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Year : 2023  |  Volume : 14  |  Issue : 1  |  Page : 29-31

Antibacterial Activity of Juglans regia L. Dry Husk Extract against Streptococcus mutans and Lactobacillus: An In Vitro Study

Department of Pedodontics and Preventive Dentistry, College of Dentistry, University of Baghdad, Baghdad, Iraq

Date of Submission20-Nov-2022
Date of Decision12-Jan-2023
Date of Acceptance15-Jan-2023
Date of Web Publication20-Mar-2023

Correspondence Address:
Ola Hameed Turki
Department of Pedodontics and Preventive Dentistry, College of Dentistry, University of Baghdad, Baghdad
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/denthyp.denthyp_144_22

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Introduction: We aimed to evaluate the anti-bacterial effect of the Juglans regia dry husk ethanolic extract against Streptococcus mutans and Lactobacillus. Methods: A quantity of 200 g of dry husk was ground into powder and sieved until no large particles were found, then mixed with 1 L of 70% ethanol, yielding 20 g of J. regia paste after ethanol exclusion by vacuum evaporator and lyophilization. The Agar well diffusion method was used to detect the antibacterial activity. Statistical analysis was carried out using Welch’s t-test (unequal variances t-test) via R software. Results: The J. regia dry husk extract showed statistically significant higher bacterial growth inhibition in comparison with prophylaxis pumice paste for both S. mutans and Lactobacillus. Conclusion: Ethanolic dry husk extract has antibacterial activity against both S. mutans and Lactobacillus.

Keywords: antibacterial, dry husk, ethanolic extract, Juglans regia, Lactobacillus, Streptococus mutans

How to cite this article:
Turki OH, Jafar ZJ. Antibacterial Activity of Juglans regia L. Dry Husk Extract against Streptococcus mutans and Lactobacillus: An In Vitro Study. Dent Hypotheses 2023;14:29-31

How to cite this URL:
Turki OH, Jafar ZJ. Antibacterial Activity of Juglans regia L. Dry Husk Extract against Streptococcus mutans and Lactobacillus: An In Vitro Study. Dent Hypotheses [serial online] 2023 [cited 2023 May 30];14:29-31. Available from:

  Introduction Top

Tooth caries is one of the most commom infectious deceases worldwide, posing a significant economic challenge to goverments’ health-care systems. It is widely believed that some species of lactic acid-producing bacteria, especially Streptococcus mutans and Lactobacillus, settle on the tooth surface and cause tooth caries.

S. mutans is a facultatively anaerobic, gram-positive, biofilm-forming cocci that contains 2000 genes. It has the capability to metabolize various types of carbohydrates, relies entirely on glycolysis, and produces lactic acid. Its other virulence aspects included adherence to tooth surfaces, construction of glycogen reserves, and the capability to produce glue-like extracellular polysaccharides.[1],[2] S. mutans is one of the main pathogens related to dental caries.[3]

Lactobacillus is an anaerobe or microaerophilic gram-positive, bacilliform bacteria containing 1953 genes. According to recent research, it is not an indigenous microorganism of the oral cavity. Because of planktonic and opportunistic features, Lactobacillus required the following three things: (1) a retentive anaerobic niche; (2) a low pH environment; and (3) availability of carbohydrates, all of which are present in caries lesions. Lactobacillus is a chief contributor to tooth decay advancement.[4]

Medicinal plants have active components that can be used directly without industrial processing.[5] In recent years, consumer demand and awareness on the subject of plant-based pharmaceuticals have grown fast worldwide. Persian walnut or Juglans regia L. is one of the most widespread tree nuts, broadening from the Balkans eastward to the Middle East and southwest China. Its husk, the outer green thick layer, is an agricultural waste product that is abundant. It contains various useful chemicals such as hydrolysable tannins, sesquiterpenes, triterpenoids, steroids, alkanes, ceramides, etc. The dry husk extract of J. regia had been used as heavy metal remover, natural hair dye, natural antioxidants, antimicrobial, and antiplatelet.[6]

Nevertheless, we aimed to evaluate the anti-bacterial effect of the dry husk ethanolic extract of the J. regia against S. mutans and Lactobacillus.

  Materials and methods Top

The ethical approval was obtained from the central committee of ethics in the College of Dentistry/ (project number: 474322). Using G Power software ( with a power of 80%, two sided alpha error probability of 0.05, and Cohen’s d effect size of 0.8, the sample size was 26 for each group (total sample size = 52).

