquinones with antifungal 2006;6:1-7. activity. In: 14th Brazilian Meeting on 18. Ara A, Saleh-e-In M, Ahmed NU, Ahmed Organic Synthesis; 2011. M, Bachar SC. Phytochemical screening, 29. Odds FC, Brown AJP, Gow NAR. analgesic, antimicrobial and anti-oxidant Antifungal agents: Mechanisms of action. activities of bark extracts of Adenanthera Trends Microbiol. 2003;11:272-279. pavonina L. (Fabaceae). Adv Nat Appl Sci. 30. Pirbalouti AG, Asadpor A, Hamedi B. 2010;4:352-360. Bioactivity of Iranian medicinal plants 19. O’Neill, J. Tackling drug-resistant against Yersinia enterocolitica. Nutr & infections globally: Final report and Food Sci. 2010;40:515-522. recommendations. The review on 31. Navarro-Garcia VM, Gonzalez A, Fuentes antimicrobial resistance; London: HM M, Aviles M, Rios MY, Zepeda G and Government and the Wellcome Trust; Rojas MG. Antifungal activities of nine 2016. traditional Mexican medicinal plants. J 20. Robinson TP, Bub DP, Carrique-Masc J, Ethnopharmacol. 2003; 87:85-88. Fèvred EM, Gilberte M, Gracea D, Hay SI, 32. Lu Y, Knoo TJ, Wiart C. Phytochemical Jiwakanonh J, Kakkari M, Kariukij S, analysis and antioxidant activity Laxminarayank R, Lubrothl J, determination of crude extracts of Magnussonm U, Thi Ngocn P, Van Melodinus eugeniifolus barks and leaves Boeckelo TP, Woolhousep MEJ. Antibiotic from Malaysia. Pharmacology & resistance is the quintessential one health Pharmacy. 2014; 5:773-780. issue. Trans R Soc Trop Med Hyg. 2016; 33. Panti AB, Aguda RM, Razal RA, Belina- 00:1–4. Aldemita MD, Tongco JVV. Proximate 21. Babu PD, Subhasree RS. Antimicrobial analysis, phytochemical screening and activities of Lawsonia inermis- a review. total phenolic and flavonoid content of the Acad J Plant Sci. 2009;2:231-232. ethanolic extract of molave Vitex parviflora 22. Daeseleire E, De Graef E, Rasschaert G, Juss. leaves. J Chem Pharm Res. 2014; De Mulder T, Van den Meersche T, Van 6:1538-1542. Coillie E, Dewulf J, Heyndrickx M. 34. Nnamdi NL, Egbuonu ACC, Ukoha PO, Antibiotic use and resistance in animals: Ejikeme PM. Comparative phytochemical Belgian initiatives. Drug Test. Analysis. and antimicrobial screening of some 2016;8:549–555. solvent extracts of Samanea saman 23. Markey B, Leonard F, Archambault M, (Fabaceae or Mimosaceae) pods. Afr J Cullinaru A and Maguire D. Clinical Pure Appl Chem. 2010;4:206-212. Veterinary Microbiology. Elsevier, London; 35. Evans WC. Trease and Evans 2013. Pharmacognosy, 16th ed. London: 24. Hirsh DC and Zee YC. Veterinary Elsevier; 2009. Microbiology, First ed. Blackwell Science, 36. Usman H, Abdulrahman FI, Usman A. Malden; 1999. Qualitative phytochemical screening and in 25. Ulricha N, Vonberg RP, Gastmeiera P. vitro antimicrobial effects of methanol stem Outbreaks caused by vancomycin-resistant bark extract of Ficus thonningii Enterococcus faecium in hematology and (Moraceae). Afr J Tradit Complement oncology departments: A systematic Altern Med. 2009;6:289-295. review. Heliyon. 2017;e00473:1-19. 37. Harborne JB. Phytochemical methods: A 26. Zeana C, Kubin CJ, Della-Latta D, Guide to modern techniques of plant Hammer SM. Vancomycin-resistant analysis, Third ed. London: Chapman and Enterococcus faecium meningitis Hall; 1973. successfully managed with linezolid: Case 38. Hernandez T, Canales M, Teran B, Avila report and review of the literature. Clinical O, Duran A, Garcia AM, Hernandez H, Infectious Diseases. 2001;33:477-482. Angeles-Lopez O, Fernandez-Araiza M, 27. Riffel A, Medina LF, Stefani V, Santos RC, Avila G. Antimicrobial activity of the Bizani D, Brandelli A. In vitro antimicrobial essential oil and extracts of Cordia activity of a new series of 1,4- curassavica (Boraginaceae). J Ethno- naphthoquinones. Braz J Med Biol Res. pharmacol. 2007;111:137-141. 2002;35:811-818. 39. Okusa PN, Penge O, Devleeschouwer M, 28. Roldi LL, Greco SJ, Lacerda V, Bezerra Duez P. Direct and indirect antimicrobial dos Santos R, de Castro E. Synthesis of effects and antioxidant activity of Cordia

