Antibacterial Activity of Plant Polyphenols

This chapter focuses on methods of polyphenol isolation and on the antibacterial activity of different polyphenols found in herbs, spices, fruits and vegetables. Polyphenols are secondary metabolites which protect plants from different pathogens, such as viruses, bacteria, fungi, insects, and herbivores. Currently, about 9000 polyphenols found in more than 480 plants are known. Their amount fluctuates across different species and varieties. This chapter describes conventional and novel methods for extraction, the influence of the type of solvents, solvent concentration and temperature on the yield. The highest yield is obtained at 70% of methanol and ethanol, and at 90% of acetone. Extraction at 80°C leads to higher amounts of polyphenols than extraction at 100°C. Polyphenols are usually metabolized in the human liver but can also remain unaf-fected as they pass through the gastrointestinal tract. The main location for their uptake is the colon. They exhibit a wide range of antibacterial activity against Pseudomonas aeruginosa , Staphylococcus aureus , Streptococcus epidermidis , Klebsiella pneumoniae, E. coli , Listeria monocytogenes , Acinetobacter sp., Proteus sp., Micrococcus sp., and Bacillus sp. All these plants, rich in antimicrobial polyphenols, represent a promising and powerful source of highly effective novel antibacterial substances in the current era of ubiquitous antibiotic resistance.


Introduction
The widespread antibiotic resistance in the last 20 years has become one of the biggest worldwide threats to mankind. Plants are valuable reservoir of novel antimicrobials and their secondary metabolites as polyphenols demonstrate strong antimicrobial activity at extremely low concentrations. Precursor of polyphenols is phenol which consist of one aromatic ring and a hydroxyl group. Polyphenols as more complex substances are polyaromatic and contain a few hydroxyl groups. They are divided into four main groups: Flavonoids, Lignans, Phenolic acids, and Stilbenes. Among them, flavonoids are the most numerous. All polyphenols play an important role in the defense of plants against bacteria, viruses, fungi, insects and herbivores. Polyphenol synthesis derives from two aromatic amino acids -tyrosine and phenylalanine. As secondary plant metabolites, their amount is estimated at only around 10% of plant metabolites [1].
Among the 300,000 plant species that exist in the world only 15% have been investigated for their pharmacological potential, the rest of them are a potential source  6 19  Hydroxycinnamic acids (4), known also as coumaric acid, contains cinnamic acid, where the benzene ring is hydroxylated at C-4. Inside the cell, hydroxycinnamic acids are located in the cytoplasm and mitochondria. Similarly to salicylic acid, trans-4-coumaric acid is present in all eukaryotes. Plants like green pepper, apricot, and blueberry are excellent source of this acid. Like gallic acid, hydroxycinnamic acids can be detected in human feces, urine, and blood.
Ferulic acid (6), a tyrosin-similar compound comprises of the water soluble trans-cinnamic acid. Main location of the substance is the plant cell is the cell wall. Its name originates from the giant fennel (Ferula communis). Rich in ferulic acid are root vegetables and sweet poprcorn. Being a constituent of the plant biopolymer lignocellulose the ferulic acid is involved in accumulation of this most abundant biowaste on Earth. Pronounced apoptosis inhibitor and cardioprotector, ferulate also helps the skin aging retardation inhibiting melanin formation. Used widely as food preservative [5] it can be successfully excreted via human epidermis. Warfarin (7) falls in the class of 4-hydroxycoumarins and is one of the best synthetic oral anticoagulants. It constrains the synthesis of blood clotting factors which depends on Vitamin K. The key role of Vitamin K is the synthesis of one unusual for proteins amino acid -gamma-carboxyglutamic acid, an important for the biological activity of clotting proteins component. Warfarin is applied in the treatment of various types of embolism like cerebral or lung embolism. Vit. K exists in two forms: Vit. K1 (phylloquinone) which is synthesized by plants and can be found mainly in green leafy vegetables, and Vit. K2 (menaquinon) which is synthesized by the probiotic lactic acid bacteria in the human intestine and is also abundant in the fermented dairy products.
1,4-Benzoquinone (8) is a member of p-benzoquinones and a metabolite of benzene. 1,4-Benzoquinone possess two C=O groups attached at the 1-and 4-positions at of the aromatic ring. Inside the cell, mitochondria and the cytoplasm are the cell structures docking quinone which enforce specific enzymatic reactions. Quinone is capable to turn into orotic acid and when accumulated in the human blood orotic acid leads to aciduria resulting in quick liberation of ammonia. Often 1,4-Benzoquinone is transformed to glycerol 3-phosphate, a key substance of Glycolysis. Quinone is also responsible for the Vit. B 12 (ciancobalamin) catabolism. Vit. B 12 is synthesized by probiotic bacteria in the human colon and its deficiency cause anemia. Anise is one of the richest sources of 1,4-Benzoquinone.
Flavone (9) is a lipid molecule and member of the class of flavanones. In the cell it is harbored in the cytoplasmic membrane and among the human tissues the most abundant is placenta. Amid fruits pomegranate is excessive in this substance while out of spices rosemary is the wealthiest one.
3-Hydroxyflavone (10) belongs to flavonols [5]. Similarly to flavanone, it is spotted in the cell membrane and is a water insoluble compound. 3-Hydroxyflavone is precursor of tambulin known an anti-aging and anti-Parkinsonian medicine. Foods abundant in this polyphenol are brassicas and papaya.

