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Investigation of the antimicrobial effects of Sapindus mukorossi on endodontic pathogens

Yıl 2020, Cilt: 37 Sayı: 4, 111 - 118, 11.09.2020

Öz

Endodontic infections have a polymicrobial nature. Thus, eliminating the microorganisms from infected root canals without damaging healthy surrounding tissue is a major concern. Sapindus mukorossi (S. mukorossi) is a natural product with potential antimicrobial effects. The aim of this study was to evaluate whether various extract solutions of S. mukorossi have an antimicrobial activity against specific endodontic pathogens. Extracts were obtained from S. mukorossi fruit pericarps using methanol, ethanol, butanol and distilled water solvents. The inhibition zone, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were determined with disc diffusion assay, broth microdilution assay and agar dilution assay. Antimicrobial inhibitory activity was observed with all four different solvent extracts of S. mukorossi against Fusobacterium nucleatum American Type Culture Collection (ATCC) 25586, Porphyromonas gingivalis ATCC 33277 and Actinomyces odontolyticus (clinical isolate). The MIC values were ranged 10.24-10.24, 0.01-0.64 and 1.28- 2.56 mg/mL, respectively. The MBC value was not detected for Fusobacterium nucleatum ATCC 25586. The MBC values were 0.02-1.25 and 2.56-5.12 mg/ mL for Porphyromonas gingivalis ATCC 33277 and Actinomyces odontolyticus (clinical isolate), respectively. Antifungal activities were also observed with the four different solvent extracts of S. mukorossi against Candida albicans ATCC 10231 and C. albicans clinical isolates 1, 2 and 3. The inhibition zone diameter values were in the range of 18-21 mm. The MIC values for C. albicans ATCC 10231 and C. albicans clinical isolates 1, 2 and 3 were 0.2-0.4 and MFC values were 0.4-0.8 mg/mL, respectively. The antimicrobial effects of the S. mukorossi fruit pericarp extract inhibited the growth of P. gingivalis, A. odontolyticus, F. nucleatum, and especially the C. albicans strains. S. mukorossi extract has interesting potential as an antimicrobial agent against endodontic pathogens.

Destekleyen Kurum

This study was supported by from the Scientific and Technological Research Council of Turkey (TUBITAK; project no. 215S627).

Proje Numarası

TUBITAK; project no. 215S627

Teşekkür

The authors thank Prof. Meral Dilara Ogunc for her contributions to the methodological assays and Akdeniz University Faculty of Chemistry Department, for technical support of this research.

