Antibacterial effect of ethanolic Morus alba Linn. leaf extract against mastitis-causing Escherichia coli and Staphylococcus aureus in vitro

Main Article Content

Xayngeun Phengvongsone
Chaiyapas Thamrongyoswittayakul
Peerapol Sukon
Jareerat Aimsaard
Raktham Mektrirat


This study was conducted to determine the in vitro antibacterial effect of an ethanolic Morus alba L. (mulberry) leaf extract against Escherichia coli and Staphylococcus aureus isolated from mastitis dairy cows and against E. coli ATCC 25922 and S. aureus ATCC 25923 reference strains. The antibacterial efficacy was evaluated by a microdilution method to determine the MIC, MBC, and a time-kill assay by using azithromycin as a positive control. The data were analyzed nonparametric tests with a significance level of p<0.05. The quercetin content of the mulberry leaf extract was 83.187 ± 0.272 µg/g dried plant material, as determined by HPLC. The extract had MICs of 10, 20, 10, and 10 mg/mL and MBCs of 20, 20, 20, and 10 mg/mL for E. coli ATCC 25922, E. coli ECCM62, S. aureus ATCC 25923, and S. aureus SACM62, respectively. Quercetin had relevant MIC of 10 mg/ml and MBC of 20 mg/mL to the extract for both ATCC strains. Time-kill tests showed a complete elimination of S. aureus ATCC 25923 and S. aureus SACM62 after a 30-min exposure to 10 and 20 mg/mL M. alba extract. E. coli ATCC 25922 and E. coli ECCM62 showed reductions of 6.8 log CFU/mL after a 360 min exposure to 40 mg/mL M. alba extract. In this study, the inhibitory effect was stronger against S. aureus than against E. coli (p<0.05). Overall, the M. alba L. extract showed appreciable in vitro antibacterial efficacy against mastitis-causing E. coli and S. aureus strains and against reference ATCC strains


Download data is not yet available.

Article Details

How to Cite
Phengvongsone, X. ., Thamrongyoswittayakul, C. ., Sukon, P. ., Aimsaard, J. ., & Mektrirat, R. . (2022). Antibacterial effect of ethanolic Morus alba Linn. leaf extract against mastitis-causing Escherichia coli and Staphylococcus aureus in vitro: Veterinary Integrative Sciences, 20(3), 517–529. Retrieved from
Research Articles


Abdel-Hamid, N.S., Abdel-Khalek, H.H., Mattar, Z.A., Abou-Taleb, K.A. and Ramadan, E.M., 2017. Screening of some Egyptian plant extracts for biological activity against some pathogenic bacteria. Arab Univ. J. Agric. Sci. 25, 377-386.

Aelenei, P., Luca, S.V., Horhogea, C.E., Rimbu, C.M., Dimitriu, G., Macovei, I., Silion M, Aprotosoaie, A.C. and Miron, A., 2019. Morus- alba leaf extract: Metabolite profiling and interactions with antibiotics against Staphylococcus spp. including MRSA. Phytochem. Lett. 31, 217-224.

Amin, M.U., Khurram, M., Khattak, B. and Khan, J., 2015. Antibiotic additive and synergistic action of rutin, morin and quercetin against methicillin resistant Staphylococcus aureus. BMC Complement. Med. Ther. 15, 59. doi: 10.1186/s12906-015-0580-0.

Appiah, T., Boakye, Y.D. and Agyare, C., 2017. Antimicrobial activities and time-kill kinetics of extracts of selected ghanaian mushrooms. Evid. Based Complement. Alternat. Med. 2017.

Bi, Y., Wang, Y.J., Qin, Y., Vallverdu, R.G., Garcia, J.M., Sun, W. and Cao, Z., 2016. Prevalence of bovine mastitis pathogens in bulk tank milk in China. PLoS One. 11, 155-621.

Budiman, A., Aulifa, D.L., Kusuma, A.S.W. and Sulastri, A., 2017. Antibacterial and antioxidant activity of black mulberry (Morus nigra L.) extract for acne treatment. Pharmacogn. J. 9, 1-14.

Cervinkova, D., Vlkova, H., Borodacova, I., Makovcova, J., Babak, V., Lorencova, A., Vrtkova, I., Marosevic, D. and Jaglic, Z., 2013. Prevalence of mastitis pathogens in milk from clinically healthy cows. Vet. Med. 58, 567-575.

Chen, H., Yu, W., Chen, G., Meng, S., Xiang, Z. and He, N., 2018. Antinociceptive and antibacterial properties of anthocyanins and flavanols from fruits of black and non-black mulberries. Molecules. 23, 4. doi: 10.3390/molecules23010004.

