Comprehensive chemical analysis of polyphenols in the ethyl acetate extract from the roots of Ephedra sinica Stapf and evaluation of its therapeutic effects on SU5416/hypoxia-induced pulmonary arterial hypertension rats

Abstract

Pulmonary hypertension (PH) is a deadly disease with limited treatment options and poor long-term survival, necessitating the discovery of novel therapeutics. Our previous study has revealed that dimeric proanthocyanidins (PACs) mainly existed in the ethyl acetate extract from the roots of Ephedra sinica Stapf (ERE), however, its therapeutic effects on SU5416/hypoxia-induced pulmonary hypertension (PH) rats remain elusive. In this study, column chromatography combined with UPLC-LTQ-Orbitrap-HRMS analysis was performed to comprehensively characterize polyphenols in ERE. The therapeutic effects of ERE were investigated using the SU5416/hypoxia rat model, in which the rats were injected with SU5416 (20 mg/kg), followed by a three-week hypoxia exposure (10% O₂). Hemodynamic indicators determined by right heart catheterization, pulmonary arterial morphological changes assessed by histopathological analysis, cardiac function and pulmonary hemodynamics using echocardiography, as well as oxidative stress markers measured by corresponding kits were used to test the therapeutic effects of ERE. Moreover, 16S rRNA sequencing combined with untargeted metabolomics was employed to capture changes in gut microbiota and serum metabolites after ERE treatment. Comprehensive chemical analysis of polyphenols in ERE revealed various levels of proanthocyanidin monomers, dimers and trimers, especially A-type dimers. In vivo experiments showed that ERE decreased pulmonary arterial pressure, right ventricular hypertrophy, right ventricular free wall (RVFW) thickness and oxidative stress levels, increased pulmonary acceleration time (PAT) and alleviated pulmonary vascular remodeling in rats exposed to SU5416/hypoxia treatment. Meanwhile, ERE improved gut microbial dysbiosis and the disturbed glycerophospholipid metabolism. Collectively, this study presents the first report on the efficacy of A-type PACs from Ephedra sinica for the treatment of PH through regulating gut microbiota and host metabolism.

Keywords

Polyphenols / Root of Ephedra sinica Stapf / SU5416 / Pulmonary arterial hypertension / Gut microbiota

References

[1] Alotaibi BS, Ijaz M, Buabeid M, Kharaba ZJ, Yaseen HS, Murtaza G. Therapeutic effects and safe uses of plant-derived polyphenolic compounds in cardiovascular diseases: a review. Drug des Devel Ther, 2021, 15: 4713-4732

[2] Boucherat O, Agrawal V, Lawrie A, Bonnet S. The latest in animal models of pulmonary hypertension and right ventricular failure. Circ Res, 2022, 130(9): 1466-1486

[3] Chang MC, Lee JJ, Chen YJ, Lin SI, Lin LD, Liou EJW, Huang WL, Chan CP, Huang CC, Jeng JH. Lysophosphatidylcholine induces cytotoxicity/apoptosis and IL-8 production of human endothelial cells: related mechanisms. Oncotarget, 2017, 8(63): 106177-106189

[4] Chen R, Zhong W, Shao C, Liu PJ, Wang CP, Wang ZQ, Jiang MP, Lu Y, Yan JC. Docosahexaenoic acid inhibits monocrotaline-induced pulmonary hypertension via attenuating endoplasmic reticulum stress and inflammation. Am J Physiol Lung Cell Mol Physiol, 2017, 314(2): L243-L255

[5] Chen YQ, Kuang MD, Liu SY, Hou C, Duan X, Yang K, He WJ, Liao J, Zheng QY, Zou GF, Chen HX, Yan H, Chen JY, Li Y, Zhou Y, Luo XY, Jiang Q, Tang HY, Lu WJ, Wang J. A novel rat model of pulmonary hypertension induced by mono treatment with SU5416. Hypertens Res, 2020, 43(8): 754-764

[6] Fan WD, Zong HR, Zhao T, Deng JJ, Yang HX. Bioactivities and mechanisms of dietary proanthocyanidins on blood pressure lowering: a critical review of in vivo and clinical studies. Crit Rev Food Sci Nutr, 2022, 64(11): 3522-3538

[7] Feldman F, Koudoufio M, Sané AT, Marcil V, Sauvé MF, Butcher J, Patey N, Martel C, Spahis S, Duan H, Figeys D, Desjardins Y, Stintzi A, Levy E. Therapeutic potential of cranberry proanthocyanidins in addressing the pathophysiology of metabolic syndrome: a scrutiny of select mechanisms of action. Antioxidants, 2025, 14(3): 268