Kaynakça

  • Abou-Rass, M., & Bogen, G, 1998. Microorganisms in closed periapical lesions. International Endodontic Journal, 31(1), 39–47.
  • Ali, M., Khan, T., Fatima, K., Ali, Q.U.A., Ovais, M., Khalil AT., Ullah I., Raza A., Shinvari Z.K., Idrees, M, 2018. Selected hepatoprotective herbal medicines: Evidence from ethnomedicinal applications, animal models, and possible mechanism of actions. Phytotherapy Research, 32, 199–215.
  • Aligiannis, N., Kaplotzakis, E., Mitaku, S., & Chinou, I.B., 2001. Composition and antimicrobial activity of the essential oils of two Origanum species. Journal of Agriculture and Food Chemistry, 49, 4168–4170.
  • Alves, E.G., Vinholis, A.H.C., Casemiro, L.A., Furtado, N.A.J.C., Silva, M.L.A., Cunha, W.R., 2008. Comparative study of screening techniques for antibacterial activity evaluation of plant crude extracts and pure compounds. Quimica Nova, 31(5), 1224-1229.
  • Aneja, K.R., Joshi, R., & Sharma, C., 2010. In vitro antimicrobial activity of Sapindus mukorossi and Emblica officinalis against dental caries pathogens. Ethnobotanical Leaflets, 14, 402–412.
  • Caetano da Silva S.D., Mendes de Souza, M.G., Oliveira Cardoso, M.J., da Silva Moraes, T., Ambrósio, S.R., Sola Veneziani, R.C., & Martins, C.H., 2014. Antibacterial activity of Pinus elliottii against anaerobic bacteria present in primary endodontic infections. Anaerobe, 30, 146–152.
  • Castilho, A.L., Saraceni, C.H., Díaz, I.E., Paciencia, M.L., & Suffredini, I.B., 2013. New trends in dentistry: Plant extracts against Enterococcus faecalis. The efficacy compared to chlorhexidine. Brazilian Oral Research, 27(2), 109–115.
  • Clinical and Laboratory Standards Institute., 2015. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Twelfth Edition. M02-A12. Clinical and Laboratory Standards Institute, Wayne, PA.
  • Clinical and Laboratory Standards Institute, 2009. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. CLSI document M44-A2. 2nd ed. Wayne: Clinical and Laboratory Standards Institute.
  • Clinical and Laboratory Standards Institute, 2015. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-tenth edition, CLSI document M07-A9. Wayne: Clinical and Laboratory Standards Institute.
  • Clinical and Laboratory Standards Institute, 2008. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast; Approved Standard-Third Edition. CLSI document M27-A3. Wayne: Clinical and Laboratory Standards Institute.
  • Clinical and Laboratory Standards Institute, 2012. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard-Eighth Edition. CLSI document M11-A8. Wayne: Clinical and Laboratory Standards Institute.
  • Duarte, M.C.T., Figueira, G.M., Sartoratto, A., Rehder, V.L.G., & Delarmelina, C., 2005. Anti-Candida activity of Brazilian medicinal plants. Journal of Ethnopharmacology, 97, 305–311.
  • Dutta, A., & Kundabala, M., 2013. Antimicrobial efficacy of endodontic irrigants from Azadirachta indica: An in vitro study. Acta Odontologica Scandinavica, 71, 1594–1598.
  • Gatot, A., Arbelle, J., Lieberman, A., & Yanai-Inbar, I.,1991. Effects of sodium-hypochlorite on soft-tissues after its inadvertent injection beyond the root apex. Journal of Endodontics, 17, 573–574.
  • Herrera, D.R., Tay, L.Y., Rezende, E.C., Kozlowski, V.A., Jr, & Santos, E.B., 2010. In vitro antimicrobial activity of phytotherapic Uncaria tomentosa against endodontic pathogens. Journal of Oral Science, 52(3), 473–476.
  • Hu, Q, Chen, Y.Y., Jiao, Q.Y., Khan, A., Li, F., Han, D.F., Cao G.D., Lou, H.X., 2018. Triterpenoid saponins from the pulp of Sapindus mukorossi and their antifungal activities. Phytochemistry, 147, 1–8.
  • Ibrahim, M., Khan, A.A., Tiwari, S.K., Habeeb, M.A., Khaja, M.N., & Habibullah, C.M., 2006. Antimicrobial activity of Sapindus mukorossi and Rheumemodi extracts against H pylori: In vitro and in vivo studies. World Journal of Gastroenterology, 12(44), 7136–7142.
