Antimicrobial Bioactive Phytocompounds from Extraction to Identification Process Standardization

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Different approaches to drug discovery using higher plants can be distinguished: random selection followed by chemical screening; random selection followed by one or more biological assays; biological activity reports and ethnomedical use of plants [14]. The latter approach includes plants used in traditional medical systems; herbalism, folklore, and shamanism; and the use of databases. The objective is the targeted isolation of new bioactive phytocompounds. When an active extract has been identified, the first task to be taken is the identification of the bioactive phytocompounds, and this can mean either a full identification of a bioactive phy-tocompound after purification or partial identification to the level of a family of known compounds [15].

In Fig. 1.2 an extraction-to-identification flowchart is proposed in order to optimize bioactive phytocompound identification. For screening selection, plants are collected either randomly or by following leads supplied by local healers in geographical areas where the plants are found. Initial screening of plants for possible antimicrobial activities typically begins by using crude aqueous or alcohol extractions followed by various organic extraction methods [16]. Plant material can be used fresh or dried. The aspects of plant collection and identification will be discussed further in this chapter. Other relevant plant materials related to antimicrobial activity are the essential oils. Essential oils are complex natural mixtures of volatile secondary metabolites, isolated from plants by hydro or steam distillation and by expression (citrus peel oils). The main constituents of essential oils (mono and sesquiterpenes), along with carbohydrates, alcohols, ethers, aldehydes, and ke-

tones, are responsible for the fragrant and biological properties of aromatic and medicinal plants. Due to these properties, since ancient times spices and herbs have been added to food, not only as flavoring agents but also as preservatives. For centuries essential oils have been isolated from different parts of plants and are also used for similar purposes.

The activities of essential oils cover a broad spectrum. Various essential oils produce pharmacological effects, demonstrating anti-inflammatory, antioxidant, and anticancerogenic properties [17-19]. Others are biocides against a broad range of organisms such as bacteria, fungi, protozoa, insects, plants, and viruses [20-22].

The dispersion of the hydrophobic components of essential oils in the growth medium is the main problem in testing the activity of essential oils. Different organic solvents must be used as solubilizing agents, which may interfere with the results of antimicrobial assays. The solution to this problem is the use of nonionic emulsifiers, such as Tween 20 and Tween 80. These molecules are relatively inactive and are widely applied as emulsifying agents. Control tests must guarantee that these emulsifying agents do not interfere in the experiments.

Plants can be dried in a number of ways: in the open air (shaded from direct sunlight); placed in thin layers on drying frames, wire-screened rooms, or in buildings; by direct sunlight, if appropriate; in drying ovens/rooms and solar dryers; by indirect fire; baking; lyophilization; microwave; or infrared devices. Where possible, temperature and humidity should be controlled to avoid damage to the active chemical constituents. The method and temperature used for drying may have a considerable impact on the quality of the resulting medicinal plant materials. For example, shade drying is preferred to maintain or minimize loss of color of leaves and flowers; and lower temperatures should be employed in the case of medicinal plant materials containing volatile substances [23]. The drying conditions should be recorded. In the case of natural drying in the open air, medicinal plant materials should be spread out in thin layers on drying frames and stirred or turned frequently. In order to secure adequate air circulation, the drying frames should be located at a sufficient height above the ground. Efforts should be made to achieve uniform drying of medicinal plant materials to avoid mold formation [24].

Drying medicinal plant material directly on bare ground should be avoided. If a concrete or cement surface is used, the plant materials should be laid on a tarpaulin or other appropriate cloth or sheeting. Insects, rodents, birds and other pests, and livestock and domestic animals should be kept away from drying sites. For indoor drying, the duration of drying, drying temperature, humidity and other conditions should be determined on the basis of the plant part concerned (root, leaf, stem, bark, flower, etc.) and any volatile natural constituents, such as essential oils. If possible, the source of heat for directs drying (fire) should be limited to butane, propane or natural gas, and temperatures should be kept below 60 °C [25]. If other sources of fire are used, contact between those materials, smoke, and the medicinal plant material should be avoided.

