Nutr Res Pract.  2023 Dec;17(6):1099-1112. 10.4162/nrp.2023.17.6.1099.

Purple perilla frutescens extracts containing α-asarone inhibit inflammatory atheroma formation and promote hepatic HDL cholesterol uptake in dyslipidemic apoE-deficient mice

• Park SH
• Sim YE
• Kang MK
• Kim DY
• Kang IJ
• Lim SS
• Kang YH

• KMID: 2548867
• DOI: http://doi.org/10.4162/nrp.2023.17.6.1099

Abstract

BACKGROUND/OBJECTIVES
Dyslipidemia causes metabolic disorders such as atherosclerosis and fatty liver syndrome due to abnormally high blood lipids. Purple perilla frutescens extract (PPE) possesses various bioactive compounds such as α-asarone, chlorogenic acid and rosmarinic acid. This study examined whether PPE and α-asarone improved dyslipidemia-associated inflammation and inhibited atheroma formation in apolipoprotein E (apoE)-deficient mice, an experimental animal model of atherosclerosis.
MATERIALS/METHODS
ApoE-deficient mice were fed on high cholesterol-diet (Paigen’s diet) and orally administrated with 10–20 mg/kg PPE and α-asarone for 10 wk.
RESULTS
The Paigen’s diet reduced body weight gain in apoE-deficient mice, which was not restored by PPE or α-asarone. PPE or α-asarone improved the plasma lipid profiles in Paigen’s diet-fed apoE-deficient mice, and despite a small increase in high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein (LDL)-cholesterol, and very LDL were significantly reduced. Paigen’s diet-induced systemic inflammation was reduced in PPE or α-asarone-treated apoE-deficient mice. Supplying PPE or α-asarone to mice lacking apoE suppressed aorta atherogenesis induced by atherogenic diet. PPE or α-asarone diminished aorta accumulation of CD68- and/or F4/80-positive macrophages induced by atherogenic diet in apoE-deficient mice. Treatment of apoE-deficient mice with PPE and α-asarone resulted in a significant decrease in plasma cholesteryl ester transfer protein level and an increase in lecithin:cholesterol acyltransferase reduced by supply of Paigen’s diet. Supplementation of PPE and α-asarone enhanced the transcription of hepatic apoA1 and SR-B1 reduced by Paigen’s diet in apoE-deficient mice.
CONCLUSIONS
α-Asarone in PPE inhibited inflammation-associated atheroma formation and promoted hepatic HDL-C trafficking in dyslipidemic mice.

Keyword

Alpha-asarone; apolipoproteins E; atheroma; high density lipoprotein; purple perilla frutescens extract

Figure

• 

  Fig. 1 Chemical structure of α-asarone (A), animal experimental design and grouping (B), and change of BWs during feeding (C). Wild type and homozygous apoE-deficient C57BL/6N mice (5 wk of age) were fed either chow diet or atherogenic Paigen’s diet. Atherogenic Paigen’s diet-fed apoE-deficient mice were divided into 5 subgroups. These apoE-deficient mice received 10–20 mg/kg PPE or 10–20 mg/kg α-asarone via gavage daily for 10 wk. The animal BW was measured at the beginning of the experiment and at 1 wk intervals for 10 wk. Values in curved linear graphs were expressed as mean ± SEM (n = 8–10).apoE, apolipoprotein E; PPE, purple perilla frutescens extract; BW, body weight.

