A Review on Worldwide Ephedra History and Story: From Fossils to Natural Products Mass Spectroscopy Characterization and Biopharmacotherapy Potential

Growing worldwide, the genus Ephedra (family Ephedraceae) had a medicinal, ecological, and economic value. The extraordinary morphological diversity suggests that Ephedra was survivor of an ancient group, and its antiquity is also supported by fossil data. It has recently been suggested that Ephedra appeared 8–32 million years ago, and a few megafossils document its presence in the Early Cretaceous. Recently, the high analytical power provided by the new mass spectrometry (MS) instruments is making the characterization of Ephedra metabolites more feasible, such as ephedrine series. In this regard, the chemical compounds isolated from crude extracts, fractions, and few isolated compounds of Ephedra species were characterized by MS-based techniques (LC-MS, LC-ESI-MS, HPLC-PDA-ESI/MS, LC-DAD-ESI/MSn, LC/Orbitrap MS, etc.). Moreover, we carry out an exhaustive review of the scientific literature on biomedicine and pharmacotherapy (anticancer, antiproliferative, anti-inflammatory, antidiabetic, antihyperlipidemic, antiarthritic, and anti-influenza activities; proapoptotic and cytotoxic potential; and so on). Equally, antimicrobial and antioxidant activities were discussed. This review is focused on all these topics, along with current studies published in the last 5 years (2015–2019) providing in-depth information for readers.


Introduction
It is becoming increasingly clear that plants, ranging from across the plant kingdom, produce a unique diversity of secondary metabolites that can be exploited for the discovery of new drugs, bio-sourced materials, nutraceuticals, or cosmetics [1][2][3][4][5]. For finding new molecules, plant natural products are undoubtedly good sources of chemical diversity [6][7][8][9][10]. It is estimated that over 200,000 primary and secondary metabolites may be present in the plant kingdom [11][12][13][14][15]. Medicinal plant is the product of long-term medical practice worldwide, with the advantages of outstanding curative properties and less side effects [16][17][18][19][20][21]. Containing many natural products and their derivatives of therapeutic value, medicinal plants are considerate as main source of remedies able to protect human body against diseases.
As medicinal plant, enclosing over 50 species, Ephedra genus belongs to the family Ephedraceae which in turn represents one of three families in the order Gnetales [22][23][24][25][26]. Ephedra is common in cold and dry places in both the Old and the New Worlds; the Gnetaceae members live in warm and humid tropical/subtropical forests of Asia, Africa, and South America [27]. e shrubs, which reach approximately one meter in height, grow in semiarid and desert conditions in both hemispheres across six continents [25,26]. Known as Ma Huang, E. sinica is one of the oldest medicinal herbs in traditional Chinese medicine [28][29][30][31]. E. sinica preparations have been used for over 5000 years as stimulants and as antiasthmatics and are traditionally used to treat cold, bronchial asthma, cough, fever, flu, headache, edema, and allergies [32,33]. It can also be used to lose weight by increasing sweating and basal metabolism and by stimulating the central nervous system [34,35]. Moreover, it has also been combined with cardiovascular drugs to treat cardiovascular diseases [36,37]. For years, ephedrine series [(-)-ephedrine, (+)-pseudoephedrine, (-)-N-methylephedrine, (+)-N-methylpseudoephedrine, (-)-norephedrine, (+)-norpseudoephedrine] were considered to be the main Ephedra constituents [38,39]. Nowadays, at the side of pharmacological effects, there has been considerable research on the phytochemistry and bioactivities of genus Ephedra, including their antibacterial and primarily antioxidant activity [40][41][42][43][44]. From the entire plant, a wide range of Ephedra natural products including alkaloids, tannins, saponins, proanthocyanidins, phenolic acids, flavonoids, and essential oils have been mentioned and the plants-derived polyphenols are of great importance for their biological and pharmacological potential [45][46][47][48][49][50].
However, these numbers may be underestimated since many metabolites have not been characterized yet and new publications appear continuously with numerous new structures. In the last two decades, there was a quick development of mass spectrometric techniques allowing analysis of Ephedra natural products. Mass spectrometry is currently one of the most versatile and sensitive instrumental methods applied to structural characterization of Ephedra metabolite [51][52][53][54][55].
Although the analysis of Ephedra natural products has been investigated for many years, there is not a review in the literature focusing on the great pharmacological and biological potential and applications of high-resolution mass spectrometry. In this review, we will provide information about these topics and their advances and applications in the last five years (2015-2019) that could be interesting for botanical, analytical chemistry, and natural products communities.

