Catechins and other phenolic compounds in herb of eight Ephedra species in comparison to Camellia sinensis

Abstract Using HPLC-DAD the presence of catechin, epicatechin, quercetin, kaempferol and protocatehuic acid was analysed in herb extracts of eight Ephedra species (Ephedra sinica, Ephedra major, Ephedra chilensis, Ephedra ciliata, Ephedra foeminea, Ephedra equisetina, Ephedra gerardiana and Ephedra distachya). For comparison purposes, the same phenolics were determined in Camellia sinensis herb, which is a common source of catechins and a medicinal plant with valuable antioxidant properties. The catechin content in Ephedra spp. ranged between 0.83 mg/g d.w.– 8.77 mg/g d.w., while the epicatechin content was between 0.11 mg/g d.w.– 3.38 mg/g d.w. In comparison to black tea, Ephedra major, Ephedra distachya and Ephedra equisetina had a higher catechin content. The selected Ephedra spp. are rich in catechins and the obtained results serve as the chemical rationale for the anti-inflammatory, antioxidant and anti-proliferative activities of Ephedra that are currently being investigated. Graphical Abstract


Introduction
The Ephedra genus consists of 69 species mainly distributed in semi-arid environments throughout both the Palearctic and Nearctic realms (Gonz alez-Ju arez et al. 2020).Ephedra species have been used in traditional Chinese medicine to treat respiratory diseases, fever, flu and headaches (Lee 2011).Ephedra sinica, E. intermedia and E. equisetina are the three Ephedra species characterised as ingredients of the Chinese drug 'Ma-Huang' and listed in each edition of the Chinese Pharmacopoeia (Miao et al. 2020).In the twentieth century Ephedra was used as a means of weight loss, however, in recent years, there has been a newly revoked interest in Ephedra spp., mainly focused on the elucidation of the chemical composition (Al-Rimawi et al. 2017).The application of novel extraction, separation and identification techniques has advanced the phytochemical research and presently at least 247 chemical components were reported in Ephedra herbs.These constituents belong to various groups of secondary metabolites and include alkaloids, flavonoids, tannins, organic acids, polysaccharides and other compounds (Miao et al. 2020).The comprehensive chemical profiling is an essential part of bioactivity studies, which have shown an overall great potential of this plant in pharmaceutical application.Extracts and monomeric compounds from Ephedra sp. were reported to possess antioxidant, antimicrobial and anti-inflammatory properties (Khan et al. 2017), enzyme-inhibitory activity (Hamoudi et al. 2022), antidiabetic (Hajleh et al. 2022) and anti-cancer properties (Danciu et al. 2018;Sioud et al. 2022), as well as wound-healing effects (Kittana et al. 2017).Most recent studies were focused on chemical elucidation (Song et al. 2022;Zhu et al. 2022), metabolomic profiling (Al-Nemi et al. 2022), the therapeutic use of Ephedra during the COVID-19 crisis (Nabeshima et al. 2022) and the evaluation of novel properties such as the toxicity and hemocompatibility of Ephedra based silver nanoparticles (Nasar et al. 2022) or molecular mechanisms of Ephedra herb in the treatment of nephrotic syndrome (Yao et al. 2022).The majority of studies report characteristics of individual Ephedra spp., which implicates the necessity for comparative outputs in the future.
Guided by previous results (Ibragic et al. 2021) that revealed overall powerful antioxidant properties and the presence of catechins in the investigated Ephedra spp., the aim of the present study was to quantify catechin (C), epicatechin (EC) and several other phenolic compounds in the herb of following Ephedra spp.: E. sinica collected in Japan, E. distachya, E. equisetina, E. gerardiana, E. chilensis and E. ciliata collected in France and E. major and E. foeminea collected in Bosnia and Herzegovina.Along with the primary catechin, epigallocatechin-3-gallate (EGCG), C and EC are also found in high amounts in Camellia sinensis that is considered a strong antioxidant with numerous beneficial effects on human health (Singh et al. 2011).The consumption of black and green tea (Camellia sinensis) has been correlated with low incidence of chronic pathologies (Tang et al. 2015).Therefore, it was of interest to compare the content of catechins in extracts of various Ephedra spp.and Camellia sinensis.Literature reports that flavonoids, dihydroflavonoids, flavonols, dihydroflavonols, flavanols, and anthocyanins make up approximately 0.29% mass of Ephedra herbs and that flavonols are mainly derived from kaempferol (K), quercetin (Q), herbacetin and their glycosides (Miao et al. 2020).However, most studies describe only the isolation and characterization of some of the selected phenolic compounds.Mighri et al. (2019) reported the presence of protocatechuic acid (PCA), C and EC in E. alata using LC/MS and the presence of C, EC, K and Q was confirmed using MS, 1H-NMR and 13 C-NMR in roots of E. sinica (Tao 2011).This is the first study to provide a quantitative profile of the selected flavonoids and protocatechuic acid in eight different Ephedra spp.The utilised method proved selective and sensitive enough and enabled a successful separation of compounds of interest in a short analysis time.

