This investigation aimed to estimate the antibacterial potency of the mixture of date syrup and aqueous extracts of different types of pepper (sweet orange, red and yellow peppers and hot red and green peppers) against Streptococcus anginosus. The bacterium was isolated from a patient in Rizgari hospital in Erbil, Iraq. It appeared that date syrup/aqueous extract mixture of different types of pepper had an inhibitory effect on growth of S. anginosus at different concentrations, and 1:4 v/v for date syrup/extract of red chili pepper gave the highest inhibitory rate of 32 mm while the inhibitory effect of red chili alone was 11 mm and date syrup alone was 10 mm, whereas the date syrup/aqueous extract of sweet orange pepper did not have any inhibitory effect on growth of the S.
anginosus at (1:1, 1: 2, 1: 3, 1: 4, 1: 5, 1:6 and 1:10 v/v). The phytochemical detetion showed presence of alkaloids, steroids and Phlobatannins in pepper extracts. The minimum inhibitory concentration of the date syrup/aqueous extract mixture of red chili was 2%.
The lowest inhibitory concentration of date syrup/rest of aqueous extracts was 3%. The results showed that the 50 °C was the best temperature for the date syrup/aqueous extract mixture of the pepper against the growth of S. anginosus. It can be concluded that the mixture of the date syrup and different types of aqueous extracts of pepper is a good antibacterial agent, and the mixture of date syrup and the aqueous extract of chili red pepper and chili green pepper is the most effective extract among the other against S.
anginosus.
Streptococcus anginosus belongs to the Streptococcus anginosus (SAG) group of bacteria (Junckerstorff et al., 2014) (previously known as the Streptococcus milleri group (Bantar et al., 1996)). These bacteria are commensal flora of the human oropharynx and gastrointestinal tract, but they are also capable of causing serious invasive pyogenic infections with a propensity for dissemination and abscess formation. Historically, S. anginosus has been found to be the most frequently isolated species from clinically significant specimens and are associated with significant morbidity and mortality (Junckerstorff et al., 2014). Recently, they have been considered a pathogen of pneumonia, pyothorax, and other infectious diseases of the respiratory system. On the other hand, the S. anginosus group displays moderate sensitivity to lactams and macrolide antibiotics, although resistance patterns are rising (Ahmed et al., 2007) found that 54.25% and 68.75% of isolates were not susceptible for Penicillin and Ampicillin respectively. In addition, Gunel and Gurler (2014) found that 16% of strains were resistant to erythromycin. 6% of the strains were resistant to clindamycin and 5%) of the strains were found to be resistant to tetracycline. In fact, excessive use of antibiotics has led to the development and spread in populations of antibiotic-resistant bacteria (Al-Snafi, 2016).
Naturally derived compounds such as aloe vera, honey and curcumin (Taleb et al., 2016), clove, thyme and other r plants (Liu et al., 2017) are becoming increasingly popular as alternative antimicrobials. In fact, the medicinal values of these plants return to the presence of certain chemical substances called secondary metabolites including alkaloids, tannins, flavonoids and other substances (Aali et al., 2018). Moreover, pepper, is a plant belonging to the genus Capsicum and it is a one of family Solanaceae. It is grown and consumed worldwide (Bello et al., 2015). It has been used for thousands of years as spices in food to improve the flavor, color and aroma of food. In addition to enhance the flavor, they are also known for their preservative and medicinal impact (Otunola et al., 2010). It contains some of the antimicrobial substances such as Terpinene, pinene, Linaleol and Terpineol (Zarringhalam et al., 2013). Crude extracts from several different C. annuum varieties have inhibited growth of species of Bacillus, Clostridium, Pseudomonas, Listeria, Salmonella, Staphylococcus, and Streptococcus (Bacon et al., 2017).
Likely, Phoenix dactylifera L., more frequently known as the date palm was used for traditional medicinal applications, where nomadic tribes in the Middle East were famed to use traditional date syrup (DS) as an agent against microbes for wound healing (Taleb et al., 2016). It was reported that palm date has a strong ability to suppress free radicals due to presence of phenolic compounds (Saleh et al., 2011). Many phenolic compounds such as polyphenols and flavonoids are antibacterial as a result of their oxidizing capacity which may provide a rationale for date fruit and DS’s medicinal use as an agent against microorganisms (Taleb et al., 2016).
This work was aimed to determine the antibacterial activity of aqueous pepper extracts in combination with the date syrup, and also to screening their activity as antibacterial under different degree of the temperature. In addition, to screening the phytochemical groups of different types of aqueous pepper extracts.
The hot (green and red) and sweet (green, yellow, orange and red) types of plant (Capsicum annuum) were bought from supermarket in Erbil city, then were washed with tap water, then with distilled H2O, then entirely air-dried, after drying were grinded into powder form and stored in polyethylene sack in refrigerator at 4 C for next processing, while the date syrup was purchased from supermarket in Erbil city.
