Search JIM Advanced Search

Journal of Integrative Medicine ›› 2016, Vol. 14 ›› Issue (6): 456-464.doi: 10.1016/S2095-4964(16)60279-0

• Research Article • Previous Articles     Next Articles

Killing of Staphylococcus aureus by allylpyrocatechol is potentiated by induction of intracellular oxidative stress and inhibition of catalase activity

Roslinah Mohamad Hussaina, Noor Faradilla Abdullaha, Zulkhairi Amomb   

  1. a Department of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA,42300 Puncak Alam, Malaysia
    b Department of Anatomy and Basic Sciences, Faculty of Health Sciences, Universiti Teknologi MARA,42300 Puncak Alam, Malaysia
  • Received:2016-06-01 Accepted:2016-08-26 Online:2016-11-15 Published:2016-11-15
  • Contact: Roslinah Mohamad Hussain, PhD; E-mail:


This study investigated the effects of allylpyrocatechol (APC), the major component in ethanolic extract of Piper betle, on key oxidative stress resistance enzymes important for the survival of Staphylococcus aureus, a major pathogen in the human host.


Effects of APC on expressions of genes encoding catalase (katA), superoxide dismutases (SODs), including sodA and sodM, and alkyl hydroperoxide reductase (ahpC) in S. aureus were quantitated by RT-qPCR in reference to gyrA and 16S rRNA. Corresponding activities of the enzymes were also investigated. The Livak analysis was performed for verification of gene-fold expression data. Effects of APC on intracellular and extracellular reactive oxygen species (ROS) levels were determined using the nitroblue tetrazolium (NBT) reduction assay.


APC-treated S. aureus cells had higher sodA and sodM transcripts at 1.5-fold and 0.7-fold expressions respectively with corresponding increase in total SOD activity of 12.24 U/mL compared to untreated cells, 10.85 U/mL (P<0.05). Expression of ahpC was highest in APC-treated cells with 5.5-fold increased expression compared to untreated cells (P<0.05). Correspondingly, ahpC activity was higher in APC-treated cells at 0.672 (A310nm) compared to untreated cells which was 0.394 (A310nm). In contrast, katA expression was 1.48-fold and 0.33-fold lower respectively relative to gyrA and 16S rRNA. Further, APC-treated cells showed decreased catalase activity of 1.8 × 10-4 (U/L or μmol/(min·L)) compared to untreated cells, which was 4.8 × 10-4 U/L (P<0.05). Absorbance readings (A575nm) for the NBT reduction assay were 0.709 and 0.695 respectively for untreated and treated cells, which indicated the presence of ROS. APC-treated S. aureus cells had lower ROS levels both extracellularly and intracellularly, but larger amounts remained intracellularly compared to extracellular levels with absorbances of 0.457 and 0.137 respectively (P<0.05).


APC induced expressions of both sodA and sodM, resulting in increased total SOD activity in S. aureus. Higher sodA expression indicated stress induced intracellularly involving O2-, presumably leading to higher intracellular pools of H2O2. A concommittant decrease in katA expression and catalase activity possibly induced ahpC expression, which was increased the highest in APC-treated cells. Our findings suggest that in the absence of catalase, cells are propelled to seek an alternate pathway involving ahpC to reduce stress invoked by O2- and H2O2. Although APC reduced levels of ROS, significant amounts eluded its antioxidative action and remained intracellularly, which adds to oxidative stress in treated cells.

Key words: Allylpyrocatechol, Catalase, Superoxide dismutase, Alkyl hydroperoxide reductase, Reactive oxygen species, Staphylococcus aureus, Piper betle

