Influence of Benincasa hispida Peel Extracts on Antioxidant and Anti-Aging Activities, including Molecular Docking Simulation.

Benincasa hispida peel, a type of postconsumer waste, is considered a source of beneficial phytochemicals. Therefore, it was subjected to investigation for biological activities in this study. B. hispida peel was extracted using 95% v/v, 50% v/v ethanol and water. The obtained extracts were B95, B50 and BW. B95 had a high flavonoid content (212.88 ± 4.73 mg QE/g extract) and phenolic content (131.52 ± 0.38 mg GAE/g extract) and possessed high antioxidant activities as confirmed by DPPH, ABTS and lipid peroxidation inhibition assays. Moreover, B95 showed inhibitory effects against collagenase and hyaluronidase with values of 41.68 ± 0.92% and 29.17 ± 0.66%, which related to anti-aging activities. Via the HPLC analysis, one of the potential compounds found in B95 was rutin. Molecular docking has provided an understanding of the molecular mechanisms underlying the interaction of extracts with collagenase and hyaluronidase. All extracts were not toxic to fibroblast cells and did not irritate the hen's egg chorioallantoic membrane, which indicated its safe use. In conclusion, B. hispida peel extracts are promising potential candidates for further use as antioxidant and anti-aging agents in the food and cosmetic industries.

Denture-Soaking Solution Containing Piper betle Extract-Loaded Polymeric Micelles; Inhibition of Candida albicans, Clinical Study, and Effects on Denture Base Resin.

Candida albicans is a common overgrowth in people wearing dentures. Long-term use of antifungal chemicals carries a risk of toxic side effects. This study focused on the edible Piper betle extract because of its safety. The broth dilution method was applied for antifungal determination of the ethyl acetate fractionated extract (fEA) and fEA-loaded polymeric micelles (PMF). The PMF was prepared by thin-film hydration using poloxamer 407 as a polymer base. The results found that the weight ratio of fEA to polymer is the main factor to obtain PMF system as a clear solution, nanoparticle sizes, narrow size distribution, negative zeta potential, and high entrapment efficiency. The activity of PMF against C. albicans is significantly higher than fEA alone, with a minimum fungicidal concentration of 1.5 mg/mL. PMF from 1:3 ratio of fEA to polymer is used to develop a denture-soaking solution contained 1.5 mg fEA/mL (PMFS). A clinical study on dentures of 15 volunteers demonstrated an 86.1 ± 9.2% reduction of C. albicans after soaking the dentures in PMFS daily for 14 days. Interestingly, PMFS did not change the hardness and roughness of the denture base resins. The developed PMFS may serve as a potential natural denture-soaking solution against candidiasis in denture wearers.

Enhancement of aqueous solubility and antibiofilm activity of 4-allylpyrocatechol by polymeric micelles.

4-Allylpyrocatechol (APC), a major active compound of Piper betle, possesses strong antimicrobial activity. However, the water-insoluble property of APC limits its clinical and pharmaceutical use. To solve this problem, APC loaded polymeric micelles (PMAC) was fabricated using the thin-film hydration method. Nanoparticles of PMAC were characterized using a photon correlation spectrophotometer and transmission electron microscope (TEM). Antibiofilm activity of PMAC was investigated using crystal violet assay and confocal laser scanning microscopy (CLSM). Cytotoxic effects of PMAC on normal cells were investigated using MTT assay. The results demonstrate that a ratio of APC to the polymer plays an important role in the physicochemical characteristics of PMAC. The most suitable PMAC formulation having a small particle size (38.8 ± 1.4 nm), narrow size distribution (0.28 ± 0.10), a high negative zeta potential (- 16.43 ± 0.55 mV), and high entrapment efficiency (86.33 ± 14.27%) can be obtained from the ratio 1:4. The water solubility of this PMAC is significantly improved, approximately 1,000-fold higher than the unentrapped APC. TEM images demonstrate that PMAC is spherical in shape. The inhibitory effects of PMAC (1.5 mg APC/mL) against Streptococcus intermedius and Streptococcus mutans biofilms are significantly stronger than chlorhexidine (0.06 mg/mL). Images from CLSM demonstrate the destruction and thickness reduction of the pathogenic biofilms after contacting with PMAC. The MTT assay confirms that PMAC at this concentration is non-toxic to normal cells. These results obviously indicate that PMAC is a promising natural and harmless antimicrobial agent suitable for use in the oral cavity for inhibition of pathogenic bacterial biofilms.

Development of Self-Nanoemulsifying Drug Delivery Systems Containing 4-Allylpyrocatechol for Treatment of Oral Infections Caused by Candida albicans.

Clinical use of 4-Allylpyrocatechol (APC), a potential antifungal agent from Piper betle, is limited because of its low water solubility. The current study explores the development of the self-nanoemulsifying drug delivery system (SNEDDS) containing APC (APC-SNEDDS) to enhance APC solubility. Results demonstrated that excipient type and concentration played an important role in the solubility of APC in the obtained SNEEDS. SNEDDS, comprising 20% Miglyol 812N, 30% Maisine 35-1, 40% Kolliphor RH40, and 10% absolute ethanol, provided the highest loading capacity and significantly increased water solubility of APC. Oil-in-water nanoemulsions (NE) with droplet sizes of less than 40 nm and a narrow size distribution were obtained after dispersing this APC-SNEDDS in water. The droplets had a negative zeta potential between -10 and -20 mV. The release kinetics of APC from APC-SNEDDS followed the Higuchi model. The NE containing 1.6 mg APC/mL had effective activity against Candida albicans with dose-dependent killing kinetics and was nontoxic to normal cells. The antifungal potential was similar to that of 1 mg nystatin/mL. These findings suggest that APC-SNEDDS are a useful system to enhance the apparent water solubility of APC and are a promising system for clinical treatment of oral infection caused by C. albicans.