Since researches are trying to identify bioactive phytocompounds in medicinal plant extracts generally used by local population to treat diseases and based on empiric knowledge that they have the searched bioactivity, the solvent chosen must be

Medicinal Plant Work Flow Chart

Fig. 1.2 Standardization flowchart: from extraction to identification of bioactive phytocompounds. (1) Plants can be chosen either randomly, based on the literature or following consulation with local healers. After choosing the right material, plant collection must be followed by botanical identification and a voucher specimen must be placed in the local herbarium. All data about the collection must be observed and documented, such as climate conditions, season, geographical localization, environmental conditions, etc. in order to elucidate future differences in bioactivity compared with other results found. Any plant part can be used but consultation of the literature or with local healers is very useful to reduce research time. (2) Collected plant material can be used fresh or dried. Several studies have started extractions with both fresh and dried material in order to compare the chemical composition of the extracts. They must be ground to optimize the solvent contact during the extraction process. Weight standardization must be used (i.e. 300 g of plant material to 1000 mL of solvent). More than 90% of the studies for antimicrobial activity in the literature start extraction with methanol, ethanol or water because it is proved that ethanol extraction is more effective in isolating the bioactive phyto-compound. The primary extractions methods are very variable but the idea is to research activity cited in popular use, and to choose the same extraction method. This is especially useful to corroborate the in vivo activity found in popular use. (3) After extraction the volume must be concentrated by lyophiliza-tion or another concentration technique before screening. Usually, after the lyophiliza-tion process ground powder is obtained. This must be resuspended in water at a higher concentration (i.e. 1 g mL-1) for initial drop test screening. The high concentration of the extract guarantees the identification of the bioactivity, if present. Using low concentrations in drop tests may lead to false negative results. (4) Due to the complex composition of the extract primary separation may be used to facilitate the identification process.

Micromolecules can be separated from macromolecules (proteins and carbohydrates) by very simple techniques such as ethanol precipitation (30% v/v), centrifuga-tion (10 000g for 10min) and filtration systems such as Centricon and Amicon (Millipore). Supernatant and precipitate phases are obtained and can be separated in drop tests. As discussed previously, antimicrobial activity is commonly present in micromolecules (supernatant) phase. (5) The antimicrobial screening by drop test (formerly disk diffusion agar assay) is the most efficient and inexpensive assay to identify antimicrobial activity. The extract is dropped (i.e. 15 |jL) onto an agar surface previously inoculated with the desired microorganism. Note that is very important to count by McFarland scale or Newbauer chamber (i.e. 105 UFCmL-1 for bacteria; 106 cells mL-1 for fungi) the microorganism inoculums; this permits the antimicrobial activity to be compared within antibiotic controls and between different microorganism groups. (6) When antimicrobial activity is detected the minimum inhibitory concentration (MIC) must be determined to continue other antimicrobial assays of interest. The MIC is usually established by the broth dilution method. The use of 96-microwell plates to minimize costs is very effective, reducing the culture media quantities drastically and enhancing the test capacity (in one plate up to eight different extracts can be tested in 10 different concentrations plus 1 negative and 1 positive controls, also see Fig. 1.3). (7) Bio-guided chromatography techniques such as bioautography preceded by solvent separation is essential to initiate the bioactive phytocompound identification process; fraction collection with HPLC or FPLC assays, preparative TLC are also valid techniques. Bio-guided fraction and purification confirms previous results leading to isolation of a bioactive phytocompound. (8) By TLC assays, Rf values can be determined and polarity or even chemical groups (use of specific dyes) elucidated (Fig. 1.3). (9) NMR, HPLC/MS, and GC/MS are used to identify a bioactive phytocompound as discussed in this chapter.

the same as that used in popular treatment. As we know, water and ethanol are by far the most commonly used, and for this reason most studies begin with water or ethanol as solvents.

Water is almost universally the solvent used to extract activity. At home, dried plants can be ingested as teas (plants steeped in hot water) or, rarely, tinctures (plants in alcoholic solutions) or inhaled via steam from boiling suspensions of the parts. Dried plant parts can be added to oils or petroleum jelly and applied externally. Poultices can also be made from concentrated teas or tinctures.

Since nearly all of the identified components from plants active against microorganisms are aromatic or saturated organic compounds, they are most often obtained initially through ethanol and water extraction [26]. Some water-soluble compounds, such as polysaccharides like starch and polypeptides, including fabatin [27] and various lectins, are commonly more effective as inhibitors of virus adsorption and would not be identified in the screening techniques commonly used [28]. Occasionally tannins and terpenoids may be found in the aqueous phase, but they are more often obtained by treatment with less polar solvents (Fig. 1.2).

