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Planta Med 2022; 88(06): 466-478
DOI: 10.1055/a-1438-2816
Natural Product Chemistry and Analytical Studies
Original Papers

Withanolides from Withania somnifera Ameliorate Neutrophil Infiltration in Endotoxin-Induced Peritonitis by Regulating Oxidative Stress and Inflammatory Cytokines

Acharya Balkrishna
1   Drug Discovery and Development Division, Patanjali Research Institute, Haridwar, Uttarakhand, India
2   Department of Allied and Applied Sciences, University of Patanjali, Patanjali Yog Peeth, Haridwar, Uttarakhand, India
3   Patanjali UK Trust, Glasgow, Scotland, United Kingdom
,
Siva Kumar Solleti‡
1   Drug Discovery and Development Division, Patanjali Research Institute, Haridwar, Uttarakhand, India
,
Hoshiyar Singh‡
1   Drug Discovery and Development Division, Patanjali Research Institute, Haridwar, Uttarakhand, India
,
Niti Sharma
1   Drug Discovery and Development Division, Patanjali Research Institute, Haridwar, Uttarakhand, India
,
Anurag Varshney 
1   Drug Discovery and Development Division, Patanjali Research Institute, Haridwar, Uttarakhand, India
2   Department of Allied and Applied Sciences, University of Patanjali, Patanjali Yog Peeth, Haridwar, Uttarakhand, India
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Abstract

Identification of novel anti-inflammatory strategies are needed to avoid the side effects associated with the currently available therapies. Use of anti-inflammatory herbal remedies is gaining attention. The purpose of the present investigation was to evaluate the pharmacological potential of the withanolide-rich root extracts of the medical plant Withania somnifera (L.) Dunal using in vivo and in vitro models of endotoxin-induced inflammation and oxidative stress. The pharmacological effects of W. somnifera root extracts were evaluated using a mouse model of endotoxin (lipopolysaccharide)-induced peritonitis and various relevant human cell lines. HPLC analysis of the W. somnifera root extracts identified the presence of various bioactive withanolides. In vivo challenge of mice with endotoxin resulted in the infiltration of various leukocytes, specifically neutrophils, along with monocytes and lymphocytes into the peritoneal cavity. Importantly, prophylactic treatment with W. somnifera inhibited the migration of neutrophils, lymphocytes, and monocytes and decreased the release of interleukin-1β, TNF-α, and interleukin-6 cytokines into the peritoneal cavity as identified by ELISA. Liver (glutathione peroxidase, glutathione, glutathione disulfide, superoxide dismutase, malondialdehyde, myeloperoxidase) and peritoneal fluid (nitrite) biochemical analysis revealed the antioxidant profile of W. somnifera. Similarly, in human HepG2 cells, W. somnifera significantly modulated the antioxidant levels. In THP-1 cells, W. somnifera decreased the secretion of interleukin-6 and TNF-α. In HEK-Blue reporter cells, W. somnifera inhibited TNF-α-induced nuclear factor-κB/activator protein 1 transcriptional activity. Our findings suggest the pharmacological effects of root extracts of W. somnifera rich in withanolides inhibit neutrophil infiltration, oxidative hepatic damage, and cytokine secretion via modulating the nuclear factor-κB/activator protein 1 pathway.

Key words

peritonitis - cytokines - oxidative stress - Ashwagandha - anti-inflammatory - antioxidant - Withania somnifera - Solanaceae


Publication History

Received: 20 November 2020

Accepted after revision: 14 March 2021

Article published online:
16 April 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
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  • References