Chotigarpa, R., Na Lampang, K., Pikulkaew, S., Okonogi, S., Silman, P. and Mektrirat, R., 2019. Antiseptic effect of natural teat dip containing lactic acid against mastitis-causing Escherichia coli. Vet. World. 12, 397-401.

de Freitas, M.M., Fontes, P.R., Souza, P.M., William Fagg, C., Neves Silva Guerra, E., de Medeiros Nóbrega, Y.K., Silveira, D., Fonseca-Bazzo, Y., Simeoni, L. A., Homem-de-Mello, M. and Oliveira Magalhães, P., 2016. Extracts of Morus nigra L. leaves standardized in chlorogenic acid, rutin and isoquercitrin: tyrosinase inhibition and cytotoxicity. PloS One. 11(9), e0163130.

Fahim, K.M., Ismael, E., Khalefa, H.S., Farag, H.S. and Hamza, D.A., 2019. Isolation and characterization of E. coli strains causing intramammary infections from dairy animals and wild birds. Int. J. Vet. Sci. Med. 7, 61-70.

Hendrix, C.M. and Sirois, M., 2007. Laboratory procedures for veterinary technicians. 5th edition. Mosby, Inc., Elsevier., St. Louis, 400 p.

Hussain, F., Rana, Z., Shafique, H., Malik, A. and Hussain, Z., 2017. Phytopharmacological potential of different species of Morus alba and their bioactive phytochemicals: A review. Asian Pac. J. Trop. Biomed. 7, 950-956.

Kampa, J., Sukolapong, V., Buttasri, A. and Charoenchai, A., 2009. Prevalence of Mycoplasma bovis and other contagious bovine mastitis pathogens in bulk tank milk of dairy cattle herds in Khon Kaen province, Thailand. Thai J. Vet. Med. 39, 275-280.

Kobus-Cisowska, J., Szczepaniak, O., Szymanowska-Powałowska, D., Piechocka, J., Szulc, P. and Dziedziński, M., 2020. Antioxidant potential of various solvent extract from Morus alba fruits and its major polyphenols composition. Cienc. Rural. 50, 1-10.

Kumar, S., Stecher, G. and Tamura, K., 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870-1874.

Kuraeiad, S., Prueksatrakun, P., Chuajeen, Y., Kooltheat, N., Sookbampen, O., Mitsuwan, W., & Kwankaew, P. (2022). Vet. Integr. Sci. 20(2): 419-430.

Lamberti, C., Gai, F., Cirrincione, S., Giribaldi, M., Purrotti, M., Manfredi, M., Marengo, E., Sicuro, B., Saviane, A., Cappellozza, S., Giuffrida, M.G. and Cavallarin, L., 2019. Investigation of the protein profile of silkworm (Bombyx mori) pupae reared on a well-calibrated artificial diet compared to mulberry leaf diet. Peer J. 7, e6723. doi: 10.7717/peerj.6723.

Lee, H.C., Chen, C.M., Wei, J.T. and Chiu, H.Y., 2018. Analysis of veterinary drug residue monitoring results for commercial livestock products in Taiwan between 2011 and 2015. J. Food Drug Anal. 26, 565-571.

Lin, S., Koh, J.J., Aung, T.T., Sin, W.L.W., Lim, F., Wang, L., Lakshminarayanan, R., Zhou, L., Tan, D.T.H., Cao, D., Beuerman, R.W., Ren, L. and Liu, S., 2017. Semisynthetic flavone derived antimicrobials with therapeutic potential against methicillin-resistant Staphylococcus aureus (MRSA). J Med. Chem. 60, 6152-6165.

Manyi-Loh, C., Mamphweli, S., Meyer, E. and Okoh, A., 2018. Antibiotic use in agriculture and its consequential resistance in environmental sources: potential public health implications. Molecules. 23, 795. doi: 10.3390/molecules23040795.

Meade, E., Savage, M., Garvey, P., Slattery, M.A. and Garvey, M., 2019. Antibiotic resistant zoonotic pathogens of bovine mastitis and possible agents of foodborne disease. Cohesive J. Microbiol. Infect. Dis. 2, 1-11.

Mesquita, A.A., Rocha, C.M.B.M., Bruhn, F.R.P., Custódio, D.A.C., Braz, M.S., Pinto, S.M., Silva, D.B. and Costa, G.M., 2019. Staphylococcus aureus and Streptococcus agalactiae: prevalence, resistance to antimicrobials, and their relationship with the milk quality of dairy cattle herds in Minas Gerais state, Brazil. Pesqui. Vet. Bras. 39, 308-316.

Miljkovic, V., Nikolic, G., Mihajlov-Krstev, T.M. and Arsic, B., 2018. Antibacterial activities of fruits extracts of three mulberry species (Morus alba L., Morus rubra L. and Morus nigra L.) and bilberry (Vacinium myrtillus L.). Acta Med. Median. 57, 5-12.

