Chemical composition, polyphenol contents and antioxidant activities of the ‘Himalayan toothache relieving tree’ (Zanthoxylum armatum DC.)

Abstract The present study carried out to investigate the bioactive chemical compounds, total polyphenol content and antioxidant potential of different extracts of the Zanthoxylum armatum leaves collected from the Nainital, Uttarakhand. The GC-MS analysis of Z. armtum leaves extract resulted in the isolation of sixty, twelve, twenty-three and nineteen phytochemical constituents in methanol, ethanol, chloroform and water extracts respectively. The leaves extracts were strongly characterised by Heneicosane, Tetratetracontane, Phytol, Fargesin, (+)- Seasmin and Paulowin. Methanol extract showed maximum DPPH (2,2-Di-phenyl-1-picryl-hydrazyl) Free radical scavenging activity (IC 50 15.63 ± 0.31), Ferric Reducing Antioxidant Activity (88.98 ± 3.34 AAE ± SD) and Metal Chelating Activity (IC 50 9.89 ± 0.83). The results showed that the methanolic extract exhibited the highest phenolic content for total phenol content (98.26 ± 0.8 mg of Gallic acid equivalent/g of dry weight), total flavonoid content (61.50 ± 1.62 mg of Quercetin equivalent/g of dry weight) and total tannin content (79.96 ± 0.81 mg of Tannic acid equivalent/g of dry weight). Graphical Abstract

Although the reports are available on the chemical composition of the leaves, however systematic investigation on the chemical composition, polyphenolic contents and antioxidant activity using different types of extract in the leaves of the species is lacking, therefore the present study was carried to i) determine the chemical composition of the species by using GC-MS ii) quantifying the total flavonoid, phenol, tannin content, and iii) antioxidant potential of Z. armatum leaves.

Total polyphenolic contents
Total phenolic content of extracts ranged from 22.03 ± 1.38 to 98.26 ± 0.8 mg of GAE/g of dw. The methanol extract of leaves showed highest amount of total phenolic content followed by ethanol, chloroform and water extract. Methanol leaf extract also possesses highest flavonoid content ranging between 61.50 ± 1.62 to 12.85 ± 1.56 QE/g of dw. The total tannin content was recorded highest in methanol extract (79.96 ± 0.81 mg TAE/g of dw ) and lowest in the aqueous extract (22.18 ± 0.22 mg TAE/g of dw) (Table S3). Barkatullah Ibrar et al. (2017) reported 16.48 ± 1.33 mg/g phenol content, 18.33 ± 1.22 mg/g flavonoid content and 34.43 ± 0.21 mg/g tannin content for the leaf of Z. armatum collected from Pakistan which is less in comparison to present study. In another study, total phenol was observed 366.3 mg of GAE/g in crude methanol extract of leaves collected from Mandi (HP), India (Guleria et al. 2013). Phenol content in leaf extract is responsible for the antioxidant activity and other biological activities (Wojdyło et al. 2007) and flavonoids are known for free radical scavenging, inhibition of hydrolytic and oxidative enzymes and anti-inflammatory action (Frankel 1995).

Antioxidant activity
The antioxidant activity of the plant extract depends on the extraction process and solvents (Aghraz et al. 2018). The minimum IC 50 value was calculated for methanol extract i.e., 15.63 ± 0.31 showing highest radical scavenging activity. In contrast aqueous extract showed lowest radical scavenging activity with maximum IC 50 value (37.62 ± 0.24) among all extract. Kanwal et al. (2015) and Alam and Ashraf (2019) also suggested that the methanol leaf extract of Z. armatum possess free radical scavenging potential.
The reducing ability of methanol extract was found to be the highest (88.98 ± 3.34 AAE) followed by ethanol, chloroform and aqueous extract (Table S3). FRAP value was observed highest in his study for leaves collected from the Shimla (55.92 ± 2.0 mM Fe (II) equivalents) in methanol extract (Gautam et al. 2021).
Among the considered parameters the FRAP and TFC assay was found to be positively significant correlated at 0.972.

Conclusion
The present study shows the presence of phytochemicals composition, polyphenols and antioxidant activities of Z. armatum leaves extracts. Phytochemical composition of the extract varied depending on the solvent types used for the extraction. In the methanolic extract, more soluble phytocompounds were isolated. Methanol extract of Z. armatum leaves showed remarkable presence of total phenol, total flavonoid and total tannin content followed by ethanol, chloroform and water. Similarly, in case of DPPH free radical scavenging activity, FRAP and MCA antioxidant activity, methanol extract showed strong antioxidant activity, capable of inhibiting free radicals to terminate the radical chain reaction, and acting as reducing agents.