  • 1 Bharti VK, Malik JK, Gupta RC. Ashwagandha: Multiple Health Benefits. In: Gupta RC. ed. Nutraceuticals: Efficacy, Safety and Toxicity. Amsterdam: Academic Press/Elsevier; 2016: 717-733
    Google Scholar
  • 2 Dar NJ, Hamid A, Ahmad M. Pharmacologic overview of Withania somnifera, the Indian Ginseng. Cell Mol Life Sci 2015; 72: 4445-4460
    CrossrefPubMedGoogle Scholar
  • 3 Winters M. Ancient medicine, modern use: Withania somnifera and its potential role in integrative oncology. Altern Med Rev 2006; 11: 269-277
    PubMedGoogle Scholar
  • 4 Pan MH, Chiou YS, Tsai ML, Ho CT. Anti-inflammatory activity of traditional Chinese medicinal herbs. J Tradit Complement Med 2011; 1: 8-24
    CrossrefPubMedGoogle Scholar
  • 5 Agarwal R, Diwanay S, Patki P, Patwardhan B. Studies on immunomodulatory activity of Withania somnifera (Ashwagandha) extracts in experimental immune inflammation. J Ethnopharmacol 1999; 67: 27-35
    CrossrefPubMedGoogle Scholar
  • 6 Sood A, Mehrotra A, Dhawan DK, Sandhir R. Indian Ginseng (Withania somnifera) supplementation ameliorates oxidative stress and mitochondrial dysfunctions in experimental model of stroke. Metab Brain Dis 2018; 33: 1261-1274
    CrossrefPubMedGoogle Scholar
  • 7 Durg S, Dhadde SB, Vandal R, Shivakumar BS, Charan CS. Withania somnifera (Ashwagandha) in neurobehavioural disorders induced by brain oxidative stress in rodents: a systematic review and meta-analysis. J Pharm Pharmacol 2015; 67: 879-899
    CrossrefPubMedGoogle Scholar
  • 8 Pawar P, Gilda S, Sharma S, Jagtap S, Paradkar A, Mahadik K, Ranjekar P, Harsulkar A. Rectal gel application of Withania somnifera root extract expounds anti-inflammatory and muco-restorative activity in TNBS-induced inflammatory bowel disease. BMC Complement Altern Med 2011; 11: 34
    CrossrefPubMedGoogle Scholar
  • 9 Minhas U, Minz R, Das P, Bhatnagar A. Therapeutic effect of Withania somnifera on pristane-induced model of SLE. Inflammopharmacology 2012; 20: 195-205
    CrossrefPubMedGoogle Scholar
  • 10 Lee W, Kim TH, Ku SK, Min KJ, Lee HS, Kwon TK, Bae JS. Barrier protective effects of withaferin A in HMGB1-induced inflammatory responses in both cellular and animal models. Toxicol Appl Pharmacol 2012; 262: 91-98
    CrossrefPubMedGoogle Scholar
  • 11 Medzhitov R. Inflammation 2010: new adventures of an old flame. Cell 2010; 140: 771-776
    CrossrefPubMedGoogle Scholar
  • 12 Scheiman JM, Hindley CE. Strategies to optimize treatment with NSAIDs in patients at risk for gastrointestinal and cardiovascular adverse events. Clin Ther 2010; 32: 667-677
    CrossrefPubMedGoogle Scholar
  • 13 Biswas SK. Does the Interdependence between Oxidative Stress and Inflammation Explain the Antioxidant Paradox?. Oxid Med Cell Longev 2016; 2016: 1-9
    CrossrefPubMedGoogle Scholar
  • 14 Netea M. Proinflammatory cytokines and sepsis syndrome: not enough, or too much of a good thing?. Trends Immunol 2003; 24: 254-258
    CrossrefPubMedGoogle Scholar
  • 15 Chen Y, Dong H, Thompson DC, Shertzer HG, Nebert DW, Vasiliou V. Glutathione defense mechanism in liver injury: insights from animal models. Food Chem Toxicol 2013; 60: 38-44
    CrossrefPubMedGoogle Scholar
  • 16 Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Signal Transduct Target Ther 2017; 2: 17023
    CrossrefPubMedGoogle Scholar
  • 17 Fujioka S, Niu J, Schmidt C, Sclabas GM, Peng B, Uwagawa T, Li Z, Evans DB, Abbruzzese JL, Chiao PJ. NF-κB and AP-1 connection: mechanism of NF-kappaB-dependent regulation of AP-1 activity. Mol Cell Biol 2004; 24: 7806-7819