Ephedra History Evolution
Containing approximately fifty species, the genus Ephedra (Family Ephedraceae) was distributed in arid and semiarid regions of Asia, Europe, northern Africa, southwestern North America, and South America [56][57][58][59][60]. In fact, Ephedra was distributed from the northern temperate zone (from the Canary Islands through the Mediterranean region and Central Asia to Shandong in China) to the arid regions of USA and Mexico, and to alpine area of the Andes in South America [26,27,[61][62][63][64]. is wide range is at least partially attributable to its effective dispersal syndromes.
Fossil evidence has been playing important roles in understanding early evolution of the Ephedra gnetophytes [65,66]. e early evolution and diversification of the Ephedra have increasingly become clear because of recently reported macrofossils from the Early Cretaceous strata of Asia, Australia, Europe, and Americas [67,68]. Ephedra macrofossils, especially female cones, provide a historical perspective for the early evolution, taxonomy, and biogeography of this genus. In this respect, by using molecular sequence data (rbcL) and assuming a constant rate of evolution calculated by landmark event calibration, the corresponding age of extant Ephedra was recently estimated to be 8-32 million years [69][70][71][72]. Macrofossils of female cones were found in the Early Cretaceous of South America [73,74], Mongolia [75,76], and adjacent Northeast China [60,[77][78][79][80]. Early Ephedra might have transformed bracts of female cones into vivid color to assist seed dispersal by birds, wind, or seed-caching rodents resulting in a wide intercontinental distribution [59,81,82].
On the other hand, phylogenetic analysis resulted in well-supported subgroups of Ephedra that correspond to geographical regions [69,[83][84][85] with African-Mediterranean species in a basal grade or clade and Asian species forming two well-supported clades [72,86]. New World species are monophyletic and comprise a South American clade [72,86] and a nonmonophyletic grade of North American species [86]. As reported by Crane [87], a possible origin of Ephedra in Africa is interesting as the diversity of ephedroid pollen grains is particularly high in Early Cretaceous palaeoequatorial regions of Africa and South America. In this context, African species constitute a basal grade or clade within Ephedra [72]: some of the basal species are limited to Africa (E. altissima); others have a broader distribution in the Old World, extending from Africa into Asia or southern Europe. In recent decades, various Ephedra and Ephedra-like meso-and macrofossils have been reported from the Early Cretaceous of South Europe, Northeast China [79], Mongolia [88], North America, and South America [25,56,89]. Seed mesofossils with in situ pollen were reported from the Early Cretaceous of North America [72] and Portugal (South Europe) [60].
In a study carried out by Danciu et al. [90] on ethanolic extracts of the aerial part of E. alata Decne., an amount equal to 156.22 mg of gallic acid equivalents/g dry sample (mg GAE/g) was reported for total polyphenol (TPC). Jaradat et al. [91] have analyzed the phytochemical composition of E. alata Decne., by using water, MeOH, and EtOH for the extraction.
e study reports that, when water was used, total polyphenols could not be detected in the extract, and the EtOH extract was 19.175 mg GAE/g. On the other hand, Alali et al. [92] and Nasar et al. [93] reported that water extracts of E. alata Decne. and E. procera C. A. Mey showed a TPC of 16.2 and 117.01 mg GAE/g, respectively.
Al-Rimawi et al. [41] analyzed extracts of E. alata Decne. collected from the southern part of the West Bank, Palestine. ese authors used three different solvents, namely, water, 100% EtOH, and 80% EtOH, in order to observe which solvent leads to the highest amounts of total flavonoid contents (TFC). e results showed that TFC was higher in case of 100% EtOH (19.5 ± 0.3 mg catechin/g dry weight). Aghdasi et al. [42] studied the variation of TFC of Iranian E. major during May to October from Bojnoord. In fact, TFC exhibited a variation during sampling period and ranged from 4.63 to 8.4 mg QE/g. Mellado et al. [94] analyzed flavonoids in E. chilensis K Presl, a Chilean endemic plant. ese authors reported significant differences in the CH 2 Cl 2 extracts (P < 0.05) compared to the hexane and EtOH extracts. e total phenolic content in both CH 2 Cl 2 extract and EtOH extract shows significant differences (P < 0.05) with the hexanoic extract. In the study of Al-Trad et al. [95], the butanolic extract from the stem of Jordanian E. alte had a phenolic content of 404.001 ± 5.53 mg/g gallic acid and flavonoids of 40.73 ± 6.59 mg/g quercetin [95].