  • Kato, H., Taguchi, Y., Tominaga, K., Umeda, M., & Tanaka, A., 2014. Porphyromonas gingivalis LPS inhibits osteoblastic differentiation and promotes-inflammatory cytokine production in human periodontal ligament stem cells. Archives of Oral Biology, 59(2), 167–175.
  • Martinho, F.C., Leite, F.R., Nóbrega, L.M., Endo, M.S., Nascimento, G.G., Darveau, R.P., & Gomes, B.P., 2016. Comparison of Fusobacterium nucleatum and Porphyromonas gingivalis lipopolysaccharides clinically isolated from root canal infection in the induction of pro-inflammatory cytokines secretion. Brazilian Dental Journal, 27(2), 202–207.
  • Nascimento G.G.F., Locatelli, N., Freitas P.C., Silva, G.L., 2000. Antibacterial activity of plant extracts and phytochemicals on antibiotic resistant bacteria. Brazilian Journal of Microbiology, 31, 247–256.
  • Onçağ, O., Hoşgör, M., Hilmioğlu, S., Zekioğlu, O., Eronat, C., & Burhanoğlu, D., 2003. Comparison of antibacterial and toxic effects of various root canal irrigants. International Endodontic Journal, 36(6), 423–432.
  • Pallotta, R.C., Ribeiro, M.S., de Lima Machado, M.E., 2007. Determination of the minimum inhibitory concentration of four medicaments used as intracanal medication. Australian Endodontic Journal, 33, 107–111.
  • Porsche, F.M., Molitor, D., Beyer, M., Charton, S., André, C., & Kollar, A., 2018. Antifungal activity of saponins from the fruit pericarp of Sapindus mukorossi against Venturia inaequalis and Botrytis cinerea Plant Disease, 102(5), 991–1000.
  • Sathyaprasad, S., Jose, B.K., & Chandra, H. S., 2015. Antimicrobial and antifungal efficacy of Spilanthes acmella as an intracanal medicament in comparison to calcium hydroxide: An in vitro study. Indian Journal of Dental Research, 26(5), 528–532.
  • Shah, M., Parveen, Z., & Khan, M. R., 2017. Evaluation of antioxidant, antiinflammatory, analgesic and antipyretic activities of the stem bark of Sapindus mukorossi. BMC Complementary and Alternative Medicine, 17, 526.
  • Sharma, A., Chandra Sati, S., Prakash Sati, O., Dobhal Sati, M., Kothiyal, S. K., Semwal, D. K. & Mehta, A., 2013. A new triterpenoid saponin and antimicrobial activity of ethanolic extract from Sapindus mukorossi. Journal of Chemistry, vol. 2013, 1-6.
  • Sharma, A., Sati, S. C., Sati, O. P., Sati, D., Maneesha & Kothiyal, S.K., 2011. Chemical constituents and bioactivities of genus Sapindus. International Journal of Research in Ayurveda & Pharmacy, 2, 403–409.
  • Simbula, G., Dettori, C., Camboni, T., & Cotti, E., 2010. Comparison of tetraacetylethylendiamine plus sodium perborate and sodium hypochlorite cytotoxicity on L929 fibroblasts. Journal of Endodontics, 36, 1516–1520.
  • Spencer, H.R., Ike, V., & Brennan, P.A., 2007. Review: the use of sodium hypochlorite in endodontics — potential complications and their management. British Dental Journal. 202, 555-559.
  • Srinivasarao, M., Lakshminarasu, M., Anjum, A., & Ibrahim, M., 2015. Comparative study on phytochemical, antimicrobial and antioxidant activity of Sapindus mukorossi Gaertn. and Rheum emodi Wall. ex Meissn.: In vitro studies. Annals of Phytomedicine, 4(2), 93–97.
  • Sundqvist, G., 1992. Ecology of the root canal flora. Journal of Endodontics. 18, 427–430.
  • Tamura Y, Miyakoshi M & Yamamoto M., 2012. Application of Saponin-Containing Plants in Foods and Cosmetics, Alternative Medicine, Hiroshi Sakagami, IntechOpen, Chapter 5, pp. 85-101.
  • Trevino, E.G., Patwardhan, A.N., Henry, M.A., Perry, G., Dybdal-Hargreaves, N., Hargreaves, K.M., & Diogenes, A., 2011. Effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. Journal of Endodontics. 37(8), 1109-1115.
  • Tsuzuki, J.K., Svidzinski, T.I., Shinobu, C.S., Silva, L.F., Rodrigues-Filho, E., Cortez, D.A., & Ferreira, I.C., 2007. Antifungal activity of the extracts and saponins from Sapindus saponaria. Anais da Academia Brasileira de Ciencias. 79(4), 577-583.
  • Upadhyay, A., & Singh, D.K., 2012. Pharmacological effects of Sapindus mukorossi. Revista do Instituto de Medicina Tropical de São Paulo. 54(5), 273–280.
Yıl 2020, Cilt: 37 Sayı: 4, 111 - 118, 11.09.2020