[8] Fiorentù G, Bernardinello N, Giulianelli G, Cocconcelli E, Balestro E, Spagnolo P. Pulmonary hypertension associated with interstitial lung disease (PH-ILD): back to the future. Adv Ther, 2025, 42(41627-1641

[9] Hadi DD, Marsool MDM, Marsool ADM, Vora N, Al-Badri SG, Al-Fatlawi NHK, Abbas Al Wssawi AF, Al-Ibraheem AMT, Hamza KA, Prajjwal P, Mateen MA, Amir O. Idiopathic pulmonary fibrosis: addressing the current and future therapeutic advances along with the role of Sotatercept in the management of pulmonary hypertension. Immun Inflamm Dis, 2023, 11(11 e1079

[10] Harbaum L, Rhodes CJ, Otero-Núñez P, Wharton J, Wilkins MR. The application of ‘omics’ to pulmonary arterial hypertension. Br J Pharmacol, 2020, 178(1): 108-120

[11] Hong J, Arneson D, Umar S, Ruffenach G, Cunningham CM, Ahn IS, Diamante G, Bhetraratana M, Park JF, Said E, Huynh C, Le T, Medzikovic L, Humbert M, Soubrier F, Montani D, Girerd B, Trégouët D-A, Channick R, Saggar R, Eghbali M, Yang X. Single-cell study of two rat models of pulmonary arterial hypertension reveals connections to human pathobiology and drug repositioning. Am J Respir Crit Care Med, 2021, 203(8): 1006-1022

[12] Hu WP, Xie L, Hao SY, Wu QH, Xiang GL, Li SQ, Liu D. Protective effects of progesterone on pulmonary artery smooth muscle cells stimulated with interleukin 6 via blocking the shuttling and transcriptional function of STAT3. Int Immunopharmacol, 2022, 102 108379

[13] Jasiewicz M, Moniuszko M, Pawlak D, Knapp M, Rusak M, Kazimierczyk R, Musial WJ, Dabrowska M, Kaminski KA. Activity of the kynurenine pathway and its interplay with immunity in patients with pulmonary arterial hypertension. Heart, 2016, 102(3): 230-237

[14] Jin HF, Liu MC, Zhang X, Pan JJ, Han JZ, Wang YD, Lei HX, Ding YC, Yuan YH. Grape seed procyanidin extract attenuates hypoxic pulmonary hypertension by inhibiting oxidative stress and pulmonary arterial smooth muscle cells proliferation. J Nutr Biochem, 2016, 36: 81-88

[15] Karoor V, Strassheim D, Sullivan T, Verin A, Umapathy NS, Dempsey EC, Frank DN, Stenmark KR, Gerasimovskaya E. The short-chain fatty acid butyrate attenuates pulmonary vascular remodeling and inflammation in hypoxia-induced pulmonary hypertension. Int J Mol Sci, 2021, 22(18 9916

[16] Kim S, Rigatto K, Gazzana MB, Knorst MM, Richards EM, Pepine CJ, Raizada MK. Altered gut microbiome profile in patients with pulmonary arterial hypertension. Hypertension, 2020, 75(4): 1063-1071

[17] Lankin VZ, Tikhaze AK, Melkumyants AM. Malondialdehyde as an important key factor of molecular mechanisms of vascular wall damage under heart diseases development. Int J Mol Sci, 2022, 24(1 128

[18] Li BL, Tian SC, Liu X, He CX, Ding ZQ, Shan YJ. Sulforaphane protected the injury of human vascular endothelial cell induced by LPC through up-regulating endogenous antioxidants and phase II enzymes. Food Funct, 2015, 6(6): 1984-1991

[19] Li XY, Wang LQ, Fang P, Sun Y, Jiang XH, Wang H, Yang XF. Lysophospholipids induce innate immune transdifferentiation of endothelial cells, resulting in prolonged endothelial activation. J Biol Chem, 2018, 293(28): 11033-11045

[20] Li YY, Deng XX, Zhou HM, Zheng X, Zhang GC, Xiong QF. Bile acid predicts congenital portosystemic venous shunt in patients with pulmonary arterial hypertension. Eur J Med Res, 2023, 28(1): 74

[21] Liu JT, Hu SL, Zhu BQ, Shao SM, Yuan LB. Grape seed procyanidin suppresses inflammation in cigarette smoke-exposed pulmonary arterial hypertension rats by the PPAR-γ/COX-2 pathway. Nutr Metab Cardiovasc Dis, 2020, 30(2347-354