Ynalvez et al.; JPRI, 23(2): 1-12, 2018; Article no.JPRI.41205

gilletii De Wild (Boraginaceae). J their antifungal mechanisms—a review. Ethnopharmacol. 2007;112:476-81. Medicinal & Aromatic Plants. 2014;3:1-6. 40. Giner MJ, Vegara S, Funes L, Marti N, 48. Shi LS, Kuo SC, Sun HD, Morris-Natschke Saura D, Micol V, Valero, M. Antimicrobial SL, Lee KH, Wu TS. Cytotoxic cardiac activity of food-compatible plant extracts glycosides and coumarins from Antiaris and chitosan against naturally occurring toxicaria. Bioorganic and Medicinal micro-organisms in tomato juice. J Sci Chemistry. 2014;22:1889-1898. Food Agr. 2011;92:1917-923. 49. Moore WN, Taylor LT. Extraction and 41. Vegara S, Funes L, Martí N, Saura D, quantitation of digoxin and acetyldigoxin Micol V, Valero M. Bactericidal activities from Digitalis lanata leaf via near- against pathogenic bacteria by selected supercritical methanol-modified carbon constituents of plant extracts in carrot dioxide. J Nat Prod. 1996;59:690-693. broth. Food Chem. 2011;128:872-77. 50. Taiz L, Zeiger E. Secondary metabolites 42. Basri DF, Nor NHM. Phytoconstitutent and plant defense, in: Plant Physiology, screening and antibacterial activity of the 5th ed. Sunderland: Sinauer Associates; leaf extracts from Canarium odontophyllum 2010. Miq. Am J Plant Sci. 2014;5:2878-2888. 51. Wink M. Modes of action of herbal 43. Ramos MFR. Bioactive phytocompounds: medicines and plant secondary New approaches in the phytosciences, in: metabolites. Medicines. 2015;2:251-286. Ahmad I, Aqil F, Owais M (Eds.), Modern 52. Arif T, Bhosale JD, Kumar N, Mandal TK, Phytomedicine: Turning Medicinal Plants Bendre RS, Lavekar GS, Dabur R. Natural into Drugs. Weinheim: Wiley-VCH. 2006; products: Anti-fungal agents derived from 1-24. plants. J Asian Nat Prod Res. 2009;7:621- 44. Agbafor KN, Akubugwo EI, Ogbashi ME, 628. Ajah PM, Ukwandu CC. Chemical and antimicrobial properties of leaf extracts of 53. Buchanan B, Gruissem W, Jones R. Zapoteca portoricensis. Res J Med Plant. Natural products (secondary metabolites) 2011;5:605-612. in Biochemistry and Molecular Biology of 45. Munyendo WLL, Orwa JA, Rukunga GM, Plants. Waldorf: Courier Companies, Inc; Bii CC. Bacteriostatic and bactericidal 2007. activities of Aspilia mossambicensis, 54. Sibi G, Chatly P, Adhikaria S, Ravikumar Ocimum gratissimum and Toddalia asiatica KR. Phytoconstituents and their influence extracts on selected pathogentic bacteria. on antimicrobial properties of Morinda Res J Med Plant. 2011;5:717-727. citrifolia L. Res J Med Plant. 2012;6:441- 46. Ani IA, Zimmermann S, Reichling J, Wink 448. M. Pharmacological synergism of bee 55. Abukakar MG, Ukwuani AN, Shehu RA. venom and melittin with antibiotics and Phytochemical screening and antibacterial plant secondary metabolites against multi- activity of Tamarindus indica pulp extract. drug resistant microbial pathogens. Asian J Biochem. 2010;5:310-314. Phytomedicine. 2015;22:245-255. 56. Cowan MM. Plant products as 47. Freiesleben SH, Jager AK. Correlations antimicrobial agents. Clin Microbiol Rev. between plant secondary metabolites and 1999;12:564-58.

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