Öz

Proje Numarası

TUBITAK; project no. 215S627

Kaynakça

  • Abou-Rass, M., & Bogen, G, 1998. Microorganisms in closed periapical lesions. International Endodontic Journal, 31(1), 39–47.
  • Ali, M., Khan, T., Fatima, K., Ali, Q.U.A., Ovais, M., Khalil AT., Ullah I., Raza A., Shinvari Z.K., Idrees, M, 2018. Selected hepatoprotective herbal medicines: Evidence from ethnomedicinal applications, animal models, and possible mechanism of actions. Phytotherapy Research, 32, 199–215.
  • Aligiannis, N., Kaplotzakis, E., Mitaku, S., & Chinou, I.B., 2001. Composition and antimicrobial activity of the essential oils of two Origanum species. Journal of Agriculture and Food Chemistry, 49, 4168–4170.
  • Alves, E.G., Vinholis, A.H.C., Casemiro, L.A., Furtado, N.A.J.C., Silva, M.L.A., Cunha, W.R., 2008. Comparative study of screening techniques for antibacterial activity evaluation of plant crude extracts and pure compounds. Quimica Nova, 31(5), 1224-1229.
  • Aneja, K.R., Joshi, R., & Sharma, C., 2010. In vitro antimicrobial activity of Sapindus mukorossi and Emblica officinalis against dental caries pathogens. Ethnobotanical Leaflets, 14, 402–412.
  • Caetano da Silva S.D., Mendes de Souza, M.G., Oliveira Cardoso, M.J., da Silva Moraes, T., Ambrósio, S.R., Sola Veneziani, R.C., & Martins, C.H., 2014. Antibacterial activity of Pinus elliottii against anaerobic bacteria present in primary endodontic infections. Anaerobe, 30, 146–152.
  • Castilho, A.L., Saraceni, C.H., Díaz, I.E., Paciencia, M.L., & Suffredini, I.B., 2013. New trends in dentistry: Plant extracts against Enterococcus faecalis. The efficacy compared to chlorhexidine. Brazilian Oral Research, 27(2), 109–115.
  • Clinical and Laboratory Standards Institute., 2015. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Twelfth Edition. M02-A12. Clinical and Laboratory Standards Institute, Wayne, PA.
  • Clinical and Laboratory Standards Institute, 2009. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. CLSI document M44-A2. 2nd ed. Wayne: Clinical and Laboratory Standards Institute.
  • Clinical and Laboratory Standards Institute, 2015. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-tenth edition, CLSI document M07-A9. Wayne: Clinical and Laboratory Standards Institute.
  • Clinical and Laboratory Standards Institute, 2008. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast; Approved Standard-Third Edition. CLSI document M27-A3. Wayne: Clinical and Laboratory Standards Institute.
  • Clinical and Laboratory Standards Institute, 2012. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard-Eighth Edition. CLSI document M11-A8. Wayne: Clinical and Laboratory Standards Institute.
  • Duarte, M.C.T., Figueira, G.M., Sartoratto, A., Rehder, V.L.G., & Delarmelina, C., 2005. Anti-Candida activity of Brazilian medicinal plants. Journal of Ethnopharmacology, 97, 305–311.
  • Dutta, A., & Kundabala, M., 2013. Antimicrobial efficacy of endodontic irrigants from Azadirachta indica: An in vitro study. Acta Odontologica Scandinavica, 71, 1594–1598.
  • Gatot, A., Arbelle, J., Lieberman, A., & Yanai-Inbar, I.,1991. Effects of sodium-hypochlorite on soft-tissues after its inadvertent injection beyond the root apex. Journal of Endodontics, 17, 573–574.
  • Herrera, D.R., Tay, L.Y., Rezende, E.C., Kozlowski, V.A., Jr, & Santos, E.B., 2010. In vitro antimicrobial activity of phytotherapic Uncaria tomentosa against endodontic pathogens. Journal of Oral Science, 52(3), 473–476.
  • Hu, Q, Chen, Y.Y., Jiao, Q.Y., Khan, A., Li, F., Han, D.F., Cao G.D., Lou, H.X., 2018. Triterpenoid saponins from the pulp of Sapindus mukorossi and their antifungal activities. Phytochemistry, 147, 1–8.
  • Ibrahim, M., Khan, A.A., Tiwari, S.K., Habeeb, M.A., Khaja, M.N., & Habibullah, C.M., 2006. Antimicrobial activity of Sapindus mukorossi and Rheumemodi extracts against H pylori: In vitro and in vivo studies. World Journal of Gastroenterology, 12(44), 7136–7142.