1 Boucher H, Miller LG, Razonable RR.Serious infections caused by methicillin-resistantStaphylococcus aureus. Clin Infect Dis. 2010; 51(Suppl 2): S183-S197.
2 Caksen H, Oztürk MK, Uzüm K, Yüksel S, Ustübaş HB.Pulmonary complications in patients with staphylococcal sepsis. Pediatr Int.2000; 42(3): 268-271.
3 Gaupp R, Ledala N, Somerville GA.Staphylococcal response to oxidative stress.Front Cell Infect Microbiol. 2012; 2: 33.
4 Poole K.Bacterial stress responses as determinants of antimicrobial resistance.J Antimicrob Chemother. 2012; 67(9): 2069-2089.
5 Horsburgh MJ, Ingham E, Foster SJ.InStaphylococcus aureus, fur is an interactive regulator with PerR, contributes to virulence, and is necessary for oxidative stress resistance through positive regulation of catalase and iron homeostasis. J Bacteriol. 2001; 183(2): 468-475.
6 Nakonieczna J, Michta E, Rybicka M, Grinholc M, Gwizdek-Wiśniewska A, Bielawski KP.Superoxide dismutase is upregulated inStaphylococcus aureus following protoporphyrin-mediated photodynamic inactivation and does not directly infl uence the response to photodynamic treatment. BMC Microbiol. 2010; 10: 323.
7 Clements MO, Watson SP, Foster SJ.Characterization of the major superoxide dismutase ofStaphylococcus aureus and its role in starvation survival, stress resistance, and pathogenicity. J Bacteriol. 1999; 181(13): 3898-3903.
8 Rocha ER, Smith CJ.Role of the alkyl hydroperoxide reductase (ahpCF) gene in oxidative stress defense of the obligate anaerobeBacteroides fragilis. J Bacteriol. 1999; 181(18): 5701-5710.
9 Chen L, Xie QW, Nathan C.Alkyl hydroperoxide reductase subunit C (AhpC) protects bacterial and human cells against reactive nitrogen intermediates.Mol Cell. 1998; 1(6): 795-805.
10 Cosgrove K, Coutts G, Jonsson IM, Tarkowski A, Kokai- Kun JF, Mond JJ, Foster SJ.Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization inStaphylococcus aureus. J Bacteriol. 2007; 189(3): 1025-1035.
11 Sharma S, Khan IA, Ali I, Ali F, Kumar M, Kumar A, Johri RK, Abdullah ST, Bani S, Pandey A, Suri KA, Gupta BD, Satti NK, Dutt P, Qazi GN.Evaluation of the antimicrobial, antioxidant, and anti-infl ammatory activities of hydroxychavicol for its potential use as an oral care agent.Antimicrob Agents Chemother. 2009; 53(1): 216-222.
12 Jesonbabu J, Spandana N, Lakshmi AK.The potential activity of hydroxychavicol against pathogenic bacteria. J Bacteriol Parasitol. 2011; 2(6): 2-5.
13 Milton PS, Muthumani M, Shagirtha K.Protective effect ofPiper betle leaf extract against cadmium-induced oxidative stress and hepatic dysfunction in rats. Saudi J Biol Sci. 2012; 19(2): 229-239.
14 Mahfuzul HM, Rattila S, Asaduzzaman SM, Bari ML, Inatsu Y, Kawamoto S.Antibacterial activity of ethanol extract of betel leaf (Piper betle L.) against some food borne pathogens. Bangladesh J Microbiol. 2011; 28(2): 58-63.
15 Chakraborty D, Shah B.Antimicrobial, anti-oxidative and anti-hemolytic activity ofPiper betle leaf extracts. Int J Pharm Pharm Sci. 2011; 3(3): 192-199.
16 Nalina T, Rahim ZHA.The crude aqueous extract ofPiper betle L. and its antibacterial effect towards Streptococcus mutans. Am J Biotechnol Biochem. 2007; 3: 10-15.
17 Sarkar D, Kundu S, De S, Hariharan C, Saha P, Manna A, Chattopadhyay S, Chatterjee M.The antioxidant activity of allylpyrocatechol is mediatedvia decreased generation of free radicals along with escalation of antioxidant mechanisms. Phytother Res. 2013; 27(3): 324-329.
18 Ramji N, Ramji N, Iyer R, Chandrasekaran S.Phenolic antibacterials fromPiper betle in the prevention of halitosis. J Ethnopharmacol. 2002; 83(1-2): 149-152.
19 Bhattacharya S, Banerjee D, Bauri AK, Chattopadhyay S, Bandyopadhyay SK.Healing property of thePiper betle phenol, allylpyrocatechol against indomethacin-induced stomach ulceration and mechanism of action. World J Gastroenterol. 2007; 13(27): 3705-3713.
