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Evaluated Bioactive Component Extracted from Punica Granatum Peel and Its Ag Nps Forms As Mouthwash Against Dental Plaque T

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Evaluated Bioactive Component Extracted from Punica Granatum Peel and Its Ag Nps Forms As Mouthwash Against Dental Plaque T Biocatalysis and Agricultural Biotechnology 18 (2019) 101073 Contents lists available at ScienceDirect Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab Evaluated bioactive component extracted from Punica granatum peel and its Ag NPs forms as mouthwash against dental plaque T ∗ Mona A. Esawya, Tamer I.M. Ragaba, , Al Shimaa Gamal Shalabya, Mona Bashab, Mahmoud Emamc a Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries Division, National Research Centre, 12622, Dokki, Giza, Egypt b Pharmaceutical Technology Department, National Research Centre, 12622, Dokki, Giza, Egypt c Phytochemistry and Plant Systematics Department, National Research Centre, 12622, Dokki, Giza, Egypt ARTICLE INFO ABSTRACT Keywords: The bioactive components of the hydroalcoholic (MeOH 70%) Punica granatum peel crude extract (PGPC) and its Punica granatum peel methanol fraction (PGPM) were extracted and tentatively identified by LCMS/MS as a number of phenolic Ag NPs + compounds which are used as capping agents fasting the reduction of Ag to Ag (0). The prepared silver nano- Antimicrobial particles Ag NPs PGPC and Ag NPs PGPM were characterized by the transmission electron microscopy (TEM) and Anti-Calculus and anti-hemorrhagic UV–vis spectra. Four mouthwashes were designed using PGPC, PGPM, Ag NPs PGPC and Ag NPs PGPM. The prepared mouthwashes exhibited a significant antimicrobial activity against the Streptococcus mutans and the three dental plaque isolates called Lysinibacillus cresolivorans, Lysinibacillus cresolivorans and Lysinibacillus bor- onitolerans. As well as, the prepared Ag NPs mouthwashes led to reduction in the cytotoxicity against BJ-1 cell line equal 35 and 23.66% with LC50 = 185.16 and 134.8 μg/mL. Finally, phenolics promote Ag NPs PGPM mouthwash to be potent anti-calculus and anti-hemorrhagic agents. 1. Introduction improve the efficacy of daily oral hygiene (Somu et al., 2012). Punica granatum L. (pomegranate) belongs to the family Punicaceae Dental plaque considered the most common disease worldwide. It is is a deciduous shrub in Iran, Spain, Italy, Afghanistan, America, India, caused by a mixture of pathogenic microorganisms and food remains. Turkey, China, Russia, Uzbekistan, Morocco, Chile and Greece (Rahimi Acid-producing bacteria, specifically Streptococcus mutans, inhabit the et al., 2012; Shaygannia et al., 2016). Therefore, the demand gradually dental surface and cause destruction to the hard tooth in the occurrence grown is due to the increasing consumer awareness of the potential of fermentable sucrose and fructose (Forssten et al., 2010). In order to health benefits of pomegranates. Its traditional uses, medicinal prop- reduce the prevalence of dental plaque, the role of the microorganisms erties and its constituents have attracted the interest of researchers and in dental diseases needs more understanding and clarification. The mentioned as fruits, seeds, peel and leaves of pomegranate contain tooth surface is enclosed by a biofilm containing a paste layer including various types of valuable ingredients and such ingredients show ther- millions of bacterial cells, salivary polymers, and food remains. This apeutics role in disease cure such as treatment and prevention of biofilm can easily reach a thickness of hundreds of cells on the surfaces cancer, cardiovascular disease, diabetes, bacterial infections and anti- of the teeth and be a good environment for colonization and growth of biotic resistance, dental conditions, erectile dysfunction, and ultraviolet many bacterial species also, the plaque provides an excellent adhesion radiation-induced skin damage. Other important applications include site for the colonization. So far, there is no sufficient information ex- infant brain ischemia, male infertility, Alzheimer's disease, arthritis, plains the mechanism which led to this conversion. Most strategies and obesity. (Ambigaipalan et al., 2016; Rahmani et al., 2017). Recent usually depended on decreasing the growth or activity of Streptococcus researches reveal that pomegranates could be implemented in oral mutans (Marsh, 2003). For controlling the dental plaque formation and health care process. Also, it was used as a successful therapy for accordingly, their pathogenic effects the mechanical oral hygiene pro- strengthening gums and fastening loose teeth. Previously, cesses were used, but in most cases, this is not enough. In order to Abdollahzadeh et al. (2011) reported in Punica Granatum Peel Extracts overcome the shortcomings of mechanical plaque control methods, as strong antibacterial against the oral pathogens like