One hundred and fifty ml of sterilized distilled water was added to 15 g of ground dried plant, then heated below the boiling point and stirred for 2 – 3 hrs. The extract was filtered with muslin cloth, then filtered with Whatman paper (No. 1) then placed in the refrigerator at 4 C for further using (Angaji and Angaji, 2009, Al-Neemy and SHKh, 2006).
Alkaloids are basic nitrogenous compounds with definite physiological and pharmacological activity. Most alkaloids are precipitated from a neutral or slightly acidic solution by Mayer’s reagent (Kumar et al., 2009). Eight ml of 1% HCl were mixed with 2 ml of the extracted plant, and warmed, then mixed with 2 ml of Mayer’s reagent. Forming of white turbidity or precipitate refers to the existence of alkaloids (Khan et al., 2011).
Two ml of extract were mixed with 2 ml of acetic anhydride followed by 2 ml of sulphuric acid. The color changed from violet to blue or is an indication of existence of steroids (Khan et al., 2011).
Four ml of 1% alcoholic aluminium chloride (methanolic) were mixed with two ml of plant extract in a test tube and the color was observed. Formation of yellow color refers to the existence of flavonols, flavones and chalcones (Khan et al., 2011, Chitravadivu et al., 2009).
Two ml of crude extract was mixed with 2 ml of 2% of FeCl3. A blue-green or black coloration indicates the existence of phenols and tannins (Yadav and Agarwala, 2011).
Two ml of plant extract was mixed with few drops of 10% of lead acetate solution, formation of white precipitate refers to the presence of phenols (Singh and Bag, 2013).
Two percent of HCl was boiled with 2 ml of extract. Red precipitate shows the presence of phlobatanins (Egwaikhide et al., 2009).
10% of sodium hydroxide and chloroform were added to the plant extract. Yellow color formation refers to the presence of coumarin (Salna et al., 2011).
Nollerэs test: The extract was warmed with Tin and Thionyl chloride. Purple coloration refers to the existence of Triterpeniods (Salna et al., 2011, Devi and Manoharan, 2011).
Sodium hydroxide was added to the plant extract. Blue green or red color formation is an indication of the existence of quinine (Salna et al., 2011).
Ten percent of HCl was added to 2 ml of plant extract and then boiled few minutes in water bath, then the mixture was filtered and allowed to cool. Equal volume of CHCl3 was added to the filtrate. Few drops of 10% NH3 were added to the mixture and heated. Rose-pink color formation refers the existence of authraquinones (Kumar et al., 2009).
Table (1) summarizes all of the results of qualitative analysis.
S. anginosus was isolated from patient in Rizgari Teaching hospital in Erbil city-Iraq. The isolate was cultured on nutrient agar to get single colonies, then identified according to the cultural, morphological and some biochemical tests (Vitek system 2). The isolate was maintained and preserved on nutrient agar slants. For every experiment, freshly prepared sterile nutrient broth (10 ml) was inoculated from the slants and incubated at 37? for 24 hours.
Two to three colonies from pure growth of tested organism were added to 5 ml of nutrient broth, and then incubated overnight at 37 C. The suspension was adjusted to 0.5 McFarland standards to obtain approximately 1*106 CFU/ ml (Alhaj et al., 2008).
Testing of antibacterial effect of plant extracts (Pepper (all mentioned types), date syrup and date syrup/Pepper 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10) v/v at 50 , and date syrup/Pepper (at concentration was made the biggest zone of inhibition), at 60 , 70, 80 and 90 , in addition to controlling represented with antibiotics ( Amoxicillin, Ampiclox and Tetracycline) then was carried out by well diffusion technique (Kivanc and Kunduhoglu, 1997). The Nutrient agar (NA) plates were inoculated with (0.1) ml of the inoculum of tested bacterium. The inoculum was spread evenly over plate. Wells with (8) mm diameter were made on the surface of the nutrient agar loop by a standard cork- borer and then left for 30 min to be absorbed on the agar, after that (100) µl of each concentration of plant extracts was added into the well, the plates were incubated at 37 ?C for 24 hrs, and the inhibition zones were measured to the nearest millimeter (mm).
The MIC of plant extracts was determined depending on agar dilution method with minor modifications. Equal volumes of bacterial strain culture containing approximately 106CFU/ml, were applied onto nutrient broth tubes supplemented with correlated plant extract at concentration 1:5 v/v (date syrup/Pepper) with concentrations ranged from 1% (v/v) to 15% (v/v) in tubes. Cultures were then incubated for 24 h at 37 °C, thereafter, 100 µl of each culture was taken and distributed on nutrient agar and further incubated for 24 h at 37 °C. MIC was defined as the lowest concentration of plant extract that inhibiting the visible growth of organism on the agar plate. The presence of one or two colonies was disregarded (Tayel and El-Tras, 2009).
All experiments were applied in triplicates.
The current study included identification of Streptococcus anginusus isolated from a patient at the Rizgari Hospital in Erbil, Iraq, based on the phenotype of the germ using a Gram stain technique, where it appeared as gram positive bacterium, coccus, and arranged in diplo or as chain (strepto); on blood agar was appeared as small colonies and non-hemolytic for blood. To verify its identity, its biochemical properties were detected using the Vitek-2 system (Table 1).