20 Sarkar D, Saha P, Gamre S, Bhattacharjee S, Hariharan C, Ganguly S, Sen R, Mandal G, Chattopadhyay S, Majumdar S, Chatterjee M.Anti-inflammatory effect of allylpyrocatechol in LPS-induced macrophages is mediated by suppression of iNOS and COX-2via the NF-κB pathway. Int Immunopharmacol. 2008; 8(9): 1264-1271.
21 Mula S, Banerjee D, Patro BS, Bhattacharya S, Barik A, Bandyopadhyay SK, Chattopadhyay S.Inhibitory property of thePiper betle phenolics against photosensitization-induced biological damages. Bioorg Med Chem. 2008; 16(6): 2932-2938.
22 Becerra MC, Albesa I.Oxidative stress induced by ciprofloxacin inStaphylococcus aureus. Biochem Biophys Res Commun. 2002; 297(4): 1003-1007.
23 Livak KJ, Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method.Methods. 2001; 25(4): 402-408.
24 Das D, Saha SS, Bishayi B.Intracellular survival ofStaphylococcus aureus: correlating production of catalase and superoxide dismutase with levels of inflammatory cytokines. Infl amm Res. 2008; 57(7): 340-349.
25 Das D, Bishayi B.Contribution of catalase and superoxide dismutase to the intracellular survival of clinical isolates ofStaphylococcus aureus in murine macrophages. Indian J Microbiol. 2010; 50(4): 375-384.
26 Rintiswati N, Wibawa T, Asmara W, Soebono H.Effect of oxidative stress on AhpC activity and virulence in katG Ser315 Thr mycobacterium tuberculosis mutant.Indones J Biotechnol. 2011; 16(2): 100-110.
27 Helmann JD, Wu MF, Gaballa A, Kobel PA, Morshedi MM, Fawcett P, Paddon C.The global transcriptional response ofBacillus subtilis to peroxide stress is coordinated by three transcription factors. J Bacteriol. 2003; 185(1): 243-253.
28 Abdullah NF, Hussain RM, Amom Z.Effect of APC on killing ofStaphylococcus aureus by oxidative stress agents. Jurnal Teknologi. 2016; 78(5): 39-44.
29 Newberry KJ, Fuangthong M, Panmanee W, Mongkolsuk S, Brennan RG.Structural mechanism of organic hydroperoxide induction of the transcription regulator OhrR.Mol Cell. 2007; 28(4): 652-664.
30 Karavolos MH, Horsburgh MJ, Ingham E, Foster SJ.Role and regulation of the superoxide dismutases ofStaphylococcus aureus. Microbiology. 2003; 149(10): 2749-2758.
31 Touati D.Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis inEscherichia coli, studied with operon and protein fusions. J Bacteriol. 1988; 170(6): 2511-2520.
32 Procházková D, Boušová I, Wilhelmová N.Antioxidant and prooxidant properties of fl avonoids.Fitoterapia. 2011; 82(4): 513-523.
33 Rahal A, Kumar A, Singh V, Yadav B, Tiwari R, Chakraborty S, Dhama K.Oxidative stress, prooxidants, and antioxidants: the interplay. Biomed Res Int. 2014; 2014: 761264.
34 Valderas MW, Hart ME.Identifi cation and characterization of a second superoxide dismutase gene (sodM) from Staphylococcus aureus. J Bacteriol. 2001; 183(11): 3399-3407.
35 Rathee JS, Patro BS, Mula S, Gamre S, Chattopadhyay S.Antioxidant activity ofPiper betle leaf extract and its constituents. J Agric Food Chem. 2006; 54(24): 9046-9054.
36 Bhalerao SA, Verma DR, Gavankar RV, Teli NC, Yatin YR, Didwana VS, Trikannad A.Phytochemistry, pharmacological profi le and therapeutic uses ofPiper betle Linn.—an overview. J Pharm Phytochem. 2013; 1(2): 10-19.
37 Hussain RM, Din NAM, Nasir NNM.Piper betle ethanolic extract reduces neutrophil scavenging ability and possibly catalase activity in S. aureus. Int J Pharm Sci Rev Res. 2013; 22(1): 35-40.
38 Abrahim NN, Kanthimathi MS, Abdul-Aziz A.Piper betle shows antioxidant activities, inhibits MCF-7 cell proliferation and increases activities of catalase and superoxide dismutase. BMC Complement Altern Med. 2012; 12(1): 220.
39 Imlay JA.Cellular defenses against superoxide and hydrogen peroxide.Annu Rev Biochem. 2008; 77: 755-776.
40 Seaver LC, Imlay JA.Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide inEscherichia coli. J Bacteriol. 2001; 183(24): 7173-7181.
[1] Bruno José Martins Da Silva, Sandro Wilson Gomes Pereira, Ana Paula Drummond Rodrigues, José Luiz Martins Do Nascimento, Edilene Oliveira Silva. In vitro antileishmanial effects of Physalis angulata root extract on Leishmania infantum. Journal of Integrative Medicine, 2018, 16(6): 404-410.