Hegazy et al. [96] observed that the total anthocyanins content (TAC) of Saudi E. foeminea, collected from Shada Mountain, southwest Saudi Arabia, was 0.14 mg cy-3-glu/ 100 g. TAC of Lebanese E. campylopoda was detected in the MeOH extracts but not in EtOH and aqueous fractions [97].

Recent Applications of High-Resolution Mass Spectrometry for Ephedra Extract Characterization
Although the analysis of natural products from Ephedra species has been investigated for many years, there is not a review in the literature focusing on the great possibilities and applications of high-resolution mass spectrometry. is review is devoted to chemical identification using mass spectrometry as the most powerful technique of qualitative analysis. It is evident in the fact that the terms of "identification" and "mass spectrometry" occur together in more than a million scientific reports returned in the search results performed by Google Scholar engine [98]. e reason for the potency of MS is that it is superior to other analytical techniques in the combination of features, such as multianalytic property, sensitivity, selectivity, possibility of compounds identification by molecular mass or formula, and possibility of combining with chromatography. e most prominent methods include MS/MS (tandem mass spectrometry); LC-ESI/MS/MSn (liquid chromatography-electrospray ionization/multistage mass spectrometry); LC-PDA (liquid chromatography coupled to photodiode array); HPLC-PDA-ESI/MS (high-performance liquid chromatography coupled to photodiode array and electrospray ionization mass spectrometric); LC-DAD-ESI/ MSn (high-performance liquid chromatography with diode array detection coupled to tandem mass spectrometry analysis with electrospray ionization); and LC/Orbitrap MS (liquid chromatography Orbitrap Fusion Tribrid tandem mass spectrometry). In this section, we will provide information about this topic and its advances and applications in the last five years (2015-2019) that could be interesting for both the analytical chemistry and the natural products communities, from Ephedra species collected from the five continents of the world. e ephedrine alkaloids (Figure 1) are considered the active constituents of plants belonging to the genus Ephedra. (−)-Ephedrine is the major isomer; the minor alkaloids include (−)-norephedrine, (+)-norpseudoephedrine, (+)-pseudoephedrine, and (−)-methylephedrine [99].
As a summary, Table 1 shows some of the most commonly used methods to identify and quantify phenolic compounds (chromatographic conditions; mobile phase and gradient, quantification and detection, and analytical method) from Ephedra species extracts.
Palestinian E. alata extracted with water, 80% ethanol, and 100% ethanol was rich in flavonoid glycosidic compounds. In fact, the full scanned LC-MS using the positive and negative electrospray ionization modes revealed the presence of luteolin-7-O-glucuronide flavonoid, myricetin 3-rhamnoside, and some other major polyphenolic compounds that share myricetin skeleton [41] Collected from Bojnoord (Iran), stems and seeds of E. major were soaked in 80% MeOH, and the amounts of ephedrine were determined by HPLC [42]. Data from HPLC analysis revealed that while root is depleted of ephedrine, the ephedrine amount in stem organ ranged from 1.50 to 2.12 mg/g dry weight. To assess the alkaloids present in Pakistani E. intermedia, the HPLC method was used for the quantitative analysis of ephedrine and pseudoephedrine [103]. is study showed that average alkaloid substance in E. intermedia was as follows: pseudoephedrine (0.209%, 0.238%, and 0.22%) and ephedrine (0.0538%, 0.0666%, and 0.0514%).