HPLC analysis of Ephedra sp. extracts
Herb extracts of eight different Ephedra spp.were prepared for quantitative analysis of C, EC, PCA, Q and K by modifying an HPLC method reported by Zuo et al. (2002).By changing the gradient, the run time was reduced from 29 min to 15 min, whereby the last analyte eluted at minute 10.The described HPLC method allowed successful, simultaneous separation and detection of PCA, C and EC at 280 nm (see Supplementary material Figure S1), as well as Q and K at 360 nm (see Supplementary material Figure S2).A slight shift of retention times for PCA (tR ¼ 3.847 min), C (tR ¼ 4.331 min) and EC (tR ¼ 5.637 min) was observed between individual standards and their mixture in a solution.Identification of phenolic compounds was based on UV spectra wherever possible and on their retention times.The retention time window was set at 5% for PCA, C and EC and at 3% for Q and K.
For quantification of the selected phenolic compounds, the calibration curves of standard substances were generated by plotting peak height (mAU) versus amount (ng), as shown in Figure S3 (Supplementary material).The limit of detection (LOD) and limit of quantification (LOQ) were determined using the signal-to-noise ratios 3:1 and 10:1, respectively (Shrivastava and Gupta 2011).
During optimization, one of the critical steps was to evaluate the effects of the extraction method on the separation efficiency.Best results were obtained by Method 2a and 2 b (see Supplementary material Figure S4), presumably due to the addition of HCl that facilitated a more complete extraction of phenolics.While both methods produced chromatograms with a stable baseline and good resolution of peaks, final advantage was given to Method 2 b due to a much shorter preparation time.Representative chromatograms are provided in Figure S5A-C (Supplementary material).Chromatograms obtained by Method 1 and 3 were unsuitable for the detection of the compounds of interest.According to the quantitative results presented in Table S1 (Supplementary material) Ephedra spp.are rich in C (0.83 mg/g d.w.À 8.77 mg/g d.w.) and EC (0.11 mg/g d.w.À 3.38 mg/g d.w.), which is a characteristic of black and green tea.Except for E. ciliata where C and EC were not detected, in extracts of all other Ephedra spp. the C content was higher than the EC content.Ephedra chilensis and E. gerardiana were the only species devoid of PCA, whereas the PCA content in the remaining Ephedra spp.extracts was rather low and ranged between 0.05 À 0.75 mg/g d.w.On the other hand, E. gerardiana was the only Ephedra species where Q and K were detected and quantified (Table 1).
By mutual comparison of the different E. spp. it can be noted that E. major, E. distachya, E. equisetina and E. gerardiana are species richer in the analysed phenolic compounds.Further on, E. major (8.77 mg/g d.w.), E. distachya (3.95 mg/g d.w.) and E. equisetina (5.62 mg/g d.w.) had a higher C content than black tea (2.57mg/g d.w.), while E. gerardiana had a higher K content (0.68 mg/g d.w.) than green tea (0.61 mg/g d.w.).
Even though EGCG is believed to be an important determinant of the antioxidant and other therapeutic qualities of Camellia sinensis (Higdon and Frei 2003;Abbas and Wink 2009;Miltonprabu and Thangapandiyan 2015), the role of other phenolic compounds cannot be disregarded either.All five phenolics were identified in samples of black tea and green tea.Black tea had the highest EC content (9.22 mg/g d.w.), while green tea was found to be richer in C (10.42 mg/g d.w.).

Conclusion
In conclusion, relative to sensitivity and resolution, the modified HPLC-DAD method proved adequate for the simultaneous identification and quantification of C, CE, PCA, Q and K in a very short-time analysis.This is the first study that provided quantitative content of the selected phenolics in eight Ephedra spp. of different geographical origin.The investigated Ephedra spp.were found to be rich in catechins, particularly E. major, E. distachya and E. equisetina.The PCA was present in rather low concentrations in all Ephedra spp.except for E. chilensis and E. gerardiana where it was not detected.On the other hand, Q and K were found in E. gerardiana only.The same phenolics were investigated in black tea and green tea.The antioxidant properties of Camellia sinensis are predominantly attributed to EGCG, however other catechins may contribute to the beneficial health effects, too.In comparison to black tea, E. major, E. distachya and E. equisetina had a higher C content.The obtained results may be used to support and facilitate further research of novel therapeutic applications of Ephedra herb that are based on their abundance of phenolic compounds and the capacity to scavenge free radicals.

Disclosure statement
The authors declare no conflict of interest.

Table 1 .
Content of PCA, C, EC, Q and K in extracts of different Ephedra spp.and Camellia sinensis expressed as mean ± SD.