Another concern during the extraction phase is that any part of the plant may contain active components. For instance, the roots of ginseng plants contain the active saponins and essential oils, while eucalyptus leaves are harvested for their essential oils and tannins. Some trees, such as the balsam poplar, yield useful substances in their bark, leaves, and shoots [29]. The choice of which part to use must be based on ethnopharmacological studies and review of the literature.

For alcoholic extractions, plant parts are dried, ground to a fine texture, and then soaked in methanol or ethanol for extended periods. The slurry is then filtered and washed, after which it may be dried under reduced pressure and redissolved in the alcohol to a determined concentration. When water is used for extractions, plants are generally soaked in distilled water, blotted dry, made into slurry through blending, and then strained or filtered. The filtrate can be centrifuged (approximately 10 000g for 10 min) multiple times for clarification [30]. Crude products can then be directly used in the drop test and broth dilution microwell assays (Fig. 1.2) to test for antifungal and antibacterial properties and in a variety of assays to screen bioactivity (Fig. 1.3).

In order to reduce or minimize the use of organic solvents and improve the extraction process, newer sample preparation methods, such as microwave-assisted extraction (MAE), supercritical fluid extraction (SFE) and accelerated solvent extraction (ASE) or pressurized liquid extraction (PLE) have been introduced for the extraction of analytes present in plant materials. Using MAE, the microwave energy is used for solution heating and results in significant reduction of extraction time (usually in less than 30 min). Other than having the advantage of high extraction speed, MAE also enables a significant reduction in the consumption of organic solvents. Other methods, such as the use of SFE that used carbon dioxide and some form of modifiers, have been used in the extraction of compounds present in medicinal plants [31].

To identify the bioactive phytocompounds, liquid chromatography with an iso-cratic/gradient elution remains the method of choice in the pharmacopeia, and re-

Fig. 1.3 Current assays to identify bioactivity and start molecule identification. (A/B) Bioauthography technique: (A) Thin-layer chromatography (TLC) of aqueous extracts of (1) Ocimun gratissimum, (2) Anadenanthera macrocarpa, (3) Croton cajucara Benth. (4) Cymbopogon citrates, and (5) Juglans regia performed in silica gel G60 F 254 aluminum plates (5 _ 8). Plates were developed with n-butanol:acetic acid:water (8:1:1, v/v) and were visualized under ultraviolet light or after staining with cerric sulfate plate. (B) Alternatively, plates were placed inside Petri dishes and covered with over solid media (10 mL BHI with 1% phenol red). After overnight incubation for diffusion of the separated components, the plate was inoculated with Candida albicans (ATCC

51501) 106 cells per plate and incubated for 48 h at 37 °C. Growth inhibition can be seen in (1, 2, and 3) after spraying with methylthiazollyltetrazolium chloride (MTT) at 5 mg mL-1. (C) Drop test at same concentrations (200 ^g mL-1) of (1) aqueous extract from Punica granatum and commercially available antifungal agents, (2) fluconazole, (3) flucytosine, and (4) anphotericin. (D) MIC microwell dilution test of (L1) Punica granatum, (L2) fluconazole, and (L3) flucytosine against Candida albicans (ATCC 51501). (C+) positive control, (C-) negative control, (1) 200 ^g mL-1, (2) 100 ^g mL-1, (3) 50 ^g mL-1, (4) 25 ^g mL-1, (5) 12.5 ^g mL-1, (6) 6.75 ^g mL-1, (7) 3.4 ^g mL-1, (8) 1.7 ^g mL-1, and (9) 0.8 ^g mL-1. (+) means fungi growth.

versed octadecyl silica (C-18) and ultraviolet detection mode is the most commonly used method. Gradient elution HPLC with reversed phase columns has also been applied for the analysis of bioactive phytocompounds present in medicinal plants extracts [32].

The advantages of liquid chromatography include its high reproducibility, good linear range, ease of automation, and its ability to analyze the number of constituents in botanicals and herbal preparation. However, for the analysis of multiple bi-oactive phytocompounds in herbal preparations with two or more medicinal plants, coeluting peaks were often observed in the chromatograms obtained due to the complexity of the matrix. The complexity of matrix may be reduced with additional sample preparation steps, such as liquid-liquid partitioning, solid-phase extraction, preparative LC and thin-layer chromatography (TLC) fractionation.