Comparative inhibitory effects of 4-allylpyrocatechol isolated from Piper betle on Streptococcus intermedius, Streptococcus mutans, and Candida albicans.

Streptococcus intermedius, Streptococcus mutans, and Candida albicans are harmful oral pathogens and prone to resist chemical antimicrobial agents. Active ingredients from plants are of increasing interest as an alternative. This study aims to compare antimicrobial effects of 4-allylpyrocatechol (APC) extracted from Piper betle on these oral pathogens. Minimum concentration of APC against the tested pathogens was determined using a broth microdilution assay. Killing kinetic study of APC was carried out within 24 h. Morphology of the pathogenic cells was assessed using scanning electron microscopy (SEM). Anti-biofilm was investigated using crystal violet assay and confocal laser scanning microscopy (CLSM). The results showed that the mechanism of inhibition of APC was bactericidal and fungicidal effects. APC at minimum concentration of 400 µg/mL could completely kill Streptococcus and Candida spp., however, the killing rate on S. intermedius and C. albicans was significantly faster than on S. mutans. APC inhibited biofilm formation of C. albicans more efficiently than that of the bacterial cells. Cell morphology from SEM indicated that APC caused bacterial cell membrane destruction and inhibited fungal budding or tubing formation. CLSM images confirmed the killing potential of APC and suggested that bacterial dead cells could be easier washed out than the fungal dead cells. It is concluded that APC potentially inhibits growth and biofilms of oral Streptococcus and Candida spp. in different mechanism of action and killing rate. APC can be considered as a promising agent for preventing and treating dental disorders caused by S. intermedius, S. mutans, and C. albicans.

Effects of Piper betle fractionated extracts on inhibition of Streptococcus mutans and Streptococcus intermedius.

The overgrowth of certain strains of normal flora in oral cavity can cause many kinds of oral infections or diseases such as carries, periodontitis, and gingivitis. Prevention and treatment of these diseases are usually achieved by chemical antiseptics. However, these chemicals are found as negative impacts of human health hazards and accession of microbial resistance. The present study explores the potential of Piper betle extracts on inhibition of two oral pathogenic bacteria; Streptococcus mutans DMST 41283 and Streptococcus intermedius DMST 42700. P. betle demonstrated significantly higher inhibitory activity against both pathogenic strains than Acacia catechu, Camellia sinensis, Coccinia grandis, Solanum indicum, and Streblus asper. Among fractionated extracts of P. betle from several solvents, the extract from ethyl acetate (Pb-EtOAc) possessed the widest inhibition zone of 11.0 ± 0.1 and 11.3 ± 0.4 mm against both bacterial strains, respectively. Pb-EtOAc showed the same minimum inhibitory concentration of 0.5 mg/mL against both strains, whereas its minimum bactericidal concentrations were 2.0 and 0.5 mg/mL against S. mutans and S. intermedius, respectively. HPLC analysis demonstrated that the major active compound of Pb-EtOAc was 4-allylpyrocatechol. It was found that the killing kinetics of Pb-EtOAc against both test strains were time and dose dependent. Scanning electron microscopy micrographs showed the morphological changes and depletion of the tested pathogens indicating cell destruction after exposure to Pb-EtOAc. It is confirmed that Pb-EtOAc is potentially effective against both oral pathogens and might be used as natural alternative agents in prevention and treatment of oral infections caused by oral pathogenic bacteria.

Potential of Piper betle extracts on inhibition of oral pathogens.

In the present study, antimicrobial activity of Piper betle crude ethanol extract against 4 strains of oral pathogens; Candida albicans DMST 8684, C. albicans DMST 5815, Streptococcus gordonii DMST 38731 and Streptococcus mutans DMST 18777 was compared with other medicinal plants. P. betle showed the strongest antimicrobial activity against all tested strains. Fractionated extracts of P. betle using hexane, ethyl acetate, and ethanol, respectively, were subjected to antimicrobial assay. The result revealed that the fractionated extract from ethyl acetate (F-EtOAc) possessed the strongest antimicrobial activity against all tested strains. Its inhibition zones against those pathogens were 23.00 ± 0.00, 24.33 ± 0.58, 12.50 ± 0.70 and 11.00 ± 0.00 mm, respectively and its minimum inhibitory concentrations were 0.50, 1.00, 0.50 and 1.00 mg/mL, respectively. Interestingly, the minimum concentration to completely kill those pathogens was the same for all strains and found to be 2.00 mg/mL. Killing kinetic study revealed that the activity of F-EtOAc was dose dependent. HPLC chromatograms of P. betle extracts were compared with its antimicrobial activity. An obvious peak at a retention time of 4.11 min was found to be a major component of F-EtOAc whereas it was a minor compound in the other extracts. This peak was considered to be an active compound of P. betle as it was consistent with the antimicrobial activity of F-EtOAc, the most potential extract against the tested pathogens. It is suggested that F-EtOAc is a promising extract of P. betle for inhibition of oral pathogens. Separation and structure elucidation of the active compound of this extract will be further investigated.