    CrossrefPubMedGoogle Scholar
  • 18 Ghasemian M, Owlia S, Owlia MB. Review of Anti-Inflammatory Herbal Medicines. Adv Pharmacol Sci 2016; 2016: 1-11
    CrossrefPubMedGoogle Scholar
  • 19 Ito Y, Kinashi H, Katsuno T, Suzuki Y, Mizuno M. Peritonitis-induced peritoneal injury models for research in peritoneal dialysis review of infectious and non-infectious models. Ren Replace Ther 2017; 3: 16
    CrossrefPubMedGoogle Scholar
  • 20 Rosa SIG, Rios-Santos F, Balogun SO, Martins DT. Vitexin reduces neutrophil migration to inflammatory focus by down-regulating pro-inflammatory mediators via inhibition of p38, ERK1/2 and JNK pathway. Phytomedicine 2016; 23: 9-17
    CrossrefPubMedGoogle Scholar
  • 21 Sun W, Liu C, Zhang Y, Qiu X, Zhang L, Zhao H, Rong Y, Sun Y. Ilexgenin A, a novel pentacyclic triterpenoid extracted from Aquifoliaceae shows reduction of LPS-induced peritonitis in mice. Eur J Pharmacol 2017; 797: 94-105
    CrossrefPubMedGoogle Scholar
  • 22 Singh A, Duggal S, Singh H, Singh J, Katekhaye S. Withanolides: Phytoconstituents with significant pharmacological activities. Int J Green Pharm 2010; 4: 224
    CrossrefPubMedGoogle Scholar
  • 23 Maurya R. Withanolides: A Prospective Drug for Infectious and Tropical Diseases. In: Kaul S, Wadhwa R. eds. Science of Ashwagandha: Preventive and Therapeutic Potentials. Cham: Springer; 2017: 105-120
    CrossrefGoogle Scholar
  • 24 White PT, Subramanian C, Motiwala HF, Cohen MS. Natural Withanolides in the Treatment of chronic Diseases. In: Gupta S, Prasad S, Aggarwal B. eds. Anti-inflammatory Nutraceuticals and chronic Diseases. Advances in experimental Medicine and Biology, vol 928. Cham: Springer; 2016: 329-373
    Google Scholar
  • 25 Uddin Q, Samiulla L, Singh VK, Jamil SS. Phytochemical and pharmacological profile of Withania somnifera Dunal: A review. J Appl Pharm Sci 2012; 2: 170-175
    PubMedGoogle Scholar
  • 26 Dar PA, Singh LR, Kamal MA, Dar TA. Unique medicinal properties of Withania somnifera: Phytochemical constituents and protein component. Curr Pharm Des 2016; 22: 535-540 doi:10.2174/1381612822666151125001751
    CrossrefPubMedGoogle Scholar
  • 27 Yun KJ, Kim JY, Kim JB, Lee KW, Jeong SY, Park HJ, Jung HJ, Cho YW, Yun K, Lee KT. Inhibition of LPS-induced NO and PGE2 production by asiatic acid via NF-κB inactivation in RAW 264.7 macrophages: possible involvement of the IKK and MAPK pathways. Int Immunopharmacol 2008; 8: 431-441
    CrossrefPubMedGoogle Scholar
  • 28 LeGrand A, Fermor B, Fink C, Pisetsky DS, Weinberg JB, Vail TP, Guilak F. Interleukin-1, tumor necrosis factor alpha, and interleukin-17 synergistically up-regulate nitric oxide and prostaglandin E2 production in explants of human osteoarthritic knee menisci. Arthritis Rheum 2001; 44: 2078-2083
    CrossrefPubMedGoogle Scholar
  • 29 Sikandan A, Shinomiya T, Nagahara Y. Ashwagandha root extract exerts anti-inflammatory effects in HaCaT cells by inhibiting the MAPK/NF-κB pathways and by regulating cytokines. Int J Mol Med 2018; 42: 425-434
    PubMedGoogle Scholar
  • 30 Lee J, Sehrawat A, Singh SV. Withaferin A causes activation of Notch2 and Notch4 in human breast cancer cells. Breast Cancer Res Treat 2012; 136: 45-56
    CrossrefPubMedGoogle Scholar
  • 31 Gao R, Shah N, Lee JS, Katiyar SP, Li L, Oh E, Sundar D, Yun CO, Wadhwa R, Kaul SC. Withanone-rich combination of Ashwagandha withanolides restricts metastasis and angiogenesis through hnRNP-K. Mol Cancer Ther 2014; 13: 2930-2940