Secondary Metabolites
Another derivative of flavone is Flavonol (11). Due to its rich yellow color it was used for centuries in wool and silk dyeing. Flavonol is capable to bind essential and heavy metal ions.
Generally, flavonoids are main contributors to the flavor of fruits and the bitterness of citruses. Naringin, tangeritin, quercetin and neohesperidin impart the bitter taste in citruses, while the bitterness of wine is due to catechins and epicatechins [6].
Pelargonidin (13) can be found in almost all berries -blueberries, blackberries, cranberries, raspberries, strawberries, and aronia. Large amounts of pelargonidin are typically found also in plums and pomegranates, a polyphenol responsible for the color of radishes.
Gallotannins (15) is a class of tannins obtained by condensation of the carboxy group of gallic acid and the hydroxy group of glucose. Rich in gallotanins are pomegranates, strawberries and gallnuts. In Table 1 are presented the foods supplying polyphenols.

Isolation of polyphenols
Isolation of polyphenols is a challenging procedure due to the instability and complex structure of these compounds. Most often polyphenols are harbored in plant leaves and gymnosperm, and within the cell in the cell wall and vacuoles associated with the nuclei. The covalent bonding of polyphenols with the plant structures is a limiting factor for their liberation [9]. Additionally, other factors influence the recovery of phenolic compounds from plant samples: location in plant tissues, extraction method, sample size, storage conditions and possible subsequent chemical conversions. A wide spectrum of plant secondary metabolites, including polyphenols can be obtained using water or organic solvents.

Conventional extraction using solvents
These methods are widely used. Washed or dried plant material is finely ground and subjected to solvent extraction. The most commonly used solvents are water, hexane, ether, chloroform, acetone, benzene, ethanol and methanol. All these solvents are effective in taking out bioactive compounds i.e. polyphenols from the cell.
EtOH dissolves alkaloids, glycosides and dyes but does not dissolve gum, waxes or fats. It easily penetrates the cell membrane and fast and effortlessly extracts the cell metabolites. A disadvantage of ethanol is its volatility and flammability.
Acetone dissolves large amount of substances both hydrophilic and lipophilic. It is excellent for tannin extraction. Its advantage is not only the prevention of microorganism growth but also the easy evaporation and low cost extraction. Like ethanol, it is flammable and volatile.
Chloroform, ether and bischloromethanol. Chloroform is suitable for tannin extraction, ether for coumarins and tannins, and bischloromethanol -for terpenoids but not for phenolic compounds. All three solvents evaporate easily and are low-cost. Their disadvantages besides volatility and flammability are explosiveness and toxicity.
According to the paper of Alothman et al. [10], who studied pineapple, banana and guava, the percentage of different solvents strongly influence the polyphenol yield. Investigating polyphenol extraction with methanol, ethanol and acetone at concentration 90, 70, and 50%, authors found the highest yield at 70% of ethanol and methanol, and at 90% of acetone. Among the three fruits, guava showed to be the most abundant in polyphenols.
The conventional methods of extraction are maceration, infusion, percolation, Soxhlet extraction, and water-alcoholic extraction via fermentation. The most preferable of these methods is the Soxhlet extraction. It operates as follows: the finely ground plant sample is placed into a filter bag, the solvent is heated using a heating device and its vapors condense in a condenser. The condensed solvent drips on the plant material (bag) and extracts the polyphenols from it. After the extraction, the solvent is discarded from the polyphenol extract using vacuum evaporation. The advantages of the Soxhlet method are the high amount of extract yielded with a small amount of solvent, the low cost and the ease of conducting the process.