• 

  Fig. 2 Effects of PPE and α-asarone on circulatory inflammation (A, B) and atheroma formation (C) in apoE-deficient mice fed atherogenic Paigen’s diet. Wild type and homozygous apoE-knockout C57BL/6N mice (5 wk of age) were fed either chow diet or atherogenic Paigen’s diet. Atherogenic Paigen’s diet-fed apoE-deficient mice were divided into 5 subgroups. These apoE-deficient mice received 10–20 mg/kg PPE or 10–20 mg/kg α-asarone via gavage daily for 10 wk. For the measurements of systemic inflammation, plasma samples were analyzed with commercial enzyme-linked immunosorbent assay kits of MCP-1 (A) and IL-1β (B). Respective values (mean ± SEM, n = 7) in bar graphs not sharing a small alphabetical letter are different at P < 0.05. For the observation of atheroma in the aorta, aorta cut-tissues were stained with oil red O, and counterstained with hematoxylin (C). Scale bar represents 100 μm. The stained tissues were observed with microscopes (n = 4).MCP-1, monocyte chemoattractant protein-1; IL-1β, interleukin-1β; apoE, apolipoprotein E; PPE, purple perilla frutescens extract; BW, body weight.

• 

  Fig. 3 Inhibition of inflammatory cell trafficking by PPE and α-asarone. Wild type and homozygous apoE-knockout C57BL/6N mice (5 wk of age) were fed either chow diet or atherogenic Paigen’s diet. Atherogenic Paigen’s diet-fed apoE-deficient mice were divided into 5 subgroups. These apoE-deficient mice received 10–20 mg/kg PPE or α-asarone via gavage daily for 10 wk. For the observation of macrophage infiltration into the aorta wall, aorta cut-tissues were immunohistochemically stained with an antibody against CD68 and F4/80. For the visualization of CD68 and F4/80, the tissue sections were stained with substrates of DAB (brown precipitate) and NovaRED (red precipitate). Scale bars represent 100 μm. Each photograph is representative of at least 4 animals. The stained tissues were observed with fluorescent microscopes.PPE, purple perilla frutescens extract; apoE, apolipoprotein E; DAB, 3,3′-diaminobenzidine

• 

  Fig. 4 Regulatory effects of PPE and α-asarone on the levels of lipid transfer proteins in atherogenesis. Wild type and homozygous apoE-knockout C57BL/6N mice (5 wk of age) were fed either chow diet or atherogenic Paigen’s diet. Atherogenic Paigen’s diet-fed apoE-deficient mice were divided into 5 subgroups. These apoE-deficient mice received 10–20 mg/kg PPE or 10–20 mg/kg α-asarone via gavage daily for 10 wk. Plasma levels of CETP, PLTP, and LCAT were analyzed by commercial enzyme-linked immunosorbent assay kits of CETP (A), PLTP (B), and LCAT (C). Respective values (mean ± SEM, n = 7) in bar graphs not sharing a small alphabetical letter are different at P < 0.05.CETP, cholesterol ester transfer protein; PPE, purple perilla frutescens extract; apoE, apolipoprotein E; PLTP, phospholipid transfer protein; LCAT, lecithin:cholesterol acyltransferase.

• 

  Fig. 5 Upregulation of hepatic transcription of apoA1 and SR-B1 by PPE and α-asarone. Wild type and homozygous apoE-knockout C57BL/6N mice (5 wk of age) were fed either chow diet or atherogenic Paigen’s diet. Atherogenic Paigen’s diet-fed apoE-deficient mice were divided into 5 subgroups. These apoE-deficient mice received 10–20 mg/kg PPE or 10–20 mg/kg α-asarone via gavage daily for 10 wk. For the measurements of the mRNA levels of hepatic apoA1 and SR-B1, the reverse transcription polymerase chain reaction analysis was conducted with mouse primers of apoA1 and SR-B1. β-Actin and GAPDH were used as internal controls. Values in bar graphs (n = 4 separate experiments) not sharing the same lower case alphabet letter indicate a significant difference at P < 0.05.Apo, apolipoprotein; SR-B1, scavenger receptor B1; PPE, purple perilla frutescens extract; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 6 Schematic diagram showing the effects of PPE and α-asarone on atherosclerosis in apoE-deficient mice. PPE and α-asarone can alleviate atherosclerosis by reducing inflammation and improving liver HDL clearance in apoE-deficient mice vulnerable to atherosclerosis.LDL, low-density lipoprotein; IDL, intermediate-density lipoprotein; VLDL, very low-density lipoprotein; HDL, high-density lipoprotein; LDLR, low-density lipoprotein receptor; CE, cholesterol ester; TG, triglycerides; PPE, purple perilla frutescens extract; apoE, apolipoprotein E.