[22] Lv MY, Chen JQ, Gao YQ, Sun JB, Zhang QQ, Zhang MH, Xu FG, Zhang ZJ. Metabolomics based on liquid chromatography with mass spectrometry reveals the chemical difference in the stems and roots derived from Ephedra sinica. J Sep Sci, 2015, 38(19): 3331-3336

[23] Lv MY, Wang Y, Wan XY, Han B, Yu W, Liang QL, Xiang J, Wang Z, Liu YQ, Xu FG. Rapid screening of proanthocyanidins from the roots of Ephedra sinica Stapf and its preventative effects on dextran-sulfate-sodium-induced ulcerative colitis. Metabolites, 2022, 12(10): 957

[24] Margalef M, Guerrero L, Pons Z, Bravo FI, Arola L, Muguerza B, Arola-Arnal A. A dose-response study of the bioavailability of grape seed proanthocyanidin in rat and lipid-lowering effects of generated metabolites in HepG2 cells. Food Res Int, 2014, 64: 500-507

[25] Mocumbi A, Humbert M, Saxena A, Jing Z-C, Sliwa K, Thienemann F, Archer SL, Stewart S. Pulmonary hypertension. Nat Rev Dis Primers, 2024, 10(1 1

[26] Naeije R, Richter MJ, Rubin LJ. The physiological basis of pulmonary arterial hypertension. Eur Respir J, 2022, 59(62102334

[27] Nagaraj C, Tang B, Nagy BM, Papp R, Jain PP, Marsh LM, Meredith AL, Ghanim B, Klepetko W, Kwapiszewska G, Weir EK, Olschewski H, Olschewski A. Docosahexaenoic acid causes rapid pulmonary arterial relaxationviaKCa channel-mediated hyperpolarisation in pulmonary hypertension. Eur Respir J, 2016, 48(41127-1136

[28] Nathan SD, Barbera JA, Gaine SP, Harari S, Martinez FJ, Olschewski H, Olsson KM, Peacock AJ, Pepke-Zaba J, Provencher S, Weissmann N, Seeger W. Pulmonary hypertension in chronic lung disease and hypoxia. Eur Respir J, 2019, 53(1): 1801914

[29] Ou KQ, Gu LW. Absorption and metabolism of proanthocyanidins. J Funct Foods, 2014, 7: 43-53

[30] Park J, Kim M, Kang SG, Jannasch AH, Cooper B, Patterson J, Kim CH. Short-chain fatty acids induce both effector and regulatory T cells by suppression of histone deacetylases and regulation of the mTOR–S6K pathway. Mucosal Immunol, 2015, 8(180-93

[31] Pi HY, Xia L, Ralph DD, Rayner SG, Shojaie A, Leary PJ, Gharib SA. Metabolomic signatures associated with pulmonary arterial hypertension outcomes. Circ Res, 2023, 132(3): 254-266

[32] Poch D, Mandel J. Pulmonary hypertension. Ann Intern Med, 2021, 174(4): ITC49-ITC64

[33] Pokharel MD, Marciano DP, Fu P, Franco MC, Unwalla H, Tieu K, Fineman JR, Wang T, Black SM. Metabolic reprogramming, oxidative stress, and pulmonary hypertension. Redox Biol, 2023, 64 102797

[34] Poyatos P, Gratacós M, Samuel K, Orriols R, Tura-Ceide O. Oxidative stress and antioxidant therapy in pulmonary hypertension. Antioxidants, 2023, 12(5): 1006

[35] Rudrapal M, Rakshit G, Singh RP, Garse S, Khan J, Chakraborty S. Dietary polyphenols: review on chemistry/sources, bioavailability/metabolism, antioxidant effects, and their role in disease management. Antioxidants, 2024, 13(4429

[36] Sintara M, Wang YF, Li L, Liu HY, Cunningham GD, Prior RR, Chen P, Chang T, Wu XL. Quantification of cranberry proanthocyanidins by normal-phase high-performance liquid chromatography using relative response factors. Phytochem Anal, 2020, 31(6874-883

[37] Sul OJ, Choi HW, Ra SW. Grape seed proanthocyanidin extract modulates cigarette smoke extract-induced epithelial cell apoptosis by inhibiting oxidative stress in chronic obstructive pulmonary disease. J Funct Foods, 2024, 112 105907

[38] Suswał K, Tomaszewski M, Romaniuk A, Świechowska-Starek P, Zygmunt W, Styczeń A, Romaniuk-Suswał M. Gut-lung axis in focus: deciphering the impact of gut microbiota on pulmonary arterial hypertension. J Pers Med, 2023, 14(1): 8