Novel methods of extraction
Ultrasonic extraction. During the process, the plant cell wall is broken by waves with a frequency of 10 kHz to 10 MHz. The polyphenol yield is improved up to 35% [9]. The best yield can be obtained at a 4:1 solvent/solute ratio for 200 ms extraction time at 400 W ultrasonic power. The disadvantage of this method is the degradation of anthocyanins due to the formation of OH radicals by the sonolysis of water and the high amplitude of treatment.
Microwave extraction. The solvent and plant sample are treated with microwaves and thus the phenolic compounds are released in the solvent. The advantages of the method are the low cost, smaller volume of solvent, temperature control and higher antioxidant activity of the product [11]. As the power increases, the amount of phenolic acids increases. In contrast, the extended treatment at 250 W leads to a decrease in total flavonoids.
Pressurized liquid extraction. Using this method, the temperature increases from 50 to 200°C and the pressure from 3.5 to 200 MPa. The advantage of the method is the better penetration of the solvent in the plant sample due to the increased pressure at high temperature.
Pulse electric field extraction. This method is used for pre-treatment of the plant material resulting in enhanced amount of polyphenols. Even under low electric field, the permeability of the plant cell is increased. Anthocyanins yield from grape pulp with grape skin is improved up to 28% when using the following conditions: 50 pulses and 1 Hz at 10 kV [12].
The best way to determine polyphenols remains HPLC-DAD (high-performance liquid chromatography with diode array detection). Many phenolic compounds, such as epicatechin, vanillic acid, quercetin, kaempferol, epigallocatechin, rutin, and myricetin were analyzed using this method. Structure elucidation of polyphenols can be implemented using gas chromatography-Gassmass spectrometry [13].

Antimicrobial activity of polyphenols from herbs and spices
Longevity of the community inhabiting Mediterranean area is due to their polyphenols rich diet. Most of Mediterranean herbs containing polyphenols were described to possess antibacterial activity against both Gram (+) and Gram (−) bacteria. Shehadi et al. [14] reported growth inhibition of Bacillus subtilis by Rosemary officinalis, Eugenia caryofillata Menta piperita, and Prunus avium. According the authors the major inhibition was observed by the extract of wild cherry (4 mg/ml) followed by cloves (1.6 mg/ ml). As described, the phenolic extracts were recovered at 80°C. An important finding of the study was the extracts' strength depends on the temperature at which they were obtained. Extracts revealed at 100°C possess lower bactericidal activity (2.4 mg/mlwild cherry and 0.6 mg/ml -cloves) compared to those eluted at 80°C. Alamri and Moustafa [15] reported action against different bacteria of Ricinnus communis L.) and Allium ampeloprasum var. porrum. Both extracts were able to inhibit the growth of Gram (+) pathogens Staphylococcus aureus, Streptococcus epidermidis, and of Gram (−) Pseudomonas aeruginosa, E. coli, Klebsiella pneumoniae, Proteus sp., and the pronounced agent of nosocomial infections -Acinetobacter. Authors discussed the abundant polyphenolic content of both extracts including at least six polyphenols and the higher activity of R. communis (27 mm) compared to Allium ampeloprasum (23 mm). Strongest inhibition was observed mainly against the pathogenic Gram (−) P. aeruginosa.
Data about the influence of different solvents for polyphenol extraction on the antibacterial activity were published by Harfouch et al. [16], Rizwana et al. [17]. Studying the effectiveness of methanolic and ethanolic extract of Martricaria aurea L. the authors found that the methanolic one is more powerfull against the Gram(+) chemolitic Streptococcus pyogenes (23 mm) and skin pathogen S. aureus (21 mm) compared to ethanolic extract. The inhibition is performed via bacterial cell wall damage.
Besides the herbs, various medicinal plants demonstrate high polyphenol concentrations [18]. Hypericum perforatum L., Origanum vulgare L., and Melissa officinalis L. along with four other medicinal plants were investigated for phenolic presence. Recently, the antibacterial activity of methanolic extracts of both flowers and leaves was proved, including wild hypericum collected from Kashmir, Himalaya.
Bactericidal effect of Geranium macrorrhizum was described by Ivancheva et al. [19]. This plant is known with its high polyphenol concentrations consisting mainly of flavonoids and tannins and in this particular study water-alcoholic extracts of the plant were investigated. The tested extracts inhibited the growth of several pathogenic bacteria S. aureus., E. coli, K. pneumonia, and P. aeruginosa.
Another plant used as medicinal plant, spice and herb -the sanogenous parsley also showed antibacterial properties. Several classes of polyphenols persist in parsley mostly flavonoids like kaempferol, apigenin and luteolin. Average content of flavonoids is approximately 100 mg/100 g fresh weight [20]. According to Tomov et al. [21] the green minced parsley leaves demonstrated weak antibacterial effect against E. coli and B. subtilis (see Table 2).
The antimicrobial activity of lavandula against Staphylococcus epidermidis was described in detail by Zou et al. [22]. The antimicrobial activity of the lavandula phenolic extract was reported by a Moroccan research team [23]. The authors described that Lavandula inhibits the growth of clinical Listeria monocytogenes and S. aureus isolates from a Moroccan hospital. Georgiev et al. [24] also reported on the antioxidant activity of Lavandula vera.
Green tea is also excessive in polyphenols and demonstratie robust antimicrobial action [26]. Green tea polyphenols consist mainly of flavonoids. Catechins are in the highest concentration of 30-40%. Four main catechins were isolated from tea: epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-gallate (EGCG), as reported by Raygaert [27]. In green tea, EGCG is the most abundant, representing approximately 59% of the total catechins. It is important to note that during the initial steaming process of tea production the enzyme polyphenol oxidase is destroyed and thus the polyphenol content is protected. Catechins of green tea damage the bacterial cell membrane, inhibit the fatty acid synthesis of  bacteria and DNA-gyrase during bacterial replication. In the same paper the inhibitory effect of green tea catechins on the binding of Helicobacter pylori to the Toll-like receptor-4 (TLR-4) on gastric epithelial cells was described.

