Phytochemical analysis, antioxidant, and anticholinesterase activities of ethanolic extracts from five ginger plants in Thailand

Abstract Five selected medicinal plants in the family Zingiberaceae, Alpinia nigra, Boesenbergia maxwellii, Etlingera araneosa, Meistera koenigii, and Wurfbainia biflora, were collected from Thailand. HPLC was used to determine the content of phenolic compounds in their ethanolic extracts. The total phenolic contents and the total flavonoid contents of M. koenigii were found to be higher than that of the others. The antioxidant activity of the extracts was assayed by two methods, namely ABTS and DPPH assays using HPTLC and a microplate spectrophotometer. Anticholinesterase activity was screened by the Ellman method against acetylcholinesterase, the chief enzyme of Alzheimer’s disease. The results showed mild acetylcholinesterase effects. The results indicated that these five ginger species, especially M. koenigii have the potential to be a good source of natural antioxidants and anticholinesterase agents that could be further applied in the pharmaceutical industry. Graphical Abstract

This work was focused to investigate the phytochemical, antioxidant, and anticholinesterase activities of five selected ginger spices in Thailand: Alpinia nigra, Boesenbergia maxwellii, Etlingera araneosa, Meistera koenigii, and Wurfbainia biflora, using high performance thin layer chromatography-compact mass spectrometry and spectrophotometric method. The quantitative analysis of individual phenolic and flavonoid compounds by HPLC was also performed. These findings were suggested its use as natural ingredients for medicinal purposes.

Determination of the phenolic compounds of the extracts by HPLC-DAD
The HPLC chromatograms of the extracts showed the presence of phenolic compounds namely, gallic acid, caffeic acid, vanillin, cyanidin chloride, rutin hydrate, quercetin, and luteolin (Figures S1-S6). The results showed the presence of caffeic acid and vanillin in all species under investigation. Cyanidin chloride was not detected in all extracts. The highest content of caffeic acid (111.55 ± 0.51 mg/100g), vanillin (35.79 ± 3.64 mg/100g), quercetin (24.90 ± 0.07), and rutin hydrate (20.55 ± 0.13) were determined in W. biflora extract. Caffeic acid was the major phenolic compound in five ginger extracts.
The HPTLC fingerprint of seven standards and five ginger extracts was shown in Figures S7 to identify phenolic compounds. Each band was confirmed by TLC/CMS analysis which compare to its standard ( Figures S8-S11).

Total phenolic contents (TPC) and total flavonoid contents (TFC)
Extraction yields of all samples were ranged from 3.81 to 7.00% (Table S1). M. koenigii extract provided the highest yield equal to 7.00%. Whereas E. araneosa gave the lowest yield of 3.81%. total phenolic contents (TPC) and total flavonoid contents (TFC) of ginger extracts were reported in Table S1. The M. koenigii extract exhibited the highest TPC (944 ± 0.0856 mg GAE/g) and TFC (531.8 ± 0.0040 mg QE/g). Considering the yield, TPC and TFC of five ginger extracts, the M. koenigii extract gave the highest values.

Antioxidant activity
The results of DPPH test in Table S1 showed that the extracts had strong effect on scavenging free radicals with the percent inhibition of 94.18 ± 0.14% to 96.02 ± 0.63% at 4.0 mg/mL. Based on IC 50 values, the scavenging effect on DPPH radical ranged from 0.63 ± 0.01 mg/mL (M. koenigii) to 1.63 ± 0.02 mg/mL (W. biflora). The extracts exhibited ABTS radical scavenging activity as mg Trolox equivalent antioxidant capacity (TEAC)/g extract ranging from 443.79 ± 26.56 mg TEAC/g to 89.43 ± 2.47 mg TEAC/g.
In the present study, the M. koenigii extract showed the highest ability to scavenge DPPH and ABTS, followed by A. nigra extract. The antioxidant results were related to TPC and TFC maybe because of the redox properties of their phenolic compounds as reducing agents, hydrogen donors and metal chelators (Rao et al. 2010). The active compounds in the extract were detected using HPTLC bioautography in Figure S12 and confirmed by TLC/CMS analysis (Figures S13-S19).

Anticholinesterase activity
The extracts showed the anticholinesterase inhibitory activity against acetylcholinesterase enzymes. It was compared with that of galantamine, as a standard drug against Alzheimer's disease (Table S1). The highest acetylcholinesterase inhibition was observed in the M. koenigii extract with 92.50 ± 1.05% (IC 50 ¼ 1.49 ± 0.04 mg/mL) followed by E. araneosa extract demonstrated 68.49 ± 0.33% (IC 50 ¼ 1.81 ± 0.07) while the galantamine gave 100% inhibition (IC 50 ¼ 0.004 mg/mL) at 12.5 mg/mL. The active compounds in the extract were also detected using HPTLC bioautography in Figure S20. The active band was luteolin in M. koenigii extract confirmed by TLC/CMS analysis ( Figure S21).

Experimental
All experimental procedures are described in the supplementary material.

Conclusions
The results of this report indicated that the ethanolic rhizome extracts of these five gingers contained bioactive compounds and possessed antioxidant and antiacetylcholinesterase properties. The bioactivities may be related to the presence of polyphenols, flavonoids, and the high antioxidant capacity. Additionally, M. koenigii had the potential to be a potential source of native antioxidants and anticholinesterase agents that could be applied as an herbal alternative source for supplementary food and drugs in the pharmaceutical industry.