Extract preparation

The dry husk was collected from Al-Sulaimanyah city in the north of Iraq in November, and this sample was dried by exposure to sunlight and then stored in room temperaturein darkness for 1 month before being prepared to make the ethanolic extract. Dry husk (200 g) was ground into powder and sieved until no large particles were found, and prepared for mixing with 1 L of 70% ethanol (Applichem Gmbh Olloweg, Darmastdt, Germany),[7] in an ultrasonic device (RPM was 350) (Kunshan Ultrasoun., Jiangsu, China) at room temperature for 50 minutes. The resultant was inserted into vacuum evaporator (Heidolph Instruments GmbH, Schwabach, Germany) to get rid of alcohol,[8] and the final resultant was 250 mL of concentrated heavy liquid, which was lyophilized to get a paste-like consistency and saved in a closed glass container, Lyophilization took about 30 minutes.[9] According to the results of a pilot study, the resultant was 20 g which was used in the experiment in its full concentration.

Test organisms

The bacteria were isolated from the saliva of many people, suspected colonies of S. mutans and Lactobacili spp. were isolated separately as a single colony, and identification of bacteria was done based on colony morphology, and confirmation was done by Gram staining and using standard microbiological test. Then, confirmed isolates were transported to sterilized brain heart Infusion broth (BHI-B) in 10 mL and incubated for 18–24 h at 35–37°C, aerobically.

Evaluation of antimicrobial activity by zone of inhibition by well diffusion

On Mueller Hinton Agar (MHA) media, the antibacterial activity of alcoholic extract was studied using the agar well diffusion technique.[10]

A single colony was added into a test tube containing 5 mL of normal saline to yield a bacterial suspension of the modest turbidity likened to the standard turbidity solution; this nearly equals to 1.5 × 108 CFU/mL (0.5 McFarland concentration). By using a sterile cotton swab, a part of bacterial suspension was transported cautiously and regularly spread on Mueller–Hinton agar medium, then left for 10 minutes. Five-mm-diameter wells were made in the previous agar layer (three wells per plate).

As Control groups, distilled water and prophylaxis pumice paste (PD dental, Vevey, Switzerland) were impregnated in 5-mm wells. The plates were incubated for 18 hours at 37°C, and then the diameter of inhibition was recorded.

This study is a part of a series of researches attempting to introduce the J. regia dry husk extract as a polishing material. Hence prophylaxis pumice paste used as control group.

Statistical methods

Statistical analysis was carried out using Welch’s t-test (unequal variances t-test) via R software (R Foundation for Statistical Computing, Vienna, Austria).

  Results Top

Levene’s test is significant (P < 0.05), hence Welch’s t-test employed for data analysis. The J. regia dry husk extract showed statistically significant higher bacterial growth inhibition in comparison with prophylaxis pumice paste for both S. mutans and Lactobacillus [Figure 1].
Figure 1 Violin plot showed summary statistics and the probability density of the data, smoothed by a kernel density estimator, related to bacterial growth inhibition zone.

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  Discussion Top

The results obtained from this investigation confirmed the anti-bacterial activity of J. regia dry husk ethanolic extract against S. mutans and Lactobacillus.

Fernandez-Agullo et al., showed aqueous extracts of the J. regia green husk had anti-bacterial activity against the tested Gram positive bacteria including B. cereus, B. subtilis, S. aureus, and S. epidermis. They reported no anti-bacterial effect against the tested gram negative bacteria including E. coli and P. aeruginosa. In this study the peak inhibition zones were reported against S. epidermis.[11] In the same way, Oliveira et al., validated antibacterial activity of aqueous extract of J. regia husk against tested Gram positive bacteria including S. aureus, B. cereus, B. subtilis.[12] Beiki et al. used acetone–water to prepare juglans regia husk extract. They reported same antibacterial activity against tested Gram positive bacteria including B. subtilis, S. aureus.[13] Moghaddam et al., showed ethyl acetate extract of J. regia husk were able to prevent the growth of S. aureus and B. cereus. In contrast with other studies they reported that aqueous extract has no antibacterial effect against B. cereus, S. aureus (Gram positive) and E. coli (Gram negative).[14]

The Juglone enzyme (5-hydroxy-1,4-napthoquinone), which is a chemical compound found predominantly in green husk,[15] was suggested to be the anti-bacterial agent.

