Quantitative analysis of tiliroside and other flavonoid glycosides in Hippophae salicifolia D. Don leaves by HPLC-PDA

Abstract A new HPLC-PDA method was developed and validated for simultaneous determination of five phenolic compounds (trans-and cis- isomers of tiliroside, quercetin-3-O-β-D-glucoside, ellagic acid, kaempferol-3-O-β-D-glucoside and isorhamnetin-3-O-glucoside) in the leaves of Hippophae salicifolia D. Don. Of the five compounds, three (tiliroside, quercetin-3-O-β-D-glucoside and ellagic acid) were isolated and characterised by spectroscopy techniques. The developed HPLC method provided a selective, sensitive and rapid analysis with good linearity (r2> 0.999), accuracy and precision. Also, the leaves of H. salicifolia were extracted by three different extraction techniques viz. reflux, microwave and ultrasound. Methanolic extracts prepared by reflux method showed the highest content of all the five compounds. Graphical Abstract


Introduction
Seabuckthorn or Leh berry belong to the genus Hippophae, which comprises seven species and eight subspecies (Singh et al. 2019) and are distributed in Himalayan region of central and Southeast Asia at an altitude of 2200-3400 m (Sharma et al.2019).A number of studies on Hippophae species from different regions suggested presence of phenolics and flavonoid glycosides in the leaves and fruits (Maheshwari et al. 2011;Saikia and Handique 2013;Dong et al. 2017).
Hippophae salicifolia D. Don. one of the species of Hippophae, is used for the treatment of various ailments as mentioned in traditional medicine (Dhyani et al. 2010).The pharmacological activities of H. salicifolia leaves and berries have been correlated to phenolics, flavonoids and fatty acids (Chakraborty et al. 2015;Sharma et al. 2015).
Tiliroside, a kaempferol-3-O-b-D-(6"-O (E)-p-coumaroyl)-glucopyranoside has been shown to possess potent antioxidant and anti-inflammatory properties when compared to other flavonoid glycosides (Zhang et al. 2015).Tiliroside exist in trans: cis isomer and the ratio of these isomers is quantified by 1 H-NMR spectroscopy.(Devi and Kumar 2020).This is the first study for qualitative and quantitative investigation of flavonoids glycosides in H. salicifolia leaves by HPLC-Photodiode Array Detector (PDA), and we report tiliroside as a major flavonoid glycoside.
The goals of this study were to isolate and characterise key phytoconstituents found in leaves of H. salicifolia, to investigate the effects of various extraction procedures on the content of phytoconstituents in H. salicifolia leaves and develop a validated HPLC-PDA method for the qualitative and quantitative analysis of phytoconstituents in H. salicifolia leaves.The ratio of isomers of tiliroside was determined in all the prepared extracts using the peak area integration method obtained from the HPLC chromatogram.S2.Total ion chromatogram of all the identified compounds is given in Figure S3.

Quantification of five compounds in leaves of H. salicifolia D. Don
The developed HPLC-PDA method was used for simultaneous quantification of five phytoconstituents (trans-and cisisomers of tiliroside, quercetin-3-O-b-D-glucoside, ellagic acid, kaempferol-3-O-b-D-glucoside and isorhamnetin-3-O-b-D-glucoside) in H. salicifolia leaves.The structures of all the analysed compounds are provided in Figure S2.Three different extraction techniques viz.reflux, microwave and ultrasonication were employed using methanol as a solvent and their qualitative as well as quantitative analysis was accomplished by HPLC-PDA method.Figure S4-S6 shows HPLC chromatogram.The results of quantitative analysis are summarised in Table S3.The % content (w/w) of the five compounds in three distinct extracts was found to be highest in the methanolic extract prepared by reflux method.Our group had reported the mentioned three extraction methods earlier (Gautam et al. 2010;Bairwa et al. 2014) for the extraction of plant materials and the obtained extracts were analysed by either by HPLC-PDA or UPLC-PDA.Results showed the highest % content of phytoconstituents in the extract prepared by reflux method.Thus, the reflux extraction method being simple, easy and quick, it can be used for preparation of samples that can be used for qualitative/quantitative analysis as compared to microwave-and ultrasonication-assisted extraction methods.Flavonoid aglycones viz.quercetin, kaempferol and isorhamnetin were found to be present below limit of detection (LOD) level in all the three extracts.
The developed HPLC-PDA method showed separation of trans:cis isomers of tiliroside with resolution of (R ¼ 4.84).As per reported literature, ratio of tiliroside isomers (trans:cis) is 3:1 (Devi and Kumar 2018) in methanolic extract of aerial parts of Malvastrum coromandelianum.We determined the ratio of tiliroside isomers as 4.5:1 in leaves of H. salicifolia and given in Table S4 by peak area integration obtained in HPLC-PDA chromatogram.Reported literature showed the ratio of isomers of tiliroside by qNMR (Devi and Kumar 2018), but our developed method can be used for the quantification of isomers of tiliroside by HPLC-PDA.

HPLC method validation
The developed HPLC method was validated as per International Council of Harmonization (ICH) Q2(R1) guideline by employing various parameters such as linearity, precision, accuracy, recovery, robustness and stability (ICH Guidelines 2005).The calibration curve for each phytoconstituent together with the LOD and Limit of Quantification (LOQ) are presented in Table S5.All the five compounds showed good linearity (r 2 ¼0.99) in relatively wide concentration range.The interday and intraday variations with % RSD values were below 2.0% (Table S6).The developed HPLC-PDA method showed acceptable accuracy with recovery of 95%-105% (Table S7).The % RSD values for the robustness study were less than 3%, which confirmed stability of the compounds during analysis (Table S8).Residual plot evaluation demonstrates that residuals were randomly distributed around zero value (Figure S7).The results revealed that the developed method was linear, sensitive, precise and accurate for simultaneous quantitative analysis of the five compounds in H. salicifolia.

Experimental
Experimental details relating to this article along with the tables and figures are available as supplementary material.

Conclusion
A rapid, precise, and accurate RP-HPLC method for qualitative and quantitative analysis of tiliroside (both transand cis-isomers), quercetin-3-O-b-D-glucoside, ellagic acid, kaempferol-3-O-b-D-glucoside and isorhamnetin-3-O-glucoside in leaves of H. salicifolia was developed.The RP-HPLC conditions ensured base line resolution of all the studied 5 phytoconstituents.Quantitative analysis revealed maximum content of the studied 5 phytoconstituents in the extract prepared by reflux method.Herein, we for the first time, are reporting the presence of both the isomers (trans and cis) of tiliroside in H. salicifolia.Ratio of both the isomers was found to be (4.5:1) by integration of peaks area observed in HPLC chromatograms.The HPLC method validation showed satisfactory statistical data for all the studied parameters.The developed HPLC method can be employed for routine quality control of crude drug samples of H. salicifolia leaves and its formulations.