  • Kato, H., Taguchi, Y., Tominaga, K., Umeda, M., & Tanaka, A., 2014. Porphyromonas gingivalis LPS inhibits osteoblastic differentiation and promotes-inflammatory cytokine production in human periodontal ligament stem cells. Archives of Oral Biology, 59(2), 167–175.
  • Martinho, F.C., Leite, F.R., Nóbrega, L.M., Endo, M.S., Nascimento, G.G., Darveau, R.P., & Gomes, B.P., 2016. Comparison of Fusobacterium nucleatum and Porphyromonas gingivalis lipopolysaccharides clinically isolated from root canal infection in the induction of pro-inflammatory cytokines secretion. Brazilian Dental Journal, 27(2), 202–207.
  • Nascimento G.G.F., Locatelli, N., Freitas P.C., Silva, G.L., 2000. Antibacterial activity of plant extracts and phytochemicals on antibiotic resistant bacteria. Brazilian Journal of Microbiology, 31, 247–256.
  • Onçağ, O., Hoşgör, M., Hilmioğlu, S., Zekioğlu, O., Eronat, C., & Burhanoğlu, D., 2003. Comparison of antibacterial and toxic effects of various root canal irrigants. International Endodontic Journal, 36(6), 423–432.
  • Pallotta, R.C., Ribeiro, M.S., de Lima Machado, M.E., 2007. Determination of the minimum inhibitory concentration of four medicaments used as intracanal medication. Australian Endodontic Journal, 33, 107–111.
  • Porsche, F.M., Molitor, D., Beyer, M., Charton, S., André, C., & Kollar, A., 2018. Antifungal activity of saponins from the fruit pericarp of Sapindus mukorossi against Venturia inaequalis and Botrytis cinerea Plant Disease, 102(5), 991–1000.
  • Sathyaprasad, S., Jose, B.K., & Chandra, H. S., 2015. Antimicrobial and antifungal efficacy of Spilanthes acmella as an intracanal medicament in comparison to calcium hydroxide: An in vitro study. Indian Journal of Dental Research, 26(5), 528–532.
  • Shah, M., Parveen, Z., & Khan, M. R., 2017. Evaluation of antioxidant, antiinflammatory, analgesic and antipyretic activities of the stem bark of Sapindus mukorossi. BMC Complementary and Alternative Medicine, 17, 526.
  • Sharma, A., Chandra Sati, S., Prakash Sati, O., Dobhal Sati, M., Kothiyal, S. K., Semwal, D. K. & Mehta, A., 2013. A new triterpenoid saponin and antimicrobial activity of ethanolic extract from Sapindus mukorossi. Journal of Chemistry, vol. 2013, 1-6.
  • Sharma, A., Sati, S. C., Sati, O. P., Sati, D., Maneesha & Kothiyal, S.K., 2011. Chemical constituents and bioactivities of genus Sapindus. International Journal of Research in Ayurveda & Pharmacy, 2, 403–409.
  • Simbula, G., Dettori, C., Camboni, T., & Cotti, E., 2010. Comparison of tetraacetylethylendiamine plus sodium perborate and sodium hypochlorite cytotoxicity on L929 fibroblasts. Journal of Endodontics, 36, 1516–1520.
  • Spencer, H.R., Ike, V., & Brennan, P.A., 2007. Review: the use of sodium hypochlorite in endodontics — potential complications and their management. British Dental Journal. 202, 555-559.
  • Srinivasarao, M., Lakshminarasu, M., Anjum, A., & Ibrahim, M., 2015. Comparative study on phytochemical, antimicrobial and antioxidant activity of Sapindus mukorossi Gaertn. and Rheum emodi Wall. ex Meissn.: In vitro studies. Annals of Phytomedicine, 4(2), 93–97.
  • Sundqvist, G., 1992. Ecology of the root canal flora. Journal of Endodontics. 18, 427–430.
  • Tamura Y, Miyakoshi M & Yamamoto M., 2012. Application of Saponin-Containing Plants in Foods and Cosmetics, Alternative Medicine, Hiroshi Sakagami, IntechOpen, Chapter 5, pp. 85-101.
  • Trevino, E.G., Patwardhan, A.N., Henry, M.A., Perry, G., Dybdal-Hargreaves, N., Hargreaves, K.M., & Diogenes, A., 2011. Effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. Journal of Endodontics. 37(8), 1109-1115.
  • Tsuzuki, J.K., Svidzinski, T.I., Shinobu, C.S., Silva, L.F., Rodrigues-Filho, E., Cortez, D.A., & Ferreira, I.C., 2007. Antifungal activity of the extracts and saponins from Sapindus saponaria. Anais da Academia Brasileira de Ciencias. 79(4), 577-583.
  • Upadhyay, A., & Singh, D.K., 2012. Pharmacological effects of Sapindus mukorossi. Revista do Instituto de Medicina Tropical de São Paulo. 54(5), 273–280.
Toplam 36 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Sağlık Kurumları Yönetimi
Bölüm Clinical Research
Yazarlar