Reference

1. Musunuru K. Atherogenic dyslipidemia: cardiovascular risk and dietary intervention. Lipids. 2010; 45:907–914. PMID: 20524075.

2. Hedayatnia M, Asadi Z, Zare-Feyzabadi R, Yaghooti-Khorasani M, Ghazizadeh H, Ghaffarian-Zirak R, Nosrati-Tirkani A, Mohammadi-Bajgiran M, Rohban M, Sadabadi F, et al. Dyslipidemia and cardiovascular disease risk among the MASHAD study population. Lipids Health Dis. 2020; 19:42. PMID: 32178672.

3. Chiu S, Williams PT, Krauss RM. Effects of a very high saturated fat diet on LDL particles in adults with atherogenic dyslipidemia: a randomized controlled trial. PLoS One. 2017; 12:e0170664. PMID: 28166253.

4. Pedro-Botet J, Ascaso JF, Blasco M, Brea Á, Díaz Á, Hernández-Mijares A, Pintó X, Millán J. Grupo de trabajo sobre Dislipidemia Aterogénica, Sociedad Española de Arteriosclerosis. Triglycerides, HDL cholesterol and atherogenic dyslipidaemia in the 2019 European guidelines for the management of dyslipidaemias. Clin Investig Arterioscler. 2020; 32:209–218.

5. Kones R, Rumana U. Current treatment of dyslipidemia: evolving roles of non-statin and newer drugs. Drugs. 2015; 75:1201–1228. PMID: 26169307.

6. Sahebkar A, Watts GF. New LDL-cholesterol lowering therapies: pharmacology, clinical trials, and relevance to acute coronary syndromes. Clin Ther. 2013; 35:1082–1098. PMID: 23932550.

7. Mahley RW, Rall SC Jr. Apolipoprotein E: far more than a lipid transport protein. Annu Rev Genomics Hum Genet. 2000; 1:507–537. PMID: 11701639.

8. Phillips MC. Apolipoprotein E isoforms and lipoprotein metabolism. IUBMB Life. 2014; 66:616–623. PMID: 25328986.

9. Getz GS, Reardon CA. Apoprotein E as a lipid transport and signaling protein in the blood, liver, and artery wall. J Lipid Res. 2009; 50(Suppl):S156–S161. PMID: 19018038.

10. Moghadasian MH, McManus BM, Nguyen LB, Shefer S, Nadji M, Godin DV, Green TJ, Hill J, Yang Y, Scudamore CH, et al. Pathophysiology of apolipoprotein E deficiency in mice: relevance to apo E-related disorders in humans. FASEB J. 2001; 15:2623–2630. PMID: 11726538.

11. Rosenson RS, Brewer HB Jr, Davidson WS, Fayad ZA, Fuster V, Goldstein J, Hellerstein M, Jiang XC, Phillips MC, Rader DJ, et al. Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport. Circulation. 2012; 125:1905–1919. PMID: 22508840.

12. Rochlani Y, Pothineni NV, Kovelamudi S, Mehta JL. Metabolic syndrome: pathophysiology, management, and modulation by natural compounds. Ther Adv Cardiovasc Dis. 2017; 11:215–225. PMID: 28639538.

13. Ji X, Shi S, Liu B, Shan M, Tang D, Zhang W, Zhang Y, Zhang L, Zhang H, Lu C, et al. Bioactive compounds from herbal medicines to manage dyslipidemia. Biomed Pharmacother. 2019; 118:109338. PMID: 31545238.

14. Hu Y, Chen X, Hu M, Zhang D, Yuan S, Li P, Feng L. Medicinal and edible plants in the treatment of dyslipidemia: advances and prospects. Chin Med. 2022; 17:113. PMID: 36175900.