[2] Shohini Chakraborty, Nashra Afaq, Neelam Singh, Sukanta Majumdar. Antimicrobial activity of Cannabis sativaThuja orientalis and Psidium guajava leaf extracts against methicillin-resistant Staphylococcus aureus. Journal of Integrative Medicine, 2018, 16(5): 350-357.
[3] Yuki Takauji, Kyoko Morino, Kensuke Miki, Mohammad Hossain, Dai Ayusawa, Michihiko Fujii. Chyawanprash, a formulation of traditional Ayurvedic medicine, shows a protective effect on skin photoaging in hairless mice. Journal of Integrative Medicine, 2016, 14(6): 473-479.
[4] Jesmin Mondal, Asmita Samadder, Anisur Rahman Khuda-Bukhsh. Psorinum 6× triggers apoptosis signals in human lung cancer cells. Journal of Integrative Medicine, 2016, 14(2): 143-153.
[5] Santu Kumar Saha, Sourav Roy, Anisur Rahman Khuda-Bukhsh. Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro. Journal of Integrative Medicine, 2015, 13(6): 400-411.
[6] Mukherjee Avinaba, Boujedaini Naoual, Rahman Khuda-Bukhsh Anisur. Homeopathic Thuja 30C ameliorates benzo(a)pyrene-induced DNA damage, stress and viability of perfused lung cells of mice in vitro. Journal of Integrative Medicine, 2013, 11(6): 397-404.
[7] Samrat Ghosh, Kausik Bishayee, Avijit Paul, Avinaba Mukherjee, Sourav Sikdar, Debrup Chakraborty, Naoual Boujedaini, Anisur Rahman Khuda-Bukhsh. Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation. Journal of Integrative Medicine, 2013, 11(2): 116-124.
[8] Yue-hua Wang, Xiao-li He, Xiao-xiu Li, Hai-lin Qin, Guan-hua Du . Effects of the effective component group of Chinese herbal medicine Xiaoxuming Decoction on brain mitochondria in rats with chronic cerebral ischemia. Journal of Chinese Integrative Medicine, 2012, 10(5): 569-576.
[9] Gao Shan, Wang Xing-hui, Huang Ling-ling, Yu Ting-ting, Du Su-ming, Guo Yan-wei, Jia Yuan, Wang Jian. Effects of a compound Chinese medicine Xinji’erkang on isoproterenol-induced ventricular remodeling in mice. Journal of Chinese Integrative Medicine, 2012, 10(3): 330-336.
[10] Das Sreemanti, Kumar Saha Santu, De Arnab, Das Durba, Rahman Khuda-Bukhsh Anisur. Potential of the homeopathic remedy, Arnica Montana 30C, to reduce DNA damage in Escherichia coli exposed to ultraviolet irradiation through up-regulation of nucleotide excision repair genes. Journal of Chinese Integrative Medicine, 2012, 10(3): 337-246.
[11] De Arnab, Das Durba, Dutta Suman, Chakraborty Debrup, Boujedaini Naoual, Rahman Khuda-Bukhsh Anisur. Potentized homeopathic drug Arsenicum Album 30C inhibits intracellular reactive oxygen species generation and up-regulates expression of arsenic resistance gene in arsenite-exposed bacteria Escherichia coli. Journal of Chinese Integrative Medicine, 2012, 10(2): 210-227.
[12] Xiao-ping Huang , Xiao-dan Liu , Chang-qing Deng . Effects of the combination of active component extracts from Astragalus membranaceus and Panax notoginseng on apoptosis, reactive oxygen species and mitochondrial membrane potential of PC12 cells with oxidative injury. Journal of Chinese Integrative Medicine, 2012, 10(10): 1127-1134.
[13] Durba Das, Arnab De, Suman Dutta, Raktim Biswas, Naoual Boujedaini, Anisur Rahman Khuda-Bukhsh. Potentized homeopathic drug Arsenicum Album 30C positively modulates protein biomarkers and gene expressions in Saccharomyces cerevisae exposed to arsenate. Journal of Chinese Integrative Medicine, 2011, 9(7): 752-760.
[14] Thanasekaran Jayakumar, Philip Aloysius Thomas, Mathivanan Isai, Pitchairaj Geraldine. An extract of the oyster mushroom, Pleurotus ostreatus, increases catalase gene expression and reduces protein oxidation during aging in rats. Journal of Chinese Integrative Medicine, 2010, 8(8): 774-780.
[15] Ding-zhu Shen , Qing Ta, Jin-xing Du, Sai-dan Ding, Gao-feng Chen, Yi-yang Ru, Ping Liu. Effects of Yiguanjian Decoction on liver cirrhosis formation: A differential proteomics study in rats. Journal of Chinese Integrative Medicine, 2010, 8(2): 158-167.
Full text