Additionally, the presence of phenolic chemicals, alkaloid, flavonoid, and steroids may be responsible for the antibacterial activities.[16]

Further research is warranted to evaluate the systemic safety of J. regia dry husk extract. Also, assessment of real mechanism of the anti-bacterial activity and evaluation of minimum inhibitory concentration recommended.

Limitations of this study should be noted. The lack of a well-known dental antibacterial compound such as chlorhexidine as an active control was one of weakness of this study. The S. mutans and Lactobacillus were not obtained from a reliable source and molecular tests was not done to confirm the bacteria isolation process.

Financial support and sponsorship


Conflicts of interest

The authors report no conflicts of interest.

  References Top

Lemos JA, Palmer SR, Zeng L et al. The biology of Streptococcus mutans. Microbiol Spectr 2019;7(1): 10.1128/microbiolspec.GPP3-0051-2018.  Back to cited text no. 1
Forssten SD, Björklund M, Ouwehand AC. Streptococcus mutans, caries and simulation models. Nutrients 2010;2:290-8.  Back to cited text no. 2
Juma’a Z, Fadhil R, Abdul-Hussain Y. Correlation between caries related microorganisms in the dental plaque and saliva with dental caries level in the upper and lower jaws in 5–9 years old children in Baghdad City. J Bagh Coll Dent 2016;28:132-6.  Back to cited text no. 3
Caufield PW, Schön CN, Saraithong P, Li Y, Argimón S. Oral Lactobacilli and dental caries: a model for Niche adaptation in humans. J Dent Res 2015;94(9 Suppl):110S–8S.  Back to cited text no. 4
Wahba M, Hashesh W, Abou-Baker N. Using some natural minerals to remove cadmium from polluted water. Baghdad Sci J 2022;19:1008.  Back to cited text no. 5
Jahanban-Esfahlan A, Ostadrahimi A, Tabibiazar M, Amarowicz R. A comprehensive review on the chemical constituents and functional uses of walnut (Juglans spp.) Husk. Int J Mol Sci 2019;20:3920.  Back to cited text no. 6
Hussien S, Juma’a Z. In vitro cytotoxic effect of annona squamosa pulp ex-tract as a mouthwash for children on human normal cell line. J Bagh Coll Dent 2022;34:60-6.  Back to cited text no. 7
Ayad A, Hamad M. Isolation of alkaloids from Papaver rhoeas (Papaveraceae) wildly grown in Iraq. Iraqi J Pharm Sci 2021;30:78-85.  Back to cited text no. 8
Wilson LL, Harris HM, Eans SO et al. Lyophilized kratom tea as a therapeutic option for opioid dependence. Drug Alcohol Depend 2020;216:108310.  Back to cited text no. 9
Lewus CB, Kaiser A, Montville TJ. Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat. Appl Environ Microbiol 1991;57:1683-8.  Back to cited text no. 10
Fernández-Agulló A, Pereira E, Freire M et al. Influence of solvent on the antioxidant and antimicrobial properties of walnut (Juglans regia L.) green husk extracts. Ind Crops Prod 2013;42:126-32.  Back to cited text no. 11
Oliveira I, Sousa A, Ferreira IC, Bento A, Estevinho L, Pereira JA. Total phenols, antioxidant potential and antimicrobial activity of walnut (Juglans regia L.) green husks. Food Chem Toxicol 2008;46:2326-31.  Back to cited text no. 12
Beiki T, Najafpour GD, Hosseini M. Evaluation of antimicrobial and dyeing properties of walnut (Juglans regia L.) green husk extract for cosmetics. Colorat Technol 2017;134:71-81.  Back to cited text no. 13
Moghaddam PZ, Mohammadi A, Feyzi P, Alesheikh P. In vitro antioxidant and antibacterial activity of various extracts from exocarps and endocarps of walnut. Pak J Pharm Sci 2017;30:1725-31.  Back to cited text no. 14
Ahmad T, Suzuki YJ. Juglone in oxidative stress and cell signaling. Antioxidants (Basel) 2019;8:91.  Back to cited text no. 15
Ara I, Shinwari MM, Rashed SA, Bakir MA. Evaluation of antimicrobial properties of two different extracts of Juglans regia tree bark and search for their compounds using gas chromatography-mass spectrum. Int J Biol 2013;5:92.  Back to cited text no. 16


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