From northern Jordan, the in vitro antioxidant activities of the butanolic extract from the stem of E alte were assessed against DPPH, ABTS, and hydroxyl radicals [95]. In fact, butanolic extract showed different levels of radicals scavenging activity in a dose-dependent manner over the range of 5-500 μg/mL concentration, indicating the high antioxidative capacity of the extract. e IC 50 values of the extract were 66.4, 50.2, 43.5, and 77.1 μg/mL for DPPH, ABTS, hydroxyl radicals, and the ferrous ion chelating activity, respectively. Hegazy et al. [96] evaluated the antioxidant activities of the five wild underutilized fruits in the mountains of southwest Saudi Arabia (Coccinia grandis (L.) Voigt, Diospyros mespiliformis Hochst. ex A. DC., Cissus rotundifolia (L.), E. foeminea Forssk., and Grewia villosa Willd.). Corresponding to this study, methanol extract of E. foeminea displayed antioxidant activity higher than 50% even at low concentration of 0.6 mg/mL. Shawarb et al. [113] and Jaradat et al. [114] found that the leaves of Palestinian E. alata showed a good antioxidant activity. is activity was evaluated by the free radical scavenging as 15.85 μg/mL (IC 50 ) and 75.02% at 100 μg/mL, respectively. Kallassy et al. [97] evaluated the antioxidant capacity of various solvent (distilled water, ethanol, and methanol) extracts of Lebanese E. campylopoda stems. e different extracts showed varying antioxidant potential, and their DPPH scavenging capacities were in the following order: ethanolic extract (IC 50 � 125 μg/ mL) >methanolic extract (IC 50 � 150 μg/mL) >aqueous extract (IC 50 � 300 μg/mL). On the other hand, this study showed that methanolic extract had the most efficient Fe 2+ chelating capacity (IC 50 � 1 mg/mL) in comparison to both the ethanolic and the aqueous extracts, which presented IC 50 values of more than 1.5 mg/mL.
Khan et al. [115] reported that the ethyl acetate fraction of Pakistani E. gerardiana (root and stem) presented more significant free radical scavenging potential than the methanol extract, chloroform, n-hexane, and n-butanol fractions, and the mean values ranged, respectively, from 21.49 to 2.96 μg/mL. It should be noted that the stem of E. gerardiana gave the maximum antioxidant yield and chloroform gave the lowest one (IC 50 � 22.73 μg/mL). Extracted by solvent mixture of ethanol/methanol/water of aerial parts of Pakistani E. intermedia, the antioxidant activity, tested by DPPH radical procedure, was 90.08 at 100 μg/mL [103].
e DPPH assay showed that the hexanoic extract had a poor activity (P < 0.05) compared with the positive controls (trolox and gallic acid). Dichloromethane (CH 2 Cl 2 ) and ethanolic extracts showed similar activities, and these activities are different from the activities of trolox and gallic acid (P < 0.05). For the FRAP assay, the CH 2 Cl 2 and EtOH extracts show better antioxidant activity than the positive controls (P < 0.05). Concerning the TRAP assay, Hex extract was the least active of all of the tested extracts compared with the positive controls (gallic acid and BHT) with significant differences (P < 0.05).

Antimicrobial Activity.
Antimicrobial efficacy of Ephedra species extracts has been described in several studies using in vitro methods such as agar disc diffusion assays and/ or minimum inhibitory concentration (MIC). e in vitro antimicrobial activity against a number of pathogenic and drug-resistant bacteria and fungi is presented in Table 3. Using both in vitro agar diffusion and MIC (minimum inhibitory concentration) Danciu et al. [90] showed that the hydroalcoholic extract of E. alata Decne, collected from Djerba (Tunisia), had a bactericidal effect against Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 51299 and fungicide impacts on Candida albicans 8 Evidence-Based Complementary and Alternative Medicine    [117] Evidence-Based Complementary and Alternative Medicine   [100]. In this study, infusions and decoctions had a weak effect, except against E. coli strains, which was the most susceptible microorganism with a MIC value of 0.625 mg/mL. On the contrary, high antibacterial and antifungal effects of this plant were previously reported in extracts prepared with water, methanol, and acetonitrile, with the latter exhibiting the most potent effect against all the microorganisms supplied by the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt [121]. Ethanolic and hydroalcoholic herb extract of Iranian E. sinica was assayed against standard and clinical Pseudomonas aeruginosa, and then the MIC and MBC (minimum bactericidal concentration) were assayed [119]. e results showed the lowest MIC values of ethanolic herb extract were, respectively, 25 and 12.5 μg/mL, but the lowest MIC values of the hydroalcoholic herb extract were 25 and 25 μg/ mL, respectively. Equally, the lowest MBC values of ethanolic herb extract on clinical and standard strains of P. aeruginosa were 50 and 25 μg/mL, respectively; however, the lowest MBC values were 25 and 25 μg/mL, respectively. e biosynthesized E. procera nanoparticles (EpNPs) exhibited considerable activity against E. coli ATCC 25922 and B. subtilis ATCC 6633 with MICs of 11.12 μg/mL and 11.33 μg/ mL, respectively. Nevertheless, EpNPs showed moderate activity against P. aeruginosa while the S. epidermidis and S. aureus strains were found resistant. Equally, EpNPs showed considerable antifungal activity against A. flavus and A. Niger, but moderate activity against Mucor spp [120]. ese authors have proven that EpNPs showed antifungal activity against A. flavus, A. Niger, and Mucor spp. with a diameter of inhibitory zone equal to 14.2, 15.8, and 11 mm, respectively. Four extracts of Pakistani E. vulgaris (CHCl 3 , MeOH, EtOH, and water) were used against three pathogen bacteria, namely, Streptococcus pneumonia, Pseudomonas aeruginosa, and Klebsiella pneumonia [122]. It was noted from the results that chloroform and aqueous extracts have no inhibition effects against P. aeruginosa. e maximum inhibitory effects (17.16 mm inhibition zone) in chloroform extract against S. pneumoniae and minimum inhibition activities (8.70 mm zone of inhibition) in the extract of ethanol against P. aeruginosa were observed.

