Phenylalkylamines, such as the plant compounds ephedrine and pseudoephedrine and the animal neurotransmitters dopamine and adrenaline, compose a large class of natural and synthetic molecules with ...important physiological functions and pharmaceutically valuable bioactivities. The final steps of ephedrine and pseudoephedrine biosynthesis in members of the plant genus Ephedra involve N-methylation of norephedrine and norpseudoephedrine, respectively. Here, using a plant transcriptome screen, we report the isolation and characterization of an N-methyltransferase (NMT) from Ephedra sinica able to catalyze the formation of (pseudo)ephedrine and other naturally occurring phenylalkylamines, including N-methylcathinone and N-methyl(pseudo)ephedrine. Phenylalkylamine N-methyltransferase (PaNMT) shares substantial amino acid sequence identity with enzymes of the NMT family involved in benzylisoquinoline alkaloid (BIA) metabolism in members of the higher plant order Ranunculales, which includes opium poppy (Papaver somniferum). PaNMT accepted a broad range of substrates with phenylalkylamine, tryptamine, β-carboline, tetrahydroisoquinoline, and BIA structural scaffolds, which is in contrast to the specificity for BIA substrates of NMT enzymes within the Ranunculales. PaNMT transcript levels were highest in young shoots of E. sinica, which corresponded to the location of NMT activity yielding (pseudo)ephedrine, N-methylcathinone, and N-methyl(pseudo)ephedrine, and with in planta accumulation of phenylalkylamines. Co-expression of recombinant genes encoding PaNMT and an ω-transaminase (PP2799) from Pseudomonas putida in Escherichia coli enabled the conversion of exogenous (R)-phenylacetylcarbinol (PAC) and (S)-PAC to ephedrine and pseudoephedrine, respectively. Our work further demonstrates the utility of plant biochemical genomics for the isolation of key enzymes that facilitate microbial engineering for the production of medicinally important metabolites.
•Structure of a new arabinan from Ephedra sinica was reported for the first time.•GC–MS, 2D-NMR and AFM were used for characterization of this polysaccharide.•The repeating unit was characterized by ...a much longer linear (1→5)-α-Araf backbone.
Plant arabinan has important biological activity. In this study, a water-soluble arabinan (Mw∼6.15kDa) isolated from the stems of Ephedra sinica was found to consist of (1→5)-Araƒ, (1→3,5)-Araƒ, T-Araƒ, (1→3)-Araƒ and (1→2,5)-Araƒ residues at proportions of 10:2:3:2:1. A tentative structure was proposed by methylation analysis, nuclear magnetic resonance (NMR) spectroscopy (1H NMR, 13C NMR, DEPT-135, 1H–1H COSY, HSQC, HMBC and ROESY) and literature. The structure proposed includes a branched (1→5)-α-Araf backbone where branching occurs at the O-2 and O-3 positions of the residues with 7.7% and 15.4% of the 1,5-linked α-Araf substituted at the O-2 and O-3 positions. The presence of a branched structure was further observed by atomic force microscopy. This polymer was characterized as having a much longer linear (1→5)-α-Araf backbone as a repeating unit. In particular, the presence of α-Araf→3)-α-Araf-(1→3)-α-Araf-(1→ attached at the O-2 is a new finding. This study may facilitate a deeper understanding of structure–activity relationships of biological polysaccharides from the stems of E. sinica.
Ephedra sinica Stapf is a widely used folk medicine in Asia to treat lung diseases, such as cold, cough and asthma. Many efforts have revealed that some traditional Chinese medicine (TCM) ...prescriptions containing Ephedra sinica could effectively alleviate the symptoms and prevent the fatal deterioration of COVID-19.
The present study aims to discover active compounds in Ephedra sinica disrupting the interaction between angiotensin-converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein receptor-binding domain (SARS-CoV-2 RBD) to inhibit SARS-CoV-2 virus infection.
The ethanol extracts of Ephedra sinica were prepared. Activity guided isolation of constituents was carried out by measuring the inhibitory activity on ACE2-RBD interaction. The structures of active compounds were identified by HPLC-Q-TOF-MS/MS and NMR. To testify the contribution of main components for the inhibitory activity, different samples were prepared by components knock-out strategy. The mechanism of compounds inhibiting protein-protein interaction (PPI) was explored by competition inhibition assays, surface plasmon resonance (SPR) assays and molecular docking. SARS-CoV-2 S protein-pseudoviruses were used to observe the viropexis effect in cells.