İmran Saglik 0000-0003-0864-4989

Öznur Güçlüer Tuncay 0000-0001-7835-0439

Betil Özhak 0000-0001-5224-1824

Proje Numarası TUBITAK; project no. 215S627
Yayımlanma Tarihi 11 Eylül 2020
Gönderilme Tarihi 21 Nisan 2020
Kabul Tarihi 12 Mayıs 2020
Yayımlandığı Sayı Yıl 2020 Cilt: 37 Sayı: 4

Kaynak Göster

APA Saglik, İ., Güçlüer Tuncay, Ö., & Özhak, B. (2020). Investigation of the antimicrobial effects of Sapindus mukorossi on endodontic pathogens. Journal of Experimental and Clinical Medicine, 37(4), 111-118.
AMA Saglik İ, Güçlüer Tuncay Ö, Özhak B. Investigation of the antimicrobial effects of Sapindus mukorossi on endodontic pathogens. J. Exp. Clin. Med. Eylül 2020;37(4):111-118.
Chicago Saglik, İmran, Öznur Güçlüer Tuncay, ve Betil Özhak. “Investigation of the Antimicrobial Effects of Sapindus Mukorossi on Endodontic Pathogens”. Journal of Experimental and Clinical Medicine 37, sy. 4 (Eylül 2020): 111-18.
EndNote Saglik İ, Güçlüer Tuncay Ö, Özhak B (01 Eylül 2020) Investigation of the antimicrobial effects of Sapindus mukorossi on endodontic pathogens. Journal of Experimental and Clinical Medicine 37 4 111–118.
IEEE İ. Saglik, Ö. Güçlüer Tuncay, ve B. Özhak, “Investigation of the antimicrobial effects of Sapindus mukorossi on endodontic pathogens”, J. Exp. Clin. Med., c. 37, sy. 4, ss. 111–118, 2020.
ISNAD Saglik, İmran vd. “Investigation of the Antimicrobial Effects of Sapindus Mukorossi on Endodontic Pathogens”. Journal of Experimental and Clinical Medicine 37/4 (Eylül 2020), 111-118.
JAMA Saglik İ, Güçlüer Tuncay Ö, Özhak B. Investigation of the antimicrobial effects of Sapindus mukorossi on endodontic pathogens. J. Exp. Clin. Med. 2020;37:111–118.
MLA Saglik, İmran vd. “Investigation of the Antimicrobial Effects of Sapindus Mukorossi on Endodontic Pathogens”. Journal of Experimental and Clinical Medicine, c. 37, sy. 4, 2020, ss. 111-8.
Vancouver Saglik İ, Güçlüer Tuncay Ö, Özhak B. Investigation of the antimicrobial effects of Sapindus mukorossi on endodontic pathogens. J. Exp. Clin. Med. 2020;37(4):111-8.