Biomedicine and Pharmacotherapy Activity
Pharmacological activities of extracts from different species of the worldwide genus Ephedra, published in the period 2015-2019, are well documented in Table 4. e evaluation of the antiproliferative, proapoptotic, and cytotoxic potential against the MCF-7 breast cancer cell line of the ethyl acetate (EA) extract of the aerial part of E. alata Decne was reported by Danciu et al. [90]. In this study, the antiproliferative activity of EA started from a concentration of 10 μg/mL, with a cell growth inhibition of 19.68%. For the highest tested concentration, 30 μg/mL, the growth inhibition percentage was 56.45. e cytotoxicity assessment revealed that the EA manifested a significant difference in cytotoxic potential, displaying a cytotoxicity percentage above 13%. e potential antimigratory activity of the EA extract on MCF-7 human breast adenocarcinoma cells was verified by means of a wound-healing technique. In this regard, on the MCF-7 cells' migration EA had a strong inhibitory effect and showed a wound-healing rate below 5% after an interval of 24 h. e cytotoxicity assessment revealed that the EA extract manifested a significant difference in the cytotoxic potential when compared to the positive control (DMSO), displaying a cytotoxicity percentage of ∼13%. To investigate the apoptotic potential of EA at the selected concentration, MCF-7 cells were treated with 30 μg/mL for 72 h, and the cells' nuclei were analyzed by DAPI (4′,6′-diamidino-2-phenylindole) staining. In this line, Danciu et al. [90] showed that the control cells exhibit a normal organization, with a large, round nucleus, a clear nucleolus, and uniform chromatin density. However, after treatment, the MCF-7 cells manifested morphological changes distinctive for apoptosis induction, such as chromatin condensation.
Jaradat et al. [114] investigated the use of herbal remedies by women living with breast cancer in the West Bank of Palestine by a questionnaire-based cross-sectional descriptive study; the questionnaire was distributed to 115 patients.
is study revealed that E. alata was the most commonly used plant species in the treatment of breast cancer. Leaves and seeds of E. alata were the most commonly used parts, and decoction was the most commonly used method of preparation. Jordanian E. aphylla extracts (methanol, chloroform, ethyl acetate, n-hexane, and water) were tested to evaluate antiproliferative potential [122]. e authors observed that all extracts displayed strong antiproliferative potential against the tested cell lines (breast cancer cell lines (T47D, MCF-7) and Vero cell line (African green monkey kidney)). Moreover, E. aphylla extracts showed a little cytotoxicity activity against the Vero normal cell line. e antiproliferative activity of various solvent extracts against MFC7 cell line was in the order of aqueous > methanol > chloroform > ethyl acetate > n-hexane [122].
Kallassy et al. [97] studied the anti-inflammatory and antiproliferative potential of ethanol, methanol, and water extracts of Lebanese E. campylopoda stems. e anti-inflammatory capacity was estimated both by evaluating RAW 264.7 murine macrophage cells-mediated secretion of PGE 2 using ELISA technique, and by quantifying the mRNA level of the proinflammatory cytokines (IL-α, IL-β, and IL-6), chemokines (CCL3 and CCL4), and inflammation-inducible COX-2 and iNOS enzymes using quantitative real-time PCR (qRT-PCR). By using the XTT viability assay, the antiproliferative potential of E. campylopoda was determined.