Streptococcus various chemotherapeutic agents have been employed and developed to mutans and Staphylococcus aureus. Al-Obaidi et al. (2017) mentioned ∗ Corresponding author. E-mail address: [email protected] (T.I.M. Ragab). https://doi.org/10.1016/j.bcab.2019.101073 Received 7 February 2019; Received in revised form 27 February 2019; Accepted 1 March 2019 Available online 08 March 2019 1878-8181/ © 2019 Elsevier Ltd. All rights reserved. M.A. Esawy, et al. Biocatalysis and Agricultural Biotechnology 18 (2019) 101073 that until this moment there are limited studies concerning the using of of g/L: wheat flour, 20; sucrose, 100; K2HPO4, 5; MgSO4.7H2O, 0.2, and pomegranate extracts as an anti-bacterial mouthwash and suggested the yeast extract, 2.5. The initial pH was also adjusted to 7.0 prior to possibility of preparing a mouthwash from pomegranate extract. Po- sterilization. megranates have polyphenols, tannins, ellagic acid and anthocyanins which are powerful antioxidants (Ashoush et al., 2013). Flavonoids, 2.4. Glucan detection ellagitannins and proanthocyanidin compounds and minerals such as calcium, magnesium, phosphorus, potassium and sodium are the most The hydrolysis products were separated chromatographically by ingredients of the peels of the pomegranate that covers around 60% of using the Whatman No. 1 filter paper and the solvent system n-butanol- the fruit (Mirdehghan and Rahemi, 2007; Rahmani et al., 2017). acetone-water (4:5:1 v/v) according to (Jayme and knolle, 1956). In previous study the treatment with Punica granatum extract sig- nificantly improved the chronic periodontal in a number of clinical 2.5. Plant collection and preparation trials (Sastravaha et al., 2005). Later, the extraction of 13 Brazilian medicinal agents was done from different plants. They were selected for The fresh Punica granatum peel (PGP) from Egyptian market was their antimicrobial activity against bacteria and yeasts. The results collected. The peels of PGP were washed several times with domestic showed that pomegranate has a promising activity on Staphylococcus water then distilled water after that dried in shade for 8 days at room aureus bacteria also, anti-candidal activity was identified (Kote et al., temperature. The dried PGP (500 g) was milled until reaching the 2011). powder form (50–100 mesh) then macerated with 70% (v/v) aqueous The present study aimed to design mouthwash had antimicrobial methanol (MeOH) for several times. The obtained PGP filtrate was fil- activity against the Streptococcus mutans and the three isolates from ® tered and dried on arotary evaporator (Rotavapor R-300, BÜCHI, dental plaque patient. Within this context, the P. granatum peel extract Switzerland). The PGPC extract was fractionated using MeOH and then (PGPC) and its methanol fraction (PGPM) were identified. Then, they ® the PGPM fraction was dried under reduced pressure using rotavapor . converted to their nano-forms which were characterized by various All samples were freeze dried till using. techniques. The antimicrobial activity for PGPC, PGPM, Ag NPs PGPC and Ag NPs PGPM were evaluated. Finally, the mouthwash containing 2.6. Total carbohydrate and phenolic content silver nitrate nano-particles (Ag NPs) using PGP extract and its me- thanol fraction were designed, also, their cytotoxicity was evaluated for The total carbohydrate content (TC) for each of the analyzing ma- the nano-forms and their mouth washes. In vitro anti-calculus and anti- terials was evaluated, after hydrolysis quantitatively as described in bleeding activities were studied. details (Ragab et al., 2014). The total phenolic content was determined according to the method mentioned by Makkar et al. (1997). One mL of 2. Material and methods the extract was taken in a test tube, and then 0.5 mL of Folin Ciocalteu reagent (1:1 with water) and 2.5 mL of sodium carbonate solution 2.1. Materials (20%) were added sequentially in each tube. After 1 h of incubation at ® room temperature, the absorbance was measured at 725 nm using the Silver nitrate (AgNO3, Merck), pluronic F-68, peppermint oil, sac- spectrophotometer and compared to a gallic acid calibration curve. charin, ethanol was purchased from Sigma Chemical Company, USA. Total phenols were determined as gallic acid equivalents (mg gallic acid thymol 99% and glycerol were obtained from ACROS Organics™. equivalent/g extract) and the values are presented as means of triplicat 2.2. Isolation and identification of bacteria 2.7. High performance liquid chromatography (HPLC-(−) ESI-MS/MS) A swab was taken from dental plaque patient. The sample was in- oculated separately into 25 mL of broth nutrient agar. The culture flasks The phytochemical analysis was done by the high-performance li- were incubated at 37 °C for 48 h. The freshly, grown culture (1 mL) quid chromatography-mass spectrometry. The LC system called from each dental plaque was diluted
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