Ephedra sinica extracts (ESE) could effectively inhibit the interaction between ACE2 and SARS-CoV-2 RBD (IC50 = 95.01 μg/mL). Three active compounds, 4,6-dihydroxyquinoline-2-carboxylic acid, 4-hydroxyquinoline-2-carboxylic acid and 4-hydroxy-6-methoxyquinoline-2-carboxylic acid were identified to inhibit ACE2-RBD interaction (IC50 = 0.58 μM, 0.07 μM and 0.15 μM respectively). And knock-out the three components could eliminate the inhibitory activity of ESE. Molecular docking calculations indicated that the hydrogen bond was the major intermolecular force. Finally, our results also showed that these compounds could inhibit the infectivity of SARS-CoV-2 S protein-pseudoviruses to 293T-ACE2 (IC50 = 0.44–1.09 μM) and Calu-3 cells.
These findings suggested that quinoline-2-carboxylic acids in Ephedra sinica could be considered as potential therapeutic agents for COVID-19. Further, this study provided some justification for the ethnomedicinal use of Ephedra sinica for COVID-19.
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•Quinoline-2-carboxylic acids in Ephedra sinica disrupt ACE2-RBD interaction.•Quinoline-2-carboxylic acids abolish the infectivity of SARS-CoV-2 pseudoviruses.•Ephedra sinica and quinoline-2-carboxylic acids may be COVID-19 therapeutic agents.
Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. ...These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using L-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.
Ephedra sinica Stapf (Mahuang) and Schisandra chinensis (Turcz.) Baill (Wuweizi) are commonly utilized in traditional Chinese medicine for the treatment of cough and asthma. The synergistic effect of ...Mahuang-Wuweizi herb pair enhances their efficacy in alleviating respiratory symptoms, making them extensively employed in the management of respiratory disorders. Although previous studies have demonstrated the therapeutic potential of Mahuang-Wuweizi in pulmonary fibrosis, the precise mechanism underlying their effectiveness against asthma remains elusive.
The objective of this study is to investigate the mechanism underlying the preventive and therapeutic effects of Mahuang-Wuweizi herb pair on asthma progression, focusing on airway inflammation and airway remodeling.
The active constituents and potential mechanisms of Mahuang-Wuweizi in the management of asthma were elucidated through network pharmacology analysis. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to detect the main components of Mahuang-Wuweizi decoction. A rat model of bronchial asthma was established, and the effects of Mahuang-Wuweizi were investigated using hematoxylin-eosin (HE) staining, immunohistochemistry (IHC) staining, enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), and real-time reverse transcription polymerase chain reaction (RT-qPCR).
The results of network pharmacological prediction showed that Mahuang had 22 active components and Wuweizi had 8 active components, with 225 potential targets. 1159 targets associated with asthma and 115 targets that overlap between drugs and diseases were identified. These include interleukin-6 (IL-6), tumor necrosis factor (TNF), Tumor Protein 53, interleukin-1β (IL-1β), as well as other essential targets. Additionally, there is a potential correlation between asthma and Phosphatidylinositol 3 kinase (PI3K)/Protein Kinase B (AKT) signaling pathway, calcium ion channels, nuclear factor-kappa B (NF-κB) signaling pathway, and other signaling pathways. The animal experiment results demonstrated that treatment with Mahuang and Wuweizi, in comparison to the model group, exhibited improvements in lung tissue pathological injury, reduction in collagen fiber accumulation around the airway and proliferation of airway smooth muscle, decrease in concentration levels of IL-6, TNF-α and IL-1β in lung tissue, as well as alleviation of airway inflammation. Furthermore, Mahuang and Wuweizi suppressed the expression of phospholipase C (PLC), transient receptor potential channel 1 (TRPC1), myosin light chain kinase (MLCK), NF-κB P
protein in ovalbumin (OVA)-sensitized rat lung tissue and downregulated the mRNA expression of PLC, TRPC1, PI3K, AKT, NF-κB P
in asthmatic rats. These findings were consistent with network pharmacological analysis.
The results show that the synergistic interaction between Mahuang and Wuweizi occur, and they can effectively reduce airway remodeling and airway inflammation induced by inhaling OVA in bronchial asthma rats by inhibiting the expression of PLC/TRPC1/PI3K/AKT/NF-κB signaling pathway. Therefore, Mahuang and Wuweizi may be potential drugs to treat asthma.