is study confirmed that the alcoholic extracts showed more potent anti-inflammatory and antiproliferative Evidence-Based Complementary and Alternative Medicine capacities than aqueous extract [97]. Hoshyar et al. [123] examined the anticancer effects of E. sarcocarpa on proliferation of breast cancer, MCF-7, and epithelial normal MCF-10A cells. ese authors evaluated the effect of E. sarcocarpa aqueous extracts on cell proliferation and investigated the cytotoxic effects at concentrations ranging between 0 and 3 mg/mL on the growth of human breast cancer (MCF-7) and normal mammalian (MCF10-A) cells after different time incubation (0-72 h) using MTT (the methylthiazolyldiphenyl-tetrazolium bromide) assay.
is study uncovers that the treatment of MCF-7 cells with the E. sarcocarpa aqueous extract (0.25, 0.5, and 0.75 mg/mL) significantly decreased cell viability and increased cell death percentage by increasing extract concentration after 72 h. Parallel treatments of the normal cells with this extract indicted a much less inhibitory effect on the viability of MCF10-A cells.
Park et al. developed E. sinica Stapf. (ES) extract-capped gold nanoparticles (ES-GNs) and investigated their antineuroinflammatory properties in microglia [116]. For this purpose, antineuroinflammatory properties of ES-GNs on production of proinflammatory mediators (nitric oxide, prostaglandin E 2 , and reactive oxygen species) and cytokines (tumor necrosis factor-α, IL-1β, and IL-6) in lipopolysaccharide-(LPS-) stimulated microglia were well investigated by ELISA and flow cytometry. In this regard, ES-GNs significantly attenuated LPS-induced production of proinflammatory mediators and cytokines, which was related to suppressed transcription and translation of inducible nitric oxide synthase and cyclooxygenase-2, determined by RT-PCR and western blotting. ese authors hypothesized that antineuroinflammatory properties of ES-GNs were mediated by AMP-activated protein kinase and nuclear erythroid 2-related factor 2/antioxidant response element signaling. In 2017, Lee et al. [130] evaluated the effects and molecular targets of methanolic extract of dried stems and leaves of E. sinica Stapf. and E. intermedia Schrenk (EHM) on high-fat diet-(HFD-) induced hyperlipidemic ICR mice. According to these authors, results showed that EHM administration for 3 weeks significantly (P < 0.05) decreased total cholesterol (TC) and triglyceride levels without altering body weight (BW) in mice, and gene expression levels in the livers of EHM-treated mice were restored at 34.0% and 48.4% of those up-or downregulated by hyperlipidaemia, respectively. Hyuga et al. [107] confirmed that ephedrine alkaloidsfree Ephedra herb extract (EFE) suppressed hepatocyte growth factor-(HGF-) induced cancer cell motility by preventing both HGF-induced phosphorylation of c-Met and its tyrosine kinase activity. Equally, this study displayed the analgesic effect of EFE and the anti-influenza virus activity by showing inhibition of MDCK cell infection in a concentration-dependent manner. All assessments of toxicity, even after repeated oral administration, suggest that EFE would be a safer alternative to Ephedra herb.
Oshima et al. [108] established a preparation method for EFE : ephedrine alkaloids-free Ephedra herb extract (EH) and revealed its chemical composition, including the content of herbacetin, a flavonoid aglycon. In addition, these authors showed the antiproliferative effects of EFE against the H1975 non-small-cell lung cancer (NSCLC) cell line. It should be noted that the antiproliferative effect of EFE against H1975 cells was comparable to that of EH extract. e Ephedra-Gypsum extracts at test dose (6, 12, 24 g/kg) significantly and dose-dependently attenuated yeast-induced fever in rats. e Ephedra-Gypsum extracts also prolonged the latent period, reduced ovalbumin-(OVA-) induced increases in eosinophils and white blood cell (WBC), and decreased the wet and dry weight ratio of the lungs in the antiasthmatic test.

Ephedra Toxicity
Although Ephedra metabolites are naturally occurring alkaloids that can be derived from evergreens worldwide and have been used as medicinals, recent studies reported that ephedrine has various adverse effects on organisms such as hepatitis, angle closure glaucoma, nephrolithiasis, neurodegenerative diseases, and cardiovascular toxicity. Few of these side effects are reversible whereas others are irreversible and may even lead to death [28]. Several recent reviews have documented the dangerous nature of using these "drugs" unsupervised, including multiple deaths, and the FDA is currently reviewing ephedrine's use in the alternative medicine industry. Powell et al. [131] reported the toxicity ephedrine nephrolithiasis in a patient using an energy supplement, Ma Huang extract, which contains ephedrine. Although previously not reported, e Louis C. Herring and Company kidney stone database shows that this is an endemic complication of ephedrine with hundreds of previous episodes.
