Antioxidant, cholinesterase inhibition activities and essential oil analysis of Nelumbo nucifera seeds

Abstract Nelumbo nucifera seeds’ essential oil (EO), crude extract and subsequent fractions were evaluated for their DPPH, ABTS and superoxide anion-free radical scavenging and cholinesterase inhibitory activities. The ethyl acetate fraction and EO showed outstanding antioxidant activities with IC50 values of 191, 450 μg/mL (DPPH), 123, 221 μg/mL (ABTS) and 69, 370 μg/mL (superoxide anion). The ethyl acetate fraction and EO also caused significant inhibition of acetylcholinesterase and butyrylcholinesterase with IC50 values of 70 ± 0.6, 64 ± 0.8 and 75 ± 0.3, 58 ± 0.2, in dose-dependent manner. The first ever gas chromatography–mass spectrometry analysis of the EO obtained from N. nucifera seeds resulted in identification of 19 constituents, mainly comprised of oxygenated sesquiterpenes responsible for their promising bioactivity. The crude and fractions revealed the presence of saponins, flavonoids, steroids, alkaloids, terpenoids and cardiac glycosides in phytochemical investigation.


Introduction
Excess of reactive oxygen species cause serious diseases like Parkinson's and Alzheimer's diseases, cause tissue damage and diabetes (Bhakta & Siva 2012). The aim of the cholinesterase inhibiters is to boost endogenous levels of acetylcholine in the brain of Alzheimer's disease patients and herby, to increase cholinergic neurotransmission (Wessler & Kirkpatrick 2008). Nelumbo nucifera Gaertn belongs to the family Nymphaeaceae and has been used to treat rheumatism, lumbago, sciatica and tumours. No study has been done so far on the antioxidant and cholinesterase activities of the essential oil (EO) obtained from seeds of this plant. In the present studies, we report the gas chromatography-mass spectrometry (GC-MS) analysis of seed essential oils along with antioxidant and cholinesterase inhibitory effects of N. nucifera seed EO, crude extract and its resultant fractions (N 1 -N 5 ) associated with the chemical composition.

In vitro antioxidant potential
N. nucifera's EO, crude extract and its fractions (N 1 -N 5 ) revealed effective scavenging activity against DPPH, ABTS and superoxide anion radicals as shown in Table 1. DPPH, ABTS and superoxide anion radicals scavenging activity results reported for crude and ethyl acetate (N 3 ) fraction with IC 50 values of 330 and 191, 183 and 123, 195 and 69 μg/mL, respectively. The remaining fractions encompass moderate to weak antioxidant effects against DPPH, ABTS and against superoxide anion radicals. EO showed very promising antioxidant effect in all the three assays with IC 50 values of 450 (DPPH), 221 (ABTS) and 370 μg/mL (superoxide anion).

Acetylcholinesterase and butyrylcholinesterase inhibitory potential
N. nucifera's crude extract and its resultant fractions revealed to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) ( Table 2). The ethyl acetate fraction (N 3 ) and crude were found to be most potent inhibitors of aforementioned enzymes, causing 70 ± 0.8 and 52 ± 1.13% inhibition of AChE, while 75 ± 0.3 and 48 ± 0.73% inhibition of BChE, respectively. On the other hand, EO showed to be non-competitive inhibitor as percentage inhibition recorded were 64 ± 0.8% (AChE) and 58 ± 0.3 (BChE), slightly more than the crude extract.

GC-MS analysis of EO
The GC obtained for leaves EO showed a total of 19 peaks which appeared at various retention indices/times ( Figure S1, Table S2) (Kilic et al. 2004;Riu-Aumatell et al. 2004). The results showed that it contained three monoterpenes hydrocarbons, six oxygenated monoterpenes, four hydrocarbon sesquiterpenes and six oxygenated sesquiterpenes. The major constituent being found was 1,8-Cineole (25.64%) while α-terpeneol, α-asarone, Borneol and γ-Gurjunene were found to be 11.11, 10.26, 7.69 and 6.84%, respectively. Because of the high content of oxygenated mono and sesquiterpenoids, the oil may be classified as a 'medicinal type' (Oyedeji et al. 1992). It seems interesting that EO showed promising antioxidant activities although it contains major constituents, i.e. 1,8-cineole (25.6%) and α-terpeneol (11.11%) are not potent antioxidants (Lee et al. 2005). The antioxidant effect of EO may be due to a relatively high content of the other oxygenated sesquiterpenoids such as α-asarone (10%). It may be concluded that the N. nucifera seed's EO could be an interesting antioxidants when applied at the highest concentration tested. EO also showed to be a non-competitive inhibitor of both acetylcholenestrase and butyrylcholinestrase. The possible effect may be due to the presence of α-asarone and other oxygenated sesquiterpenoids (Mukherjee et al. 2007).

Disclosure statement
No potential conflict of interest was reported by the authors.