    CrossrefPubMedGoogle Scholar
  • 32 Kaileh M, Vanden Berghe W, Heyerick A, Horion J, Piette J, Libert C, De Keukeleire D, Essawi T, Haegeman G. Withaferin A strongly elicits IκB kinase β hyperphosphorylation concomitant with potent inhibition of its kinase activity. J Biol Chem 2007; 282: 4253-4264
    CrossrefPubMedGoogle Scholar
  • 33 Choi BY, Kim BW. Withaferin-A inhibits colon cancer cell growth by blocking STAT3 transcriptional activity. J Cancer Prev 2015; 20: 185-192
    CrossrefPubMedGoogle Scholar
  • 34 Hunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease. Nat Immunol 2015; 16: 448-457
    CrossrefPubMedGoogle Scholar
  • 35 Azuma Y, Kosaka K, Kashimata M. Phospholipase D-dependent and -independent p 38MAPK activation pathways are required for superoxide production and chemotactic induction, respectively, in rat neutrophils stimulated by fMLP. Eur J Pharmacol 2007; 568: 260-268
    CrossrefPubMedGoogle Scholar
  • 36 Mittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB. Reactive oxygen species in inflammation and tissue injury. Antioxid Redox Signal 2014; 20: 1126-1167
    CrossrefPubMedGoogle Scholar
  • 37 Carlsen MH, Halvorsen BL, Holte K, Bøhn SK, Dragland S, Sampson L, Willey C, Senoo H, Umezono Y, Sanada C, Barikmo I, Berhe N, Willett WC, Phillips KM, Jacobs jr. DR, Blomhoff R. The total antioxidant content of more than 3100 foods, beverages, spices, herbs and supplements used worldwide. Nutr J 2010; 9: 3 doi:10.1186/1475-2891-9-3
    CrossrefPubMedGoogle Scholar
  • 38 Alok S, Jain SK, Verma A, Kumar M, Mahor A, Sabharwal M. Herbal antioxidant in clinical practice: a review. Asian Pac J Trop Biomed 2014; 4: 78-84
    CrossrefPubMedGoogle Scholar
  • 39 Alam N, Hossain M, Mottalib MA, Sulaiman SA, Gan SH, Khalil MI. Methanolic extracts of Withania somnifera leaves, fruits and roots possess antioxidant properties and antibacterial activities. BMC Complement Altern Med 2012; 12: 175
    CrossrefPubMedGoogle Scholar
  • 40 Bhattacharya SK, Satyan SK. Experimental methods for evaluation of psychotropic agents in rodents: I–Anti-anxiety agent. Indian J Exp Biol 1997; 35: 565-575
    PubMedGoogle Scholar
  • 41 Ahmad M, Saleem S, Ahmad AS, Ansari MA, Yousuf S, Hoda MN, Islam F. Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats. Hum Exp Toxicol 2005; 24: 137-147
    CrossrefPubMedGoogle Scholar
  • 42 Hörauf JA, Kany S, Janicova A, Xu B, Vrdoljak T, Sturm R, Dunay IR, Martin L, Relja B. Short exposure to ethanol diminishes caspase-1 and ASC activation in human HepG2 cells in vitro . Int J Mol Sci 2020; 21: 3196
    CrossrefPubMedGoogle Scholar
  • 43 Selim NM, Elgazar AA, Abdel-Hamid NM, El-Magd MRA, Yasri A, Hefnawy HME, Sobeh M. Chrysophanol, physcion, hesperidin and curcumin modulate the gene expression of pro-inflammatory mediators induced by LPS in HepG2: in silico and molecular studies. Antioxidants (Basel).2019; 8: 371.
    PubMedGoogle Scholar
  • 44 Shrestha A, Park PH. Globular adiponectin attenuates LPS-induced reactive oxygen species production in HepG2 cells via FoxO3A and HO-1 signaling. Life Sci 2016; 148: 71-79
    CrossrefPubMedGoogle Scholar
  • 45 Pal A, Naika M, Khanum F, Bawa AS. In-vitro studies on the antioxidant assay profiling of Withania somnifera L. (Ashwagandha) Dunal root: Part 1. Pharmacogn J 2011; 3: 47-55
    CrossrefPubMedGoogle Scholar