Capillary electrophoresis (CE) proved to be a powerful alternative to HPLC in the analysis of polar and thermally labile compounds. Reviews on the analysis of natural medicines or natural products in complex matrix by CE are well reported. Many publications showed that all variants of CE, such as capillary zone electrophoresis (CZE), micellar electrokinetic capillary chromatography (MEKC), and capillary isoelectric focusing (clEF), have been used for the separation of natural products. The separation in CZE is based on the differences in the electrophoretic mobilities resulting in different velocities of migration of ionic species in the electrophoretic buffer in the capillary. For MEKC, the main separation mechanism is based on solute partitioning between the micellar phase and the solution phase. Factors that are known to affect separation in CZE and MEKC include the pH of the running buffer, ionic strength, applied voltage, and concentration and type of micelle added. From the review articles, CE has been used to determine the amount of cate-chin and others in tea composition, phenolic acids in coffee samples and flavo-noids and alkaloids in plant materials.

Chromatographic separation with mass spectrometry for the chemical characterization and composition analysis of botanicals has been growing rapidly in popularity in recent years. Reviews on the use of mass spectrometry and high-performance liquid chromatography mass spectrometry (HPLC/MS) on botanicals have been reported. The use of hyphenated techniques, such as high-resolution gas chromatography mass spectrometry (HRGC/MS), high performance liquid chromatography/mass spectrometry (HPLC/MS), liquid chromatography tandem mass spectrometry (HPLC/MS/MS) and tandem mass spectrometry (MS/MS) to perform on-line composition and structural analyses provide rich information that is unsurpassed by other techniques.

HRGC/MS remains the method of choice for the analysis of volatile and semi-volatile components, such as essential oils and others in botanicals and herbal preparations, along with high-resolution separation with capillary column coupling with mass spectrometry using electron impact ionization (EI).

In analyzing bioactive phytocompounds, HPLC/MS has played an increasingly significant role as the technique is capable of characterizing compounds that are thermally labile, ranging from small polar molecules to macromolecules, such as peptides/proteins, carbohydrates, and nucleic acids. The most common mode of ionization in HPLC/MS includes electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Mass analyzers, such as single quadruple, triple quadruple, ion-trap, time-of-flight, quadruple time-of-flight (Q-TOF) and others, are also used. With tandem mass spectrometry, additional structural information can be obtained about the target compounds. However, methods using HPLC/MS are still limited to conditions that are suitable for MS operations. There are restrictions on pH, solvent choice, solvent additives and flow rate for LC in order to achieve optimal sensitivity.

For the identification of bioactive phytocompounds by HRGC/MS or HPLC/MS, the following conditions are useful when standards are available: a suspect peak has to show a retention time similar to the average retention time of the pure standard or control sample and mass spectra for the suspect peaks have to show relative abundance ±10% (arithmetic difference) of the relative abundance of the standard analyzed that day. With HPLC/MS, applying the right separation, with the right ionization interface and mass analyzer, significant information can be obtained with regards to the target compounds. However, for the quantification of bi-oactive phytocompounds in plant materials, the system precision will be higher compared to that obtained using HPLC with ultraviolet detection. For on-line HPLC/MS, the internal diameter of the column selected will be an important consideration.

Another important chromatography technique is bioautography (Fig. 1.3). Bio-autography is often used as an option to identify chemical groups of bioactive phy-tocompounds or even a single bioactive phytocompound when padrons are available. The complex chemical composition of plant extracts is generally a limiting obstacle to the isolation of antimicrobial compounds. Nevertheless, the use of bioautography agar overlay bioassays allows the detection of active components in a crude plant extract. This method permits the localization of antimicrobial active components that have been separated by TLC [33]. Precipitation with ethanol of plant aqueous extracts allows the separation of polymers, such as polysaccharides and proteins, from micrometabolites [34, 27]. By this technique, the solvation between molecules is changed, and in the same way, the interaction between molecules. Polymers (macromolecules) will be found in the water-soluble precipitate and micrometabolites in the supernatant. The precipitation of macromolecules can also be achieved by ammonium sulfate and acetone. The association of bioautography and ethanol precipitation techniques allows the detection of otherwise nondetectable bioactive phytocompounds [35].

An extremely important aspect of chromatography techniques is to identify non-natural molecules, such as paracetamol, that may be present in or added to health supplements and commercially available herbal preparations.

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