15. Yu H, Qiu JF, Ma LJ, Hu YJ, Li P, Wan JB. Phytochemical and phytopharmacological review of Perilla frutescens L. (Labiatae), a traditional edible-medicinal herb in China. Food Chem Toxicol. 2017; 108:375–391. PMID: 27890564.

16. Liu S, Jin X, Shang Y, Wang L, Du K, Chen S, Li J, He J, Fang S, Chang Y. A comprehensive review of the botany, ethnopharmacology, phytochemistry, pharmacology, toxicity and quality control of Perillae Fructus. J Ethnopharmacol. 2023; 304:116022. PMID: 36481246.

17. Park SH, Paek JH, Shin D, Lee JY, Lim SS, Kang YH. Purple perilla extracts with α-asarone enhance cholesterol efflux from oxidized LDL-exposed macrophages. Int J Mol Med. 2015; 35:957–965. PMID: 25673178.

18. Park SH, Shin D, Lim SS, Lee JY, Kang YH. Purple perilla extracts allay ER stress in lipid-laden macrophages. PLoS One. 2014; 9:e110581. PMID: 25333946.

19. Paek JH, Shin KH, Kang YH, Lee JY, Lim SS. Rapid identification of aldose reductase inhibitory compounds from Perilla frutescens. BioMed Res Int. 2013; 2013:679463. PMID: 24308003.

20. Wang Z, Kwon SH, Hwang SH, Kang YH, Lee JY, Lim SS. Competitive binding experiments can reduce the false positive results of affinity-based ultrafiltration-HPLC: a case study for identification of potent xanthine oxidase inhibitors from Perilla frutescens extract. J Chromatogr B Analyt Technol Biomed Life Sci. 2017; 1048:30–37.

21. Rodríguez-Páez L, Juárez-Sanchez M, Antúnez-Solís J, Baeza I, Wong C. Alpha-asarone inhibits HMG-CoA reductase, lowers serum LDL-cholesterol levels and reduces biliary CSI in hypercholesterolemic rats. Phytomedicine. 2003; 10:397–404. PMID: 12834005.

22. Hwang SH, Kwon SH, Kang YH, Lee JY, Lim SS. Rapid high performance liquid chromatography determination and optimization of extraction parameters of the α-asarone isolated from Perilla frutescens L. Molecules. 2017; 22:270. PMID: 28208640.

23. Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994; 14:133–140. PMID: 8274468.

24. Lloyd CM, Phillips ARJ, Cooper GJS, Dunbar PR. Three-colour fluorescence immunohistochemistry reveals the diversity of cells staining for macrophage markers in murine spleen and liver. J Immunol Methods. 2008; 334:70–81. PMID: 18367204.

25. Lo Sasso G, Schlage WK, Boué S, Veljkovic E, Peitsch MC, Hoeng J. The Apoe(-/-) mouse model: a suitable model to study cardiovascular and respiratory diseases in the context of cigarette smoke exposure and harm reduction. J Transl Med. 2016; 14:146. PMID: 27207171.

26. De Giorgi M, Li A, Hurley A, Barzi M, Doerfler AM, Cherayil NA, Smith HE, Brown JD, Lin CY, Bissig KD, et al. Targeting the Apoa1 locus for liver-directed gene therapy. Mol Ther Methods Clin Dev. 2021; 21:656–669. PMID: 34141821.

27. Ganesan LP, Mates JM, Cheplowitz AM, Avila CL, Zimmerer JM, Yao Z, Maiseyeu A, Rajaram MV, Robinson JM, Anderson CL. Scavenger receptor B1, the HDL receptor, is expressed abundantly in liver sinusoidal endothelial cells. Sci Rep. 2016; 6:20646. PMID: 26865459.

28. Leiva A, Verdejo H, Benítez ML, Martínez A, Busso D, Rigotti A. Mechanisms regulating hepatic SR-BI expression and their impact on HDL metabolism. Atherosclerosis. 2011; 217:299–307. PMID: 21741044.