Further, the synergistic effect of the date syrup/aqueous extract mixture of the different types of pepper plant against S. anginosus was investigated and Amoxicillin Ampicolx and Tetracycline were used as antibiotic control. The results showed (Table 2, Fig. 1) that the date syrup/aqueous extract mix of the different types of pepper had an inhibitory effect on the growth of S. anginosus at different concentrations, and 1:4 v/v for the date syrup/extract of red chili pepper gave the highest inhibitory rate of 32 mm while the inhibitory effect of red chili alone was 11 mm and date syrup alone was 10 mm (Fig. 1). Table 2 shows that the date syrup/aqueous extract of sweet orange pepper did not have any inhibitory effect on the growth of the mentioned bacterium at the following concentrations (1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1:6 and 1:10 and the aqueous extract alone). In addition, the diameter of the inhibition zone at the concentration of 1: 7, 1: 8 and 1: 9 was low compared with the other date syrup/aqueous extract mixture of the different types of pepper. The efficacy of antibacterial effect of extracts against the growth of bacteria is due to the presence of alkaloids, steroids and Phlobatannins. The results of the phytochemical group detection showed that the active groups found in most of the aqueous extracts used in the present study (Table 3). Pepper plant also contains flavonoids, tannins and other groups but their absence in the extracts used in the current study may be due to the type of solvent used in the process of extraction of raw materials, where usually the alcoholic solvents have a better inhibitory effect compared to aqueous extracts because of the extracting of most active groups of Plant (Cowan, 1999). In fact, the aim of the current study is to use a safe extract in all respects, so only the pepper types were extracted with water.
The results listed in Table (2) also reveal that the mixture of date syrup/red hot pepper/extract and the mixture of date syrup/green hot pepper/extract were more effective than the other types of pepper extracts in combination with the date syrup.
The synergistic effect of date syrup and pepper extracts is also due to the incorporation of date syrup on phenol (Farahnaky et al., 2016), which inhibits bacterial growth by inhibiting enzymes in the bacterial cell (Cowan, 1999, El Sohaimy et al., 2015) and containing flavonoids (Farahnaky et al., 2016, Bello et al., 2015), which in turn are complex with extracellular proteins and soluble proteins of bacteria, as well as complexes with the cell wall of bacteria (Cowan, 1999) and also contains tannins (Ahmed et al., 2016) known for their inhibitory effect of cell envelope transport proteins (Cowan, 1999) and have been noted to form irreversible complexes with proline-rich proteins resulting in the inhibition of the cell protein synthesis (Abdulrahman et al., 2016). On the other hand, date palm is rich in phytochemicals like phenolics, sterols, carotenoids, anthocyanins, procyanins and flavonoids (El Sohaimy et al., 2015). Nazari and Weiss (2010) reported to the antimicrobial and antifungal activity of date syrup under various conditions and they referred the antimicrobial activity to the presence of specific compounds naturally present in date fruits. It has been found that when the syrup was mixed with a range of disease-causing bacteria such as Staphylococcus aureus, Escherichia coli, Enterococcus spp. and Pseudomonas aeruginosa – it inhibited their growth in about six hours, as described by researchers is faster than manuka honey, which is known for its antibacterial and wound healing properties (Taleb et al., 2016).
It has been demonstrated that date syrup is able to inhibit (as a pro-oxidant) Gram negative and Gram positive bacteria by generating H2O2 (Taleb et al., 2016).
Table (4) shows that the minimum inhibitory concentration (MIC) of the date syrup/aqueous extract mixture of red chili was 2%. The lowest inhibitory concentration of the date syrup/rest of aqueous extracts was 3%. The lowest inhibitory concentration is the concentration that inhibits the visible growth of bacteria on agar medium (Tayel and El-Tras, 2009). Hot taste is due to the presence of Capsaicinoids, particularly Capsaicin (N-(4-hydroxy-3-methoxy-phenyl) methyl) 8-methyl-non-6-enamide) and dihydrocapsaicin (N-(4-hydroxy-3-methoxy-phenyl) methyl) 8-methyl-nonamide) which are responsible for 80-90 % of the spiciness; Capsaicin have several medicinal properties (Gayathri et al., 2016).
Table 5 shows the effective antibacterial activity of the date syrup/aqueous extract mixture of the different types of pepper plant at 1:5 v/v concentration (this concentration was selected because it recorded the highest inhibitory rate of most extracts when mixed with date syrup) against S. anginosus at different temperatures, where the results showed that the 50 °C was the best temperature for the date syrup/ aqueous extract mixture of the pepper against the growth of the mentioned bacterium (Fig. 2). It can be inferred from the results that increasing in temperature keeps the inhibition ratios low as shown in Table (5). In general, high temperature can damage or dissolve sugars in date syrup as well as damage or breakdown of the organic structure of organic compounds.
The study reveals that the mixture of the date syrup and different types of aqueous extracts of pepper is a good antibacterial agent, and the mixture of date syrup and the aqueous extract of chili red pepper and chili green pepper is the most effective extract among the other against S. anginosus.
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