Antimicrobial activity of polyphenols from fruits
Polyphenol abundant fruits also exhibit antibacterial action. In pomegranate juice (Pommes granatum L.) were obtained caffeic acid, gallic acid and epigallocatechin. Latest substance can be found as prior component also in the green tea. Divyarhree and Kunniah [28] studied its hydrochloric extract on the oral cavity inhabiting bacteria colonizing the dental plaque. Authors' research showed the extract inhibited Porphyromonas gingvinalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans, all responsible for assaultive periodontitis.
Noticeable Mediterranean fruits wealthy in polyphenols are olives (Olea europea). The general phenolic component responsible for their health beneficial effect is hydroxytyrosol [29]. Besides its antimicrobial activity, hydroxytyrosol is a superior antioxidant and radical scavenger, which induces apoptosis and arrests the cell cycle in cancer cells. Usually hydroxytyrosol is renally evacuated. Other phenolic compounds in olives are tyrosol, glycoside oleuropein, oleocanthal, and oleacein. Hydroxytyrosol and oleuropein demonstrated antimicrobial activity [30] against ATTC bacterial strains and clinical bacterial isolates.
In vitro antibacterial (MIC and MBC) effect of Sida alba, a polyphenolcontaining and typical for India and Arabian peninsula plant was obtained by Konate et al. [31].
Tannins, a common polyphenolic substances in all types of red wines were reported as natural antibacterial substances as well [33].
Polyphenols from tobacco leaves extracted with 80% ethanol manifest antibacterial activity against Escherichia coli, S. aureus and B. subtilis with inhibition zones ranging 13, 17 and to 20 mm [34].
Polyphenols play synergistic effect when applied in combinations with antibiotics [35,36]. Their mode of action is straight inhibition of the pathogenic microorganisms' virulence factors.

Antimicrobial activity of polyphenols from vegetables
Some authors report antibacterial activity of tomato. Tomato ranks second in world consumption among all vegetables [37]. Our previous research [21] showed no significant difference in the effect of raw or cooked tomato products against bacteria. The antibacterial effect was not a strong one (up to 7 mm zone). Seeds of two out of six tomato varieties slightly inhibited the growth of B. subtilis and showed no antibacterial activity against