Effect of different extraction techniques on total phenolic and flavonoid contents, and antioxidant activity of betelvine and quantification of its phenolic constituents by validated HPTLC method

Abstract

The objective of the study is to analyze the effect of different extracting methods on the polyphenolic content and antioxidant activities in Piper betle leaves. In the present research, P. betle leaf extract was prepared by sonication, Soxhlet and maceration methods using acetone (100%, v/v). The efficiency of the extraction methods was estimated by quantifying the total phenolic content (TPC) by the Folin-Ciocalteu method and total flavonoid content (TFC) by AlCl 3 colorometric methods, and antioxidant power of the various extracts was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging assay. Thin-layer chromatography (TLC) bioautography was carried out to identify antioxidants, and their amount was determined by the newly developed high-performance thin-layer chromatography (HPTLC) method. DPPH free radical scavenging capacity of the different extracts from strongest to weakest was as follows: ascorbic acid (4.27 µg/mL) > sonication (5.35 µg/mL) >, maceration (5.53 µg/mL)>, soxhlet extraction (5.83 µg/mL). Same trend was also observed for the ABTS radical scavenging capacity. Similarly, findings of this study also showed that sonication extract possessed highest phenolic and flavonoid contents followed by maceration and Soxhlet extraction. In addition, important bioactive phenolic constituents which contribute largely towards antioxidant potential such as eugenol and eugenol acetate were quantified using HPTLC (high-performance thin-layer chromatography) method. The average percent recovery of eugenol and eugenol acetate was found to be 97.28% and 98.04%, respectively. The LOD (limit of detection) and LOQ (limit of quantification) for eugenol were 5 and 15 ng/spot, whereas that of eugenol acetate were 10 and 30 ng/spot. The HPTLC densitometric determination also supported the results of antioxidant assays by revealing the presence of higher amount of identified antioxidants in sonication followed by maceration and Soxhlet extraction. The developed HPTLC chromatogram profile may be used as a reference for the standardization of P. betle leaf extracts.

Simultaneous determination of active compounds in Piper betle Linn. leaf extract and effect of extracting solvents on bioactivity

“…The highest concentration of extracted eugenol was 2.898 mg/g found in the ethanolic extract. This value is higher compared with those extracted from P. betle leaves using maceration (1.8 mg/g) and Soxhlet techniques (1.6 mg/g) reported by Das et al 17 In this study, the ratio of hydroxychavicol to eugenol was obtained at approximately 9, which is lower than the ratio of about 14 found in the previous study on betel leaves from Vellore, India. 18 It is noted that eugenol is a hydrophobic compound thus it is able to dissolve in a nonpolar solvent like hexane with a slightly lower extracted amount at 2.691 mg/g.…”

Enhancing the Potentiality of Trichoderma harzianum against Pythium Pathogen of Beans Using Chamomile (Matricaria chamomilla, L.) Flower Extract

“…To measure the total flavonoids colorimetrically of the air-dried leaves, 0.1 g of the leaves were dissolved in 1 mL of distilled water of which 0.5 mL was then taken and 1.5 mL of 95% ethyl alcohol, 0.1 mL of 10% aluminum chloride (AlCl 3 ), 0.1 mL of 1 M potassium acetate (CH 3 COOK) and 2.8 mL of distilled water was added [ 67 ]. The resulting solution was then incubated for 40 m at standard room temperature before the solution was measured using a spectrophotometer at 415 nm with distilled water used as a blank.…”

Important Flavonoids and Their Role as a Therapeutic Agent

“…The flavonoid and phenolic content, along with the antioxidant potential may vary and depends on the method of extraction used [ 98 ]. Leaves extract of piper beetle was prepared using sonication, and had a total flavonoid content of 49.79 ± 1.54 mg QE/gm extract, whereas maceration gave 32.10 ± 0.65 mg QE/gm and Soxhlet extraction gave 40.89 ± 0.87 mg QE/gm ( Table 2 ) [ 99 ]. The radical scavenging activity of the leaves extract and its role as an antioxidant agent are closely related to the flavonoid and phenolic content.…”