[39] Teunis CJ, Stroes ESG, Boekholdt SM, Wareham NJ, Murphy AJ, Nieuwdorp M, Hazen SL, Hanssen NMJ. Tryptophan metabolites and incident cardiovascular disease: the EPIC-Norfolk prospective population study. Atherosclerosis, 2023, 387 117344

[40] Wang YT, Liu HZ, McKenzie G, Witting PK, Stasch JP, Hahn M, Changsirivathanathamrong D, Wu BJ, Ball HJ, Thomas SR, Kapoor V, Celermajer DS, Mellor AL, Keaney JFJr, Hunt NH, Stocker R. Kynurenine is an endothelium-derived relaxing factor produced during inflammation. Nat Med, 2010, 16(3279-285

[41] Wang AP, Yang F, Tian Y, Su JH, Gu Q, Chen W, Gong SX, Ma XF, Qin XP, Jiang ZS. Pulmonary artery smooth muscle cell senescence promotes the proliferation of PASMCs by paracrine IL-6 in hypoxia-induced pulmonary hypertension. Front Physiol, 2021, 12 656139

[42] Wang H, Wang G, Banerjee N, Liang Y, Du X, Boor PJ, Hoffman KL, Khan MF. Aberrant gut microbiome contributes to intestinal oxidative stress, barrier dysfunction, inflammation and systemic autoimmune responses in MRL/lpr mice. Front Immunol, 2021, 12 651191

[43] Wang P, Liu XL, Jiang ZZ, Long Y, Gao CL, Huang W, Tan XZ, Ma XM, Xu Y. Effect of proanthocyanidins on blood lipids: a systematic review and meta-analysis. Phytother Res, 2024, 38(5): 2154-2164

[44] Wang XY, Wang YC, Yuan TY, Wang HJ, Zeng ZM, Tian LY, Cui LD, Guo J, Chen YC. Network pharmacology provides new insights into the mechanism of traditional Chinese medicine and natural products used to treat pulmonary hypertension. Phytomedicine, 2024, 135 156062

[45] Wang Y, Qin XG, Shuai JH, Wan XY, Yu DN, Ling L, Lu QW, Lv MY. Pristimerin alleviates DSS-induced colitis in mice by modulating intestinal barrier function, gut microbiota balance and host metabolism. Inflammation, 2025, 48(4): 2166-2181

[46] Wood JC. Pulmonary hypertension in thalassemia: a call to action. Blood, 2022, 139(13): 1937-1938

[47] Xu D, Hu YH, Gou X, Li FY, Yang XYC, Li YM, Chen F. Oxidative stress and antioxidative therapy in pulmonary arterial hypertension. Molecules, 2022, 27(12): 3724

[48] Xu C, Wang B, Li M, Dong ZF, Chen N, Duan JY, Zhou Y, Jin MF, Chen R, Yuan W. FUNDC1/USP15/Drp1 ameliorated TNF-α-induced pulmonary artery endothelial cell proliferation by regulating mitochondrial dynamics. Cell Signal, 2024, 113 110939

[49] Yan D, Sun Y, Zhou XY, Si WH, Liu JY, Li M, Wu MN. Regulatory effect of gut microbes on blood pressure. Anim Model Exp Med, 2022, 5(6513-531

[50] Yang YF, Lu Y, Wu GF, Xiao MY, Li LZ, Wu HZ. Experimental study on the hypotensive effect of Ephedra root extracts on the spontaneously hypertensive rats. Chin Hosp Pharm J, 2010, 30(17): 1434-1436

[51] Yang J, Liu JX, Gu HY, Song WL, Zhang H, Wang J, Yang PR. Gut microbiota, metabolites, and pulmonary hypertension: mutual regulation and potential therapies. Microbiol Res, 2025, 299 128245

[52] Zhang JJ, Mao M, Shao MM, Wang MC. Therapeutic potential of natural flavonoids in pulmonary arterial hypertension: a review. Phytomedicine, 2024, 128 155535

Funding

the National Natural Science Foundation of China for Youths(81903906)

the Natural Science Foundation of the Jiangsu Higher Education Institutions of China(23KJB360018)

the Administration of Traditional Chinese Medicine of Jiangsu Province(MS2022094)

the Qinglan Project of Yangzhou University(2023)

the central government guides the special project of local scince and technology development(ZYYD2023B11)

RIGHTS & PERMISSIONS

The Author(s)