The terrestrial stems of Ephedra (Ephedra spp.; including Ephedra sinica Stapf and Ephedra przewalskii Stapf) extracts are used in traditional medicines in East Asia. In Japan, the Kampo formula ...containing E. sinica extract is prescribed for the treatment of the common cold, influenza virus infections, and mild symptoms of coronavirus disease 2019 (COVID-19). Although ephedrine alkaloids in E. sinica exert antitussive effects, they may have side effects associated with the sympathetic nervous system. E. przewalskii extract, a drug used in traditional Uyghur and Mongolian medicine, is considered to be free of ephedrine alkaloids and is a promising candidate for the treatment of infectious diseases. However, its use is currently limited because evidence of its antiviral efficacy remains inconclusive.
Aim of the study: We compared the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) effects of E. przewalskii and E. sinica extracts in vitro. Additionally, we examined the differences in their antiviral effects against different SARS-CoV-2 strains.
VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 (Conventional, Delta, and Omicron strains—BA.1, BA.2, BA.4, and BA.5), and lysates prepared from each herbal extract were added. The infectious titer was determined using the 50% tissue culture infectious dose (TCID50) method; in turn, the half-maximal inhibitory concentration (IC50) was calculated for each extract to compare the antiviral efficacy of E. sinica and E. przewalskii extracts. Further, the extracts were compared with remdesivir for their antiviral efficacy against the conventional viral strain. To verify the effect of the inactivation of virus particles, these extracts were added to each SARS-CoV-2 strain, and the infectious titers were determined using the TCID50 method.
The antiviral efficacy (i.e., IC50) of the E. przewalskii extract against each SARS-CoV-2 strain was 2.7–10.8-fold greater than that of the E. sinica extract. The antiviral efficacy of the E. przewalskii extract against conventional viral strains was compared with that of remdesivir, which was 1/27.6 of remdesivir's efficacy. The E. sinica extract showed minimal inactivation of virus particles of each strain, whereas the E. przewalskii extract resulted in substantial viral inactivation.
The E. przewalskii extract showed higher antiviral activity against SARS-CoV-2 than the E. sinica extract. Overall, our study suggests that E. przewalskii extract can be used for the treatment of viral infections, including COVID-19.
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•E. przewalskii is free of ephedrine alkaloids and is safer for infectious diseases.•Anti-SARS-CoV-2 effects of E. przewalskii and E. sinica extracts were compared.•E. przewalskii extract shows 2.7–10.8-fold greater antiviral activity than E. sinica.•E. przewalskii extract shows promise against viral infections, including COVID-19.
In our previous study, we found that the acidic polysaccharides of Ephedra sinica had immunosuppressive effect to treat rheumatoid arthritis and the pure polysaccharide ESP-B4 was the main ...composition of the acidic polysaccharides. At present, the exact molecular mechanism of ESP-B4 on treating arthritis is unclear. We are thus evaluating the properties of ESP-B4 on LPS-induced THP-1 pro-monocytic cells and adjuvant-induced arthritis in Wistar rats via TLR4. In vitro, ESP-B4 decreased the production of cytokines induced by LPS. In addition, ESP-B4 reduced the LPS-stimulated nuclear translocation of p65 subunit of NF-κB. Pretreatment with ESP-B4 significantly down-regulated the phosphorylation of MAPKs induced by LPS. Furthermore, in vivo, after 12 days of disease induced by adjuvant, rats were treated with ESP-B4 for 16 days. ESP-B4 significantly improved all parameters of inflammation. ESP-B4 reduced the release of inflammatory factors and cytokines by inhibiting the TLR4 signaling pathway to treat rheumatoid arthritis.
Traditional Chinese medicines played an important role in the treatment of COVID-19 in 2020.