[1] Zhao-guo Li. Discussion on English translation of commonly used sentences in traditional Chinese medicine: part one. Journal of Chinese Integrative Medicine, 2008, 6(1): 107-110
[2] Jun Hu, Jian-ping Liu. Non-invasive physical treatments for chronic/recurrent headache. Journal of Chinese Integrative Medicine, 2008, 6(1): 31
[3] Dong Yang, Yong-ping Du, Qing Shen, Wei Chen, Yan Yu, Guang-lei Chen. Expression of alpha-smooth muscle actin in renal tubulointerstitium in patients with kidney collateral stasis. Journal of Chinese Integrative Medicine, 2008, 6(1): 41-44
[4] Zhi-chun Jin. Problems in establishing clinical guideline for integrated traditional Chinese and Western medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 5-8
[5] Jin-zhou Tian, Jing Shi, Xin-qing Zhang, Qi Bi, Xin Ma, Zhi-liang Wang, Xiao-bin Li, Shu-li Shen, Lin Li, Zhen-yun Wu, Li-yan Fang, Xiao-dong Zhao, Ying-chun Miao, Peng-wen Wang, Ying Ren, Jun-xiang Yin, Yong-yan Wang, Beijing United Study Group on MCI of the Capital Foundation of Medical Developments. Guiding principles of clinical research on mild cognitive impairment (protocol). Journal of Chinese Integrative Medicine, 2008, 6(1): 9-14
[6] Daniel Weber, Janelle M Wheat, Geoffrey M Currie. Inflammation and cancer: Tumor initiation, progression and metastasis,and Chinese botanical medicines. Journal of Chinese Integrative Medicine, 2010, 8(11): 1006-1013
[7] Xin-jun Wang, Ling-ling Wang . A mechanism of endogenous opioid peptides for rapid onset of acupuncture effect in treatment of depression. Journal of Chinese Integrative Medicine, 2010, 8(11): 1014-1017
[8] Bo Wang , Wei Yan , Li-hui Hou, Xiao-ke Wu. Disorder of Tiangui (kidney essence) and reproductive dysfunction in patients with polycystic ovary syndrome. Journal of Chinese Integrative Medicine, 2010, 8(11): 1018-1022
[9] Chan Chen, Yong-mei Meng , Peng Zhang, Juan Wang, Hui-hui Zhao, Shu-zhen Guo , Wei Wang. Diagnosis and treatment rule of traditional Chinese medicine for syndrome factors of chronic congestive heart failure: a study based on Shannon entropy method. Journal of Chinese Integrative Medicine, 2010, 8(11): 1080-1084
[10] Liang-ping Hu , Xiao-lei Bao, Xue Guan. Statistical inference and experimental design of univariate qualitative data of single-group design. Journal of Chinese Integrative Medicine, 2010, 8(11): 1085-1089