Pang, D., Wang, W., Li, E., Shen, W., Mu, L., Liao, S. and Liu, F., 2019. Sanggenon D from root bark of mulberry inhibits the growth of Staphylococcus aureus by moderating the fatty acid biosynthesis system. Ind. Crops Prod. 140, 111719.

Pangprasit, N., Srithanasuwan, A., Suriyasathaporn, W. and Chaisri, W. 2021. Antibacterial properties of lauric acid in combination with organic acids against major pathogens causing dairy mastitis. Vet. Integr. Sci. 19(1), 37-44.

Peanparkdee, M., Iwamoto, S., Borompichaichartkul, C., Duangmal, K. and Yamauchi, R., 2016. Microencapsulation of bioactive compounds from mulberry (Morus alba L.) leaf extracts by protein-polysaccharide interactions. Int. J. Food Sci. 51, 649-655.

Polumackanycz, M., Sledzinski, T., Goyke, E., Wesolowski, M., Viapiana, A., 2019. A comparative study on the phenolic composition and biological activities of Morus alba L. commercial samples. Molecules. 24, 2-19.

Pothinuch, P. and Tongchitpakdee S., 2019. Phenolic analysis for classification of mulberry (Morus spp.) leaves according to cultivar and leaf age. J. Food Qual. 2019.

Pulbutr, P., Rattanakiat, S., Khunawattanakul, W., Saramunee, K. and Sungthong, B., 2018. Development of chewing gum containing mulberry leaf extract with anti-cariogenic activity against Streptococcus mutans. J. Biol. Sci. 18, 407-414.

Quinn, P.J., Markey, B.K., Carter, M.E., Donnelly, W.J. and Leonard, F.C., 2001. Veterinary Microbiology and Microbial Disease. Blackwell Science, Oxford, UK, 544 p.

Rahman, H.S., Muhammad, K.S., Sattar, F.A., Abdullah, R., Othman, H.H., Yeap, S.K., Chartrand, M.S. and Amin, K.M., 2018. The effectiveness of super ovulation and multiple pregnancies in sprague dawley rat using Morus alba Linn. fruit. Int. J. Med. Res. Health Sci. 7, 17-26.

Rohela, G.K., Shukla, P., Muttanna, Kumar, R. and Chowdhury, S.R. 2020. Mulberry (Morus spp.): An ideal plant for sustainable development. Trees For. People. 2, 100011.

Royster, E. and Wagner, S., 2015. Treatment of mastitis in cattle. Vet. Clin. North Am. Food Anim. Pract. 31, 17-46.

Strommenger, B., Kettlitz, C., Werner, G. and Witte, W. 2003. Multiplex PCR assay for Simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J. Clin. Microbiol. 41, 4089-4094.

Suriyaprom, S., Kaewkod, T., Promputtha, I., Desvaux, M. and Tragoolpua, Y. 2021. Evaluation of antioxidant and antibacterial activities of white mulberry (Morus alba L.) fruit extracts. Plants. 10(12), 2736.

Takahashi, T., Kaneko, M., Mori, Y., Tsuji, M., Kikuchi, N. and Hiramune, T. 1997. Phylogenetic analyses of Staphylococcus based on the 16S rDNA sequence and assignment of clinical isolates from animals. J. Vet. Med. Sci. 59(9), 775-83.

Tallini, L.R., Pedrazza, G.P., Bordignon, S.A.L., Costa, A.C., Steppe, M., Fuentefria, A. and Zuanazzi. J.A., 2015. Analysis of flavonoids in Rubus erythrocladus and Morus nigra leaves extracts by liquid chromatography and capillary electrophoresis. Rev. Bras. Farmacogn. 25, 219-227.

Wang, G., Clark, C.G., Rodgers, F.G., 2002. Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157:H7 serotype, and components of the type 2 Shiga toxin family by multiplex PCR. J. Clin. Microbiol. 40, 3613-3619.

Wu, S.C., Han, F., Song, M.R., Chen, S., Li, Q., Zhang, Q., Zhu, K. and Shen, J.Z., 2019. Natural flavones from Morus alba against methicillin-resistant Staphylococcus aureus via Targeting the Proton Motive Force and Membrane Permeability. J. Agric. Food Chem. 67, 10222-10234.

Yu, L., Shang, F., Chen, X., Ni, J., Yu, L., Zhang, M., Sun, D. and Xue, T., 2018. The anti-biofilm effect of silver-nanoparticle-decorated quercetin nanoparticles on a multi-drug resistant Escherichia coli strain isolated from a dairy cow with mastitis. Peer J. 6, e5711. doi: 10.7717/peerj.5711.

Zhai, K.F., Duan, H., Shi, S.X., Liu, L.L., Cao, W.G., Gao, G.Z., Shan, L.L., 2018. Synchronised determination of chlorogenic acid and five flavonoids in mulberry leaves using HPLC with photodiode array detection. Qual. Assur. Saf. Crop. Foods. 10, 175-182.