Ephedra sinica
, one of the major constituent herbs of multi-component herbal formula, has been widely ...used to treat COVID-19 in China. However, its active components are still unclear. The objectives of this study are to screen and evaluate active components from the traditional Chinese medicine
Ephedra sinica
for the treatment of COVID-19. In our study, we established an ACE2/CMC bioaffinity chromatography model, and then developed an ACE2/CMC-HPLC-IT-TOF-MS system for the active compounds screening and identification from
Ephedra sinica
extract. We performed molecular docking and surface plasmon resonance (SPR) assays to assess the binding characteristics (binding mode and
K
D
value). We used CCK-8 staining to assess the toxicity of screened compounds, and also used SARS-CoV-2 pseudovirus to observe the viropexis effect of screened compounds in ACE2
h
cells. In this current work, one fraction was fished out, separated and identified as ephedrine (EP), pseudoephedrine (PEP), and methylephedrine (MEP). Binding assays showed that the three compounds could bind with ACE2 in a special way to some amino acid residues, similar to the way SARS-CoV-2 bound with ACE2. Additionally, the three compounds, especially EP, can inhibit the entrance of SARS-CoV-2 spike pseudovirus into ACE2
h
cells because they can reduce the entrance ratio of pseudovirus in the pseudovirus model. Overall, the ACE2/CMC-HPLC-IT-TOF-MS system was established and verified to be suitable for ACE2-targeted bioactive compound screening. EP, PEP, and MEP with ACE2-binding features were screened out from
Ephedra sinica
, and acted as blockers inhibiting SARS-CoV-2 spike pseudovirus entering ACE2
h
cells.
Graphical abstract
Ephedra sinica (Ma Huang) has been used in traditional Chinese medicine for more than 5000 years for treatment of various conditions, including modern-day obesity. Ephedra has been used as a ...supplement for weight loss and its effects have been reported. The current study investigated the influence of ephedra on the composition of gut microbiota, and its correlation with weight loss.
Clinical data of subjects were measured at pre- and post-intake of ephedra (4g of water extract, roughly equivalent of 24g of crude herb), and analysis of the alteration of gut microbiota was performed simultaneously using 16S rRNA gene based pyrosequencing.
Body weight (BW), body mass index (BMI), and body fat percentage of subjects were reduced after intake (p<0.05). In correlation analysis, Subdoligranulum, Oscillibacter, and Akkermansia showed an association with changes of BW and BMI (p<0.05). However, the alteration of gut microbiota varied by indigenous microbiota of each subject, and the dissimilarity between microbiota of subjects at pre- and post-intake were different.
The influences of gut microbiota are unique according to indigenous microbiota and differences in individual sensitivity to ephedra. Alteration of gut microbiota by ephedra intake showed correlation with loss of BW and BMI.
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1-Phenylpropane-1,2-dione and (
S)-cathinone are biosynthetic precursors of ephedrine alkaloids in
Ephedra sinica. (
S)-Cathinone reductase activities determine the stereochemical branching of the ...pathway.
Ephedra sinica Stapf (Ephedraceae) is a widely used Chinese medicinal plant (Chinese name:
Ma Huang). The main active constituents of
E. sinica are the unique and taxonomically restricted adrenergic agonists phenylpropylamino alkaloids, also known as ephedrine alkaloids: (1
R,2
S)-norephedrine (1
S,2
S)-norpseudoephedrine, (1
R,2
S)-ephedrine, (1
S,2
S)-pseudoephedrine, (1
R,2
S)-
N-methylephedrine and (1
S,2
S)-
N-methylpseudoephedrine. GC–MS analysis of freshly picked young
E. sinica stems enabled the detection of 1-phenylpropane-1,2-dione and (
S)-cathinone, the first two putative committed biosynthetic precursors to the ephedrine alkaloids. These metabolites are only present in young
E. sinica stems and not in mature stems or roots. The related
Ephedra foemina and
Ephedra foliata also lack ephedrine alkaloids and their metabolic precursors in their aerial parts. A marked diversity in the ephedrine alkaloids content and stereochemical composition in 16 different
E. sinica accessions growing under the same environmental conditions was revealed, indicating genetic control of these traits. The accessions can be classified into two groups according to the stereochemistry of the products accumulated: a group that displayed only 1
R stereoisomers, and a group that displayed both 1
S and 1
R stereoisomers. (
S)-cathinone reductase activities were detected in
E. sinica stems capable of reducing (
S)-cathinone to (1
R,2
S)-norephedrine and (1
S,2
S)-norpseudoephedrine in the presence of NADH. The proportion of the diastereoisomers formed varied according to the accession tested. A (1
R,2
S)-norephedrine
N-methyltransferase capable of converting (1
R,2
S)-norephedrine to (1
R,2
S)-ephedrine in the presence of
S-adenosylmethionine (SAM) was also detected in
E. sinica stems. Our studies further support the notion that 1-phenylpropane-1,2-dione and (
S)-cathinone are biosynthetic precursors of the ephedrine alkaloids in
E. sinica stems and that the activity of (
S)-cathinone reductases directs and determines the stereochemical branching of the pathway. Further methylations are likely due to
N-methyltransferase activities.
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