Nauffal and Gabardi [132] found that the use of 40-3,000 mg/day of E. sinica for several months can cause nephrolithiasis with flank pain, hematuria, and renal dysfunction. On the other hand, recent studies have reported that Ephedra herb containing many pharmacologically active alkaloids, principally ephedrine, has been reported to cause acute hepatitis. In this context, Lee et al. [133] investigated hepatotoxicity and key regulation of mitophagy in ephedrine-treated LX-2 cells. However, mitochondrial swelling and autolysosome were observed in ephedrinetreated cells. Also, ephedrine inhibited mitochondrial biogenesis, and the mitochondrial copy number was decreased. Moreover, antioxidants can serve as therapeutic targets for ephedrine-induced hepatotoxicity [133]. Equally, it is important to note that Ephedra species have been implicated in causing liver injury in case reports [134]. Similarly, speculation has also indicated that the alkaloid ephedrine may be hepatotoxic, based upon case reports, and associated with liver injury [135]. An in vitro assay using human hepatoblastoma cells (HepG2) demonstrated that when Ephedra (Ma Huang) extracts were normalized for their ephedrine content, they displayed greater cytotoxicity relative to ephedrine itself, indicating that there may be other constituents responsible for toxicity [133]. While ephedrine and pseudoephedrine showed cytotoxicity in the HepG2 cell line, the concentrations required (i.e., >300 μg/mL) again indicate that such data are likely irrelevant to in vivo administration. e reported adverse reactions principally involve the cardiovascular system and are, in general, similar to other 16 Evidence-Based Complementary and Alternative Medicine sympathomimetics.
e most common side effect is hypertension with a risk of hemorrhagic stroke. Also ischemic stroke due to vasoconstriction and likely platelet aggregation can occur after its consumption [136]. Although the risk of hemorrhagic stroke with pseudoephedrine seems to be lower, it can occur and might result in death. e adverse reactions after Ephedra administration can more easily occur when it is used in combination with caffeine. is combination increases the effect of sympathomimetics, and the mechanisms will be discussed later [136].
Ephedrine is also found in dietary supplements that promote short-term weight loss, but those are now illegal in the USA. However, in traditional Chinese medicines that contain ephedrine, it is legal. e ephedrine quantity in dietary supplements taken orally is about 20 mg per serving, and doses are taken up to two to three times per day. It has been shown that labels of dietary supplements do not list ephedrine content or incorrectly report the amount of ephedrine in these products. Ephedrine has been associated with cardiovascular dysfunction such as myocardial infarction, severe hypertension, myocarditis, and lethal cardiac arrhythmias. e typical dose of ephedrine used for bronchodilation is 25-50 mg, but ephedrine doses associated with adverse events were less than this amount [137]. e use of drug-herb interaction causes irreversible neurodegenerative diseases. For example, a 29-year-old male has been injected with an intravenous mixture of pseudoephedrine (extracted from Ephedra), potassium permanganate, and acetylsalicylic acid two to three times a day for 9 years and a half. roughout these years, the patient developed many symptoms including speech disturbance, inability to walk independently, postural instability, tremor, and dystonia. e patient was diagnosed with manganese toxicity which leads to irreversible neurodegenerative disorder due to the long exposure to E. sinica [28].

Conclusion
Ephedra natural products have attracted more and more attention since they can exhibit complementary biological and therapeutic effects against diseases. Historically, Ephedra may even have been diverse and widespread at that time and the corresponding fossils document that Ephedra was already present in the Early Cretaceous. Further testing and development of methods for molecular dating will be needed to clarify conflicts between molecular signals and the fossil record.
In this review, we summarized the chemical components isolated and identified by MS. Further instrument sophistication in coupling several systems such as multidimensional chromatography with NMR and MS in series is already occurring. e prediction of the future for promising approaches involves the application of HPLC with ESI time of flight mass spectrometers and ESI FT ion cyclotron resonance mass spectrometers. An increased emphasis on microcapillary columns with nanotechnology ESI systems driven partly by environmental issues seems inevitable.
e relationship between the Ephedra natural products structure and its pharmacological activity needs to be further studied. In this context, the mechanisms of action of phytochemical Ephedra content can provide guidance for its clinical application.

Conflicts of Interest
e authors declare no conflicts of interest.