  • 46 Vidyashankar S, Thiyagarajan OS, Varma RS, Kumar LMS, Babu UV, Patki PS. Ashwagandha (Withania somnifera) supercritical CO2 extract derived withanolides mitigates Bisphenol A induced mitochondrial toxicity in HepG2 cells. Toxicol Reports 2014; 1: 1004-1012
    CrossrefPubMedGoogle Scholar
  • 47 Kuczynski B, Reo NV. Evidence that plasmalogen is protective against oxidative stress in the rat brain. Neurochem Res 2006; 31: 639-656
    CrossrefPubMedGoogle Scholar
  • 48 Vaishnavi K, Saxena N, Shah N, Singh R, Manjunath K, Uthayakumar M, Kanaujia SP, Kaul SC, Sekar K, Wadhwa R. Differential activities of the two closely related withanolides, Withaferin A and Withanone: bioinformatics and experimental evidences. PLoS One 2012; 7: e44419
    CrossrefPubMedGoogle Scholar
  • 49 Baitharu I, Jain V, Deep SN, Shroff S, Sahu JK, Naik PK, Ilavazhagan G. Withanolide A prevents neurodegeneration by modulating hippocampal glutathione biosynthesis during hypoxia. PLoS One 2014; 9: e105311
    CrossrefPubMedGoogle Scholar
  • 50 Nair A, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 2016; 7: 27-31
    CrossrefPubMedGoogle Scholar
  • 51 Qin X, Jiang X, Jiang X, Wang Y, Miao Z, He W, Yang G, Lv Z, Yu Y, Zheng Y. Micheliolide inhibits LPS-induced inflammatory response and protects mice from LPS challenge. Sci Rep 2016; 6: 23240
    CrossrefPubMedGoogle Scholar
  • 52 Solleti SK, Simon DM, Srisuma S, Arikan MC, Bhattacharya S, Rangasamy T, Bijli KM, Rahman A, Crossno jr. JT, Shapiro SD, Mariani TJ. Airway epithelial cell PPARγ modulates cigarette smoke-induced chemokine expression and emphysema susceptibility in mice. Am J Physiol Lung Cell Mol Physiol 2015; 309: L293-L304
    CrossrefPubMedGoogle Scholar
  • 53 Shackelford C, Long G, Wolf J, Okerberg C, Herbert R. Qualitative and quantitative analysis of nonneoplastic lesions in toxicology studies. Toxicol Pathol 2002; 30: 93-96
    CrossrefPubMedGoogle Scholar
  • 54 Mann PC, Vahle J, Keenan CM, Baker JF, Bradley AE, Goodman DG, Harada T, Herbert R, Kaufmann W, Kellner R, Nolte T, Rittinghausen S, Tanaka T. International harmonization of toxicologic pathology nomenclature: an overview and review of basic principles. Toxicol Pathol 2012; 40: 7S-13S
    CrossrefPubMedGoogle Scholar
  • 55 Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: biochemical role as a component of glatathione peroxidase. Science 1973; 179: 588-590
    CrossrefPubMedGoogle Scholar
  • 56 Hissin PJ, Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 1976; 74: 214-226
    CrossrefPubMedGoogle Scholar
  • 57 Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971; 44: 276-287
    CrossrefPubMedGoogle Scholar
  • 58 Heath RL, Packer L. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 1968; 125: 189-198
    CrossrefPubMedGoogle Scholar
  • 59 Bozeman PM, Learn DB, Thomas EL. Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase. J Immunol Methods 1990; 126: 125-133
    CrossrefPubMedGoogle Scholar
  • 60 Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura T. Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Anal Biochem 1983; 132: 345-352
    CrossrefPubMedGoogle Scholar
  • 61 Griess P. Bemerkungen zu der Abhandlung der HH. Weselsky und Benedikt „Ueber einige Azoverbindungen“. Berichte der Dtsch Chem Gesellschaft 1879; 12: 426-428
    CrossrefPubMedGoogle Scholar
  • 62 Góth L. A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta 1991; 196: 143-151
    CrossrefPubMedGoogle Scholar