29. Mahley RW. Apolipoprotein E: from cardiovascular disease to neurodegenerative disorders. J Mol Med (Berl). 2016; 94:739–746. PMID: 27277824.

30. Chowaniec Z, Skoczyńska A. Plasma lipid transfer proteins: the role of PLTP and CETP in atherogenesis. Adv Clin Exp Med. 2018; 27:429–436. PMID: 29558025.

31. Manthei KA, Patra D, Wilson CJ, Fawaz MV, Piersimoni L, Shenkar JC, Yuan W, Andrews PC, Engen JR, Schwendeman A, et al. Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles. Commun Biol. 2020; 3:28. PMID: 31942029.

32. Rong S, Zhao S, Kai X, Zhang L, Zhao Y, Xiao X, Bao W, Liu L. Procyanidins extracted from the litchi pericarp attenuate atherosclerosis and hyperlipidemia associated with consumption of a high fat diet in apolipoprotein-E knockout mice. Biomed Pharmacother. 2018; 97:1639–1644. PMID: 29793326.

33. Li J, Zhong Z, Yuan J, Chen X, Huang Z, Wu Z. Resveratrol improves endothelial dysfunction and attenuates atherogenesis in apolipoprotein E-deficient mice. J Nutr Biochem. 2019; 67:63–71. PMID: 30856465.

34. Wu C, Luan H, Zhang X, Wang S, Zhang X, Sun X, Guo P. Chlorogenic acid protects against atherosclerosis in ApoE-/- mice and promotes cholesterol efflux from RAW264.7 macrophages. PLoS One. 2014; 9:e95452. PMID: 25187964.

35. Park SH, Kang MK, Choi YJ, Kim YH, Antika LD, Kim DY, Lee EJ, Lim SS, Kang YH. α-Asarone blocks 7β-hydroxycholesterol-exposed macrophage injury through blocking elF2α phosphorylation and prompting beclin-1-dependent autophagy. Oncotarget. 2017; 8:7370–7383. PMID: 28088783.

36. Park SH, Kang MK, Choi YJ, Kim YH, Antika LD, Lim SS, Kang YH. Dietary compound α-asarone alleviates ER stress-mediated apoptosis in 7β-hydroxycholesterol-challenged macrophages. Mol Nutr Food Res. 2016; 60:1033–1047. PMID: 26893256.

37. Farrell N, Norris G, Lee SG, Chun OK, Blesso CN. Anthocyanin-rich black elderberry extract improves markers of HDL function and reduces aortic cholesterol in hyperlipidemic mice. Food Funct. 2015; 6:1278–1287. PMID: 25758596.

38. Xu M, Lv C, Wang H, Lu Q, Ye M, Zhu X, Liu R. Peanut skin extract ameliorates high-fat diet-induced atherosclerosis by regulating lipid metabolism, inflammation reaction and gut microbiota in ApoE^-/- mice. Food Res Int. 2022; 154:111014. PMID: 35337573.

39. Olszanecki R, Jawień J, Gajda M, Mateuszuk L, Gebska A, Korabiowska M, Chłopicki S, Korbut R. Effect of curcumin on atherosclerosis in apoE/LDLR-double knockout mice. J Physiol Pharmacol. 2005; 56:627–635. PMID: 16391419.

40. Huang W, Zeng Z, Lang Y, Xiang X, Qi G, Lu G, Yang X. Cannabis seed oil alleviates experimental atherosclerosis by ameliorating vascular inflammation in apolipoprotein-E-deficient mice. J Agric Food Chem. 2021; 69:9102–9110. PMID: 34037390.

41. Peng Y, Xu J, Zeng Y, Chen L, Xu XL. Polydatin attenuates atherosclerosis in apolipoprotein E-deficient mice: role of reverse cholesterol transport. Phytomedicine. 2019; 62:152935. PMID: 31085374.

42. Millar CL, Duclos Q, Blesso CN. Effects of dietary flavonoids on reverse cholesterol transport, HDL metabolism, and HDL function. Adv Nutr. 2017; 8:226–239. PMID: 28298268.