Isocratic elution based analytical method for quantification of phytoconstituents in Hedychium spicatum and evaluation of their α-glucosidase inhibitory activity

Abstract An efficient, fast, selective and simple isocratic elution method has been established using ultra performance liquid chromatography coupled to photodiode array detection and electrospray ionisation mass spectrometry (UPLC-PDA-ESI-MS) techniques for the quantification of isolated molecules from Hedychium spicatum rhizomes and assess their α-glucosidase inhibitory activity. Developed analytical method was validated as per the regulatory guidelines such as linearity, selectivity, the limits of detection (LOD) and quantification (LOQ), repeatability and accuracy. Highest concentration of 7-hydroxy hedychenone (4) (76.7 mg/g of dried plant material) was detected in rhizomes. Present results demonstrated parent extract (ethanolic) had the greatest inhibitory effect on α-glucosidase with IC50 values of 4.0 µg/mL. Developed method can be used as a new analytical approach for quality assessment of H. spicatum based herbal formulations and other products. Furthermore, current findings showed that the extract, fraction and isolated metabolites may be a good natural antidiabetic. Graphical Abstract


Introduction
Hedychium spicatum Buch.-Ham.ex Sm. (Family Zingiberaceae), commonly called as spiked ginger lily, van haldi, kapoor kachari and shati is considered as one of the most valued medicinal plants having a number of pharmaceutical, oriental medicine and food applications (Flora of China 2000;Mishra et al. 2016;Rawat et al. 2018;Maurya et al. 2022a;2022b).The plant possesses anti-inflammatory, anti-asthmatic, anti-allergic, analgesic, ulcer protection, hepatoprotective, antihyperglycaemic, hair growth promoting, anticancer, anthelmintic, tranquillising, insect-repelling properties and antioxidant activities (Koundal et al. 2015;Rawat et al. 2018).Previously reported methods demonstrated for the quantification of H. spicatum (methoxy cinnamic acid ethyl ester, hedychenone) based on HPTLC (Srinivas et al. 2007;Arora and Jain 2010).However, to the best of our knowledge there is no literature report regarding the UPLC methodology and quantification of H. spicatum phytoconstituents.
The current objective of our research was to develop a new simple isocratic elution method for quantitative estimation of isolated compounds and assess their a-glucosidase inhibitory activity.Developed method will be helpful to assess the quality of H. spicatum raw material and their derived products.

Optimisation of UPLC condition
Appropriate UPLC chromatographic parameters were optimised with mobile phase composition, such as acetonitrile: methanol, acetonitrile: water, methanol: water and water (0.1% formic acid): acetonitrile with column temperatures 25, 30, 35, 40 C to achieve better resolution, separation and good peak shape with standard mixture and sample solutions.Isolated compounds such as 5 (retention time, t R : 4.91), 2 (t R : 5.28), 3 (t R : 10.17), 1 (t R : 16.79) and 4 (t R : 19.72) were separated.Figures S17 and S18 were presented the chromatograms of reference compounds and sample with standards.During our initial method screening, the reversed phase UPLC-PDA-ESI-MS based method was developed for the simultaneous analysis of the isolated molecules and optimised by using different compositions of the acetonitrile, methanol and water with the formic acid as eluent.Linear gradient trials showed that methanol was found to be better resolution of closest eluting compounds and proper baseline as comparison to acetonitrile (Figures S19 and S20).So, for further optimisation methanol and water with the 0.1% formic acid were taken as mobile phase eluent.The aforesaid mobile phase with different combinations, both with gradient elution and isocratic elution program with flow rate and the column temperature were optimised.After several trials, the reported method was found to be best suitable method for the simultaneous analysis of metabolites.

Validation of parameters
A fast and sensitive UPLC-PDA-ESI-MS method was established for the simultaneous quantitative analysis of five components in the rhizomes of H. spicatum.All calibration curves of the five compounds were plotted between peak area ratio (y), versus their concentration (x) which gained with good regression coefficients (R 2 ) ranging from 1.0 to 0.98 (Table S2).LOD (limits of detection) and LOQ (limits of quantification) for all components were found to be 0.78-25.0and 2.5-82.5 mg/mL, respectively (Table S2).

Repeatability and recovery
Current findings (Table S2) revealed the established analytical technique was accurate and precise.Recovery for isolated molecules determined at selected concentrations by spiking ethanolic extract with reference working solutions.Recovery was calculated and found to be 100.1-109.6%for five reference component.Also, the intra-and interday precisions; showed as relative standard deviation (%RSD) obtained in the range of 0.36-4.31%and 2.27-2.65%,respectively which demonstrated good recovery (100.1-109.3%)(Table S2).The overall findings of UPLC-PDA demonstrated established method was sufficiently reliable, accurate and acceptable; therefore developed method could be applied for the quantitative analysis of other diterpenes containing plants.

Quantitative estimation using UPLC-PDA
Quantity of all isolated components was evaluated in ethanolic extract of H. spicatum rhizomes.Compound 4 was recorded in higher concentration (76.7 mg/g of dry plant material).However, isolated compound 5, 3, 1 and 2 exhibited 47.6, 38.9, 21.4 and 18.7 mg/g of dry plant material respectively (Table S2).

a-Glucosidase inhibition activity of extract, fraction and isolated compounds
The inhibitory activity of the extract, fractions and isolated compounds on a-glucosidase activity was evaluated and the results are presented in Table S3.These findings suggested that parent extract found to be greatest inhibitory effect on a-glucosidase with IC 50 values of 4.0 ± 0.08 mg/mL, which was close to the positive control, acarbose.In addition, compound 1 (hedychenone) showed good a-glucosidase inhibition activity with IC 50 value13.24± 0.29 mg/mL.In this study, the observed a-glucosidase inhibitor activity of the parent extract (ethanolic) was may be due to the synergistic effect of the metabolites present in the parent extract (Maurya et al. 2022b).

Conclusion
In the current study, a new analytical method was developed for the separation and quantification of isolated molecules.Compound 4 was recorded in higher concentration as compared to other metabolites.Developed analytical method will be helpful to assess the quality of H. spicatum raw material and their derived products.Moreover, the current study explained that the extract, fraction and isolated compounds from H. spicatum possess good a-glucosidase inhibitory activity.Findings of above results showed that the extract, fraction and isolated metabolites can be used as nutraceutical supplements for the management of type 2 diabetes mellitus and its complications.